Preparation method and detection method of gaultheria yunnanensis extract and preparation of gaultheria yunnanensis extract

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

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine modernization, and specifically relates to a preparation method and a detection method for an extract of Gaultheria yunnanensis and its preparations. Background Art

[0002] Gaultheria yunnanensis is the dried whole plant of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu of the Ericaceae family. It is dug in summer and autumn, impurities are removed, and it is dried, or processed while it is fresh. The stems are cut into sections and the roots are cut into pieces. It is included in the "Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province" in 2019 edition; it has the effects of dispelling wind and dampness, promoting blood circulation to remove stasis, and lowering qi and relieving asthma. It is used for dizziness, wind-cold cold, cough and asthma, amenorrhea, swelling and pain from falls and bruises, and rheumatic arthralgia. It is distributed in Yunnan, Sichuan, Guizhou, Guangxi and other places. It grows in wild grasslands and forest edges at low altitudes to about 3500 m above sea level. However, at present, the evaluation system of the quality standards for extracts of Gaultheria yunnanensis and its preparations is still imperfect, and its indicators cannot be effectively evaluated, restricting its application and development. Summary of the Invention

[0003] Aiming at the above technical problems, the purpose of the present invention is to provide a preparation method and a detection method for an extract of Gaultheria yunnanensis and its preparations.

[0004] Specifically, the present invention provides the following technical solutions:

[0005] In the first aspect, the present invention provides a preparation method for an extract of Gaultheria yunnanensis, including the following steps:

[0006] Take the cut pieces of Gaultheria yunnanensis, decoct with water, separate the solid and liquid to obtain a decoction, which is the extract of Gaultheria yunnanensis.

[0007] Preferably, the number of times of decocting with water is 1 to 3 times.

[0008] Preferably, the number of times of decocting with water is 2 times; among them, 9 to 14 times the amount of water is added for the first decoction, and 7 to 12 times the amount of water is added for the second decoction; and / or, the first decoction time is 30 to 120 min, and the second decoction time is 20 to 80 min.

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

[0010] And / or, preferably, the extract of Gaultheria yunnanensis further includes the following step: concentrating the obtained decoction; more preferably, the concentration temperature is 55 to 70 °C; and / or, more preferably, the vacuum degree of concentration is -0.08 to -0.09 MPa; and / or, more preferably, the density of the concentrated solution is 1 to 1.1 g / mL (45 ± 5 °C);

[0011] And / or, preferably, the concentrated liquid obtained by concentration is dried; more preferably, the drying is freeze-drying, vacuum drying, spray drying, microwave drying or infrared drying.

[0012] And / or, preferably, the concentrated liquid after concentration is further freeze-dried to obtain Elsholtzia blanda extract; wherein, the freeze-drying is divided into three stages: a. Pre-freezing: the pre-freezing temperature is -50°C to -45°C; b. Primary drying: the primary drying temperature is -30°C to 0°C; c. Secondary drying: the secondary drying temperature is 5 to 25°C.

[0013] And / or, preferably, the pre-freezing time is 3 to 6 h; more preferably, the pre-freezing time is 3 to 5 h; the primary drying time is 10 to 14 h; more preferably, the primary drying time is 13 to 14 h; and / or the secondary drying time is 5 to 7 h; more preferably, the secondary drying time is 6 to 7 h.

[0014] And / or, preferably, the primary drying includes the following steps: first drying at a temperature of -30 to -28°C; then drying at a temperature of -21 to -19°C; then drying at a temperature of -11 to -9°C; and finally drying at a temperature of -1 to 1°C; more preferably, the primary drying includes the following steps: first drying at a temperature of -30 to -28°C for 6.5 to 7.5 h; then drying at a temperature of -21 to -19°C for 1.5 to 2.5 h; then drying at a temperature of -11 to -9°C for 1.5 to 2.5 h; and finally drying at a temperature of -1 to 1°C for 2.5 to 3.5 h.

[0015] And / or, preferably, the secondary drying includes the following steps: first drying at a temperature of 4 to 6°C; then drying at a temperature of 14 to 16°C; and finally drying at a temperature of 24 to 26°C; more preferably, the secondary drying includes the following steps: first drying at a temperature of 4 to 6°C for 1.5 to 2.5 h; then drying at a temperature of 14 to 16°C for 1.5 to 2.5 h; and finally drying at a temperature of 24 to 26°C for 2.5 to 3.5 h.

[0016] In a second aspect, the present invention provides a method for preparing an Elsholtzia blanda extract preparation. After adding a first auxiliary material to the Elsholtzia blanda extract, drying is carried out; preferably, the drying is spray drying; more preferably, the inlet air temperature is 165 to 195°C and the outlet air temperature is 85 to 105°C;

[0017] And / or, preferably, the first auxiliary material contains maltodextrin. More preferably, the addition amount of the first auxiliary material is 5% to 15% of the feeding amount of Elsholtzia blanda slices; even more preferably, the addition amount of the first auxiliary material is 6% to 9% of the feeding amount of Elsholtzia blanda slices.

[0018] And / or, preferably, a second adjuvant is added to the dried material for mixing; more preferably, the second adjuvant contains silica; still more preferably, the percentage of the silica in the dried material by weight is greater than 0 and less than or equal to 0.3%.

[0019] In a third aspect, the present invention provides a Gaultheria yunnanensis extract or a Gaultheria yunnanensis extract preparation, wherein the Gaultheria yunnanensis extract is prepared by the preparation method of the Gaultheria yunnanensis extract described above; or, the Gaultheria yunnanensis extract preparation is prepared by the preparation method of the Gaultheria yunnanensis extract preparation described above.

[0020] Preferably, the Gaultheria yunnanensis extract is a standard decoction of Gaultheria yunnanensis.

[0021] And / or, preferably, the Gaultheria yunnanensis extract preparation is a formula granule of Gaultheria yunnanensis.

[0022] More preferably, the production amount of the formula granule of Gaultheria yunnanensis is 13% - 14%, wherein the production amount refers to the ratio of the mass of the prepared formula granule to the mass of the crude drug input; still more preferably, the production amount of the formula granule of Gaultheria yunnanensis is 13.1% - 13.5%.

[0023] In a fourth aspect, the present invention provides a detection method for the content of quercetin-3-O-β-D-glucuronide in the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation, comprising the following steps:

[0024] Step 1: Preparation of the reference substance solution

[0025] Weigh the quercetin-3-O-β-D-glucuronide reference substance and make it into a solution with a solvent;

[0026] Step 2: Preparation of the test solution

[0027] Take the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation, add a solvent for extraction;

[0028] Step 3: Liquid chromatography analysis

[0029] Using octadecylsilane chemically bonded silica as the filler, mobile phase A is the organic phase, mobile phase B is the aqueous phase, aspirate the reference substance solution and the test solution and inject them into the liquid chromatograph, and perform gradient elution for detection.

[0030] Preferably, 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.

[0031] And / or, preferably, in step 2, the preparation of the test solution comprises the following steps: taking the extract of Elsholtzia blanda or the preparation thereof, adding the solvent for extraction, weighing, performing extraction, weighing again, making up the lost weight with the solvent for extraction, and the liquid obtained by solid-liquid separation is the test solution; more preferably, in step 2, the solvent for extraction is selected from one or more of the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; and / or, more preferably, in step 2, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; further preferably, the extraction time is 15 - 60 min; still further preferably, the volume of the solvent for extraction is 10 - 40 mL.

[0032] And / or, preferably, in step 2, the preparation method of the test solution of the standard decoction of Elsholtzia blanda or the formula granules of Elsholtzia blanda comprises the following steps: taking the standard decoction of Elsholtzia blanda or the formula granules of Elsholtzia blanda, adding the solvent for extraction, weighing, performing extraction, weighing again, making up the lost weight with the solvent for extraction, and the liquid obtained by solid-liquid separation is the test solution; preferably, the ratio of the mass of the standard decoction of Elsholtzia blanda or the formula granules of Elsholtzia blanda to the volume of the solvent for extraction is 0.1:10 - 40, wherein the unit of mass is g and the unit of volume is mL.

[0033] Preferably, in step 3, the detection wavelength of the chromatography is 190 - 400 nm; more preferably, the detection wavelength is 254 - 258 nm.

[0034] And / or, preferably, in step 3, the flow rate is 0.2 - 0.3 mL / min.

[0035] And / or, preferably, in step 3, the column temperature is 25 - 35 °C.

[0036] And / or, preferably, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; more preferably, the chromatographic column is selected from ACQUITY HSS T3, AmethSep C18AQ or Chromcore AQ C18.

[0037] And / or, preferably, in step 3, the organic phase is selected from methanol or acetonitrile; the aqueous phase is an aqueous solution containing 0.1% phosphoric acid.

[0038] And / or, preferably, in step 3, the gradient elution program is as follows:

[0039] 0 - 20 min, the volume percentage of mobile phase A increases from 5% to 14%, and the volume percentage of mobile phase B decreases from 95% to 86%;

[0040] For 20 - 45 min, the volume percentage of mobile phase A is increased from 14% to 28%, and the volume percentage of mobile phase B is decreased from 86% to 72%.

[0041] And / or, preferably, in the osmanthus extract or osmanthus extract preparation, the content of quercetin - 3 - O - β - D - glucuronide is 2.62 - 31.69 mg / g; and / or, in the osmanthus extract or osmanthus extract preparation, the transfer rate of quercetin - 3 - O - β - D - glucuronide is 24.62% - 91.70%.

[0042] Fifthly, the present invention provides a method for detecting the characteristic fingerprint of the osmanthus extract or the osmanthus extract preparation, comprising the following steps:

[0043] Step 1: Preparation of the reference substance solution

[0044] Weigh the reference substance of quercetin - 3 - O - β - D - glucuronide and make it into a solution with a solvent;

[0045] Step 2: Preparation of the test sample solution

[0046] Take the osmanthus extract or osmanthus extract preparation, add a solvent for extraction;

[0047] Step 3: Liquid chromatography analysis

[0048] Using octadecylsilane chemically bonded silica gel as the filler, mobile phase A as the organic phase, and mobile phase B as the aqueous phase, aspirate the reference substance solution and the test sample solution and inject them into the liquid chromatograph, and perform gradient elution for detection.

[0049] Step 4: Establish the characteristic fingerprint of the osmanthus extract or osmanthus extract preparation.

[0050] Preferably, 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.

[0051] And / or, preferably, in step 2, the preparation of the test solution comprises the following steps: taking the extract of Gaultheria yunnanensis (Franch.) Rehd. or the preparation thereof, adding the solvent used for extraction, weighing, performing extraction, weighing 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 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 any one of shaking extraction, ultrasonic extraction or reflux extraction; even more preferably, the extraction time is 15 - 60 min; even further preferably, the volume of the solvent used for extraction is 10 - 40 mL.

[0052] And / or, preferably, in step 2, the preparation method of the test solution of the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. or the formula granules of Gaultheria yunnanensis (Franch.) Rehd. comprises the following steps: taking the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. or the formula granules of Gaultheria yunnanensis (Franch.) Rehd., adding the solvent used for extraction, weighing, performing extraction, weighing again, making up the lost weight with the solvent used for extraction, and the liquid obtained by solid-liquid separation is the test solution; preferably, the ratio of the mass of the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. or the formula granules of Gaultheria yunnanensis (Franch.) Rehd. to the volume of the solvent used for extraction is 0.1:10 - 40, wherein the unit of mass is g and the unit of volume is mL.

[0053] And / or, preferably, in step 3, the detection wavelength of the chromatography is 190 - 400 nm; more preferably, the detection wavelength is 254 - 258 nm.

[0054] And / or, preferably, in step 3, the flow rate is 0.2 - 0.3 mL / min.

[0055] And / or, preferably, in step 3, the column temperature is 25 - 35 °C.

[0056] And / or, preferably, in step 3, the specifications of the chromatographic column are: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; more preferably, the chromatographic column is selected from ACQUITY HSS T3, AmethSep C18AQ or Chromcore AQ C18.

