Analytical method for litsea lancilimba formula granules

Abstract

The present invention relates to the technical field of drug analysis, and specifically relates to an analytical method for Litsea lancilimba formula granules. The Litsea lancilimba formula granules are formed by decocting Litsea lancilimba decoction pieces in water while collecting volatile oil and carrying out concentration, drying, and granulation. The analytical method comprises the following items: descriptions, identification, tests, extractives, characteristic chromatographic fingerprints, and assay. The identification method uses thin-layer chromatography for identification; in the characteristic chromatographic fingerprints, by taking the peak corresponding to the reference peak of magnoflorine reference substance as an S peak, high performance liquid chromatography is used to mark four characteristic peaks; and the assay is to determine the content of magnoflorine. The analytical method established in the present invention improves the analytical requirements for medicinal materials, decoction pieces, standard decoctions and formula granules of Litsea lancilimba, which helps to establish a perfect quality control system for various products of Litsea lancilimba, and helps to ensure the quality and clinical efficacy of the Litsea lancilimba formula granules.

Description

A detection method for Litsea cubeba formulation granules Technical Field

[0001] This invention belongs to the field of drug detection technology, specifically relating to a detection method for large-fruited Litsea cubeba formula granules. Background Technology

[0002] Traditional Chinese medicine (TCM) granules are made from single-herb medicinal slices through one or more extraction and concentration processes, and are intended for use in TCM clinical prescriptions. They have the advantages of being stable and controllable, safe and efficient, requiring no decoction, easy to prepare, convenient to carry, and ready to take with a simple dissolving process.

[0003] Litsea cubeba, also known as Cinnamomum migao HWLi, is the dried, mature fruit of the plant Cinnamomum migao (family Lauraceae). It is currently distributed in Guizhou, Yunnan, Guangxi, and other provinces. It is commonly used to treat abdominal pain, stomach pain, chest tightness, vomiting, and other ailments, and has shown good efficacy in treating arrhythmias. Clinically, it is primarily used to treat stomach pain due to cold stagnation, qi stagnation, and angina pectoris caused by deficiency-cold syndrome in coronary heart disease.

[0004] The chemical composition of Litsea cubeba is diverse, including volatile oils, fatty oils, fatty acids, flavonoids and inorganic elements, among which volatile oils are the main component and are widely used in clinical practice.

[0005] Currently, the main quality control methods for Litsea cubeba medicinal materials are morphological properties and thin-layer chromatography. With the increasing clinical use of formulated granules, there is an urgent need for comprehensive, convenient, and controllable testing methods to control the quality of Litsea cubeba formulated granules. Summary of the Invention

[0006] This invention addresses the shortcomings of existing detection methods for Litsea cubeba formula granules by providing a quality detection method for Litsea cubeba formula granules, specifically:

[0007] The testing items for the Litsea cubeba formula granules include morphology, identification, inspection, extractives, characteristic chromatograms, and content determination.

[0008] In some embodiments, the granules of Litsea cubeba are: yellowish-brown granules; slightly fragrant odor and bitter taste.

[0009] In some embodiments, the granules of Litsea cubeba are: yellowish-brown granules; slightly fragrant odor and bitter taste.

[0010] Identification: Take 2g of *Litsea cubeba* granules, grind them finely, add 20ml of ether, sonicate for 30 minutes, filter, and concentrate the filtrate to about 1ml as the test solution. Separately, take 0.5g of *Litsea cubeba* reference material and prepare a reference material solution using the same method. Perform thin-layer chromatography (Chinese Pharmacopoeia 2020 General Chapter 0502). Apply 5μl of each of the above two solutions separately to the same silica gel G thin-layer plate, using cyclohexane-ethyl acetate (8:2) as the developing solvent. Develop, remove, air dry, spray with vanillin-sulfuric acid reagent, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference material.

[0011] Extractives: Take 2g of Litsea cubeba granules, grind them into a fine powder, accurately weigh them, add 100ml of ethanol, and determine the extractives according to the hot extraction method under the "Determination of Alcohol-Soluble Extractives" section of the Chinese Pharmacopoeia.

[0012] Inspection: It should comply with all relevant regulations under the Granules section.

[0013] Characteristic chromatograms: determined by high performance liquid chromatography according to the general principles of the Chinese Pharmacopoeia;

[0014] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.1%–0.5% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.2–1 ml per minute; the column temperature was 30–40℃; the detection wavelength was 250–350 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should be no less than 5000–10000. The gradient elution conditions were as follows:

[0015] The detection method includes the following operations:

[0016] Preparation of reference solution: Take 0.5-2.0 g of Litsea cubeba reference material, add 25-50 ml of 70% methanol, heat under reflux for 30-60 min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of magnoflorine reference standard, accurately weigh it, add 70% methanol to prepare a solution containing 20-30 μg per ml, as the reference solution of the reference standard;

[0017] Preparation of the test solution: Take the formula granules of Litsea cubeba, grind them finely, take about 0.1-1g, weigh accurately, add 25-50ml of 70% methanol accurately, weigh accurately, sonicate for 30-60 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0018] For the determination method, accurately pipette 2-10 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography.

