Detection method of fructus cinnamoii formula granules

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a detection method of fructus cinnamoii formula granules, the fructus cinnamoii formula granules are prepared by decocting fructus cinnamoii decoction pieces with water, collecting volatile oil, concentrating, drying and granulating, and the detection method comprises the items of character determination, identification, inspection, extract determination, characteristic spectrum determination and content determination. According to the identification method, thin-layer chromatography is adopted for identification; the characteristic chromatogram is characterized in that a high performance liquid chromatography is adopted, a peak corresponding to a magnoflorine reference substance peak is taken as an S peak, and four characteristic peaks are marked; and the content determination is to detect the content of magnoflorine. According to the detection method established by the invention, the detection requirements on medicinal materials, decoction pieces, standard decoction and formula granules of the fructus cinnamoii are improved, establishment of a perfect quality control system for each product of the fructus cinnamoii is facilitated, and the quality and clinical curative effect of the fructus cinnamoii formula granules are guaranteed.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a detection method for Litsea cubeba formula granules. Background Art

[0002] Chinese medicine formula granules are made from single Chinese medicine pieces through one or more processes to extract and concentrate, and are used in Chinese medicine clinical prescriptions. They have the advantages of being stable and controllable, safe and efficient, no need for decoction, easy to prepare, convenient to carry, and ready to take.

[0003] Litsea cubeba, also known as migao (Cinnamomum migao HWLi), is the dried mature fruit of migao, a plant of the Lauraceae family. It is now distributed in Guizhou Province, Yunnan Province, Guangxi Zhuang Autonomous Region, etc. It is often used to treat abdominal pain, stomach pain, chest tightness, vomiting and other symptoms, and has a good effect in treating arrhythmia. In clinical practice, it is mainly used to treat angina pectoris caused by cold stagnation, qi stagnation and deficiency-cold syndrome caused by coronary heart disease.

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

[0005] At present, the quality control methods in the standard of Litsea cubeba medicinal materials mainly use properties and thin-layer chromatography. With the increasing clinical application of formula granules, there is an urgent need for comprehensive, convenient and controllable detection methods to control the quality of Litsea cubeba formula granules. Summary of the invention

[0006] In view of the imperfect detection methods in the prior art of Litsea cubeba formula granules, the present invention provides a quality detection method for Litsea cubeba formula granules, which is specifically:

[0007] The testing items of the Litsea cubeba formula granules include properties, identification, inspection, extracts, characteristic spectra, and content determination.

[0008] In some embodiments, the Litsea cubeba formula granules have the following properties: yellow-brown granules; slightly fragrant and bitter.

[0009] In some embodiments, the Litsea cubeba formula granules have the following properties: yellow-brown granules; slightly fragrant and bitter.

[0010] Identification: Take 2g of Litsea cubeba formula granules, grind them, add 20ml of ether, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to about 1ml as the test solution. Take another 0.5g of Litsea cubeba reference medicinal material and prepare the reference medicinal material solution in the same way. According to the thin layer chromatography method (General Rules 0502 of the 2020 Edition of the Chinese Pharmacopoeia), 5μl of each of the above two solutions were taken and spotted on the same silica gel G thin layer plate, using cyclohexane-ethyl acetate (8:2) as the developing agent, develop, take out, dry, spray with vanillin sulfuric acid test solution, and heat at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding position of the chromatogram of the reference medicinal material.

[0011] Extract: Take the formula granules of Litsea cubeba, grind them into powder, take 2g, weigh accurately, add 100ml of ethanol accurately, and determine it according to the hot soaking method under the item of determination method of alcohol-soluble extract in the general rules of the Chinese Pharmacopoeia.

[0012] Inspection: It should comply with the relevant provisions under granules.

[0013] Characteristic spectrum: Determined by high performance liquid chromatography according to the general rules of the Chinese Pharmacopoeia;

[0014] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 0.1% to 0.5% formic acid solution is used as mobile phase B for gradient elution, the flow rate is 0.2ml to 1ml per minute; the column temperature is 30 to 40°C; the detection wavelength is 250 to 350nm, and the theoretical plate number calculated based on the magnolia alkaloid peak should be not less than 5000 to 10000; the gradient elution conditions are:

[0015]

[0016] The detection method comprises the following operations:

[0017] Preparation of reference solution: Take 0.5-2.0g of Litsea cubeba reference medicinal material, add 25-50ml of 70% methanol, heat and reflux for 30-60min, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; take an appropriate amount of magnolia alkaloid reference substance, accurately weigh it, add 70% methanol to make a solution containing 20-30μg per 1ml, as the reference substance solution;

[0018] Preparation of the test solution: Take the Litsea cubeba formula granules, grind them into powder, take about 0.1-1g, weigh accurately, add 25-50ml of 70% methanol accurately, weigh the weight, ultrasonically treat for 30-60 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain;

[0019] Determination method: Accurately pipette 2 to 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 the high performance liquid chromatography.

