Usnea diffracta extract and preparation method and detection method of preparation thereof

Abstract

The invention belongs to the field of modernization of traditional Chinese medicines, and particularly relates to a preparation method and a detection method of a Chinese usnea extract and a preparation thereof. The preparation method of the usnea diffracta extract comprises the following steps: taking usnea diffracta decoction pieces, adding water, decocting, and carrying out solid-liquid separation to obtain decoction, namely the usnea diffracta extract. The usnea diffracta extract preparation is prepared from the usnea diffracta extract. According to the method for detecting the content of the index components in the usnea extract and the preparation thereof, the specific chromatogram detection method and the thin-layer identification method provided by the invention, the quality of the usnea extract and the preparation thereof can be more effectively evaluated, and meanwhile, a scientific and reasonable basis is provided for formulating process standards and quality standards of the usnea extract and the preparation thereof.

Description

Technical Field

[0001] The invention belongs to the field of modernization of traditional Chinese medicine, and particularly relates to a preparation method and a detection method of a usnea extract and a preparation thereof. Background Art

[0002] Usnea is the dried whole plant of the Usnea diffracta Vain. It is also known as female vine, pine, dragon beard grass, cloud grass, Laojun beard or mountain dragon, etc. It is flat in nature, sweet and bitter in taste, and enters the heart, kidney and lung meridians. It has the effects of clearing away heat and detoxifying, removing phlegm and relieving cough, removing dampness and unblocking meridians, stopping bleeding and relieving pain. It is used for phlegm-heat warm malaria, lung heat cough, wind-cold dampness arthralgia, limb pain, muscle and bone cramps. Modern pharmacological studies have shown that Usnea has antibacterial, analgesic and antipyretic, insecticidal, anti-tumor, anti-allergic, antiviral, and wound healing effects. Usnea contains a variety of chemical components such as phenolic acid, steroids, triterpenes and polysaccharides, among which phenolic acid is its main active ingredient, such as usnic acid, diffractoic acid or ramic acid.

[0003] However, at present, there is a widespread phenomenon of confusion in the use of raw materials of Usnea on the market, and the evaluation system of quality standards for Usnea extracts and their preparations is still imperfect, which cannot effectively evaluate their indicators, limiting their application and development. Summary of the invention

[0004] In view of the above technical problems, the object of the present invention is to provide a preparation method and a detection method of a Usnea extract and a preparation thereof.

[0005] Specifically, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing a usnea extract, comprising the following steps:

[0007] Take Usnea slices, add water and boil, separate the solid and liquid, and obtain the decoction, which is the Usnea extract.

[0008] Preferably, the number of times of adding water for decoction is 1 to 3 times; more preferably, the number of times of adding water for decoction is 2 times; wherein, 10 to 15 times the amount of water is added for the first decoction; and 8 to 14 times the amount of water is added for the second decoction; and / or, more preferably, the first decoction time is 30 to 120 minutes; and the second decoction time is 20 to 80 minutes.

[0009] And / or, preferably, the solid-liquid separation is filtration; more preferably, filtration is performed using a 100-300 mesh sieve.

[0010] And / or, preferably, the Usnea extract also includes the following steps: concentrating and drying the obtained decoction; more preferably, the concentration temperature is 55-70°C; and / or, more preferably, the concentration vacuum degree is -0.080-0.090MPa; and / or, more preferably, the concentration of the concentrate is 1-1.1g / mL.

[0011] And / or, more preferably, the drying is freeze drying, vacuum drying, spray drying, microwave drying or infrared drying.

[0012] In a second aspect, the present invention provides a method for preparing a usnea extract preparation, comprising the following steps:

[0013] After adding the usnea extract to the first auxiliary material, drying is performed. Further preferably, the first auxiliary material comprises maltodextrin. Further preferably, the addition amount of the first auxiliary material is 12% to 18% of the amount of the usnea decoction piece.

[0014] Preferably, a second auxiliary material is added to the dried material; more preferably, the second auxiliary material comprises silicon dioxide; further preferably, the percentage of silicon dioxide in the weight of the dried material is greater than 0 and less than or equal to 0.3%.

[0015] In a third aspect, the present invention provides a usnea extract or a usnea extract preparation, wherein the usnea extract is prepared by the usnea extract preparation method, or the usnea extract preparation is prepared by the usnea extract preparation preparation method.

[0016] Preferably, the usnea extract preparation is usnea formula granules; or, preferably, the usnea extract is usnea standard decoction;

[0017] Further preferably, the preparation amount of the Usnea formula granules is 27% to 31%, wherein the preparation amount refers to the ratio of the mass of the prepared formula granules to the mass of the medicinal materials fed; even more preferably, the preparation amount of the Usnea formula granules is 28% to 31%.

[0018] And / or, preferably, in the standard usnea decoction, the content of usnic acid is 0.32-3.26 mg / g; and / or, in the standard usnea decoction, the transfer rate of usnic acid is 2.31%-27.15%.

[0019] In a fourth aspect, the present invention provides a method for detecting the content of usnic acid in the usnea extract or the usnea extract preparation, comprising the following steps:

[0020] Step 1: Preparation of reference solution

[0021] Weigh the usnic acid reference substance and add solvent to make a solution;

[0022] Step 2: Preparation of test solution

[0023] Take a usnea extract or a usnea extract preparation, add a solvent to extract;

[0024] Step 3: Liquid chromatography analysis

[0025] Octadecylsilane bonded silica gel was used as filler, mobile phase A was organic phase, mobile phase B was aqueous phase, reference solution and test solution were injected into liquid chromatograph, and gradient elution was used for detection.

[0026] Preferably, in step 1, the solvent comprises one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water.

[0027] And / or, preferably, in step 2, the preparation of the test solution comprises the following steps: taking a usnea extract or a usnea extract preparation, adding an extraction solvent, weighing the weight, performing extraction, weighing the weight again, making up the lost weight with the extraction solvent, and obtaining the liquid by solid-liquid separation to obtain the test solution.

[0028] And / or, preferably, in step 2, the extraction solvent includes one or more than two selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water.

[0029] And / or, preferably, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; more preferably, the extraction time is 15 to 60 minutes.

[0030] And / or, preferably, the method for preparing the test solution of the Usnea standard decoction or Usnea formula granules comprises the following steps: taking the Usnea standard decoction or Usnea formula granules, adding an extraction solvent, weighing the weight, extracting, weighing the weight again, making up the lost weight with the extraction solvent, and obtaining the liquid by solid-liquid separation to obtain the test solution; preferably, the ratio of the mass of the Usnea standard decoction or Usnea formula granules to the volume of the extraction solvent is 0.1-1:10-25, wherein the unit of mass is g and the unit of volume is mL.

[0031] And / or, preferably, in step 3, the detection wavelength of the chromatogram is 190-400 nm; preferably, the detection wavelength of the chromatogram is 230-235 nm; and / or, in step 3, the flow rate is 0.2-0.3 mL / min; and / or, in step 3, the column temperature is 25-35° C.; and / or, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; preferably, the chromatographic column is selected from ACQUITY T3 (or ACQUITY UPLC HSS T3), AmethSep C18AQ or Chromcore AQC18.

[0032] And / or, preferably, in step 3, the organic phase is selected from acetonitrile; and the aqueous phase is an aqueous solution containing 0.1% phosphoric acid.

[0033] And / or, preferably, in step 3, the gradient elution procedure is as follows:

[0034] From 0 to 20 min, the volume percentage of mobile phase A increased from 5% to 65%, and the volume percentage of mobile phase B decreased from 95% to 35%;

[0035] 20-35 min, the volume percentage of mobile phase A is 65%, and the volume percentage of mobile phase B is 35%;

[0036] From 35 to 40 min, the volume percentage of mobile phase A increased from 65% to 80%, and the volume percentage of mobile phase B decreased from 35% to 20%.

[0037] In a fifth aspect, the present invention provides a method for detecting a characteristic spectrum of the usnea extract or the usnea extract preparation, characterized in that it comprises the following steps:

[0038] Step 1: Preparation of reference solution

[0039] Weigh the usnic acid reference substance and add solvent to make a solution;

[0040] Step 2: Preparation of test solution

[0041] Take a usnea extract or a usnea extract preparation, add a solvent to extract;

[0042] Step 3: Liquid chromatography analysis

[0043] Octadecylsilane bonded silica gel was used as filler, mobile phase A was organic phase, mobile phase B was aqueous phase, reference solution and test solution were injected into liquid chromatograph, gradient elution was used for detection;

[0044] Step 4: Establish a characteristic spectrum of the usnea extract or usnea extract preparation.

[0045] Preferably, in step 1, the extraction solvent includes one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water.

[0046] And / or, preferably, in step 2, the preparation of the test solution comprises the following steps: taking a usnea extract or a usnea extract preparation, adding a solvent used for extraction, weighing the weight, performing extraction, weighing the weight again, making up the lost weight with the solvent used for extraction, and the liquid obtained by solid-liquid separation is the test solution; more preferably, in step 2, the extraction solvent comprises one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; and / or, more preferably, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; further preferably, the extraction time is 15 to 60 min.

[0047] And / or, preferably, the method for preparing the test solution of the Usnea standard decoction or Usnea formula granules comprises the following steps: taking the Usnea standard decoction or Usnea formula granules, adding an extraction solvent, weighing the weight, extracting, weighing the weight again, making up the lost weight with the extraction solvent, and obtaining the liquid by solid-liquid separation to obtain the test solution; preferably, the ratio of the mass of the Usnea standard decoction or Usnea formula granules to the volume of the extraction solvent is 0.1-1:10-25, wherein the unit of mass is g and the unit of volume is mL.

[0048] And / or, preferably, in step 3, the detection wavelength of the chromatogram is 190-400 nm; preferably, the detection wavelength of the chromatogram is 230-235 nm; and / or, in step 3, the flow rate is 0.2-0.3 mL / min; and / or, in step 3, the column temperature is 25-35° C.; and / or, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; preferably, the chromatographic column is selected from ACQUITY T3 (or ACQUITY UPLC HSS T3), AmethSep C18 AQ or Chromcore AQC18; and / or, in step 3, the organic phase is selected from acetonitrile; and the aqueous phase is an aqueous solution containing 0.1% phosphoric acid.

[0049] And / or, preferably, in step 3, the gradient elution procedure is as follows:

[0050] From 0 to 20 min, the volume percentage of mobile phase A increased from 5% to 65%, and the volume percentage of mobile phase B decreased from 95% to 35%;

[0051] 20-35 min, the volume percentage of mobile phase A is 65%, and the volume percentage of mobile phase B is 35%;

[0052] From 35 to 40 min, the volume percentage of mobile phase A increased from 65% to 80%, and the volume percentage of mobile phase B decreased from 35% to 20%.

[0053] And / or, preferably, the characteristic spectrum of the usnea extract or usnea extract preparation presents 5 characteristic peaks, among which peak 5 is consistent with the retention time of the usnea acid reference peak, which is the S peak, and the relative retention time of each characteristic peak and the S peak is within ±10% of the specified value, and the specified value is: 0.25 (peak 1), 0.31 (peak 2), 0.42 (peak 3) and 0.47 (peak 4).