[0019] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, the peak corresponding to the peak of the magnoflorine reference standard is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: relative retention time of peak 1 is 0.33, relative retention time of peak 2 is 0.48, and relative retention time of peak 3 is 0.63.

[0020] In some embodiments, the characteristic chromatogram detection method employs ultra-high performance liquid chromatography (UHPLC).

[0021] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.3 ml / min; the column temperature was 35℃; the detection wavelength was 300 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 10,000. The gradient elution conditions were as follows:

[0022] The detection method includes the following operations:

[0023] Preparation of reference solution: Take 0.5g of Litsea cubeba reference material, add 25ml of 70% methanol, heat under reflux for 30min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of magnoflorine reference standard, accurately weigh it, add 70% methanol to prepare a solution containing 20μg per ml, as the reference solution of the reference standard.

[0024] Preparation of the test solution: Take the formula granules of Litsea cubeba, grind them finely, take 0.2g, weigh accurately, add 25ml of 70% methanol accurately, weigh accurately, sonicate for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0025] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography. Record the chromatograms from 0 to 22 min.

[0026] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, the peak corresponding to the peak of the magnoflorine reference standard is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: relative retention time of peak 1 is 0.33, relative retention time of peak 2 is 0.48, and relative retention time of peak 3 is 0.63.

[0027] Content determination: determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020).

[0028] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.3 ml / min; the column temperature was 35℃; the detection wavelength was 300 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 10,000. The gradient elution conditions were as follows:

[0029] Preparation of the reference solution: Take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 20 μg per ml.

[0030] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0031] The assay involves precisely pipetting 2 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0032] The formula for Litsea cubeba granules is prepared by taking 4500g of Litsea cubeba slices, adding water and decocting, collecting the volatile oil (encapsulated with β-cyclodextrin for later use), filtering the decoction, concentrating the filtrate into a clear paste, drying (or drying and pulverizing), adding the volatile oil inclusion complex and appropriate amount of excipients, mixing well, granulating, and making 1000g of the product. Beneficial effects:

[0033] 1. This invention is a detection method for Litsea cubeba formula granules. The detection method has comprehensive detection items, simple operation, high precision, strong specificity and good reproducibility.

[0034] 2. In addition to the conventional detection items, this invention adds the detection of characteristic chromatograms, which can more comprehensively reflect the types and quantities of medicinal chemical components contained in the product. In particular, the characteristic chromatograms are detected by UPLC, which has the characteristics of simple operation and good reproducibility. At the same time, the detection method also includes a content determination method, which is consistent with the chromatographic conditions of the characteristic chromatograms. The quantitative determination of magnoflorine can be carried out when detecting the characteristic chromatograms, saving time and effort.

[0035] 3. In existing technologies for Litsea cubeba, the content of ammonia olein in the volatile oil is used as the indicator for content determination. However, Litsea cubeba formulation granules are extracted by decoction. This invention uses the water-soluble component magnolia alkaloid as the content indicator, which is the first time it has been used and is an inevitable trend in the quality control of Litsea cubeba formulation granules.

[0036] 4. Through testing, the detection method of this invention is also applicable to Litsea cubeba medicinal materials, decoction pieces, standard decoctions, etc., and has a wide range of applications. Attached Figure Description

[0037] Figure 1. Characteristic spectrum of Litsea cubeba formulation granules and its specificity investigation.

[0038] Figure 2. Results of the overall evaluation of the formulation granules of Litsea cubeba.

[0039] Figure 3. Separation results of different chromatographic columns.

[0040] Figure 4 shows the characteristic spectrum of the formula granules of Litsea cubeba, where peak 4 is magnoflorine.

[0041] Figure 5 shows the characteristic spectrum of Litsea cubeba medicinal material, where peak 4 is magnoflorine.

[0042] Figure 6. Characteristic spectrum of standard decoction of Litsea cubeba, where peak 4 is magnoflorine.

[0043] Figure 7. Thin-layer chromatogram of *Litsea cubeba* formulation granules, where 1-3 are three batches of *Litsea cubeba* formulation granules samples, and 4 is the *Litsea cubeba* reference material. Detailed Implementation

[0044] To make the technical solutions and effects of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. The embodiments described below are some embodiments of the present invention, but not all embodiments. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Example 1: Establishment of a method for detecting the characteristic spectral features of Litsea cubeba granules.