[0020] There should be four characteristic peaks in the chromatogram of the test sample, and the retention time 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 magnolia alkaloid reference material is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: the relative retention time of peak 1 is 0.33, the relative retention time of peak 2 is 0.48, and the relative retention time of peak 3 is 0.63.

[0021] In some embodiments, the characteristic spectrum detection method is to use ultra-high performance liquid chromatography to measure:

[0022] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 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°C; the detection wavelength is 300nm, and the theoretical plate number calculated based on the magnolia alkaloid peak should be not less than 10000; the gradient elution conditions are:

[0023]

[0024] The detection method comprises the following operations:

[0025] Preparation of reference solution: Take 0.5g of Litsea cubeba reference medicinal material, add 25ml of 70% methanol, heat and reflux for 30min, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; take an appropriate amount of magnolia alkaloid reference substance, accurately weigh, add 70% methanol to make a solution containing 20μg per 1ml, as the reference substance solution;

[0026] Preparation of test solution: Take Litsea cubeba formula granules, grind them into powder, take 0.2g, accurately weigh, accurately add 25ml of 70% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0027] Determination method: Accurately pipette 2 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure according to HPLC working conditions, and record the chromatogram from 0 to 22 min.

[0028] There should be four characteristic peaks in the chromatogram of the test sample, and the retention time 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 magnolia alkaloid reference material is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: the relative retention time of peak 1 is 0.33, the relative retention time of peak 2 is 0.48, and the relative retention time of peak 3 is 0.63.

[0029] Content determination: Determined by high performance liquid chromatography (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0030] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 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°C; the detection wavelength is 300nm, and the theoretical plate number calculated based on the magnolia alkaloid peak should be not less than 10000; the gradient elution conditions are:

[0031]

[0032] Preparation of reference solution: Take an appropriate amount of magnolia alkaloid reference substance, weigh it accurately, and add 70% methanol to make a solution containing 20 μg per 1 ml.

[0033] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0034] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0035] Among them, the formula granules of Litsea cubeba are prepared by taking 4500g of Litsea cubeba decoction pieces, adding water for decoction, collecting volatile oil (encapsulated with β-cyclodextrin for standby use), filtering the decoction, concentrating the filtrate into a clear paste, drying (or drying and crushing), adding appropriate amounts of volatile oil inclusion compound and auxiliary materials, mixing well, granulating, and making 1000g.

[0036] Beneficial effects:

[0037] 1. The present invention is a detection method developed for the Litsea cubeba formula granules. The detection method has comprehensive detection items, simple operation method, high precision, strong specificity and good reproducibility.

[0038] 2. The present invention also adds the detection of characteristic spectrum on the basis of conventional detection items, which can more comprehensively reflect the types and quantities of chemical components of medicinal materials contained in the product. In particular, the characteristic spectrum adopts UPLC detection method, which has the characteristics of simple operation and good reproducibility. At the same time, the content determination method is also formulated in the detection method, and its method is consistent with the chromatographic conditions of the characteristic spectrum. When detecting the characteristic spectrum, the quantitative determination of magnolidine can be carried out, which saves time and effort.

[0039] 3. In the prior art of Litsea cubeba, the content determination index is ammoniacal alcohol in the volatile oil, but the Litsea cubeba formula granules are extracted by decoction in water. The present invention uses the water-soluble component magnolamine as the content index for the first time, which is an inevitable trend in the quality control of the Litsea cubeba formula granules.

