Fingerprint spectrum and multi-component simultaneous determination method for atractylodes macrocephala koidz medicinal material and extract thereof

Abstract

The invention belongs to the technical field of quality control of traditional Chinese medicines, and relates to a method for simultaneously determining fingerprints and multiple components of a rhizoma atractylodis macrocephalae medicinal material and an extract of the rhizoma atractylodis macrocephalae medicinal material. The invention provides the detection method of the fingerprint spectrum of the rhizoma atractylodis macrocephalae medicinal material and the extract thereof and the construction method of the standard fingerprint spectrum, which are of great significance in enhancing qualitative and quantitative control of the rhizoma atractylodis macrocephalae and ensuring that the medicinal effect of the rhizoma atractylodis macrocephalae medicinal material is maintained in the process from production, transportation to clinical application; on the basis of the existing fingerprint spectrum research, the common peak distribution characteristics of the fingerprint spectrum of the rhizoma atractylodis macrocephalae medicinal material and the volatile oil are further determined, and an HPLC simultaneous determination method for the five components of atractylone, atractylenolides I-III and beta-caryophyllene is established by a wavelength gradient method, so that a solid foundation is laid for drawing up the quality standard of the rhizoma atractylodis macrocephalae medicinal material, the rhizoma atractylodis macrocephalae volatile oil and the preparation of the rhizoma atractylodis macrocephalae medicinal material and the rhizoma atractylodis macrocephalae volatile oil.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of quality control of traditional Chinese medicine, and relates to a method for fingerprint spectrum and simultaneous determination of multiple components of Atractylodes macrocephala Koidz. BACKGROUND

[0002] Atractylodes macrocephala Koidz. is a dry rhizome of Atractylodes macrocephala Koidz. of the family Asteraceae, with bitter and sweet taste, warm nature, and meridian entering spleen and stomach, and has the effects of invigorating the spleen and replenishing qi, drying dampness and promoting water metabolism. It is the most frequently used medicinal material in the treatment of gastrointestinal diseases in traditional Chinese medicine. Atractylodes macrocephala Koidz. Rhizoma’s Essential Oil (AO) is the main effective part of Atractylodes macrocephala Koidz., and its content in the rhizome can be as high as 0.3-1.0%, including 78 terpenes, 5 aliphatic, 3 aromatic, 1 steroidal and 1 alkaloid compounds. Among them, the content of terpenes-atractylodin in Atractylodes macrocephala Koidz. Rhizoma’s Essential Oil is often >50%. In the 2025 edition of Chinese Pharmacopoeia, Part I, atactylodin is used as the only compound spot for qualitative identification of Atractylodes macrocephala Koidz. medicinal materials by thin layer chromatography. Chinese patent CN202210215758 shows that Atractylodes macrocephala Koidz. Rhizoma’s Essential Oil and its main component atactylodin have obvious improvement effect on the pathological structure of gastrointestinal mucosa of zebrafish with Helicobacter pylori infection, revealing the important contribution of atactylodin to the efficacy of Atractylodes macrocephala Koidz.

[0003] However, atractylone is easily converted into other components such as atractylenolides under the influence of environmental factors such as moisture, heat, light, etc. In the quality research of Atractylodes Rhizoma and even its volatile oil and preparations, it is of great significance to adopt a multi-component control strategy and focus on the distribution monitoring of atractylone and its conversion products atractylenolides. Chinese patent CN201010174361.X uses supercritical extraction method to obtain Atractylodes Rhizoma volatile oil, water vapor distillation method to obtain Atractylodes Rhizoma volatile oil + Atractylodes Rhizoma water extract components, water vapor distillation method to obtain Atractylodes Rhizoma volatile oil + Atractylodes Rhizoma alcohol extract components and their preparations to treat neostigmine-induced small intestine hyperfunction in mice, atropine-induced small intestine dysfunction in mice, Dahuang-induced spleen deficiency in mice, and barium chloride-induced intestinal spasm in rabbits, all of which have significant improvement effects, showing the overall and synergistic pharmacological effects of Atractylodes Rhizoma active substance basis in regulating gastrointestinal motility, improving intestinal microbiota, anti-digestive tract inflammation, and antioxidant effects. Earlier Chinese patent CN113899844A established a one-sample multi-evaluation method for Atractylodes Rhizoma based on UPLC, achieving rapid determination of 4 components including atractylone, atractylenolide I-III, and Chinese patent CN202310525773.0 established an HPLC fingerprint of Atractylodes Rhizoma using HPLC-DAD multi-wavelength switching method, identifying 9 common peaks, and identifying atractylone, atractylenolide I-III and an unknown component (characteristic peak 9) as the marker components for quality differences. It can be seen that although the significance of atractylone and its conversion products atractylenolides as index components has been established, there is a lack of attention to other coexisting components, and the number of identified components in common peaks is small, indicating that the Atractylodes Rhizoma quality evaluation system still needs to be further improved.

[0004] In addition, atractylenolide I-III has been confirmed to have anti-inflammatory, anticancer and other pharmacological activities, but terpenoid small molecules in atractylodes volatile oil except alkanone are widely distributed in different traditional Chinese medicine volatile oils at a low content level, which is not easy to detect and control. Previous statistics found that the content range of compounds in atractylodes volatile oil except atractylenolide III was large, and the ratio of the maximum and minimum content of alkanone was 5.4 times to 276.3 times of atractylenolide I. In fact, terpenoid small molecules have pharmacological activities and contribute to the efficacy of atractylodes volatile oil. As a representative of β-caryophyllene, Chinese patent CN202110418474.8 found that its interference effect on skin barrier was significantly better than that of the classic chemical transdermal absorption enhancer, and Chinese patent CN202410478360.6 found that its high, medium and low hypoglycemic and hypolipidemic activities were good. While β-elemene was recently reported by Chinese patent CN202411265244.2 for the first time to inhibit the expression of ferritin FTH1 and FTL in ectopic endometrial cells, which can effectively treat endometriosis. CN202510160452.4 clearly showed that β-eudesmol monomer component had a therapeutic effect on S. aureus pneumonia. At the same time, large root geraniol B is commonly involved in anticancer and anti-inflammatory pharmacological effects in volatile oils of many plants such as atractylodes and Prangos platychlaena Boiss. Obviously, the above components as components in atractylodes volatile oil also participate in the efficacy of atractylodes volatile oil. However, the quality control of the above components in atractylodes volatile oil is ignored. Therefore, based on the integrity and synergy of the pharmacodynamic material basis of atractylodes, strengthening the qualitative and quantitative control of atractylodes, and perfecting the HPLC fingerprint and simultaneous determination method are of great significance to maintain the efficacy of atractylodes medicinal materials and their extracts and preparations from production, transportation to clinical application. SUMMARY

[0005] In view of the above-mentioned shortcomings of the prior art, the present application further clarifies the common peak distribution characteristics of atractylodes medicinal materials and atractylodes volatile oil fingerprint, and establishes an HPLC simultaneous determination method of five components including alkanone, atractylenolide I-III and β-caryophyllene by wavelength gradient method, which lays a solid foundation for formulating quality standards of atractylodes medicinal materials, atractylodes volatile oil and its preparations.

[0006] To achieve the above-mentioned purpose and other related purposes, the first aspect of the present application provides a detection method of atractylodes medicinal materials or atractylodes volatile oil fingerprint, comprising the following steps:

[0007] (1) providing the test sample solution: when the detection object is Atractylodes lancea, take Atractylodes lancea powder, add organic solvent, weigh the total weight, then ultrasonic treatment, make up the evaporated organic solvent to the original weight, filter, and the test sample solution is obtained; when the detection object is Atractylodes lancea volatile oil, take Atractylodes lancea volatile oil, add organic solvent, ultrasonic treatment, filter, and the test sample solution is obtained;

[0008] (2) providing the control sample solution: the control sample solution includes atractylodin, atractyloides lactone I, atractyloides lactone II, atractyloides lactone III, β-eudesmol, β-elemene, β-caryophyllene and geranylgeraniol B control sample and organic solvent;

[0009] (3) determination: the high performance liquid chromatography with the same chromatographic condition is used to determine the test sample solution and the control sample solution respectively, the fingerprint of the test sample solution and the fingerprint of the control sample solution are obtained, the fingerprint of the test sample solution is compared with the fingerprint of the control sample solution, the index component in the fingerprint of the test sample solution is attributed and positioned, and thus the fingerprint of Atractylodes lancea or Atractylodes lancea volatile oil is obtained.

[0010] Preferably, the particle size of the Atractylodes lancea powder in step (1) is ≤355 μm, and most of the particles have a particle size of 150-355 μm. Usually, the Atractylodes lancea powder is passed through a No. 3 sieve.

[0011] Preferably, the organic solvent in step (1) is selected from methanol; when the detection object is Atractylodes lancea, the mass of the Atractylodes lancea powder to the volume of the added organic solvent is 1g:20-40mL, preferably 1g:30mL; when the detection object is Atractylodes lancea volatile oil, the mass of the added Atractylodes lancea volatile oil to the volume of the added organic solvent is 40-60mg:20-30mL, preferably 50mg:25mL.

[0012] Preferably, in step (1), when the detection object is Atractylodes lancea, the ultrasonic treatment is carried out by flowing water, the ultrasonic treatment time is 20-40min, preferably 30min, and the sample is placed at room temperature after the ultrasonic treatment; when the detection object is Atractylodes lancea volatile oil, the ultrasonic treatment is carried out under ice water bath, and the ultrasonic treatment time is 10-20min, preferably 15min.

[0013] In the present application, the filtration without special instructions is all filtered through 0.22 μm or 0.45 μm microporous filter membrane.