[0054] In a sixth aspect, the present invention provides a thin layer identification method of the usnea extract or the usnea extract preparation, comprising the following steps:

[0055] Preparation of a test solution of usnea extract or usnea extract preparation, spotting on a thin layer plate, developing and coloring; wherein the method for preparing the test solution of usnea extract or usnea extract preparation comprises the following steps: extracting the usnea extract or usnea extract preparation with methanol, and the liquid obtained by solid-liquid separation is the test solution; and / or the developing agent is a mixed solvent of chloroform, toluene, methanol and glacial acetic acid.

[0056] Preferably, the volume ratio of chloroform, toluene, methanol and glacial acetic acid is 7.8-8.2:11.8-12.2:0.8-1.2:0.1-0.3; more preferably, the volume ratio of chloroform, toluene, methanol and glacial acetic acid is 8:12:1:0.2.

[0057] In a seventh aspect, the present invention provides an application of a method for detecting the content of usnic acid in usnea extracts and preparations thereof.

[0058] In an eighth aspect, the present invention provides an application of the characteristic spectrum detection method in Usnea extracts and preparations thereof.

[0059] In a ninth aspect, the present invention provides an application of the thin layer identification method in Usnea extracts and preparations thereof.

[0060] In a tenth aspect, the present invention provides an application of any one or more of the usnic acid content detection method, the characteristic spectrum detection method and the thin layer identification method in the quality evaluation of usnea extracts and preparations thereof.

[0061] Beneficial effects of the present invention:

[0062] (1) The present invention provides a preparation method and a detection method of a usnea extract. The usnea extract and the detection method provide a standard for the quality control of the usnea extract and its preparations, thereby achieving quality control and effective supervision.

[0063] (2) The present invention provides a Usnea formula granule and a preparation method thereof. The preparation method is simple and is decocted according to the traditional method, which conforms to the characteristics of traditional Chinese medicine.

[0064] (3) The present invention provides a method for detecting the content of usnic acid in a usnea extract and its preparation (the usnea extract is a usnea standard decoction, and the usnea extract preparation is a usnea formula granule). The detection method has good specificity, precision, stability and repeatability.

[0065] (4) The present invention provides a characteristic spectrum detection method for a usnea extract and its preparation (the usnea extract is a usnea standard decoction, and the usnea extract preparation is a usnea formula granule). The detection method has good specificity, precision, stability and repeatability.

[0066] (5) The present invention can more effectively evaluate the quality of usnea extract and its preparations through thin layer chromatography identification method, usnic acid content detection method and characteristic spectrum detection method, and at the same time provide a scientific and reasonable basis for formulating process standards and quality standards for usnea extract and its preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is the peak purity chromatogram of the verification of the content determination methodology of Usnea standard decoction.

[0068] Figure 2 It is the regression equation for the linear investigation of usnic acid in the methodological verification of the content determination of usnea standard decoction.

[0069] Figure 3 The effect of different chromatographic columns on the target components contained in the standard decoction of Usnea.

[0070] Figure 4 These are the chromatogram results of the target components in the standard decoction of Usnea at different column temperatures.

[0071] Figure 5 These are the chromatographic results of the target components in the standard decoction of Usnea at different flow rates.

[0072] Figure 6 This is the DAD diagram of the standard decoction of Usnea.

[0073] Figure 7 These are UPLC spectra of different mobile phase systems.

[0074] Figure 8It is the UPLC spectrum of the characteristic spectrum of Usnea standard decoction in aqueous phase.

[0075] Fig. 9 The characteristic spectrum of Usnea standard decoction was investigated with different extraction solvents.

[0076] Fig.10 It is the characteristic spectrum of the standard decoction of Usnea.

[0077] Fig.11 This is a comprehensive investigation of the methodological validation of the characteristic spectrum analysis method of Usnea standard decoction.

[0078] Fig.12 This is a UPLC comparison chart of different chromatographic columns for methodological validation of the characteristic spectrum analysis method of Usnea standard decoction.

[0079] Fig.13 This is a UPLC comparison chart of different column temperatures for methodological validation of the characteristic spectrum analysis method of Usnea standard decoction.

[0080] Fig.14 This is a UPLC comparison chart of different flow rates for methodological validation of the characteristic spectrum analysis method of Usnea standard decoction.

[0081] Fig.15 It is the peak purity chromatogram of the verification of the content determination methodology of Usnea formula granules.

[0082] Fig.16 It is the regression equation for the linear investigation of usnic acid in the methodological verification of the content determination of usnea formula granules.

[0083] Fig.17 It is the influence of different chromatographic columns on the target components contained in Usnea formula granules.

[0084] Fig.18 These are the chromatogram results of the target components in Usnea formula granules at different column temperatures.

[0085] Fig.19 These are the chromatogram results of the target components in the Usnea formula granules at different flow rates.

[0086] Fig. 20 This is the characteristic spectrum of Usnea formula granules.

[0087] Fig.21 This is a thin layer chromatogram of the sample quantity inspection of Usnea formula granules.

[0088] Fig. 22 It is a TLC-specific formula of Usnea granules.

[0089] Fig.23 This is the thin layer identification chromatogram of Usnea formula granules under different temperature conditions.

[0090] Fig.24This is the thin layer identification chromatogram of Usnea formula granules under different humidity conditions.

[0091] Fig.25 This is a chromatogram of silica gel plates from different manufacturers.

[0092] Fig.26 It is 3 batches of Usnea formula granules TLC. DETAILED DESCRIPTION

[0093] As described above, the object of the present invention is to provide a preparation method and detection method of a usnea extract and its preparation. Among them, the usnea decoction pieces are boiled in water for extraction and concentration to obtain a usnea standard decoction, and the usnea decoction pieces are boiled in water for extraction, concentration, auxiliary materials are added and dried to obtain a usnea formula granule. The present invention standardizes the quality control and standard research of the usnea extract and its preparation, realizes the overall quality control and effective supervision of the usnea standard decoction and formula granules, and provides a reference for the quality control of the usnea extract and its preparation.

[0094] Standard decoction, also known as standard decoction, is a traditional form of medication widely used in clinical practice. Standard decoctions are made in accordance with the theories of traditional Chinese medicine and are standardized according to clinical decoction methods, with solid-liquid separation, and are prepared by appropriate concentration or drying by suitable methods. The medicinal substances of Chinese medicine formula granules are consistent with those of Chinese medicine decoction slices, and they are a continuation of Chinese medicine decoction slices. Standard decoctions can be used as standard reference materials to measure whether Chinese medicine formula granules are basically consistent with clinical decoctions.

[0095] Since standard decoctions are the "bridge" connecting traditional Chinese medicine pieces and modern Chinese medicine preparations, they provide a reference for controlling the quality of Chinese medicine terminal products, standardizing different forms of Chinese medicine, ensuring the uniformity of quality and consistency of efficacy, and evaluating the consistency of product quality of different manufacturers. Therefore, the formulation of quality standards for standard Chinese medicine decoctions will provide a basis for the formulation of quality standards for all final products derived from decoctions of pieces. However, at present, there are many cases of confusion in the use of the original medicinal materials of Usnea in the market. Therefore, establishing an effective and reliable method for Usnea standard decoctions and formula granules is conducive to ensuring the authenticity and accuracy of the drug, clinical effectiveness and the development of subsequent formula granule work.

[0096] The index component described in the present invention refers to usnic acid.

[0097] In the present invention, the reagents used in the examples are conventional reagents unless otherwise specified. The information of the raw materials and experimental equipment used in the examples are shown in Table 1 and Table 2, respectively:

[0098] Table 1 Raw material information used in the present invention

[0099]

[0100]

[0101] Table 2 Information of experimental equipment used in the present invention

[0102]

[0103] In the present invention, the Usnea medicinal material is the dried whole plant of the Usnea diffracta Vain. It can be harvested and dried throughout the year.

[0104] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0105] Example 1: Preparation method of usnea standard decoction, determination and analysis method of usnea acid content and establishment of characteristic spectrum analysis method

[0106] Example 1-1: Preparation method of Usnea standard decoction

[0107] The preparation method of Usnea slices comprises the following steps: taking the original Usnea medicinal material, cleaning it, selecting it, removing non-medicinal parts, impurities and deteriorated products, etc., the impurity content shall not exceed 3%, cutting it into sections and sieving it to remove ash. The origin of the Usnea slices is Wudang District, Guiyang City, Guizhou Province.

[0108] Step 1: Take 100g of Usnea japonica slices, weigh them, put them in an electric ceramic pot, add water and boil them twice. For the first decoction, add 14 times the amount of water, soak for 30 minutes, boil them over high heat (500W), and keep them slightly boiling for 40 minutes over low heat (200W). Filter the decoction through a 300-mesh sieve while hot, and cool the filtrate. For the second decoction, add 12 times the amount of water, heat and boil them over high heat (500W), and keep them slightly boiling for 30 minutes over low heat (200W). Filter the decoction through a 300-mesh sieve while hot, and cool the filtrate. Combine the two decoctions.

[0109] Step 2: Transfer the decoction to a 2000mL round-bottom flask and use a rotary evaporator to reduce pressure and concentrate to 500mL of concentrated solution (extract). The concentration temperature is 65°C, the concentration vacuum degree is -0.080~-0.090MPa, the measured extract rate is 19.74%, and the concentration of the concentrate is 1.00~1.03g / mL. The Usnea standard decoction product is obtained and named N24052313. Among them, the usnic acid content in the decoction piece is 63.83mg / g, the usnic acid content in the standard decoction is 1.75mg / g, and the usnic acid content transfer rate is 13.69%.

[0110] In the present invention, the method for determining the paste yield comprises the following steps: the constant weight of the evaporating dish is m 1, take 10g of the concentrated solution into an evaporating dish with constant weight, dry it in a water bath, and then dry it in a drying oven at 105℃ for 3 hours, cool it in a desiccator for half an hour, and weigh it quickly, which is m 2 The calculation formula of the paste yield is as follows:

[0111]

[0112] Where: X---paste yield, numerical form expressed in %; m 2 ---Total weight of the sample and evaporating dish after drying, in g; m 1 ---The weight of the evaporating dish, in g; the total weight of the concentrate is the total mass of the concentrate obtained in step 2, in g; the sampling amount of the concentrate is the sampling amount of the concentrate when determining the paste yield, in g; the sampling amount of the decoction pieces is the sampling amount of the decoction pieces in step 1, in g.

[0113] Example 1-2: Establishment of an analytical method for determining the content of usnic acid in a standard decoction of usnea

[0114] 2.1 Determination of chromatographic conditions

[0115] Chromatographic conditions and system suitability test

[0116] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.25 mL per minute; the column temperature was 30°C; the detection wavelength was 232 nm. The theoretical plate number calculated based on the usnic acid peak should be no less than 3000.

