Method for detecting Xiaoganzi water extract and method for constructing characteristic chromatogram

Abstract

The present application relates to the field of traditional Chinese medicine analysis, in particular to the preparation method, detection method and characteristic spectrum construction method of small canthium extract. The preparation method comprises the following steps: decocting small canthium medicinal materials twice and filtering to obtain decoction, and concentrating and drying the decoction to obtain small canthium extract. The small canthium extract has high extract yield, and the mass content and transfer rate of protocatechuic acid are both high. The protocatechuic acid mass content and / or transfer rate detection method and the characteristic spectrum construction method provided by the present application have good specificity, good repeatability and good stability. The present application provides a standard for the quality control of small canthium extract, and realizes the overall quality control and effective supervision of small canthium extract and its preparations.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of traditional Chinese medicine analysis, in particular to the preparation method, detection method and characteristic spectrum construction method of Cyrtomium fortunei extract. BACKGROUND

[0002] Cyrtomium fortunei is the dried rhizome and petiole base of Cyrtomium fortunei J. Sm. of Dryopteridaceae. It is collected in spring and autumn, and then the leaves, root hairs and mud are removed and dried. It is also a minority medicine in our province. It is included in the 2003 edition of "Guizhou Province Chinese Herbal Medicine, Quality Standard of National Medicine": clearing heat and resolving toxicity, cooling blood and stopping bleeding, killing insects. It is used for cold headache, jaundice, hematemesis, epistaxis, menorrhagia, intestinal parasites. It is distributed in East China, Central South, Southwest and Hebei, Shanxi, Shaanxi, Gansu and other places. It mainly contains cyrtomin, isoquercitrin, astragalin, cyrtominetin and other components.

[0003] At present, the commodity Cyrtomium involves little Cyrtomium fortunei, and is only used in folk in some areas. There is no national medicine standard at present, and only the local Chinese herbal medicine standard such as "Guizhou Province Chinese Herbal Medicine, Quality Standard of National Medicine" is included, but it is rough and difficult to effectively evaluate the quality of Cyrtomium fortunei extract and its preparation.

[0004] So far, there is no literature report on the chemical composition and detection method of Cyrtomium fortunei. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a preparation method of Cyrtomium fortunei extract and a detection method and a characteristic spectrum construction method, which can provide quality control means for quality evaluation of Cyrtomium fortunei extract and its extract preparation.

[0006] To achieve the above object, the present application provides the following technical scheme:

[0007] In the first aspect, the present application provides a Cyrtomium fortunei extract, which comprises twice water decocting and filtering Cyrtomium fortunei medicinal materials to obtain decocting liquid, and concentrating and drying the decocting liquid to obtain Cyrtomium fortunei extract.

[0008] In some embodiments of the present application, 6-9 times the amount of water is added for the first time water decocting, and the first time water decocting time is preferably 36-50 min; 5-7 times the amount of water is added for the second time water decocting, and the second time water decocting time is preferably 25-35 min.

[0009] In some embodiments of the present application, the drying is freeze-drying, which is divided into three stages: a. pre-freezing: the pre-freezing temperature is -55 to -50°C; b. primary drying: the drying temperature is -45 to 0°C; c. secondary drying for desorption: the drying temperature is 10 to 30°C.

[0010] Preferably, the pre-freezing time is 2 to 5 hours, preferably 3 hours; the primary drying time is 35 to 45 hours, preferably 39 hours; and the secondary drying time is 5 to 7 hours, preferably 6 hours.

[0011] More preferably, the vacuum degree of the primary drying is 0 to 0.2 mbar.

[0012] In some embodiments of the present application, the concentration temperature is 60 to 65°C; and preferably, the concentration is to a concentrated liquid density of 1.05 to 1.08 g / mL.

[0013] In a second aspect, the present application further provides a small-leafed euonymus extract prepared by the above preparation method.

[0014] Preferably, the mass content of protocatechuic acid in the small-leafed euonymus extract is 1.82 to 5.00 mg / g, and more preferably, the transfer rate of protocatechuic acid is 20.14 to 56.56%.

[0015] In a third aspect, the present application further provides a detection method for the mass content of protocatechuic acid and / or the transfer rate of protocatechuic acid in the above small-leafed euonymus extract, which comprises the following steps:

[0016] (1) Preparation of reference solution

[0017] Weigh protocatechuic acid reference substance and add solvent to prepare a solution;

[0018] (2) Preparation of test solution

[0019] Weigh small-leafed euonymus extract and add solvent for extraction;

[0020] (3) Ultra-high performance liquid chromatography analysis

[0021] Use octadecylsilane-bonded silica gel as the filler, organic solvent as the mobile phase A, and 0.1% phosphoric acid aqueous solution as the mobile phase B for gradient elution, and then suck the reference solution and the test solution into the ultra-high performance liquid chromatograph for analysis.