[0046] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0047] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.3 ml / min; the column temperature was 35℃; the detection wavelength was 300 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 10,000. The gradient elution conditions were as follows:

[0048] Preparation of reference solution: Take 0.5g of Litsea cubeba reference material, add 25ml of 70% methanol, heat under reflux for 30min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of magnoflorine reference standard, accurately weigh it, add 70% methanol to prepare a solution containing 20μg per ml, as the reference solution of the reference standard.

[0049] Preparation of the test solution: Take the formula granules of Litsea cubeba, grind them finely, take 0.2g, weigh accurately, add 25ml of 70% methanol accurately, weigh accurately, sonicate for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0050] Determination method: Accurately pipette 2 μl of the reference solution and the test solution into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography. Record the chromatograms from 0 to 22 min.

[0051] The chromatogram of the test sample should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, the peak corresponding to the peak of the magnoflorine reference standard is the S peak. Calculate the relative retention time of each characteristic peak and the S peak. The relative retention time should be within ±10% of the specified value. The specified values ​​are: relative retention time of peak 1 is 0.33, relative retention time of peak 2 is 0.48, and relative retention time of peak 3 is 0.63.

[0052] Example 2: Methodological validation of the spectral characteristic detection method for Litsea cubeba formulation granules

[0053] The practicality and accuracy of the feature map detection method in Example 1 were verified by examining its specificity, integrity, precision, repeatability, solution stability, and robustness.

[0054] In the embodiments, all original reagent materials are commercially available, and experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0055] 2.1 Instruments, reagents, and reagents used in the examples

[0056] Instruments: Agilent 1290 Infinity high-performance liquid chromatograph (Agilent Technologies, Inc., USA); 0.001 g balance (EL-204, Mettler Toledo Instruments Ltd.); 0.001 g balance (AE-240, Mettler Toledo Instruments Ltd.); micropipette (Eppendorf AG, Germany); CNC ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instruments Co., Ltd.); electric thermostatic water bath (SYG-1-7, Tianjin Test Instruments Co., Ltd.).

[0057] Reagents: Acetonitrile, methanol (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); anhydrous formic acid (analytical grade, Aladdin Reagent).

[0058] Test drug: Magnolia alkaloid (batch number: AFBJ1903, purity: 99.62%, Chengdu Efa Biotechnology Co., Ltd.); Litsea cubeba reference material (batch number: 121300~201802, China National Institute for Food and Drug Control).

[0059] 2.2 Specificity Test

[0060] Blank solvent test solution: Take 70% methanol solution.

[0061] Preparation of reference solution: Prepared according to the method of "Preparation of reference solution" in Example 1.

[0062] Preparation of test solution: Prepared according to the method of "Preparation of test solution" in Example 1.

[0063] Take blank solution, reference solution, and test solution, and inject them for analysis according to the chromatographic conditions of Example 1. Record the chromatograms as shown in Figure 1.

[0064] The results showed that the chromatogram of the test sample had the same chromatographic peak at the corresponding retention time as the chromatogram of the reference sample, and the blank solvent did not interfere, indicating that the method has good specificity.

[0065] 2.3 Holistic Examination

[0066] Take an appropriate amount of Litsea cubeba granules, grind them into a fine powder, take about 0.2g, and prepare the test solution according to the method of "Preparation of Test Solution" in Example 1. Based on the chromatographic conditions of Example 1, maintain the elution gradient at the initial ratio, double the elution time, and record the chromatogram, as shown in Figure 2.

[0067] The results showed that no obvious chromatographic peaks eluted after the initial gradient elution, indicating that the chromatographic conditions basically met the principle of maximizing information content.

[0068] 2.4 Precision Test

[0069] The granules of Litsea cubeba were used to prepare a test solution according to the method described in Example 1, "Preparation of Test Solution". The solution was then injected and analyzed under the chromatographic conditions of Example 1, with six repeated injections. Using the magnoflorin chromatographic peak as the reference S peak, the relative retention time and relative peak area of ​​each characteristic peak relative to the S peak were calculated, and the relative standard deviation was also calculated. The results showed that for the same sample, after six consecutive injections, the RSD values ​​of the relative retention time between each characteristic peak and the S peak were in the range of 0.04–0.09%, and the relative peak area was in the range of 0.25–1.14%, all less than 2%, indicating good instrument precision.