[0014] Preferably, in the reference solution described in step (2), the concentration range of atractylone, atractylenolide I, atractylenolide II, atractylenolide III, β-eucalyptol, β-elemene, β-caryophyllene, and geraniol B is 1 μg / mL to 2 mg / mL; preferably, in the reference solution, the concentration range of atractylone is 92–94 μg / mL, the concentration range of atractylenolide I is 50–52 μg / mL, the concentration range of atractylenolide II is 74–76 μg / mL, the concentration range of atractylenolide III is 93–95 μg / mL, the concentration range of β-eucalyptol is 26–28 μg / mL, the concentration range of β-elemene is 50–52 μg / mL, and the concentration range of β-caryophyllene is 38–40 μg / mL. The concentration of geraniol B ranges from 9 to 11 μg / mL. More preferably, in the reference solution, the concentration of atractylone ranges from 93.0 to 93.5 μg / mL, the concentration of atractylodes lactone I ranges from 50.5 to 51.5 μg / mL, the concentration of atractylodes lactone II ranges from 74.5 to 75.0 μg / mL, the concentration of atractylodes lactone III ranges from 93.5 to 94.0 μg / mL, the concentration of β-cineole ranges from 27.0 to 27.5 μg / mL, the concentration of β-elemene ranges from 50.5 to 51.5 μg / mL, the concentration of β-caryophyllene ranges from 38.5 to 39.0 μg / mL, and the concentration of geraniol B ranges from 9.5 to 10.5 μg / mL.

[0015] Preferably, the chromatographic conditions in step (3) are as follows: a C18 column is used; the column temperature is 25-40℃; the detection wavelength is 200-220nm; the flow rate is 0.8-1.2mL / min; the injection volume is 2-20μL; in the mobile phase, phase A is 0.05%-0.1% (v / v) phosphoric acid aqueous solution, and phase B is acetonitrile; the analysis time is 70min, with gradient elution; the detector is a DAD or VWD detector.

[0016] The specific procedure for gradient elution is as follows:

[0017] 0 min → 4 to 6 min, the volume ratio of phase A to phase B is 37-39%: 61-63%;

[0018] 4~6min→9~11min, A phase:B phase volume ratio is 37-39%:61-63%→33-35%:65-67%;

[0019] 9–11 min → 29–31 min, A phase: B phase volume ratio is 33–35%: 65–67% → 31–33%: 67–69%;

[0020] 29–31 min → 34–36 min, A phase: B phase volume ratio is 31–33%: 67–69% → 24–26%: 74–76%;

[0021] 34-36 min → 39-41 min, A phase: B phase volume ratio is 24-26%:74-76% → 14-16%:84-86%;

[0022] 39~41min→44~46min, A phase:B phase volume ratio is 14-16%:84-86%;

[0023] 44~46min→54~56min, A phase:B phase volume ratio is 14-16%:84-86%→9-11%:89-91%;

[0024] 54-56 min → 69-70 min, A phase: B phase volume ratio is 9-11%: 89-91%.

[0025] More preferably, in the chromatographic conditions, a BP-C18 Plus column, model 5AEILM179, is used, with column dimensions of 250mm × 4.6mm and a packing diameter of 5μm; the column temperature is 35℃; the detection wavelength is 203nm; the flow rate is 1mL / min; the injection volume is 5μL; in the mobile phase, phase A is 0.05% (v / v) phosphoric acid aqueous solution, and phase B is acetonitrile; the specific procedure for the gradient elution is as follows:

[0026] 0 min → 5 min, the volume ratio of phase A to phase B is 38%:62%;

[0027] From 5 min to 10 min, the volume ratio of phase A to phase B changed from 38%:62% to 34%:66%.

[0028] From 10 min to 30 min, the volume ratio of phase A to phase B was 34%:66% to 32%:68%.

[0029] 30min→35min, the volume ratio of phase A: phase B is 32%:68%→25%:75%;

[0030] 35min→40min, the volume ratio of phase A: phase B is 25%:75%→15%:85%;

[0031] 40min→45min, A phase:B phase volume ratio is 15%:85%;

[0032] 45min→55min, A phase:B phase volume ratio is 15%:85%→10%:90%;

[0033] 55min→70min, A phase:B phase volume ratio is 10%:90%.

[0034] The second aspect of this invention provides a method for constructing a standard fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, comprising: detecting multiple batches of Atractylodes macrocephala medicinal material samples or Atractylodes macrocephala volatile oil samples respectively using the above-mentioned detection method for fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, obtaining fingerprint spectra of multiple batches of Atractylodes macrocephala medicinal material samples or Atractylodes macrocephala volatile oil samples, generating a common pattern control spectrum, taking the chromatographic peaks present in all spectra as common characteristic peaks, determining the relative retention time of the common characteristic peaks, the ratio of the area of ​​each common characteristic peak to the total peak area, and assigning and locating the index components in the fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil according to the relative retention time, and establishing a standard fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, wherein the similarity result of the common characteristic peaks is ≥0.9.

[0035] Preferably, the number of batches of Atractylodes macrocephala medicinal material samples or Atractylodes macrocephala volatile oil samples tested is 10 to 20, more preferably 15.

[0036] Preferably, the generation of the common pattern comparison spectrum includes: using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission to compare the similarity of fingerprint spectra of multiple batches of Atractylodes macrocephala medicinal material samples or Atractylodes macrocephala volatile oil samples, and using the median method to generate the superimposed spectrum and the common pattern comparison spectrum.

[0037] When the sample being tested is Atractylodes macrocephala, the number of common characteristic peaks across multiple batches is 18. Taking peak 11 as the reference peak (S-peak) with a relative retention time of 1.000, the relative retention times of the other 17 common characteristic peaks are as follows: Peak 1: 0.186, Peak 2: 0.256, Peak 3: 0.2827, Peak 4: 0.297, Peak 5: 0.312, Peak 6: 0.433, Peak 7: 0.500, Peak 8: 0.523, Peak 9: 0.542, Peak 10: 0.978, and Peak 12: 1.0. Peaks 65 and 13 have a relative retention time of 1.226, peak 14 has a relative retention time of 1.246, peak 15 has a relative retention time of 1.336, peak 16 has a relative retention time of 1.373, and peak 17 has a relative retention time of 1.413. The relative deviation of the relative retention times of the 17 common characteristic peaks (excluding peak 11) is ≤ ±10%. The index components include atractylodes lactone III (peak 1), atractylodes lactone II (peak 5), atractylodes lactone I (peak 6), β-cineole (peak 7), atractylone (peak 11), β-elemene (peak 13), geraniol B (peak 15), and β-caryophyllene (peak 16). Preferably, the similarity between the fingerprint spectra of the multiple batches of Atractylodes macrocephala samples and the control spectra is > 0.95.

[0038] When the sample being tested is Atractylodes macrocephala volatile oil, the number of common characteristic peaks across multiple batches is 15. Taking peak 9 as the reference peak (S peak) with a relative retention time of 1.000, the relative retention times of the other 14 common characteristic peaks are as follows: peak 1: 0.179, peak 2: 0.304, peak 3: 0.378, peak 4: 0.406, peak 5: 0.426, peak 6: 0.488, peak 7: 0.528, peak 8: 0.976, peak 10: 1.131, 1... Peak 2 has a retention time of 1.240, peak 13 has a retention time of 1.261, peak 14 has a retention time of 1.399, and peak 15 has a retention time of 1.441. The relative deviations of the relative retention times of the 14 common characteristic peaks (excluding peak 9) are all ≤ ±10%. The index components include atractylodes lactone III (peak 1), atractylodes lactone II (peak 2), atractylodes lactone I (peak 5), β-cineole (peak 6), atractylone (peak 9), β-elemene (peak 10), geraniol B (peak 12), and β-caryophyllene (peak 13). Preferably, the similarity between the fingerprint spectra of multiple batches of Atractylodes macrocephala volatile oil samples and the control spectra is > 0.97.

[0039] A third aspect of this invention provides a method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala, comprising the following steps:

[0040] S1. Provide the test solution: Take Atractylodes macrocephala powder, add organic reagents for ultrasonic extraction, cool, centrifuge and take the supernatant to obtain the test solution;

[0041] S2. Provide a reference solution: the reference solution includes a certain amount of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene and an organic solvent;

[0042] S3. Determination: Using high performance liquid chromatography under the same chromatographic conditions, the test solution of step S1 and the reference solution of step S2 are determined respectively. The contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the test solution are calculated by external standard method.

[0043] Preferably, the particle size of the Atractylodes macrocephala powder in step S1 is ≤355μm, and most of the particles have a particle size of 150-355μm.

[0044] Preferably, the organic solvent in steps S1 and S2 is selected from methanol; the mass ratio of Atractylodes macrocephala powder to the volume of organic solvent added in step S2 is 1g:12-18mL, preferably 1g:15mL.

[0045] Preferably, the ultrasonic treatment in step S1 is performed using running water ultrasonic treatment, and the ultrasonic treatment time is 12 to 18 minutes, preferably 15 minutes.

[0046] Preferably, after ultrasonic treatment in step S1, the mixture is placed at room temperature.

[0047] (h) Preferably, in the reference solution described in step S2, the concentration range of atractylone is 883.80–883.90 μg / mL, the concentration range of atractylodes lactone I is 216.90–216.95 μg / mL, the concentration range of atractylodes lactone II is 203.20–203.30 μg / mL, the concentration range of atractylodes lactone III is 209.90–210.00 μg / mL, and the concentration range of β-caryophyllene is 277.65–277.75 μg / mL;

[0048] Preferably, the chromatographic conditions in step S3 are as follows: a C18 column is used; the column temperature is 35℃; the flow rate is 1 mL / min; the injection volume is 5 μL; in the mobile phase, phase A is 0.05% (v / v) phosphoric acid aqueous solution, and phase B is acetonitrile; the analysis time is 70 min, with gradient elution; the detector is a photodiode array detector (DAD).