[0117] Table 3 Gradient elution program

[0118] Time (min) Mobile phase A (volume %) Mobile phase B (volume %) 0～20 5→65 95→35 20～35 65 35 35～40 65→80 35→20

[0119] The calculation formula of usnic acid content is as follows:

[0120]

[0121] Where: c x ---The content of usnic acid in the sample, in mg / g; c r ---Concentration of usnic acid in the reference substance, in mg / mL; A x ---Peak area of ​​usnic acid in the sample; A r ---Peak area of ​​reference substance usnic acid; V x ---The volume of solvent used for extraction, in mL; m x ---Weighing weight of sample, in g.

[0122] The calculation formula of the transfer rate of usnic acid (the component to be tested) is as follows:

[0123]

[0124] Wherein: X---usnic acid transfer rate, unit is %; content of the component to be tested in the standard decoction (dry basis), unit is mg / g; content of the component to be tested in the decoction piece (dry basis), unit is mg / g.

[0125] 2.2 Preparation of reference solution

[0126] Take an appropriate amount of usnic acid reference substance, weigh it accurately, and add methanol to prepare a solution containing 60 μg usnic acid per 1 mL, which is used as the reference substance solution.

[0127] 2.3 Preparation of test solution

[0128] 2.3.1 Investigation of extraction solvent

[0129] Take an appropriate amount of Usnea standard decoction N24052313, a total of 9 portions, each with 2 parallel samples, and place them in stoppered conical flasks respectively. Accurately add 20mL of methanol (anhydrous methanol), 75 volume% methanol, 50 volume% methanol, 25 volume% methanol, ethanol (anhydrous ethanol), 75 volume% ethanol, 50 volume% ethanol, 25 volume% ethanol, and water in sequence, weigh the weight, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, collect the filtrate, and obtain each test sample solution.

[0130] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0131] Table 4 Effects of different extraction solvents on the content determination results in Usnea standard decoction

[0132]

[0133]

[0134] From the experimental results in Table 4, it can be seen that considering the content differences, methanol was selected as the best extraction solvent for subsequent research.

[0135] 2.3.2 Investigation of extraction methods

[0136] Take an appropriate amount of Usnea standard decoction N24052313, a total of 2 portions, each with 2 parallel samples, respectively place them in stoppered conical samples, accurately add 20 mL of methanol in turn, weigh the weight, ultrasonically and reflux for 30 minutes respectively, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain each test sample solution.

[0137] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0138] Table 5 Effects of different extraction methods on the content determination results of Usnea standard decoction

[0139]

[0140] From the experimental results in Table 5, it can be seen that: considering the content differences comprehensively, ultrasonic treatment (power 500W, frequency 40kHz) extraction was selected as the extraction method for subsequent research.

[0141] 2.3.3 Investigation of extraction solvent volume

[0142] Take an appropriate amount of Usnea standard decoction N24052313, a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 10mL, 20mL, 30mL and 40mL of methanol in turn and weigh them. Ultrasonicate (power 500W, frequency 40kHz) for 30 minutes respectively. Cool and weigh them again. Make up the lost weight with methanol. Take the filtrate and pass it through a 0.22μm microporous filter membrane to obtain the test sample solution.

[0143] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0144] Table 6 Effect of different extraction solvent volumes on the content determination results of Usnea standard decoction

[0145]

[0146] From the experimental results in Table 6, it can be seen that considering the solvent cost and its content difference, the extraction volume of 25 mL was selected for subsequent research.

[0147] 2.3.4 Investigation of sampling volume

[0148] Take an appropriate amount of Usnea standard decoction N24052313, take about 0.25, 0.50, 0.75 and 1.0 g respectively, weigh accurately, put 2 parallel samples in stoppered cones respectively, add 25 mL of methanol accurately in turn, weigh the weight, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes respectively, cool, weigh again, make up the lost weight with methanol, take an appropriate amount of filtrate and pass it through a 0.22 μm microporous filter membrane to obtain each test sample solution.

[0149] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0150] Table 7 Effect of different sampling volumes on the content determination results of Usnea standard decoction

[0151]

[0152] From the experimental results in Table 7, it can be seen that: considering the content differences, a sampling amount of 1.0g was selected for subsequent research.

[0153] 2.3.5 Investigation of extraction time

[0154] Take an appropriate amount of Usnea standard decoction N24052313, a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 25 mL of methanol in sequence and weigh the weight. Ultrasonicate (power 500 W, frequency 40 kHz) for 15, 30, 45 and 60 minutes respectively. Cool and weigh again. Make up the lost weight with methanol, take an appropriate amount of the filtrate and pass it through a 0.22 μm microporous filter membrane to obtain the test sample solution.

[0155] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0156] Table 8 Effect of different extraction time on the content determination results of Usnea standard decoction

[0157]

[0158] From the experimental results in Table 8, it can be seen that considering the time cost and the difference in content, ultrasonic treatment (power 500 W, frequency 40 kHz) for 60 minutes was selected for subsequent research.

[0159] 2.3.6 Determination of the preparation method of the test solution

[0160] According to the experimental results of sample pretreatment, the preparation method of the test sample can be determined as follows: Take an appropriate amount of this product, about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 mL of methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0161] 2.4 Methodology Validation of Content Determination

[0162] 2.4.1 Peak Purity

[0163] Precisely pipette 1 μL each of the test sample solution and the reference solution prepared according to 2.3.6 from the Usnea standard decoction (N24052313) into an ultra-high performance liquid chromatograph, and perform on-machine detection according to the chromatographic conditions in 2.1 above. Record the chromatographic effluent spectrum to obtain the peak purity of the target peak. The results are shown in Figure 1 . The peak purity matching value of usnic acid is 999.99, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0164] 2.4.2 Linearity

[0165] Precisely weigh an appropriate amount of usnic acid reference substance, place it in a numbered 50 mL volumetric flask, add methanol to make a solution containing 209.6000 μg of usnic acid per 1 mL, shake well to obtain the usnic acid reference substance stock solution, and store it in the refrigerator for later use. Dilute the usnic acid reference substance stock solution by 1.25, 1.56, 1.95, 2.44, 3.05, 3.81, 4.77, 5.96, 7.45, 14.90, 29.80, and 59.60 times to obtain reference solutions of usnic acid at different concentrations. Perform on-machine detection according to the chromatographic conditions in 2.1 above. With the concentration as the abscissa and the peak area value as the ordinate, investigate the linear range of usnic acid. The results of the linear investigation are shown in Table 9 below and Figure 2 .

[0166] Table 9 Linear Investigation of Usnic Acid

[0167] serial number Usnic acid concentration (μg / mL) Peak area value (mAU) Usnic acid-1 300.2230 6206.745 Usnic acid-2 240.1784 4949.729 Usnic acid-3 192.1427 3950.412 Usnic acid-4 153.7142 3153.866 Usnic acid-5 122.9713 2500.205 Usnic acid-6 98.3771 1942.327 Usnic acid-7 49.1885 969.976 Usnic acid-8 24.5943 485.078 Usnic acid-9 12.2971 242.562 Usnic Acid-10 6.1486 121.223 Usnic acid-11 3.0743 60.795 Usnic Acid-12 1.5371 30.385

[0168] From the experimental results in Table 9 and Figure 2 , it can be known that there is a good linear relationship between the concentration of usnic acid and the peak area value in the range of 1.5371 - 300.2230 μg / mL for usnic acid. The correlation coefficient R 2 = 0.9998, and the linear regression equation is: y = 20.673x - 23.856.

[0169] 2.4.3 Precision Test

[0170] 2.4.3.1 Instrument Precision Test

[0171] 1 μL of the test solution and usnic acid reference solution prepared according to 2.3.6 of the Usnea Standard Decoction (Batch No.: N24052313) were accurately drawn and injected into the ultra-high performance liquid chromatograph. The chromatographic conditions in 2.1 were followed and the chromatographic elution profile was recorded. The RSD (%) value of the target peak was calculated using the external standard one-point method in terms of usnic acid. The specific results are shown in the table below.

[0172] Table 10 Precision test results of the content determination method of Usnea standard decoction

[0173]

[0174]

[0175] From the experimental results in Table 10, it can be seen that the RSD (%) value of the target peak usnic acid is 0.21%<2.0%, indicating that the method has good precision.

[0176] 2.4.3.2 Repeatability test

[0177] Take about 1.0g of the same batch of Usnea standard decoction (N24052313), accurately weigh, and prepare 6 portions in parallel. Prepare 6 portions of the test solution according to the above 2.3.6 for use. Detect on the machine according to the chromatographic conditions in the above 2.1, and calculate the RSD (%) value of the target peak content by the external standard one-point method based on the usnic acid in the test solution. The specific results are shown in the table below.

[0178] Table 11 Results of repeatability test of content determination method of Usnea standard decoction

[0179]

[0180] From the experimental results in Table 11, it can be seen that the RSD (%) value of the target peak usnic acid content is 1.48%<2.0%, indicating that the repeatability of this method is good.

[0181] 2.4.4 Accuracy test

[0182] Take 0.5 g of known usnea standard decoction (batch number N24052313, usnea acid content 1.81 mg / g), accurately weigh a total of 6 portions, accurately add 25 mL of usnea acid reference solution prepared in methanol (concentration 0.036026 mg / mL); weigh the weight, prepare the test solution according to the test solution preparation method in 2.3.6 above, test on the machine according to the chromatographic conditions in 2.1 above, inject 1 μL respectively, and record the chromatogram. In terms of usnea acid, the content of the target peak is calculated by the external standard one-point method, and the recovery rate and relative standard deviation are calculated according to the following formula. The results are shown in the table below.

[0183]

[0184] In the formula: measured amount refers to the content of the indicator component measured after adding the indicator component to the standard decoction, and the unit is mg; spiked amount refers to the content of the indicator component added to the standard decoction, and the unit is mg; sample content refers to the content of the indicator component in the standard decoction, and the unit is mg.

[0185] Table 12 Usnea acid addition recovery test results of the method for determining the content of Usnea standard decoction

[0186]

[0187] From the experimental results in Table 12, it can be seen that the recovery rate of usnic acid in the standard decoction is within the range of 92%-105%, and the RSD% (1.25%) is less than 2.0%, indicating that the accuracy of the determination method is good.

[0188] 2.4.5 Durability test

[0189] 2.4.5.1 Investigation of different chromatographic columns

[0190] The effects of three chromatographic columns, ACQUITY UPLC HSS T3 (2.1*150mm, 1.8μm), AmethSep C18AQ (2.1*150mm, 1.8μm), and Chromcore AQ C18 (2.1*150mm, 1.8μm), on the peak shape and separation of usnic acid in usnea standard decoction were compared.

[0191] Take the Usnea standard decoction (batch number N24052313) and prepare the test solution according to the preparation method of the test solution in 2.3.6 above, and measure it according to the chromatographic conditions in 2.1 above, and record the chromatographic data in terms of usnic acid. The experimental results are shown in Table 13 and Figure 3 shown.

[0192] Table 13 Effects of different chromatographic columns on the test results of Usnea standard decoction

[0193] Chromatographic columns Indicator ingredients Separation / R Theoretical plates ACQUITY UPLC HSST3 Usnic acid 27.28 140457 AmethSepC18AQ Usnic acid 19.67 236721 ChromcoreAQC18 Usnic acid 19.92 243398

[0194] From Table 13 and Figure 3 The experimental results show that the separation degree and theoretical plate number of ACQUITY UPLC HSS T3 column are better than those of AmethSep C18 AQ and Chromcore AQ C18 columns. Therefore, ACQUITY UPLC HSS T3 was selected as the preferred column for this experiment.