[0070] 2.5 Repeatability Test

[0071] Take an appropriate amount of *Litsea cubeba* granules, grind them finely, and accurately weigh approximately 0.2 g. Prepare a test solution according to the method described in Example 1, "Preparation of Test Solution," in six parallel batches. Using the magnoflorine chromatographic peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak relative to the S peak, and calculate the relative standard deviation. The results show that when six parallel batches of the same sample are prepared, the relative retention time RSD values ​​of each characteristic peak are in the range of 0.23–0.27%, and the relative peak area is in the range of 0.51–0.85%, all less than 1%, indicating that the method has good repeatability.

[0072] 2.5 Stability Test

[0073] Take an appropriate amount of *Litsea cubeba* granules, grind them finely, and take about 0.2 g. Prepare a test solution according to the method described in Example 1, "Preparation of Test Solution". Inject 2 μL at 0, 2, 4, 8, 12, and 24 hours, and record the chromatograms. Using the magnoflorine chromatographic peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the relative standard deviation. The results show that within 24 hours, the RSD values ​​of the relative retention times of each characteristic peak and the S peak are in the range of 0.32–0.36%, and the relative retention times are in the range of 0.63–1.09%, all less than 3%, indicating that the test solution is stable within 24 hours.

[0074] 2.6 Durability

[0075] 2.6.1 Column Investigation

[0076] Three chromatographic columns were used to analyze the Litsea cubeba formulation granules: 1. ZORBAX Eclipse Plus C18 (Agilent, 2.1 mm × 100 mm, 1.8 μm); 2. ACQUITY UHPLC CSH Phenyl-Hexyl Column (Waters, 1.7 μm, 2.1 mm × 100 mm); 3. ACQUITY UHPLC BEH C18 Column (Waters, 1.7 μm, 2.1 mm × 100 mm). The results are shown in Figure 3.

[0077] The results showed that magnoflorine in Litsea cubeba was well separated using the ZORBAX Eclipse Plus C18 column among the three different brands of columns. Subsequent studies selected the ZORBAX Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8 μm).

[0078] 2.6.2 Flow velocity investigation

[0079] A ZORBAX Eclipse Plus C18 (2.1 mm × 100 mm, 1.8 μm) column was used to investigate the sample separation performance at flow rates of 0.25 mL / min, 0.3 mL / min, and 0.35 mL / min. The results showed that the analytical method achieved good separation of all characteristic peaks within a flow rate range of 0.25–0.30 mL / min. Small flow rate variations were sufficient to meet the system suitability requirements; a flow rate of 0.3 mL / min was used in subsequent studies.

[0080] 2.6.3 Column Temperature Investigation

[0081] The results showed that the peak separation was better at column temperatures between 30℃ and 40℃, indicating good column temperature robustness. Based on the sample peak elution, 35℃ was selected as the detection temperature.

[0082] 2.7 Sample Determination

[0083] Take an appropriate amount of Litsea cubeba granules from three batches, grind them into a fine powder, take 0.2g, and prepare a test solution according to the "Preparation Method of Test Solution" in Example 1. Inject the sample and determine the chromatogram according to the chromatographic conditions in Example 1. The results are shown in the table below.

[0084] Relative retention time of characteristic spectra of three batches of Litsea cubeba formulation granules

[0085] The UHPLC characteristic chromatograms of the three batches of Litsea cubeba formula granules were matched using the "Similarity Evaluation System for Chromatographic Characteristic Spectra of Traditional Chinese Medicine". Using peak No. 4, magnoflorine, as the reference peak S, a control chromatogram was generated by the average method, and a control characteristic chromatogram of Litsea cubeba formula granules was established, as shown in Figure 4.

[0086] The final standard for the characteristic chromatogram of Litsea cubeba is determined as follows: the test sample chromatogram should show four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the reference chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak. The peak corresponding to the magnoflorine reference peak is peak S. The relative retention times of peaks 1-3 with peak S are calculated, and their relative retention times should all be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.48 (peak 2), and 0.63 (peak 3).

[0087] Example 3: Establishment of a method for determining the content of Litsea cubeba granules in a formula

[0088] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0089] Chromatographic conditions and system suitability experiments: The chromatographic column was packed with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B for gradient elution. The flow rate was 0.3 ml / min; the column temperature was 35℃; the detection wavelength was 300 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 10,000. The gradient elution conditions were as follows:

[0090] Preparation of the reference solution: Take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 20 μg per ml.

[0091] Preparation of the test solution: Take an appropriate amount of this product, grind it into a fine powder, take about 0.2g, weigh it accurately, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30 minutes, take it out, let it cool, weigh it again, make up the weight loss with 70% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0092] The assay involves precisely pipetting 2 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0093] Example 4: Methodological Validation of the Method for Determining the Granular Content of Litsea cubeba Formula

[0094] The practicality and accuracy of the content determination method in Example 3 were verified by examining the linear range, precision, repeatability, solution stability, and recovery rate of the detection method.

