Quality detection method for standard decoction of rhizoma alismatis decoction

Abstract

The invention provides a quality detection method for a standard decoction of rhizoma alismatis decoction. The HPLC fingerprint spectrum, the content determination method and the thin-layer chromatography identification method of the rhizoma alismatis decoction standard decoction are established on the basis of early-stage literature examination and medicinal material producing area screening research results by taking reduction of'one bowl of decoction 'as a starting point, so that a method and a basis are provided for quality evaluation of the rhizoma alismatis decoction standard decoction; meanwhile, a foundation is laid for development and research of a compound granular preparation.

Description

Technical Field

[0001] The invention relates to a quality detection method for a standard decoction of Zexie decoction, belonging to the field of medicines. Background Art

[0002] Zexie Decoction was first recorded in the twelfth chapter of "Pulse and Symptoms of Phlegm and Fluid Cough Disease and Treatment" in Zhang Zhongjing's "Golden Chamber" in the Eastern Han Dynasty. It is now included in the "Catalogue of Ancient Classic Prescriptions (First Batch)" issued by the State Administration of Traditional Chinese Medicine. Zexie Decoction is an important prescription for treating phlegm and fluid diseases in ancient times. The original text records: "There is fluid under the heart, and the person suffers from dizziness and vertigo. Zexie Decoction is the main treatment. Zexie Decoction 5 liang, Atractylodes macrocephala 2 liang. The above two ingredients are boiled with 2 liters of water to obtain 1 liter, and then divided into portions and taken warm." It has the effects of diuresis, invigorating the spleen and controlling water. It is mainly used to treat fluid retention under the heart, dizziness, chest fullness, cough and edema. Modern pharmacological studies have shown that Zexie Decoction has the effects of diuresis, lowering blood pressure, lowering blood lipids, anti-atherosclerosis, anti-inflammatory, and regulating metabolism. Clinically, Zexie Decoction is used in its original form or modified form to treat hypertension, hyperlipidemia, dizziness, otitis media, non-alcoholic fatty liver disease and other diseases, and the effect is remarkable, with broad market application prospects.

[0003] The "Technical Guidelines for Pharmaceutical Research of Traditional Chinese Medicine Compound Preparations Managed According to the Catalog of Ancient Classic Prescriptions (Trial)" requires that standard decoctions should be studied and prepared in accordance with the key information of ancient classic prescriptions released by the state and the records in ancient books, and quality research of standard decoctions should be carried out, including specificity identification, dry paste rate, extract / total solids, content of multiple index components, fingerprint / characteristic spectrum, etc. for overall quality evaluation to characterize its quality. Standard decoctions are generally decoctions, concentrated extracts or dry products. Ancient books record that Zexie Decoction is boiled and then taken warm. Using its standard decoction as the benchmark sample for preparation research may restore its general appearance to the greatest extent and better reflect respect for clinical practice. At present, there are few studies on the quality of Zexie Decoction standard decoction. Cai Luyao et al. used the freeze-dried powder of Zexie Decoction as the standard decoction and clarified the transmission law of medicinal materials-decoction pieces-standard decoction.

[0004] CN201910065029.0, invention name: Quality detection method and quality evaluation method for standard decoction of Alisma orientalis decoction, discloses a quality detection method and evaluation method for standard decoction of Alisma orientalis decoction, the quality detection method is to first make Alisma orientalis and Atractylodes macrocephala into Alisma orientalis decoction extract, and then make it into standard decoction of Alisma orientalis decoction, and perform quality detection on the standard decoction of Alisma orientalis decoction, and the quality detection respectively includes detection of paste yield, content, HPLC fingerprint spectrum, physical fingerprint spectrum and UV-Vis fingerprint spectrum.

[0005] CN202310575929.6, invention name: detection method of Alisma decoction, discloses a detection method of Alisma decoction, which comprises: taking Atractylodes lactone III as internal standard, establishing relative correction factors of epoxy alismaene, alisma alcohol C, 23-acetyl alisma alcohol C, Atractylodes lactone II, alisma alcohol A, alisma alcohol B, 23-acetyl alisma alcohol B, and 11-deoxy alisma alcohol B; using liquid chromatography to determine the content of Atractylodes lactone III in Alisma decoction; calculating the contents of epoxy alismaene, alisma alcohol C, 23-acetyl alisma alcohol C, Atractylodes lactone II, alisma alcohol A, alisma alcohol B, 23-acetyl alisma alcohol B, and 11-deoxy alisma alcohol B in Alisma decoction according to each relative correction factor.

[0006] CN202010424127.1, invention name: A method for detecting a UPLC characteristic spectrum of Zexie decoction and an application of the characteristic spectrum, utilizing ultra-high performance liquid chromatography, taking multiple reference substances as reference peaks, and taking acetonitrile-phosphoric acid aqueous solution as the mobile phase for gradient elution, and through systematic and reasonable control of the chromatographic conditions, 19 common peaks can be identified at the same time, constituting the full picture of the characteristic spectrum of Zexie decoction. There are 7 identified common peaks, which enables the quality control of Zexie decoction to be upgraded from the original content determination of two components or the detection of a few common peaks to the control of the intrinsic quality of the entire Zexie decoction, and can more comprehensively reflect the characteristics of Zexie decoction, avoiding the method defects caused by detecting only a single or a few common peaks. Application number 202010139733.9, invention name: Method for establishing fingerprint of Zexie decoction and its fingerprint, discloses a method for establishing fingerprint of Zexie decoction and its fingerprint, comprising the following steps: step 1, using Zexie and Atractylodes macrocephala from different origins and batches to prepare Zexie decoction, using methanol solution to extract the decoction of Zexie decoction to obtain a test solution; using methanol solution to dissolve Atractylodes lactone I, Atractylodes lactone II, Atractylodes lactone III, Atractylalaxol A, Atractylalaxol B, and 23-acetylalaxol B to prepare a reference solution; step 2, performing ultra high performance liquid chromatography analysis on the test solution and the reference solution, and recording the spectra.