[0049] The specific procedure for gradient elution is as follows:

[0050] 0 min → 5-6 min, A phase: B phase volume ratio is 37-39%: 61-63%;

[0051] 5-6 min → 9-11 min, A phase: B phase volume ratio is 37-39%: 61-63% → 33-35%: 65-67%;

[0052] 9–11 min → 29–31 min, A phase: B phase volume ratio is 33–35%: 65–67% → 31–33%: 67–69%;

[0053] 29-31 min → 34-36 min, A phase: B phase volume ratio is 31-33%: 67-69% → 24-26%: 74-76%;

[0054] 34-36min→39~41min, A phase:B phase volume ratio is 24-26%:74-76%→14-16%:84-86%;

[0055] 39~41min→44~46min, A phase:B phase volume ratio is 14-16%:84-86%;

[0056] 44~46min→54~56min, A phase:B phase volume ratio is 14-16%:84-86%→9-11%:89-91%;

[0057] 54~56min→69~70min, A phase:B phase volume ratio is 9-11%:89-91%;

[0058] The detection is wavelength gradient detection, and the wavelength gradient procedure is as follows:

[0059] 0 min to 15 min, wavelength 220 nm;

[0060] 15 min to 17 min, wavelength 250 nm;

[0061] 17 min to 22 min, wavelength 278 nm;

[0062] 22 min to 26 min, wavelength 240 nm;

[0063] 26 min to 35 min, wavelength 220 nm;

[0064] 35 min to 70 min, wavelength 203 nm;

[0065] Preferably, the external standard method in step S3 includes: preparing reference solutions of gradient concentrations in parallel according to step S2, analyzing them using high performance liquid chromatography (HPLC), establishing standard curve equations for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene by establishing the correspondence between the peak area and the known content, then performing HPLC detection on the test solution, recording the peak area of ​​each component, and calculating the content of each component in the test solution based on the standard curve equation.

[0066] The fourth aspect of this invention provides a method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the volatile oil of Atractylodes macrocephala, comprising the following steps:

[0067] S1. Provide the test solution: Take the volatile oil of Atractylodes macrocephala, add organic reagents, sonicate, filter, and the test solution is obtained.

[0068] S2. Provide a reference solution: the reference solution includes a certain amount of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene and an organic solvent;

[0069] S3. Determination: Using high performance liquid chromatography under the same chromatographic conditions, the test solution of step S1 and the reference solution of step S2 are determined respectively. The contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the test solution are calculated by external standard method.

[0070] Preferably, the ratio of the mass of Atractylodes macrocephala volatile oil added in step S1 to the volume of the organic solvent added is 40-60 mg: 20-30 mL, more preferably 50 mg: 25 mL.

[0071] Preferably, the organic solvents used in steps S1 and S2 are both methanol.

[0072] Preferably, the ultrasonic treatment in step S1 is performed in an ice-water bath for 10 to 20 minutes.

[0073] Preferably, in the reference solution described in step S2, the concentration range of atractylone is 1017.5–1017.6 μg / mL, the concentration range of atractylodes lactone I is 1009.7–1009.8 μg / mL, the concentration range of atractylodes lactone II is 1033.2–1033.3 μg / mL, the concentration range of atractylodes lactone III is 3986.6–3986.7 μg / mL, and the concentration range of β-caryophyllene is 2120.6–2120.7 μg / mL, with a concentration range of 9.5–10.5 μg / mL.

[0074] Preferably, the chromatographic conditions described in step S3 are the same as those preferred in step S3 of the method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala in the third aspect described above.

[0075] Preferably, the external standard method in step S3 includes: preparing reference solutions of gradient concentrations in parallel according to step S2, analyzing them using high performance liquid chromatography (HPLC), establishing standard curve equations for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene by establishing the correspondence between the peak area and the known content; subsequently, performing HPLC detection on the test solution, recording the peak area of ​​each component, and calculating the content of each component in the test solution based on the standard curve equation.

[0076] The fifth aspect of this invention provides the use of the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil obtained by the method for constructing the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil, the method for simultaneous detection of the contents of atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III, atractylone and β-caryophyllene in the above-mentioned Atractylodes macrocephala, or the method for simultaneous detection of the contents of atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III, atractylone and β-caryophyllene in the above-mentioned Atractylodes macrocephala volatile oil in the quality inspection and quality control of Atractylodes macrocephala products.

[0077] The Atractylodes macrocephala products include Atractylodes macrocephala raw materials, Atractylodes macrocephala extracts, and Atractylodes macrocephala preparations. Atractylodes macrocephala raw materials refer to the dried rhizomes of the Atractylodes macrocephala plant (Atractylodes macrocephala, belonging to the Asteraceae family), including raw Atractylodes macrocephala, stir-fried Atractylodes macrocephala with wheat bran, stir-fried Atractylodes macrocephala with soil, and charred Atractylodes macrocephala. Atractylodes macrocephala extracts are refined products obtained and enriched from Atractylodes macrocephala raw materials using modern extraction and separation techniques (such as solvent extraction, steam distillation, supercritical fluid extraction, column chromatography, etc.) to selectively obtain and enrich specific groups or single components of its effective components. Atractylodes macrocephala preparations are finished drugs made from Atractylodes macrocephala raw materials or Atractylodes macrocephala extracts as the main raw material, processed using modern pharmaceutical processes, and can be directly used for disease prevention or treatment, including but not limited to tablets, capsules, granules, pills, powders, oral liquids, injections, or external application preparations.

[0078] The method for quality control using the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil according to the present invention includes: obtaining a fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil sample using the above-mentioned detection method for fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil; comparing the similarity with the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil obtained using the above-mentioned method for constructing the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil; if the similarity is ≥0.90, it is considered qualified.

[0079] Compared with the prior art, the advantages and beneficial effects of the present invention include:

[0080] 1. This invention optimizes and screens the solvent preparation process and HPLC chromatographic conditions of the test samples, establishing a standard fingerprint chromatogram of Atractylodes macrocephala with high resolution and good reproducibility. Eighteen common peaks with good stability and peak shape were identified, accounting for over 90% of the total peak area. By comparing with the reference standard, eight components were identified: peak 1 (atractylenolide III), peak 5 (atractylenolide II), peak 6 (atractylenolide I), peak 7 (β-eudesminol), peak 11 (atractylone), peak 13 (β-elemene), peak 15 (geraniol B), and peak 16 (β-caryophyllene). The similarity between each batch of samples and the reference chromatogram was greater than 0.95, indicating that the method for constructing the fingerprint chromatogram is stable and the results are reliable. This is of great significance for strengthening the qualitative and quantitative control of Atractylodes macrocephala and ensuring the maintenance of its efficacy from production and transportation to clinical application.

[0081] 2. This invention optimizes and screens the solvent preparation process and HPLC chromatographic conditions of the test sample, establishing a standard fingerprint spectrum of Atractylodes macrocephala volatile oil with high resolution and good reproducibility. A total of 15 common peaks were identified, and 8 chromatographic peaks were designated: peak 1 (atractylenolide III), peak 2 (atractylenolide II), peak 5 (atractylenolide I), peak 6 (β-eucalyptol), peak 9 (atractylone, S peak), peak 10 (β-elemene), peak 12 (geranylene B), and peak 13 (β-caryophyllene). The similarity between the fingerprint spectrum and the control spectrum is greater than 0.97, indicating that the method for constructing the fingerprint spectrum is stable and the results are reliable. This is of great significance for strengthening the qualitative and quantitative control of Atractylodes macrocephala volatile oil and ensuring the maintenance of its efficacy from production and transportation to clinical application.

[0082] 3. This invention establishes an HPLC method for the simultaneous determination of five components—atractylone, atractylone I-III, and β-caryophyllene—in Atractylodes macrocephala and its volatile oil using a wavelength gradient method, laying a solid foundation for formulating quality standards for Atractylodes macrocephala, its volatile oil, and its preparations. Attached Figure Description

[0083] Figure 1 The fingerprint chromatograms of different extraction solvents for Atractylodes macrocephala are shown, where (a) is methanol and (b) is ethanol.

[0084] Figure 2 The fingerprint chromatograms of different mass fractions of extraction solvents for Atractylodes macrocephala are shown, where (a) is 100% methanol, (b) is 80% methanol, and (c) is 60% methanol.

[0085] Figure 3 The fingerprint chromatograms are shown for different material-liquid ratios, where (a) is a material-liquid ratio of 1:15, (b) is a material-liquid ratio of 1:30, and (c) is a material-liquid ratio of 1:45.

[0086] Figure 4 The results of fingerprint spectrum measurements at different detection wavelengths are shown, where (a) is 203 nm, (b) is 220 nm, and (c) is 278 nm.

[0087] Figure 5 The fingerprint spectral results are shown for different types of mobile phases, where (a) is 0.1% phosphoric acid-acetonitrile, (b) is 0.1% phosphoric acid-methanol, (c) is water-acetonitrile, and (d) is water-methanol.

[0088] Figure 6 The fingerprint spectral results are for different concentrations and types of acidic water, where (a) is 0.05% phosphate-acetonitrile, (b) is 0.1% phosphate-acetonitrile, (c) is 0.05% acetic acid-acetonitrile, and (d) is 0.1% acetic acid-acetonitrile.

[0089] Figure 7 The fingerprint spectral results are shown for different concentrations and types of acidic water, where (a) represents gradient I, (b) represents gradient II, and (c) represents gradient III.

[0090] Figure 8 The results of fingerprint analysis at different flow rates are shown, where (a) is 0.4 mL / min, (b) is 0.6 mL / min, (c) is 0.8 mL / min, (d) is 1 mL / min, and (e) is 1.2 mL / min.

[0091] Figure 9 The fingerprint chromatograms are measured with different injection volumes, where (a) is 2 μL, (b) is 5 μL, (c) is 10 μL, and (d) is 20 μL.

[0092] Figure 10 The HPLC overlay chromatograms of 15 batches of Atractylodes macrocephala samples are shown.