[0057] And / or, preferably, in step 3, the organic phase is selected from methanol or acetonitrile; the aqueous phase is an aqueous solution containing 0.1% phosphoric acid.

[0058] And / or, preferably, in step 3, the gradient elution program is as follows:

[0059] From 0 to 20 min, the volume percentage of mobile phase A increases from 5% to 14%, and the volume percentage of mobile phase B decreases from 95% to 86%.

[0060] From 20 to 45 min, the volume percentage of mobile phase A increases from 14% to 28%, and the volume percentage of mobile phase B decreases from 86% to 72%.

[0061] And / or, preferably, the characteristic chromatogram of the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation shows 3 characteristic peaks, among which the retention time of peak 2 is consistent with that of the quercetin-3-O-β-D-glucuronide reference peak and is the S peak. The relative retention times of each characteristic peak and the S peak are within the range of ±10% of the specified value, and the specified values are: 0.25 (peak 1) and 1.13 (peak 3).

[0062] In the sixth aspect, the present invention provides a thin-layer identification method for the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation, including the following steps:

[0063] Preparation of the test solution of the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation, spotting on a thin-layer plate, development, and color development; wherein, the preparation method of the test solution of the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation includes the following steps: extracting the Gaultheria yunnanensis extract or the Gaultheria yunnanensis extract preparation with methanol, and the obtained liquid after solid-liquid separation is the test solution; and / or, the developing agent is a mixed solvent of toluene, ethyl acetate, methanol, formic acid, and water.

[0064] Preferably, the volume ratio of toluene, ethyl acetate, methanol, formic acid, and water is 0.8 - 1.2:7.8 - 8.2:0.8 - 1.2:0.8 - 1.2:0.8 - 1.2; more preferably, the volume ratio of toluene, ethyl acetate, methanol, formic acid, and water is 1:8:1:1:1.

[0065] In the seventh aspect, the present invention provides an application of the detection method for the content of the quercetin-3-O-β-D-glucuronide component in the Gaultheria yunnanensis extract and its preparation.

[0066] In the eighth aspect, the present invention provides an application of the characteristic chromatogram detection method in the Gaultheria yunnanensis extract and its preparation.

[0067] In the ninth aspect, the present invention provides an application of the thin-layer identification method in the Gaultheria yunnanensis extract and its preparation.

[0068] In the tenth aspect, the present invention provides an application of any one or more of the detection method for the content of the index components in the Gaultheria yunnanensis and its preparation, the characteristic chromatogram detection method for the Gaultheria yunnanensis and its preparation, and the thin-layer identification method for the Gaultheria yunnanensis and its preparation in the quality evaluation of the Gaultheria yunnanensis extract and its preparation.

[0069] Advantages of the present invention:

[0070] (1) The present invention provides an extract of Elsholtzia blanda, a preparation method thereof, and a detection method thereof. The extract of the Elsholtzia blanda decoction pieces is concentrated and freeze-dried to obtain a standard decoction, which provides a standard for the quality of the extract of Elsholtzia blanda and its preparations, and realizes quality control and effective supervision.

[0071] (2) The present invention provides a formula granule of Elsholtzia blanda and a preparation method thereof. The preparation method is simple and decocted together according to the traditional method, which conforms to the characteristics of traditional Chinese medicine medication.

[0072] (3) The present invention provides a detection method for the content of quercetin-3-O-β-D-glucuronide in the extract of Elsholtzia blanda and its preparations. The detection method has good specificity, precision, stability and repeatability.

[0073] (4) The present invention provides a characteristic chromatogram detection method for the extract of Elsholtzia blanda and its preparations. The detection method has good specificity, precision, stability and repeatability.

[0074] (5) Through thin layer chromatography identification, determination of the content of quercetin-3-O-β-D-glucuronide, and establishment of a characteristic chromatogram, the present invention can more effectively evaluate the quality of the extract of Elsholtzia blanda and its preparations, and at the same time provides a scientific and reasonable basis for formulating the process standard and quality standard of the extract of Elsholtzia blanda and its preparations. Description of the drawings

[0075] Figure 1 is the peak purity chromatogram of the methodological verification of the content determination method of the standard decoction of Elsholtzia blanda.

[0076] Figure 2 is the regression equation of the linear investigation of quercetin-3-O-β-D-glucuronide in the methodological verification of the content determination method of the standard decoction of Elsholtzia blanda.

[0077] Figure 3 is the influence of different chromatographic columns on the target components for content determination in the standard decoction of Elsholtzia blanda.

[0078] Figure 4 is the chromatogram result of the target components for content determination in the standard decoction of Elsholtzia blanda at different column temperatures.

[0079] Figure 5 is the chromatogram result of the target components for content determination in the standard decoction of Elsholtzia blanda at different flow rates.

[0080] Figure 6 is the DAD diagram of the standard decoction of Elsholtzia blanda.

[0081] Figure 7 are UPLC chromatograms of different mobile phase systems.

[0082] Figure 8 is the UPLC chromatogram for the investigation of the aqueous phase of the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0083] Figure 9 is the investigation of the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland with different extraction solvents.

[0084] Figure 10 is the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0085] Figure 11 is the overall investigation of the methodology validation of the analysis method for the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0086] Figure 12 is the UPLC comparison chart for the investigation of different chromatographic columns in the methodology validation of the analysis method for the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0087] Figure 13 is the UPLC comparison chart for the investigation of different column temperatures in the methodology validation of the analysis method for the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0088] Figure 14 is the UPLC comparison chart for the investigation of different flow rates in the methodology validation of the analysis method for the characteristic chromatogram of the standard decoction of Elsholtzia ciliata (Thunb.) Hyland.

[0089] Figure 15 is the peak purity chromatogram for the methodology validation of the content determination of Elsholtzia ciliata (Thunb.) Hyland formula granules.

[0090] Figure 16 is the regression equation for the linear investigation of quercetin-3-O-β-D-glucuronide in the methodology validation of the content determination of Elsholtzia ciliata (Thunb.) Hyland formula granules.

[0091] Figure 17 is the influence of different chromatographic columns on the target components for the content determination of Elsholtzia ciliata (Thunb.) Hyland formula granules.

[0092] Figure 18 are the chromatogram results of the target components for the content determination of Elsholtzia ciliata (Thunb.) Hyland formula granules at different column temperatures.

[0093] Figure 19 are the chromatogram results of the target components for the content determination of Elsholtzia ciliata (Thunb.) Hyland formula granules at different flow rates.

[0094] Figure 20 is the thin layer chromatogram for the investigation of the sample application amount of Elsholtzia ciliata (Thunb.) Hyland formula granules.

[0095] Figure 21 is the specificity of the thin layer chromatography of Elsholtzia ciliata (Thunb.) Hyland formula granules.

[0096] Figure 22It is the thin-layer identification chromatogram of Elsholtzia rugulosa formula granules under different temperature conditions.

[0097] Figure 23 It is the thin-layer identification chromatogram of Elsholtzia rugulosa formula granules under different humidity conditions.

[0098] Figure 24 It is the chromatogram for investigating silica gel plates from different manufacturers.

[0099] Figure 25 It is the thin-layer chromatogram of 3 batches of Elsholtzia rugulosa formula granules. Specific implementation manners

[0100] As described above, the object of the present invention is to provide a preparation method and a detection method for an Elsholtzia rugulosa extract and its preparation. Among them, the standard decoction is obtained by decocting and extracting the Elsholtzia rugulosa decoction pieces, concentrating and freeze-drying; the Elsholtzia rugulosa formula granules are obtained by decocting and extracting the Elsholtzia rugulosa decoction pieces, concentrating, adding excipients and then drying. The content detection method, characteristic fingerprint detection method and thin-layer chromatography identification method of the index components described in the present invention standardize the quality control and standard research of the standard decoction and formula granules of Elsholtzia rugulosa, thereby realizing the quality control and effective supervision of it, and providing a reference for the quality control of the standard decoction and formula granules of Elsholtzia rugulosa.

[0101] The standard decoction, also known as the standard decocted liquid, is a traditional form of medicine widely used clinically. The effective substances of traditional Chinese medicine formula granules are the same as those of the water decoction of traditional Chinese medicine decoction pieces, which is an inheritance of traditional Chinese medicine decoction pieces; the standard decoction can be used as a standard reference to measure whether the traditional Chinese medicine formula granules are basically consistent with the clinical decoction.

[0102] Since the standard decoction is a "bridge" connecting traditional Chinese medicine decoction pieces and modern Chinese medicine preparations, providing a reference for controlling the quality of Chinese medicine terminal products, standardizing different dosage forms of Chinese medicine, ensuring the uniformity of quality and the consistency of curative effects, and providing a reference for evaluating the consistency of product quality of different manufacturers. Therefore, the formulation of the quality standard of the Chinese medicine standard decoction will provide a basis for the formulation of the quality standards of all final products of water decoctions derived from decoction pieces. Therefore, establishing an effective and reliable method for the standard decoction and formula granules of Elsholtzia rugulosa is beneficial to ensuring the authenticity and accuracy of drugs, clinical effectiveness and the development of subsequent formula granule work.

[0103] The index component described in the present invention refers to quercetin-3-O-β-D-glucuronide.

[0104] In the present invention, for the reagents used in the examples, unless otherwise specified, they are all conventional reagents. Among them, the information of the raw materials and experimental equipment used in the examples are shown in Table 1 and Table 2 respectively:

[0105] Table 1 Information of raw materials used in the present invention

[0106]

[0107] Table 2 Information on the experimental equipment used in the present invention

[0108]

[0109] In the present invention, the medicinal material of Gaultheria yunnanensis is the dried whole plant of Gaultheria leucocarpa Bl. var. crenulata (Kurz) T. Z. Hsu of the Ericaceae family. It is dug in summer and autumn, impurities are removed, and it is dried, or processed while it is fresh. The stem is cut into sections and the root is cut into pieces.

[0110] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0111] Example 1: Preparation method of the standard decoction of Gaultheria yunnanensis, determination and analysis method of the content of quercetin-3-O-β-D-glucuronide, and establishment of the characteristic fingerprint analysis method

[0112] Example 1-1: Preparation method of the standard decoction of Gaultheria yunnanensis

[0113] The preparation method of the Gaultheria yunnanensis decoction pieces includes the following steps: Take the original Gaultheria yunnanensis medicinal material for cleaning, selection, remove non-medicinal parts, impurities and deteriorated products, etc. The impurity content shall not exceed 3%. Spray an appropriate amount of clear water, moisten thoroughly, cut the root into thick slices or pieces, cut the stem into sections (root pieces < 6 cm, stem < 5 cm), cut the leaves into pieces, and dry. Among them, the place of origin of the original Gaultheria yunnanensis medicinal material is Shuangliu Town, Kaiyang County, Guiyang City, Guizhou Province.

[0114] Step 1: Take 100 g of the Gaultheria yunnanensis decoction pieces, weigh them, place them in an electric ceramic pot, add water and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500 W), and then keep it simmering over low heat (200 W) for 60 minutes. Filter the decoction while it is hot through a 300-mesh sieve, and cool the filtrate for later use. For the second decoction, add 8 times the amount of water, bring to a boil over high heat (500 W), and then keep it simmering over low heat (200 W) for 40 minutes. Filter the decoction while it is hot through a 300-mesh sieve, cool the filtrate, and combine the two decoctions.

[0115] Step 2: Transfer the decoction to a 5000 mL round-bottom flask, and concentrate it under reduced pressure to a 100 mL concentrated solution (extract) using a rotary evaporator. The concentration temperature is 65 °C, and the vacuum degree for concentration is -0.080 to -0.090 MPa. Measure the density and extract yield of the concentrated solution. The density of the concentrated solution is 1.01 to 1.03 g / mL, and the extract yield is 8.94%.