[0007] Since Zexie decoction contains both water-soluble and fat-soluble components, the currently reported methods cannot fully take both into account. Summary of the invention

[0008] The present invention establishes a qualitative and quantitative detection method for the standard decoction of the classic prescription Zexie decoction, provides a basis for its quality evaluation, and lays a research foundation for the development of compound preparations. This study preliminarily established the HPLC fingerprint spectrum, content determination method, and thin-layer chromatography identification method of the standard decoction of the classic prescription Zexie decoction, which can provide a method and basis for the quality evaluation of the standard decoction of Zexie decoction, and lay a foundation for the later research of Zexie decoction compound preparations.

[0009] The invention provides a quality detection method for a standard decoction of Zexie decoction, which comprises qualitative identification, fingerprint spectrum and content determination, wherein the chromatographic conditions of the fingerprint spectrum and the content determination are as follows: chromatographic column: Agilent Polaris 5C18-A (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-water (B), gradient elution: 14%-16% A (0-10min), 16% A (10-20min), 16%-51% A (20-64min), 51%-52% A (64-84min), 52%-70% A (84-102min), 70% A (102-117min); detection wavelength: 208nm-276nm; flow rate: 1mL / min; column temperature: 30℃; injection volume: 20μL;

[0010] The preparation method of the standard decoction is as follows: take 69g of Alisma orientalis and 27.6g of Atractylodes macrocephala, put them in a ceramic pot, add 400ml of water, cover, soak for 60min, heat to boiling over high heat, turn to low heat and boil until slightly boiling, boil until 200ml, remove, filter through a 120-mesh Chinese medicine filter, and obtain the decoction.

[0011] It adopts fingerprint spectrum and content determination to detect 23-acetyl alismatol B, 23-acetyl alismatol C, epoxy alismatene, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III.

[0012] Wherein, the detection steps of the fingerprint spectrum and content determination are:

[0013] a. Preparation of test solution

[0014] Accurately pipette 1 ml of the standard decoction of Zexie decoction, add methanol to make up to 2 ml, sonicate for 30 min, and filter through a 0.45 μm microporous membrane to obtain the solution;

[0015] b. Preparation of negative test solution

[0016] Weigh 69.0 g of Rhizoma Alismatis and 27.6 g of Atractylodes macrocephala to prepare negative sample solutions lacking Atractylodes macrocephala and Rhizoma Alismatis respectively;

[0017] c. Preparation of reference solution

[0018] Take the reference substances 23-acetyl alismatol B, 23-acetyl alismatol C, epoxy alismatene, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III respectively, weigh accurately, add 50% methanol to dissolve and make up to 10 mL, shake well, and obtain;

[0019] d. Detect according to the chromatographic conditions described.

[0020] The detection wavelength is 208 nm. The fingerprint spectrum has 20 common peaks, of which 6 peaks are epoxy alismatene (peak 11), atractylodes lactone III (peak 12), atractylodes lactone II (peak 13), alismatol A (peak 14), alismatol B (peak 18), and 23-acetyl alismatol B (peak 19). The relative retention times are 0.678, 1.000, 1.165, 1.192, 1.537, and 1.706, respectively.

[0021] The 8 chemical components in the standard decoction of Alisma orientalis decoction are respectively the contents of epoxyalismen, 23-acetylalismenol B, 23-acetylalismenol C, alisol A, alisol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III, with average values ​​of 34.74, 180.86, 18.65, 17.65, 238.19, 2.85, 6.38 and 15.42 μg / g, respectively; the detection wavelengths are: 208 nm for epoxyalismen, alisol A, atractylodes lactone B and 23-acetylalismenol B, 246 nm for 23-acetylalismenol C, 276 nm for atractylodes lactone I and 220 nm for atractylodes lactone III.

[0022] It adopts thin layer chromatography to detect 23-acetyl alismatol C, atractylodes lactone II and atractylodes lactone III.

[0023] The detection method of the thin layer chromatography is:

[0024] a. Preparation of test solution

[0025] Take 30ml of standard decoction of Zexie decoction, extract with n-hexane for 3 times, 20ml each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 1ml of methanol as the test solution; prepare Zexie negative test solution and Atractylodes macrocephala negative test solution in the same way;

[0026] b. Preparation of reference solution

[0027] Take 23-acetyl alismatol C reference substance, weigh it accurately, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution I; take atractylodes lactone II reference substance and atractylodes lactone III reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution II and reference solution III respectively;

[0028] c. Identification method

[0029] Identification of Alisma orientalis: Take 10μl of the test solution and 5μl of the reference solution I, spot them on the same silica gel G thin layer plate, use chloroform-acetone-formic acid (10:1:0.2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored; check under ultraviolet light (365nm), the test solution will show spots of the same color at the corresponding position of the reference solution chromatogram;

[0030] Identification of Atractylodes macrocephala: Pipette 10μl of the test solution and 5μl of each of the reference solutions II and III, spot them separately on the same silica gel G thin layer plate, use chloroform-acetone-formic acid (9.5:0.5:0.05) as the developing agent, develop, take out, dry, spray with 10% ethanolic sulfuric acid solution, heat at 105℃ until the spots are clearly colored; examine under ultraviolet light (365nm), the test solution will show spots of the same color at the corresponding position of the reference solution chromatogram.