[0022] In some embodiments of the present application, in the above preparation method, the solvent in steps (1) and (2) is selected from one or more of the following: dilute ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol, and 100% methanol, preferably dilute ethanol, and / or,

[0023] The extraction in step (2) is one of reflux extraction, shaking extraction or ultrasonic extraction, preferably ultrasonic extraction; preferably, the extraction time in step (2) is 15-60 min, preferably 30 min, and / or,

[0024] The organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; preferably, the gradient elution conditions in step (3) are as follows: 0-5 min, mobile phase A is 5%, mobile phase B is 95%; 5-15 min, mobile phase A is 5%→9%, mobile phase B is 95%→91%; 15-15.1 min, mobile phase A is 9%→5%, mobile phase B is 91%→95%; 15.1-20 min, mobile phase A is 5%, mobile phase B is 95%.

[0025] In a fourth aspect, the present application further provides a method for constructing the characteristic map of the extract of smallleaf rhubarb as described above, which comprises the following steps:

[0026] (1) Preparation of reference solution

[0027] The protocatechuic acid control sample is weighed and dissolved in a solvent to prepare a solution;

[0028] (2) Preparation of test solution

[0029] The smallleaf rhubarb extract is weighed and extracted with a solvent;

[0030] (3) Analysis by ultra-high performance liquid chromatography

[0031] The reference solution and the test solution are injected into an ultra-high performance liquid chromatograph for analysis, using octadecylsilane-bonded silica gel as the filler, organic solvent as mobile phase A, and aqueous phase as mobile phase B for gradient elution.

[0032] In some embodiments of the present application, in the preparation method described above, the solvent in steps (1) and (2) is selected from one or more of water, 50% ethanol, 75% ethanol, 95% ethanol, 50% methanol, 75% methanol and 100% methanol, preferably 100% methanol, and / or,

[0033] The extraction in step (2) is one of reflux extraction, shaking extraction or ultrasonic extraction, preferably ultrasonic extraction; preferably, the extraction time in step (2) is 15-60 min, preferably 30 min, and / or,

[0034] The organic solvent in step (3) is selected from acetonitrile or methanol, preferably acetonitrile; preferably, the aqueous phase in step (3) is selected from one of water, 0.1% formic acid solution, 0.1% phosphoric acid solution and 0.1% acetic acid solution, preferably 0.1% phosphoric acid solution; more preferably, the gradient elution conditions in step (3) are as follows: 0-5 min, mobile phase A is 5%, mobile phase B is 95%; 5-21 min, mobile phase A is 5%→12%, mobile phase B is 95%→88%; 21-24 min, mobile phase A is 12%→16%, mobile phase B is 88%→84%; 24-34 min, mobile phase A is 16%→18%, mobile phase B is 84→82%; 34-38 min, mobile phase A is 18%→60%, mobile phase B is 82%→40%; 38-38.2 min, mobile phase A is 60%→5%, mobile phase B is 40%→95%; 38.2-40 min, mobile phase A is 5%, mobile phase B is 95%; 40 min, mobile phase A is 5%.

[0035] In a fifth aspect, the present application further provides a method for identifying the extract of smallleaf galangal, comprising identifying the extract of smallleaf galangal by thin layer chromatography; the thin layer chromatography comprises the following steps: a. preparing an extract of smallleaf galangal test sample solution and a smallleaf galangal control sample solution; b. spotting the extract of smallleaf galangal test sample solution and the smallleaf galangal control sample solution on the same silica gel G thin layer plate, developing with a developing agent, taking out and air-drying, spraying with a color developing agent to develop the spots, and detecting, to obtain the identification result.

[0036] Preferably, the extract of smallleaf galangal test sample solution is prepared by the following steps: taking 0.1-1 g of the extract of smallleaf galangal, dissolving with methanol, filtering after ultrasonic treatment, to obtain the extract of smallleaf galangal test sample solution; preferably, the smallleaf galangal control sample solution is prepared by the following steps: taking 1-3 g of smallleaf galangal medicinal material, adding water and heating to reflux, filtering, evaporating the filtrate to dryness to obtain a residue, and dissolving the residue with methanol, to obtain the smallleaf galangal control sample solution.

[0037] More preferably, the developing agent is toluene-ethyl acetate-formic acid-water, and further preferably, the volume ratio of toluene, ethyl acetate, formic acid and water is 1:12:2.5:3.

[0038] In a sixth aspect, the present application further provides the use of the extract of smallleaf galangal in the preparation of health food and traditional Chinese medicine; preferably, the traditional Chinese medicine is a standard decoction, a capsule, a tablet, a granule or a formula granule.