[0116] Step 3: Under magnetic stirring, the concentrated solution was dispensed into 5 mL brown vials, with 2 mL in each vial, semi-capped, and transferred to a vacuum freeze dryer after dispensing for freeze-drying; the freeze-drying parameters were as follows: the pre-freezing temperature was -45 °C, the pre-freezing time was 240 minutes, the primary drying (sublimation drying) temperature was -30 to 0 °C, the primary drying time was 840 minutes, and the vacuum degree was 0.2 mbar (the specific process of primary drying was drying at -30 °C for 420 min; drying at -20 °C for 120 min; drying at -10 °C for 120 min and drying at 0 °C for 180 min); the secondary drying (desorption drying) temperature was 5 to 25 °C, the secondary drying time was 420 minutes, and the vacuum degree was 0.2 mbar (the specific process of secondary drying was drying at 5 °C for 120 min; drying at 15 °C for 120 min and drying at 25 °C for 180 min). The freeze-dried powder of the standard decoction of Elsholtzia blanda was named BT2104-13. Among them, in the Chinese herbal pieces, the content of quercetin-3-O-β-D-glucuronide was 2.76 mg / g, in the standard decoction, the content of quercetin-3-O-β-D-glucuronide was 19.89 mg / g, and the transfer rate of quercetin-3-O-β-D-glucuronide content was 64.42%.

[0117] In the present invention, the method for determining the extract yield comprises the following steps: The constant weight of the evaporating dish is denoted as m 1 , take 10 g of the concentrated solution in the pre-weighed evaporating dish, dry it on a water bath and then dry it in a drying oven at 105 °C for 3 hours, cool it in a desiccator for half an hour and quickly weigh it, denoted as m 2 . The calculation formula for the extract yield is as follows:

[0118]

[0119] In the formula: X---extract yield, expressed in percentage; m 2 ---total weight of the dried sample and the evaporating dish, unit: g; m 1 ---weight of the evaporating dish, unit: g; the total weight of the concentrated solution is the total mass of the concentrated solution obtained in Step 2, unit: g; the sampling amount of the concentrated solution is the sampling amount of the concentrated solution when determining the extract yield, unit: g; the sampling amount of the Chinese herbal pieces is the sampling amount of the Chinese herbal pieces in Step 1, unit: g.

[0120] Examples 1-2: Establishment of a determination and analysis method for the content of quercetin-3-O-β-D-glucuronide in the standard decoction of Elsholtzia blanda

[0121] 2.1 Determination of chromatographic conditions

[0122] Chromatographic conditions and system suitability test: Use octadecylsilyl silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); use acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution (the mass of phosphoric acid contained in every 100 mL of the solution is 0.1 g) as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.25 mL / min; the column temperature is 30 °C; the detection wavelength is 256 nm. The number of theoretical plates calculated based on the quercetin-3-O-β-D-glucuronide peak should be not less than 3000.

[0123] Table 3 Gradient elution program

[0124] Time (min) Mobile Phase A (volume%) Mobile Phase B (volume%) 0～20 5→14 95→86 20～45 14→28 86→72

[0125] Among them, the calculation formula for the content of quercetin-3-O-β-D-glucuronide is as follows:

[0126]

[0127] In the formula: c x --- The concentration of quercetin-3-O-β-D-glucuronide in the sample, unit: mg / g; c r --- The concentration of quercetin-3-O-β-D-glucuronide in the reference substance, unit: mg / mL; A x --- The peak area of quercetin-3-O-β-D-glucuronide in the sample; A r --- The peak area of quercetin-3-O-β-D-glucuronide in the reference substance; V x --- The volume of the extraction solvent, unit: mL; m x --- The weighed amount of the sample, unit: g.

[0128] Among them, the calculation formula for the transfer rate of quercetin-3-O-β-D-glucuronide (the component to be measured) is as follows:

[0129]

[0130] In the formula: X --- The transfer rate of quercetin-3-O-β-D-glucuronide in the standard decoction, expressed in percentage; the content of the component to be measured in the standard decoction (calculated on a dry basis), unit: mg / g; the content of the component to be measured in the medicinal material (calculated on a dry basis), unit: mg / g.

[0131] 2.2 Preparation of the reference substance solution

[0132] Take an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance, dissolve it in methanol to prepare a solution containing 50 μg of quercetin-3-O-β-D-glucuronide per 1 mL as the reference substance solution.

[0133] 2.3 Preparation of test solution

[0134] 2.3.1 Investigation of extraction solvents

[0135] Take an appropriate amount of the standard decoction of Elsholtzia blanda BT2104-13, a total of 9 portions, with 2 samples in parallel for each portion. Respectively place them in stoppered conical flasks, and successively add 20 mL of methanol (anhydrous methanol), 75% (v / v) methanol, 50% (v / v) methanol, 25% (v / v) methanol, ethanol (anhydrous ethanol), 75% (v / v) ethanol, 50% (v / v) ethanol, 25% (v / v) ethanol, and water. Weigh accurately, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 minutes, let it cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter through a 0.22 μm microporous membrane, and collect the subsequent filtrate to obtain each test solution.

[0136] According to the chromatographic conditions in 2.1 above, accurately pipette 1 μL of the reference substance solution and the test solution respectively, inject them into an ultra-high performance liquid chromatograph for determination. The experimental results are calculated by the external standard single-point method, and the specific results are shown in the following table.

[0137] Table 4 Influence of different extraction solvents on the content determination results of the standard decoction of Elsholtzia blanda

[0138]

[0139]

[0140] It can be seen from the experimental results in Table 4 that considering the content difference of quercetin-3-O-β-D-glucuronide comprehensively, 75% methanol is selected as the best extraction solvent for subsequent research.

[0141] 2.3.2 Investigation of extraction methods

[0142] Take an appropriate amount of the standard decoction of Elsholtzia blanda BT2104-13, a total of 2 portions, with 2 samples in parallel for each portion. Respectively place them in stoppered conical flasks, accurately add 20 mL of 75% methanol successively, weigh accurately, ultrasonically treat and reflux for 30 minutes respectively, let it cool, weigh again, make up the lost weight with 75% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain each test solution.

[0143] According to the chromatographic conditions in 2.1 above, accurately pipette 1 μL of the reference substance solution and the test solution respectively, inject them into an ultra-high performance liquid chromatograph for determination. The experimental results are calculated by the external standard single-point method, and the specific results are shown in the following table.

[0144] Table 5 Effects of different extraction methods on the content determination results of standard decoction of Touguxiang

[0145]

[0146] From the experimental results in Table 5, it can be seen that considering the difference in the content of quercetin-3-O-β-D-pyranoglucopyranosyl glucoside, ultrasonic extraction (power 500 W, frequency 40 kHz) was selected as the extraction method.

[0147] 2.3.3 Investigation of extraction time

[0148] Take an appropriate amount of 4 portions of Touguxiang standard decoction BT2104-13, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 20 mL of 75% methanol in sequence, weigh the weight, and ultrasonically treat (power 500 W, frequency 40 kHz) for 15, 30, 45 and 60 minutes respectively. Cool, weigh again, make up the lost weight with 75% methanol, shake well, filter with 0.22 μm microporous filter membrane, and take the filtrate 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 aspirated and injected into the ultra-high performance liquid chromatography instrument for determination. The experimental results were calculated using the external standard one-point method, and the specific results are shown in the table below.

[0150] Table 6 Effect of different extraction time on the content determination results in standard decoction of Touguxiang

[0151]

[0152] From the experimental results in Table 6, it can be seen that considering the time cost and the difference in the content of quercetin-3-O-β-D-pyranoglucopyranoside, ultrasonic treatment for 30 minutes was selected for subsequent research.

[0153] 2.3.4 Investigation of extraction solvent volume

[0154] Take an appropriate amount of Touguxiang standard decoction BT2104-13, a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones respectively. Accurately add 75% methanol 10mL, 20mL, 30mL and 40mL in sequence to weigh the weight, and ultrasonically treat (power 500W, frequency 40kHz) for 30 minutes respectively. Cool and weigh again, make up the lost weight with 75% methanol, shake well, filter with 0.22μm microporous filter membrane, and take the filtrate to obtain each test sample solution.

[0155] According to the chromatographic conditions in 2.1 above, 1 μL of the reference solution and the test solution were respectively taken and injected into the ultra-high performance liquid chromatography for determination. The results were calculated using the external standard one-point method, as shown in the following table.

[0156] Table 7 Effects of Different Extraction Solvent Volumes on the Determination Results of the Contents in the Standard Decoction of Elsholtzia blanda

[0157]

[0158]

[0159] It can be seen from the experimental results in Table 7 that considering the solvent cost and the content difference of quercetin-3-O-β-D-glucuronide comprehensively, an extraction volume of 20 mL was selected for subsequent research.

[0160] 2.3.5 Determination of the Preparation Method of the Test Solution

[0161] According to the above experimental results, the preparation method of the test solution was determined as follows: Take about 0.1 g of the standard decoction of Elsholtzia blanda, weigh it accurately, place it in a stoppered conical flask, accurately add 20 mL of 75% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0162] 2.4 Methodology Verification of Content Determination

[0163] 2.4.1 Peak Purity

[0164] Respectively pipette 1 μL of the test solution and the reference solution of the standard decoction of Elsholtzia blanda (BT2104-13) prepared according to 2.3.5 and inject them into an ultra-high performance liquid chromatograph, and perform on-machine detection according to the chromatographic conditions in 2.1 above. The results are shown in Figure 1 . The peak purity matching value of quercetin-3-O-β-D-glucuronide is 998.45, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0165] 2.4.2 Linearity

[0166] Accurately weigh an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance, place it in a numbered 50 mL volumetric flask, add methanol to prepare a solution containing 209.6 μg of quercetin-3-O-β-D-glucuronide per 1 mL, shake well, and obtain the quercetin-3-O-β-D-glucuronide reference substance stock solution, which is stored in the refrigerator for later use. Dilute the quercetin-3-O-β-D-glucuronide 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 substance solutions of different concentrations of quercetin-3-O-β-D-glucuronide. Detect according to the above 2.1 chromatographic conditions. Using the concentration as the abscissa and the peak area value as the ordinate, investigate the linear range of quercetin-3-O-β-D-glucuronide. The results of the linear investigation are shown in Table 8 below and Figure 2 。

[0167] Table 8 Linear investigation of quercetin-3-O-β-D-glucuronide

[0168]

[0169] From Table 8 and Figure 3 the experimental results show that: quercetin-3-O-β-D-glucuronide has a good linear relationship with the peak area value in the concentration range of 3.5165 - 209.6 μg / mL, and the correlation coefficient R 2 = 0.9993, and the linear regression equation is: y = 9.9574x + 3.5233.

[0170] 2.4.3 Precision test

[0171] 2.4.3.1 Instrument precision test

[0172] Respectively pipette 1 μL of the test solution and the reference solution of the standard decoction of Gaultheria yunnanensis (BT2104-13) prepared according to 2.3.5, inject them into an ultra-high performance liquid chromatograph, and perform on-machine detection according to the above 2.1 chromatographic conditions. Calculate the RSD (%) value of the target peak by the external standard single-point method based on quercetin-3-O-β-D-glucuronide. The specific results are shown in the following table.

[0173] Table 9 Results of the instrument precision test for the content determination method of the standard decoction of Gaultheria yunnanensis

[0174]

[0175] As can be seen from the experimental results in Table 9, the RSD (%) value of the target peak quercetin-3-O-β-D-glucuronide was 0.52%, which was less than 1.5%, indicating that the method had good precision.

[0176] 2.4.3.2 Repeatability Test

[0177] Take about 0.1 g of the same batch of the standard decoction of Elsholtzia blanda (BT2104-13), in parallel for 6 portions, and prepare 6 portions of the test solution according to 2.3.5. Detect according to the chromatographic conditions in 2.1 above. Calculate the RSD (%) value of the content of the target peak by the external standard single-point method with quercetin-3-O-β-D-glucuronide as the reference. The results are shown in the following table.

[0178] Table 10 Results of the repeatability test for the content determination method of the standard decoction of Elsholtzia blanda

[0179]

[0180] As can be seen from the experimental results in Table 10, the RSD (%) value of the content of the target peak quercetin-3-O-β-D-glucuronide was 1.01%, which was less than 1.5%, indicating that the method had good repeatability.

