A HPLC fingerprint of Atractylodes macrocephala and its detection method, and a method for screening quality-marking components of Atractylodes macrocephala

Abstract

The present invention provides a method for detecting the HPLC fingerprint of Atractylodes macrocephala. The present invention provides an Atractylodes macrocephala medicinal material, which uses the above-mentioned detection method to detect the content of four components: atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone. Each gram of Atractylodes macrocephala medicinal material contains 0.21-0.32 mg of atractylodes lactone I, 0.13-0.25 mg of atractylodes lactone II, 0.1-0.19 mg of atractylodes lactone III, and 5.21-14.08 mg of atractylodes ketone. The present invention also provides an HPLC fingerprint of Atractylodes macrocephala and a method for determining the quality of Atractylodes macrocephala medicinal material. The present invention also provides a method for screening quality-marking components of Atractylodes macrocephala medicinal material. The present invention adopts the HPLC-DAD multi-wavelength switching method to establish the HPLC fingerprint of Atractylodes macrocephala, conducts chemometric research at the same time, and determines the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and atractylodesone. On the one hand, it can comprehensively evaluate the quality of Atractylodes macrocephala, provide a reference for the quality evaluation and control of Atractylodes macrocephala medicinal materials and decoction pieces, and also lay the foundation for the research on its pharmacological substance basis and related compound research; on the other hand, it can provide a reference for the improvement of the quality standard of Atractylodes macrocephala.

Description

Technical Field

[0001] The invention relates to an HPLC fingerprint of Atractylodes macrocephala and a detection method thereof, and a method for screening quality-marking components of Atractylodes macrocephala medicinal materials, and belongs to the field of medicinal material quality detection. Background Art

[0002] Atractylodes macrocephala is the dried rhizome of the Asteraceae family. It is warm in nature, bitter and sweet in flavor, and enters the spleen and stomach meridians. It has the effects of invigorating the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and stabilizing pregnancy. It is a major medicinal herb in my country, primarily distributed in Zhejiang, Anhui, Hubei, and Hunan. It is a component ingredient in many famous prescriptions. Thirteen of the 100 prescriptions in the first batch of classic prescriptions published by the State Administration of Traditional Chinese Medicine contain Atractylodes macrocephala, and three of the seven prescriptions in the second batch of classic prescriptions (pediatric section) contain Atractylodes macrocephala. Modern research indicates that Atractylodes macrocephala contains chemical components such as volatile oils, polysaccharides, and lactones, and possesses multiple pharmacological effects, including anti-tumor, anti-inflammatory, antidepressant, and gastrointestinal regulation. It holds broad market application prospects, but quality standards for Atractylodes macrocephala remain incomplete. The 2020 edition of the Chinese Pharmacopoeia does not include content determination and fingerprint analysis for Atractylodes macrocephala. Research on establishing fingerprint and content determination methods for Atractylodes macrocephala is of great significance, and the selection of quality control indicators is particularly critical. Atractylodes lactones and atractylodes ketones are the main active ingredients of Atractylodes macrocephala. It is of great significance to use atractylodes ketones and atractylodes lactones as detection indicators to control the quality of Atractylodes macrocephala. Regarding the quality research of Atractylodes macrocephala, most of them focus on the research of Atractylodes macrocephala fingerprint [Chen Xiangdong, Zhang Guangda, Lan Xiaoyong, et al. Study on the quality evaluation of Atractylodes macrocephala by HPLC fingerprint [J]. Chinese Materia Medica, 2013, 36(02): 208-212. DOI: 10.13863 / j.issn1001-4454.2013.02.007. Sun Xue, Wen Hongmei, Cui Xiaobing, et al. Qualitative identification and evaluation of the quality of Atractylodes macrocephala from different origins by HPLC-PDA fingerprint combined with UFLC-Q-TOF / MS [J [J]. Chinese Herbal Medicine, 2016, 47(19): 3494-3501.], or content determination research [Yin Hua, Wang Zhiqing, Wang Ling, etc. Simultaneous determination of the content of atractylodes lactone I, II, III and atractylodesone in Atractylodes macrocephala by HPLC-DAD wavelength switching method [J]. Chinese Journal of Traditional Chinese Medicine, 2013, 28(01): 233-236.], or determination of its fingerprint and content under a single wavelength [Lin Shuang, Wang Jie, Gao Shanshan, etc. Evaluation of the quality of Atractylodes macrocephala based on chemometrics combined with UPLC [J]. Modern Chinese Medicine Research and Practice,