[0093] Figure 11 The chromatograms are shown in (a) for the reference material of Atractylodes macrocephala and (b) for the mixed reference solution.

[0094] Figure 12 The fingerprint chromatograms of different mass fractions of extraction solvents for Atractylodes macrocephala volatile oil are shown, where (a) is 100% methanol; (b) is 80% methanol; and (c) is 60% methanol.

[0095] Figure 13 HPLC superimposed chromatograms of volatile oil samples from 15 batches of Atractylodes macrocephala.

[0096] Figure 14 The reference chromatograms are shown in (a) and (b) for the mixed reference standards.

[0097] Figure 15 The images show full-wavelength scanning chromatograms of different components in Atractylodes macrocephala under the simultaneous determination method of HPLC content. Among them, (a) is atractylodes macrocephala lactone I; (b) is atractylodes macrocephala lactone II; (c) is atractylodes macrocephala lactone III; (d) is atractylone; and (e) is β-caryophyllene. Detailed Implementation

[0098] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0099] The instrument information used in the embodiments is shown in Table 1 below.

[0100] Table 1 Instrument Information

[0101]

[0102] The reagent information used in the examples is shown in Table 2 below.

[0103] Table 2 Reagent Information

[0104]

[0105]

[0106] The information on the reference standards and reference medicinal materials in the examples is shown in Table 3 below.

[0107] Table 3 Information on reference standards and reference medicinal materials

[0108]

[0109] Example 1: Determination of the preparation method for HPLC fingerprint chromatogram of Atractylodes macrocephala test sample

[0110] 1.1 Preparation of mixed reference solution

[0111] Accurately weigh appropriate amounts of atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, β-cineole, β-elemene, β-caryophyllene, and geraniol B, and add methanol to prepare stock solutions of each reference standard. Take an appropriate amount of the stock solution of the reference standard, dilute with methanol, and prepare mixed reference standard solutions with concentrations of 93.4, 51.0, 74.9, 93.7, 27.2, 51.0, 38.2, and 10.0 μg / mL, respectively.

[0112] 1.2 Preparation of the test sample

[0113] Take about 1g of Atractylodes macrocephala powder (passed through a No. 3 sieve), place it in a stoppered conical flask, accurately add 30mL of methanol, weigh it, sonicate it under running water for 30min, remove it, let it stand at room temperature, weigh it again, make up the weight loss, shake it well, filter it, and you will get the product.

[0114] 1.3 Fingerprint Spectrum Determination Conditions

[0115] Chromatographic column: BP-C18 Plus column (Serial Number: 5AEILM179) (250mm×4.6mm, 5μm), detection wavelength: 203nm, flow rate: 1.0mL / min, column temperature: 35℃, injection volume: 5μL, mobile phase: 0.05% phosphoric acid water (A)-acetonitrile (B), gradient elution program is shown in Table 4.

[0116] Table 4. HPLC elution procedure for fingerprint chromatograms

[0117]

[0118] 1.4 Effects of different extraction solvent types, solvent mass fractions, extraction methods, and solid-liquid ratios on chromatographic peaks

[0119] Based on single-factor experiments, the effects of different extraction solvent types (methanol, ethanol), different extraction solvent mass fractions (100%, 80%, 60% methanol), different extraction methods (running water ultrasonication, water bath reflux), different extraction times (15, 30, and 45 min), and different solid-liquid ratios (1:15, 1:30, 1:45; g:mL) on chromatographic peaks were investigated sequentially.

[0120] Among different types of extraction solvents, such as Figure 1 As shown, the chromatographic peak baseline of the test solution prepared by methanol extraction was relatively stable, with good resolution, and the peak area of ​​the main component, atractylone, was relatively large. Therefore, methanol was ultimately chosen for further optimization of its mass fraction. Among different extraction solvent mass fractions, such as... Figure 2 As shown, the chromatographic peak resolution of the test solution prepared by 100% methanol extraction was good, and the peak area of ​​the main component, atractylone, was relatively large. Therefore, methanol was ultimately selected for further extraction method optimization. Among different extraction methods, the test solution prepared by methanol-water ultrasonic extraction had a more stable chromatographic peak baseline, good resolution, and a larger ratio of characteristic peak area to total peak area. The peak area of ​​the main component, atractylone, was also relatively large. Therefore, water ultrasonic extraction was ultimately selected for further extraction time optimization. Among different extraction times, the test solution prepared with 30 min had a more stable chromatographic peak baseline, good resolution, and a relatively large peak area of ​​the main component, atractylone. Therefore, an extraction time of 30 min was ultimately selected for further solid-liquid ratio optimization. Among different solid-liquid ratios, such as... Figure 3 As shown, the chromatographic peak baseline of the test sample solution prepared with a solid-liquid ratio of 1:30 is relatively stable, with good resolution, moderate peak shape, and a large ratio of characteristic peak area to total peak area. Therefore, a solid-liquid ratio of 1:30 was ultimately selected as the preparation condition for the test sample.

[0121] 1.5 Summary

[0122] The optimal preparation method for the Atractylodes macrocephala medicinal material test sample is as follows: Take about 1g of Atractylodes macrocephala medicinal material powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 30mL of methanol, weigh it, extract it with running water using ultrasound for 30min, take it out, place it at room temperature, weigh it again, make up the weight loss, shake it well, filter it, and the sample is obtained.

[0123] Example 2: Determination of chromatographic conditions for HPLC fingerprinting of Atractylodes macrocephala.

[0124] Referring to Example 1, based on single-factor experiments, the effects of different absorption wavelengths (203, 250, 278 nm), different mobile phase types (AB; 0.1% phosphoric acid-methanol, water-methanol, 0.1% phosphoric acid-acetonitrile, water-acetonitrile), different acid-water types (AB; 0.05% phosphoric acid, 0.05% acetic acid, 0.1% phosphoric acid, 0.1% acetic acid), different mobile phase ratios (gradients I to III, as shown in Table 5 below), different flow rates (0.4, 0.6, 0.8, 1, 1.2 mL / min), different injection volumes (2, 5, 10, 20 μL), and different column temperatures (25, 30, 35, 40 °C) on chromatographic peaks were investigated sequentially.

[0125] Table 5 Elution program settings for mobile phase ratio analysis

[0126]

[0127] like Figure 4 As shown, by comparing the absorption chromatograms at three different wavelengths, the Atractylodes macrocephala herbal sample solution showed more detectable chromatographic peaks around 203 nm, with good response values ​​for each peak, achieving baseline separation and producing a neat and aesthetically pleasing chromatogram. Therefore, 203 nm was selected as the optimal absorption wavelength. Figure 5 As shown, the chromatogram of 0.1 phosphate-acetonitrile exhibits a more stable baseline and achieves better baseline separation; therefore, 0.1 phosphate-acetonitrile was chosen as the mobile phase. Figure 6 As shown, using phosphoric acid achieves baseline separation. The characteristic peak area of ​​0.05% phosphoric acid has a high ratio to the total peak area, indicating good separation. Therefore, 0.05% phosphoric acid-acetonitrile was ultimately chosen as the mobile phase. Figure 7 As shown, gradient I has the highest ratio of characteristic peak area to total peak area, indicating better separation; therefore, gradient I is chosen for elution. Figure 8 As shown, the ratio of the characteristic peak area to the total peak area is highest at a elution rate of 1 mL / min, indicating good separation; therefore, a elution rate of 1 mL / min was chosen for elution. Figure 9 As shown, the ratio of characteristic peak area to total peak area was highest at 5 μL, indicating good separation; therefore, an injection volume of 5 μL was chosen. Furthermore, the ratio of characteristic peak area to total peak area was highest at 35℃, indicating good separation and a more stable baseline; therefore, a column temperature of 35℃ was selected.

[0128] The final chromatographic conditions were determined as follows: 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution according to gradient I; detection wavelength 203 nm; flow rate 1 mL / min; column temperature 35 ℃; injection volume 5 μL; total time 70 min.

[0129] Example 3: Construction, methodological investigation, and similarity evaluation of HPLC standard fingerprint of Atractylodes macrocephala.

[0130] 3.1 Precision Test

[0131] The same sample was injected six times consecutively under the chromatographic conditions determined in Example 2. The RSD values ​​of the relative retention times of the common peaks were 0.07% to 1.55%, indicating good instrument precision.

[0132] 3.2 Repeatability Test

[0133] Take 6 test solutions and inject each one once consecutively. The RSD value of the relative retention time of each common peak is 0.05% to 0.16%, indicating that the method has good repeatability.

[0134] 3.3 Stability Test

[0135] The same sample was injected at 0, 2, 4, 6, 8, 12, 16, 24, 30 and 36 h according to the chromatographic conditions determined in Example 2. The RSD of the relative retention time of each common peak was 0.08% to 1.53%, indicating that the sample was stable within 36 h.