[0040] 4. Through experiments, the detection method of the present invention is also applicable to Litsea cubeba medicinal materials, decoction pieces, standard decoctions, etc., and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Characteristic spectrum of Litsea cubeba formula granules, specificity survey spectrum

[0042] Figure 2 Results of the overall investigation of Litsea cubeba formula granules

[0043] Figure 3 Separation effect diagram of different chromatographic columns

[0044] Figure 4 Comparative characteristic spectrum of Litsea cubeba formula granules, where peak 4 is magnolamine

[0045] Figure 5 Comparative characteristic spectrum of Litsea cubeba medicinal materials, where peak 4 is magnolamine

[0046] Figure 6 Comparative characteristic spectrum of Litsea cubeba standard decoction, where peak 4 is magnolamine

[0047] Figure 7 Thin layer chromatogram of Litsea cubeba formula granules, where 1 to 3 are samples of 3 batches of Litsea cubeba formula granules, and 4 is the reference material of Litsea cubeba DETAILED DESCRIPTION

[0048] In order to make the technical scheme and technical effect of the present invention clearer, the technical scheme in the embodiment of the present invention is described clearly and completely. The embodiments described below are part of the embodiments of the present invention, rather than all of the embodiments. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Example 1 Establishment of a method for detecting the characteristic spectrum of Litsea cubeba formula granules

[0050] Determined by high performance liquid chromatography (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0051] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 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°C; the detection wavelength is 300nm, and the theoretical plate number calculated based on the magnolia alkaloid peak should be not less than 10000; the gradient elution conditions are:

[0052]

[0053] Preparation of reference solution: Take 0.5g of Litsea cubeba reference medicinal material, add 25ml of 70% methanol, heat and reflux for 30min, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; take an appropriate amount of magnolia alkaloid reference substance, accurately weigh, add 70% methanol to make a solution containing 20μg per 1ml, as the reference substance solution;

[0054] Preparation of test solution: Take Litsea cubeba formula granules, grind them into powder, take 0.2g, accurately weigh, accurately add 25ml of 70% methanol, weigh the weight, ultrasonically treat for 30 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the solution.

[0055] Determination method: Accurately pipette 2 μl of reference solution and test solution respectively, inject into liquid chromatograph, measure according to HPLC working conditions, and record the chromatogram from 0 to 22 min.

[0056] There should be four characteristic peaks in the chromatogram of the test sample, and the retention time 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 magnolia alkaloid reference material is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: the relative retention time of peak 1 is 0.33, the relative retention time of peak 2 is 0.48, and the relative retention time of peak 3 is 0.63.

[0057] Example 2: Methodological verification of the characteristic spectrum detection method of Litsea cubeba formula granules

[0058] The practicability and accuracy of the characteristic spectrum detection method of Example 1 were verified from the aspects of specificity, integrity, precision, repeatability, solution stability, durability, etc. of the detection method.

[0059] In the examples, all raw reagents and materials are commercially available, and the experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or are based on the conditions recommended by the instrument manufacturer.

[0060] 2.1 Instruments, reagents and reagents used in the examples

[0061] Instruments: Agilent 1290 Infinity high performance liquid chromatograph (Agilent Technologies, Inc., USA); 1 / 10,000 balance (EL-204, Mettler-Toledo Instrument Co., Ltd.); 1 / 100,000 balance (AE-240, Mettler-Toledo Instrument Co., Ltd.); micropipette (Eppendorf AG, Germany); CNC ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instrument Co., Ltd.); electric constant temperature water bath (SYG-1-7, Tianjin Test Instrument Co., Ltd.).

[0062] Reagents: acetonitrile, methanol (chromatographic grade, Sinopharm Chemical Reagent Co., Ltd.); anhydrous formic acid (analytical grade, Aladdin Reagent).

[0063] Test drugs: magnolia alkaloids (batch number: AFBJ1903, purity: 99.62%, Chengdu Aifa Biotechnology Co., Ltd.); Litsea cubeba reference medicinal material (batch number: 121300-201802, China Food and Drug Identification Institute).

[0064] 2.2 Specificity test

[0065] Blank solvent for test sample solution: Take 70% methanol solution.

[0066] Preparation of reference solution: Prepare according to the method of Example 1 “Preparation of reference solution”.

[0067] Preparation of test solution: Prepare according to the method of Example 1 "Preparation of test solution".

[0068] Take blank solution, reference substance solution and test solution, and analyze them according to the chromatographic conditions of Example 1, and record the chromatogram. Figure 1 .

[0069] The results showed that the test sample chromatogram had the same chromatographic peak at the corresponding retention time as the reference chromatogram, and there was no interference from the blank solvent, indicating that the method had good specificity.

[0070] 2.3 Overall Examination

[0071] Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take about 0.2g, prepare the test solution according to the method of "Preparation of test solution" in Example 1, on the basis of the chromatographic conditions in Example 1, keep the elution gradient at the initial ratio, extend the elution time by one time, record the chromatogram, see Figure 2 .