[0003] 2022, 36(05): 54-59. DOI: 10.13728 / j.1673-6427.2022.05.010.]. There are few studies on the quality markers of Atractylodes macrocephala. Lin Shuang et al. used UPLC to establish the fingerprint of Atractylodes macrocephala. They screened out 9 differential components affecting the quality of Atractylodes macrocephala by principal component analysis and partial least squares discriminant analysis, identified 5 compounds (neochlorogenic acid, cryptochlorogenic acid, chlorogenic acid, atractylodes lactone III and atractylodes lactone II) and determined their contents [Lin Shuang, Wang Jie, Gao Shanshan et al. Evaluation of the quality of Atractylodes macrocephala based on chemometrics combined with UPLC [J]. Modern Chinese Medicine Research and Practice, 2022, 36(05): 54-59. DOI: 10.13728 / j.1673-6427.2022.05.010.]. The content of lactone components in Atractylodes macrocephala is low, and the spectral absorption is different. Existing detection methods are difficult to effectively control the quality of Atractylodes macrocephala. Compared with a single wavelength, multi-wavelength switching can make the chromatographic peak reach the ideal response value and more accurately and intuitively reflect the information of the chromatographic peak under multiple components [Shi Chongjing, Li Yuke, Liu Xiaomei, et al. HPLC-DAD multi-wavelength switching fingerprint combined with chemometrics to evaluate the medicinal quality of Sichuan Cyathula from different origins [J]. Chinese Journal of Traditional Chinese Medicine, 2019, 34(06): 2431-2436.]. Using multi-wavelength switching to establish the fingerprint of traditional Chinese medicine with low content of index components and simultaneously determine their content can effectively control the quality of the traditional Chinese medicine and reduce the detection cost.

[0004] According to current reports, it is still difficult to achieve quality control of Atractylodes macrocephala. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides an HPLC fingerprint of Atractylodes macrocephala and a detection method thereof, as well as a method for screening quality-marking components of Atractylodes macrocephala medicinal materials.

[0006] The present invention provides a method for detecting the HPLC fingerprint of Atractylodes macrocephala, which comprises the following steps:

[0007] a. Weigh the Atractylodes macrocephala, add water or organic solvent and ultrasonically extract for 15-60 min at a material-liquid ratio of 1:10-50, filter, and collect the filtered product;

[0008] b. Prepare reference solution: Take reference substances Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III, and Atractylodes ketone, dissolve them in methanol and dilute to volume to prepare reference solution;

[0009] c. Establishment of Atractylodes macrocephala fingerprint using high performance liquid chromatography-diode array detector (HPLC-DAD) multi-wavelength switching method: Chromatographic conditions: Chromatographic column: Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-0.05% phosphoric acid water (B), gradient elution: 12%~20% A (0~7min), 20%~58% A (7~19min), 58%~63% A (19~29min), 63%~68% A (29~43min), 68%~75% A (43~48min), 75%~95% A (48~51min), 95%~95% A (51~66min); flow rate: 1mL / min; detection wavelength: 0~32min, 220nm; 32~38.5min, 276nm: 38.5~54min, 242nm: 54~66min, 220nm; column temperature: 25-35℃; injection volume: 10μL.

[0010] The organic solvent in step a is methanol, ethanol, acetonitrile, ethyl acetate, 50% methanol, or 70% methanol; the ultrasonic extraction is performed for 30 min; the material-liquid ratio is 1:25; and the column temperature in step c is 25° C. Furthermore, preferably, the organic solvent in step a is methanol.

[0011] The present invention provides a medicinal material of Atractylodes macrocephala, which adopts the detection method to detect the contents of four components, namely, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone. Each gram of the medicinal material contains 0.21-0.32 mg of atractylodes lactone I, 0.13-0.25 mg of atractylodes lactone II, 0.1-0.19 mg of atractylodes lactone III, and 5.21-14.08 mg of atractylodes ketone.

[0012] The present invention provides an HPLC fingerprint of Atractylodes macrocephala, which is obtained by the detection method, including the following characteristic peaks and relative retention times:

[0013] Peak 1: 0.39±0.05; Peak 2: 0.42±0.05; Peak 3: 0.50±0.05; Peak 4: 0.54±0.05; Peak 5: 0.65±0.05; Peak 6: 0.96±0.05; Peak 7: 0.99±0.05; Peak 8: 1.00±0.05; Peak 9: 1.13±0.05.

[0014] The fingerprint is as follows Figure 1 shown.

[0015] The present invention provides a method for judging the quality of Atractylodes macrocephala, which comprises the following steps:

[0016] a. Using the detection method to obtain the fingerprint of Atractylodes macrocephala;

[0017] b. The peak areas of the nine common peaks were imported into IBM SPSS Statistics 25.0 software, and principal component analysis was performed on the above 37 × 9 order data matrix to determine the three principal components;

[0018] c. Establish a comprehensive scoring model: F = (45.580F1 + 32.000F2 + 15.569F3) / 93.148, where F1, F2, and F3 represent the information expressed by the three principal components as the components of Atractylodes macrocephala;

[0019] d. Judge the quality of Atractylodes macrocephala based on the score.

[0020] The present invention also provides a method for screening quality-marking components of Atractylodes macrocephala, which comprises the following steps:

[0021] a. Obtaining a fingerprint of Atractylodes macrocephala using the detection method according to any one of claims 1 to 3;

[0022] b. Hierarchical cluster analysis (HCA): The relative peak areas of the common peaks were imported into IBM SPSS Statistics 25.0 software, and the median clustering method was used to perform a hierarchical cluster analysis with cosine distance as the classification basis.

