Quality control method for stir-fried rhizoma atractylodis macrocephalae with fructus aurantii

Abstract

The invention belongs to the technical field of quality detection of traditional Chinese medicines, and discloses a quality detection method of rhizoma atractylodis macrocephalae fried with fructus aurantii, which is characterized by comprising the following steps: performing qualitative analysis on rhizoma atractylodis macrocephalae fried with fructus aurantii by respectively adopting a thin-layer chromatography and an HPLC (High Performance Liquid Chromatography) characteristic chromatogram; a high performance liquid phase external standard curve method is adopted to carry out quantitative analysis on atractylenolide I, atractylenolide II, atractylenolide III, naringin and neohesperidin in the fructus aurantii fried bighead atractylodes rhizome. The invention establishes a thin-layer identification method for rhizoma atractylodis macrocephalae and fructus aurantii in a fructus aurantii fried rhizoma atractylodis macrocephalae processed product; according to the method for establishing the fingerprint spectrum, the similarity of 10 batches of rhizoma atractylodis macrocephalae fried with fructus aurantii ranges from 0.991 to 1.000; the method for determining the content of atractylenolide I, atractylenolide II, atractylenolide III, naringin and neohesperidin is established, scientific support is provided for formulating the quality standard of the characteristic processing decoction pieces of the temporary prescription, and effective guarantee is provided for clinical application of the decoction pieces.

Description

Technical Field

[0001] The present invention belongs to the field of quality inspection of traditional Chinese medicines and relates to a quality control method for stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus. Background Art

[0002] Atractylodes macrocephala is the dried rhizome of the plant Atractylodes macrocephala Koidz. of the Compositae family. It is bitter, sweet, and warm in nature, and belongs to the spleen and stomach meridians. It has the effects of invigorating the spleen and replenishing qi, drying dampness and promoting diuresis, stopping sweating, and preventing miscarriage. It is mainly used for treating anorexia due to spleen deficiency, abdominal distension and diarrhea, dizziness and palpitation due to phlegm retention, edema, spontaneous sweating, and restlessness during pregnancy [1] 。Atractylodes macrocephala was first recorded in "Shennong Ben Cao Jing" under the name of "Zhu", and was listed as a superior grade. However, after textual research, it was found that the "Zhu" in "Ben Jing" was actually Atractylodes lancea, and Atractylodes macrocephala was only regarded as a source of Atractylodes lancea and was classified under Atractylodes lancea. It was not until "Ben Cao Jing Ji Zhu" by Tao Hongjing in the Liang Dynasty that the two were distinguished according to their different plant morphologies [2] 。As a commonly used large-batch medicinal material, Atractylodes macrocephala is widely used clinically. Since the Tang and Song dynasties, Atractylodes macrocephala has been processed and used as medicine, and it has been widely spread to this day

[0003] Stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus is a processed variety with the characteristic of "processing medicine with medicine" in the Menghe medical school. It appears many times in "Medical Records of Ma Peizhi" and is mainly used for treating digestive system diseases such as spleen deficiency, phlegm retention, and dysentery. For example, when combined with processed Pinellia ternata and Shenxiang Powder, it can warm the middle-jiao and resolve turbidity, regulate the yang of the spleen and stomach, and treat syndromes such as deficiency of both the spleen and kidney, discomfort in the chest and epigastrium, vomiting acid and nausea; when combined with Ginseng Radix and Rhizoma Cimicifugae, it can treat syndromes of phlegm retention in the middle-jiao due to damp-phlegm, failure of the spleen yang to rise, and failure of the stomach turbidity to descend. "Yao Xing Ge Jue" records: "Atractylodes macrocephala, dries the deep dampness, tonifies the deficiency of the middle earth; Fructus Aurantii Immaturus, moves the intestines and stomach, breaks qi to attack masses; expels this phlegm and saliva, relieves distension and stops pain. Strengthens the spleen and promotes digestion, and all cases of vomiting and coughing are cured..." [3] , Atractylodes macrocephala can invigorate the spleen and replenish qi, dry dampness and promote diuresis; Fructus Aurantii Immaturus can regulate qi and soothe the middle-jiao, promote qi movement and relieve distension [4] ; Atractylodes macrocephala is warm in nature and tends to tonify, while Fructus Aurantii Immaturus is slightly cold in nature and tends to move. Stir-frying Atractylodes macrocephala with the juice of Fructus Aurantii Immaturus, the two herbs complement and restrict each other, with the combination of movement and tonification, and can achieve the effects of promoting qi movement and invigorating the spleen, drying dampness and resolving phlegm. Modern pharmacological research has confirmed that the lactone components contained in Atractylodes macrocephala have significant therapeutic effects on diseases such as malignant tumors, neuronal apoptosis, gastrointestinal inflammatory reactions, and liver fibrosis [5] .

[0004] At present, the overall research on stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus is relatively blank, especially the qualitative and quantitative research related to quality control is particularly lacking. Therefore, establishing a systematic analysis and detection method and clarifying its material basis and key quality attributes are the key tasks for improving the quality evaluation standard of stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus

[0005] References

[0006] [1] Pharmacopoeia Commission of the People's Republic of China. Pharmacopoeia of the People's Republic of China: Volume I [S]. Beijing: China Medical Science and Technology Press, 2020: 155-168.

[0007] [2] Yang Yang, Zhao Rong, Liu Chang, et al. Textual Research on Rhizoma Atractylodis Macrocephalae and Rhizoma Atractylodis [J]. Information on Traditional Chinese Medicine, 2023, 40(02): 18-22.

[0008] [3] Wang Hui, Zhong Guoyue, Zhang Shouwen, He Junwei, Zeng Jinxiang, et al. Research Progress on Chemical Constituents, Pharmacological Effects of Fructus Aurantii Immaturus and Prediction Analysis of Its Quality Markers [J]. Chinese Archives of Traditional Chinese Medicine, 40(9) (in 2022): 184-192, 284.

[0009] [4] Xie Dongyu. Research on the Academic Thought, Clinical Application and Inheritance Law of the Spleen and Stomach of the Ma School of the Menghe Medical School [D]. Nanjing University of Chinese Medicine, 2009.

[0010] [5] Li Tianle, Wang Zhengjiao, Li Yayu, et al. Research Progress on Pharmacological Effects of Atractylenolide in the Past 10 Years [J]. Chinese Medicine Modern Distance Education of China, 2025, 23(01): 172-174. Summary of the Invention

[0011] In order to overcome the disadvantages of the above-mentioned prior art, the purpose of the present invention is to provide a quality control method for stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus.

[0012] Stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus is prepared by using Fructus Aurantii Immaturus juice as an auxiliary material to moisten and stir-fry Atractylodes macrocephala slices. The peak areas of atractylenolide II and atractylenolide III in stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus are significantly higher than those in stir-fried Atractylodes macrocephala with bran.

[0013] Stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus is prepared by the following preparation process, including: taking Fructus Aurantii Immaturus slices according to the mass of Fructus Aurantii Immaturus slices being 5-15% of the mass of Atractylodes macrocephala slices, and preparing Fructus Aurantii Immaturus juice; mixing Atractylodes macrocephala slices with Fructus Aurantii Immaturus juice, moistening at room temperature for 20-30 min, and then stir-frying at a temperature of 120-150 °C for 10-20 min to obtain stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus.

[0014] Preferably, taking Fructus Aurantii Immaturus slices according to the mass of Fructus Aurantii Immaturus slices being 15% of the mass of Atractylodes macrocephala slices, preparing Fructus Aurantii Immaturus juice; mixing Fructus Aurantii Immaturus juice and Atractylodes macrocephala slices, moistening at room temperature for 25 min, and then stir-frying at a temperature of 130-140 °C for 15 min to obtain stir-fried Atractylodes macrocephala with Fructus Aurantii Immaturus.

[0015] The Fructus Aurantii Immaturus juice is prepared by the following method: weighing Fructus Aurantii Immaturus slices, using water as an extraction solvent, heating under reflux for extraction 1-3 times, each extraction for 0.5-1 h, filtering after each extraction, combining the decoction, and concentrating the decoction under reduced pressure to a concentration of 0.3 g / mL to obtain Fructus Aurantii Immaturus juice.

[0016] Preferably, the Fructus Aurantii juice is prepared by the following method: Weigh the Fructus Aurantii decoction pieces, use water as the extraction solvent, add water according to the mass ratio of Fructus Aurantii decoction pieces to water of 1:14 each time, heat under reflux for 3 times in total, extract for 1 h each time, filter after each extraction, combine the decoction liquid, and concentrate the decoction liquid under reduced pressure to a concentration of 0.3 g / mL to obtain the Fructus Aurantii juice.