[0039] In a seventh aspect, the present application further provides a standard decoction prepared from the extract of smallleaf galangal.

[0040] In an eighth aspect, the present application further provides a formula granule prepared from the extract of smallleaf galangal by adding excipients.

[0041] In a ninth aspect, the application further provides application of the method for determining the mass content of protocatechuic acid and / or the protocatechuic acid transfer rate in the above-mentioned smallleaf caryopteris extract or the method for constructing the characteristic atlas of the above-mentioned smallleaf caryopteris extract in the smallleaf caryopteris extract and preparation thereof.

[0042] The application has the following beneficial effects:

[0043] 1. The preparation method of the smallleaf caryopteris extract has a higher extract yield of 6.3-16.8%, the mass content of protocatechuic acid in the prepared smallleaf caryopteris extract is 1.82-5.00 mg / g, and the protocatechuic acid transfer rate is 20.14-56.56%, both the content of protocatechuic acid and the protocatechuic acid transfer rate are higher, and the accelerated stability and long-term stability test results show that the prepared smallleaf caryopteris extract has very stable indexes.

[0044] 2. The method for constructing the characteristic atlas established by the application has the characteristics of simplicity, stability, high precision and good reproducibility, and the obtained extract fingerprint atlas has more peaks and good peak type, is easy to identify, and is accurate and reliable. The method can provide a quality control means for quality evaluation of the smallleaf caryopteris extract and preparation thereof. Moreover, the method for constructing the characteristic atlas is simple, time-saving and environmentally friendly, the analysis of a batch of preparations only takes 30 min, can greatly shorten the detection and analysis time, improve the production efficiency, and make it possible to sample test a large sample and quickly monitor the quality of products.

[0045] 3. The application provides a standard for quality control of the smallleaf caryopteris extract, and realizes overall quality control and effective supervision of the smallleaf caryopteris extract and preparation thereof. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is an ultra-high performance liquid chromatogram for determining the mass content of protocatechuic acid and the protocatechuic acid transfer rate in the smallleaf caryopteris extract prepared in Example 1;

[0047] Figure 2 It is an ultra-high performance liquid chromatogram for investigating the specificity of the method for determining the mass content of protocatechuic acid and the protocatechuic acid transfer rate in the smallleaf caryopteris extract;

[0048] Figure 3 It is an ultra-high performance liquid chromatogram for investigating the peak purity of the method for determining the mass content of protocatechuic acid and the protocatechuic acid transfer rate in the smallleaf caryopteris extract;

[0049] Figure 4 It is a graph for investigating the linearity of the method for determining the mass content of protocatechuic acid and the protocatechuic acid transfer rate in the smallleaf caryopteris extract;

[0050] Figure 5 It is an ultra-high performance liquid chromatogram of different chromatographic columns in the method for detecting the mass content of protocatechuic acid and the protocatechuic acid transfer rate in the smallleaf caryopteris extract in Example 1;

[0051] Figure 6 UPLC chromatogram of the extract of Smilax riparia for the determination of the mass content and transfer rate of protocatechuic acid in Example 1 at different column temperatures;

[0052] Figure 7 UPLC chromatogram of the extract of Smilax riparia for the determination of the mass content and transfer rate of protocatechuic acid in Example 1 at different column temperatures;

[0053] Figure 8 UPLC chromatogram of the extract of Smilax riparia for the determination of the mass content and transfer rate of protocatechuic acid in Example 1 at different flow rates;

[0054] Figure 9 DAD spectrum of the extract of Smilax riparia in the range of 190-400 nm;

[0055] Figure 10 UPLC chromatogram of the extract of Smilax riparia for the determination of the characteristic spectrum in Example 1 with mobile phase of acetonitrile-0.1% phosphoric acid solution and methanol-0.1% phosphoric acid solution;

[0056] Figure 11 UPLC chromatogram of the extract of Smilax riparia for the determination of the characteristic spectrum in Example 1 with different mobile phases;

[0057] Figure 12 Characteristic spectrum of the extract of Smilax riparia prepared in Example 1;

[0058] Figure 13 Chromatogram for the specificity investigation of the characteristic spectrum determination method of the extract of Smilax riparia;

[0059] Figure 14 Chromatogram for the integrity investigation of the characteristic spectrum determination method of the extract of Smilax riparia;

[0060] Figure 15 UPLC chromatogram of the extract of Smilax riparia for the determination of the characteristic spectrum in Example 1 with different column temperatures;

[0061] Figure 16 UPLC chromatogram of the extract of Smilax riparia for the determination of the characteristic spectrum in Example 1 with different column temperatures;