[0072] The results showed that no obvious chromatographic peaks were eluted after the initial gradient elution, indicating that the chromatographic conditions basically met the principle of maximum information content.

[0073] 2.4 Precision test

[0074] Take the Litsea cubeba formula granules, prepare the test solution according to the method of "Preparation of test solution" in Example 1, perform sample injection analysis according to the chromatographic conditions in Example 1, and repeat the injection 6 times; take the magnolia alkaloid chromatographic peak as the reference S peak, calculate the relative retention time and relative peak area of ​​each characteristic peak and S peak, and calculate the relative standard deviation. The results show that the same sample is injected 6 times continuously, and the relative retention time RSD value of each characteristic peak and S peak is in the range of 0.04-0.09%, and the relative peak area is in the range of 0.25-1.14%, which is less than 2%, indicating that the instrument has good precision.

[0075] 2.5 Repeatability test

[0076] Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take about 0.2g, weigh accurately, and prepare the test solution according to the method of "Preparation of test solution" in Example 1, and parallel 6 portions. With the chromatographic peak of magnolia alkaloids 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 6 portions of the same batch of samples were prepared in parallel, and the RSD values ​​of the relative retention time of each characteristic peak were in the range of 0.23-0.27%, and the relative peak area was in the range of 0.51-0.85%, all less than 1%, indicating that the method has good repeatability.

[0077] 2.5 Stability test

[0078] Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take about 0.2g, and prepare the test solution according to the method of "Preparation of test solution" in Example 1. Inject 2μL at 0, 2, 4, 8, 12, and 24h, and record the chromatogram. Take the magnolia alkaloid 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 value of the relative retention time of each characteristic peak and the S peak is in the range of 0.32-0.36%, and the relative retention time is in the range of 0.63-1.09%, which is less than 3%, indicating that the test solution is stable within 24h.

[0079] 2.6 Durability

[0080] 2.6.1 Chromatographic column investigation

[0081] Three chromatographic columns were used to analyze the Litsea cubeba formula granule samples: 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 Table 1. Figure 3 .

[0082] The results showed that among the three different brands of chromatographic columns, the separation of magnolone in Litsea cubeba was better under ZORBAX EclipsePlus C18. ZORBAX Eclipse Plus C18 (2.1mm×100mm, 1.8μm) chromatographic column was selected for subsequent research.

[0083] 2.6.2 Flow rate investigation

[0084] The ZORBAX Eclipse Plus C18 (2.1mm×100mm, 1.8μm) column was used to investigate the sample separation effect at flow rates of 0.25mL / min, 0.3mL / min, and 0.35mL / min. The results showed that the separation of each characteristic peak was good when the flow rate varied in the range of 0.25-0.30mL / min. Smaller flow rate changes could meet the system suitability requirements, and 0.3mL / min was used in subsequent studies.

[0085] 2.6.3 Column temperature investigation

[0086] Three temperatures of 30℃, 35℃ and 40℃ were investigated respectively. The results showed that the peak separation was good when the column temperature was between 30℃ and 40℃, indicating that the column temperature had good durability. According to the peak elution of the sample, 35℃ was selected as the detection temperature.

[0087] 2.7 Sample determination

[0088] Take an appropriate amount of 3 batches of Litsea cubeba formula granules, grind them into powder, take 0.2 g, prepare the test solution according to the "test solution preparation method" of Example 1, inject and measure according to the chromatographic conditions of Example 1, record the spectrum, and the results are shown in the table below.

[0089] Relative retention time of characteristic spectra of three batches of Litsea cubeba formula granules

[0090]

[0091] The UHPLC characteristic spectra of the three batches of Litsea cubeba formula granules were matched using the "Chinese Herbal Medicine Chromatographic Characteristic Spectrum Similarity Evaluation System", and the No. 4 peak magnolia alkaloids were used as the reference peak S. The reference characteristic spectrum of the Litsea cubeba formula granules was established by the average method. Figure 4 .

[0092] The final standard for the characteristic spectrum of Litsea cubeba is: there should be 4 characteristic peaks in the chromatogram of the test sample, and the retention time should correspond to the 4 characteristic peaks in the chromatogram of the reference medicinal material, among which peak 4 should correspond to the retention time of the reference peak of the reference substance. The peak corresponding to the peak of the magnolia alkaloid reference substance is the S peak, and the relative retention time of peaks 1 to 3 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, and the specified value is: 0.33 (peak 1), 0.48 (peak 2), 0.63 (peak 3).