[0181] 2.4.4 Accuracy Test

[0182] Take the standard decoction of Elsholtzia blanda with known content (batch number BT2104-13, quercetin-3-O-β-D-glucuronide content 19.25 mg / g), 0.05 g, accurately weighed in 6 portions in total. Accurately add 4.7 mL of the quercetin-3-O-β-D-glucuronide reference substance solution with 75% methanol as the solvent (concentration 0.2096 mg / mL), and another 15.3 mL of 75% methanol was accurately added. Detect on the machine according to the chromatographic conditions in 2.1 above, inject 1 μL respectively, and record the chromatogram. Calculate the content of the target peak by the external standard single-point method with quercetin-3-O-β-D-glucuronide as the reference, and calculate the recovery rate and relative standard deviation according to the following formula. The results are shown in the following table.

[0183]

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

[0185] Table 11 Results of the spike recovery experiment for the content determination method of the standard decoction of Elsholtzia blanda

[0186]

[0187] As can be seen from the experimental results in Table 11, the recovery rate range of quercetin-3-O-β-D-glucuronide in the standard decoction is within 95% - 102%, and the RSD(%) value is 1.25%, which is less than 1.5%, indicating that the accuracy of this content detection method is good.

[0188] 2.4.5 Durability investigation

[0189] 2.4.5.1 Investigation of different chromatographic columns

[0190] Compared the effects of three chromatographic columns, namely ACQUITY HSS T3 (2.1*150mm, 1.8μm); chromatographic column AmethSep C18AQ (2.1*150mm, 1.8μm); chromatographic column Chromcore AQ C18 (2.1*150mm, 1.8μm), on the peak shape and resolution of the quercetin-3-O-β-D-glucuronide chromatographic peak in the standard decoction of Gaultheria yunnanensis (Franch.) Rehd.

[0191] Take the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. (batch number BT2104 - 13) to prepare the test solution according to 2.3.5, and determine it according to the chromatographic conditions in 2.1 above. The results are shown in Table 12 and Figure 3 as follows.

[0192] Table 12 Effects of different chromatographic columns on the content determination results of the standard decoction of Gaultheria yunnanensis (Franch.) Rehd.

[0193]

[0194] As can be seen from the experimental results in Table 12 and Figure 3 it can be known that the ACQUITY HSS T3 chromatographic column has better resolution and theoretical plate number, so this chromatographic column was selected for subsequent research.

[0195] 2.4.5.2 Investigation of different column temperatures

[0196] Compared the effects of different column temperatures of 25°C, 30°C and 35°C on the peak shape and resolution of the quercetin-3-O-β-D-glucuronide chromatographic peak in the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. Take the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. (batch number BT2104 - 13) to prepare the test solution according to 2.3.5 above, and determine it according to the chromatographic conditions in 2.1 above. The experimental results are shown in Table 13 and Figure 4 as follows.

[0197] Table 13 Detection results of the content determination method of the standard decoction of Gaultheria yunnanensis (Franch.) Rehd. at different column temperatures

[0198] Column Temperature Index Component Resolution / R Theoretical Plate Number 25℃ Quercetin-3-O-β-D-glucuronide 1.77 65928 30℃ Quercetin-3-O-β-D-glucuronide 1.7 61806 35℃ Quercetin-3-O-β-D-glucuronide 1.78 66244

[0199] From Table 13 and Figure 4 the experimental results show that: Considering the tolerance of the chromatographic column and the analysis time required, the column temperature was selected to be 30 °C.

[0200] 2.4.5.3 Investigation of different flow rates

[0201] Compare the effects of different flow rates of 0.20 mL / min, 0.25 mL / min, and 0.30 mL / min on the peak shape and resolution of the quercetin-3-O-β-D-glucuronide chromatographic peak in the standard decoction of Gaultheria yunnanensis. Take the standard decoction of Gaultheria yunnanensis (batch number BT2104-13) to prepare the test solution according to the above 2.3.5, and determine it according to the chromatographic conditions in the above 2.1. The results are shown in Table 14 and Figure 5 as follows.

[0202] Table 14 Detection results of the content determination method of the standard decoction of Gaultheria yunnanensis at different flow rates

[0203] Flow Rate Index Component Resolution / R Theoretical Plate Number 0.20 mL / min Quercetin-3-O-β-D-glucuronide 1.56 58646 0.25 mL / min Quercetin-3-O-β-D-glucuronide 1.7 61806 0.30 mL / min Quercetin-3-O-β-D-glucuronide 1.75 57430

[0204] From Table 14 and Figure 5 the experimental results show that: When the flow rate is 0.25 mL / min, the resolution of the target component is good and the baseline has no drift. In this experiment, the flow rate of 0.25 mL / min was selected.

[0205] Examples 1-3: Establishment of the characteristic chromatogram analysis of the standard decoction of Gaultheria yunnanensis

[0206] 3.1 Preparation of the reference solution

[0207] Take an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance, weigh it accurately, and dissolve it in methanol to make a solution containing 50 mg of quercetin-3-O-β-D-glucuronide per 1 mL, and shake well.

[0208] 3.2 Determination of the chromatographic conditions

[0209] (1) Determination of the detection wavelength: Inject the test solution of the standard decoction of Gaultheria yunnanensis for analysis, and record the absorption spectrum in the range of 190-400 nm. As Figure 6 shown, at a wavelength of 256 nm, more chromatographic peak information was detected in the sample solution of the standard decoction of Gaultheria yunnanensis and the baseline noise interference was small. Therefore, 256 nm was selected as the detection wavelength.

[0210] (2) Optimization of the mobile phase

[0211] ① Investigate that mobile phase A is the organic phase and mobile phase B is the aqueous phase. The UPLC chromatograms of different mobile phases are as Figure 7 shown. The gradient is shown in Table 15 below.

[0212] Table 15 Gradient Elution Program

[0213] Time (min) Mobile Phase A (volume%) Mobile Phase B (volume%) 0～20 5→14 95→86 20～45 14→28 86→72

[0214] Experimental results: The elution ability of mobile phase A being acetonitrile and mobile phase B being 0.1% phosphoric acid solution is stronger, so it is selected as the mobile phase for subsequent research.

[0215] ② Investigate the cases with and without adding acid: The UPLC chromatograms of the aqueous phase are as shown in Figure 8 : The peak shape is better with acid addition than without acid addition. When mobile phase A is acetonitrile and mobile phase B is 0.1% aqueous phosphoric acid solution, the peak shape is the best. Therefore, mobile phase A being acetonitrile and mobile phase B being 0.1% aqueous phosphoric acid solution are selected for elution.

[0216] (3) Determination of chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); using acetonitrile as mobile phase A and 0.1% aqueous phosphoric acid solution as mobile phase B, perform gradient elution according to the regulations in the following table; the flow rate is 0.25 mL / min; the column temperature is 30 °C; the detection wavelength is 256 nm. The number of theoretical plates calculated based on the quercetin-3-O-β-D-glucuronide peak should be not less than 3000.

[0217] Table 16 Gradient Elution Program

[0218] Time (min) Mobile Phase A (volume%) Mobile Phase B (volume%) 0～20 5→14 95→86 20～45 14→28 86→72

[0219] 3.3 Preparation of test solution

[0220] Take an appropriate amount of the standard decoction of Gaultheria yunnanensis (BT2104-13), take about 0.1 g, place it in a stoppered conical flask, accurately add methanol (anhydrous methanol), 75% methanol, 50% methanol, 25% methanol, ethanol (anhydrous ethanol), 75% ethanol, 50% ethanol, 25% ethanol and water, each 20 mL, weigh, ultrasonically treat (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh again, make up the lost weight with the corresponding reagent, shake well, filter, and take the subsequent filtrate, that is obtained. 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 chromatogram of the standard decoction of Gaultheria yunnanensis are shown in Figure 9 . Experimental results: When the extraction solvent is 75% methanol, the extraction efficiency is relatively high. Therefore, 75% methanol is selected as the extraction solvent.

[0221] According to the above experimental results, the sample pretreatment method for the characteristic chromatogram of the standard decoction of Elsholtzia blanda can be determined as follows: Take about 0.1 g of the standard decoction of Elsholtzia blanda (BT2104-13), accurately weigh it, place it in a stoppered conical flask, accurately add 20 mL of 75% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, which is the required solution.

[0222] 3.4 Determination and identification of common peaks

[0223] Determine the UPLC chromatograms of the standard decoction samples of Elsholtzia blanda in different batches, and use the "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China for result analysis to select their common peaks. The results are as Figure 10 shown. There are 3 relatively obvious common peaks in the characteristic chromatogram of the standard decoction of Elsholtzia blanda. Among them, the retention time of peak 2 should be consistent with that of the reference substance peak of quercetin-3-O-β-D-glucuronide. The peak corresponding to the reference substance peak of quercetin-3-O-β-D-glucuronide is the S peak. Calculate the relative retention time of each characteristic peak and the S peak, and its relative retention time should be within the range of ±10% of the specified value. The specified values are: 0.25 (peak 1), 1.13 (peak 3).

[0224] 3.5 Methodology verification of the characteristic chromatogram analysis method

[0225] (1) Overall inspection: Take the test solution prepared according to 3.3 from the standard decoction of Elsholtzia blanda (BT2104-13), 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 chromatogram. The results are as Figure 11 shown. There are no obvious chromatographic peaks after extending the elution time by one time under this chromatographic condition, indicating that this chromatographic condition basically meets the principle of maximum information content.

[0226] (2) Precision inspection: Take the test solution prepared according to 3.3 from the standard decoction of Elsholtzia blanda (BT2104-13), inject it repeatedly 6 times, and the injection volume is 1 μL. Tentatively calibrate 3 characteristic peaks, take peak 2 as the reference peak, and calculate the relative retention time. The experimental results are shown in Table 17.

[0227] Table 17 Precision results of the characteristic chromatogram of the standard decoction of Elsholtzia blanda (relative retention time)

[0228] Sample Peak 1 Peak 2 Peak 3 1 0.25 1.00 1.13 2 0.25 1.00 1.13 3 0.25 1.00 1.13 4 0.25 1.00 1.13 5 0.25 1.00 1.13 6 0.25 1.00 1.13 Average 0.25 1.00 1.13 RSD % 0.06 0.00 0.03

[0229] The experimental results show that the relative standard deviation (RSD) of the relative retention time of each chromatographic peak is < 3.0%, indicating that the precision of this instrument is good.

[0230] (3) Stability investigation: Take the standard decoction of Gaultheria yunnanensis (BT2104-13), prepare the test solution according to 3.3, inject samples at 0, 2, 4, 8, 12, 16, 20, and 24 hours respectively, and the injection volume is 1 μL. Tentatively calibrate 3 characteristic peaks, take peak 2 as the reference peak, and calculate the relative retention time. The results are shown in Table 18 below.

[0231] Table 18 Stability results of the characteristic chromatogram of the standard decoction of Gaultheria yunnanensis (relative retention time)

[0232] Sample Peak 1 Peak 2 Peak 3 0h 0.25 1.00 1.13 2h 0.25 1.00 1.13 4h 0.25 1.00 1.13 8h 0.25 1.00 1.13 12h 0.25 1.00 1.13 16h 0.25 1.00 1.13 20h 0.25 1.00 1.13 24h 0.25 1.00 1.13 Average 0.25 1.00 1.13 RSD (%) 0.37 0.00 0.05

[0233] The experimental results show that the RSD of the relative retention time of the chromatographic peaks is in the range of 0.00% - 1.05%, and the RSD is less than 3.0%, indicating that the sample solution is relatively stable.

[0234] (4) Repeatability investigation: Take about 0.1 g of the standard decoction of Gaultheria yunnanensis (BT2104-13), accurately weigh it, prepare 6 parallel samples, prepare the test solution according to 3.3, and inject 1 μL respectively. Tentatively calibrate 3 characteristic peaks, take peak 2 as the reference peak, and calculate the relative retention time.