[0062] Figure 17 UPLC chromatogram of the extract of Smilax riparia for the determination of the characteristic spectrum in Example 1 with different flow rates;

[0063] Figure 18 Determination results of the common peaks of the extract of Smilax riparia;

[0064] Figure 19 Characteristic spectrum of the Smilax riparia formula granules prepared in Example 5;

[0065] Figure 20 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts;

[0066] Figure 21 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts;

[0067] Figure 22-1 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 22-2 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 22-3 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts;

[0068] Figure 23-1 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 23-2 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 23-3 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts;

[0069] Figure 24-1 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 24-2 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts; Figure 24-3 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts;

[0070] Figure 25 Thin layer chromatogram of Xiaoguanchun Dispensing Granules for the identification method with different sample loading amounts. DETAILED DESCRIPTION

[0071] In order to make the purpose, technical scheme and technical effects of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application is described clearly and completely. The embodiments described below are part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art without creative labor on the basis of the embodiments in the present application belong to the protection scope of the present application.

[0072] The term "diluted ethanol" is prepared by using the test solution in Appendix XVB of Chinese Pharmacopoeia Part I, i.e. 529 ml of ethanol is diluted to 1000 ml with water, and the obtained solution contains C2H5OH of 49.5%-50.5% (ml / ml) at 20℃.

[0073] In a specific embodiment of the present application, the present application provides a preparation method of Xuanfuzong extract, comprising twice water decocting and filtering Xuanfuzong medicinal materials to obtain decocting liquid, and concentrating and freeze-drying the decocting liquid to obtain Xuanfuzong extract.

[0074] In some embodiments of the present application, the Xuanfuzong medicinal materials are moderately crushed and soaked before being decocted with water, and the soaking time is 20-40 min.

[0075] According to the present application, the filtering is hot filtering, and the filtering is performed using a 100-300 mesh sieve, preferably a 300 mesh sieve.

[0076] The sieve is a Tyler sieve (American Tyler Company) or filter cloth (Xinxiang Zhongxin Chemical Co., Ltd.).

[0077] In some embodiments of the present application, 6-9 times the amount of water is added for the first water decocting, preferably 8 times the amount of water, and preferably the first water decocting time is 36-50 min, preferably 40 min; 5-7 times the amount of water is added for the second water decocting, preferably 6 times the amount of water; and preferably the second water decocting time is 25-35 min, preferably 30 min.

[0078] In some embodiments of the present application, the concentration of the decocting liquid is performed by reduced pressure concentration, and specifically, the concentration is performed at a temperature of 60-65℃ until the density is 1.05-1.08 g / mL.

[0079] In some embodiments of the present application, the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is -55 to -50℃; b. primary drying: the drying temperature is -45 to 0℃; and c. secondary drying for desorption: the drying temperature is 10-30℃.

[0080] The pre-freezing time is 2-5 hours, preferably 3 hours; the primary drying time is 35-45 hours, preferably 39 hours, and the vacuum degree of the primary drying is 0-0.2 mbar; the secondary drying time is 5-7 hours, preferably 6 hours, and the vacuum degree of the secondary drying is 0 mbar.

[0081] In order to facilitate the freeze-dried powder to have a good shape, uniform color and retain effective substances, and improve the stability of the Xuanfuzong extract, during the primary drying, the freezing is performed at -45℃ for 2-3 h, at -35℃ for 6-7 h, at -30℃ for 18-20 h, at -25℃ for 9-10 h, at -20℃ for 2-3 h, at -10℃ for 1-2 h, and at 0℃ for 1-2 h.

[0082] In a second aspect, the present application further provides a small-leafed stachys extract prepared by the above method.

[0083] In some embodiments of the present application, the content of protocatechuic acid in the small-leafed stachys extract is 1.82-5.00 mg / g, and more preferably, the transfer rate of protocatechuic acid is 20.14-56.56%.

[0084] It can be understood that the small-leafed stachys extract can be directly used as a standard decoction, or can be added with other excipients to prepare a formula granule.

[0085] In a third aspect, the present application further provides a method for detecting the content of protocatechuic acid and / or the transfer rate of protocatechuic acid in a small-leafed stachys extract, comprising the following steps:

[0086] (1) Preparation of reference solution

[0087] A protocatechuic acid reference substance is weighed and dissolved in a solvent to prepare a solution;

[0088] (2) Preparation of test solution

[0089] A protocatechuic acid extract is weighed and extracted with a solvent;

[0090] (3) Ultra-high performance liquid chromatography analysis

[0091] Octadecylsilane-bonded silica gel is used as a filler, an organic solvent is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B for gradient elution, and the reference solution and the test solution are respectively taken and injected into an ultra-high performance liquid chromatograph for analysis.