[0095] In this embodiment, the instruments, reagents, and reagents used are the same as in Example 2.

[0096] 4.1 Linear Examination

[0097] Accurately weigh magnoflorine reference standard and place it in a 100 mL volumetric flask. Add methanol to prepare a stock solution containing 264.99 μg of magnoflorine reference standard per mL. Accurately measure 0.1 mL, 1.0 mL, 2.0 mL, 3.0 mL, 4.0 mL, and 5.0 mL of the above mixed stock solution and place them in separate 10 mL volumetric flasks. Add methanol to the mark and shake well to obtain working solutions of different concentrations of reference standard. Accurately pipette 2 μL of each working solution of different concentrations of reference standard and determine them sequentially according to the chromatographic conditions of Example 3, recording the peak area. Plot a standard curve with peak area as the ordinate (y) and reference standard concentration as the abscissa (x). The results show that the linear regression equation for magnoflorine is: y = 1.9805x + 0.3105, R 2 =0.9987. This indicates a good linear relationship between the concentration of magnoflorine and the peak area within the concentration range of 2.65–132.49 μg / mL.

[0098] 4.2 Precision Test

[0099] Methods of investigation: Take an appropriate amount of Litsea cubeba granules, grind them into a fine powder, take 0.2g, and prepare a test solution according to the method of "Preparation of Test Solution" in Example 3. Inject the sample for analysis according to the chromatographic conditions of Example 3, inject the sample 6 times consecutively, record the chromatographic peak area, and calculate the relative standard deviation.

[0100] The results showed that when the same sample solution was injected repeatedly 6 times, the RSD value of the magnoflorine peak area was 0.46%, which is less than 1%, indicating that the instrument has good precision.

[0101] 4.3 Repeatability Test

[0102] Methods of investigation: Take an appropriate amount of Litsea cubeba granules, grind them into a fine powder, take 0.2g, and prepare 6 parallel samples. Prepare the test solution according to the test solution preparation method in Example 3. Inject the sample for analysis according to the chromatographic conditions in Example 3. Inject the sample 6 times consecutively, record the chromatographic peak area, and calculate its content and RSD.

[0103] The results showed that the content of magnoflorine in the six test solutions injected consecutively ranged from 2.02 to 2.14 mg / g, with an average of 2.088 mg / g and an RSD of 1.90%, which was less than 2%, indicating good repeatability.

[0104] 4.4 Stability Test

[0105] Methods of analysis: Take an appropriate amount of Litsea cubeba granules, grind them finely, take 0.2g, and prepare a test solution according to the test solution preparation method in Example 3. Analyze the solution under the chromatographic conditions of Example 3 at 0h, 2h, 4h, 8h, 12h, and 24h, and record the peak areas. Calculate the peak area RSD values ​​for each peak.

[0106] The results showed that when the test solution was injected at 0, 2, 4, 8, 12, and 24 hours, the RSD value of the magnoflorine peak area was 1.54%, which was less than 2%, indicating that the method had good stability within 24 hours.

[0107] 4.5 Recovery Rate Test

[0108] Methods: An appropriate amount of *Litsea cubeba* granules with a known content (magnolianine content: 2.0887 mg / g) was ground finely. Six 0.1g portions were accurately weighed and added to each portion. A reference solution containing the same amount of magnolianine as the 0.1g sample was added to each portion. The sample was prepared according to the method described in Example 3. 2 μL of each sample was injected under the chromatographic conditions described in Example 3. The recovery rate and RSD were calculated using the following formulas. Results are shown in the table below.

[0109] Results of the recovery test for the content of Litsea cubeba granules in the formulation

[0110] Results: The recoveries of magnoflorine were all between 96.19% and 100.94%, and the peak area RSD of magnoflorine was 1.24%, which was less than 3%, indicating good recovery.

[0111] 4.6 Sample Determination

[0112] Take an appropriate amount of three batches of Litsea cubeba granules, grind them finely, take 0.2g, and prepare a test solution according to the "Preparation Method of Test Solution" in Example 3. Inject the sample under the chromatographic conditions of Example 3 to determine the content of magnoflorine. The results are shown in the table below.

[0113] Example 5: Establishment of Characteristic Spectrum and Content Determination Method for Litsea cubeba Medicinal Material

[0114] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0521, Part IV, Chinese Pharmacopoeia 2020 Edition).

[0115] Chromatographic conditions and system suitability tests are the same as those in the [Assay] section.