[0023] c. Based on the cluster analysis results, partial least squares discriminant analysis (PLS-DA) was performed on the medicinal samples, and a two-dimensional scatter plot was drawn;

[0024] d. Screen the characteristic components that cause differences between Atractylodes macrocephala samples.

[0025] The quality-marking components of the Atractylodes macrocephala medicinal material screened by the screening method are characterized in that they include the following five components: atractylodes lactone III, atractylodes lactone II, atractylodes lactone I, atractylodes ketone, and an unknown component corresponding to the chromatographic peak No. 9.

[0026] The present invention adopts the HPLC-DAD multi-wavelength switching method to establish the HPLC fingerprint of Atractylodes macrocephala, and simultaneously conducts chemometric research and determines the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and atractylodesone. On the one hand, the quality of Atractylodes macrocephala can be comprehensively evaluated, providing a reference for the quality evaluation and control of Atractylodes macrocephala medicinal materials and decoction pieces, and also laying the foundation for the research on the basic efficacy substances and related compound prescriptions; on the other hand, it can provide a reference for the improvement of the quality standard of Atractylodes macrocephala. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 HPLC chromatogram of ethyl acetate extraction

[0028] Figure 2 HPLC chromatogram of acetonitrile extraction

[0029] Figure 3 HPLC chromatogram of ethanol extraction

[0030] Figure 4 Methanol extraction HPLC chromatogram

[0031] Figure 5 Water extraction HPLC chromatogram

[0032] Figure 6 HPLC chromatogram of acetone extraction

[0033] Figure 7 HPLC chromatogram after 70% methanol extraction

[0034] Figure 8 HPLC chromatogram after 50% methanol extraction

[0035] Figure 9 Phenoment Gemini C18 column chromatogram

[0036] Figure 10 Agilent ZORBAX Eclipse XDB-C18 column chromatogram

[0037] Figure 11 Agilent ZORBAX Extend-C18 column chromatogram

[0038] Figure 12 Methanol-water HPLC chromatogram

[0039] Figure 13 Acetonitrile-water HPLC chromatogram

[0040] Figure 14 HPLC chromatogram of acetonitrile-0.05% phosphoric acid water

[0041] Figure 15 HPLC chromatogram at 25℃

[0042] Figure 16 HPLC chromatogram at 30℃

[0043] Figure 17 35℃ HPLC chromatogram

[0044] Figure 18 Ultrasonic extraction HPLC chromatogram

[0045] Figure 19 Reflux extraction HPLC chromatogram

[0046] Figure 20 HPLC chromatogram of material-liquid ratio investigation

[0047] Figure 21Ultrasound time study

[0048] Figure 22 HPLC fingerprint and reference spectrum of Atractylodes macrocephala slices

[0049] Figure 23 HPLC fingerprint and reference spectrum of Atractylodes macrocephala

[0050] Figure 24 Reference Atlas R

[0051] Figure 25 HPLC chromatogram of mixed reference solution (including 2. Atractylodes lactone III; 4. Atractylodes lactone II; 5. Atractylodes lactone I; 8. Atractylodes ketone)

[0052] Figure 26 Dendrogram of cluster analysis of 37 batches of Atractylodes macrocephala

[0053] Figure 27 Principal component scree plot of Atractylodes macrocephala samples

[0054] Figure 28 PLS-DA score graph of Atractylodes macrocephala samples

[0055] Figure 29 Variable Importance Projection Plot DETAILED DESCRIPTION

[0056] Example 1 Chromatographic Condition Screening Test of Atractylodes macrocephala HPLC Fingerprint of the Present Invention

[0057] 1. Extraction solvent investigation

[0058] 1.1 Preparation of test solution Take about 1.00g of Atractylodes macrocephala powder, accurately weigh it, and place it in a stoppered centrifuge tube. Accurately add 25mL each of methanol, ethanol, acetonitrile, ethyl acetate, water, acetone, 50% methanol, and 70% methanol, weigh the mass, and ultrasonically extract for 30min. Cool, weigh the mass, add the corresponding solvent to make up the weight loss, shake well, filter through a 0.45μm microporous membrane, and take the filtrate to prepare the test solution prepared with the corresponding solvent. Analyze according to the chromatographic conditions in 1.2. The chromatogram is shown in Figures 1-8 The results showed that methanol extraction resulted in more chromatographic peaks and a relatively stable baseline, so methanol was determined to be the extraction solvent.

[0059] 1.2 Chromatographic conditions: Agilent ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) water (B), gradient elution: 5% A (0-10 min), 5%-14% A (10-15 min), 14%-55% A (15-20 min), 55%-66% A (20-35 min), 66% A (35-50 min), 66%-95% A (50-60 min), 95% A (60-70 min); flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 10 μL.