[0017] Specifically, the Fructus Aurantii juice is prepared by the following method: Weigh the Fructus Aurantii decoction pieces, add water according to the mass ratio of Fructus Aurantii decoction pieces to water of 1:14, bring to a boil over high heat, then change to low heat to keep it slightly boiling, reflux and extract for 1 h in the slightly boiling state, and filter while it is hot with a filter cloth (120 meshes); according to the mass ratio of Fructus Aurantii decoction pieces to water of 1:14, add water to the medicinal residues and repeat the extraction 2 times. Each time, bring to a boil over high heat, then change to low heat to keep it slightly boiling, reflux and extract for 1 h in the slightly boiling state, and filter while it is hot with a filter cloth (120 meshes); combine the decoction liquid of the three extractions, and concentrate it under reduced pressure to a concentration of 0.3 g / mL to obtain the Fructus Aurantii juice.

[0018] The present invention is realized by the following technical solutions:

[0019] A quality detection method for stir-fried Atractylodes macrocephala with Fructus Aurantii includes: respectively using thin-layer chromatography and HPLC characteristic fingerprints to conduct qualitative analysis on stir-fried Atractylodes macrocephala with Fructus Aurantii, and using the external standard standard curve method of high-performance liquid chromatography to conduct quantitative analysis on atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin in stir-fried Atractylodes macrocephala with Fructus Aurantii;

[0020] Conducting qualitative analysis on stir-fried Atractylodes macrocephala with Fructus Aurantii by using HPLC characteristic fingerprints includes:

[0021] Step (1), establish the HPLC dual-wavelength segmented fusion fingerprint of stir-fried Atractylodes macrocephala with Fructus Aurantii: Weigh at least 10 batches of stir-fried Atractylodes macrocephala with Fructus Aurantii, prepare the test solution and the reference solution respectively, inject them into the high-performance liquid chromatograph for qualitative analysis. The HPLC chromatographic conditions are: chromatographic column: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm); mobile phase: 0.1% formic acid water (mobile phase A) - acetonitrile (mobile phase B), gradient elution: 0 - 5 min, 20% - 25% B; 5 - 15 min, 25% - 55% B; 15 - 28 min, 55% - 65% B; 28 - 35 min, 65% - 70% B; detection wavelength: 240 nm and 280 nm; flow rate 1 mL·min -1Column temperature: 40°C; Injection volume: 10 μL; Record the chromatograms at detection wavelengths of 240 nm and 280 nm. Select the data at the detection wavelength of 240 nm from 10 - 29 min, and select the data at the detection wavelength of 280 nm from 0 - 10 min and 29 - 35 min. Combine the data to obtain the dual-wavelength segmented fusion chromatograms of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, and analyze them using the similarity evaluation system software for traditional Chinese medicine chromatographic fingerprints to obtain the HPLC dual-wavelength fusion fingerprint of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, and calibrate the common characteristic peaks;

[0022] Step (2): Take stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus to prepare a test solution. Inject 10 μL of the test solution into a high-performance liquid chromatograph, and perform qualitative analysis according to the HPLC chromatographic conditions in step (1). Record the chromatograms at detection wavelengths of 240 nm and 280 nm, and use the HPLC dual-wavelength fusion fingerprint of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus obtained in step (1) as a reference to perform qualitative analysis on stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus.

[0023] When performing qualitative analysis on stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus using HPLC characteristic fingerprints, preparation of the test solution: Take 10 g of the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, add water at 10 - 14 times the weight of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, soak at room temperature for 15 min - 1 h, perform reflux extraction 3 times, with the weight ratio of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus to water being 1:10 - 1:14 each time, extract for 1.5 h each time, filter after each extraction, combine the decoctions, accurately measure 20 mL, freeze-dry to obtain the freeze-dried powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, accurately add 20 mL of methanol for reconstitution, perform ultrasonic treatment, centrifuge, take the supernatant, and filter to obtain the test solution.

[0024] The power of the ultrasonic treatment is 254 W, and the frequency is 40 kHz.

[0025] The rotation speed of the centrifugation is 12000 r·min -1 , and the centrifugation time is 10 min.

[0026] When performing qualitative analysis on stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus using HPLC characteristic fingerprints, the reference solution is a mixed reference solution of naringin, neohesperidin, atractylenolide I, atractylenolide II, and atractylenolide III.

[0027] Preparation of the reference solution: Accurately weigh a certain amount of naringin reference substance, neohesperidin reference substance, atractylenolide I reference substance, atractylenolide II reference substance, and atractylenolide III reference substance respectively, dissolve them with methanol to prepare a mixed reference solution of naringin, neohesperidin, atractylenolide I, atractylenolide II, and atractylenolide III.

[0028] Preferably, in the reference solution, the concentration of naringin is 563.96 μg·mL -1 , and the concentration of neohesperidin is 379.86 μg·mL -1, the concentration of atractylenolide I is 11.98 μg·mL -1 , the concentration of atractylenolide II is 42.01 μg·mL -1 , the concentration of atractylenolide III is 94.96 μg·mL -1 .

[0029] The HPLC dual-wavelength fusion fingerprint of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus calibrates 11 common characteristic peaks, and the characteristic peaks and their retention times are as follows:

[0030]

[0031]

[0032] The quantitative analysis described above includes: weighing the freeze-dried powder of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus to prepare a test solution; taking reference substances of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin, and preparing a series of concentration mixed reference substance solutions with methanol. Precisely pipette 10 μL of the test solution and the mixed reference substance solution respectively, inject them into a high-performance liquid chromatograph, draw a standard curve, and calculate the contents of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin according to the external standard curve method.

[0033] During quantitative analysis, the preparation of the test solution: weigh about 0.5 g of the freeze-dried powder of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus, accurately weigh it, place it in a conical flask, add 25 mL of methanol, ultrasonically extract for 30 min, centrifuge, take the supernatant, filter, and take the continuous filtrate to obtain the test solution.

[0034] The preparation of the freeze-dried powder of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus: add water with a weight 10 - 14 times that of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus to the powder of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus, soak for 15 min, heat under reflux for extraction 3 times, the weight ratio of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus to water is 1:10 - 1:14 each time, 1.5 h each time, filter after each extraction, combine the decoctions, and freeze-dry the decoctions to obtain the freeze-dried powder of the stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus.

[0035] For quantitative analysis, preparation of the series of concentration mixed reference substance solutions: Weigh appropriate amounts of atractylenolide I reference substance, atractylenolide II reference substance, atractylenolide III reference substance, naringin reference substance, and neohesperidin reference substance accurately, dissolve them with methanol and dilute to prepare solutions containing 1.47 mg, 1.09 mg, 1.06 mg, 1.04 mg, and 1.56 mg of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin respectively in each 1 mL, and that's how you get them. The preparation method of the mixed reference substance solution is as follows: Accurately measure appropriate amounts of the stock solutions of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin reference substances respectively, dilute them with methanol to prepare a mixed reference substance solution containing 15.85 μg of atractylenolide I, 31.42 μg of atractylenolide II, 75.97 μg of atractylenolide III, 502.26 μg of naringin, and 538.36 μg of neohesperidin in each 1 mL, and then dilute with methanol to obtain the series of concentration mixed reference substance solutions.

[0036] For quantitative analysis, the HPLC chromatographic conditions are as follows: Chromatographic column: Agilent ZORBAX SB - AQ (4.6×250 mm, 5 μm); Using 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution; The flow rate is 1.0 mL per minute; The column temperature is 40 °C; The detection wavelengths are 240 nm (for atractylenolide II and III) and 280 nm (for atractylenolide I, naringin, and neohesperidin).

[0037] The gradient elution program is as follows:

[0038]

[0039] The qualitative analysis of stir - fried Atractylodes macrocephala Koidz. with Fructus aurantii using thin - layer chromatography includes thin - layer chromatography identification of Atractylodes macrocephala Koidz. and thin - layer chromatography identification of Fructus aurantii.

[0040] The thin - layer chromatography identification of Atractylodes macrocephala Koidz. includes:

[0041] Preparation of the test solution: Take 0.5 g of the powder of stir - fried Atractylodes macrocephala Koidz. with Fructus aurantii, add 2 mL of n - hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the test solution.

[0042] Preparation of the reference medicinal material solution: Take 0.5 g of the powder of the reference medicinal material of Atractylodes macrocephala Koidz., add 2 mL of n - hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the reference medicinal material solution.

[0043] Thin layer chromatography identification: The sample application volume of the test solution is 10 - 15 μL, and that of the control crude drug solution is 10 - 15 μL. They are respectively spotted on the same silica gel thin layer plate; using a mixed solvent of petroleum ether (60 - 90 °C) - ethyl acetate with a volume ratio of 50:1 as the developing solvent: the developing distance is 9 cm; take out, inspect: air dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clearly developed. In the test solution chromatogram, pink spots appear at the corresponding positions of the Atractylodes macrocephala Koidz. control crude drug chromatogram.