[0031] Compared with patent CN201910065029.0, the present invention prepares the standard decoction of Zexie Decoction in strict accordance with the ancient decoction method based on the verification of dosage, amount of water added, etc.; methanol (making the methanol concentration 50%) is added to the standard decoction as an extraction solvent to prepare HPLC fingerprint and test solution for content determination, taking into account the extraction of water-soluble and fat-soluble components in Zexie Decoction; the established fingerprint spectrum identified 20 common peaks, which can better reflect the overview of Zexie Decoction; the established fingerprint spectrum chromatographic conditions can be used for content determination at the same time, and the contents of 8 compounds can be determined simultaneously, and the method is simple, fast and accurate.

[0032] Compared with patent CN202310575929.6, the present invention simultaneously establishes the fingerprint spectrum, content determination and thin layer identification method of the standard decoction of Alisma decoction, which can provide methods and basis for comprehensively evaluating and controlling the quality of Alisma decoction.

[0033] Compared with patents CN202010424127.1 and 202010139733.9, the present invention uses high performance liquid chromatography to establish the fingerprint of Alisma decoction. The instrument is more universal. The present invention also establishes the content determination and thin layer identification methods of Alisma decoction, which can more comprehensively evaluate and control the quality of Alisma decoction.

[0034] Based on previous literature research and screening of medicinal materials origin, the present invention takes the restoration of "a bowl of soup" as the starting point, and establishes the HPLC fingerprint, content determination method and thin layer chromatography identification method of the standard decoction of Zexie Decoction, in order to provide a method and basis for the quality evaluation of the standard decoction of Zexie Decoction, and at the same time lay a foundation for the development and research of its compound granule preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1HPLC fingerprint of standard decoction of Zexie decoction;

[0036] Figure 2 Comparison of fingerprints of the standard decoction of Zexie decoction (Note: 11. Epoxy alismatene; 12. Atractylodes lactone III; 13. Atractylodes lactone II; 14. Alismatol A; 18. Alismatol B; 19. 23-acetyl alismatol B);

[0037] Figure 3 Comparison chart of the standard decoction of Zexie decoction and negative samples of each single herb (Note: 11. Epoxy alismatene; 12. Atractylolactone III; 13. Atractylolactone II; 14. Alismatol A; 18. Alismatol B; 19. 23-acetyl alismatol B; a. 23-acetyl alismatol C; b. Atractylolactone I);

[0038] Figure 4 Thin layer chromatogram of the specificity investigation of Alisma orientalis (from left to right: Alisma orientalis decoction test sample, Alisma orientalis decoction test sample, Alisma orientalis negative sample, 23-acetyl alisma orientalis alcohol C);

[0039] Figure 5 Thin layer chromatogram of the specificity investigation of Atractylodes macrocephala (from left to right: Zexie decoction test sample, Zexie decoction test sample, Atractylodes macrocephala negative, Atractylodes lactone II, Atractylodes lactone III);

[0040] Figure 6 Alisma orientalis thin layer identification chromatogram (among which, A is Alisma orientalis thin layer identification chromatogram (Merck silica gel G plate) (from left to right are Alisma orientalis decoction compound, Alisma orientalis decoction compound, Alisma orientalis decoction compound, Alisma orientalis negative, 23-acetyl alisma alcohol C); B is Alisma orientalis thin layer identification chromatogram (Qingdao Ocean silica gel G plate) (from left to right are Alisma orientalis decoction compound, Alisma orientalis decoction compound, Alisma orientalis negative, 23-acetyl alisma alcohol C); C is Alisma orientalis thin layer identification chromatogram (Qingdao Ocean high-efficiency silica gel G plate) (from left to right are Alisma orientalis decoction compound, Alisma orientalis decoction compound, Alisma orientalis negative, 23-acetyl alisma alcohol B, 23-acetyl alisma alcohol C, Atractylodes macrocephala negative, Atractylodes macrocephala lactone II, Atractylodes macrocephala lactone III);

[0041] Figure 7 Atractylodes macrocephala thin layer identification chromatogram (wherein, A is the Atractylodes macrocephala thin layer identification chromatogram (Merck silica gel G plate) (from left to right are Alisma decoction compound, Alisma decoction compound, Alisma decoction compound, Atractylodes macrocephala negative, Atractylodes macrocephala lactone II, Atractylodes macrocephala lactone III); B is the Atractylodes macrocephala thin layer identification chromatogram (Qingdao Ocean high-efficiency silica gel G plate) (from left to right are Alisma decoction compound, Alisma decoction compound, Alisma decoction negative, 23-acetyl alism alcohol B, 23-acetyl alism alcohol C, Atractylodes macrocephala negative, Atractylodes macrocephala lactone II, Atractylodes macrocephala lactone III));

[0042] Figure 8TLC identification chromatogram of the standard decoction of Zexie decoction (where A from left to right is S1-S4, 23-acetylzexetol C, S5-S8; B from left to right is S9-S12, 23-acetylzexetol C, S13-S15);

[0043] Fig. 9 TLC identification chromatogram of Atractylodes macrocephala in the standard decoction of Zexie decoction (A from left to right is S1-S4, Atractylodes lactone II, Atractylodes lactone III, S5-S8; B from left to right is S9-S12, Atractylodes lactone II, Atractylodes lactone III, S13-S15);

[0044] Fig.10 HPLC chromatograms of standard decoction of Zexie decoction on different chromatographic columns;

[0045] Fig.11 Mobile phase investigation HPLC chromatogram;

[0046] Fig.12 HPLC chromatograms with different elution programs;

[0047] Fig.13 HPLC chromatograms of standard decoction of Zexie decoction at different column temperatures;

[0048] Fig.14 Investigate the chromatogram at different flow rates;

[0049] Fig.15 HPLC chromatograms of standard decoction of Zexie decoction with different extraction solvents;

[0050] Fig.16 HPLC chromatograms of test sample solutions of standard decoction of Zexie decoction at different ultrasonic times. DETAILED DESCRIPTION

[0051] Example 1 Quality testing method for the standard decoction of Zexie decoction of the present invention

[0052] 1 Materials

[0053] 1.1 Instruments Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); Buch R-100 rotary evaporator (Buch, Switzerland); XS205 electronic balance (Mettler Toledo, Germany); KH7200E ultrasonic cleaner (Kunshan Hechuang Ultrasonic Instrument Co., Ltd.); Heraeus Mμltifuge X1R high-speed refrigerated centrifuge (Thermo Fisher Scientific (China) Co., Ltd.).