[0060] 2. Chromatographic column inspection

[0061] 2.1 Chromatographic conditions: Mobile phase: acetonitrile (A) + water (B), gradient elution: 5% to 95% A (0 to 60 min), 95% A (60 to 80 min), flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 20 μL.

[0062] The same sample solution was measured using Phenoment Gemini C18 (250mm×4.6mm, 5μm), Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm), and Agilent ZORBAX Eclipse XDB-C18 (250mm×4.6mm, 5μm) columns according to "2.1 Chromatographic Conditions". After comparison, the Agilent ZORBAX Extend-C18 column showed better separation performance. See the chromatogram for details. Figures 9-11 .

[0063] 3. Mobile phase investigation

[0064] Take the same test solution and measure it under the following chromatographic conditions:

[0065] 1) Chromatographic column: Agilent ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: methanol (A)-water (B), gradient elution: 5%–21% A (0–10 min), 21%–68% A (10–15 min), 68%–100% A (15–60 min), 100% A (60–75 min); flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 20 μL.

[0066] 2) Chromatographic column: Agilent ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A) water (B), gradient elution: 5% A (0–10 min), 5%–14% A (10–15 min), 14%–55% A (15–20 min), 55%–66% A (20–35 min), 66% A (35–50 min), 66%–95% A (50–60 min), 95% A (60–70 min); flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 20 μL.

[0067] 3) Chromatographic column: Agilent ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.05% phosphoric acid (B), gradient elution: 5% A (0-10 min), 5%-14% A (10-15 min), 14%-55% A (15-20 min), 55%-66% A (20-35 min), 66% A (35-50 min), 66%-95% A (50-60 min), 95% A (60-70 min); flow rate: 1 mL / min; detection wavelength: 220 nm; column temperature: 30°C; injection volume: 20 μL.

[0068] The results showed that when acetonitrile-0.05% phosphoric acid water was used as the mobile phase, the chromatogram had more chromatographic peaks and better peak shape. Figure 12-14 .

[0069] 4. Column temperature inspection

[0070] 4.1 Chromatographic conditions

[0071] Chromatographic column: Agilent ZORBAX Extend-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: acetonitrile (A)-0.05% phosphoric acid water (B), gradient elution: 12%-20% A (0-7 min), 20%-58% A (7-24 min), 58%-70% A (24-49 min), 70%-90% A (49-69 min), 90% A (69-84 min); flow rate: 1 mL / min; detection wavelength: 0-36 min, 222 nm; 36-41 min, 276 nm: 41-56 min, 242 nm: 56-84 min, 222 nm; injection volume: 5 μL.

[0072] Take the same test solution and measure it under the above chromatographic conditions at column temperatures of 25℃, 30℃, and 35℃. After comparison, the chromatographic peak separation effect is better when the column temperature is 25℃, so the column temperature is determined to be 25℃. Figure 15-17 .

[0073] 5. Investigation of extraction methods

[0074] Take about 1.00g of Atractylodes macrocephala powder, make two copies, accurately weigh, place in a stoppered centrifuge tube, accurately add 25mL of methanol, weigh the mass, reflux or ultrasonic extraction for 60min, cool, weigh the mass, make up the weight loss with methanol, shake well, filter through a 0.45μm microporous membrane, take the filtrate, and prepare the test solution. Analyze according to the chromatographic conditions of 1.2 (detection wavelength is 220nm), the chromatogram is shown in Figure 18-19 The results showed that the chromatogram peak shapes of the two extraction methods were similar, and there was no significant difference in the peak areas of the same chemical components, so ultrasonic extraction was selected.

[0075] 6. Material-liquid ratio investigation

[0076] Take about 1.00g of Atractylodes macrocephala powder, accurately weigh it, and place it in four copies in a stoppered centrifuge tube. Accurately add 10, 20, 25, and 50mL of methanol, weigh it, ultrasonicate it for 30 minutes, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it through a 0.45μm microporous membrane, and take the filtrate to prepare the test solution. Analyze it according to the chromatographic conditions in 4.1. The chromatogram is shown in the figure. Figure 20 After comparison, there was no significant difference in the peak area of ​​each chromatographic peak (after conversion to volume) between the material-liquid ratio of 1:25 and 1:50, and it was higher than the peak area of ​​each chromatographic peak at the material-liquid ratio of 1:10 and 1:20, so the extraction material-liquid ratio was determined to be 1:25.

[0077] 7. Ultrasound Time Investigation

[0078] Take about 1.00g of Atractylodes macrocephala powder, accurately weigh it, and place it in triplicate in a stoppered centrifuge tube. Accurately add 25mL of methanol to each tube and weigh it. Ultrasonicate for 15, 30, and 60 minutes, cool it, weigh it, add methanol to make up the weight loss, shake it well, filter it through a 0.45μm microporous membrane, and take the filtrate to prepare the test solution. Analyze according to the chromatographic conditions in 4.1. The chromatogram is shown in the figure. Figure 21 After comparison, the peak areas of each chromatographic peak were the lowest after 15 minutes of ultrasound, and there was no significant difference in the peak areas of each chromatographic peak between 30 minutes and 60 minutes of ultrasound, so the extraction ultrasound time was determined to be 30 minutes.