[0044] Preferably, the silica gel thin layer plate is a silica gel G thin layer plate or a silica gel F thin layer plate.

[0045] Preferably, the sample application volume of the test solution is 10 μL, and that of the control crude drug solution is 10 μL.

[0046] Preferably, the developing tank used for thin layer chromatography identification is a double - groove developing tank, 20 cm × 10 cm. The developing tank is pre - equilibrated for 15 minutes, developed by ascending development, and the developing distance is 9 cm.

[0047] Thin layer chromatography identification of Fructus Aurantii Immaturus includes:

[0048] Preparation of the test solution: Take 0.5 g of stir - fried Atractylodes macrocephala Koidz. and Fructus Aurantii Immaturus powder, add 25 mL of methanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution;

[0049] Preparation of the control crude drug solution: Take 0.2 g of Fructus Aurantii Immaturus control crude drug powder, add 10 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol to obtain the control crude drug solution;

[0050] Preparation of the naringin reference substance solution: Take naringin reference substance, add methanol to make a solution containing 1 mg per 1 mL as the naringin reference substance solution.

[0051] Preparation of the neohesperidin reference substance solution: Take neohesperidin reference substance, add methanol to make a solution containing 1 mg per 1 mL as the neohesperidin reference substance solution.

[0052] Thin layer chromatography identification: The sample application volume of the test solution is 15 - 20 μL, and the sample application volumes of the control crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution are each 10 μL. They are respectively spotted on the same silica gel thin layer plate; using the lower layer solution of chloroform - methanol - water (volume ratio = 13:6:2) as the developing solvent, the developing distance is 9 cm; take out, inspect: air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and inspect under an ultraviolet lamp (365 nm). In the test solution chromatogram, fluorescent spots of the same color appear at the corresponding positions of the naringin reference substance and neohesperidin reference substance chromatograms.

[0053] Preferably, the silica gel thin layer plate is a silica gel G thin layer plate, a silica gel F thin layer plate or a silica gel HSG thin layer plate.

[0054] Preferably, the sample solution spotting volume is 15 μL.

[0055] Preferably, the developing tank used for thin layer chromatography identification is a double groove developing tank, 20 cm × 10 cm. The developing tank is pre-equilibrated for 15 minutes, developed upward, and the developing distance is 9 cm.

[0056] The stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii powder is the coarse powder obtained by crushing stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii and passing through a No. 2 sieve.

[0057] The Atractylodes macrocephala Koidz. control medicinal material powder is the powder obtained by crushing Atractylodes macrocephala Koidz. and passing through a No. 2 sieve.

[0058] The Fructus aurantii control medicinal material powder is the powder obtained by crushing Fructus aurantii and passing through a No. 2 sieve.

[0059] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0060] In view of the deficiencies in the current existing research, the present invention establishes a thin layer identification method for Atractylodes macrocephala Koidz. and Fructus aurantii in the stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii processed product; establishes a fingerprint method, and the similarity of 10 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii is between 0.991 and 1.000; establishes a content determination method for atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin. The present invention provides scientific support for the formulation of the quality standard of the clinically prepared special processed cut crude drugs, and also provides an effective guarantee for their clinical application. Description of the Drawings

[0061] Figure 1 It is the finished product drawing of the cut crude drug of the stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii sample 2.

[0062] Figure 2 It is the thin layer chromatogram of 20 batches of samples; among them, the control medicinal material is the Atractylodes macrocephala Koidz. control medicinal material solution, and samples 1 - 20 are the test solution of 20 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii.

[0063] Figure 3 It is the thin layer chromatogram for comparing different spotting volumes; among them, the control medicinal material is the Atractylodes macrocephala Koidz. control medicinal material solution, and sample 2 is the test solution of one of the 20 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii.

[0064] Figure 4 It is the thin layer chromatogram of different thin layer plates; among them, the left figure is the Qingdao Ocean G plate (t: 26 °C, RH: 50%), the right figure is the Merck F plate (t: 26 °C, RH: 50%), the control medicinal material is the Atractylodes macrocephala Koidz. control medicinal material solution, and sample 2 is the test solution of one of the 20 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii.

[0065] Figure 5Thin layer chromatography (TLC) diagrams at different temperatures and humidities; among them, the left diagram is from Merck (t: 5°C, RH: 45%), the right diagram is from Merck (t: 26°C, RH: 50%), the control medicinal material is the solution of Atractylodis macrocephalae control medicinal material, and sample 2 is the test solution of stir-fried Atractylodis macrocephalae with Fructus aurantii from one of the 20 batches.

[0066] Figure 6 TLC diagrams of 20 batches of samples; among them, the control medicinal material is the solution of Fructus aurantii control medicinal material, and samples 1 - 20 are the test solutions of stir-fried Atractylodis macrocephalae with Fructus aurantii from 20 batches.

[0067] Figure 7 TLC diagrams for comparison of different sample application amounts; among them, the control medicinal material is the solution of Fructus aurantii control medicinal material, and sample 2 is the test solution of stir-fried Atractylodis macrocephalae with Fructus aurantii from one of the 20 batches.

[0068] Figure 8 TLC diagrams on different thin layer plates; among them, A is Huanghai HSG plate (t: 26°C, RH: 44%), B is Merck F plate (t: 26°C, RH: 60%), C diagram is Qingdao Ocean G plate (t: 26°C, RH: 44%), D is Yinlong HSG plate (t: 26°C, RH: 60%), the control medicinal material is the solution of Fructus aurantii control medicinal material, and sample 2 is the test solution of stir-fried Atractylodis macrocephalae with Fructus aurantii from one of the 20 batches.

[0069] Figure 9 TLC diagrams at different temperatures and humidities; among them, the left diagram is from Merck (t: 5°C, RH: 44%), the right diagram is from Merck (t: 26°C, RH: 60%), the control medicinal material is the solution of Fructus aurantii control medicinal material, and sample 2 is the test solution of stir-fried Atractylodis macrocephalae with Fructus aurantii from one of the 20 batches.

[0070] Figure 10 High performance liquid chromatography (HPLC) diagrams of stir-fried Atractylodis macrocephalae with Fructus aurantii; among them, A is the HPLC diagram at a detection wavelength of 240 nm; B is the HPLC diagram at a detection wavelength of 280 nm.

[0071] Figure 11 HPLC fingerprint diagrams of stir-fried Atractylodis macrocephalae with Fructus aurantii; among them, A is the superimposed fingerprint diagram of 10 batches of samples; B is the reference diagram; C is the HPLC diagram of the mixed reference substances at a detection wavelength of 280 nm; D is the HPLC diagram of the mixed reference substances at a detection wavelength of 240 nm; 3. Naringin; 5. Neohesperidin; 8. Atractylenolide III; 10. Atractylenolide II; 11. Atractylenolide I.

[0072] Figure 12 HPLC chromatogram of the reference substance; among them, the upper diagram is the chromatogram showing the peaks of atractylenolide III and atractylenolide II (240 nm), and the lower diagram is the chromatogram showing the peaks of naringin, neohesperidin, and atractylenolide I (280 nm).

[0073] Figure 13 It is the HPLC chromatogram of Sample 1. Among them, the upper figure is the chromatogram of the peaks of atractylenolide III and atractylenolide II (240 nm), and the lower figure is the chromatogram of the peaks of naringin, neohesperidin, and atractylenolide I (280 nm). Specific implementation manners

[0074] The following details the solutions of the present invention through specific implementation manners. It should be noted that the said implementation manners are only for explaining the technical solutions of the present invention and do not constitute a limitation on the protection scope. Reasonable adjustments or equivalent replacements made by those skilled in the art based on the content of the present invention all fall within the protection scope defined by the claims of the present invention.

[0075] Example 1

[0076] The preparation process of the bitter orange juice is as follows: Weigh a certain amount of bitter orange slices (the dried immature fruits of the Rutaceae plant Citrus aurantium L. and its cultivated varieties), and according to the mass ratio of bitter orange slices to water of 1:14, add 14 times the amount of water to the bitter orange slices; Place the mixture in an electric heating mantle, turn on the heating reflux extraction device, first quickly heat it to the boiling state with strong fire, start timing after the medicinal liquid boils, and immediately turn the fire to gentle fire to keep the medicinal liquid in a slightly boiling state, and continuously extract for 1 hour in the slightly boiling state. After the extraction is completed, filter it while it is hot with a 120-mesh filter cloth to obtain the decoction; According to the mass ratio of slices to water of 1:14, add 14 times the amount of water to the medicinal residues again, and repeat the extraction 2 times according to the above method. Combine the decoctions obtained from the 3 extractions, and use a vacuum concentration device for concentration until the concentration of the concentrated liquid reaches 0.3 g / mL, that is, each 1 mL of the medicinal juice contains 0.3 g of the active ingredients of the bitter orange slices, thus obtaining the bitter orange juice.