[0054] 1.2 Reagents and drugs Atractylodes lactone I (batch number: 111975-201501), Atractylodes lactone II (batch number: 111976-201501), Atractylodes lactone III (batch number: 111978-201501), 23-acetyl alismatol B (batch number: 111846-202006, purity 98.3%), 23-acetyl alismatol C (batch number: 112062-202102, purity 99.2%) ), all purchased from China Food and Drug Inspection Institute; alismatol A (batch number: MUST-22110115), alismatol B (batch number: MUST-22070410), alismatol F (batch number: MUST-22060704), alismatene epoxy (batch number: MUST-22112709), all purchased from Chengdu Munster Biotechnology Co., Ltd.; acetonitrile was chromatographically pure; water was ultrapure water; the rest of the reagents were of analytical grade.

[0055] 1.3 Medicinal materials and decoction pieces

[0056] All medicinal materials were collected or collected from the origin after selecting the origin. 15 batches of Alisma medicinal materials were from Xiejia Town, Pengshan, Sichuan, and 15 batches of Atractylodes medicinal materials were from Pan'an County, Zhejiang Province. They were identified by Fang Qingmao, a researcher at the Institute of Chinese Medicinal Resources and Planting of Sichuan Academy of Chinese Medicine, as the dried tubers of Alisma planta go-aquatica Linn., a plant of the Alismataceae family, and the dried rhizomes of Atractylodes macrocephala Koidz., a plant of the Asteraceae family. The processing of 15 batches of Alisma decoction pieces and 15 batches of Atractylodes decoction pieces was prepared according to the processing methods of Alisma and Atractylodes decoction pieces in the Chinese Pharmacopoeia (2020 edition).

[0057] 2HPLC fingerprint and content determination study

[0058] 2.1 Chromatographic conditions

[0059] Chromatographic column: Agilent Polaris 5C18-A (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-water (B), gradient elution: 14%~16%A (0~10min), 16%A (10~20min), 16%~51%A (20~64min), 51%~52%A (64~84min), 52%~70%A (84~102min), 70%A (102~117min); detection wavelength: 208nm; flow rate 1mL / min: column temperature: 30℃; injection volume: 20μL.

[0060] 2.2 Preparation of standard decoction of Zexie decoction

[0061] 15 batches of standard decoctions of Zexie decoction were decocted from the corresponding batches of medicinal pieces according to the ancient decoction method, numbered ZXT1~ZXT15. The decoction method was as follows: take 69g of Zexie and 27.6g of Atractylodes macrocephala, put them in a ceramic pot, add 400ml of water, cover, soak for 60min, heat to boiling over high heat, then simmer over low heat until slightly boiling, decocted to 200ml, removed, and filtered through a 120-mesh Chinese medicine filter.

[0062] 2.3 Preparation of test solution

[0063] Accurately pipette 1 ml of the standard decoction of Zexie decoction, add methanol to make up to 2 ml, sonicate for 30 min, and filter through a 0.45 μm microporous membrane to obtain the corresponding numbers S1 to S15.

[0064] 2.4 Preparation of negative test solution

[0065] Weigh 69.0 g of Alisma orientalis and 27.6 g of Atractylodes macrocephala respectively, and prepare negative sample solutions lacking Atractylodes macrocephala and Alisma orientalis according to the methods in 2.2 and 2.3.

[0066] 2.5 Preparation of reference solution

[0067] Take reference substances 23-acetyl alismatol B, 23-acetyl alismatol C, epoxy alismatene, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III respectively, weigh them accurately, add 50% methanol to dissolve and make up to 10 mL, shake well to obtain.

[0068] 2.6 HPLC fingerprint establishment

[0069] 2.6.1 Precision test Take the same test solution (S1) and inject it 6 times continuously according to the chromatographic conditions under "2.1" and record the peak area. Take Atractylodes lactone III with good separation and medium retention time as the reference peak, and calculate that the relative retention time RSD values ​​of each common peak are all less than 0.1%, and the relative peak area RSD values ​​are all less than 3.04%, indicating that the instrument has good precision.

[0070] 2.6.2 Stability test Take the same batch of test solution (S1) and measure the samples at 0, 4, 8, 12, 24 and 48 h according to the chromatographic conditions under "2.1". Record the peak area. Take Atractylodes lactone III as the reference peak. The relative retention time RSD values ​​of each common peak are calculated to be less than 0.3%, and the relative peak area RSD values ​​are less than 3.82%, indicating that the test solution has good stability within 48 h.

[0071] 2.6.3 Repeatability test Take 6 portions of standard decoction of Zexie decoction (S1) and prepare 6 portions of test sample solutions in parallel. Inject and analyze according to the chromatographic conditions under "2.1", record the peak area, and use Atractylodes lactone III as the reference peak. The relative retention time RSD values ​​of each common peak are calculated to be less than 0.2%, and the relative peak area RSD values ​​are less than 3.19%, indicating that the method has good repeatability.