[0235] Table 19 Repeatability results of the characteristic chromatogram of the standard decoction of Gaultheria yunnanensis (relative retention time)

[0236] Sample Peak 1 Peak 2 (S) Peak 3 1 0.25 1.00 1.13 2 0.25 1.00 1.13 3 0.25 1.00 1.13 4 0.25 1.00 1.13 5 0.25 1.00 1.13 6 0.25 1.00 1.13 Average 0.25 1.00 1.13 RSD % 0.06 0.00 0.01

[0237] The experimental results show that the RSD of the relative retention time of each chromatographic peak is between 0.00% - 0.10%, and the RSD value is less than 3.0%, indicating that the method has good repeatability.

[0238] (5) Durability investigation

[0239] ① Investigation of chromatographic column: Investigate the influence of 3 kinds of chromatographic columns on the peak emergence of the characteristic chromatogram of the standard decoction of Gaultheria yunnanensis, which are: chromatographic column ACQUITY HSS T3 C18 (2.1 * 150 mm, 1.8 μm); chromatographic column AmethSep C18 AQ (2.1 * 150 mm, 1.8 μm); chromatographic column Chromcore AQ C18 (2.1 * 150 mm, 1.8 μm). The results are as Figure 12 shown in Table 20.

[0240] Table 20 Results of the investigation of chromatographic columns for the characteristic chromatogram of the standard decoction of Gaultheria yunnanensis (relative retention time)

[0241]

[0242] The experimental results show that different chromatographic columns have a certain influence on the peak emergence. Using the chromatographic column ACQUITY Elution was carried out using a hyper - efficient liquid chromatography column of HSS T3C18. The chromatographic peak shape was good and the separation effect was the best. Therefore, it was selected as the chromatographic column for subsequent research.

[0243] ② Column temperature investigation: The elution conditions at different column temperatures (25 °C, 30 °C, and 35 °C) were investigated. The results are as Figure 13 shown in Table 21.

[0244] Table 21 Results of column temperature investigation for the characteristic chromatogram of the Herba Gaultheriae Yunnanensis decoction (relative retention time)

[0245] Column Temperature Peak 1 Peak 2 (S) Peak 3 25℃ 0.26 1.00 1.12 30℃ 0.25 1.00 1.13 35℃ 0.28 1.00 1.13

[0246] The experimental results showed that the separation effect was the best and the peak shape was good when the column temperature was 30 °C. Therefore, it was used as the determination column temperature.

[0247] ③ Flow rate investigation: The elution conditions at different flow rates (0.20 mL / min, 0.25 mL / min, and 0.30 mL / min) were investigated. The results are as Figure 14 shown in Table 22.

[0248] Table 22 Results of flow rate investigation for the characteristic chromatogram of the Herba Gaultheriae Yunnanensis decoction (relative retention time)

[0249] Flow Rate Peak 1 Peak 2 (S) Peak 3 0.20 mL / min 0.29 1.00 1.12 0.25 mL / min 0.25 1.00 1.13 0.30 mL / min 0.22 1.00 1.13

[0250] The experimental results showed that the peak shape was the best when the flow rate was 0.25 mL / min. Therefore, it was used as the determination flow rate.

[0251] Example 2: Preparation method of Herba Gaultheriae Yunnanensis formula granules, determination and analysis method for the content of quercetin - 3 - O - β - D - glucuronide, and establishment of characteristic chromatogram analysis method

[0252] Example 2 - 1 Preparation method of Herba Gaultheriae Yunnanensis formula granules

[0253] The preparation method of Herba Gaultheriae Yunnanensis decoction pieces includes the following steps: Take the original Herba Gaultheriae Yunnanensis medicinal materials for cleaning, selection, removing non - medicinal parts, impurities, deteriorated products, etc. The impurity content shall not exceed 3%. Spray an appropriate amount of clear water, moisten thoroughly, cut the roots into thick slices or blocks, cut the stems into sections (root blocks < 6 cm, stems < 5 cm), cut the leaves into pieces, and dry. Among them, the origin of the original Herba Gaultheriae Yunnanensis medicinal materials with the batch number TGX210410 - 11 is Jiangkou County, Tongren City, Guizhou Province; the origin of the original Herba Gaultheriae Yunnanensis medicinal materials with the batch number TGX210410 - 13 is Shuangliu Town, Kaiyang County, Guiyang City, Guizhou Province; the origin of the original Herba Gaultheriae Yunnanensis medicinal materials with the batch number TGX210410 - 15 is Wantanhe Town, Longli County, Guiyang City, Guizhou Province.

[0254] Step 1: Extraction and concentration: Take the Herba Elsholtziae ciliatae slices, decoct them twice with water. Soak them for 30 minutes before decocting. Add 12 times the amount of water for the first time, heat to boiling, maintain gentle boiling for 60 minutes, filter (200 mesh), and reserve the filtrate. Add 10 times the amount of water for the second time, heat to boiling, maintain gentle boiling for 40 minutes, filter (200 mesh), and combine the filtrates obtained from the two decoctions.

[0255] Adopt vacuum decompression concentration. The concentration temperature range is 60 - 70°C, and the vacuum degree is -0.080 to -0.090 MPa. The filtrate is concentrated under reduced pressure to a clear paste with a relative density of 1.03 - 1.06 g / mL (45 ± 5°C).

[0256] Step 2: Drying and powder collection: Add 6% - 9% of maltodextrin based on the total weight of the slices to the clear paste, mix well, and pass through a No. 6 sieve (100 mesh), then perform spray drying. The inlet air temperature is 165 - 195°C, the outlet air temperature is 85 - 105°C, collect the powder, and the yield of the dry paste powder is 12.28%.

[0257] Step 3: Ingredient mixing, granulation and total mixing: Add silicon dioxide with a weight ≤ 0.3% of the intermediate (dry paste powder containing excipients), sieve and mix well, then granulate to make 1000 g, and it is ready.

[0258] Table 23 Summary table of research data results

[0259]

[0260]

[0261] Investigation on the preparation process of Herba Elsholtziae ciliatae formula granules

[0262] 4.1 Selection of extraction process parameters

[0263] 4.1.1 Investigation on the multiple of water addition

[0264] Since Herba Elsholtziae ciliatae belongs to the whole herb medicine, referring to the preparation process of standard decoction, investigate the multiple of water addition and extraction time parameters. The results are shown in Tables 24 and 25.

[0265] Table 24 Investigation results of different multiples of water addition for Herba Elsholtziae ciliatae

[0266]

[0267] From the experimental results in Table 24, it can be seen that: based on the comprehensive judgment of the paste yield results and the content of index components, the multiples of water addition are determined to be 12 times and 10 times.

[0268] 4.1.2 Investigation on extraction time

[0269] Table 25 Investigation results of different extraction times for Herba Elsholtziae ciliatae

[0270]

[0271]

[0272] In summary, the finally selected extraction process parameters are as follows: the number of decoction extractions is two times. The amount of water added for the first time is 12 times the amount of crude drug charged. The soaking time is 30 minutes, and the decoction time for the first time is 60 minutes (in the boiling state). The amount of water added for the second time is 10 times the amount of crude drug charged, and the decoction time is 40 minutes.

[0273] 4.2 Drying Process Research

[0274] Take an appropriate amount of 4 portions of well-extracted and concentrated clear paste, each portion being 1 kg (each portion converted to the amount of crude drug charged is equivalent to 1 kg of crude drug). Select reasonable spray drying process parameters according to the design scheme in the following table, and conduct an investigation test on the addition amount of spray drying excipients. The test results are shown in Table 26.

[0275] Table 26 Investigation Results of the Amount of Excipients Used in Spray Drying of Elsholtzia ciliata Concentrated Clear Paste

[0276]

[0277] Note: The amount of dry extract powder obtained is the amount of dry extract powder containing excipients, and the yield of dry extract powder refers to the yield of dry extract powder without excipients.

[0278] As can be seen from Table 26: After adding 6% - 9% of the excipient maltodextrin based on the amount of crude drug charged, it is easy to dry, the moisture content is low, and the drying effect is good. This shows that adding a certain amount of maltodextrin to the Elsholtzia ciliata concentrated clear paste can significantly improve the spray drying effect and make it easier to dry.

[0279] 4.3 Granulation Process Research

[0280] In view of the fact that a certain proportion of maltodextrin has been added to the dry extract powder of Elsholtzia ciliata formula granules, considering that the fluidity of the intermediate product (dry extract powder) of Elsholtzia ciliata affects the granulation efficiency of dry granulation and the filling difference of the prepared formula granules during packaging, the commonly used glidants in the process of traditional Chinese medicine preparation are mainly silicon dioxide. Therefore, in this experiment, the intermediate product of Elsholtzia ciliata prepared by pilot-scale extraction research is taken to conduct an investigation on the excipient amount and granulation process. Each time, 1.0 kg of dry extract powder of the Elsholtzia ciliata intermediate product is taken, and the excipients are added according to the experimental design ratio and mixed evenly. Then, it is put into a GZL100 - 30L dry granulator to study the effects of process parameters such as excipient amount, roller speed, and pressure on the granulation effect. The screen mesh size is selected as 12 - 40 meshes for sizing, and the excipient amount and granulation and forming process are evaluated with indicators such as particle size, fluidity, primary product yield, bulk density, solubility, and fine powder rate. The test results of the granulation process parameters of Elsholtzia ciliata are shown in Table 27, and the screening of excipient amounts is shown in Table 28.

[0281] Table 27 Production Equipment Parameter Table for Investigation of Granulation and Forming Process

[0282] Stirring Speed (r / min) 20.0 Feeding Speed (r / min) 75.0 Pressing Wheel Speed (r / min) 7.5 Crushing Speed (r / min) 80.0 Final Granulation Speed (r / min) 72.0 Pressing Wheel Spacing (mm) 0.70 Hydraulic Pressure (MPa) 6.6 Granulation Screen Mesh Size (mesh) 18 Upper Sieve Mesh Size (mesh) 10 Lower Sieve Mesh Size (mesh) 40 Mixing Time (min) 30 Mixer Speed (r / min) 10

[0283] Table 28 Investigation Table of Process Parameters for Preparing Granules of Gaultheria yunnanensis (Franch.) Rehd.

[0284]

[0285] It can be seen from the experimental results in Table 27 and Table 28 that: the feeding speed of the intermediate of Gaultheria yunnanensis (Franch.) Rehd. should not be too fast, otherwise it will affect the primary product rate and particle strength of the granules. Adding a certain amount of glidant to the intermediate before dry granulation of the intermediate of Gaultheria yunnanensis (Franch.) Rehd. can significantly improve the fluidity of the granules. Adding auxiliary materials with a dosage of ≤ 0.3% during dry granulation has good granulation effects.

[0286] Example 2-2: Establishment of the Content Determination and Analysis Method for Granules of Gaultheria yunnanensis (Franch.) Rehd.

[0287] 5.1 Determination of Chromatographic Conditions

[0288] Using octadecylsilane chemically bonded silica gel as the filler (column length is 150 mm, inner diameter is 2.1 mm, particle size is 1.8 μm); using acetonitrile as mobile phase A and using an aqueous solution containing 0.1% phosphoric acid as mobile phase B, and performing gradient elution according to the regulations in the following table; the flow rate is 0.25 mL per minute; the column temperature is 30 °C; the detection wavelength is 256 nm. The number of theoretical plates calculated by quercetin-3-O-β-D-glucuronide should not be less than 3000.

[0289] Table 29 Gradient Elution Program

[0290] Time (min) Mobile Phase A (volume%) Mobile Phase B (volume%) 0～20 5→14 95→86 20～45 14→28 86→72

[0291] 5.2 Preparation of Reference Substance Solution

[0292] Weigh an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance accurately, add methanol to make a solution containing 50 μg of quercetin-3-O-β-D-glucuronide per 1 mL, and shake well to obtain.