[0077] The preparation process of stir-fried atractylodes macrocephala with bitter orange is as follows: According to the mass of the bitter orange slices being 15% of the mass of the atractylodes macrocephala slices, take the bitter orange slices and prepare the bitter orange juice with a concentration of 0.3 g / mL according to the "preparation process of the bitter orange juice"; Take the cleaned atractylodes macrocephala slices, add the bitter orange juice, mix well, and then moisten it (i.e., seal and soak) at room temperature for 25 minutes; Preheat the frying pan to 130 - 140 °C, keep it on gentle fire (130 - 140 °C), add the moistened atractylodes macrocephala, and continuously stir-fry for 15 minutes. At this time, the surface of the slices is uniformly dark yellow, and the texture is loose and crispy and does not stick to the hand; Place the stir-fried atractylodes macrocephala with bitter orange slices ( Figure 1 ) in a dry and clean container, and let it cool naturally to room temperature. The stir-fried atractylodes macrocephala with bitter orange slices is processed by a pulverizer and sieved through a No. 2 sieve to obtain light yellowish-brown powder of stir-fried atractylodes macrocephala with bitter orange.

[0078] The preparation process of the freeze-dried powder of Citrus aurantium and stir-fried Atractylodes macrocephala is as follows: add 14 times the weight of water to the Citrus aurantium and stir-fried Atractylodes macrocephala powder, soak at room temperature for 15 minutes, heat and reflux to extract 3 times, each time the weight ratio of Citrus aurantium and stir-fried Atractylodes macrocephala to water is 1:14, each extraction is 1.5 hours, filter after each extraction, combine the decoction, accurately measure 20 mL of the decoction, freeze it at -80℃ for 48 hours, take it out, and immediately put it into a freeze dryer for sublimation analysis to obtain the freeze-dried powder of Citrus aurantium and stir-fried Atractylodes macrocephala.

[0079] The inventors prepared 20 batches of stir-fried Atractylodes macrocephala with Fructus Aurantii I (denoted as S1-S20), used thin layer chromatography and fingerprint to identify the index components of Atractylodes macrocephala slices, used high performance liquid chromatography to determine the contents of five components, including atractylodes lactone I, atractylodes lactone II, atractylodes lactone III, naringin, and neohesperidin, and proposed a quality control method for stir-fried Atractylodes macrocephala with Fructus Aurantii I.

[0080] 1 Establish a thin layer identification study on Atractylodes macrocephala in fried Atractylodes macrocephala with Citrus aurantium

[0081] 1.1 Materials

[0082] CAMAG ATS 4 automatic spotter, CAMAG TLC Visualizer thin layer chromatography digital imaging system (Swiss Kama (China) Technology Co., Ltd.), METTLER TOLEDO AB204-N electronic balance (1 / 10,000, Mettler-Toledo Co., Ltd., Switzerland). Atractylodes macrocephala (China Food and Drug Inspection Institute, batch number: 120925-202114). Reagents such as n-hexane and ethyl acetate were of analytical grade.

[0083] High-efficiency silica gel F prefabricated thin layer plate (TLC silica gel 60, Merck, batch number HX31898026), high-efficiency silica gel G prefabricated thin layer plate (Ocean, Qingdao Ocean Chemical Co., Ltd., batch number 20230718).

[0084] 1.2 Solution preparation

[0085] Preparation of the test solution: Take 0.5 g of the powder of the stir-fried Atractylodes macrocephala and Fructus Aurantii Immaturus sample (the stir-fried Atractylodes macrocephala and Fructus Aurantii Immaturus are processed by a grinder and passed through a No. 2 sieve), add 2 mL of n-hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the test solution.

[0086] Preparation of control medicinal material solution: Take 0.5 g of Atractylodes macrocephala control medicinal material powder (Atractylodes macrocephala is processed by a grinder and passed through a No. 2 sieve), add 2 mL of n-hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the control medicinal material solution.

[0087] 1.3 Thin layer chromatography conditions

[0088] Thin layer board: high-efficiency silica gel G prefabricated thin layer board, high-efficiency silica gel F prefabricated thin layer board;

[0089] Sample application volume: Pipette 10 μL of the test solution and 10 μL of the control crude drug solution; spot them on the same silica gel thin layer plate respectively;

[0090] Developing solvent: Petroleum ether (60 - 90 °C) - ethyl acetate (volume ratio = 50∶1);

[0091] Developing tank: Double - trough developing tank, 20 cm × 10 cm;

[0092] Development: Control t: 28 °C, RH: 40%, pre - equilibrate the developing tank for 15 minutes, develop in an ascending manner, development distance 9 cm;

[0093] Inspection: Air - dry, spray with 5% vanillin sulfuric acid solution, heat until the spots develop clearly.

[0094] The thin layer chromatograms of 20 batches of Atractylodis Macrocephalae samples are shown in Figure 2 . In the chromatogram of the test sample, a pink spot appears at the corresponding position of the chromatogram of the control crude drug of Atractylodis Macrocephalae, which is atractylon.

[0095] 1.4 System suitability

[0096] 1.4.1 Comparison of different sample application volumes

[0097] Take the stir - fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus sample (sample number 2), prepare the test solution (test solution of sample 2) and the control crude drug solution according to the above - mentioned thin layer chromatography conditions and "1.2 Preparation of solutions". According to different sample application volumes of the test solution (2.5 μL, 10 μL, 15 μL, 20 μL), spot them on the same pre - cast high - performance silica gel F thin layer plate (TLC silica gel 60, Merck, batch number HX31898026). Except for the different sample application volumes, the others are all carried out according to "1.3 Thin layer chromatography conditions", spot, develop, take out, air - dry, spray with 5% vanillin sulfuric acid solution, heat until the spots develop clearly. The experimental results are shown in Figure 3 . When the sample application volume of the test solution is 10 μL and 15 μL, the chromatographic spots are relatively clear; when the sample application volume of the test solution is 20 μL, due to the excessive sample application volume, it is easy to smear the adjacent spots.

[0098] 1.4.2 Comparison of different thin layer plates

[0099] Take the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus sample (numbered sample 2) and the corresponding unprepared Atractylodis Macrocephalae decoction pieces (negative), and prepare the test solution and the control crude drug solution according to "1.2 Preparation of Solutions". Pipette 10 μL of the above test solution and 10 μL of the control crude drug solution, and spot them on the same precast high-efficiency silica gel G thin-layer plate (Ocean, Qingdao Ocean Chemical Co., Ltd., batch number 20230718) and the precast high-efficiency silica gel F thin-layer plate (TLC silica gel 60, Merck, batch number HX31898026) respectively. Except for the different thin-layer plates, the rest are carried out according to "1.3 Thin-layer Chromatography Conditions", spot the samples, develop, take out, dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clearly visible. The experimental results are shown in Figure 4 , the thin-layer silica gel plates from different manufacturers have no significant effect on the thin-layer identification of Atractylodis Macrocephalae in the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus.

[0100] 1.4.3 Comparison under different temperature and humidity

[0101] Take the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus sample (numbered sample 2) and the corresponding unprepared Atractylodis Macrocephalae decoction pieces (negative), and prepare the test solution and the control crude drug solution according to "1.2 Preparation of Solutions". Spot them on the same precast high-efficiency silica gel F thin-layer plate (TLC silica gel 60, Merck, batch number HX31898026). Except for the different temperature and humidity (t: 5 °C, RH: 45%; t: 26 °C, RH: 50%), the rest are carried out according to "1.3 Thin-layer Chromatography Conditions", develop them separately under different temperature and humidity conditions, take out, dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clearly visible. The experimental results are shown in Figure 5 , the separation of the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus is relatively clear under different temperature and humidity conditions, and the temperature and humidity have no obvious effect on the thin-layer separation effect of the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus.

[0102] 1.5 Summary

[0103] The experimental results show that the thin-layer chromatography method has good consistency in the identification of Atractylodis Macrocephalae in 20 batches of stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus samples. For the thin-layer chromatography identification of Atractylodis Macrocephalae, the thin-layer chromatography conditions are as follows: Thin-layer plate: Precast high-efficiency silica gel G thin-layer plate or precast high-efficiency silica gel F thin-layer plate; Sample application volume: The sample application volume of the test solution is 10 μL - 15 μL, preferably 10 μL, and the sample application volume of the control crude drug solution is 10 μL - 15 μL, preferably 10 μL; Spot them separately on the same precast high-efficiency silica gel G thin-layer plate or precast high-efficiency silica gel F thin-layer plate; Developing agent: Petroleum ether (60 - 90 °C) - ethyl acetate (volume ratio = 50:1); Development distance: 9 cm; Take out, dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clearly visible. In the test solution chromatogram, a pink spot appears at the corresponding position of the Atractylodis Macrocephalae control crude drug chromatogram, which is atractylone.