[0072] 2.6.4 Fingerprint establishment and similarity evaluation The 15 batches of standard decoction of Zexie decoction were injected and tested according to the chromatographic conditions under "2.1", and the chromatograms were recorded. The data were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", and the S15 sample spectrum was used as the reference spectrum. The time window width was set to 0.1 min, and the median method was used. The superimposed spectrum and the reference spectrum R were established through multi-point calibration and Mark peak matching. Figure 1 , Figure 2 The fingerprint was assigned to the chromatographic peaks, and a total of 20 common peaks were calibrated. Figure 3 ) were compared, and 6 chromatographic peaks were identified, namely epoxy alismatene (peak 11), atractyl lactone III (peak 12), atractyl lactone II (peak 13), alismatol A (peak 14), alismatol B (peak 18), and 23-acetyl alismatol B (peak 19). By comparing with the negative samples of Alisma and Atractylodes, peaks 1 to 7 and peak 10 are common peaks of Alisma and Atractylodes, peaks 9, 11, 14, 16, 17, 18, 19, and 20 are from the monarch drug Alisma, and peaks 8, 12, 13, and 15 are from the minister drug Atractylodes.

[0073] The chromatographic peak No. 12, Atractylodes lactone III, which has good separation, medium retention time and is the effective component of Atractylodes macrocephala, was used as the reference peak (S), and the relative retention time RSD of other peaks was calculated to be <0.3%. The reference spectrum R was used as the reference spectrum for similarity evaluation. The similarities between the HPLC chromatograms of the 15 batches of Zexie decoction standard decoction samples and the reference spectrum were 0.995, 0.978, 0.979, 0.981, 0.996, 0.973, 0.992, 0.999, 0.997, 0.988, 0.980, 0.975, 0.963, 0.998, and 0.984, respectively, all greater than 0.960, indicating that the preparation process of Zexie decoction standard decoction is relatively stable, and the quality differences of different batches of samples are small.

[0074] 2.7 Determination of chemical composition content

[0075] 2.7.1 Chromatographic conditions The contents of 8 chemical components in the standard decoction of Alisma orientalis decoction were determined. The detection wavelengths were 208 nm for epoxyalaxene, alisma alcohol A, alisma alcohol B, 23-acetyl alisma alcohol B, 246 nm for 23-acetyl alisma alcohol C, 276 nm for atractylodes lactone I, 220 nm for atractylodes lactone II and III, and the other chromatographic conditions were the same as those in “2.1 Chromatographic conditions”.

[0076] 2.7.2 Investigation of linear relationship Take an appropriate amount of reference solution and dilute it with 50% methanol to prepare a mixed reference solution of a series of concentrations. Inject and measure according to the chromatographic conditions under "2.7.1". Draw a standard curve with the concentration of the reference solution as the horizontal axis and the peak area as the vertical axis. The results are shown in Table 1.

[0077] Table 1 Linear relationship investigation results

[0078]

[0079] 2.7.3 Precision test Take the mixed reference solution and inject it continuously for 6 times according to the chromatographic conditions under "2.7.1". Record the peak areas and calculate the RSDs of the peak areas of epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III, which are 0.93%, 0.17%, 0.21%, 3.30%, 0.47%, 0.89%, 0.31% and 0.21%, respectively, indicating that the precision of the instrument is good.

[0080] 2.7.4 Stability test The same batch of test solution was taken and sampled at 0, 4, 8, 12, 24 and 48 h according to the chromatographic conditions under "2.7.1". The RSDs of the peak areas of epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III were calculated to be 3.58%, 0.45%, 0.47%, 2.76%, 0.50%, 0.68%, 0.58% and 0.56%, respectively, indicating that the test solution had good stability within 48 h.

[0081] 2.7.5 Repeatability test Take 6 portions of the same standard decoction of the compound, prepare the test solution according to the method under "2.2", and analyze according to the chromatographic conditions under "2.7.1". The RSDs of the peak areas of epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III were calculated to be 3.02%, 2.73%, 2.03%, 2.63%, 1.32%, 1.59%, 1.04%, and 1.10%, respectively, indicating that the method has good repeatability.

[0082] 2.7.6 Sample addition recovery test Take S11 standard decoction with known component content in 6 parallel portions, add reference substances equivalent to the sample content to each other, prepare test solution according to the method under "2.3", inject and measure, and the average sample addition recoveries of epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III are calculated to be 100.22%, 98.42%, 97.16%, 81.11%, 91.26%, 95.29%, 93.61% and 80.34%, respectively, and the RSDs are 5.35%, 3.55%, 7.02%, 4.60%, 4.08%, 3.87%, 5.16% and 5.08%, respectively, indicating that the method has good accuracy.

[0083] 2.7.7 Content determination Take the test sample solutions of 15 batches of standard decoction of Zexie Decoction under "2.3" and determine the contents of epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractyl lactone I, atractyl lactone II, and atractyl lactone III in 15 batches of standard decoction of Zexie Decoction according to the chromatographic conditions under "2.7.1" (calculated as target ingredient content / prescription amount). The results are shown in Table 2.

[0084] Table 2 Active ingredient content of standard decoction of Zexie decoction

[0085]

[0086] 3 Thin layer chromatography identification study

[0087] 3.1 Preparation of test solution

[0088] Take 30ml of standard decoction of Zexie decoction, extract with n-hexane 3 times, 20ml each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 1ml of methanol as the test solution. Prepare Zexie negative test solution and Atractylodes macrocephala negative test solution in the same way.

[0089] 3.2 Preparation of reference solution

[0090] Take 23-acetyl alismatol C reference substance, weigh it accurately, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution I. Take atractylodes lactone II reference substance and atractylodes lactone III reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution II and reference solution III, respectively.

[0091] 3.3 Identification methods

[0092] Identification of Alisma orientalis: 10 μl of the test solution and 5 μl of the reference solution I were taken and spotted on the same silica gel G thin layer plate, respectively, and chloroform-acetone-formic acid (10:1:0.2) was used as the developing agent. After development, the plate was taken out, dried, and sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The plate was examined under ultraviolet light (365nm), and the test solution showed spots of the same color at the corresponding positions of the reference solution chromatogram.