[0116] Preparation of the reference solution: Take 0.5g of Litsea cubeba reference material, place it in a stoppered conical flask, add 25ml of 70% methanol, heat under reflux for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 30μg of magnoflorine per 1ml, as the reference solution.

[0117] The preparation of the test solution is the same as in the [Assay] section.

[0118] The determination method involves precisely pipetting 2 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0119] The chromatogram of the test sample should show four characteristic peaks, which should correspond to the retention times of the four characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak. The peak corresponding to the magnoflorine reference peak is peak S. The relative retention times of peaks 1-3 with peak S should be calculated, and their relative retention times should all be within ±10% of the specified values. The specified values ​​are: 0.34 (peak 1), 0.48 (peak 2), and 0.64 (peak 3). The characteristic chromatogram of *Litsea cubeba* (large-fruited ginger) is shown in Figure 5.

[0120] [Content Determination] Magnolia alkaloids were determined by high performance liquid chromatography (General Chapter 0521, Chinese Pharmacopoeia 2020 Edition).

[0121] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 35 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the magnoflorine peak, should be no less than 10,000.

[0122] Preparation of the reference solution: Take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 30 μg per 1 mL.

[0123] Preparation of the test solution: Take about 1g of Litsea cubeba powder (passed through a No. 3 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, weigh it, heat under reflux for 30 minutes, cool it, weigh it again, make up the lost weight with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0124] The assay involves precisely pipetting 2 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0125] Example 6: Establishment of Characteristic Chromatography and Content Determination Method for Standard Decoction of Litsea cubeba

[0126] [Characteristic chromatogram] Determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0127] Chromatographic conditions and system suitability tests are the same as those for the [content determination] section on magnoflorine.

[0128] Preparation of the reference solution: Take 0.5g of Litsea cubeba reference material, place it in a stoppered conical flask, add 25ml of 70% methanol, heat under reflux for 30 minutes, cool, shake well, filter, and collect the filtrate as the reference solution. Separately, take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add methanol to prepare a solution containing 30μg of magnoflorine per 1ml, as the reference solution.

[0129] The preparation of the test solution is the same as that for the [content determination] section on magnoflorine.

[0130] The assay involves precisely pipetting 2 μl each of the reference solution and the test solution into a liquid chromatograph and measuring the concentration.

[0131] The chromatogram of the test sample should show four characteristic peaks, which should correspond to the retention times of the four characteristic peaks in the chromatogram of the reference medicinal material. Peak 4 should correspond to the retention time of the reference peak. The peak corresponding to the magnoflorine reference peak is peak S. The relative retention times of peaks 1-3 with peak S should be calculated, and their relative retention times should all be within ±10% of the specified values. The specified values ​​are: 0.33 (peak 1), 0.47 (peak 2), and 0.63 (peak 3). The characteristic chromatogram of the Litsea cubeba standard decoction is shown in Figure 6.

[0132] [Content Determination] Magnolia alkaloids were determined by high performance liquid chromatography (General Chapter 0512, Chinese Pharmacopoeia 2020 Edition).

[0133] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A and 0.2% formic acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate 0.3 mL / min; column temperature 35 °C; detection wavelength 300 nm. The theoretical plate number, calculated based on the magnoflorine peak, should be no less than 10,000.

[0134] Preparation of the reference solution: Take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 30 μg per 1 mL.

[0135] Preparation of the test solution: Take about 0.2g of Litsea cubeba standard decoction powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 70% methanol, stopper tightly, weigh it, sonicate (power 500W, frequency 40kHz) for 30 minutes, remove it, cool it, weigh it again, make up the weight loss with 70% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0136] The assay involves precisely pipetting 2 μl of both the reference solution and the test solution into a liquid chromatograph and measuring the results.

[0137] Example 7: Establishment of a thin-layer chromatography method for identifying granules of Litsea cubeba.

[0138] Preparation of test solution: Take 2g of Litsea cubeba formula granules, grind them into a fine powder, add 20ml of ether, sonicate for 30 minutes, filter, concentrate the filtrate to about 1ml, and use it as the test solution.

[0139] Preparation of the reference medicinal material solution: Take 0.5g of Litsea cubeba reference medicinal material and prepare the reference medicinal material solution according to the preparation method of the test sample solution;

[0140] Developing solvent: cyclohexane-ethyl acetate (8:2);

[0141] Colorimetric reagent: Vanillin-sulfuric acid solution;

[0142] Detection method: Apply 5 μl each of the test sample and reference medicinal material solutions to the same silica gel G thin-layer plate. Develop with the above-mentioned developing solvent, remove, air dry, spray with the above-mentioned colorimetric reagent, and heat at 105℃ until the spots are clearly visible. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions as in the chromatogram of the reference medicinal material.