[0104] 2 Establishment of the thin-layer identification study of Fructus Aurantii Immaturus in the stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus

[0105] 2.1 Materials

[0106] CAMAG ATS 4 fully automatic spotting instrument, CAMAG TLC Visualizer thin layer chromatography digital imaging system (CAMAG (China) Technology Co., Ltd.), METTLER TOLEDO XPR204 electronic balance (one ten-thousandth, METTLER TOLEDO Co., Ltd., Switzerland), METTLER TOLEDO XP105DR electronic balance (one hundred-thousandth, METTLER TOLEDO Co., Ltd., Switzerland).

[0107] Naringin (National Institutes for Food and Drug Control, batch number: 110722 - 202116, content 93.5%), neohesperidin (National Institutes for Food and Drug Control, batch number: 111857 - 202305, content 99.6%), Fructus Aurantii (National Institutes for Food and Drug Control, batch number: 120981 - 202106). Water is purified water, and other reagents such as methanol are all of analytical grade.

[0108] High - efficiency silica gel F pre - coated thin layer plate (TLC silica gel 60, Merck, batch number HX31898026), high - efficiency silica gel G pre - coated thin layer plate (Ocean, Qingdao Ocean Chemical Industry Co., Ltd., batch number 20230718), high - efficiency silica gel HSG pre - coated thin layer plate (Huanghai, Yantai Jiangyou Silica Gel Development Co., Ltd., batch number 20170221), high - efficiency silica gel HSG pre - coated thin layer plate (Yinlong, Yantai Chemical Industry Research Institute, batch number 20170818).

[0109] 2.2 Preparation of Solutions

[0110] Preparation of test solution: Take 0.5 g of the powder of stir - fried Atractylodes macrocephala Koidz. with Fructus Aurantii (the stir - fried Atractylodes macrocephala Koidz. with Fructus Aurantii is processed by a pulverizer and passed through a No. 2 sieve), add 25 mL of methanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution.

[0111] Preparation of control crude drug solution: Take 0.2 g of the powder of control crude drug of Fructus Aurantii (the Fructus Aurantii is processed by a pulverizer and passed through a No. 2 sieve), add 10 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol to obtain the control crude drug solution.

[0112] Preparation of naringin reference substance solution: Take naringin reference substance, add methanol to make a solution containing 1 mg per 1 mL as the naringin reference substance solution.

[0113] Preparation of neohesperidin reference substance solution: Take neohesperidin reference substance, add methanol to make a solution containing 1 mg per 1 mL as the neohesperidin reference substance solution.

[0114] 2.3 Thin - layer Chromatography Conditions

[0115] Thin layer plates: High - efficiency silica gel G pre - prepared thin layer plates, high - efficiency silica gel F pre - prepared thin layer plates, high - efficiency silica gel HSG pre - prepared thin layer plates;

[0116] Sample application volume: Pipette 15 μL of the test solution, 10 μL each of the reference crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution;

[0117] Developing solvent: Lower layer solution of chloroform - methanol - water (volume ratio = 13∶6∶2);

[0118] Developing tank: Double - groove developing tank, 20 cm×10 cm;

[0119] Development: Pre - balance the developing tank for 15 minutes, develop by ascending development, development distance 9 cm;

[0120] Inspection: Air - dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, inspect under ultraviolet lamp (365 nm). The thin - layer chromatograms of 20 batches of Fructus Aurantii Immaturus samples (t: 28 °C, RH: 40%) are shown in Figure 6 , in the chromatogram of the test sample, at the positions corresponding to the chromatograms of naringin reference substance and neohesperidin reference substance, there are fluorescent spots of the same color.

[0121] 2.4 System suitability

[0122] 2.4.1 Comparison of different sample application volumes

[0123] Take the Fructus Aurantii Immaturus stir - fried Atractylodes macrocephala sample (sample No. 2), Fructus Aurantii Immaturus reference crude drug, naringin reference substance, neohesperidin reference substance, prepare the test solution (sample 2), reference crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution according to "2.2 Preparation of solutions". Additionally, take the corresponding unprocessed Atractylodes macrocephala slices (negative without processing), and prepare the Atractylodes macrocephala negative test solution according to the "Preparation of test solution" in "2.2 Preparation of solutions"; According to different sample application volumes (5, 10, 15, 20 μL) of the test solution and the Atractylodes macrocephala negative test solution, spot them on the same high - efficiency silica gel F pre - prepared thin layer plate (TLC silica gel 60, Merck, batch number HX31898026) respectively, and according to "2.3 Thin - layer chromatography conditions", spot, develop, air - dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and inspect under ultraviolet lamp (365 nm). The experimental results are shown in Figure 7 , when the sample application volumes of the test solution and the Atractylodes macrocephala negative test solution are both 15 μL and 20 μL, the chromatographic spots are relatively clear, the Rf value is moderate, and there is no interference from the negative sample. Therefore, the optimal sample application volume of the test solution is determined to be 15 μL.

[0124] 2.4.2 Comparison of different thin layer plates

[0125] Take the stir-fried Atractylodis Macrocephalae and Fructus Aurantii Immaturus sample (sample No. 2), the control crude drug of Fructus Aurantii Immaturus, naringin reference substance, and neohesperidin reference substance. Prepare the test solution, control crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution according to "2.2 Preparation of Solutions". Additionally, take the corresponding unprocessed Atractylodis Macrocephalae decoction pieces (negative without processing) and prepare the negative test solution of Atractylodis Macrocephalae according to the "Preparation of Test Solution" in "2.2 Preparation of Solutions". Spot them respectively on the same pre-coated high-efficiency silica gel HSG thin-layer plate (Huanghai, Yantai Jiangyou Silica Gel Development Co., Ltd., batch number 20170221), high-efficiency silica gel F thin-layer plate (TLC silica gel 60, Merck, batch number HX31898026), high-efficiency silica gel G thin-layer plate (Haiyang, Qingdao Ocean Chemical Industry Co., Ltd., batch number 20230718), and high-efficiency silica gel HSG thin-layer plate (Yinlong, Yantai Chemical Industry Research Institute, batch number 20170818). Except for the different thin-layer plates, the rest are carried out according to "2.3 Thin-Layer Chromatography Conditions". Spot the samples, develop, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and examine under an ultraviolet lamp (365 nm). The experimental results are shown in Figure 8 , the thin-layer silica gel plates from different manufacturers have no significant effect on the thin-layer identification of Fructus Aurantii Immaturus in stir-fried Atractylodis Macrocephalae and Fructus Aurantii Immaturus.

[0126] 2.4.3 Comparison under different temperature and humidity conditions

[0127] Take the stir-fried Atractylodis Macrocephalae and Fructus Aurantii Immaturus sample (sample No. 2), the control crude drug of Fructus Aurantii Immaturus, naringin reference substance, and neohesperidin reference substance. Prepare the test solution, control crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution according to "2.2 Preparation of Solutions". Additionally, take the corresponding unprocessed Atractylodis Macrocephalae decoction pieces (negative without processing) and prepare the negative test solution of Atractylodis Macrocephalae according to the "Preparation of Test Solution" in "2.2 Preparation of Solutions". Spot them on the same pre-coated high-efficiency silica gel F thin-layer plate (TLC silica gel 60, Merck, batch number HX31898026). Except for the different temperature and humidity conditions (t: 5 °C, RH: 44%; t: 26 °C, RH: 60%), the rest are carried out according to "2.3 Thin-Layer Chromatography Conditions". Develop them respectively under different temperature and humidity conditions, take out, air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and examine under an ultraviolet lamp (365 nm). The experimental results are shown in Figure 9 , the separation of stir-fried Atractylodis Macrocephalae and Fructus Aurantii Immaturus is relatively clear under different temperature and humidity conditions, and the temperature and humidity have no obvious effect on the thin-layer separation effect of stir-fried Atractylodis Macrocephalae and Fructus Aurantii Immaturus.