[0093] Identification of Atractylodes macrocephala: 10 μl of the test solution and 5 μl of the reference solution II and III were respectively spotted on the same silica gel G thin layer plate, and chloroform-acetone-formic acid (9.5:0.5:0.05) was used as the developing agent. After development, the plate was taken out, dried, sprayed with 10% sulfuric acid ethanol solution, and heated at 105°C until the spots were clearly colored. The plate was placed under ultraviolet light (365nm) for inspection, and the test solution showed spots of the same color at the corresponding positions of the reference solution chromatogram.

[0094] 3.4 Exclusivity Investigation

[0095] 10 μl of each of the test solution and negative test solution of Zexie decoction standard decoction and 5 μl of reference solution I were taken and examined according to the method under "3.3 Identification of Zexie". The results showed that under ultraviolet light at 365nm, the test solution showed spots of the same color at the corresponding positions of the reference solution chromatogram, indicating that the negative Zexie sample had no interference. See the chromatogram for details. Figure 4 .

[0096] 10 μl of the test solution of Zexie decoction standard decoction, 10 μl of the negative test solution of Atractylodes macrocephala, and 5 μl of the reference solution II and III were taken, and the test was carried out according to the method under "3.3 Identification of Atractylodes macrocephala". The results showed that under ultraviolet light at 365 nm, the test solution showed spots of the same color at the corresponding positions of the reference solution chromatogram, indicating that the negative test solution of Atractylodes macrocephala had no interference. See the chromatogram for details. Figure 5 .

[0097] 3.5 Durability test

[0098] According to the thin layer identification method of Zexie and Atractylodes macrocephala under item "3.3", the separation effect of different brands of thin layer silica gel G plates on Zexie decoction compound samples was compared. The results showed that the Zexie decoction compound samples were well adapted to different silica gel G plate identification systems. Figure 6 , Figure 7 .

[0099] 3.6 Thin layer chromatography identification of standard decoction of Zexie decoction

[0100] According to the TLC identification method of Zexie and Atractylodes macrocephala under item "3.3", 15 batches of Zexie decoction standard decoctions were identified, respectively. Figure 8 and Fig. 9The results showed that the test solution showed spots of the same color at the corresponding positions on the chromatogram of the reference solution.

[0101] 4. Discussion

[0102] 4.1 Standard decoction dosage and preparation method

[0103] Zexie decoction comes from the book "Golden Chamber Synopsis·Tanyin cough disease pulse and treatment Chapter 12" by Zhang Zhongjing, the medical saint: "There is fluid under the heart, and the person suffers from dizziness, and Zexie decoction is the main treatment." Zexie 5 liang, Baizhu 2 liang. The above two ingredients are boiled with 2 liters of water to obtain 1 liter, and then divided into warm portions for serving. The preparation method of standard decoction should be basically consistent with the records in ancient books. Guided by the "Key Information Table of Ancient Classic Prescriptions (7 Prescriptions)" (from "Golden Chamber Synopsis") issued by the State Administration of Traditional Chinese Medicine, it is determined that 1 liang is equivalent to 13.8g in modern times, and 1 liter corresponds to 200ml in modern times. Therefore, the preparation method of Zexie decoction is: take 69g Zexie, 27.6g Baizhu, add 400ml of water, boil until 200ml, and then divide into warm portions for serving.

[0104] 4.2 Analysis of chromatographic conditions

[0105] This study investigated the separation effects of different chromatographic columns (Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm), Agilent Polaris 5C18-A (250mm×4.6mm, 5μm)), different mobile phases (methanol-water, acetonitrile-water, acetonitrile-0.05% phosphoric acid water), different elution procedures, different column temperatures (25℃, 30℃, 35℃), and different flow rates (0.8, 1.0, 1.2mL / min) on the standard decoction of Zexie decoction, and finally determined its chromatographic conditions. In addition, the content of each chemical component in the standard decoction is generally low, so the content of each chemical component is determined at the maximum absorption wavelength, that is, epoxy alisma, alisma alcohol A, alisma alcohol B, 23-acetyl alisma alcohol B: 208nm, 23-acetyl alisma alcohol C: 246nm, atractyl lactone I: 276nm, atractyl lactone II, atractyl lactone III: 220nm.

[0106] The decoction process of Zexie decoction is water decoction. The extracted medicinal ingredients contain more water-soluble components. The standard decoction was prepared with methanol water, acetonitrile water and ethanol water of different concentrations to determine the content of the test solution. The number of chromatographic peaks, peak area and chromatogram baseline were used as evaluation criteria, and the extraction solvent was finally determined to be 50% methanol.

[0107] 4.3 Results Analysis

[0108] In this study, the HPLC fingerprint, HPLC content determination method of index components and thin layer identification method of the standard decoction of Zexie decoction were established. The thin layer chromatography identification method is simple and feasible, and can quickly identify Zexie and Atractylodes macrocephala in the standard decoction; the similarity of HPLC fingerprints of 15 batches of Zexie decoction standard decoctions ranged from 0.960 to 0.999, all greater than 0.90, indicating that there was no significant difference in the main component groups of each batch of Zexie decoction standard decoction. The chemical components epoxy alismaene, 23-acetyl alisma alcohol B, 23-acetyl alisma alcohol C, alisma alcohol A, and alisma alcohol B are the effective components of Zexie decoction, which have anti-tumor, anti-allergic, anti-osteoporosis, and anti-allergic pharmacological effects. Atractylodes lactone Ⅰ, atractylodes lactone Ⅱ, and atractylodes lactone Ⅲ are the main effective components of Atractylodes macrocephala, which have multiple pharmacological effects such as anti-inflammatory, liver protection, spleen strengthening, and anti-tumor. The determination of their component contents can lay a material foundation for the study of the pharmacological and pharmacodynamic effects of Zexie decoction.