[0143] The thin-layer chromatography identification method was validated under different temperatures and humidity levels, different sample amounts, and different manufacturers' thin-layer chromatography plates. The method has good robustness. The thin-layer chromatograms of three batches of Litsea cubeba formulation granules are shown in Figure 7.

[0144] Example 8: Preparation method of Litsea cubeba formulation granules

[0145] Pre-processing: The large-fruited Litsea cubeba is cut using a linear reciprocating herb cutter at a fixed cutting frequency of 180 times / min; it should be cut and cooked immediately.

[0146] Extraction process: First, soak the raw material in 12 times the amount of water for 30 minutes, decoct for 1 hour, collect the volatile oil, filter the decoction, then add 10 times the amount of water for 1 hour, collect the volatile oil, filter the decoction, and combine the filtrates; concentrate the filtrate under reduced pressure, vacuum dry, and pulverize to obtain a dry extract powder.

[0147] Inclusion process: Mix Litsea cubeba oil with an equal volume of anhydrous ethanol. Accurately weigh an appropriate amount of β-cyclodextrin (8 times the amount of the volatile oil) and place it in a round-bottom flask. Add 200 mL of ultrapure water and heat to make a saturated aqueous solution. Transfer to a constant-temperature stirrer and slowly add the Litsea cubeba volatile oil ethanol solution dropwise, maintaining a speed of 500 rpm and stirring at 40°C for 60 min. After inclusion, cool to room temperature, refrigerate at 4°C for 24 h, filter, dry, and pulverize to obtain the volatile oil inclusion complex.

[0148] Molding process: Take dry paste powder and volatile oil inclusion complex, add appropriate amount of silicon dioxide and maltodextrin, mix well, granulate and package to obtain the final product.

[0149] Example 9: Preparation method of standard decoction of Litsea cubeba

[0150] Weigh 100g of Litsea cubeba slices, add 8 times the amount of water and soak for 30 minutes. First, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter while hot and cool the filtrate in a cold water bath. For the second decoction, add 9 times the amount of water, bring to a boil over high heat, then simmer over low heat for 15 minutes. Filter while hot and cool the filtrate in a cold water bath. Combine the two filtrates and concentrate under reduced pressure at 65℃ to a relative density of 1.04-1.10 (65℃). Dispense the concentrate into vials and freeze dry in a freeze dryer to obtain the standard Litsea cubeba decoction.

[0151] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting large-fruited Litsea cubeba granules, comprising morphology, identification, inspection, extractives, characteristic chromatograms, and content determination, characterized in that, The characteristic chromatogram and content determination method are performed using high-performance liquid chromatography (HPLC). The HPLC operating conditions are as follows: the chromatographic column is packed with octadecylsilane-bonded silica gel; acetonitrile is used as mobile phase A; and 0.1%–0.5% formic acid solution is used as mobile phase B for gradient elution; the flow rate is 0.2–1 ml per minute; the column temperature is 30–40℃; the detection wavelength is 250–350 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should be no less than 5000–10000. The gradient elution conditions are as follows: From 0 to 1 min, mobile phase A increased from 5% to 7%, and mobile phase B increased from 95% to 93%. 1–5 min, mobile phase A 7 → 8%, mobile phase B 93 → 92%; 5–7 min, mobile phase A 8→9%, mobile phase B 92→91%; 7–11 min, mobile phase A 9→11%, mobile phase B 91→89%; From 11 to 19 min, mobile phase A decreased by 11% to 17%, and mobile phase B decreased by 89% to 83%. 19–20 min, mobile phase A 17%, mobile phase B 83%; 20–22 min, mobile phase A 17→19%, mobile phase B 83→81%; The characteristic chromatogram and content determination method further includes preparing a reference solution or a standard solution, preparing a test solution, and injection determination, wherein: Preparation of reference solution: Take 0.5-2.0 g of Litsea cubeba reference material, add 25-50 ml of 70% methanol, heat under reflux for 30-60 min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of neomagnolicin reference standard, accurately weigh it, add 70% methanol to prepare a mixed solution containing 20 μg per ml, as the reference solution of the reference standard; Preparation of reference solution: Take an appropriate amount of magnoflorine reference standard, accurately weigh it, and add 70% methanol to prepare a solution containing 20-30 μg per ml. Preparation of the test solution: Take the formula granules of Litsea cubeba, grind them finely, take about 0.1-1g, weigh accurately, add 25-50ml of 70% methanol accurately, weigh accurately, sonicate for 30-60 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution. Determination method: Accurately pipette 2-10 μl each of the reference solution or standard solution and the test solution, inject them into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography.