[0079] 8. Relative retention time of common peaks

[0080]

[0081] 9. Establishment of HPLC fingerprint of Atractylodes macrocephala slices

[0082] Take 15 batches of raw Atractylodes macrocephala slices samples, prepare the test solution according to the method under "2.3" of Example 2, and determine the fingerprint of Atractylodes macrocephala slices according to the method under "2.1" of Example 2. Figure 22 (Comparison spectrum after magnification). The similarity between the chromatograms of 15 batches of Atractylodes macrocephala samples and the reference spectrum ranged from 0.994 to 1, and the relative retention times of the nine common peaks were 0.39, 0.42, 0.50, 0.54, 0.65, 0.96, 0.99, 1.00, and 1.13, respectively, which were consistent with the relative retention times of the common peaks in the fingerprint of Atractylodes macrocephala.

[0083] Example 2 Detection method of Atractylodes macrocephala HPLC fingerprint of the present invention

[0084] 1 Instruments and Materials

[0085] 1.1 Instruments: Agilent 1200 high-performance liquid chromatograph (Agilent Technologies, USA); XS205 electronic balance (Mettler Toledo, Germany); KH7200E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); Heraeus Multifuge X1R high-speed refrigerated centrifuge (Thermo Fisher Scientific (China) Co., Ltd.).

[0086] 1.2 Materials and Reference Substances: Atractylodes lactone I (Batch No. 111975-201501), Atractylodes lactone II (Batch No. 111976-201501), and Atractylodes lactone III (Batch No. 111978-201501) were purchased from the China Food and Drug Administration; atractylone (Batch No. MUST-22050910) was purchased from Chengdu Munster Biotechnology Co., Ltd. Acetonitrile was chromatographically pure; water was ultrapure; all other reagents were analytically pure. All 37 batches of Atractylodes macrocephala were collected from the local production area and identified by Fang Qingmao, Director of the Resource Institute of the Sichuan Academy of Traditional Chinese Medicine, as the dried rhizome of Atractylodes macrocephala Koidz. (Asteraceae). Sample source information is provided in Table 1.

[0087] Table 1 Information on the source of Atractylodes macrocephala

[0088]

[0089]

[0090] 2 Methods and Results

[0091] 2.1 Chromatographic conditions Column: Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-0.05% phosphoric acid water (B), gradient elution: 12%~20% A (0~7min), 20%~58% A (7~19min), 58%~63% A (19~29min), 63%~68% A (29~43min), 68%~75% A (43~48min), 75%~95% A (48~51min), 95%~95% A (51~66min); flow rate: 1mL / min; detection wavelength: 0~32min, 220nm; 32~38.5min, 276nm: 38.5~54min, 242nm: 54~66min, 220nm; column temperature: 25℃; injection volume: 10μL.

[0092] 2.2 Preparation of reference solution Take appropriate amount of reference substances Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III and Atractylodes ketone, weigh accurately, dissolve in methanol and make up to 10 mL to obtain reference solution with concentrations of 1.384, 1.500, 1.825 and 2.008 mg / mL respectively.

[0093] 2.3 Preparation of test solution: Take about 1.00 g of Atractylodes macrocephala powder, accurately weigh it, place it in a stoppered centrifuge tube, accurately add 25 mL of methanol, weigh it, extract it by ultrasonic for 30 min, cool it, weigh it, make up the weight loss with methanol, shake it well, filter it through a 0.45 μm microporous membrane, and take the filtrate.

[0094] 2.4 Methodological Investigation

[0095] 2.4.1 Precision Test: Take the same batch of test sample solution (S19) and inject it six times continuously according to the chromatographic conditions in "2.1". Record the chromatogram. Using atractylodesone (peak 8) as the reference peak, calculate the relative retention time RSD of each common peak to be less than 0.07%, and the relative peak area RSD is less than 1.13%, indicating good instrument precision.

[0096] 2.4.2 Stability test Take the same batch of test solution (S19) and inject the sample at 0, 4, 8, 12, 24 and 48 h according to the chromatographic conditions under "2.1". Record the chromatogram. Using atractylodesone as the reference peak, the relative retention time RSD of each common peak is calculated to be less than 0.09%, and the relative peak area RSD is less than 3.43%, indicating that the test solution has good stability within 48 h.

[0097] 2.4.3 Repeatability test Six portions of the same Atractylodes macrocephala sample (S19) were taken, and the test solution was prepared according to the method under "2.3". The solution was analyzed according to the chromatographic conditions under "2.1". The chromatogram was recorded. Using atractylodesone as the reference peak, the relative retention time RSD of each common peak was calculated to be less than 0.05%, and the relative peak area RSD was less than 3.70%, indicating that the method had good repeatability.