[0109] The average contents of 8 chemical components, including epoxy alismatene, 23-acetyl alismatol B, 23-acetyl alismatol C, alismatol A, alismatol B, atractyl lactone I, atractyl lactone II, and atractyl lactone III, in 15 batches of standard decoctions of Alisma decoction were 34.74, 180.86, 18.65, 17.65, 238.19, 2.85, 6.38, and 15.42 μg / g, respectively. The fluctuation range of alismatol A, alismatol B, and 23-acetyl alismatol B was larger than that of other chemical components (exceeding the average value ± 30%), which may be related to the thermal instability of alismatol components or the differences in the contents of chemical components between different batches. Therefore, when producing preparations, the decoction specifications should be strictly followed, and the representativeness and uniformity of sampling should be emphasized to ensure the consistency of the quality of preparations, intermediate products, and reference samples.

[0110] Example 2 Chromatographic conditions investigation:

[0111] 1. Chromatographic column inspection

[0112] Using acetonitrile (A)-water (B) as the mobile phase, gradient elution, 2%~21%A (0~24min), 21%A (24~34min), 21%~52%A (34~64min), 52%A (64~74min), 52%~68%A (74~104min), 68%A (104~119min), flow rate: 1mL / min; detection wavelength: 208nm; column temperature: 30℃; injection volume: 20μL, the separation effects of different chromatographic columns Agilent ZORBAX Extend-C18 (250mm×4.6mm, 5μm) and Agilent Polaris 5C18-A (250mm×4.6mm, 5μm) on the standard decoction of Alisma Orientalis Decoction were investigated. The chromatographic column Agilent Polaris 5C18-A has a better separation effect on the samples, and the chromatogram baseline is relatively flat. The chromatographic column is determined to be Agilent Polaris 5C18-A (see Fig.10 ).

[0113] 2. Mobile phase investigation

[0114] The separation effect of the standard decoction of Zexie decoction with methanol-water, acetonitrile-water, and acetonitrile-0.05% phosphoric acid water as the mobile phase was investigated. The chromatographic column was Agilent Polaris 5C18-A (250mm×4.6mm, 5μm), flow rate: 1mL / min; column temperature: 30℃; detection wavelength: 208nm; injection volume: 20μL, gradient elution conditions were methanol-water, acetonitrile-water, and acetonitrile-0.05% phosphoric acid water, respectively. The gradient elution program is shown in Tables 3-5. When acetonitrile-water was used as the mobile phase, the chromatogram had more chromatographic peaks and better peak shape, and the mobile phase was determined to be acetonitrile-water (see Fig.11 ).

[0115] Table 3 Methanol-water elution procedure

[0116]

[0117] Table 4 Acetonitrile-water elution procedure

[0118]

[0119] Table 5 Acetonitrile-0.05% phosphoric acid elution program

[0120]

[0121]

[0122] 3. Elution procedure investigation

[0123] Take the same test solution, use acetonitrile-water as the mobile phase, and examine the chromatographic peak separation effect of different gradient elution programs. Take three elution programs as examples. The gradient elution programs are shown in Tables 6-8, and the chromatograms are shown in Fig.12 The chromatogram of the gradient 3 elution program has better chromatographic peak separation effect and better peak shape, so the elution program is determined to be gradient 3.

[0124] Table 6 Gradient elution program 1

[0125]

[0126] Table 7 Gradient elution program 2

[0127]

[0128] Table 8 Gradient elution program 3

[0129]

[0130]

[0131] 4. Column temperature inspection

[0132] The separation effect of different column temperatures of 25℃, 30℃ and 35℃ on the standard decoction samples of Zexie decoction was investigated. Mobile phase: acetonitrile (A)-water (B), gradient elution: 4%~16%A (0~10min), 16%A (10~20min), 16%~51%A (20~64min), 51%~52%A (64~84min), 52%~70%A (84~102min), 70%A (102~117min); flow rate: 1mL / min; detection wavelength: 208nm; injection volume: 20μL. See the chromatogram for details. Fig.13 When the column temperature is 30℃, the chromatographic peak separation effect is better, so the column temperature is determined to be 30℃.

[0133] 5. Flow rate inspection

[0134] The separation effect of different flow rates of 0.8, 1.0, and 1.2 mL / min on the standard decoction samples of Zexie decoction was investigated. Mobile phase: acetonitrile (A)-water (B), gradient elution: 4%-16% A (0-10 min), 16% A (10-20 min), 16%-51% A (20-64 min), 51%-52% A (64-84 min), 52%-70% A (84-102 min), 70% A (102-117 min); detection wavelength: 208 nm; injection volume: 20 μL. See the chromatogram for details. Fig.14 When the flow rate is 1 mL / min, the peaks in the chromatogram are better separated and the peak shape is better, so the elution flow rate is determined to be 1 mL / min.

[0135] 6. Investigation on the preparation method of the standard decoction of Zexie decoction:

[0136] ① Extraction solvent investigation: Prepare 50% methanol, 50% acetonitrile, 70% methanol, 90% methanol solutions and aqueous solutions of the same concentration for the standard decoction of Zexie decoction, compare the chromatogram information, and determine the extraction solvent concentration of the test sample. The investigation results are shown in Fig.15 The chromatogram of the 50% methanol solution of the standard decoction of Zexie Decoction has richer chromatographic peak information, so it was determined that the test solution of the standard decoction of Zexie Decoction was an extraction solution of 50% methanol.

[0137] ② Ultrasonic time investigation The extraction effect of different ultrasonic times (15min, 30min, 60min) was investigated. There was no significant difference in the peak area of ​​the chromatograms of ultrasonic 30min and 60min, which was slightly higher than the peak area of ​​the chromatogram of ultrasonic 15min, so the ultrasonic time was determined to be 30min. The results of the investigation are shown in Fig.16 .