[0093] Example 3 Establishment of a method for determining the content of Litsea cubeba formula granules

[0094] Determined by high performance liquid chromatography (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0095] Chromatographic conditions and system suitability experiment The chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 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°C; the detection wavelength is 300nm, and the theoretical plate number calculated based on the magnolia alkaloid peak should be not less than 10000; the gradient elution conditions are:

[0096]

[0097] Preparation of reference solution: Take an appropriate amount of magnolia alkaloid reference substance, weigh it accurately, and add 70% methanol to make a solution containing 20 μg per 1 ml.

[0098] Preparation of test solution: Take an appropriate amount of the product, grind it into powder, take about 0.2 g, weigh it accurately, put it in a stoppered conical flask, accurately add 25 ml of 70% methanol, weigh it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0099] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0100] Example 4 Methodological Verification of the Content Determination Method of Litsea glutinosa Formula Granules

[0101] The practicability and accuracy of the content determination method in Example 3 were verified from the aspects of the linear range, precision, repeatability, solution stability, and sample recovery rate of the detection method.

[0102] In the embodiment, the instruments, reagents and drugs used are the same as those in Example 2.

[0103] 4.1 Linear investigation

[0104] Accurately weigh the magnolia alkaloid reference substance, place it in a 100mL volumetric flask, and add methanol to make a stock solution containing 264.99μg of magnolia alkaloid reference substance per 1mL. Accurately measure 0.1mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, and 5.0mL of the above-mentioned reference substance mixed stock solution and place them in 10mL volumetric flasks, add methanol to the scale, and shake well to obtain reference substance application solutions of different concentrations. Accurately aspirate 2μL of each reference substance application solution of different concentrations, measure them in turn according to the chromatographic conditions of Example 3, and record the chromatographic peak area. Use the peak area as the ordinate (y) and the reference substance concentration as the abscissa (x), and draw a standard curve. The results show that the linear regression equation for magnolia alkaloid is: y=1.9805x+0.3105, R 2 =0.9987. This indicates that the linear relationship between the concentration of magnolia alkaloids and the peak area is good within the concentration range of 2.65 to 132.49 μg / mL.

[0105] 4.2 Precision test

[0106] Investigation method: Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take 0.2g, and prepare the test solution according to the method of "Preparation of test solution" in Example 3. According to the chromatographic conditions of Example 3, perform sample injection analysis, inject samples continuously for 6 times, record the chromatographic peak area, and calculate the relative standard deviation.

[0107] The results showed that the same sample solution was injected six times, and the RSD value of the magnolia alkaloid peak area was 0.46%, which was less than 1%, indicating that the instrument had good precision.

[0108] 4.3 Repeatability test

[0109] Investigation method: Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take 0.2g, and divide them into 6 parallel portions. According to the test solution preparation method in Example 3, a test solution is prepared. According to the chromatographic conditions in Example 3, the sample is injected and analyzed. The sample is injected 6 times continuously, the chromatographic peak area is recorded, and its content and RSD are calculated.

[0110] The results showed that the content of magnolia alkaloids in 6 test solutions was 2.02-2.14 mg / g, with an average of 2.088 mg / g. The RSD value was 1.90%, which was less than 2%, indicating good repeatability.

[0111] 4.4 Stability test

[0112] Investigation method: Take an appropriate amount of Litsea cubeba formula granules, grind them into powder, take 0.2g, prepare the test solution according to the test solution preparation method in Example 3, and analyze the samples at 0h, 2h, 4h, 8h, 12h, and 24h according to the chromatographic conditions in Example 3, and record the chromatographic peak areas. Calculate the peak area RSD values ​​respectively.

[0113] The results showed that when the test solution was injected at 0, 2, 4, 8, 12, and 24 hours, the RSD value of the peak area of ​​magnolia alkaloids was 1.54%, which was less than 2%, indicating that the method had good stability within 24 hours.

[0114] 4.5 Recovery test

[0115] Investigation method: Take an appropriate amount of a sample of Litsea cubeba formula granules with a known content (magnolia alkaloid content: 2.0887 mg / g), grind it into powder, take 0.1g, and weigh six parallel portions accurately. Add a reference solution of magnolia alkaloids with the same content as 0.1g of the sample, and prepare the sample recovery solution according to the preparation method of the sample solution in Example 3. According to the chromatographic conditions in Example 3, inject 2μL of the sample, and calculate the recovery rate and RSD with the following formula. The results are shown in the table below.