[0098] 2.5 Fingerprint establishment and similarity evaluation Take 37 batches of Atractylodes macrocephala samples, prepare the test solution according to the method under "2.3", inject the samples in sequence according to the chromatographic conditions under "2.1", record the chromatograms, and import the data into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Use the S12 sample spectrum as the reference spectrum, set the time window width to 0.1 min, use the median method, and establish the superimposed spectrum and the reference spectrum R through multi-point calibration and Mark peak matching. Figure 23 , Figure 24 A total of 9 common peaks were calibrated and compared with the reference substance ( Figure 25 ) were compared with the reference peaks, and four chromatographic peaks were identified: atractylodes lactone III (peak 2), atractylodes lactone II (peak 4), atractylodes lactone I (peak 5), and atractylodesone (peak 8). Similarity evaluation was performed using the reference spectrum as a reference. The similarity evaluation results are shown in Table 2. The similarity between the 37 batches of Atractylodes samples and the reference spectrum ranged from 0.539 to 0.996. With the exception of S16, the similarity between the remaining samples and the reference spectrum was above 0.84, indicating that the established fingerprint method can be used for overall quality control of Atractylodes macrocephala. Using atractylodesone, peak 8, which has a large peak area and high resolution and is the active ingredient of Atractylodes macrocephala, as the reference peak (S), the relative retention time RSDs of the common peaks were calculated to be less than 0.2%, and the relative peak area RSDs ranged from 32.59% to 96.17% (Table 3), indicating that the chemical components contained in different batches of samples were similar in type but with varying contents.

[0099] Table 2 Similarity evaluation results of 37 batches of samples

[0100]

[0101] Table 3 Relative peak areas and RSD values ​​of common peaks in 37 batches of samples

[0102]

[0103]

[0104] 2.6 Chemometric analysis

[0105] 2.6.1 Hierarchical Cluster Analysis (HCA) The relative peak areas of the common peaks were imported into IBM SPSS Statistics 25.0 software, and the median clustering method was used to perform a systematic cluster analysis with cosine distance as the classification basis. The results are shown in Figure 26 When the discriminant distance was 5, 37 batches of Atractylodes macrocephala samples were clustered into 3 categories, S23 to S37 were clustered into 1 category, S16 was clustered into 1 category alone, and the rest of the samples were clustered into 1 category. When the discriminant distance was 10, 37 batches of Atractylodes macrocephala samples were clustered into 2 categories, S16 was clustered into 1 category alone, and the rest of the samples were clustered into 1 category, which was consistent with the similarity evaluation results.

[0106] 2.6.2 Principal Component Analysis (PCA)

[0107] The peak areas of the nine common peaks of 37 batches of Atractylodes macrocephala samples were imported into IBM SPSS Statistics 25.0 software, and principal component analysis was performed on the above 37 × 9 order data matrix. The KMO test statistic was 0.708, and the Bartlett's sphericity test statistic was 0.000, which was suitable for principal component analysis. The principal component eigenvalues ​​and variance contribution rates, component matrix, and principal component factor score matrix are shown in Tables 4, 5, and 6, respectively. The scree plot is shown in Table 4. Figure 27 Three principal components were extracted using the eigenvalue λ>1 as the standard. The cumulative variance contribution rate of the three principal components was 93.1481%, indicating that the first three principal components can represent 93.1481% of the sample information. As shown in Table 6, peaks 6, 7, 8 (atractylone), and 9 contribute most to principal component 1, peaks 2 (atractylodes lactone III), 4 (atractylodes lactone II), and 5 (atractylodes lactone I) contribute most to principal component 2, and peak 1 contributes most to principal component 3. Therefore, these components are important factors affecting the quality of Atractylodes macrocephala.

[0108] The information expressed by the components of the 37 batches of Atractylodes macrocephala samples was expressed by the three principal components F1, F2, and F3. The principal component scores of the Atractylodes macrocephala samples were further calculated by combining the data in Tables 4 and 5, thereby obtaining a comprehensive scoring model: F = (45.580F1 + 32.000F2 + 15.569F3) / 93.148. The results are shown in Table 7. The samples collected in April 2022 (S23-S37) scored higher and ranked concentrated and high, indicating that the newly collected Atractylodes macrocephala samples in Pan'an, Zhejiang are of good quality, which may be related to the storage status of the samples. For the remaining samples, S16 (Sichuan Atractylodes macrocephala) ranked fifth. The average ranking of the three origin samples S1-S15 and S17-S22 was highest in Hubei, and Zhejiang was slightly higher than Anhui, indicating that the quality of this batch of Atractylodes macrocephala samples was the best in Hubei, in the middle, and in Anhui.