[0128] 2.5 Summary

[0129] The experimental results show that the thin-layer chromatography method has good consistency in the identification of Aurantii Fructus in 20 batches of stir-fried Atractylodis Macrocephalae with Aurantii Fructus samples. For the thin-layer chromatography identification of Aurantii Fructus, the thin-layer chromatography conditions are as follows: thin-layer plate: pre-coated high-efficiency silica gel G thin-layer plate, pre-coated high-efficiency silica gel F thin-layer plate or pre-coated high-efficiency silica gel HSG thin-layer plate; sample application volume: the sample solution is applied in an amount of 15 - 20 μL, preferably 15 μL, and 10 μL each of the reference crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution; they are respectively spotted on the same pre-coated high-efficiency silica gel G thin-layer plate, pre-coated high-efficiency silica gel F thin-layer plate or pre-coated high-efficiency silica gel HSG thin-layer plate; developing solvent: the lower layer solution of chloroform - methanol - water (volume ratio = 13∶6∶2); developing distance: 9 cm; take out, dry in air, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and examine under an ultraviolet lamp (365 nm). In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions of the chromatograms of the naringin reference substance and the neohesperidin reference substance.

[0130] 3 Establishment of the fingerprint of stir-fried Atractylodis Macrocephalae with Aurantii Fructus

[0131] 3.1 Instruments

[0132] Table 1. Experimental instruments

[0133]

[0134]

[0135] 3.2 Test drugs

[0136] Slices of Atractylodis Macrocephalae (batch number: 231202, Suzhou Tianling Sliced ​​Herbs Co., Ltd.), slices of Aurantii Fructus (batch number: 231055, Suzhou Tianling Sliced ​​Herbs Co., Ltd.); reference substances: naringin reference substance (purity 98.12%, batch number: MUST - 22062911), neohesperidin reference substance (purity 98.63%, batch number: MUST - 22072810), atractylenolide I reference substance (purity 99.82%, batch number: MUST - 22092811), atractylenolide II reference substance (purity 99.41%, batch number: MUST - 22111604), atractylenolide III reference substance (purity 99.79%, batch number: MUST - 22101715); water is ultrapure water; acetonitrile and methanol are chromatographically pure; the rest of the reagents are of analytical grade. 10 batches of Atractylodis Macrocephalae herbs (detailed source information is shown in Table 2).

[0137] Table 2. Information of 10 batches of Atractylodis Macrocephalae herbs

[0138]

[0139] 3.3 Chromatographic conditions

[0140] HPLC Chromatographic Conditions 1: Chromatographic column: Agilent ZORBAX SB-C 18 (250 mm × 4.6 mm, 5 μm); Mobile phase: 0.1% formic acid in water (A) - acetonitrile (B), gradient elution: 0 - 5 min, 20% - 25% B; 5 - 15 min, 25% - 55% B; 15 - 28 min, 55% - 65% B; 28 - 35 min, 65% - 70% B; Detection wavelength: 240 nm and 280 nm; Flow rate 1 mL·min -1 ; Column temperature: 40 °C; Injection volume: 10 μL.

[0141] HPLC Chromatographic Conditions 2: Chromatographic column: Agilent ZORBAX SB-C 18 (250 mm × 4.6 mm, 5 μm); Mobile phase: 0.1% formic acid in water (A) - acetonitrile (B), gradient elution: 0 - 5 min, 20% - 30% B; 5 - 10 min, 30% - 55% B; 10 - 15 min, 55% - 70% B; 15 - 22 min, 70% - 90% B; Detection wavelength: 240 nm and 280 nm; Flow rate 1 mL·min -1 ; Column temperature: 40 °C; Injection volume: 10 μL.

[0142] The HPLC chromatographic conditions for stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus were investigated. By comparing different elution ratios of 0.1% formic acid in water - acetonitrile, it was found that in the HPLC chromatogram obtained by gradient elution under Chromatographic Conditions 2, the resolution (<1.5) and peak shape of the chromatographic peaks were poor ( Figure 10 ); in the HPLC chromatogram obtained under Chromatographic Conditions 1, the resolution (>1.5) and peak shape of the chromatographic peaks were good ( Figure 11 ). Therefore, the gradient elution method of the mobile phase was determined as Chromatographic Conditions 1.

[0143] 3.4 Solution Preparation

[0144] Preparation of reference solution: Weigh accurately a certain amount of naringin reference substance, neohesperidin reference substance, atractylenolide I reference substance, atractylenolide II reference substance, and atractylenolide III reference substance respectively, dissolve them with methanol, and prepare a mixed reference solution with the concentration of naringin being 563.96 μg·mL -1 , the concentration of neohesperidin being 379.86 μg·mL -1 , the concentration of atractylenolide I being 11.98 μg·mL -1 , the concentration of atractylenolide II being 42.01 μg·mL -1 , and the concentration of atractylenolide III being 94.96 μg·mL -1 .

[0145] Preparation of the test solution: Take 10 g of the crude powder of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus (sieved through No. 2 sieve), add 14 times the amount (500 g) of water, soak at room temperature for 15 min, extract by heating under reflux 3 times. Each time, the weight ratio of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus to water is 1:14, extract for 1.5 h each time. Filter after each extraction, combine the decoctions, accurately measure 20 mL, place in a freezer at -80 °C for 48 h, take out, immediately put into a freeze dryer for sublimation and analysis, obtain the freeze-dried powder of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus, accurately add 20 mL of methanol for re-dissolution, and perform ultrasonic treatment (power 254 W, frequency 40 kHz), 12000 r·min -1 Centrifuge for 10 min, take the supernatant, filter, and obtain the test solution.

[0146] 3.5 Methodology investigation of fingerprint

[0147] 3.5.1 Precision

[0148] Take the test solution of the same batch of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus decoction pieces, and inject samples continuously for determination 6 times. Using neohesperidin as the reference peak, the calculated RSD values of the relative retention times of each common peak are less than 1.03%, and the RSD values of the relative peak areas are less than 1.57%, indicating good precision of the instrument.

[0149] 3.5.2 Repeatability

[0150] Take the same batch of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus decoction pieces, prepare 6 parallel test solutions, and inject samples for determination. Using neohesperidin as the reference peak, the calculated RSD values of the relative retention times of each common peak are less than 0.98%, and the RSD values of the relative peak areas are less than 3.90%, indicating good repeatability of this method.

[0151] 3.5.3 Stability

[0152] Take the test solution of the same batch of stir-fried Atractylodis Macrocephalae Rhizoma and Fructus Aurantii Immaturus decoction pieces, and inject samples for determination at 0, 2, 4, 8, 12, and 24 h respectively. Using neohesperidin as the reference peak, the calculated RSD values of the relative retention times of each common peak are less than 1.06%, and the RSD values of the relative peak areas are less than 4.21%, indicating good stability of the test solution within 24 h.

[0153] 3.6 Establishment of fingerprint and similarity evaluation

[0154] Ten batches of processed Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus decoction pieces were used to prepare test solutions. After injection and determination, the chromatograms at detection wavelengths of 240 nm and 280 nm were recorded. The data at 240 nm were selected for the time range of 10 - 29 min, and the data at 280 nm were selected for the time ranges of 0 - 10 min and 29 - 35 min. The data were combined to obtain the dual-wavelength segmented fusion chromatograms of each batch of processed Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus. These chromatograms were imported into the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" software. Using sample S10 as the reference chromatogram, with the time window width set at 0.1 min, and adopting the median method, through multi-point calibration and manual identification of Mark peaks, the dual-wavelength fusion fingerprint chromatogram and the reference chromatogram of processed Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus were obtained, as shown in Figure 11 A and B of Figure 11 C and D). A total of 11 common peaks were calibrated. By comparing with the chromatogram of the mixed reference substance solution (

[0155] Table 3. Each characteristic peak and its retention time

[0156] Peak Number Substance Name Retention Time 1 Unknown 5.262 - 5.575 min 2 Unknown 6.466 - 6.815 min 3 Naringin 7.089 - 7.446 min 4 Unknown 7.655 - 7.947 min 5 Neohesperidin 8.317 - 8.679 min 6 Unknown 10.083 - 10.297 min 7 Unknown 14.664 - 14.785 min 8 Atractylenolide III 19.972 - 20.095 min 9 Unknown 24.865 - 25.007 min 10 Atractylenolide II 26.116 - 26.253 min 11 Atractylenolide I 31.080 - 31.193 min

[0157] Table 4. Similarity evaluation of fingerprint chromatograms

[0158]

[0159] 4 Determination of the contents of 5 components in processed Atractylodis Macrocephalae Rhizoma and Aurantii Fructus Immaturus

[0160] 4.1 Instruments, materials and reagents

[0161] High performance liquid chromatograph (LC-40D X3, Shimadzu Corporation); Ultrasonic instrument (KQ5200DE, Kunshan Ultrasonic Instruments Co., Ltd.); XPE205 electronic balance (one in one hundred thousand, Mettler-Toledo AG, Switzerland); AB204-N electronic balance (one in ten thousand, Mettler-Toledo AG, Switzerland).