[0116]

[0117] Results of the recovery test of the content determination of Litsea cubeba formula granules

[0118]

[0119] Results: The recovery rates of magnolia alkaloids were between 96.19% and 100.94%, and the RSD value of the peak area of ​​magnolia alkaloids was 1.24%, which was less than 3%. The recovery rate test was good.

[0120] 4.6 Sample determination

[0121] Take an appropriate amount of three batches of Litsea cubeba formula granules, grind them into powder, take 0.2 g, prepare the test solution according to the "test solution preparation method" of Example 3, and measure the content of magnolidine according to the chromatographic conditions of Example 3. The results are shown in the following table.

[0122]

[0123] Example 5 Establishment of characteristic spectrum and content determination method of Litsea cubeba

[0124] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0521 of Part IV of the 2020 edition of the Chinese Pharmacopoeia).

[0125] Chromatographic conditions and system suitability test are the same as [Content determination]

[0126] Preparation of reference solution Take 0.5g of Litsea cubeba reference medicinal material, place it in a stoppered conical flask, add 25ml of 70% methanol, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of magnolia alkaloid reference substance, accurately weigh it, add methanol to make a solution containing 30μg of magnolia alkaloid per 1ml, as the reference substance solution.

[0127] Preparation of test solution is the same as that of [Content determination]

[0128] The determination method is to accurately pipette 2 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and determine the result.

[0129] The test sample chromatogram should show 4 characteristic peaks, and the retention time should correspond to the 4 characteristic peaks in the reference chromatogram of the control medicinal material. Peak 4 should correspond to the retention time of the reference peak of the control. The peak corresponding to the peak of the magnolia alkaloid reference is the S peak. The relative retention time of peaks 1 to 3 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.34 (peak 1), 0.48 (peak 2), 0.64 (peak 3). The reference characteristic spectrum of Litsea cubeba is shown in Figure 5 .

[0130] [Content Determination] Magnolia alkaloids were determined by high performance liquid chromatography (General Rules 0521 of the 2020 edition of the Chinese Pharmacopoeia).

[0131] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); acetonitrile is used as mobile phase A, 0.2% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; flow rate is 0.3 ml per minute; column temperature is 35°C; detection wavelength is 300 nm. The theoretical plate number calculated based on the magnolia alkaloid peak should not be less than 10,000.

[0132]

[0133] Preparation of reference solution: Take an appropriate amount of magnolia alkaloid reference substance, weigh it accurately, and add methanol to make a solution containing 30 μg per 1 mL.

[0134] Preparation of test solution: Take about 1 g of Litsea cubeba powder (passed through No. 3 sieve), weigh it accurately, put it in a stoppered conical flask, add 25 ml of 70% methanol accurately, weigh it, heat and reflux for 30 minutes, cool, weigh it again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain the test solution.

[0135] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0136] Example 6 Establishment of characteristic spectrum and content determination method of standard decoction of Litsea cubeba

[0137] [Characteristic spectrum] Determined by high performance liquid chromatography (General Rules 0512 of the Chinese Pharmacopoeia 2020 Edition).

[0138] Chromatographic conditions and system suitability test are the same as those for [Content Determination] Magnolia alkaloids

[0139] Preparation of reference solution Take 0.5g of Litsea cubeba reference medicinal material, place it in a stoppered conical flask, add 25ml of 70% methanol, heat and reflux for 30 minutes, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution. Take an appropriate amount of magnolia alkaloid reference substance, accurately weigh it, add methanol to make a solution containing 30μg of magnolia alkaloid per 1ml, as the reference substance solution.

[0140] Preparation of test solution is the same as that of Magnololidine

[0141] Determination method: Accurately pipette 2μl of reference solution and test solution, inject into liquid chromatograph, and determine.

[0142] The test sample chromatogram should show 4 characteristic peaks, and the retention time should correspond to the 4 characteristic peaks in the reference drug chromatogram. Peak 4 should correspond to the retention time of the reference drug peak. The peak corresponding to the magnolia alkaloid reference peak is the S peak. The relative retention time of peaks 1 to 3 and S peak is calculated. The relative retention time should be within the range of ±10% of the specified value. The specified value is: 0.33 (peak 1), 0.47 (peak 2), 0.63 (peak 3). The reference characteristic spectrum of the standard decoction of Litsea cubeba is shown in Figure 6 .