[0293] 5.3 Preparation of Test Solution

[0294] 5.3.1 Investigation of Different Extraction Solvents

[0295] Take an appropriate amount of Touguxiang formula granules (batch number: 240101), a total of 9 portions, each with 2 parallel samples, and place them in stoppered conical bottles, and accurately add 20mL 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 500W, frequency 40kHz) 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 solution. According to the chromatographic conditions in 5.1 above, accurately take 1μL of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and determine them. Based on the content of quercetin-3-O-β-D-pyranoglucopyranose, calculate the effect of different extraction solvents on its content and determine the best extraction solvent. The experimental results were calculated using the external standard one-point method. The specific results are shown in the table below.

[0296] Table 30 Effect of different extraction solvents on the content determination results of Touguxiang formula granules

[0297]

[0298]

[0299] From the experimental results in Table 30, it can be seen that: considering the content differences, 75 volume % methanol was selected as the best extraction solvent for subsequent research.

[0300] 5.3.2 Investigation of extraction methods

[0301] Take an appropriate amount of Touguxiang formula granules (batch number: 240101), a total of 2 portions, each with 2 parallel samples, respectively placed in a stoppered cone, 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 each test solution. According to the chromatographic conditions in 5.1 above, accurately aspirate 1μL of the reference solution and the test solution, inject into the ultra-high performance liquid chromatograph, and determine. Based on the content of quercetin-3-O-β-D-pyranoglucopyranose, calculate the effects of different extraction methods on its content and determine the best extraction method. The experimental results were calculated using the external standard one-point method, and the specific results are shown in the table below.

[0302] Table 31 Effect of different extraction methods on the content determination results of Touguxiang formula granules

[0303]

[0304] From the experimental results in Table 31, it can be seen that: taking into account the differences in their contents, combined with the experimental conditions of this laboratory and the ease of actual operation, ultrasonic treatment (power 500W, frequency 40kHz) extraction was selected as the extraction method for subsequent research.

[0305] 5.3.3 Investigation of extraction solvent volume

[0306] Take an appropriate amount of Touguxiang formula granules (batch number: 240101), a total of 7 portions, each with 2 parallel samples, and place them in stoppered cones, accurately add 75% methanol 10mL, 20mL, 30mL and 40mL in sequence to weigh the weight, respectively, 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 through a 0.22μm microporous filter membrane, place in a high liquid sample bottle, and obtain each test solution for standby use. According to the chromatographic conditions in 5.1 above, accurately draw 1μL of the reference solution and the test solution, inject into the ultra-high performance liquid chromatograph, and determine. Based on the content of quercetin-3-O-β-D-pyranoglucopyranose, calculate the effect of different extraction times on its content and determine the optimal extraction time. The experimental results were calculated using the external standard one-point method, and the specific results are shown in the table below.

[0307] Table 32 Effect of different extraction volumes on the content determination results of Touguxiang formula granules

[0308]

[0309] From the experimental results in Table 32, it can be seen that considering the solvent cost and its content difference, the extraction volume of 20 mL was selected for subsequent research.

[0310] 5.3.4 Investigation of extraction time

[0311] Take an appropriate amount of Touguxiang formula granules (batch number: 240101), a total of 4 portions, each with 2 parallel samples, and place them in stoppered cones, accurately add 20mL of 75% methanol in sequence, weigh the weight, and ultrasonically treat (power 500W, frequency 40kHz) for 15, 30, 45, and 60 minutes respectively, cool, weigh the weight again, and use 75% methanol to make up the lost weight. Take an appropriate amount of the filtrate to pass through a 0.22μm microporous filter membrane and place it in a high-liquid sample bottle to obtain each test solution for use. According to the chromatographic conditions in 5.1 above, accurately aspirate 1μL of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and determine them. The experimental results were calculated using the external standard one-point method. The specific results are shown in the table below.

[0312] Table 33 Effect of different extraction time on the content determination results of Touguxiang formula granules

[0313]

[0314]

[0315] As can be seen from the experimental results in Table 33: Considering the time cost and its content difference comprehensively, (power 500W, frequency 40kHz) for 30 minutes was selected for subsequent research.

[0316] 5.3.5 Determination of the preparation method of the test solution

[0317] According to the experimental results of sample pretreatment and referring to the method for the determination item of the Herba Elsholtziae ciliatae formula granules, the preparation method of the test solution can be determined as follows: Take an appropriate amount of this product, grind it finely and mix well. Take about 0.1g, weigh it accurately, place it in a stoppered conical flask, accurately add 20 mL of 75% methanol, weigh it, ultrasonically treat it (power 500W, frequency 40kHz) for 30 min, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0318] 5.4 Method validation

[0319] 5.4.1 Peak purity

[0320] Precisely pipette 10 μL each of the test solution and the reference solution prepared according to 5.3.5 from the Herba Elsholtziae ciliatae formula granules (batch number: 240101) into an ultra-high performance liquid chromatograph for determination, and the peak purity of the target peak can be obtained. The results are shown in Figure 15 . The peak purity matching value of quercetin-3-O-β-D-glucuronide is 996.93, which is greater than 960, indicating that its peak purity meets the analysis requirements.

[0321] 5.4.2 Investigation of linear relationship

[0322] Precisely weigh an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance, place it in a numbered 50 mL volumetric flask, add methanol to make a solution containing 209.6 μg of quercetin-3-O-β-D-glucuronide per 1 mL, shake well, and that is the quercetin-3-O-β-D-glucuronide reference substance stock solution, and store it in the refrigerator for later use. Dilute the quercetin-3-O-β-D-glucuronide 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 respectively, and that is the reference solutions of quercetin-3-O-β-D-glucuronide with different concentrations. Carry out on-machine detection according to the chromatographic conditions in 5.1 above. With the concentration as the abscissa and the peak area value as the ordinate, investigate the linear range of quercetin-3-O-β-D-glucuronide. The results of the linear investigation are shown in Table 34 below and Figure 16。

[0323] Table 34 Linear investigation of quercetin-3-O-β-D-glucuronide

[0324]

[0325] From Table 34 and Figure 16 the experimental results show that: in the concentration range of 3.5165 - 209.6 μg / mL for quercetin-3-O-β-D-glucuronide, there is a good linear relationship between the concentration of quercetin-3-O-β-D-glucuronide and the peak area value, and the correlation coefficient R 2 = 0.9993, and the linear regression equation is: y = 9.9574x + 3.5233.

[0326] 5.4.3 Precision investigation

[0327] Precisely pipette 1 μL each of the test solution prepared according to 5.3.5 from the formula granules of Elsholtzia blanda (batch number: 240101) and the reference solution of quercetin-3-O-β-D-glucuronide, inject them into the ultra-high performance liquid chromatograph for determination. Detect on the machine according to the chromatographic conditions in 5.1 above. Calculated by the external standard single-point method based on quercetin-3-O-β-D-glucuronide, the RSD(%) value of the target peak is obtained. The specific results are shown in the following table.

[0328] Table 35 Experimental results of the precision of the content determination method for the formula granules of Elsholtzia blanda

[0329]

[0330]

[0331] From the experimental results in Table 35, it can be seen that the RSD(%) value of the target peak of quercetin-3-O-β-D-glucuronide is 0.37%, which is less than 1.5%, indicating that the precision of this method is good.

[0332] 5.4.4 Repeatability investigation

[0333] Take about 0.1 g of the same batch of formula granules of Elsholtzia blanda (batch number: 240101), accurately weigh, and prepare 6 parallel portions of the test solution according to 5.3.5 above for standby. Detect on the machine according to the chromatographic conditions in 5.1 above. Calculated by the external standard single-point method based on quercetin-3-O-β-D-glucuronide, the RSD(%) value of the content of the target peak is obtained. The specific results are shown in the following table.

[0334] Table 36 Experimental results of the repeatability of the content determination method for the formula granules of Elsholtzia blanda

[0335]

[0336] As can be seen from the experimental results in Table 36, the RSD (%) value of the content of the target peak quercetin-3-O-β-D-glucuronide is 1.44% < 1.5%, indicating that the repeatability of this method is good.

[0337] 5.4.5 Accuracy test

[0338] Take 0.05 g of the known-content Gaultheria yunnanensis formula granules (batch number: 240102, quercetin-3-O-β-D-glucuronide content 9.66 mg / g), accurately weigh a total of 6 portions, and sequentially and accurately add 2.3 mL of the quercetin-3-O-β-D-glucuronide reference substance solution prepared with 75% methanol (concentration 0.2096 mg / mL), and accurately add another 17.7 mL of 75% methanol. Detect on the machine according to the chromatographic conditions in 5.1 above, inject 1 μL respectively, and calculate the content of the target peak by the external standard one-point method based on quercetin-3-O-β-D-glucuronide.

[0339] Table 37 Experimental results of the recovery test of quercetin-3-O-β-D-glucuronide in the content determination method

[0340]

[0341]

[0342] As can be seen from the experimental results in Table 37, the recovery rate range of quercetin-3-O-β-D-glucuronide in the Gaultheria yunnanensis formula granules is within the range of 95% - 102%, and the RSD% (1.16%) is less than 1.5%, indicating that the accuracy of this content determination method is good.

[0343] 5.4.6 Robustness investigation

[0344] 5.4.6.1 Investigation with different chromatographic columns

[0345] Compared the effects of three chromatographic columns, namely AmethSep C18 AQ (2.1 * 150 mm, 1.8 μm); Chromcore AQ C18 (2.1 * 150 mm, 1.8 μm), and ACQUITY HSS T3 (2.1 * 150 mm, 1.8 μm) on the peak shape and resolution of the quercetin-3-O-β-D-glucuronide chromatographic peak in the Gaultheria yunnanensis formula granules.

[0346] Take the test solution prepared from the Touguxiang formula granules (batch number: 240101) according to 5.3.5, and determine it under the chromatographic conditions in 5.3 above. Record the chromatographic data based on quercetin-3-O-β-D-glucuronide. The experimental results are as follows Figure 17 and shown in Table 38

[0347] Table 38 Influence of different chromatographic columns on the content determination results

[0348]

[0349] From Figure 17 and the experimental results in Table 38, it can be seen that: the separation effects of the three chromatographic columns are quite different. The chromatographic column ACQUITY UPLC HSS T3 can meet the requirements for the content determination of quercetin-3-O-β-D-glucuronide in Touguxiang. Its resolution, symmetry factor, and theoretical plate number are superior to those of the AmethSep C18 AQ and Chromcore AQ C18 chromatographic columns. Therefore, ACQUITY UPLC HSS T3 is selected as the preferred chromatographic column for this experiment

[0350] 5.4.6.2 Investigation of different column temperatures

[0351] Compare the influence of different column temperatures of 25 °C, 30 °C, and 35 °C on the peak shape of the quercetin-3-O-β-D-glucuronide chromatographic peak in the Touguxiang formula granules

[0352] Take the test solution prepared from the Touguxiang formula granules (batch number: 240101) according to 5.3.5, and determine it under the chromatographic conditions in 5.3 above. Record the chromatographic data based on quercetin-3-O-β-D-glucuronide. The experimental results are as Figure 18 and shown in Table 39

[0353] Table 39 Detection results of the content determination method of Touguxiang formula granules at different column temperatures

[0354]

[0355] From Figure 18 and the experimental results in Table 39, it can be seen that: the peak shapes and separation effects of the chromatographic peaks are all good at the three column temperatures. The baseline of the chromatographic elution diagram has no drift at 30 °C, and there is no significant difference in the retention time compared with the other two temperatures. Considering the tolerance of the chromatographic column and the analysis time required, the column temperature of 30 °C is selected

[0356] 5.4.6.3 Investigation of different flow rates

[0357] Compare the effects of different flow rates of 0.20 mL / min, 0.25 mL / min, and 0.30 mL / min on the peak shape and resolution of the quercetin-3-O-β-D-glucuronide chromatographic peak in the formula granules of Elsholtzia blanda Benth.

[0358] Take the test solution prepared from the formula granules of Elsholtzia blanda Benth. (batch number: 240101) according to 5.3.5, and determine it under the chromatographic conditions in 5.3 above. Record the chromatographic data based on quercetin-3-O-β-D-glucuronide. The experimental results are as Figure 19 shown in Table 40.