[0092] In some embodiments of the present application, when performing ultra-high performance liquid chromatography analysis, the gradient elution conditions are as follows: 0-5 min, 5% of mobile phase A and 95% of mobile phase B, 5-15 min, 5%→9% of mobile phase A and 95%→91% of mobile phase B, 15.0-15.1 min, 9%→5% of mobile phase A and 91%→95% of mobile phase B, 15.1-20 min, 5% of mobile phase A and 95% of mobile phase B.

[0093] In some embodiments of the present application, the preparation method of the reference solution is as follows: a protocatechuic acid reference substance is weighed and precisely measured, and a solvent is added to prepare a solution containing 16 μg of protocatechuic acid per 1 mL. The solvent is selected from dilute ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol and 100% methanol, and preferably is dilute ethanol.

[0094] In some embodiments of the present invention, the preparation method of the above-mentioned test solution is as follows: Take 0.1-0.15 g of *Dryopteris crassirhizoma* extract, accurately weigh it, add 25 mL of solvent, weigh it, and extract it by reflux, shaking, or ultrasonication, preferably ultrasonic extraction. Weigh it again, replenish the weight loss with solvent, shake well, and filter to obtain the filtrate. The solvent is selected from dilute ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% methanol (v / v), and 100% methanol, preferably dilute ethanol.

[0095] In some embodiments of the present invention, the chromatographic column used for ultra-high performance liquid chromatography analysis can be a Shimadzu Shim-pack GIST-HP C10 column. 18 -AQ (column length 150mm, inner diameter 2.1mm, particle size 1.9μm)), Dikma Endeavorsil C 18 (Column length 150mm, inner diameter 2.1mm, particle size 1.8μm) or Thermo Fisher Acclaim vanquish C 18 (Column length 150mm, inner diameter 2.1mm, particle size 1.8μm); preferably Shimadzu Shim-pack GIST-HP C 18 -AQ (column length 150mm, inner diameter 2.1mm, particle size 1.9μm).

[0096] In some embodiments of the present invention, when performing ultra-high performance liquid chromatography analysis, the column temperature of the chromatographic column used is 30-40°C, preferably 40°C.

[0097] In some embodiments of the present invention, the flow rate used for ultra-high performance liquid chromatography analysis can be 0.25-0.35 ml / min, preferably 0.3 ml / min.

[0098] This invention also provides a method for constructing a characteristic map of an extract of Dryopteris crassirhizoma, comprising the following steps:

[0099] (1) Preparation of reference solution

[0100] Weigh out the protocatechuic acid reference standard and add it to a solvent to prepare a solution;

[0101] (2) Preparation of the test solution

[0102] Take the extract of Dryopteris crassirhizoma and add a solvent to extract it;

[0103] (3) Ultra-high performance liquid chromatography analysis

[0104] The reference solution and the sample solution are injected into the ultra-high performance liquid chromatograph for analysis by gradient elution with octadecylsilane silyl-bonded silica gel as the filler, organic solvent as the mobile phase A, and aqueous phase as the mobile phase B.

[0105] In some embodiments of the present application, the reference solution is prepared by accurately measuring a proper amount of protocatechuic acid control, and adding a solvent to prepare a solution containing 16 μg of protocatechuic acid per 1 mL. The solvent is selected from one or more of water, 50% (v / v) ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol, and 100% methanol, and is preferably 100% methanol.

[0106] The sample solution is prepared by accurately weighing 0.1-0.15 g of the extract of smallleaf lamiophlomis, adding 25 mL of a solvent, weighing, extracting by reflux or oscillation, or ultrasonic extraction, preferably ultrasonic extraction, weighing again, supplementing the weight loss with the solvent, shaking, and filtering to obtain the filtrate. The solvent is selected from one or more of water, 50% (v / v) ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol, and 100% methanol, and is preferably 100% methanol.

[0107] In some embodiments of the present application, the mobile phase A is acetonitrile or methanol, preferably acetonitrile, when performing ultra-high performance liquid chromatography.

[0108] In some embodiments of the present application, the aqueous phase is one of water, 0.1% formic acid solution, 0.1% phosphoric acid solution, and 0.1% acetic acid solution, preferably 0.1% phosphoric acid solution, when performing ultra-high performance liquid chromatography, and the gradient elution conditions are as follows: 0-5 min, 5% of the mobile phase A and 95% of the mobile phase B; 5-21 min, 5%→12% of the mobile phase A and 95%→88% of the mobile phase B; 21-24 min, 12%→16% of the mobile phase A and 88%→84% of the mobile phase B; 24-34 min, 16%→18% of the mobile phase A and 84%→82% of the mobile phase B; 34-38 min, 18%→60% of the mobile phase A and 82%→40% of the mobile phase B; 38-38.2 min, 60%→5% of the mobile phase A and 40%→95% of the mobile phase B; 38.2-40 min, 5% of the mobile phase A and 95% of the mobile phase B.