[0162] Naringin (National Institutes for Food and Drug Control, batch number: 110722 - 202116, content 93.5%), Neohesperidin (National Institutes for Food and Drug Control, batch number: 111857 - 202305, content 99.6%), Atractylenolide I (National Institutes for Food and Drug Control, batch number: 111975 - 201501, content 99.9%), Atractylenolide II (National Institutes for Food and Drug Control, batch number: 111976 - 201501, content 99.9%), Atractylenolide III (National Institutes for Food and Drug Control, batch number: 111978 - 202302, content 100.0%). Acetonitrile was of chromatographic purity, water was ultrapure water, and other reagents such as methanol and phosphoric acid were of analytical purity.

[0163] 4.2 Experimental Method

[0164] HPLC chromatographic conditions: The filler was octadecylsilane-bonded silica gel (Agilent ZORBAX SB - AQ 4.6×250mm, 5μm); 0.1% phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B, and gradient elution was carried out according to Table 5; the flow rate was 1.0 mL per minute; the column temperature was 40°C; the detection wavelengths were 240 nm (for Atractylenolide II and III) and 280 nm (for Atractylenolide I, Naringin, and Neohesperidin). The typical chromatogram is shown in Figure 12 and Figure 13 。

[0165] Table 5. Gradient Elution Table

[0166]

[0167] 4.3 Solution Preparation

[0168] Preparation of reference substance stock solution: Appropriate amounts of reference substances of Atractylenolide I, Atractylenolide II, Atractylenolide III, Naringin, and Neohesperidin were accurately weighed, dissolved and diluted with methanol to prepare Atractylenolide I reference substance stock solution containing 1.47 mg of Atractylenolide I per 1 mL, Atractylenolide II reference substance stock solution containing 1.09 mg of Atractylenolide II per 1 mL, Atractylenolide III reference substance stock solution containing 1.06 mg of Atractylenolide III per 1 mL, Naringin reference substance stock solution containing 1.04 mg of Naringin per 1 mL, and Neohesperidin reference substance stock solution containing 1.56 mg per 1 mL.

[0169] Preparation of mixed reference substance solution: Accurately measure appropriate amounts of the stock solutions of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin reference substances respectively, and dilute them with methanol to prepare a mixed reference substance solution containing 15.85 μg of atractylenolide I, 31.42 μg of atractylenolide II, 75.97 μg of atractylenolide III, 502.26 μg of naringin, and 538.36 μg of neohesperidin per 1 mL respectively.

[0170] Take an appropriate amount of the mixed reference substance solution and dilute it with methanol to prepare a series of concentration mixed reference substance solutions with the concentrations of atractylenolide I being 0.16 μg / mL, 0.32 μg / mL, 0.79 μg / mL, 1.59 μg / mL, 3.17 μg / mL, 7.93 μg / mL, the concentrations of atractylenolide II being 0.31 μg / mL, 0.63 μg / mL, 1.57 μg / mL, 3.14 μg / mL, 6.28 μg / mL, 15.71 μg / mL, the concentrations of atractylenolide III being 0.76 μg / mL, 1.52 μg / mL, 3.80 μg / mL, 7.60 μg / mL, 15.19 μg / mL, 37.99 μg / mL, the concentrations of naringin being 5.02 μg / mL, 10.05 μg / mL, 25.11 μg / mL, 50.23 μg / mL, 100.45 μg / mL, 251.13 μg / mL, and the concentrations of neohesperidin being 5.38 μg / mL, 10.77 μg / mL, 26.92 μg / mL, 53.84 μg / mL, 107.67 μg / mL, 269.18 μg / mL.

[0171] Preparation of test solution: Take about 0.5 g of the freeze-dried powder of stir-fried Atractylodis Macrocephalae with Fructus Aurantii Immaturus, accurately weigh it, place it in a conical flask, add 25 mL of methanol, extract ultrasonically for 30 min, cool, centrifuge to obtain the supernatant, filter, and take the subsequent filtrate to obtain the test solution.

[0172] Accurately pipette 10 μL of the mixed reference substance solution and the test solution respectively, inject them into a high performance liquid chromatograph, and calculate according to the external standard curve method to obtain the contents of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin.

[0173] 4.4 Method validation

[0174] 4.4.1 Linear range

[0175] Take the series of mixed reference substance solutions, determine them according to the above method, draw a standard curve, and the linear regression equations are shown in Table 6. The test results show that the linear correlation coefficients are all above 0.995, and the linear relationship is good.

[0176] Table 6. Linear equations and correlation coefficients of 5 components

[0177]

[0178] 4.4.2 Detection Limit and Quantitation Limit

[0179] Take the first concentration point of the standard curve, continuously dilute it, and determine it according to the above analysis method. The detection limit is determined by the corresponding concentration when the signal-to-noise ratio is greater than 3:1, and the quantitation limit is determined by the corresponding concentration when the signal-to-noise ratio is greater than 10:1. The results are shown in Table 7 for details.

[0180] Table 7. Detection Limit and Quantitation Limit of 5 Components

[0181]

[0182]

[0183] 4.4.3 Precision

[0184] Take the fourth concentration point of the standard curve, and repeat the determination 6 times according to the above analysis method. Calculate the RSDs of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin to be 0.13%, 0.06%, 0.11%, 0.93%, and 0.83% respectively according to the measured values. See Table 8 for details. The test results show that the precision of the instrument is good.

[0185] Table 8. Precision Test Results of 5 Components (Peak Area)

[0186]

[0187] 4.4.4 Repeatability

[0188] Take the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus of sample No. 2, prepare 6 portions of the test solution according to the above method and analyze. The determination results of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin are shown in Table 9. The results show that the repeatability of this method is good.

[0189] Table 9. Calculation Results of Repeatability Test of 5 Components (mg / g)

[0190]

[0191]

[0192] 4.4.5 Recovery Rate of Added Samples

[0193] Take the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus sample No. 2, place it in a conical flask, accurately add 2.5 ml of the mixed reference substance solution, add 22.5 ml of methanol, and prepare 6 portions of the test solution in parallel according to the above method for preparing the test solution and analyze. The contents of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin in sample 2 are 0.215 mg / g, 0.116 mg / g, 0.142 mg / g, 0 mg / g, and 11.140 mg / g respectively. Calculate its content, average recovery rate, and relative standard deviation (RSD%) according to the test results. The results are shown in Table 10 in detail. The calculation results show that the accuracy of this test is good.

[0194] Table 10. Results of the recovery test of five components

[0195]

[0196]

[0197] 4.4.6 Stability

[0198] Take the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus sample No. 2, prepare the test solution for stability determination according to the above method, and inject samples multiple times within 48 h for analysis. Calculate the RSDs of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin to be 0.53%, 0.67%, 0.58%, 1.37%, and 1.39% respectively according to the measured values. See Table 11 for details. The results show that the stability of this method is good within 48 h.

[0199] Table 11. Results of the stability test of five components (peak area)

[0200]

[0201] 4.5 Results and Discussion of Sample Determination

[0202] Determine the 20 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus samples this time. The results are shown in Table 12 in detail. There are large differences in the contents among different components, and the overall content is relatively low, which is not suitable for limit requirements for the time being.

[0203] Table 12. Determination results of five components in stir-fried Atractylodes macrocephala Koidz. with Fructus Aurantii Immaturus samples (mg / g)

[0204]

[0205]

Claims

1. A quality detection method for stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii, characterized in that: Including: Qualitative analysis of stir-fried Atractylodis Macrocephalae with Aurantii Fructus was performed by thin-layer chromatography and HPLC characteristic fingerprint respectively, and quantitative analysis of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin in stir-fried Atractylodis Macrocephalae with Aurantii Fructus was performed by the external standard curve method of high performance liquid chromatography. Qualitative analysis of stir-fried Atractylodis Macrocephalae with Aurantii Fructus was performed by HPLC characteristic fingerprint, including: Step (1): Establish the HPLC dual-wavelength segmented fusion fingerprint of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus: Weigh at least 10 batches of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, prepare the test solution and reference solution respectively, inject them into the high-performance liquid chromatograph for qualitative analysis. The HPLC chromatographic conditions are as follows: Chromatographic column: Agilent ZORBAX SB-C18 (250 mm × 4.6 mm, 5 μm); Mobile phase: 0.1% formic acid water is used as mobile phase A, and acetonitrile is used as mobile phase B. Gradient elution: 0 - 5 min, 20% - 25% B; 5 - 15 min, 25% - 55% B; 15 - 28 min, 55% - 65% B; 28 - 35 min, 65% - 70% B; Detection wavelength: 240 nm and 280 nm; Flow rate: 1 mL·min -1 ; Column temperature: 40 °C; Injection volume: 10 μL; Record the chromatograms at detection wavelengths of 240 nm and 280 nm. Select the data at the detection wavelength of 240 nm from 10 - 29 min, and select the data at the detection wavelength of 280 nm from 0 - 10 min and 29 - 35 min. Combine the data to obtain the dual-wavelength segmented fusion chromatogram of each batch of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus. Analyze it using the similarity evaluation system software for traditional Chinese medicine chromatographic fingerprints to obtain the HPLC dual-wavelength fusion fingerprint of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus, and calibrate the common characteristic peaks; Step (2): Take stir-fried Atractylodis Macrocephalae with Aurantii Fructus, prepare the test solution, take 10 μL of the test solution, inject it into the high performance liquid chromatograph, and perform qualitative analysis according to the HPLC chromatographic conditions in step (1). Record the chromatograms at detection wavelengths of 240 nm and 280 nm, and use the HPLC dual-wavelength fusion fingerprint of stir-fried Atractylodis Macrocephalae with Aurantii Fructus obtained in step (1) as a reference to perform qualitative analysis on stir-fried Atractylodis Macrocephalae with Aurantii Fructus.