[0143] [Content Determination] Magnolia alkaloids were determined by high performance liquid chromatography (General Rules 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0144] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel is used as filler (column length is 100 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); acetonitrile is used as mobile phase A, 0.2% formic acid solution is used as mobile phase B, and gradient elution is performed according to the provisions in the following table; flow rate is 0.3 ml per minute; column temperature is 35°C; detection wavelength is 300 nm. The theoretical plate number calculated based on the magnolia alkaloid peak should not be less than 10,000.

[0145]

[0146] Preparation of reference solution: Take an appropriate amount of magnolia alkaloid reference substance, weigh it accurately, and add 70% methanol to make a solution containing 30 μg per 1 mL.

[0147] Preparation of test solution: Take about 0.2 g of powder of standard decoction of Litsea cubeba, weigh it accurately, put it in a stoppered conical flask, add 25 ml of 70% methanol accurately, stopper it tightly, weigh it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with 70% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0148] Determination method: Accurately pipette 2μl of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0149] Example 7 Establishment of Thin Layer Identification Method for Litsea glutinosa Formula Granules

[0150] Preparation of test solution: Take 2g of Litsea cubeba granules, grind them into powder, add 20ml of ether, ultrasonically treat for 30 minutes, filter, and concentrate the filtrate to about 1ml as the test solution;

[0151] Preparation of control medicinal material solution: Take 0.5 g of Litsea cubeba control medicinal material and prepare the control medicinal material solution according to the preparation method of the test solution;

[0152] Developing solvent: cyclohexane-ethyl acetate (8:2);

[0153] Color developer: vanillin sulfuric acid test solution;

[0154] Detection method: Take 5 μl of each solution of the test sample and the control medicinal material, spot them on the same silica gel G thin layer plate, develop them with the above-mentioned developer, take them out, dry them, spray them with the above-mentioned developer, and heat them at 105°C until the spots are clearly colored. In the chromatogram of the test sample, spots of the same color appear at the corresponding positions of the chromatogram of the control medicinal material.

[0155] The TLC identification method was verified by TLC plates of different manufacturers at different temperatures and humidities, with different sample volumes. The method had good durability. The TLC of three batches of Litsea cubeba formula granules showed Figure 7 .

[0156] Example 8 Preparation method of Litsea cubeba formula granules

[0157] Pre-treatment process: Use a linear reciprocating herb cutter to cut the large-fruited Litsea cubeba, and the cutting frequency is fixed at 180 times / min; it should be cut and cooked immediately;

[0158] Extraction process: add 12 times the amount of water to soak for 30 minutes, decoct for 1 hour, collect volatile oil, filter the decoction, add 10 times the amount of water to soak for 1 hour, collect volatile oil, filter the decoction, and combine the filtrate; the filtrate is concentrated under reduced pressure, vacuum dried, and crushed to obtain dry paste powder;

[0159] Inclusion process: Mix the oil of Litsea cubeba with the same volume of anhydrous ethanol, accurately weigh an appropriate amount of β-cyclodextrin 8 times the amount of volatile oil, place it in a round-bottom flask, add 200mL of ultrapure water, and heat it to make it a saturated aqueous solution. Transfer it to a constant temperature stirrer, slowly drip the ethanol solution of Litsea cubeba volatile oil, maintain 500r / min, and stir at 40℃ for 60min. After the inclusion is completed, cool to room temperature, refrigerate at 4℃ for 24h, filter, dry, and crush to obtain the volatile oil inclusion compound.

[0160] Molding process: Take dry paste powder and volatile oil inclusion compound, add appropriate amount of silicon dioxide and maltodextrin, mix, granulate and package.

[0161] Example 9 Preparation method of Litsea cubeba standard decoction

[0162] Take 100g of Litsea cubeba slices, weigh it, add 8 times water to soak for 30min, boil it with high heat first, then simmer it with low heat for 20min, filter it while hot, and cool the filtrate in a cold water bath; add 9 times water for the second decoction, boil it with high heat, then simmer it with low heat for 15min, filter it while hot, and cool the filtrate in a cold water bath; combine the two filtrates and concentrate them under reduced pressure at 65°C to a relative density of 1.04-1.10 (65°C), divide the concentrate into vials, place them in a freeze dryer, and freeze-dry them to obtain a standard decoction of Litsea cubeba.