[0136] 3.4 Establishment and Similarity Evaluation of Fingerprint Spectrum of Atractylodes macrocephala

[0137] Fifteen batches of Atractylodes macrocephala medicinal materials were used to prepare test samples. Chromatographic analysis was performed under the conditions determined in Example 2. Data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using the chromatogram of sample S2 as the reference chromatogram, the median method was employed, with a time window width of 0.1 min. Multi-point correction and automatic matching were performed to generate overlay chromatograms and control chromatograms (see...). Figures 10-11Using sample S2 as a reference, a standard fingerprint spectrum (R) was established, identifying 18 common peaks with good stability and peak shape. The area of ​​these common peaks accounted for more than 90% of the total peak area. By comparing with the reference standard, eight components were identified: peak 1 (atractylodes methylphenidate III), peak 5 (atractylodes methylphenidate II), peak 6 (atractylodes methylphenidate I), peak 7 (β-eucalyptol), peak 11 (atractylone), peak 13 (β-elemene), peak 15 (geranylene B), and peak 16 (β-caryophyllene). Atractylone peak had a relatively large area and moderate retention time, therefore it was selected as the reference peak S. The relative retention times of the other 17 common characteristic peaks were as follows: peak 1: 0.186, peak 2: 0.256, peak 3: 0.2827, peak 4: 0.297, peak 5: 0.312, peak 6: 0.433, peak 7: 0.500, peak 8: 0.523, peak 9: 0.542, peak 10: 0.978, peak 12: 1.065, peak 13: 1.226, peak 14: 1.246, peak 15: 1.336, peak 16: 1.373, and peak 17: 1.413. The relative deviations of the relative retention times of the above 17 common characteristic peaks, except for peak 11, were all ≤ ±10%. The similarity scores between the 15 batches of samples and the control chromatograms were 0.994, 0.993, 0.994, 0.993, 0.996, 0.988, 0.955, 0.964, 0.982, 0.997, 0.996, 0.996, 0.996, 0.997, and 0.996, respectively, all greater than 0.95. This indicates that the method for constructing fingerprint chromatograms in this embodiment is stable and the results are reliable.

[0138] 3.5 Summary

[0139] The HPLC method for fingerprinting Atractylodes macrocephala established in this invention exhibits good precision and repeatability, and the test sample is stable for 36 hours. A total of 18 common peaks were identified in 15 batches of Atractylodes macrocephala. Eight chromatographic peaks were designated as peak 1 (atractylenolide III), peak 5 (atractylenolide II), peak 6 (atractylenolide I), peak 7 (β-eucalyptol), peak 11 (atractylone, S peak), peak 13 (β-elemene), peak 15 (geraniol B), and peak 16 (β-caryophyllene). The similarity between these peaks and the reference chromatograms was greater than 0.95.

[0140] Example 4: Construction, methodological investigation, and similarity evaluation of HPLC standard fingerprint of Atractylodes macrocephala volatile oil

[0141] 4.1 Preparation of reference standards and test samples and chromatographic conditions

[0142] Preparation of the test sample: Accurately weigh about 50 mg of Atractylodes macrocephala volatile oil, dilute to 25 mL with methanol, sonicate in an ice-water bath for 15 min, remove, place at room temperature, filter, and the sample is obtained.

[0143] The rest of the procedure is the same as the HPLC fingerprinting procedure for Atractylodes macrocephala.

[0144] 4.2 Effects of different extraction solvent types, extraction solvent mass fractions, and extraction solvent volumes on chromatographic peaks

[0145] Based on single-factor experiments, the effects of different types of extraction solvents (methanol, ethanol), different mass fractions of extraction solvents (100%, 80%, 60% methanol), and different volumes of extraction solvents (10, 25, 40 mL) on chromatographic peaks were investigated sequentially.

[0146] Among different extraction solvents, the test sample solution prepared by methanol extraction showed a more stable chromatographic peak baseline, better resolution, and a larger peak area for the main component, atractylone. Therefore, methanol was selected as the extraction solvent for the test sample. Regarding the mass fraction of different extraction solvents, such as... Figure 12 As shown, the chromatographic peak baseline of the test sample solution prepared by 100% methanol extraction was relatively stable, with good resolution and a large number of characteristic peaks. The peak area of ​​the main component, atractylone, was relatively large. Therefore, the mass fraction of methanol as the extraction solvent for the test sample was determined to be 100%. Among different extraction solvent volumes, the chromatographic peak baseline of the test sample solution prepared by 25 mL methanol extraction was relatively stable, with good resolution and moderate peak shape. Therefore, the extraction solvent volume was determined to be 25 mL.

[0147] 4.3 Precision Test

[0148] The same sample was injected six times consecutively, and the RSD values ​​of the relative retention times of each common peak were calculated to be 0.02% to 0.07%, indicating that the instrument has good precision.

[0149] 4.4 Repeatability Test

[0150] Six test solutions were taken and each was injected once consecutively. The RSD of the relative retention time of each common peak was calculated to be 0.12% to 0.25%, indicating that the method has good repeatability.

[0151] 4.5 Stability Test

[0152] The same test sample was injected at 0, 2, 4, 6, 8, 12, 16, 24, 30, and 36 hours. The RSD of the relative retention time of each common peak was calculated to be 0.09% to 0.18%, indicating that the test sample was stable within 36 hours.

[0153] 4.6 Establishment and Similarity Evaluation of Fingerprint Spectrum of Atractylodes macrocephala Volatile Oil

[0154] Fifteen batches of Atractylodes macrocephala volatile oil samples were tested and injected under the chromatographic conditions determined in Example 2. The data were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)". Using the chromatogram of sample S2 as the reference chromatogram, the median method was used, with a time window width of 0.1 min. Multi-point correction and automatic matching were performed to generate overlay chromatograms and reference chromatograms (see...). Figures 13-14 Using the chromatogram of sample S11 as a reference, a superimposed chromatogram and a control chromatogram were generated, identifying 15 common peaks. These 15 common peaks accounted for over 95% of the total peak area. By comparing with the reference standard, eight components were identified: peak 1 (atractylenolide III), peak 2 (atractylenolide II), peak 5 (atractylenolide I), peak 6 (β-eucalyptol), peak 9 (atractylone, S peak), peak 10 (β-elemene), peak 12 (geraniol B), and peak 13 (β-caryophyllene). (See [link to relevant documentation]). Figure 13 and Figure 14 Except for peak S, the relative retention times of the other 14 common characteristic peaks are as follows: peak 1 is 0.179, peak 2 is 0.304, peak 3 is 0.378, peak 4 is 0.406, peak 5 is 0.426, peak 6 is 0.488, peak 7 is 0.528, peak 8 is 0.976, peak 10 is 1.131, peak 12 is 1.240, peak 13 is 1.261, peak 14 is 1.399, and peak 15 is 1.441. The relative deviations of the relative retention times of the above 14 common characteristic peaks, except for peak 9, are all ≤±10%. The similarity scores between the 15 batches of samples and the control chromatograms were 0.994, 0.999, 0.999, 0.994, 0.996, 0.991, 0.972, 0.975, 0.991, 0.999, 0.997, 0.997, 0.997, and 0.998, respectively, all greater than 0.97, indicating that the established fingerprinting method is feasible.

[0155] Compared with the HPLC fingerprint of Atractylodes macrocephala, although the number of common peaks decreased, the ratio of peak area to total peak area increased, the number of identified components was consistent, and the similarity evaluation was good, indicating that the HPLC method established in this invention is suitable for Atractylodes macrocephala volatile oil.

[0156] 4.7 Summary

[0157] The HPLC fingerprinting method for Atractylodes macrocephala volatile oil established in this invention exhibits good precision and repeatability, and the test sample is stable for 36 hours. Fifteen batches of Atractylodes macrocephala volatile oil were analyzed, identifying 15 common peaks. Eight chromatographic peaks were identified as peak 1 (atractylenolide III), peak 2 (atractylenolide II), peak 5 (atractylenolide I), peak 6 (β-eucalyptol), peak 9 (atractylone, S peak), peak 10 (β-elemene), peak 12 (geraniol B), and peak 13 (β-caryophyllene). The similarity between these peaks and the reference chromatograms was greater than 0.97.

[0158] Example 5: Establishment of a simultaneous HPLC method for the determination of Atractylodes macrocephala content and determination of the test sample preparation method.

[0159] 5.1 Establishment of HPLC content determination method

[0160] By establishing a fingerprint chromatogram of Atractylodes macrocephala, and based on the resolution, peak area, pharmacological activity, and practical considerations of the identified components in the chromatogram, five index components for the determination of Atractylodes macrocephala content were identified as atractylenolide I, atractylenolide II, atractylenolide III, atractylone, and β-caryophyllene. A BP-C18 Plus column (Serial Number: 5AEILM179) (250mm × 4.6mm, 5μm) and a DAD detector were used to perform full-wavelength scanning of the reference solutions of these five components. The remaining chromatographic conditions were the same as those in Examples 1-3: 0.05% phosphoric acid as mobile phase A, acetonitrile as mobile phase B, gradient elution according to Table 6; flow rate 1 mL / min; column temperature 35℃; injection volume 5 μL; total duration 70 min.

[0161] Table 6. Mobile Phase Gradient Elution Program

[0162]

[0163] Full-wavelength scanning results showed that atractylone, atractylenolide I, atractylenolide II, atractylenolide III, and β-caryophyllene had higher peaks at 203 nm, 278 nm, 220 nm, 220 nm, and 203 nm, respectively. Figure 15 Therefore, based on the liquid chromatography conditions of the fingerprint spectrum of Atractylodes macrocephala, a wavelength gradient was added, as shown in Table 7.

[0164] Table 7 Wavelength gradient program

[0165]

[0166] 5.2 Preparation of mixed reference solution

[0167] Accurately weigh appropriate amounts of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene, and prepare stock solutions of each reference standard with methanol. Take an appropriate amount of the stock solution of the reference standard, dilute with methanol, and prepare mixed reference standard solutions with concentrations of 216.91, 203.23, 209.95, 883.85, and 277.70 μg / mL, respectively.

[0168] 5.3 Preparation of the test sample

[0169] Based on single-factor experiments, referring to the optimal sample preparation method in Example 1, the effects of different extraction solvent types and mass fractions (100% methanol, 100% ethanol, 80% methanol, 60% methanol), different extraction methods (hot maceration, ultrasonication, reflux), different extraction times (15, 30, 45 min), and different solid-liquid ratios (g:mL; 1:15, 1:30, 1:45) on the content determination results of Atractylodes macrocephala. The sample was analyzed using the content determination conditions determined in section 1.1 of Example 5. 1 g of Atractylodes macrocephala was used, and the optimal sample preparation method was determined by comparing the comprehensive scores of the five index components under each investigation item. The weights were allocated according to the content ratio of each component in the sample, and the final comprehensive score was determined as: (average content of atractylodes macrocephala I × 0.1) + (average content of atractylodes macrocephala II × 0.1) + (average content of atractylodes macrocephala III × 0.1) + (average content of atractylone × 0.6) + (average content of β-caryophyllene × 0.1).