2. The detection method according to claim 1, characterized in that, The operating conditions for the high performance liquid chromatography method are as follows: an ultra-high performance liquid chromatograph is used; the chromatographic column is packed with octadecylsilane-bonded silica gel; acetonitrile is used as mobile phase A; and 0.2% formic acid solution is used as mobile phase B for gradient elution; the flow rate is 0.3 ml per minute; the column temperature is 35℃; the detection wavelength is 300 nm; and the theoretical plate number, calculated based on the magnoflorine peak, should not be less than 10,000. The preparation method of the reference solution is as follows: Take 0.5g of Litsea cubeba reference material, add 25ml of 70% methanol, heat under reflux for 30min, cool, shake well, filter, and take the filtrate as the reference solution of the reference material; separately take an appropriate amount of magnoflorine reference standard, accurately weigh it, add 70% methanol to prepare a solution containing 20μg per ml, as the reference solution of the reference standard. The preparation method of the test solution is as follows: take the formula granules of Litsea cubeba, grind them finely, take 0.2g, weigh accurately, add 25ml of 70% methanol accurately, weigh accurately, sonicate for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

3. The detection method according to claim 1, characterized in that, The method for determining the characteristic chromatogram is as follows: accurately pipette 2 μl of the reference solution and the test solution, inject them into the liquid chromatograph, and perform the determination according to the working conditions of high performance liquid chromatography, recording the chromatograms from 0 to 22 min.

4. The detection method according to any one of claims 1 to 3, characterized in that, The chromatogram of the test sample should show four characteristic peaks, which should correspond to the retention times of the four characteristic peaks in the chromatogram of the reference medicinal material. Among them, the peak corresponding to the peak of the magnoflorine reference standard is the S peak. The relative retention times of each characteristic peak and the S peak should be calculated, and the relative retention times should be within ±10% of the specified values. The specified values ​​are: relative retention time of peak 1 is 0.33, relative retention time of peak 2 is 0.48, and relative retention time of peak 3 is 0.

63.

5. The detection method according to claim 1, characterized in that, The determination method for the content is as follows: accurately pipette 2 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of magnoflorine by external standard method.

6. The detection method according to claim 1, characterized in that, The identification method is as follows: Take 1-2g of Litsea cubeba granules, grind them finely, add 20-30ml of ether, sonicate for 20-60 minutes, filter, and concentrate the filtrate to about 1ml as the test solution; separately take 0.5-1g of Litsea cubeba reference material and prepare a reference material solution using the same method; according to the thin-layer chromatography method of the Chinese Pharmacopoeia, take 5μl of each of the above two solutions and spot them separately on the same silica gel G thin-layer plate, use cyclohexane-ethyl acetate = 8:2 as the developing solvent, develop, remove, air dry, spray with vanillin-sulfuric acid test solution, and heat at 105℃ until the spots are clearly visible; in the chromatogram of the test sample, spots of the same color should appear at the corresponding positions as in the chromatogram of the reference material.

7. The detection method according to claim 1, characterized in that, The method for detecting the extract is as follows: Take 2g of Litsea cubeba granules, grind them into a fine powder, accurately weigh them, add 100ml of ethanol, and determine the extract using the hot extraction method under the "Determination of Alcohol-Soluble Extracts" section of the Chinese Pharmacopoeia.

8. The detection method according to any one of claims 1 to 7, characterized in that, The Litsea cubeba formula granules are made by taking Litsea cubeba slices, adding water and boiling them, collecting the volatile oil, encapsulating the volatile oil with β-cyclodextrin, and setting it aside; filtering the decoction, concentrating the filtrate into a clear paste, drying or drying and pulverizing it, adding the volatile oil inclusion complex and pharmaceutically acceptable excipients, mixing well, and granulating. Each 1g of formula granules is equivalent to 4.5g of the slices.

9. The detection method according to claim 8, characterized in that, The specific method for encapsulating the volatile oil with β-cyclodextrin is as follows: Mix the volatile oil of Litsea cubeba with an equal volume of anhydrous ethanol, weigh 8 times the amount of β-cyclodextrin, add 200 mL of water, heat to make a saturated aqueous solution, transfer to a constant temperature stirrer, slowly drip in the ethanol solution of Litsea cubeba volatile oil, maintain 500 r / min, stir at 40℃ for 60 min, after the encapsulation is completed, cool to room temperature, refrigerate at 4℃ for 24 h, filter, dry, and pulverize to obtain the final product.

10. The application of the detection method according to any one of claims 1 to 9, characterized in that, The detection method is applied to the quality testing of Litsea cubeba medicinal materials, Litsea cubeba slices, Litsea cubeba standard decoction, and Litsea cubeba formula granules.

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