[0109] In some embodiments of the present application, when performing the ultra-high performance liquid chromatography analysis, the chromatographic column used can be Dikma C18-A (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm), Waters ACQUITY HSS T3 (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm) or Shim-pack GIST-HP C18-AQ (column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm), preferably Shim-pack GIST-HP C18-AQ (column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm).

[0110] In some embodiments of the present application, when performing the ultra-high performance liquid chromatography analysis, the column temperature of the chromatographic column used is 30-40°C, preferably 40°C.

[0111] In some embodiments of the present application, when performing the ultra-high performance liquid chromatography analysis, the flow rate used can be 0.2-0.3 ml / min, preferably 0.3 ml / min.

[0112] The present application also provides a thin layer chromatography identification method of the Xiaogan extract as described above, which comprises the following steps: a. preparing a Xiaogan extract sample solution and a Xiaogan reference solution; b. spotting the Xiaogan extract sample solution and the Xiaogan reference solution on the same silica gel G thin layer plate, developing with a developing agent, taking out and air-drying, spraying with a color developing agent to develop the spots, and detecting, to obtain the thin layer chromatography identification result.

[0113] Preferably, the Xiaogan extract sample solution is prepared by the following steps: taking 0.1-1 g of the Xiaogan extract, dissolving in methanol, and filtering after ultrasonic treatment to obtain the Xiaogan extract sample solution; and preferably the Xiaogan reference solution is prepared by the following steps: taking 1-3 g of Xiaogan medicinal material, adding water and heating to reflux, filtering, evaporating the filtrate to dryness to obtain a residue, and dissolving the residue in methanol to obtain the Xiaogan reference solution.

[0114] More preferably, the developing agent is toluene-ethyl acetate-formic acid-water, and further preferably, the volume ratio of toluene, ethyl acetate, formic acid and water is 1:12:2.5:3.

[0115] The present application also provides the use of the Xiaogan extract as described above in the preparation of health food and traditional Chinese medicine products; preferably, the traditional Chinese medicine product is a standard decoction, a capsule, a tablet, a granule or a formula granule.

[0116] The present application also provides a standard decoction prepared by the Xiaogan extract as described above.

[0117] The present application also provides a Xiaogan formula granule, which comprises the Xiaogan extract and pharmaceutical excipients prepared by dry granulation.

[0118] Dry granulation mainly refers to the granules prepared without wetting agents or liquid binders, which is a method of mixing drugs and excipients uniformly, compressing into large sheet or strip, and then crushing into granules of desired size. This method relies on compression force to produce binding force between ions, and its preparation methods include heavy pressure method and roller pressure method. Compared with the traditional wet granulation, the process is simple, the amount of excipients is reduced, and the drug loading capacity is improved. However, the relevant regulations of traditional Chinese medicine formula granules require that the finished product of traditional Chinese medicine formula granules should not or should only add a small amount of excipients, so it is appropriate to choose dry granulation as the granulation method of traditional Chinese medicine formula granules. The pharmaceutical excipients include but are not limited to malt dextrin, magnesium stearate, silicon dioxide, talc or microcrystalline cellulose, which can change the properties of powders, improve the appearance and flowability of materials, and control the storage and transportation, solubility, porosity and specific surface area. Considering the comprehensive performance of particle size, flowability, first product yield, bulk density, solubility and the like, the amount of excipients added in dry granulation should be ≦0.3% (based on the weight of the clear paste). Too much material will bring some performance defects, such as too much magnesium stearate, which will make the dissolution slow due to its hydrophobicity.

[0119] The application also provides application of the determination method of the mass content of protocatechuic acid and / or the protocatechuic acid transfer rate or the construction method of the characteristic spectrum of the extract of smallleaf figwort in the extract of smallleaf figwort and preparations thereof.

[0120] The advantageous effects of the present application will be further illustrated by specific examples.

[0121] The raw materials or reagents used in the present application are purchased from mainstream manufacturers in the market, and those without specified manufacturers or concentrations are analytical grade raw materials or reagents that can be commonly obtained, without special restrictions as long as they can achieve the intended effect.