[0170] The results are shown in Table 8. The option with the higher overall score was preferred. Methanol was the preferred solvent in the selection of extraction solvent types, and the solvent with the highest mass fraction was also preferred.

[0171] Table 8. Single-factor experimental results of the test sample preparation method

[0172]

[0173] 5.4 Summary

[0174] Based on the fingerprint chromatogram, a wavelength gradient elution program as shown in Table 17 was established as a simultaneous HPLC method for the determination of Atractylodes macrocephala content in Atractylodes macrocephala. This method is applicable to the determination of atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III, atractylone, and β-caryophyllene in Atractylodes macrocephala.

[0175] Based on the results of single-factor experiments, the optimal preparation method for the Atractylodes macrocephala medicinal material test sample was determined as follows: Take about 1g of Atractylodes macrocephala medicinal material powder (passed through a No. 3 sieve), weigh it accurately, place it in a stoppered conical flask, add 15mL of methanol accurately, weigh it, extract it with running water using ultrasound for 15min, take it out, place it at room temperature, weigh it again, make up the weight loss, shake it well, filter it, and the product is obtained.

[0176] Example 6: Methodological investigation and determination of the content of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala by HPLC

[0177] 6.1 Preparation of reference standards and test samples and chromatographic conditions

[0178] Refer to Example 5.

[0179] 6.2 Linear

[0180] Accurately weigh appropriate amounts of atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and β-caryophyllene, dissolve them in methanol to prepare stock solutions containing 1767.709, 2169.132, 2032.321, 2099.529, and 5554.043 μg of each reference standard per 1 mL. Shake well and use as stock solutions. Take 1 mL of each of the stock solutions of atractylodes lactone I, II, and III, 5 mL of the stock solution of atractylone, and 0.5 mL of the stock solution of β-caryophyllene, and dilute to 10 mL with methanol to obtain a high-concentration mixed standard solution. This mixed standard solution is serially diluted to prepare a series of reference standard solutions, which are then analyzed. A standard curve is plotted with concentration (μg / mL) as the abscissa (X) and peak area as the ordinate (Y). The linear regression equations and correlation coefficients for each index component are calculated. The results are shown in Table 9.

[0181] Table 9. Linear Range, Regression Equation, and Correlation Coefficient

[0182]

[0183] 6.3 Precision Test

[0184] A mixed reference solution of high, medium, and low linear concentrations, as well as a test solution prepared according to the method for preparing the test solution, were injected six times consecutively. The results showed that the RSDs were all less than 3%, indicating that the reference solution and the test solution had good precision.

[0185] 6.4 Repeatability Test

[0186] Six test samples were taken and injected once each. The peak areas were recorded, and the RSD values ​​of the peak areas were calculated. The average contents of atractylone, atractylenolide I, atractylenolide II, atractylenolide III, and β-caryophyllene in the samples were 5.5091, 0.1744, 0.2346, 0.2628, and 0.0825 mg / g, respectively, with RSDs of 0.41, 0.51, 0.89, 2.11, and 0.81%, respectively. This indicates that the method has good repeatability.

[0187] 6.5 Stability Test

[0188] The same test sample was injected at 0, 2, 4, 6, 10, 14, 18, and 24 hours. The results showed that the peak area RSD of each component was less than 1%, indicating that the reference solution and the test solution had good stability within 24 hours.

[0189] 6.6 Recovery rate

[0190] Approximately 0.5 g of a sample of Atractylodes macrocephala with known content (BZ-10, with contents of atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and β-caryophyllene of 5.5091, 0.1744, 0.2346, 0.2628, and 0.0825 mg / g, respectively) was accurately weighed and placed in a stoppered conical flask. Reference solutions equivalent to 50%, 100%, and 150% of the specified content were added to prepare test solutions. The solutions were then injected for determination, and the recovery rate (%) was calculated as (measured amount - known amount) / added amount * 100%. The results are shown in Table 10. The recovery limits all met the requirements, and the RSDs were all less than 3%, indicating that the method established in this invention has good accuracy.

[0191] Table 10 Results of Sample Recovery Rate

[0192]

[0193] 6.7 Durability Test

[0194] The same sample was used, and the effects of different column temperatures (25℃, 30℃, 35℃), flow rates (0.9 mL / min, 1.0 mL / min, 1.1 mL / min), acid concentrations (0.03%, 0.06%, 0.09%), different chromatographic columns (BP-C18 Plus, XB-C18, XDB-C18), and different instruments (Agilent 1260VWD, Agilent 1260DAD) on the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene in the sample were investigated. The results are shown in Table 11.

[0195] Under different conditions, the RSD values ​​of atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III in the test samples were all less than 5%, with no significant difference. The RSD values ​​of atractylone content in the test samples were greater than 5% under different column temperatures, with atractylone content relatively lower at 40℃, and RSD values ​​less than 5% under other conditions, showing no significant difference. Under different column temperatures, flow rates, column types, and instrument conditions, the RSD values ​​of β-caryophyllene content in the test samples were all greater than 5%. Analysis of the results showed that β-caryophyllene content was relatively low at a column temperature of 30℃, and connected to a small peak on the right at a flow rate of 0.9 mL / min, while connected to a peak on the left in the XBC18 column. Because its content in Atractylodes macrocephala is relatively low, its robustness range is relatively narrow.

[0196] Therefore, it can be concluded that, within the column temperature range of 30–40℃, flow rate of 0.9 mL / min–1.1 mL / min, and acid concentration of 0.03%–0.07%, and under different chromatographic columns (BP C18, XB C18, XDB C18) and different instruments (Agilent 1260VWD, Agilent 1260DAD), this method exhibits good robustness for the three atractylodes lactones; within the column temperature range of 30–35℃, and under other different conditions, this method exhibits good robustness for atractylone; and within the column temperature range of 35–40℃, flow rate of 1.0 mL / min–1.1 mL / min, and under different chromatographic columns (BP C18, XDB C18) and other different conditions, this method exhibits good robustness for β-caryophyllene.

[0197] Table 11 Content of each component under different conditions

[0198]

[0199] 6.8 Determination of Atractylodes macrocephala content in medicinal materials

[0200] The established content determination method was used to determine the contents of the above five components in 15 batches of Atractylodes macrocephala samples. As shown in Table 12, the contents of atractylodes macrocephala lactone I, atractylodes macrocephala lactone II, atractylodes macrocephala lactone III, atractylone, and β-caryophyllene in the 15 batches of Atractylodes macrocephala samples ranged from 0.062 to 0.535, 0.024 to 0.662, 0.039 to 0.704, 3.427 to 6.230, and 0.027 to 0.186 mg / g, respectively. The RSD values ​​of each component were all less than 5%, indicating that this method can be used for the content determination of multiple indicators of components in multiple batches of Atractylodes macrocephala.

[0201] Table 1215: Determination of Atractylodes macrocephala content (mg / g) in medicinal materials.

[0202]

[0203]

[0204] 6.9 Summary

[0205] The linear relationships, precision, repeatability, and accuracy of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene were good. The stability of the reference solution and the test solution within 24 hours and the robustness within a certain range were good, indicating that the established content determination method is reliable and suitable for the simultaneous determination of the content of five components in Atractylodes macrocephala.

[0206] Example 7: Methodological investigation and determination of the content of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala volatile oil by HPLC

[0207] 7.1 Preparation of mixed reference solution

[0208] Accurately weigh appropriate amounts of atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, and β-caryophyllene reference standards, dissolve each in methanol and dilute to volume to prepare reference stock solutions with mass concentrations of 1017.593, 1009.701, 1033.276, 3986.640, and 2120.688 μg / mL, respectively, and store at 4℃ for later use.

[0209] 7.2 Preparation of the test sample

[0210] Accurately weigh approximately 50 mg of Atractylodes macrocephala volatile oil, place it in a stoppered conical flask, add 25 mL of methanol, weigh the mixture, sonicate for 15 min, remove the flask, allow it to cool to room temperature, weigh the mixture again, add methanol to make up the mass, shake well, filter, and the product is obtained.

[0211] 7.3 Chromatographic conditions

[0212] A BP-C18 Plus column (Serial Number: 5AEILM179) (250 mm × 4.6 mm, 5 μm) was used. 0.05% phosphoric acid was used as mobile phase A and acetonitrile was used as mobile phase B. Gradient elution and gradient wavelength detection were performed according to Table 13. The flow rate was 1 mL / min, the column temperature was 35 ℃, the injection volume was 5 μL, and the total time was 70 min.

[0213] Table 13 Gradient elution and detection wavelength settings for content determination

[0214]

[0215]

[0216] 7.4 Linear

[0217] Take appropriate amounts of the stock solutions of each component reference standard, prepare a mixed reference standard solution, and dilute it stepwise to obtain a series of reference standard solutions for injection and determination. Plot a standard curve with the reference standard concentration (μg / mL) as the abscissa (X) and the peak area as the ordinate (Y). Calculate the linear regression equations and correlation coefficients for atractylone, atractylenolide I, atractylenolide II, atractylenolide III, and β-caryophyllene. The results are shown in Table 14, indicating good linearity for each component.

[0218] Table 14 Linear Range, Regression Equation, and Correlation Coefficient

[0219]

[0220] 7.5 Precision Test

[0221] A mixed reference solution with linear high, medium and low concentrations and the test solution were injected six times consecutively. The results showed that the RSD was less than 3% for all of them, indicating that the reference solution and the test solution had good precision.