[0195] 2.4.5.2 Investigation of different column temperatures

[0196] The effects of different column temperatures of 25℃, 30℃ and 35℃ on the chromatographic peak shape of usnic acid in standard usnea decoction were compared.

[0197] Take the Usnea standard decoction (batch number N24052313) and prepare the test solution according to the above 2.3.6. Determine it according to the chromatographic conditions in the above 2.1, and record the chromatographic data in terms of Usnea acid. The experimental results are shown in Table 14 and Figure 4 shown.

[0198] Table 14 Test results of different column temperatures on the content determination method of Songluo Biao Tang

[0199] Column temperature Indicator ingredients Separation / R Theoretical plates 25℃ Usnic acid 27.76 141338 30℃ Usnic acid 27.53 149295 35℃ Usnic acid 27.12 157047

[0200] From Table 14 and Figure 4 The experimental results show that considering the tolerance of the chromatographic column and the time required for analysis, the column temperature was selected to be 30°C.

[0201] 2.4.5.3 Investigation of different flow rates

[0202] The effects of different flow rates of 0.20mL / min, 0.25mL / min and 0.30mL / min on the chromatographic peak shape and separation of usnic acid in standard usnea decoction were compared.

[0203] Take the Usnea standard decoction (batch number N24052313) and prepare the test solution according to the above 2.3.6. Determine it according to the chromatographic conditions in the above 2.1, and record the chromatographic data in terms of Usnea acid. The experimental results are shown in Table 15 and Figure 5 shown.

[0204] Table 15 Test results of different flow rates for the determination method of the content of Songluo Biao Tang

[0205] Flow rate Indicator ingredients Separation / R Theoretical plates 0.20mL / min Usnic acid 27.48 114624 0.25mL / min Usnic acid 27.53 149295 0.30mL / min Usnic acid 21.32 184249

[0206] From Table 15 and Figure 5 The experimental results show that when the flow rate is 0.25 mL per minute, the separation of the target components is good and there is no baseline drift. The flow rate of 0.25 mL per minute was selected in this experiment.

[0207] Example 1-3: Establishment of characteristic spectrum analysis of Usnea standard decoction

[0208] 3.1 Preparation of reference solution

[0209] Take an appropriate amount of usnic acid reference substance, weigh it accurately, add methanol to make a solution containing 60 μg of usnic acid per 1 mL, shake well, and the solution is ready.

[0210] 3.2 Determination of chromatographic conditions

[0211] (1) Determination of detection wavelength

[0212] Take the sample solution of Usnea standard decoction for analysis and record the absorption spectrum in the range of 190-400nm. Figure 6 The experimental results showed that at a wavelength of 232 nm, the Usnea standard decoction sample solution could detect more chromatographic peak information and less baseline noise interference, so 232 nm was selected as the detection wavelength.

[0213] (2) Optimization of mobile phase

[0214] ① Investigation A is the organic phase, methanol and acetonitrile, respectively, and B is an aqueous solution containing 0.1% phosphoric acid. The gradient is shown in Table 16 below, and the chromatogram is shown in Figure 7 shown.

[0215] Table 16 Gradient elution program

[0216] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→65 95→35 20～35 65→65 35→35 35～40 65→80 35→20

[0217] like Fig.16 As shown: the elution ability of acetonitrile-0.1% phosphoric acid aqueous solution is stronger than that of methanol-0.1% phosphoric acid aqueous solution, so acetonitrile-0.1% phosphoric acid aqueous solution was selected for subsequent research.

[0218] ②Investigate the situation of adding acid and not adding acid

[0219] like Figure 8 As shown: the peak shape is better when adding acid than when not adding acid. The peak shape is best when the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution system, so acetonitrile-0.1% phosphoric acid aqueous solution is selected for elution.

[0220] (3) Determination of chromatographic conditions

[0221] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.25 mL per minute; the column temperature was 30°C; the detection wavelength was 232 nm. The theoretical plate number calculated based on the usnic acid peak should be no less than 3000.

[0222] Table 17 Gradient elution program

[0223] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→65 95→35 20～35 65 35 35～40 65→80 35→20

[0224] 3.3 Preparation of test solution

[0225] This experiment investigated the effects of different extraction solvents on the characteristic spectra of Usnea standard decoction. Methanol (anhydrous methanol), 75 volume% methanol, 50 volume% methanol, 25 volume% methanol, ethanol (anhydrous ethanol), 75 volume% ethanol, 50 volume% ethanol, 25 volume% ethanol, and water were selected as extraction solvents. The characteristic spectra results of different extraction solvents were compared by temporarily determining the total peak area / sample weight of three chromatographic peaks and the chromatograms.

[0226] Take an appropriate amount of Usnea standard decoction (N24052313), take about 1.0g, weigh accurately, put it in a stoppered conical flask, accurately add methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol, water, 20mL each, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding reagent, shake well, filter, and take the filtrate. According to the chromatographic conditions determined in 3.2 above, inject the sample and record the chromatogram. The results of the investigation of different extraction solvents for the characteristic spectrum of Usnea standard decoction are shown in Fig. 9 .

[0227] Experimental results: When the extraction solvent is methanol, the extraction efficiency is higher, so methanol is selected as the extraction solvent.

[0228] According to the above experimental results, the sample pretreatment method of the characteristic spectrum of the standard decoction of Usnea can be determined as follows:

[0229] Take about 1.0 g of Usnea standard decoction (N24052313), accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of methanol, weigh it, treat it with ultrasound (power 500 W, frequency 40 kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with methanol, shake it well, filter it, and take the filtrate to obtain it.

[0230] 3.4 Determination and identification of common peaks

[0231] The UPLC spectra of different batches of Usnea standard decoction samples were determined, and the results were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the Chinese Pharmacopoeia Committee, and the common peaks were selected. Fig.10 As shown in the figure, there are 5 obvious common peaks in the characteristic spectrum of the standard decoction of usnea. The chromatogram of the test sample should show 5 characteristic peaks, among which peak 5 has the same retention time as the peak of the usnic acid reference substance. The peak corresponding to the peak of the usnic acid reference substance is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time should be within the range of ±10% of the specified value, which is: 0.25 (peak 1), 0.31 (peak 2), 0.42 (peak 3) and 0.47 (peak 4).

[0232] 3.5 Methodological validation of the characteristic profile analysis method

[0233] (1) Overall investigation

[0234] Take the test solution prepared according to 3.3 of Usnea standard decoction (N24052313), inject it into the liquid chromatograph, extend the elution time by one time at the mobile phase ratio at the gradient end point, and analyze the characteristic spectrum.

[0235] The results are as follows Fig.11 As shown, there is no obvious chromatographic peak after doubling the elution time under this chromatographic condition, indicating that this chromatographic condition basically meets the principle of maximum information content.

[0236] (2) Precision inspection

[0237] Take the test solution prepared according to 3.3 of Usnea Standard Decoction (N24052313), repeat the injection 6 times, and the injection volume is 1 μL. Temporarily calibrate 5 characteristic peaks, take peak 5 as the reference peak, calculate the relative retention time, and the experimental results are shown in Table 18 below.

[0238] Table 18 Precision results of characteristic spectrum of standard decoction of Usnea (relative retention time)

[0239] sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) 1 0.252 0.306 0.415 0.473 1.000 2 0.252 0.306 0.415 0.473 1.000 3 0.252 0.307 0.415 0.473 1.000 4 0.252 0.307 0.415 0.473 1.000 5 0.252 0.306 0.415 0.473 1.000 6 0.252 0.307 0.415 0.473 1.000 average value 0.25 0.31 0.41 0.47 1.00 RSD% 0.03 0.04 0.03 0.03 0.00

[0240] The experimental results show that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, and the instrument has good precision.

[0241] (3) Stability investigation

[0242] Take the test solution prepared according to 3.3 of Usnea Standard Decoction (N24052313), and inject it at 0, 2, 4, 8, 12, 16, 20, and 24 hours, respectively, with an injection volume of 1 μL. Temporarily calibrate 5 characteristic peaks, take peak 5 as the reference peak, and calculate the relative retention time. The results are shown in Table 19 below.

[0243] Table 19 Characteristic spectrum stability results of Usnea standard decoction (relative retention time)

[0244] sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) 0h 0.249 0.303 0.410 0.467 1.000 2h 0.252 0.306 0.415 0.473 1.000 4h 0.252 0.306 0.415 0.473 1.000 8h 0.253 0.307 0.415 0.472 1.000 12h 0.252 0.307 0.415 0.473 1.000 16h 0.256 0.311 0.416 0.474 1.000 20h 0.252 0.307 0.415 0.473 1.000 24h 0.249 0.303 0.414 0.473 1.000 average value 0.25 0.31 0.41 0.47 1.00 RSD(%) 0.82 0.76 0.46 0.44 0.00

[0245] The experimental results show that the RSD of the relative retention time of the chromatographic peak is in the range of 0.00% to 0.82%, and the RSD is less than 3.0%, indicating that the sample solution is relatively stable.

[0246] (4) Repeatability study

[0247] Take about 0.1g of the same batch of Usnea standard decoction (N24052313), accurately weigh it, and prepare 6 parallel portions into test solution according to the test solution preparation method obtained in 3.3, and inject 1μL of each. Temporarily calibrate 5 characteristic peaks, take peak 5 as the reference peak, and calculate the relative retention time.

[0248] Table 20 Repeatability results of characteristic spectrum of Usnea standard decoction (relative retention time)

[0249] sample Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) 1 0.249 0.303 0.410 0.467 1.000 2 0.253 0.307 0.415 0.473 1.000 3 0.252 0.307 0.415 0.473 1.000 4 0.252 0.307 0.415 0.473 1.000 5 0.252 0.307 0.415 0.473 1.000 6 0.252 0.306 0.414 0.472 1.000 average value 0.25 0.31 0.41 0.47 1.00 RSD% 0.47 0.46 0.50 0.49 0.00

[0250] The experimental results showed that the RSD of the relative retention time of each chromatographic peak was between 0.00% and 0.50%, and the RSD values ​​were all <3.0%, indicating that the method had good repeatability.

[0251] (5) Durability test

[0252] ①Chromatographic column inspection

[0253] Three chromatographic columns were investigated, namely: ACQUITY UPLC HSS T3 C18 (2.1*150mm, 1.8μm), AmethSep C18 AQ (2.1*150mm, 1.8μm), and Chromcore AQ C18 (2.1*150mm, 1.8μm). The effects of the three chromatographic columns on the peak elution of the characteristic spectrum of Usnea standard decoction were investigated.

[0254] Take the Usnea standard decoction (batch number N24052313) and prepare the test solution according to the preparation method of the test solution in 3.3 above. Determine it according to the chromatographic conditions in 3.2 above, and record the chromatographic data in terms of usnic acid. The experimental results are as follows Fig.12 As shown in Table 21.