[0163] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting Litsea cubeba formula granules, comprising properties, identification, inspection, extract, characteristic spectrum, and content determination, characterized in that: The characteristic spectrum and content determination detection method is to use high performance liquid chromatography for detection. The working conditions of the high performance liquid chromatography are: the chromatographic column is filled with octadecylsilane bonded silica gel, acetonitrile is used as mobile phase A, 0.1% to 0.5% formic acid solution is used as mobile phase B for gradient elution, and the flow rate is 0.2 ml to 1 ml per minute; the column temperature is 30 to 40°C; the detection wavelength is 250 to 350 nm, and the theoretical plate number calculated according to the magnolia alkaloid peak should be not less than 5000 to 10000; the gradient elution conditions are: 0-1min, mobile phase A 5→7%, mobile phase B 95→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%; 11-19 min, mobile phase A 11→17%, mobile phase B 89→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 spectrum and content determination detection method further comprises preparing a reference solution or preparing a control solution, preparing a test solution and injecting a sample for determination, wherein: Preparation of reference solution Take 0.5-2.0g of Litsea cubeba reference medicinal material, add 25-50ml of 70% methanol, heat and reflux for 30-60min, cool, shake well, filter, and take the filtrate as the reference medicinal material reference solution; take an appropriate amount of new magnolia alkaloid reference substance, accurately weigh it, add 70% methanol to make a mixed solution containing 20μg per 1ml, as the reference substance solution; Preparation of reference solution Take an appropriate amount of magnolia alkaloid reference substance, weigh accurately, and add 70% methanol to make a solution containing 20-30 μg per 1 ml. Preparation of the test solution: Take the Litsea cubeba formula granules, grind them into powder, take about 0.1-1g, weigh accurately, add 25-50ml of 70% methanol accurately, weigh the weight, ultrasonically treat for 30-60 minutes, cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and take the filtrate to obtain; Determination method: Accurately pipette 2-10 μl of reference solution or control solution and test solution respectively, inject into liquid chromatograph, and perform determination according to HPLC working conditions.

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

3. The detection method according to claim 1, characterized in that: The characteristic spectrum determination method is: accurately aspirate 2 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, perform determination according to the working conditions of the high performance liquid chromatography, and record the chromatogram from 0 to 22 minutes.

4. The detection method according to any one of claims 1 to 3, characterized in that: The test sample chromatogram of the characteristic spectrum should present four characteristic peaks, and the retention times should correspond to the four characteristic peaks in the chromatogram of the control medicinal material reference substance. Among them, the peak corresponding to the magnolia alkaloid reference substance peak is the S peak. The relative retention time of each characteristic peak and the S peak is calculated, and the relative retention time should be within ±10% of the specified value; the specified value is: the relative retention time of peak 1 is 0.33, the relative retention time of peak 2 is 0.48, and the relative retention time of peak 3 is 0.

63.

5. The detection method according to claim 1, characterized in that: The content determination method is: accurately aspirate 2 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, record the chromatogram, and calculate the content of magnolidine by the external standard method.

6. The detection method according to claim 1, characterized in that The identification method is as follows: 1-2g of Litsea cubeba formula granules are taken, ground, 20-30ml of ether is added, ultrasonic treatment is performed for 20-60 minutes, filtered, and the filtrate is concentrated to about 1ml as the test solution; 0.5-1g of Litsea cubeba reference medicinal material is taken separately, and the reference medicinal material solution is prepared in the same way; according to the thin layer chromatography test of the Chinese Pharmacopoeia, 5μl of each of the above two solutions are taken, and they are respectively spotted on the same silica gel G thin layer plate, cyclohexane-ethyl acetate = 8:2 is used as the developing solvent, the plate is developed, taken out, dried, sprayed with vanillin sulfuric acid test solution, and heated at 105°C until the spots are clearly colored; in the chromatogram of the test sample, spots of the same color should appear at the corresponding positions of the chromatogram of the reference medicinal material.

7. The detection method according to claim 1, characterized in that: The extract detection method is as follows: take the Litsea cubeba formula granules, grind them into powder, take 2g, accurately weigh, accurately add 100ml of ethanol, and determine it by the hot soaking method under the alcohol-soluble extract determination method 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 prepared by taking Litsea cubeba slices, adding water to boil, collecting volatile oil, encapsulating the volatile oil with β-cyclodextrin and setting aside; filtering the decoction, concentrating the filtrate into a clear paste, drying or dry-crushing, adding the volatile oil inclusion compound and pharmaceutically acceptable auxiliary materials, mixing, and granulating, wherein 1 gram of the formula granules is equivalent to 4.5 grams of the slices.

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

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

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