[0122] In this example, the specific techniques or conditions are not specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0123] Hereinafter, the present application will be more specifically described by using examples and comparative examples, but the technical scope of the present application is not limited to these examples. It should be noted that all percentages, parts, and ratios used in the present application are based on mass, unless otherwise specifically stated. The information of raw materials and information of experimental equipment used in the examples and comparative examples are shown in Tables 1 and 2, respectively:

[0124] Table 1 Information of raw materials used in the present application

[0125] Raw material name Purity / batch number Sales company Xiaoguanzhong decoction pieces YP2106-1 Jianhe Jiujia village, Guizhou Xiaoguanzhong decoction pieces YP2106-2 Jianhe Jujixu village, Guizhou Xiaoguanzhong decoction pieces YP2106-3 Jianhe Jiyouxu village, Guizhou Xiaoguanzhong decoction pieces YP2106-4 Jianhe Zhanfeng village, Guizhou Xiaoguanzhong decoction pieces YP2106-5 Jianhe Wuliang village, Guizhou Xiaoguanzhong decoction pieces YP2106-6 Jianhe Pingxia village, Guizhou Xiaoguanzhong decoction pieces YP2106-7 Jianhe Yanme village, Guizhou Xiaoguanzhong decoction pieces YP2106-8 Jianhe Zhanggou village, Guizhou Xiaoguanzhong decoction pieces YP2106-9 Jianhe Pingwu village, Guizhou Xiaoguanzhong decoction pieces YP2106-10 Jianhe Mingdong village, Guizhou Xiaoguanzhong decoction pieces YP2106-11 Jianhe Mafeng village, Guizhou Xiaoguanzhong decoction pieces YP2106-12 Jianhe Nanming town, Guizhou Xiaoguanzhong decoction pieces YP2106-13 Jianhe Shengdong village, Guizhou Xiaoguanzhong decoction pieces YP2106-14 Jianhe Pingjiao village, Guizhou Xiaoguanzhong decoction pieces YP2106-15 Jiuzhai village, Changshun, Guizhou Protocatechuic acid 111809-201906 China Institute for Food and Drug Control

[0126] Table 2 Information of experimental equipment used in the present application

[0127]

[0128]

[0129] The solid content of Xiaogan Zhong concentrated solution in the following examples was determined by the following method:

[0130] According to the determination of extract content by hot extraction method in the 2020 edition of Chinese Pharmacopoeia (Volume IV) General Rules 2201: accurately weigh 10 g of concentrated solution, place it in an evaporating dish with constant weight, evaporate on a water bath, dry at 105°C for 3 hours, cool in a desiccator for 30 minutes, and accurately weigh the weight, calculate the solid content of the concentrated solution.

[0131] Example 1

[0132] 1. Preparation method of Xiaogan Zhong extract

[0133] (1) Take Xiaogan Zhong decoction pieces (batch number YP2106-1) 100 g, place in an electric ceramic pot, add water and decoct twice, the first time, add 8 times the amount of water, soak for 30 minutes, boil with strong fire (500W), then keep the fire small with weak fire (200W) for 40 minutes, filter the decoction with a 300 mesh sieve while hot; the second time, add 6 times the amount of water, heat to boil with strong fire, then keep the fire small with weak fire for 30 minutes, filter the decoction with a 300 mesh sieve while hot, and combine the two filtrates;

[0134] (2) Transfer the filtrate to a 2000 ml round-bottom flask, use a rotary evaporator to concentrate under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.080 to -0.090 MPa) to 100 ml of extract, measure its density as 1.06 g / mL and extract yield as 13.62%; under magnetic stirring, divide into 10 ml brown schlenk bottles, each bottle contains 1 ml, half-capped, after the division is complete, transfer to a vacuum freeze dryer for freeze-drying, first pre-freeze at -50°C for 3 hours, then dry once at -45°C, -35°C, -30°C, -25°C, -20°C, -10°C and 0°C, vacuum degree is 0.2 mbar, drying time is 2 hours, 6 hours, 18 hours, 9 hours, 2 hours, 1 hour and 1 hour respectively, then dry twice at 10°C, 20°C and 30°C, vacuum degree is 0 mbar, drying time is 1 hour, 1 hour and 4 hours respectively, take out, roll aluminum cap, get Xiaogan Zhong extract, the extract yield of Xiaogan Zhong extract is calculated by the following formula,

[0135] Extract yield = extract weight * solid content of extract / decoction piece feed amount * 100%.

[0136] 2. Determination method of the mass content of protocatechuic acid in Xiaoganzi extract and its transfer rate

[0137] 2.1 Optimization of the conditions for the detection method

[0138] 2.1.1 Preparation of the reference solution

[0139] Take the protocatechuic acid reference substance, accurately weigh, and place it in a numbered 10 ml volumetric flask. Add dilute ethanol to make a reference solution containing 16 μg of protocatechuic acid per 1 ml, and shake well to obtain it.