[0255] Table 21 Chromatographic column investigation results of Songluo Biao decoction characteristic spectrum (relative retention time)

[0256]

[0257] The experimental results show that the chromatographic peak shape is better and the separation effect is best when the chromatographic column ACQUITY UPLC HSS T3 C18 is used for elution. Therefore, it is recommended that this type of chromatographic column be used for subsequent research.

[0258] ② Column temperature investigation

[0259] The elution conditions at different column temperatures (25°C, 30°C and 35°C) were investigated. The test solution was prepared according to the preparation method of the test solution in 3.3 above, and the chromatographic conditions in 3.2 above were used to measure the elution. The chromatographic data were recorded in terms of usnic acid. The experimental results are shown in Figure 2. Fig.13 and as shown in Table 22.

[0260] Table 22 Column temperature investigation results of characteristic spectrum of Songluo Biao Tang (relative retention time)

[0261] Column temperature Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) 25℃ 0.253 0.307 0.411 0.469 1.000 30℃ 0.252 0.307 0.415 0.473 1.000 35℃ 0.252 0.305 0.418 0.477 1.000

[0262] The experimental results show that the column temperature has a certain influence on the peak elution, but the separation effect is best and the peak shape is better when the column temperature is 30℃, so it is recommended to use a column temperature of 30℃ for measurement.

[0263] ③ Flow rate investigation: Investigate the elution conditions at different flow rates (0.20mL / min, 0.25mL / min and 0.30mL / min). Prepare the test solution using the Usnea Standard Decoction (batch number N24052313) according to the preparation method of the test solution in 3.3 above, and measure according to the chromatographic conditions in 3.2 above, with Usnea acid as the calculation method, and record the chromatographic data. The results are as follows: Fig.14 As shown in Table 23.

[0264] Table 23 Flow rate investigation results of characteristic spectrum of Songluo Xiangbiao Decoction (relative retention time)

[0265] Flow rate Peak 1 Peak 2 Peak 3 Peak 4 Peak 5(S) 0.20mL / min 0.259 0.308 0.410 0.460 1.000 0.25mL / min 0.252 0.307 0.415 0.473 1.000 0.30mL / min 0.245 0.303 0.416 0.480 1.000

[0266] The experimental results show that the flow rate has a certain influence on the peak appearance, but the peak shape is best when the flow rate is 0.25mL / min. Therefore, it is recommended to use 0.25mL / min as the measurement flow rate.

[0267] Example 2: Preparation method of usnea formula granules, determination and analysis method of usnea acid content, and establishment of characteristic spectrum analysis method

[0268] Example 2-1 Preparation method of Usnea formula granules

[0269] The preparation method of Usnea slices includes the following steps: taking the original Usnea medicinal material, cleaning it, selecting it, removing non-medicinal parts, impurities and deteriorated products, etc., the impurity content shall not exceed 3%, cutting it into sections and sieving it to remove ash. Among them, the origin of the Usnea medicinal material with the raw material batch number of P-SL-240112-QGY-YC02 is Liuchang Town, Qingzhen City, Guiyang City, Guizhou Province; the origin of the Usnea medicinal material with the raw material batch number of P-SL-240112-QGY-YC06 is Wantanhe Town, Longli County, Guiyang City, Guizhou Province; the origin of the Usnea medicinal material with the raw material batch number of P-SL-240112-QGY-YC07 is Shuangliu Town, Kaiyang County, Guiyang City, Guizhou Province.

[0270] Step 1: Extraction and concentration: Take the Usnea slices and boil them twice with water. Soak for 30 minutes before boiling. Add 12 times the water for the first time, heat to boiling, keep boiling for 60 minutes, filter (200 mesh), and set aside the filtrate. Add 10 times the water for the second time, heat to boiling, keep boiling for 40 minutes, filter (200 mesh), and combine the two filtrates.

[0271] The filtrate is concentrated by vacuum decompression, with a concentration temperature range of 60 to 70°C and a vacuum degree of -0.04 to -0.08 MPa. The filtrate is concentrated into a clear paste with a relative density of 1.00 to 1.03 (45±5°C).

[0272] Step 2: Drying and powdering: Add 12% to 18% of maltodextrin based on the total weight of the slices to the clear paste, mix well, pass through a No. 6 sieve (100 mesh), spray dry, with an inlet air temperature of 165 to 195°C and an outlet air temperature of 85 to 105°C, and collect powder. The dry paste powder yield is 12.28%.

[0273] Step 3: Ingredients for granulation and total mixing: Add silicon dioxide in an amount of ≤0.3% by weight of the intermediate (dry paste powder containing auxiliary materials), sieve and mix well, and granulate to obtain.

[0274] Table 24 Summary of research data results

[0275]

[0276]

[0277] Investigation on the preparation technology of Usnea formula granules

[0278] 4.1 Selection of extraction process parameters

[0279] 4.1.1 Investigation of water addition multiples

[0280] Because Usnea is a whole-herb medicinal material, referring to the standard soup preparation process, combined with the differences in extraction performance and appearance between the standard soup decoction equipment and the pilot production equipment, the equipment matching and the actual situation of large-scale production, the water addition multiple and extraction time parameters were examined. The results are shown in Tables 25 and 26.

[0281] Table 25 Investigation results of different water addition multiples of Usnea

[0282]

[0283] From the experimental results in Table 25, it can be seen that the water addition multiples are determined to be 12 times and 10 times based on a comprehensive judgment based on the paste yield results and the content of index components.

[0284] 4.1.2 Investigation of extraction time

[0285] Table 26 Results of investigation on different extraction times of Usnea

[0286]

[0287]

[0288] In summary, the final extraction process parameters were selected as follows: extraction and decoction times were two, the first water addition amount was 12 times the amount of the decoction piece, the soaking time was 30 minutes, and the first decoction time was 60 minutes. The second water addition amount was 10 times the amount of the decoction piece, and the decoction time was 40 minutes.

[0289] 4.2 Selection of solid-liquid separation method

[0290] In combination with the actual situation of large-scale production, the extraction liquid was used to observe and compare the difficulty of filtration at different mesh sizes. The results are shown in Table 27.

[0291] Table 27 Observation results of the difficulty of filtering with different filter mesh sizes of Usnea

[0292]

[0293] From the experimental results in Table 27, it can be seen that after the extract was filtered through 100, 200 and 300 meshes, the filtrate was relatively clear except for 100 mesh, without obvious insoluble impurities, and a good solid-liquid separation effect was achieved. However, 300 mesh filtration was slower. Referring to the separation method of the standard soup and combining it with the actual situation of large-scale production, the confirmed solid-liquid separation method was selected as: hot filtration method, with a filter screen mesh of 200 mesh.

[0294] 4.3 Drying process research

[0295] Take 4 portions of extracted and concentrated clear paste, 4 kg each (each portion is equivalent to 1.5 kg of medicinal material), select reasonable spray drying process parameters according to the design scheme in the table below, and conduct an experiment on the amount of spray drying auxiliary materials added. The test results are shown in Table 28.

[0296] Table 28 Results of the investigation on the dosage of auxiliary materials for spray drying of Usnea concentrated clear paste

[0297]

[0298]

[0299] Note: The amount of dry paste powder refers to the amount of dry paste powder containing auxiliary materials, and the dry paste powder yield refers to the dry paste powder yield without auxiliary materials.

[0300] From the experimental results in Table 28, it can be seen that without adding auxiliary materials, there is a wall-sticking phenomenon and it is not easy to dry. After adding 12% to 18% of the auxiliary material maltodextrin in the amount of the decoction pieces, it is easy to dry, the moisture content is low, and the drying effect is good, indicating that adding a certain amount of maltodextrin to the concentrated usnea paste can significantly improve the spray drying effect and make it easier to dry. From the observation results of different proportions of auxiliary material addition, it is more reasonable to add maltodextrin in the drying process according to 12% to 18% of the amount of the decoction pieces.

[0301] 4.4 Molding process research

[0302] In view of the fact that a certain proportion of maltodextrin has been added to the dry paste powder of the Usnea formula granules, and considering the influence of the fluidity of the Usnea intermediate (dry paste powder) on the efficiency of dry granulation and the influence of the prepared formula granules on the difference in the loading amount of the packaging, the commonly used flow aids in the process of Chinese medicine preparation are mainly silicon dioxide. Therefore, this experiment took a pilot extraction study to prepare the Usnea intermediate, and investigated the amount of excipients and granulation process. Each time, 1.0 kg of Usnea intermediate dry paste powder was taken, and the excipients were added according to the experimental design ratio and fully mixed. After that, the GZL100-30L dry granulator was placed to study the effects of process parameters such as excipient amount, roller speed and pressure on the granulation effect. The mesh size of the sieve was selected to be 12-40 for granulation. The excipient amount and granulation molding process were evaluated based on indicators such as particle size, fluidity, one-time finished product rate, bulk density, solubility, and fine powder rate. The test results of Usnea granulation process parameters are shown in Table 29, and the screening of excipient amount is shown in Table 30.

[0303] Table 29 Granulation and molding process inspection production equipment parameters

[0304] Stirring speed (r / min) 30.0 Feeding speed (r / min) 80.0 Roller speed (r / min) 7.0 Crushing speed (r / min) 80.0 Final granulation speed (r / min) 80.0 Press wheel spacing (mm) 0.75 Hydraulic pressure (MPa) 6.5 Screen mesh number for whole grains (mesh) 18 Upper sieve mesh number (mesh) 10 The number of meshes of the lower screen (mesh) 40 Mixing time (min) 30 Mixer speed (r / min) 10

[0305] Table 30 Usnea granulation process parameter investigation table

[0306]

[0307]

[0308] From the experimental results in Tables 29 and 30, it can be seen that the amount of auxiliary materials and the granulation molding process were evaluated. The results showed that the amount of auxiliary materials to be added in dry granulation should be ≤ 0.3%.

[0309] Example 2-2: Establishment of analytical method for content determination of Usnea granules

[0310] 5.1 Determination of chromatographic conditions

[0311] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile was used as the mobile phase A, 0.1% phosphoric acid aqueous solution was used as the mobile phase B, and gradient elution was performed according to the provisions in Table 31 below; the flow rate was 0.25 mL per minute; the column temperature was 30°C; the detection wavelength was 232 nm. The theoretical plate number calculated based on the usnic acid peak should be no less than 3000.

[0312] Table 31 Gradient elution program

[0313] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→65 95→35 20～35 65 35 35～40 65→80 35→20

[0314] 5.2 Preparation of reference solution

[0315] Take an appropriate amount of usnic acid reference substance, weigh it accurately, add 75 volume % methanol to make a solution containing 60 μg usnic acid per 1 mL, and shake well.

[0316] 5.3 Preparation of test solution

[0317] 5.3.1 Investigation of different extraction solvents

[0318] Take 0.1 g of Usnea formula granules (batch number: 240702), a total of 9 portions, each with 2 parallel samples, and place them in stoppered conical flasks respectively. Accurately add 20 mL of methanol (anhydrous methanol), 75 volume% methanol, 50 volume% methanol, 25 volume% methanol, ethanol (anhydrous ethanol), 75 volume% ethanol, 50 volume% ethanol, 25 volume% ethanol and water in sequence, weigh the weight, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, collect the filtrate, and obtain each test sample solution.