[0359] Table 40 Detection results of the content determination method of formula granules of Elsholtzia blanda Benth. at different flow rates

[0360]

[0361] From Figure 19 and the experimental results in Table 40, it can be seen that the peak shapes and separation effects of the chromatographic peaks are all good at the three flow rates. When the flow rate is 0.25 mL per minute, the resolution of each component is good and the baseline has no drift. In this experiment, the flow rate of 0.25 mL per minute is selected.

[0362] 5.5 Determination of the content of 3 batches of production granule samples

[0363] Take appropriate amounts of 3 batches of samples of the formula granules of Elsholtzia blanda Benth., grind them finely and mix them evenly. Take about 0.1 g, in duplicate for each batch, and prepare the test solutions of 3 batches of samples according to 5.3.5. According to the chromatographic conditions in 5.1 above, accurately pipette 1 μL of the reference solution and the test solution respectively, inject them into the ultra-high performance liquid chromatograph for determination, and thus obtain. Based on quercetin-3-O-β-D-glucuronide, the external standard method is used for calculation, and the experimental results are shown in Table 41 below.

[0364] Table 41 Determination results of the content of 3 batches of Elsholtzia blanda Benth. granules

[0365]

[0366] Examples 2-3: Establishment of the determination and analysis method for the characteristic fingerprint of Elsholtzia blanda Benth. granules

[0367] Step 1: Preparation of the reference solution

[0368] Take an appropriate amount of quercetin-3-O-β-D-glucuronide reference substance, accurately weigh it, and dissolve it in methanol to make a solution containing 50 μg of quercetin-3-O-β-D-glucuronide per 1 mL, and thus obtain.

[0369] Step 2: Preparation of the test solution

[0370] Take an appropriate amount of this product, grind it finely, take about 0.1 g, weigh it precisely, add 20 mL of 75% methanol, weigh it, ultrasonically treat it (power 500 W, frequency 40 kHz) for 30 min, let it cool, weigh it again, make up the lost weight with 75% methanol, shake well, filter, and take the subsequent filtrate, that is obtained.

[0371] Step 3: Determination of chromatographic conditions

[0372] Use octadecylsilane chemically bonded silica as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); use acetonitrile as mobile phase A and an aqueous solution containing 0.1% phosphoric acid as mobile phase B, and perform gradient elution according to the regulations in the following table; the flow rate is 0.25 mL per minute; the column temperature is 30 °C; the detection wavelength is 256 nm. The number of theoretical plates calculated with quercetin-3-O-β-D-glucuronide should be not less than 3000.

[0373] Table 42 Gradient elution program

[0374] Time (min) Mobile Phase A (volume%) Mobile Phase B (volume%) 0～20 5→14 95→86 20～45 14→28 86→72

[0375] Establishment of characteristic chromatogram

[0376] Take 3 batches of samples of Gaultheria yunnanensis formula granules, 0.1 g for each, prepare the test solution, and then detect it on the machine according to the chromatographic conditions in Step 3. The determination results are shown in the following table.

[0377] Table 43 Determination results of samples (relative retention time)

[0378] Serial Number Batch Number Peak 1 Peak 2 (S) Peak 3 1 240101 0.25 1.00 1.13 2 240102 0.25 1.00 1.13 3 240103 0.25 1.00 1.13 Average 0.25 1.00 1.13 RSD Value 0.15 0.00 0.03

[0379] The experimental results show that: the ratio of the retention times of the characteristic peaks in the 3 batches of samples is relatively stable, and the RSDs are all less than 3.0%.

[0380] To sum up, determine the UPLC chromatograms of different batches of Gaultheria yunnanensis formula granules, analyze the results using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines (2012 Edition)" recommended by the Pharmacopoeia Commission of the People's Republic of China, select their common peaks, and then use the relatively stable Peak 2 as the reference peak (i.e., the S peak), and select the relatively stable common peaks among them as the characteristic peaks.

[0381] The determination method of characteristic fingerprints was established by ultra-high performance liquid chromatography and the methodology was investigated. The characteristic fingerprints of 3 batches of Rubus parvifolius formula granules were determined, and finally the standard of characteristic fingerprints of Rubus parvifolius formula granules was determined: three characteristic peaks should appear in the characteristic fingerprints of the test sample, and the retention time of peak 2 corresponds to that of the reference substance characteristic peak of quercetin-3-O-β-D-glucuronide. The peak corresponding to the reference substance of quercetin-3-O-β-D-glucuronide is the S peak. Calculate the relative retention time of other peaks to the S peak, and the relative retention time should be within ±10% of the specified value, and the specified values are 0.25 (peak 1) and 1.13 (peak 3).

[0382] Examples 2-4: TLC identification of Rubus parvifolius formula granules

[0383] 6.1 Instruments, reagents and test drugs

[0384] Instruments: Automatic TLC imaging instrument (TlC VISUAlIZER2, CAMAG, Switzerland), double-tank developing tank, automatic TLC sampler (AUTOMATIC TlC SAMPlER4, CAMAG, Switzerland), AL-104 type electronic balance [METTLER TOLEDO Instruments (Shanghai) Co., Ltd.], KQ-500DA type ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), silica gel G TLC plates (10 cm × 10 cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20231106), silica gel G TLC plates (10 cm × 10 cm, Yantai Huayang New Materials Technology Co., Ltd., batch number: 20231204), silica gel G TLC plates (10 cm × 10 cm, Merck KGaA), silica gel G high-efficiency plates (10 cm × 10 cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20230315).

[0385] Reagents: Ethyl acetate (batch number: 20231001), methanol (batch number: 20230301), toluene (batch number: 20230201) were all purchased from Chongqing Chuandong Chemical (Group) Co., Ltd. Formic acid (batch number: 20220802) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0386] Test drugs: 3 batches of Rubus parvifolius formula granules (batch numbers: 240101, 240102 and 240103); Rubus parvifolius control crude drug (batch number: 121292-201203, purchased from: National Institutes for Food and Drug Control).

[0387] 6.2 Preparation of solutions

[0388] 6.2.1 Preparation of test sample solution

[0389] Take 0.5 g of this product, grind it finely, add 10 mL of methanol, ultrasonically treat for 10 minutes, filter, and use it as the test sample solution.

[0390] 6.2.2 Preparation of Reference Substance Solution

[0391] Take 0.5 g of the reference crude drug of Elsholtzia blanda, add 10 mL of water, reflux under heating for 60 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 5 mL of methanol, ultrasonically treat for 10 minutes, and filter to obtain the reference crude drug solution.

[0392] 6.2.3 Preparation of Negative Sample Solution

[0393] Take 0.5 g of the negative sample of the Elsholtzia blanda formula granule, grind it finely, and process it in the same method as the test sample to obtain the negative sample solution.

[0394] 6.3 Thin Layer Chromatography Conditions

[0395] Thin layer plate: Silica gel G thin layer plate. Developing solvent: Toluene, ethyl acetate, methanol, formic acid and water in a volume ratio of 1:8:1:1:1. Spotting method: Spray strip-like spotting. Development method: Develop using a double-tank developing chamber. Visual inspection: Spray with 1% aluminum trichloride ethanol solution, let stand for 10 min, and then inspect under an ultraviolet lamp (365 nm).

[0396] 6.4 Investigation of Spotting Volume

[0397] Respectively absorb the test sample solution of the Elsholtzia blanda formula granule and the reference crude drug solution of Elsholtzia blanda and spot them on the same silica gel G thin layer plate. Use toluene - ethyl acetate - methanol - formic acid - water (volume ratio of 1:8:1:1:1) as the developing solvent, develop, take out, dry in air, spray with 1% aluminum trichloride ethanol solution, let stand for 10 min, and then inspect under an ultraviolet lamp (365 nm). The results are shown in Figure 20 . In the figure, 1 is the spotting volume of 1 μL of the Elsholtzia blanda formula granule; 2 is the spotting volume of 2 μL of the Elsholtzia blanda formula granule; 3 is the spotting volume of 3 μL of the Elsholtzia blanda formula granule; 4 is the spotting volume of 1 μL of the reference crude drug of Elsholtzia blanda; 5 is the spotting volume of 2 μL of the reference crude drug of Elsholtzia blanda; 6 is the spotting volume of 3 μL of the reference crude drug of Elsholtzia blanda. It can be seen from Figure 20 that when the spotting volume is 2 μL, the key spots at the corresponding positions of the test sample and the reference crude drug are clearer and the spot shape is better. Therefore, the spotting volume of 2 μL is selected.

[0398] 6.5 Specificity Investigation of the Elsholtzia blanda Formula Granule

[0399] Respectively absorb 2 μL of the test sample solution of the Elsholtzia blanda formula granule, the reference crude drug solution of Elsholtzia blanda and the negative sample solution and spot them on the same silica gel G thin layer plate. Use toluene - ethyl acetate - methanol - formic acid - water (volume ratio of 1:8:1:1:1) as the developing solvent, develop, take out, dry in air, spray with 1% aluminum trichloride ethanol solution, let stand for 10 min, and then inspect under an ultraviolet lamp (365 nm). The experimental results are shown below Figure 21Detection conditions in the figure: Temperature: 25°C, RH: 66%; Qingdao Marine silica gel G plate; among them, 1-3 are 2 μL of the formula granules of Elsholtzia blanda (240101-240103) respectively; 4 is 2 μL of the control medicinal material of Elsholtzia blanda; 5 is 2 μL of the negative sample.

[0400] 6.6 Investigation at different temperatures

[0401] Respectively pipette 2 μL of the test solution of the formula granules of Elsholtzia blanda and the solution of the control medicinal material of Elsholtzia blanda and spot them on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-methanol-formic acid-water (volume ratio 1:8:1:1:1) as the developing agent, develop at different temperatures (5°C, 25°C and 40°C), take out, dry, spray with 1% aluminum trichloride ethanol solution, and examine under an ultraviolet lamp (365 nm) after standing for 10 min. The experimental results are as follows Figure 22 In the figure, the detection conditions of (a) are: Temperature: 40°C, RH: 66%, Qingdao Marine silica gel G plate; the detection conditions of (b) are: Temperature: 25°C, RH: 66%, Qingdao Marine silica gel G plate; the detection conditions of (c) are: Temperature: 5°C, RH: 66%; Qingdao Marine silica gel G plate; among them, 1-3 are 2 μL of the formula granules of Elsholtzia blanda (240101-240103) respectively; 4 is 2 μL of the control medicinal material of Elsholtzia blanda.

[0402] It can be seen from Figure 22 that under different temperature conditions, the corresponding positions of the chromatograms of the test solution of the formula granules of Elsholtzia blanda and the chromatogram of the control medicinal material show fluorescent main spots of the same color, with clear color development, good resolution, no trailing phenomenon, no interference in the background, and only with the increase of temperature, the corresponding spot formation positions have no obvious change, indicating that temperature has no obvious influence on the thin-layer identification of the formula granules of Elsholtzia blanda, indicating that the thin-layer identification method has good durability to temperature.

[0403] 6.7 Investigation at different humidities

[0404] Respectively pipette 2 μL of the test solution of the formula granules of Elsholtzia blanda and the solution of the control medicinal material of Elsholtzia blanda and spot them on the same silica gel G thin-layer plate. Use toluene-ethyl acetate-methanol-formic acid-water (volume ratio 1:8:1:1:1) as the developing agent, develop at different relative humidities (33%, 66% and 88%), take out, dry, spray with 1% aluminum trichloride ethanol solution, and examine under an ultraviolet lamp (365 nm) after standing for 10 min. The experimental results are as follows Figure 23In the figure, the detection conditions for (a) are: temperature: 25°C, RH: 88%, Qingdao Ocean silica gel G plate; the detection conditions for (b) are: temperature: 25°C, RH: 66%, Qingdao Ocean silica gel G plate; the detection conditions for (c) are: temperature: 25°C, RH: 33%, Qingdao Ocean silica gel G plate; among them, 1-3 are 2 μL of the formula granules of Elsholtzia ciliata (240101-240103) respectively; 4 is 2 μL of the control medicinal material of Elsholtzia ciliata.