2. The quality detection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 1, characterized in that: Stir-fried Atractylodis Macrocephalae with Aurantii Fructus is prepared by the following preparation process, including: taking Aurantii Fructus pieces according to the quality of Aurantii Fructus pieces being 5-15% of the quality of Atractylodis Macrocephalae pieces, and preparing Aurantii Fructus juice; mixing Atractylodis Macrocephalae pieces with Aurantii Fructus juice, moistening at room temperature for 20-30 min, and then stir-frying at a temperature of 120-150 °C for 10-20 min to obtain stir-fried Atractylodis Macrocephalae with Aurantii Fructus; wherein, the Aurantii Fructus juice is prepared by the following method: weighing Aurantii Fructus pieces, using water as the extraction solvent, heating under reflux for extraction 1-3 times, each extraction for 0.5-1 h, filtering after each extraction, combining the decoction, and concentrating the decoction under reduced pressure to a concentration of 0.3 g / mL to obtain Aurantii Fructus juice.

3. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii Immaturus according to claim 1, characterized in that: When performing qualitative analysis of stir-fried Atractylodis Macrocephalae with Aurantii Fructus by HPLC characteristic fingerprint, the preparation of the test solution: take 10 g of stir-fried Atractylodis Macrocephalae with Aurantii Fructus powder, add 10-14 times the weight of stir-fried Atractylodis Macrocephalae with Aurantii Fructus of water, soak at room temperature for 15 min-1 h, heat under reflux for extraction 3 times, the weight ratio of stir-fried Atractylodis Macrocephalae with Aurantii Fructus to water each time is 1:10-1:14, each extraction for 1.5 h, filtering after each extraction, combining the decoction, precisely measuring 20 mL, freeze-drying to obtain the freeze-dried powder of stir-fried Atractylodis Macrocephalae with Aurantii Fructus, precisely adding 20 mL of methanol for reconstitution, performing ultrasonic treatment, centrifuging, taking the supernatant, and filtering to obtain the test solution.

4. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 1, characterized in that: When performing qualitative analysis of stir-fried Atractylodis Macrocephalae with Aurantii Fructus by HPLC characteristic fingerprint, the reference solution is a mixed reference solution of naringin, neohesperidin, atractylenolide I, atractylenolide II, and atractylenolide III.

5. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 1, characterized in that: The HPLC dual-wavelength fusion fingerprint of the stir-fried Atractylodis Macrocephalae with Aurantii Fructus calibrates 11 common characteristic peaks, and the characteristic peaks and their retention times are as follows: 。 6. The quality detection method of stir-fried Atractylodes macrocephala with Fructus aurantii immaturus according to claim 1, characterized in that: The quantitative analysis includes: weighing the freeze-dried powder of stir-fried Atractylodis Macrocephalae with Aurantii Fructus, preparing the test solution; taking atractylenolide I reference substance, atractylenolide II reference substance, atractylenolide III reference substance, naringin reference substance, and neohesperidin reference substance, preparing a series of concentration mixed reference solutions with methanol, precisely sucking 10 μL of the test solution and the mixed reference solution respectively, injecting them into the high performance liquid chromatograph, drawing a standard curve, and calculating the contents of atractylenolide I, atractylenolide II, atractylenolide III, naringin, and neohesperidin according to the external standard curve method.

7. The quality detection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 6, characterized in that: For quantitative analysis, preparation of the test solution: Weigh approximately 0.5 g of the freeze-dried powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus, accurately weigh, place in a conical flask, add 25 mL of methanol, extract ultrasonically for 30 min, centrifuge, take the supernatant, filter, and take the subsequent filtrate to obtain the test solution. Preparation of the freeze-dried powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus: Add water in an amount 10 - 14 times the weight of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus to the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus, soak for 15 min, reflux extract with heating 3 times, with the weight ratio of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus to water being 1:10 - 1:14 each time, 1.5 h each time. Filter after each extraction, combine the decoctions, and lyophilize the decoctions to obtain the freeze-dried powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus.

8. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 6, characterized in that: For quantitative analysis, the HPLC chromatographic conditions are as follows: Chromatographic column: Agilent ZORBAX SB - AQ (4.6×250 mm, 5 μm); Use 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B for gradient elution; Flow rate is 1.0 mL per minute; Column temperature is 40 °C; The detection wavelength for atractylenolide II and III is 240 nm, and the detection wavelength for atractylenolide I, naringin, and neohesperidin is 280 nm. The gradient elution program is as follows:

9. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 1, characterized in that: Qualitative analysis of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus by thin-layer chromatography includes thin-layer chromatography identification of Atractylodes macrocephala Koidz. and thin-layer chromatography identification of Fructus aurantii immaturus. Thin-layer chromatography identification of Atractylodes macrocephala Koidz. includes: Preparation of the test solution: Take 0.5 g of the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus, add 2 mL of n-hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the test solution. Preparation of the reference crude drug solution: Take 0.5 g of the powder of the reference crude drug of Atractylodes macrocephala Koidz., add 2 mL of n-hexane, ultrasonically treat for 15 minutes, filter, and take the filtrate as the reference crude drug solution. Thin-layer chromatography identification: The sample application amount of the test solution is 10 - 15 μL, and the sample application amount of the reference crude drug solution is 10 - 15 μL. Spot them on the same silica gel thin-layer plate respectively; Use a mixed solvent of petroleum ether - ethyl acetate with a boiling range of 60 - 90 °C and a volume ratio of 50:1 as the developing agent: Develop a distance of 9 cm; Take out, examine: Air dry, spray with 5% vanillin sulfuric acid solution, heat until the spots are clearly colored. In the test solution chromatogram, a pink spot appears at the corresponding position to the chromatogram of the reference crude drug of Atractylodes macrocephala Koidz. Thin-layer chromatography identification of Fructus aurantii immaturus includes: Preparation of the test solution: Take 0.5 g of the powder of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus, add 25 mL of methanol, ultrasonically treat for 15 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 2 mL of methanol to obtain the test solution. Preparation of the reference crude drug solution: Take 0.2 g of the powder of the reference crude drug of Fructus aurantii immaturus, add 10 mL of methanol, ultrasonically treat for 30 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol to obtain the reference crude drug solution. Preparation of the naringin reference substance solution: Take the naringin reference substance, dissolve it in methanol to make a solution containing 1 mg per 1 mL as the naringin reference substance solution. Preparation of the neohesperidin reference substance solution: Take the neohesperidin reference substance, dissolve it in methanol to make a solution containing 1 mg per 1 mL as the neohesperidin reference substance solution. Thin layer chromatography identification: The sample application volume of the test solution is 15 - 20 μL, and the sample application volumes of the control crude drug solution, naringin reference substance solution, and neohesperidin reference substance solution are each 10 μL. They are respectively spotted on the same silica gel thin layer plate; using the lower layer solution with a volume ratio of chloroform - methanol - water = 13:6:2 as the developing agent, the developing distance is 9 cm; take out, inspect: air dry, spray with 3% aluminum trichloride ethanol solution, heat at 105 °C for about 5 minutes, and inspect under an ultraviolet lamp at 365 nm. In the test sample chromatogram, at the positions corresponding to the chromatograms of naringin reference substance and neohesperidin reference substance, there are fluorescent spots of the same color.

10. The quality inspection method of stir-fried Atractylodes macrocephala Koidz. with Fructus aurantii immaturus according to claim 9, characterized in that: When identifying Atractylodes macrocephala Koidz. by thin layer chromatography, the silica gel thin layer plate mentioned is silica gel G thin layer plate or silica gel F thin layer plate; the sample application volume of the test solution is 10 μL, and the sample application volume of the control crude drug solution is 10 μL; when identifying Fructus Aurantii by thin layer chromatography, the silica gel thin layer plate mentioned is silica gel G thin layer plate or silica gel F thin layer plate or silica gel HSG thin layer plate; the sample application volume of the test solution is 15 μL.

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