[0319] According to the chromatographic conditions in 5.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0320] Table 32 Effect of different extraction solvents on the content determination results of Usnea formula granules

[0321]

[0322] From the experimental results in Table 32, it can be seen that: considering the content differences comprehensively, 75% methanol was selected as the best extraction solvent for subsequent research.

[0323] 5.3.2 Investigation of extraction methods

[0324] Take an appropriate amount of Usnea formula granules (batch number: 240702), grind and mix well, take about 0.1g, a total of 2 portions, each with 2 parallel samples, place them in stoppered cones, accurately add 20mL of 75% methanol in turn, weigh the weight, ultrasonically and reflux for 30 minutes respectively, cool, weigh again, make up the lost weight with water, shake well, filter, and take the filtrate to obtain the test sample solution.

[0325] According to the chromatographic conditions in 5.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0326] Table 33 Effect of different extraction methods on the content determination results of Usnea formula granules

[0327]

[0328] From the experimental results in Table 33, it can be seen that: taking into account the differences in their contents, ultrasonic treatment (power 500 W, frequency 40 kHz) extraction was used as the extraction method for subsequent research.

[0329] 5.3.3 Investigation of extraction solvent volume

[0330] Take an appropriate amount of Usnea formula granules (batch number: 240702), grind and mix well, take about 0.1g, a total of 4 portions, each with 2 parallel samples, place them in stoppered cones, accurately add 10mL, 15mL, 20mL and 25mL of 75 volume% methanol in turn, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes, cool, weigh again, make up the lost weight with water, take an appropriate amount of filtrate and pass it through a 0.22μm microporous filter membrane, place it in a high liquid sample bottle, and obtain the test sample solution for use.

[0331] According to the chromatographic conditions in 5.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0332] Table 34 Effect of different extraction volumes on the content determination results of Usnea formula granules

[0333]

[0334] From the experimental results in Table 34, it can be seen that considering the solvent cost and its content difference, the extraction volume of 20 mL was selected for subsequent research.

[0335] 5.3.4 Investigation of extraction time

[0336] Take an appropriate amount of Usnea formula granules (batch number: 240702), grind and mix well, take about 0.1g, a total of 4 portions, each with 2 parallel samples, place them in stoppered cones, accurately add 20mL of 75% methanol in turn, weigh the weight, ultrasonically treat (power 500W, frequency 40kHz) for 15, 30, 45 and 60 minutes respectively, cool, weigh again, make up the lost weight with 75% methanol, take an appropriate amount of filtrate and pass it through a 0.22μm microporous filter membrane, place it in a high liquid sample bottle, and obtain the test sample solution for use.

[0337] According to the chromatographic conditions in 5.1 above, 1 μL of the reference solution and the test solution were accurately drawn, injected into the ultra-high performance liquid chromatograph, and determined. The experimental results were calculated using the external standard one-point method based on the usnic acid content. The specific results are shown in the table below.

[0338] Table 35 Effect of different extraction time on the content determination results of Usnea formula granules

[0339]

[0340] From the experimental results in Table 35, it can be seen that: considering the time cost and the difference in content, (power 500W, frequency 40kHz) 60 minutes was selected for subsequent research.

[0341] 5.3.5 Determination of the preparation method of the test solution

[0342] According to the results of the sample pretreatment experiment and referring to the content determination method of Usnea formula granules, the preparation method of the test sample can be determined as follows: take an appropriate amount of this product, grind it finely and mix it evenly, take about 0.1g, accurately weigh it, put it in a stoppered conical flask, accurately add 20mL of 75% methanol, weigh it, ultrasonically treat it (power 500W, frequency 40kHz) for 60 minutes, cool it, weigh it again, make up the lost weight with 75% methanol, shake it evenly, filter it, and take the filtrate to obtain it.

[0343] 5.4 Methodological Validation

[0344] 5.4.1 Peak purity

[0345] 1 μL of the test solution and reference solution prepared according to 5.3.5 of Usnea Formula Granules (Batch No.: 240702) were accurately taken and injected into the ultra-high performance liquid chromatograph. The peak purity of the target peak was obtained according to the chromatographic conditions in 5.1 above. The results are shown in Fig.15 The peak purity matching value of usnic acid is 1000.00, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0346] 5.4.2 Investigation of linear relationship

[0347] Accurately weigh an appropriate amount of usnic acid reference substance, place it in a numbered 50mL volumetric flask, add methanol to make a solution containing 300.2230μg of usnic acid per 1mL, shake well, and obtain the usnic acid reference substance stock solution, which is kept in the refrigerator for later use. Dilute the usnic acid reference substance stock solution 1.25, 1.56, 1.95, 2.44, 3.05, 6.10, 12.21, 24.41, 48.83, 97.66, and 195.32 times to obtain reference substance solutions of different concentrations of usnic acid, and perform on-machine testing according to the chromatographic conditions in 5.1 above, using concentration as the horizontal coordinate and peak area value as the vertical coordinate to examine the linear range of usnic acid. The results of the linear investigation are shown in Tables 36 and 36 below. Fig.16 .

[0348] Table 36 Linearity study of usnic acid

[0349]

[0350] From Table 36 and Fig.16 The experimental results show that the usnic acid concentration and peak area value have a good linear relationship in the concentration range of 1.5371-300.2230 μg / mL, and the correlation coefficient R 2 =0.9998, the linear regression equation is: y=20.673x-23.856.

[0351] 5.4.3 Precision investigation

[0352] 1 μL of the test solution and usnic acid reference solution prepared in accordance with 5.3.5 of Usnea Formula Granules (Batch No.: 240702) were accurately drawn into the ultra-high performance liquid chromatograph and determined according to the chromatographic conditions in 5.1 above. The RSD (%) value of the target peak was calculated using the external standard one-point method in terms of usnic acid. The specific results are shown in the table below.

[0353] Table 37 Usnea formula granule content determination method precision test results

[0354]

[0355]

[0356] From the experimental results in Table 37, it can be seen that the RSD (%) value of the target peak usnic acid is 0.37% < 2.0%, indicating that the method has good precision.

[0357] 5.4.4 Repeatability study

[0358] Take about 0.1g of the same batch of Usnea Formula Granules (Batch No.: 240702), accurately weigh, and prepare 6 portions in parallel. Prepare 6 portions of the test solution according to the above 5.3.5 test solution preparation method for use. Detect on the machine according to the chromatographic conditions in 5.1 above, and calculate the RSD (%) value of the target peak content based on the Usnea acid in the test solution using the external standard one-point method. The specific results are shown in the table below.

[0359] Table 38 Repeatability test results of the content determination method of Usnea formula granules

[0360]

[0361] From the experimental results in Table 38, it can be seen that the RSD (%) value of the target peak usnic acid content is 0.97%<2.0%, indicating that the repeatability of this method is good.

[0362] 5.4.5 Accuracy test

[0363] Take 0.05 g of usnea formula granules with known content (batch number: 240702, usnea acid content 5.94 mg / g), accurately weigh a total of 6 portions, and accurately add 20 mL of usnea acid reference solution prepared with 75% methanol (concentration 0.015011 mg / mL) to each of them; weigh the weight, prepare the test solution according to the test solution preparation method in 5.3.5, and detect on the machine according to the chromatographic conditions in 5.1 above, inject 1 μL of each solution, calculate the content of the target peak in terms of usnea acid, and calculate the recovery rate and RSD%. The results are shown in Table 39 below.

[0364] Table 39 Content determination method usnic acid sample recovery test results

[0365]

[0366]

[0367] From the experimental results in Table 39, it can be seen that the recovery rate of usnic acid in the usnea formula granules is within the range of 92%-105%, and the RSD% is less than 2.0%, indicating that the accuracy of the determination method is good.

[0368] 5.4.6 Durability test

[0369] 5.4.6.1 Investigation of different chromatographic columns

[0370] The chromatographic columns AmethSep C18 AQ (2.1*150mm, 1.8μm), Chromcore AQ C18 (2.1*150mm, 1.8μm), and ACQUITY T3 (2.1*150mm, 1.8μm); Effects of three chromatographic columns on the peak shape and separation of usnic acid in standard usnea decoction.

[0371] Take Usnea Formula Granules (Batch No.: 240702) and prepare the test solution according to 5.3.5. Determine according to the chromatographic conditions in 5.1 above, and record the chromatographic data in terms of Usnea acid. The experimental results are as follows Fig.17 As shown in Table 40.

[0372] Table 40 Effects of different chromatographic columns on the test results

[0373]

[0374] Depend on Fig.17 The experimental results in Table 40 show that the separation effects of the three chromatographic columns are quite different. T3 (2.1*150mm, 1.8μm) can meet the requirements of usnea usnic acid content determination. Its resolution, symmetry factor and theoretical plate number are better than those of AmethSep C18 AQ (2.1*150mm, 1.8μm) and Chromcore AQ C18 (2.1*150mm, 1.8μm) columns. Therefore, ACQUITY T3 (2.1*150mm, 1.8μm) was selected as the preferred chromatographic column for this experiment.

[0375] 5.4.6.2 Investigation of different column temperatures

[0376] The effects of different column temperatures of 25℃, 30℃ and 35℃ on the chromatographic peak shape of usnic acid in usnea formula granules were compared.

[0377] Take Usnea Formula Granules (Batch No.: 240702) and prepare the test solution according to 5.3.5. Determine according to the chromatographic conditions in 5.1 above, and record the chromatographic data in terms of Usnea acid. Fig.18 As shown in Table 41.

[0378] Table 41 Test results of different column temperatures for the determination of the content of Usnea formula granules

[0379]

[0380] Depend on Fig.18 The experimental results in Table 41 show that the chromatographic peak shape and separation effect are good under the three column temperatures. The baseline of the chromatographic elution graph at 30°C has no drift, and there is no significant difference in retention time compared with the other two temperatures. Considering the tolerance of the chromatographic column and the time required for analysis, the column temperature is selected to be 30°C.

[0381] 5.4.6.3 Investigation of different flow rates

[0382] The effects of different flow rates of 0.20 mL / min, 0.25 mL / min and 0.30 mL / min on the chromatographic peak shape and separation of usnic acid in usnea formula granules were compared.

[0383] Take Usnea Formula Granules (Batch No.: 240702) and prepare the test solution according to 5.3.5. Determine according to the chromatographic conditions in 5.1 above, and record the chromatographic data in terms of Usnea acid. Fig.19 As shown in Table 42.

[0384] Table 42 Test results of different flow rates for the determination of the content of Usnea formula particles

[0385]

[0386] Depend on Fig.19 The experimental results in Table 42 show that the chromatographic peak shape and separation effect are good at the three flow rates. When the flow rate is 0.25 mL per minute, the separation of the components is good and the baseline does not drift. The flow rate of 0.25 mL per minute was selected in this experiment.