[0405] It can be seen from Figure 23 that under different humidity conditions, the fluorescence spots of the same color appear at the corresponding positions of the chromatograms of the test samples of the formula granules of Elsholtzia ciliata and the chromatogram of the control medicinal material, and the main spots are all clearly colored, with good resolution and no tailing phenomenon, indicating that the change in humidity has no obvious effect on the thin-layer identification of the formula granules of Elsholtzia ciliata, which shows that the thin-layer identification method has good durability to humidity.

[0406] 6.8 Investigation of thin-layer plates from different manufacturers

[0407] 2 μL of the test sample solution of the formula granules of Elsholtzia ciliata and 2 μL of the solution of the control medicinal material of Elsholtzia ciliata were respectively spotted on silica gel G thin-layer plates from different manufacturers (Ocean silica gel G plate, Yantai Yinlong prefabricated silica gel G plate, Merck silica gel G plate, high-efficiency G plate), using toluene-ethyl acetate-methanol-formic acid-water (volume ratio 1:8:1:1:1) as the developing agent, developing, taking out, air-drying, spraying with 1% aluminum trichloride ethanol solution, and examining under an ultraviolet lamp (365 nm) after standing for 10 min. The experimental results are as follows Figure 24 In the figure, the detection conditions for (a) are: temperature: 25°C, RH: 66%, Qingdao Ocean silica gel G plate; the detection conditions for (b) are: temperature: 25°C, RH: 66%, Yantai Yinlong prefabricated silica gel G plate; the detection conditions for (c) are: temperature: 25°C, RH: 66%, Merck silica gel G plate; the detection conditions for (d) are: temperature: 25°C, RH: 66%, high-efficiency silica gel G plate; among them, 1-3 are 2 μL of the formula granules of Elsholtzia ciliata (240101-240103) respectively; 4 is 2 μL of the control medicinal material of Elsholtzia ciliata.

[0408] It can be seen from Figure 24 that when using silica gel thin-layer plates from different manufacturers (Qingdao Ocean silica gel G plate, Yantai Yinlong prefabricated silica gel G plate, German Merck plate and Qingdao Ocean high-efficiency silica gel G plate), the main spots of the chromatograms of the test samples of the formula granules of Elsholtzia ciliata and the chromatogram of the control medicinal material can correspond to each other, with no significant effect, indicating that the thin-layer identification method has good durability to silica gel G thin-layer plates from different manufacturers.

[0409] 6.9 Thin-layer identification chromatogram of the formula granules of Elsholtzia ciliata

[0410] 2 μL each of the test solution of Elsholtzia ciliata formula granules and the control medicinal material solution of Elsholtzia ciliata were respectively spotted on the same silica gel G thin-layer plate, developed with toluene-ethyl acetate-methanol-formic acid-water (volume ratio 1∶8∶1∶1∶1) as the developing solvent, taken out, air-dried, sprayed with 1% aluminum trichloride ethanol solution, left for 10 min, and then examined under an ultraviolet lamp (365 nm). The experimental results are as follows. Figure 25 . In the figure, the detection conditions were: temperature: 25 °C, RH: 66%; Qingdao Ocean silica gel G plate; among them, 1-3 were respectively 2 μL of Elsholtzia ciliata formula granules (240101-240103); 4 was 2 μL of the control medicinal material of Elsholtzia ciliata; 5 was 2 μL of the negative sample. From Figure 25 it can be seen that the test product chromatograms in the 3 batches of Elsholtzia ciliata formula granules showed fluorescent spots of the same color at the corresponding positions of the chromatogram of the control medicinal material, indicating that the thin-layer identification of the 3 batches of Elsholtzia ciliata formula granules all met the requirements.

[0411] Summary: Judging from the spots in the chromatogram, the separation effect of the fluorescent spots of Elsholtzia ciliata formula granules was good, and the main fluorescent spots of the same color appeared at the corresponding positions of the chromatogram of Elsholtzia ciliata formula granules and the chromatogram of the control medicinal material of Elsholtzia ciliata, and they could correspond one by one. This method can effectively identify the Elsholtzia ciliata formula granules that have lost the characteristics of the cut pieces. Through the investigation of the thin-layer chromatography methodology, the specificity and durability of this method were good, and it was applicable to the thin-layer chromatography identification of Elsholtzia ciliata formula granules.

[0412] Although the specific implementation manners of the present invention have been described above, it does not limit the protection scope of the present invention. Based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A method for preparing a radix spatholobi extract, characterized in that: The steps include: Take the Tougaxiang decoction pieces, add water and boil them, separate the solid and liquid, and obtain the decoction, which is the Tougaxiang extract.

2. The method for preparing the extract of Rhizoma Cibotii 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, 9 to 14 times the amount of water is added for the first decoction, and 7 to 12 times the amount of water is added for the second decoction; and / or, 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 extract of Rhizoma Cibotii according to claim 1 or 2, wherein: The extract of Radix Angelicae Sinensis also includes the following steps: concentrating the obtained decoction; 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 density of the concentrated solution is 1 to 1.1 g / mL; More preferably, the concentrated solution obtained by concentration is dried; further preferably, the drying is freeze drying, vacuum drying, spray drying, microwave drying or infrared drying.

4. The method for preparing the extract of Rhizoma Cibotii according to claim 3, wherein: The drying is freeze drying, which is divided into three stages: a. pre-freezing: the pre-freezing temperature is -50°C to -45°C; b. primary drying: the primary drying temperature is -30°C to 0°C; c. secondary drying: the secondary drying temperature is 5 to 25°C; Preferably, the pre-freezing time is 3 to 6 hours; more preferably, the pre-freezing time is 3 to 5 hours; the primary drying time is 10 to 14 hours; more preferably, the primary drying time is 13 to 14 hours; and / or, the secondary drying time is 5 to 7 hours; more preferably, the secondary drying time is 6 to 7 hours.

5. A method for preparing a preparation of a Radix Angelicae Pubescentis extract, characterized in that: After adding the first auxiliary material to the extract of any one of claims 1 to 3, drying is performed; preferably, the drying is spray drying; More preferably, the first auxiliary material comprises maltodextrin; More preferably, the added amount of the first auxiliary material is 5% to 15% of the amount of Touguxiangyin tablets.

6. The method for preparing the extract preparation of the Herba Lycopodii flavescentis according to claim 5, wherein: Adding the second auxiliary material to the dried material and mixing; 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%.

7. A Radix Angelicae Sinensis extract or Radix Angelicae Sinensis extract preparation, characterized in that: The extract of Radix Angelicae Sinensis is prepared by the method for preparing the extract of Radix Angelicae Sinensis according to any one of claims 1 to 4; or, the preparation of the extract of Radix Angelicae Sinensis is prepared by the method for preparing the preparation of the extract of Radix Angelicae Sinensis according to claim 5 or 6; Preferably, the extract preparation of the Herba Lycopodiellae is a Herba Lycopodiellae formula granules; more preferably, the preparation amount of the Herba Lycopodiellae formula granules is 13% to 14%, wherein the preparation amount refers to the ratio of the mass of the prepared formula granules to the mass of the raw materials of the herbal medicine; further preferably, the preparation amount of the Herba Lycopodiellae formula granules is 13.1% to 13.5%.

8. A method for detecting the content of quercetin-3-O-β-D-pyranoglucopyranoside in the Radix Angelicae Sinensis extract or the Radix Angelicae Sinensis extract preparation according to claim 7, characterized in that: The steps include: Step 1: Preparation of reference solution Weigh the quercetin-3-O-β-D-pyranoglucopyranoside reference substance and add solvent to make a solution; Step 2: Preparation of test solution Take the Radix Angelicae Sinensis extract or Radix Angelicae Sinensis extract preparation, add a solvent to perform extraction; 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.

9. The method for detecting the content of quercetin-3-O-β-D-pyranoglucopyranoside according to claim 8, 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 Radix Angelicae Pubescentis extract or a Radix Angelicae Pubescentis 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 is selected from one or more of the group consisting of methanol, 75% methanol, 50% methanol, 25% methanol, ethanol, 75% ethanol, 50% ethanol, 25% ethanol and water; And / or, preferably, in step 2, the extraction method is selected from shaking extraction, ultrasonic extraction or reflux extraction; more preferably, the extraction time is 15 to 60 minutes.

10. The method for detecting the content of quercetin-3-O-β-D-pyranoglucopyranoside according to claim 8 or 9, wherein: In step 3, the detection wavelength of the chromatogram is 190 to 400 nm; preferably, the detection wavelength is 254 to 258 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 C18 AQ or Chromcore AQ C18; And / or, in step 3, the organic phase is selected from methanol or 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 14%, and the volume percentage of mobile phase B decreased from 95% to 86%; From 20 to 45 min, the volume percentage of mobile phase A increased from 14% to 28%, and the volume percentage of mobile phase B decreased from 86% to 72%.

11. A characteristic spectrum detection method for the Radix Angelicae Sinensis extract or the Radix Angelicae Sinensis extract preparation according to claim 7, characterized in that: The steps include: Step 1: Preparation of reference solution Weigh the quercetin-3-O-β-D-pyranoglucopyranoside reference substance and add solvent to make a solution; Step 2: Preparation of test solution Take the Radix Angelicae Sinensis extract or Radix Angelicae Sinensis extract preparation, add a solvent to perform extraction; 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 the characteristic spectrum of the Radix Angelicae Pubescentis extract or the Radix Angelicae Pubescentis extract preparation.

12. The characteristic spectrum detection method according to claim 11, 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 the Radix Angelicae Pubescentis extract or the Radix Angelicae Pubescentis extract preparation, adding the 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 the liquid obtained by solid-liquid separation to obtain the test solution; Preferably, in step 2, 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; And / or, preferably, the extraction method is selected from any one of shaking extraction, ultrasonic extraction or reflux extraction; more preferably, the extraction time is 15 to 60 minutes.

13. The characteristic spectrum detection method according to claim 11 or 12, wherein: In step 3, the detection wavelength of the chromatogram is 190 to 400 nm; preferably, the detection wavelength is 254 to 258 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 C18 AQ or Chromcore AQ C18; And / or, in step 3, the organic phase is selected from methanol or 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 14%, and the volume percentage of mobile phase B decreased from 95% to 86%; From 20 to 45 min, the volume percentage of mobile phase A increased from 14% to 28%, and the volume percentage of mobile phase B decreased from 86% to 72%; Preferably, the characteristic spectrum of the extract of Radix Angelicae Sinensis or the preparation of the extract of Radix Angelicae Sinensis presents three characteristic peaks, wherein peak 2 is consistent with the retention time of the reference peak of the quercetin-3-O-β-D-pyranoglucopyranosidase reference substance, which is 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) and 1.13 (peak 3).

14. A thin layer identification method for the Radix Angelicae Pubescentis extract or the Radix Angelicae Pubescentis extract preparation according to claim 7, characterized in that: The steps include: Preparation of test solution of Radix Angelicae Pubescentis extract or Radix Angelicae Pubescentis extract preparation, spotting on thin layer plate, developing and color development; The method for preparing the test solution of the Radix Angelicae Sinensis extract or Radix Angelicae Sinensis extract preparation comprises the following steps: extracting the Radix Angelicae Sinensis extract or Radix Angelicae Sinensis 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 toluene, ethyl acetate, methanol, formic acid and water; preferably, the volume ratio of toluene, ethyl acetate, methanol, formic acid and water is 0.8-1.2:7.8-8.2:0.8-1.2:0.8-1.2:0.8-1.2; more preferably, the volume ratio of toluene, ethyl acetate, methanol, formic acid and water is 1:8:1:1:

1.

15. Use of the method for detecting the content of quercetin-3-O-β-D-pyranoglucopyranoside as claimed in any one of claims 8 to 10 in extracts of Radix Angelicae Pubescentis and preparations thereof.

16. Application of the characteristic spectrum detection method according to any one of claims 11 to 13 in the extract of Radix Angelicae Pubescentis and its preparation.

17. Application of the thin layer identification method according to claim 14 in extracts of Radix Angelicae Pubescentis and preparations thereof.

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