[0222] 7.6 Repeatability Test

[0223] Six aliquots of the test solution were taken and each was injected once. The results showed that the average contents of atractylone, atractylenolide I, atractylenolide II, atractylenolide III, and β-caryophyllene in the Atractylodes macrocephala volatile oil samples were 488.276, 12.658, 1.844, 1.006, and 49.186 mg / g, respectively, with RSDs all less than 3%. This indicates that the method has good reproducibility.

[0224] 7.7 Stability Test

[0225] The same test sample was injected at 0, 2, 4, 6, 10, 14, 18 and 24 h. The results showed that the RSD of the peak area of ​​each component was less than 1.5%, and the test sample solution had good stability within 24 h.

[0226] 7.8 Recovery rate

[0227] Approximately 25 mg of a sample of Atractylodes macrocephala volatile oil with known content (containing 488.28, 12.66, 1.84, 0.43, and 49.19 mg / g of atractylodes macrocephala volatile oil, respectively) was accurately weighed and placed in a stoppered conical flask. Reference solutions equivalent to 50%, 100%, and 150% of the specified content were added to prepare test solutions. The solutions were then injected for determination, and the recovery rate (%) was calculated as (measured amount - known amount) / added amount * 100%. The results are shown in Table 15. The recovery limits all met the requirements, and the RSDs were all less than 3%, indicating that the method established in this invention has good accuracy.

[0228] Table 15 Recovery Results

[0229]

[0230]

[0231] 7.9 Determination of Volatile Oil Content in Atractylodes macrocephala

[0232] The contents of the above five components in 15 batches of Atractylodes macrocephala volatile oil samples were determined using the above-mentioned content determination method.

[0233] As shown in Table 16, the content ranges of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene in 15 batches of Atractylodes macrocephala volatile oil under this content determination method were 1.089–106.357, 0.258–25.183, 0.047–23.084, 20.271–1979.600, and 0.443–216.192 mg / g, respectively. The RSD values ​​of all components were less than 5%. This indicates that this method can be used for the content determination of multiple components in Atractylodes macrocephala volatile oil.

[0234] Table 16. Determination of volatile oil content (mg / g) in batch 15 of Atractylodes macrocephala.

[0235]

[0236] 7.10 Summary

[0237] The results showed that the HPLC method established in this embodiment exhibited good linearity between the peak area and the concentration of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone, and β-caryophyllene within a certain concentration range. The instrument demonstrated good precision and the method showed good repeatability. Both the reference solution and the test solution showed good stability within 24 hours, and the recoveries of all five components were within the limits. Furthermore, this method could detect all five components in multiple batches of Atractylodes macrocephala volatile oil, with RSD values ​​all less than 5%.

[0238] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for detecting the fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, characterized in that, Includes the following steps: (1) Provide test solution: When the test object is Atractylodes macrocephala, take Atractylodes macrocephala powder, add organic solvent, weigh the total weight, sonicate, replenish the volatile organic solvent to the original weight, filter, and the test solution is obtained; when the test object is Atractylodes macrocephala volatile oil, take Atractylodes macrocephala volatile oil, add organic solvent, sonicate, filter, and the test solution is obtained. (2) Provide a reference solution: the reference solution includes atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, β-eucalyptol, β-elemene, β-caryophyllene and geraniol B reference standards and organic solvents; (3) Determination: The test solution and the reference solution were determined by high performance liquid chromatography under the same chromatographic conditions to obtain the fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution. The fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution were compared to determine the index components in the fingerprint spectrum of the test solution, thereby obtaining the fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil.

2. The method for detecting the fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil as described in claim 1, characterized in that, Includes one or more of the following characteristics: (a) In step (1), the organic solvent is selected from methanol; when the target of detection is Atractylodes macrocephala, the ratio of the mass of Atractylodes macrocephala powder to the volume of organic solvent added is 1g:15-45mL, preferably 1g:30mL; when the target of detection is Atractylodes macrocephala volatile oil, the ratio of the mass of Atractylodes macrocephala volatile oil added to the volume of organic solvent added is 40-60mg:20-30mL, preferably 50mg:25mL; (b) In step (1), when the object to be tested is Atractylodes macrocephala, the ultrasonic treatment is performed by running water ultrasonic treatment for a time of 10 to 60 minutes, preferably 30 minutes; when the object to be tested is Atractylodes macrocephala volatile oil, the ultrasonic treatment is performed in an ice water bath for a time of 10 to 20 minutes, preferably 15 minutes. (c) In the reference solution described in step (2), the concentration range of atractylone, atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, β-eucalyptol, β-elemene, β-caryophyllene and geraniol B is 1 μg / mL to 2 mg / mL. (d) The chromatographic conditions described in step (3) are as follows: C18 column is used; column temperature is 25-40℃; detection wavelength is 200-220nm; flow rate is 0.8-1.2mL / min; injection volume is 2-20μL; in the mobile phase, phase A is 0.05%-0.1% (v / v) phosphoric acid aqueous solution, and phase B is acetonitrile; the analysis time is 70min, gradient elution is performed; the detector is DAD or VWD detector. The specific procedure for gradient elution is as follows: 0 min → 4 to 6 min, the volume ratio of phase A to phase B is 37-39%: 61-63%; 4~6min→9~11min, A phase:B phase volume ratio is 37-39%:61-63%→33-35%:65-67%; 9–11 min → 29–31 min, A phase: B phase volume ratio is 33–35%: 65–67% → 31–33%: 67–69%; 29–31 min → 34–36 min, A phase: B phase volume ratio is 31–33%: 67–69% → 24–26%: 74–76%; 34-36 min → 39-41 min, A phase: B phase volume ratio is 24-26%:74-76% → 14-16%:84-86%; 39~41min→44~46min, A phase:B phase volume ratio is 14-16%:84-86%; 44~46min→54~56min, A phase:B phase volume ratio is 14-16%:84-86%→9-11%:89-91%; 54–56 min → 69–70 min, A phase: B phase volume ratio is 9–11%: 89–91%.

3. The method for detecting the fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil as described in claim 2, characterized in that, Includes one or more of the following characteristics: (c1) In step (2), the concentration range of atractylone in the reference solution is 92-94 μg / mL, the concentration range of atractylodes lactone I is 50-52 μg / mL, the concentration range of atractylodes lactone II is 74-76 μg / mL, the concentration range of atractylodes lactone III is 93-95 μg / mL, the concentration range of β-eudesmol is 26-28 μg / mL, the concentration range of β-elemene is 50-52 μg / mL, the concentration range of β-caryophyllene is 38-40 μg / mL, and the concentration range of geraniol B is 9-11 μg / mL. (d1) The chromatographic conditions described use a C18 column, model BP-C18 Plus, with column dimensions of 250mm × 4.6mm and a packing diameter of 5μm. The column temperature is 35℃. A DAD detector is used; the detection wavelength is 203nm; the flow rate is 1mL / min; the injection volume is 5μL; the mobile phase consists of phase A being 0.05% (v / v) phosphoric acid aqueous solution and phase B being acetonitrile. The specific gradient elution procedure is as follows: 0 min → 5 min, the volume ratio of phase A to phase B is 38%:62%; From 5 min to 10 min, the volume ratio of phase A to phase B changed from 38%:62% to 34%:66%. From 10 min to 30 min, the volume ratio of phase A to phase B changed from 34%:66% to 32%:68%. 30min→35min, the volume ratio of phase A: phase B is 32%:68%→25%:75%; 35min→40min, the volume ratio of phase A: phase B is 25%:75%→15%:85%; 40min→45min, A phase:B phase volume ratio is 15%:85%; 45min→55min, A phase:B phase volume ratio 15%:85%→10%:90%; 55min→70min, A phase:B phase volume ratio is 10%:90%.

4. A method for constructing a standard fingerprint spectrum of Atractylodes macrocephala medicinal material or Atractylodes macrocephala volatile oil, characterized in that, include: By employing the fingerprint chromatogram detection method for Atractylodes macrocephala or Atractylodes macrocephala volatile oil as described in any one of claims 1 or 2, multiple batches of Atractylodes macrocephala or Atractylodes macrocephala volatile oil samples are detected to obtain fingerprint chromatograms of multiple batches of Atractylodes macrocephala or Atractylodes macrocephala volatile oil samples. A common pattern control chromatogram is generated, and the chromatographic peaks present in all chromatograms are taken as common characteristic peaks. The relative retention time of the common characteristic peaks and the ratio of the area of ​​each common characteristic peak to the total peak area are determined. Based on the relative retention time, the index components in the fingerprint chromatograms of Atractylodes macrocephala or Atractylodes macrocephala volatile oil are assigned and located, and a standard fingerprint chromatogram of Atractylodes macrocephala or Atractylodes macrocephala volatile oil is established. The similarity result of the common characteristic peaks is ≥0.

9.

5. The method for constructing the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil as described in claim 4, characterized in that, Includes one or more of the following characteristics: (1) The number of batches of Atractylodes macrocephala medicinal material samples or Atractylodes macrocephala volatile oil samples tested is 10 to 20; (2) The generation of the common pattern comparison spectrum includes: using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" software issued by the National Pharmacopoeia Commission to compare the similarity of fingerprint spectra of multiple batches of Atractylodes macrocephala medicinal materials or fingerprint spectra of Atractylodes macrocephala volatile oil samples, and using the median method to generate the superimposed spectrum and the common pattern comparison spectrum. (3) When the test object is a sample of Atractylodes macrocephala, the number of common characteristic peaks in the multiple batches is 18. Taking peak No. 11 as the reference peak S, the relative retention time is 1.

000. The relative retention times of the other 17 common characteristic peaks are as follows: peak No. 1 is 0.186, peak No. 2 is 0.256, peak No. 3 is 0.2827, peak No. 4 is 0.297, peak No. 5 is 0.312, and peak No. 6 is 0.