[0387] 5.5 Content determination of 3 batches of granule samples

[0388] Take appropriate amount of 3 batches of samples of Usnea Formula Granules, grind and mix, take about 0.1g, and prepare two parallel portions of each batch. According to the above 5.3.5 test solution preparation method, prepare 3 batches of Usnea Formula Granules sample test solution. According to the above 5.1 chromatographic conditions, accurately pipette 1μL of reference solution and test solution respectively, inject into ultra-high performance liquid chromatography, and measure. In terms of Usnea acid, the surface one-point method is used for calculation. The experimental results are shown in Table 43 below.

[0389] Table 43 Content determination results of 3 batches of Usnea granules

[0390]

[0391] Example 2-3: Establishment of the determination and analysis method of the characteristic spectrum of Usnea granules

[0392] Step 1: Preparation of reference solution

[0393] Take an appropriate amount of usnic acid reference substance, weigh it accurately, and add 75% methanol to make it contain 60 μg usnic acid per 1 mL.

[0394] Step 2: Preparation of test solution

[0395] Take an appropriate amount of this product, grind it into powder, take about 0.1g, weigh it accurately, add 20mL of 75% methanol, weigh it, treat it with ultrasound (power 500W, frequency 40kHz) for 60 minutes, let it cool, weigh it again, make up the lost weight with 75% methanol, shake it well, filter it, and take the filtrate to obtain it.

[0396] Step 3: Determination of chromatographic conditions

[0397] Octadecylsilane bonded silica gel was used as the filler (column length: 150 mm, inner diameter: 2.1 mm, particle size: 1.8 μm); acetonitrile was used as mobile phase A, 0.1% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the provisions in the following table; the flow rate was 0.25 mL per minute; the column temperature was 30°C; the detection wavelength was 232 nm. The theoretical plate number calculated based on the usnic acid peak should be no less than 3000.

[0398] Table 44 Gradient elution program

[0399] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0～20 5→65 95→35 20～35 65 35 35～40 65→80 35→20

[0400] Establishment of feature maps

[0401] Take 3 batches of Usnea formula granules, 0.1g each, to prepare the test solution, and then test it on the machine according to the chromatographic conditions in step 3. The test results are shown in the following table and Fig. 20 .

[0402] Table 45 Sample determination results (relative retention time)

[0403]

[0404]

[0405] The experimental results show that the retention time ratios of the characteristic peaks in the three batches of samples are relatively stable, and the RSDs are all less than 3.0%.

[0406] In summary, the UPLC spectra of different batches of Usnea formula granules were measured, and the results were analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" recommended by the National Pharmacopoeia Committee. The common peak was selected, and then the peak No. 5 with a relatively stable retention time was used as the reference peak (i.e., S peak), and the common peak with a relatively stable retention time was selected as the characteristic peak.

[0407] The characteristic spectrum determination method was established by ultra-high performance liquid chromatography and a methodological investigation was conducted. The characteristic spectrum of 5 batches of Usnea Formula Granules was determined, and the characteristic spectrum standard of Usnea Formula Granules was finally determined: 5 characteristic peaks should be present in the characteristic spectrum of the test sample, among which Peak 5 corresponds to the retention time of the characteristic peak of the Usnea Acid Reference Standard. The peak corresponding to the Usnea Acid Reference Standard is the S peak. The relative retention time of other peaks to the S peak is calculated, and the relative retention time should be within ±10% of the specified value, 0.25 (peak 1), 0.31 (peak 2), 0.42 (peak 3) and 0.47 (peak 4).

[0408] Example 2-4: Thin layer chromatography identification of Usnea formula granules

[0409] 6.1 Instruments, reagents and test drugs

[0410] Instruments: TLC automatic imager (TLC VISUAlIZER2, CAMAG, Switzerland), double-slot development cylinder, fully automatic TLC spotter (AUTOMATIC TLC SAMPlER4, CAMAG, Switzerland), AL-104 electronic balance (Mettler-Toledo Instrument (Shanghai) Co., Ltd.), KQ-500DA ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.), silica gel GF 254 Thin layer plate (10 cm × 10 cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20231106), silica gel GF 254 Thin layer board (10cm×10cm, Yantai Huayang New Material Technology Co., Ltd., batch number: 20231204), silica gel GF 254 Thin layer plate (10 cm×10 cm, Merck KGaA).

[0411] Reagents: Chloroform (batch number: 20240601), methanol (batch number: 20230301), toluene (batch number: 20240701), and glacial acetic acid (batch number: 20240101) were all purchased from Chongqing Chuandong Chemical (Group) Co., Ltd.

[0412] Test drugs: 3 batches of Usnea formula granules (batch numbers: 240701, 240702, 240703; source: Sinopharm Group Tongjitang (Guizhou) Pharmaceutical Co., Ltd.; Usnea control medicinal materials (batch number: 2024-073001, Chengdu Glip Biotechnology Co., Ltd.).

[0413] 6.2 Solution preparation

[0414] 6.2.1 Preparation of test solution

[0415] Take 0.25 g of this product, grind it into powder, add 10 mL of chloroform, treat it with ultrasound for 10 minutes, filter it, evaporate the filtrate to dryness, add 2 mL of chloroform to the residue to dissolve it, and use it as the test solution.

[0416] 6.2.2 Preparation of reference solution

[0417] Take 0.5g of Usnea control medicinal material, add 10mL of water, heat and reflux for 60 minutes, filter, evaporate the filtrate to dryness, add 5mL of methanol to the residue, ultrasonically treat for 10 minutes, filter, and use it as a control medicinal material solution. Take 5.0g of Usnea control medicinal material, add 50mL of water, heat and reflux for 30 minutes, filter, evaporate the filtrate to dryness, add 10mL of chloroform to the residue, ultrasonically treat for 10 minutes, filter, evaporate the filtrate to dryness, add 2mL of chloroform to the residue to dissolve it, and use it as a control medicinal material solution.

[0418] 6.2.3 Preparation of negative sample solution

[0419] Take 0.25 g of the negative sample of Usnea formula granules, grind it into powder, and treat it in the same way as the test sample to serve as the negative sample solution.

[0420] 6.3 Thin layer chromatography conditions

[0421] Thin layer board: Silicone GF 254 Thin layer plate. Developing agent: the volume ratio of chloroform, toluene, methanol and glacial acetic acid is 8:12:1:0.2. Spotting method: spray strip spotting. Development method: develop with double-slot development cylinder. Inspection: inspect under ultraviolet light (254nm).

[0422] 6.4 Sampling quantity inspection

[0423] Pipette the Usnea formula granule test solution and Usnea control medicinal material solution onto the same silica gel GF2 54 The thin layer plate was developed with chloroform-toluene-methanol-acetic acid (volume ratio of 8:12:1:0.2) as the developing agent, taken out, dried, and examined under ultraviolet light (254nm). The results are shown in Fig.21 In the figure, 1 is the sample volume of Usnea Formula Granules 1μL; 2 is the sample volume of Usnea Formula Granules 2μL; 3 is the sample volume of Usnea Formula Granules 3μL; 4 is the sample volume of Usnea Control Medicinal Material 6μL; 5 is the sample volume of Usnea Control Medicinal Material 8μL; 6 is the sample volume of Usnea Control Medicinal Material 10μL. Fig.21 It can be seen that when the spotting volume of the test sample solution is 2-3 μL, the spotting volume of the control medicinal material solution is 8-10 μL, the key spots at the corresponding positions of the test sample and the control medicinal material are clearer and have better point properties. Therefore, the spotting volume is selected as 2 μL for the test sample solution and 8 μL for the control medicinal material solution.

[0424] 6.5 Investigation on the specificity of Usnea formula granules

[0425] 2 μL of Usnea formula granule test solution, 8 μL of Usnea control medicinal material solution and 2 μL of negative sample solution were respectively applied to the same silica gel GF 254On the thin layer plate, chloroform-toluene-methanol-glacial acetic acid (volume ratio of 8:12:1:0.2) was used as the developing agent, developed, taken out, dried, and examined under ultraviolet light (254nm). The experimental results are shown below Fig. 22 In the figure, the test conditions are: temperature: 25℃, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; 1 is the negative sample 2 μL; 2-4 are Usnea formula granules (240701-240703) 2 μL; 5 is Usnea control medicinal material 8 μL. Fig. 22 It can be seen that the chromatogram of the Usnea formula granules showed spots of the same color at the corresponding positions of the chromatogram of the control medicinal material, and there was no interference from the negative sample, which indicated that the TLC method had good specificity.

[0426] 6.6 Investigation at different temperatures

[0427] 2 μL of Usnea formula granule test solution, 8 μL of Usnea control medicinal material solution and 2 μL of negative sample solution were respectively applied to the same silica gel GF 254 On the thin layer plate, chloroform-toluene-methanol-glacial acetic acid (volume ratio of 8:12:1:0.2) was used as the developing agent, and it was developed at different temperatures (5°C, 25°C and 40°C), taken out, dried, and examined under ultraviolet light (254nm). The experimental results are shown below Fig.23 In the figure, the test conditions of (a) are: temperature: 40°C, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; (b) test conditions: temperature: 25°C, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; (c) test conditions: temperature: 5°C, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; 1 is negative sample 2 μL; 2-4 are Usnea formula granules (240701-240703) 2 μL; 5 is Usnea control medicinal material 8 μL. Fig.23 It can be seen that under different temperature conditions, the main spots of the same color appear at the corresponding positions of the chromatograms of the test samples and the control medicinal materials of the Usnea formula granules, with clear color development, good separation, no tailing phenomenon, and no background interference. Only with the increase of temperature, the positions of the corresponding spots do not change significantly, indicating that temperature has no obvious effect on the thin-layer identification of the Usnea formula granules, indicating that the thin-layer identification method has good durability to temperature.

[0428] 6.7 Investigation of different humidity

[0429] 2 μL of Usnea formula granule test solution, 8 μL of Usnea control medicinal material solution and 2 μL of negative sample solution were respectively applied to the same silica gel GF 254On the thin layer plate, chloroform-toluene-methanol-glacial acetic acid (volume ratio of 8:12:1:0.2) was used as the developing agent, and it was developed at different relative humidity (33%, 66% and 88%), taken out, dried, and examined under ultraviolet light (254nm). The experimental results are shown below Fig.24 In the figure, the test conditions of (a) are: temperature: 25°C, RH: 88%; Qingdao Ocean Silica Gel GF 254 Plate; (b) test conditions: temperature: 25°C, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; (c) detection conditions: temperature: 25°C, RH: 33%; Qingdao Marine Silica G Plate; 1 is negative sample 2 μL; 2-4 are Usnea Formula Granules (240701-240703) 2 μL; 5 is Usnea control medicinal material 8 μL. Fig.24 It can be seen that under different humidity conditions, spots of the same color appear at corresponding positions of the chromatogram of the test sample of the Usnea formula granules and the chromatogram of the control medicinal materials, and the main spots are clearly colored, with good separation and no tailing phenomenon, indicating that changes in humidity have no obvious effect on the thin-layer identification of the Usnea formula granules, indicating that the thin-layer identification method has good durability to humidity.