[0109] Table 4 Eigenvalues ​​and variance contribution rates

[0110]

[0111] Table 5 Component matrix

[0112]

[0113] Table 6 Principal component factor score matrix

[0114]

[0115]

[0116] Table 7 Principal component scores and comprehensive scores of Atractylodes macrocephala samples

[0117]

[0118]

[0119] 2.6.3 Partial Least Squares Discriminant Analysis (PLS-DA)

[0120] According to the cluster analysis results, supervised PLS-DA discriminant analysis was performed on 37 batches of samples, and a two-dimensional scatter plot was drawn. Figure 28 The deviation of S16 is large, and the remaining samples can still be divided into two categories except S24 and S34, but the degree of dispersion is large, indicating that there are large differences in quality between the samples. In order to further screen the characteristic components that cause the differences between Atractylodes macrocephala samples, VIP value > 1 was used as the evaluation standard, and 5 chemical components including chromatographic peaks 2 (Atractylodes lactone III), 4 (Atractylodes lactone II), 5 (Atractylodes lactone I), 8 (Atractylodes ketone), and 9 were screened out. Figure 29 , these five components are the characteristic components that affect the quality differences of Atractylodes macrocephala samples.

[0121] 2.7 Content determination

[0122] 2.7.1 Investigation of Linear Relationship Take an appropriate amount of reference solution and dilute it with methanol to prepare a series of mixed reference solutions. Inject and measure according to the chromatographic conditions in "2.1". Draw a standard curve with the concentration of the reference solution as the abscissa and the peak area as the ordinate. The results are shown in Table 8.

[0123] Table 8 Results of linear relationship investigation of the four components in Atractylodes macrocephala

[0124]

[0125] 2.7.2 Precision test: Take the mixed reference solution and inject it continuously for 6 times according to the chromatographic conditions under "2.1". Record the peak areas and calculate the RSDs of the peak areas of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and atractylodes ketone to be 0.30%, 0.51%, 0.33% and 0.34%, respectively, indicating that the instrument has good precision.

[0126] 2.7.3 Stability test: Take the same batch of test solution and measure the sample at 0, 4, 8, 12, 24 and 48 hours according to the chromatographic conditions under "2.1". The RSDs of the peak areas of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and atractylodes ketone are calculated to be 1.03%, 1.01%, 0.87% and 0.87%, respectively, indicating that the test solution has good stability within 48 hours.

[0127] 2.7.4 Repeatability test Six portions of the same Atractylodes macrocephala sample (S23) were taken, and the test solution was prepared according to the method under "2.3". The samples were analyzed according to the chromatographic conditions under "2.1". The average contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone were calculated to be 0.1811, 0.2264, 0.2594, and 4.7449 mg / g, respectively, with RSDs of 1.71%, 1.33%, 2.20%, and 1.95%, respectively, indicating that the method had good repeatability.

[0128] 2.7.5 Recovery test of sample addition The powder of Atractylodes macrocephala sample with known content was accurately weighed and divided into 6 parallel portions. A reference substance equivalent to the sample content was added to each of them. The test solution was prepared according to the method under "2.3". The samples were injected and determined. The average recovery rates of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III and atractylodes ketone were calculated to be 94.63%, 97.88%, 95.65% and 96.48%, respectively, with RSDs of 3.58%, 4.44%, 3.67% and 2.36%, respectively.

[0129] 2.7.6 Sample content determination: 37 batches of Atractylodes macrocephala sample solutions were taken and injected for determination. The contents of the four components in the samples were calculated. The results are shown in Table 9.

[0130] Table 9 Determination results of the contents of four components in 37 batches of Atractylodes macrocephala samples (mg / g, n=2)

[0131]

[0132] 3. Discussion

[0133] 3.1 Sample pretreatment and chromatographic conditions The present invention investigated the extraction solvents (methanol, ethanol, acetonitrile, ethyl acetate, 50% methanol, 70% methanol, water), material-liquid ratio (1:10; 1:20; 1:25; 1:50), and extraction time (15min, 30min, 60min). Finally, the material-liquid ratio of 1:25 and methanol ultrasonic treatment for 30min were selected as the optimal extraction conditions. The effects of different chromatographic columns (Phenoment Gemini C18 (250mm×4.6mm, 5μm), Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm), Agilent ZORBAX Eclipse XDB-C18 (250mm×4.6mm, 5μm), different mobile phases (methanol-water, acetonitrile-water, acetonitrile-0.05% phosphoric acid water) and different column temperatures (25℃, 30℃, 35℃) on the chromatogram were compared. When using Agilent ZORBAX Using an Extend-C18 column with an acetonitrile-0.05% phosphoric acid mobile phase, the chromatographic peaks were well separated and the baseline was relatively stable. Full-wavelength scanning of the test solution using a DAD detector revealed that the components in Atractylodes macrocephala exhibited strong absorption at wavelengths of 220 nm, 242 nm, and 276 nm. Atractylodes lactone I exhibited the strongest absorption at 276 nm, while atractylodes lactone III, atractylodes lactone II, and atractylodes ketone exhibited the strongest absorption at 220 nm.