[0138] According to the above experimental results, the preparation method of the test solution of the standard decoction of Zexie Decoction was determined as follows: accurately aspirate 1 ml of the standard decoction of Zexie Decoction, add methanol to make the volume to 2 ml, ultrasonicate for 30 min, and filter through a 0.45 μm microporous filter membrane.

Claims

1. A quality detection method for a standard decoction of Zexie decoction, characterized in that: It includes qualitative identification, fingerprint spectrum, and content determination, wherein the chromatographic conditions of fingerprint spectrum and content determination are as follows: chromatographic column: Agilent Polaris 5C18-A (250mm×4.6mm, 5μm); mobile phase: acetonitrile (A)-water (B), gradient elution: 14%-16% A (0-10min), 16% A (10-20min), 16%-51% A (20-64min), 51%-52% A (64-84min), 52%-70% A (84-102min), 70% A (102-117min); detection wavelength: 208nm-276nm; flow rate 1mL / min: column temperature: 30℃; injection volume: 20μL; The preparation method of the standard decoction is as follows: take 69g of Alisma orientalis and 27.6g of Atractylodes macrocephala, put them in a ceramic pot, add 400ml of water, cover, soak for 60min, heat to boiling over high heat, turn to low heat and boil until slightly boiling, boil until 200ml, remove, filter through a 120-mesh Chinese medicine filter, and obtain the decoction.

2. The quality detection method of the standard decoction of Zexie decoction according to claim 1, characterized in that: It adopts fingerprint spectrum and content determination to detect 23-acetyl alismatol B, 23-acetyl alismatol C, epoxy alismatene, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III.

3. The quality detection method of the standard decoction of Zexie decoction according to claim 1 or 2, characterized in that: The detection steps of the fingerprint and content determination are as follows: a. Preparation of test solution Accurately pipette 1 ml of the standard decoction of Zexie decoction, add methanol to make up to 2 ml, sonicate for 30 min, and filter through a 0.45 μm microporous membrane to obtain the solution; b. Preparation of negative test solution Weigh 69.0 g of Rhizoma Alismatis and 27.6 g of Atractylodes macrocephala to prepare negative sample solutions lacking Atractylodes macrocephala and Rhizoma Alismatis respectively; c. Preparation of reference solution Take the reference substances 23-acetyl alismatol B, 23-acetyl alismatol C, epoxy alismatene, alismatol A, alismatol B, atractylodes lactone I, atractylodes lactone II, and atractylodes lactone III respectively, weigh accurately, add 50% methanol to dissolve and make up to 10 mL, shake well, and obtain; d. Detection according to the chromatographic conditions of claim 1.

4. The quality detection method of the standard decoction of Zexie decoction according to claim 3, characterized in that: The detection wavelength is 208 nm. The fingerprint spectrum has 20 common peaks, of which 6 peaks are epoxy alismatene (peak 11), atractylodes lactone III (peak 12), atractylodes lactone II (peak 13), alismatol A (peak 14), alismatol B (peak 18), and 23-acetyl alismatol B (peak 19). The relative retention times are 0.678, 1.000, 1.165, 1.192, 1.537, and 1.706, respectively.

5. The quality detection method of the standard decoction of Zexie decoction according to any one of claims 2 to 4, characterized in that: The 8 chemical components in the standard decoction of Alisma orientalis decoction are respectively the contents of epoxyalismen, 23-acetylalismenol B, 23-acetylalismenol C, alismenol A, alismenol B, atractylodes lactone I, atractylodes lactone II and atractylodes lactone III, with average values ​​of 34.74, 180.86, 18.65, 17.65, 238.19, 2.85, 6.38 and 15.42 μg / g, respectively; the detection wavelengths are respectively: 208 nm for epoxyalismen, atractylodes lactone A, atractylodes lactone B and 23-acetylalismenol B, 246 nm for 23-acetylalismenol C, 276 nm for atractylodes lactone I and 220 nm for atractylodes lactone III.

6. The quality detection method of the standard decoction of Zexie decoction according to claim 1, characterized in that: It adopts thin layer chromatography to detect 23-acetyl alismatol C, atractylodes lactone II and atractylodes lactone III.

7. The quality detection method of the standard decoction of Zexie decoction according to claim 6, characterized in that: The detection method of the thin layer chromatography is: a. Preparation of test solution Take 30ml of standard decoction of Zexie decoction, extract it with n-hexane for 3 times, 20ml each time, combine the extracts, evaporate to dryness in a water bath, dissolve the residue in 1ml of methanol, and use it as the test solution; Prepare Alisma orientalis negative test solution and Atractylodes macrocephala negative test solution in the same way; b. Preparation of reference solution Take 23-acetyl alismatol C reference substance, weigh it accurately, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution I; take atractylodes lactone II reference substance and atractylodes lactone III reference substance, add methanol to make a solution containing 0.5 mg per 1 mL, as reference solution II and reference solution III respectively; c. Identification method Identification of Alisma orientalis: Take 10μl of the test solution and 5μl of the reference solution I, spot them on the same silica gel G thin layer plate, use chloroform-acetone-formic acid (10:1:0.2) as the developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105℃ until the spots are clearly colored; check under ultraviolet light (365nm), the test solution will show spots of the same color at the corresponding position of the reference solution chromatogram; Identification of Atractylodes macrocephala: Pipette 10μl of the test solution and 5μl of each of the reference solutions II and III, spot them separately on the same silica gel G thin layer plate, use chloroform-acetone-formic acid (9.5:0.5:0.05) as the developing agent, develop, take out, dry, spray with 10% ethanolic sulfuric acid solution, heat at 105℃ until the spots are clearly colored; examine under ultraviolet light (365nm), the test solution will show spots of the same color at the corresponding position of the reference solution chromatogram.

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