[0430] 6.8 Inspection of thin-layer boards from different manufacturers

[0431] 2 μL of Usnea formula granule test solution, 8 μL of Usnea control medicinal material solution and 2 μL of negative sample solution were respectively applied to silica gel GF 254 On a thin layer plate (Qingdao Ocean, Yantai Yinlong, Merck), chloroform-toluene-methanol-glacial acetic acid (volume ratio of 8:12:1:0.2) was used as the developing agent, developed, taken out, dried, and examined under ultraviolet light (254nm). The experimental results are shown below Fig.25 In the figure, the test conditions of (a) are: temperature: 25°C, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; (b) test conditions: temperature: 25 ° C, RH: 66%; Yantai Yinlong silica gel prefabricated silica gel GF 254 Plate; (c) test conditions: temperature: 25 ° C, RH: 66%; Merck silica gel GF 254 Plate; 1 is negative sample 2 μL; 2-4 are Usnea formula granules (240701-240703) 2 μL; 5 is Usnea control medicinal material 8 μL. Fig.25 It can be seen that by using silica gel thin layer plates from different manufacturers (Qingdao Ocean Silica G plate, Yantai Yinlong Silica G prefabricated silica gel G plate, German Merck plate), the main spots of the chromatogram of the test sample and the chromatogram of the reference medicinal material of the Usnea formula granules can correspond to each other without significant influence.

[0432] 6.9 Thin layer identification chromatogram of Usnea formula granules

[0433] 2 μL of Usnea formula granule test solution, 8 μL of Usnea control medicinal material solution and 2 μL of negative sample solution were respectively applied to the same silica gel GF 254 On the thin layer plate, chloroform-toluene-methanol-glacial acetic acid (volume ratio of 8:12:1:0.2) was used as the developing agent, developed, taken out, dried, and examined under ultraviolet light (254nm). The experimental results are shown below Fig.26 In the figure, the test conditions are: temperature: 25℃, RH: 66%; Qingdao Ocean Silica Gel GF 254 Plate; 1 is negative sample 2 μL; 2-4 are Usnea formula granules (240701-240703) 2 μL; 5 is Usnea control medicinal material 8 μL. Fig.26 It can be seen that the chromatograms of the test samples in the three batches of Usnea formula granules show fluorescent spots of the same color at the corresponding positions of the chromatograms of the control medicinal materials, indicating that the thin layer identifications of the three batches of Usnea formula granules all meet the requirements.

[0434] Summary: From the chromatogram spots, the separation effect of the fluorescent spots of Usnea Formula Granules is good, and the chromatograms of Usnea Formula Granules and Usnea Control Herbal Material show the same color of fluorescent main spots at the corresponding positions, and they can correspond one to one. This method can effectively qualitatively identify Usnea Formula Granules that have lost their medicinal properties. Through the thin layer chromatography methodology, the method has good specificity and durability, and is suitable for the chromatographic identification of Usnea Formula Granules.

[0435] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A method for preparing a usnea extract, characterized in that: The steps include: Take Usnea slices, add water and boil, separate the solid and liquid, and obtain the decoction, which is the Usnea extract.

2. The method for preparing the usnea extract according to claim 1, wherein: The number of times of adding water for decoction is 1 to 3 times; preferably, the number of times of adding water for decoction is 2 times; wherein, 10 to 15 times the amount of water is added for the first decoction; and 8 to 14 times the amount of water is added for the second decoction; and / or, preferably, the first decoction time is 30 to 120 minutes; and the second decoction time is 20 to 80 minutes; And / or, the solid-liquid separation is filtration; preferably, filtration is performed using a 100-300 mesh sieve.

3. The method for preparing the usnea extract according to claim 1 or 2, wherein: The obtained decoction is concentrated; Preferably, the concentration temperature is 55 to 70° C.; and / or, preferably, the concentration vacuum is -0.08 to -0.09 MPa; and / or, preferably, the concentration of the concentrated solution is 1 to 1.1 g / mL.

4. A method for preparing a usnea extract preparation, characterized in that: Adding the usnea extract according to any one of claims 1 to 3 to the first auxiliary material and drying the mixture; Preferably, the drying is vacuum drying, spray drying, microwave drying or infrared drying; More preferably, the first auxiliary material comprises maltodextrin; More preferably, the added amount of the first auxiliary material is 12% to 18% of the amount of Usnea decoction pieces added.

5. The method for preparing the usnea extract preparation according to claim 4, wherein: adding the second auxiliary material to the dried material; Preferably, the second auxiliary material comprises silicon dioxide; More preferably, the percentage of silicon dioxide in the weight of the dried material is greater than 0 and less than or equal to 0.3%.

6. A usnea extract or a usnea extract preparation, characterized in that: The usnea extract is prepared by the method for preparing the usnea extract according to any one of claims 1 to 3; or, the usnea extract preparation is prepared by the method for preparing the usnea extract preparation according to claim 4 or 5; Preferably, the usnea extract preparation is usnea formula granules; More preferably, the preparation amount of the Usnea formula granules is 27% to 31%, wherein the preparation amount refers to the ratio of the mass of the prepared formula granules to the mass of the medicinal materials; further preferably, the preparation amount of the Usnea formula granules is 28% to 31%.

7. A method for detecting the usnic acid content in the usnea extract according to claim 6 or the usnea extract preparation according to claim 6, characterized in that: The following steps are included: Step 1: Preparation of reference solution Weigh the usnic acid reference substance and add solvent to make a solution; Step 2: Preparation of test solution Take a usnea extract or a usnea extract preparation, add a solvent to extract; Step 3: Liquid chromatography analysis Octadecylsilane bonded silica gel was used as filler, mobile phase A was organic phase, mobile phase B was aqueous phase, reference solution and test solution were injected into liquid chromatograph, and gradient elution was used for detection.

8. The method for detecting the content of usnic acid according to claim 7, wherein: In step 1, the solvent includes one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; And / or, in step 2, the preparation of the test solution comprises the following steps: taking a usnea extract or a usnea extract preparation, adding an extraction solvent, weighing the weight, performing extraction, weighing the weight again, supplementing the lost weight with the extraction solvent, and obtaining a liquid by solid-liquid separation to obtain the test solution; Preferably, in step 2, the extraction solvent includes one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; And / or, preferably, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; more preferably, the extraction time is 15 to 60 minutes.

9. The method for detecting the content of usnic acid according to claim 7 or 8, wherein: In step 3, the detection wavelength of the chromatogram is 190 to 400 nm; preferably, the detection wavelength of the chromatogram is 230 to 235 nm; and / or, in step 3, the flow rate is 0.2 to 0.3 mL / min; and / or, in step 3, the column temperature is 25 to 35°C; And / or, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; preferably, the chromatographic column is selected from ACQUITY HSS T3, AmethSep C18AQ or Chromcore AQ C18; And / or, in step 3, the organic phase is selected from acetonitrile; the aqueous phase is an aqueous solution containing 0.1% phosphoric acid; And / or, in step 3, the gradient elution procedure is as follows: From 0 to 20 min, the volume percentage of mobile phase A increased from 5% to 65%, and the volume percentage of mobile phase B decreased from 95% to 35%; 20-35 min, the volume percentage of mobile phase A is 65%, and the volume percentage of mobile phase B is 35%; From 35 to 40 min, the volume percentage of mobile phase A increased from 65% to 80%, and the volume percentage of mobile phase B decreased from 35% to 20%.

10. A method for detecting a characteristic spectrum of the usnea extract or the usnea extract preparation according to claim 6, characterized in that: The steps include: Step 1: Preparation of reference solution Weigh the usnic acid reference substance and add solvent to make a solution; Step 2: Preparation of test solution Take a usnea extract or a usnea extract preparation, add a solvent to extract; Step 3: Liquid chromatography analysis Octadecylsilane bonded silica gel was used as filler, mobile phase A was organic phase, mobile phase B was aqueous phase, reference solution and test solution were injected into liquid chromatograph, gradient elution was used for detection; Step 4: Establish a characteristic spectrum of the usnea extract or usnea extract preparation.

11. The characteristic spectrum detection method according to claim 10, wherein: In step 1, the extraction solvent includes one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; And / or, in step 2, the preparation of the test solution comprises the following steps: taking a usnea extract or a usnea extract preparation, adding a solvent used for extraction, weighing the weight, performing extraction, weighing the weight again, supplementing the lost weight with the solvent used for extraction, and obtaining a liquid by solid-liquid separation to obtain the test solution; Preferably, in step 2, the extraction solvent includes one or more selected from the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; And / or, preferably, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; more preferably, the extraction time is 15 to 60 minutes.

12. The characteristic spectrum detection method according to claim 10 or 11, wherein: In step 3, the detection wavelength of the chromatogram is 190 to 400 nm; preferably, the detection wavelength of the chromatogram is 230 to 235 nm; and / or, in step 3, the flow rate is 0.2 to 0.3 mL / min; and / or, in step 3, the column temperature is 25 to 35°C; And / or, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; preferably, the chromatographic column is selected from ACQUITY HSS T3, AmethSep C18AQ or Chromcore AQ C18; And / or, in step 3, the organic phase is selected from acetonitrile; the aqueous phase is an aqueous solution containing 0.1% phosphoric acid; And / or, in step 3, the gradient elution procedure is as follows: From 0 to 20 min, the volume percentage of mobile phase A increased from 5% to 65%, and the volume percentage of mobile phase B decreased from 95% to 35%; 20-35 min, the volume percentage of mobile phase A is 65%, and the volume percentage of mobile phase B is 35%; From 35 to 40 min, the volume percentage of mobile phase A increased from 65% to 80%, and the volume percentage of mobile phase B decreased from 35% to 20%; Preferably, the characteristic spectrum of the usnea extract or usnea extract preparation presents 5 characteristic peaks, wherein peak 5 is consistent with the retention time of the usneic acid reference peak, namely, the S peak, and the relative retention time of each characteristic peak and the S peak is within the range of ±10% of the specified value, and the specified value is: 0.25 (peak 1), 0.31 (peak 2), 0.42 (peak 3) and 0.47 (peak 4).

13. A thin layer identification method for the usnea extract or the usnea extract preparation according to claim 6, characterized in that: The steps include: Preparation of test solution of usnea extract or usnea extract preparation, spotting on thin layer plate, developing and color development; The method for preparing the test solution of the usnea extract or usnea extract preparation comprises the following steps: extracting the usnea extract or usnea extract preparation with methanol, and obtaining the liquid obtained by solid-liquid separation as the test solution; And / or, the developing agent is a mixed solvent of chloroform, toluene, methanol and glacial acetic acid; preferably, the volume ratio of chloroform, toluene, methanol and glacial acetic acid is 7.8-8.2:11.8-12.2:0.8-1.2:0.1-0.3; more preferably, the volume ratio of chloroform, toluene, methanol and glacial acetic acid is 8:12:1:0.

2.

14. Use of the method for detecting the content of usnic acid according to any one of claims 7 to 9 in usnea extracts and preparations thereof.

15. Application of the characteristic spectrum detection method according to any one of claims 10 to 12 in Usnea extract and its preparation.

16. Application of the thin layer identification method according to claim 13 in Usnea extract and its preparation.

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