[0134] 3.2 Data Processing and Analysis Similarity evaluation results showed that the chemical components of different Atractylodes samples were similar in type, but their contents varied to some extent. HCA, PCA, and PLS-DA analyses revealed that the classification of 37 batches of samples showed overlap in origin, and that the quality of Atractylodes samples from the same origin varied. Atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and the unknown component peak 9 were likely the main factors affecting the quality of Atractylodes. With the exception of samples S16, S24, and S34, the remaining 34 batches of Atractylodes could be divided into two categories: S1-S15 and S17-S22 were classified as category 1, and the remaining samples were classified as category 2. PCA ranking of principal components revealed that among the samples from Anhui, Hubei, and Zhejiang, the quality of Atractylodes from Hubei was the highest, that from Anhui was the lowest, and that from Zhejiang was in the middle. The Atractylodes samples from Zhejiang in category 2 all ranked higher than those from category 1. The results of content determination showed that the average contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone in the second-category Atractylodes samples were 1.37, 2.36, 1.95, and 2.85 times those in the first-category samples, respectively. The average contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone in the second-category Atractylodes samples were 1.35, 2.45, 2.08, and 3.24 times those in the first-category Zhejiang Atractylodes samples, respectively. This indicates that atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone are all components that contribute to the quality of Atractylodes, which is consistent with the results of PCA and PLS-DA analysis. According to literature reports, atractylodes ketone in Atractylodes is unstable and easily decomposes and transforms into other components. The atractylodes ketone content decreased significantly 8 hours after the Atractylodes was pulverized, but remained relatively stable after the powder was prepared into a test solution. In this study, S1 to S22 were crushed and stored in powder form after collection. The lower content of atractylodesone may be related to this. However, whether the changes in the contents of atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III are related to the storage status of the samples requires further study.

[0135] The present invention establishes an HPLC fingerprint of Atractylodes macrocephala and simultaneously determines the contents of multiple indicators. The method is simple, accurate and reliable, and can be used for quality evaluation and control of Atractylodes macrocephala and its decoction pieces. The research results can lay a foundation for the research on its pharmacological substance basis and related compound research, and can also provide a reference for improving the quality standards of Atractylodes macrocephala.

Claims

1. A method for detecting the HPLC fingerprint of Atractylodes macrocephala, characterized in that: It includes the following steps: a. Weigh Atractylodes macrocephala, add methanol and ultrasonically extract for 30 min at a material-liquid ratio of 1 g:25 ml, filter and collect the filtrate; b. Prepare reference solution: Take reference substances Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III, and Atractylodes ketone, dissolve them in methanol and dilute to volume to prepare reference solution; c. The fingerprint of Atractylodes macrocephala was established by high performance liquid chromatography-diode array detector (HPLC-DAD) multi-wavelength switching method: Chromatographic conditions: chromatographic column: Agilent ZORBAX Extend-C18 250 mm×4.6 mm, 5 μm; mobile phase: acetonitrile (A)-0.05% phosphoric acid water (B), gradient elution: 12%-20%A 0-7 min, 20%-58%A 7-19 min, 58%-63%A 19-29 min, 63%-68%A 29-43 min, 68%-75%A 43-48 min, 75%-95%A 48-51 min, 95%-95%A 51-66 min; flow rate: 1 mL / min; detection wavelength: 220 nm, 0-32 min; 276 nm, 32-38.5 min. nm: 38.5-54 min, 242 nm: 54-66 min, 220 nm; column temperature: 25 ℃; injection volume: 10 μL.

2. A medicinal material of Atractylodes macrocephala, characterized in that: The method adopts the detection method described in claim 1 to detect the contents of four components, namely, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and atractylodes ketone. Each gram of the drug contains 0.21-0.32 mg of atractylodes lactone I, 0.13-0.25 mg of atractylodes lactone II, 0.1-0.19 mg of atractylodes lactone III, and 5.21-14.08 mg of atractylodes ketone.

3. A HPLC fingerprint of Atractylodes macrocephala, characterized by: It is obtained by the detection method according to claim 1, and includes the following characteristic peaks and relative retention times: Peak 1: 0.39±0.05; Peak 2: 0.42±0.05; Peak 3: 0.50±0.05; Peak 4: 0.54±0.05; Peak 5: 0.65±0.05; Peak 6: 0.96±0.05; Peak 7: 0.99±0.05; Peak 8: 1.00±0.05; Peak 9: 1.13±0.

05.

4. A method for screening quality-signaling components of Atractylodes macrocephala, characterized by: It includes the following steps: a. Obtaining the fingerprint of Atractylodes macrocephala using the detection method according to claim 1; b. Hierarchical cluster analysis (HCA): The relative peak areas of the common peaks were imported into IBM SPSS Statistics 25.0 software, and the median clustering method was used to perform a hierarchical cluster analysis with cosine distance as the classification basis. c. Based on the cluster analysis results, partial least squares discriminant analysis (PLS-DA) was performed on the medicinal samples, and a two-dimensional scatter plot was drawn; d. Screen the characteristic components that cause differences between Atractylodes macrocephala samples.

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