433. Peak 7 is 0.500, Peak 8 is 0.523, Peak 9 is 0.542, Peak 10 is 0.978, Peak 12 is 1.065, Peak 13 is 1.226, Peak 14 is 1.246, Peak 15 is 1.336, Peak 16 is 1.373, and Peak 17 is 1.413; the relative deviation of the relative retention time of the above 17 common characteristic peaks, except for Peak 11, is ≤ ±10%. (4) When the test object is a sample of Atractylodes macrocephala, the index components include peak 1 as atractylodes macrocephala III, peak 5 as atractylodes macrocephala II, peak 6 as atractylodes macrocephala I, peak 7 as β-eucalyptol, peak 11 as atractylone, peak 13 as β-elemene, peak 15 as geraniol B, and peak 16 as β-caryophyllene. (5) When the test object is Atractylodes macrocephala volatile oil sample, the number of common characteristic peaks in the multiple batches is 15. Taking peak No. 9 as the reference peak S peak, the relative retention time is 1.

000. The relative retention times of the other 14 common characteristic peaks are as follows: peak No. 1 is 0.179, peak No. 2 is 0.304, peak No. 3 is 0.378, peak No. 4 is 0.406, peak No. 5 is 0.426, peak No. 6 is 0.488, peak No. 7 is 0.528, peak No. 8 is 0.976, peak No. 10 is 1.131, peak No. 12 is 1.240, peak No. 13 is 1.261, peak No. 14 is 1.399, and peak No. 15 is 1.

441. The relative deviation of the relative retention times of the above 14 common characteristic peaks except for peak No. 9 is ≤±10%. (6) When the test object is a sample of Atractylodes macrocephala volatile oil, the index components include peak 1 as atractylodes macrocephala III, peak 2 as atractylodes macrocephala II, peak 5 as atractylodes macrocephala I, peak 6 as β-eucalyptol, peak 9 as atractylone, peak 10 as β-elemene, peak 12 as geraniol B, and peak 13 as β-caryophyllene. (7) When the test object is Atractylodes macrocephala volatile oil sample, the similarity between the fingerprint spectrum of multiple batches of Atractylodes macrocephala volatile oil sample and the control spectrum is >0.

97.

6. A method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala, characterized in that, Includes the following steps: S1. Provide the test solution: Take Atractylodes macrocephala powder, add organic reagents for ultrasonic extraction, cool, centrifuge and take the supernatant to obtain the test solution; S2. Provide a reference solution: the reference solution includes a certain amount of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene and an organic solvent; S3. Determination: Using high performance liquid chromatography under the same chromatographic conditions, the test solution of step S1 and the reference solution of step S2 are determined respectively. The contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the test solution are calculated by external standard method.

7. The method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in Atractylodes macrocephala as described in claim 6, characterized in that, Includes one or more of the following characteristics: (a) The particle size of the Atractylodes macrocephala powder in step S1 is ≤355μm; (b) The organic solvent mentioned in steps S1 and S2 is selected from methanol; the ratio of the mass of Atractylodes macrocephala powder to the volume of organic solvent added in step S1 is 1g:12-18mL, preferably 1g:15mL. (c) The ultrasonic treatment in step S1 is performed by running water ultrasonic treatment, and the ultrasonic treatment time is 12 to 18 minutes, preferably 15 minutes. (d) After ultrasonic treatment in step S1, place the sample at room temperature; (e) In the reference solution described in step S2, the concentration range of atractylone is 883.80–883.90 μg / mL, the concentration range of atractylodes lactone I is 216.90–216.95 μg / mL, the concentration range of atractylodes lactone II is 203.20–203.30 μg / mL, the concentration range of atractylodes lactone III is 209.90–210.00 μg / mL, and the concentration range of β-caryophyllene is 277.65–277.75 μg / mL; (f) The chromatographic conditions described in step S3 are as follows: a C18 column is used; the column temperature is 35℃; the flow rate is 1 mL / min; the injection volume is 5 μL; in the mobile phase, phase A is 0.05% (v / v) phosphoric acid aqueous solution and phase B is acetonitrile; the analysis time is 70 min, gradient elution is performed; the detector is a photodiode array detector. The specific procedure for gradient elution is as follows: 0 min → 4 to 6 min, the volume ratio of phase A to phase B is 37-39%: 61-63%; 4~6min→9~11min, A phase:B phase volume ratio is 37-39%:61-63%→33-35%:65-67%; 9–11 min → 29–31 min, A phase: B phase volume ratio is 33–35%: 65–67% → 31–33%: 67–69%; 29–31 min → 34–36 min, A phase: B phase volume ratio is 31–33%: 67–69% → 24–26%: 74–76%; 34-36 min → 39-41 min, A phase: B phase volume ratio is 24-26%:74-76% → 14-16%:84-86%; 39~41min→44~46min, A phase:B phase volume ratio is 14-16%:84-86%; 44~46min→54~56min, A phase:B phase volume ratio is 14-16%:84-86%→9-11%:89-91%; 54~56min→69~70min, A phase:B phase volume ratio is 9-11%:89-91%; The detection is wavelength gradient detection, and the wavelength gradient procedure is as follows: 0 min to 15 min, wavelength 220 nm; 15 min to 17 min, wavelength 250 nm; 17 min to 22 min, wavelength 278 nm; 22 min to 26 min, wavelength 240 nm; 26 min to 35 min, wavelength 220 nm; 35 min to 70 min, wavelength 203 nm; (g) The external standard method described in step S3 includes: preparing reference solutions of gradient concentrations in parallel according to step S2, analyzing them by high performance liquid chromatography, establishing standard curve equations for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene by the correspondence between the peak area and the known content, and then performing high performance liquid chromatography on the test solution, recording the peak area of ​​each component, and calculating the content of each component in the test solution according to the standard curve equation.

8. A method for simultaneous detection of the contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the volatile oil of Atractylodes macrocephala, characterized in that, Includes the following steps: S1. Provide the test solution: Take the volatile oil of Atractylodes macrocephala, add organic reagents, sonicate, filter, and the test solution is obtained. S2. Provide a reference solution: the reference solution includes a certain amount of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene and an organic solvent; S3. Determination: Using high performance liquid chromatography under the same chromatographic conditions, the test solution of step S1 and the reference solution of step S2 are determined respectively. The contents of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the test solution are calculated by external standard method.

9. The method for simultaneous detection of atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene in the volatile oil of Atractylodes macrocephala as described in claim 8, characterized in that, Includes one or more of the following characteristics: (a) The ratio of the mass of Atractylodes macrocephala volatile oil added in step S1 to the volume of organic solvent added is 40-60 mg: 20-30 mL, preferably 50 mg: 25 mL; (b) The organic solvents used in steps S1 and S2 are both methanol; (c) The ultrasonic treatment described in step S1 is performed in an ice water bath for 10 to 20 minutes. (d) In the reference solution described in step S2, the concentration range of atractylone is 1017.5–1017.6 μg / mL, the concentration range of atractylodes lactone I is 1009.7–1009.8 μg / mL, the concentration range of atractylodes lactone II is 1033.2–1033.3 μg / mL, the concentration range of atractylodes lactone III is 3986.6–3986.7 μg / mL, and the concentration range of β-caryophyllene is 2120.6–2120.7 μg / mL; (e) The chromatographic conditions described in step (3) are as follows: C18 column is used; column temperature is 35℃; flow rate is 1mL / min; injection volume is 5μL; in the mobile phase, phase A is 0.05% (v / v) phosphoric acid aqueous solution and phase B is acetonitrile; analysis time is 70min, gradient elution is performed; detector is photodiode array detector. The specific procedure for gradient elution is as follows: 0 min → 4 to 6 min, the volume ratio of phase A to phase B is 37-39%: 61-63%; 4~6min→9~11min, A phase:B phase volume ratio is 37-39%:61-63%→33-35%:65-67%; 9–11 min → 29–31 min, A phase: B phase volume ratio is 33–35%: 65–67% → 31–33%: 67–69%; 29-31 min → 34-36 min, A phase: B phase volume ratio is 31-33%: 67-69% → 24-26%: 74-76%; 34-36min→39~41min, A phase:B phase volume ratio is 24-26%:74-76%→14-16%:84-86%; 39~41min→44~46min, A phase:B phase volume ratio is 14-16%:84-86%; 44~46min→54~56min, A phase:B phase volume ratio is 14-16%:84-86%→9-11%:89-91%; 54~56min→69~70min, A phase:B phase volume ratio is 9-11%:89-91%; The detection is wavelength gradient detection, and the wavelength gradient procedure is as follows: 0 min to 15 min, wavelength 220 nm; 15 min to 17 min, wavelength 250 nm; 17 min to 22 min, wavelength 278 nm; 22 minutes, wavelength from 278nm to 240nm; 22 min to 26 min, wavelength 240 nm; 26 minutes, wavelength from 240nm to 220nm; 26 min to 35 min, wavelength 220 nm; 35 minutes, wavelength from 220nm to 203nm; 35 min to 70 min, wavelength 203 nm; (f) The external standard method described in step S3 includes: preparing reference solutions of gradient concentrations in parallel according to step S2, analyzing them by high performance liquid chromatography, establishing standard curve equations for atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, atractylone and β-caryophyllene by the correspondence between the peak area and the known content, then performing high performance liquid chromatography on the test solution, recording the peak area of ​​each component, and calculating the content of each component in the test solution according to the standard curve equation.

10. The use of the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil obtained by the method for constructing the standard fingerprint spectrum of Atractylodes macrocephala or Atractylodes macrocephala volatile oil as described in claim 4 or 5, the method for simultaneous detection of the contents of atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III, atractylone and β-caryophyllene in Atractylodes macrocephala as described in claim 6 or 7, or the method for simultaneous detection of the contents of atractylodes macrocephala I, atractylodes macrocephala II, atractylodes macrocephala III, atractylone and β-caryophyllene in Atractylodes macrocephala volatile oil as described in claim 8 or 9 in the quality inspection and quality control of Atractylodes macrocephala products.

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