[0140] 2.1.2 Preparation of the test solution

[0141] Take about 0.12 g of Xiaoganzi extract prepared above, accurately weigh, and place it in a stoppered conical flask. Add 25 mL of solvent, weigh, extract for a period of time, cool, re-weigh, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, take an appropriate amount of the filtrate, pass it through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle.

[0142] 2.1.3 Ultra-high performance liquid chromatography analysis

[0143] Take octadecylsilane-bonded silica gel as the filler (Shim-pack GIST-HP C 18 18, column length 150 mm, inner diameter 2.1 mm, particle size 1.9 μm); take acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, perform gradient elution according to the provisions in Table 3; the column temperature is 40°C; the flow rate is 0.3 ml / min, and the detection wavelength is 259 nm. The theoretical plate number calculated according to the protocatechuic acid peak should not be less than 3000. Take 10 μL of the reference solution in 2.1.1 and the test solution in 2.1.2, respectively, and inject them into the ultra-high performance liquid chromatograph for analysis.

[0144] Table 3 Elution gradient

[0145] Time (min) Mobile phase A (%) Mobile phase B (%) 0-5 5 95 5-15 5→9 95→91 15-15.1 9→5 91→95 15.1-20 5 95

[0146] 2.1.4 The mass content of protocatechuic acid and the transfer rate of protocatechuic acid are calculated according to the following formula:

[0147] Wherein,

[0148] w 原儿茶酸 = A 供 × C 对 × V 供 × 10 -3 / (m 供 × A 对 )

[0149] w 饮 = A 饮供 × C对 x V 饮供 x 10 -3 / (m 饮供 x A 对 )

[0150] The transfer rate of protocatechuic acid = (m 提 x w 原儿茶酸 ) / (m 饮 x w 饮 )

[0151] In the formula:

[0152] w 原儿茶酸 —mass content of protocatechuic acid in the extract of Xianhuangzong, mg / g;

[0153] w 饮 —mass content of protocatechuic acid in the decoction piece of Xianhuangzong, mg / g;

[0154] A 对 —absorption peak area of protocatechuic acid in the ultra-high performance liquid chromatogram of the reference solution;

[0155] A 供 —absorption peak area of protocatechuic acid in the ultra-high performance liquid chromatogram of the test solution;

[0156] A 饮供 —absorption peak area of protocatechuic acid in the ultra-high performance liquid chromatogram of the decoction piece test solution;

[0157] C 对 —concentration of the reference solution control, μg / mL;

[0158] m 供 —absolute dry mass of the extract of Xianhuangzong in the test solution, g;

[0159] m 饮供 —absolute dry mass of the decoction piece of Xianhuangzong in the test solution, g;

[0160] V 供 —volume of the test solution, ml;

[0161] V 饮供 —volume of the decoction piece test solution, ml;

[0162] m 提 —absolute dry mass of the extract of Xianhuangzong prepared, g;

[0163] m 饮 —absolute dry mass of the raw material decoction piece of Xianhuangzong, g.

[0164] 2.1.5 Optimization of preparation conditions

[0165] Ⅰ Investigation of different extraction solvents

[0166] Take about 0.12 g of the extract of smallleaf fig prepared in Example 1, accurately weigh, 2 samples in parallel for each, respectively placed in a conical flask with a plug, accurately add 25 ml of water, 50% (v / v) ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol and 100% methanol in turn, weigh, ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 min, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, and the test sample solution is obtained. Accurately pipette 2 μL of the control sample solution and the test sample solution respectively, inject into the ultra-high performance liquid chromatograph, and perform chromatographic analysis according to the chromatographic conditions in 2.1.3. Calculate the influence of different extraction solvents on the content according to the above method, determine the best extraction solvent, and the specific results are shown in Table 4:

[0167] Table 4 Influence of different extraction solvents on the content of protocatechuic acid in smallleaf fig extract

[0168]

[0169]

[0170] From Table 4, it can be seen that different solvents have significant influence on the mass content of protocatechuic acid in smallleaf fig extract. Considering the content and target peak shape, 50% (v / v) ethanol is selected as the extraction solvent.

[0171] Ⅱ Investigation of different extraction methods

[0172] Take about 0.12 g of the extract of smallleaf fig prepared in Example 1, accurately weigh, 2 samples in parallel for each, respectively placed in a conical flask with a plug, accurately add 25 ml of water, 50% (v / v) ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol and 100% methanol in turn, weigh, ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 min, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, and the test sample solution is obtained. Accurately pipette 2 μL of the control sample solution and the test sample solution respectively, inject into the ultra-high performance liquid chromatograph, and perform chromatographic analysis according to the chromatographic conditions in 2.1.3. Calculate the influence of different extraction solvents on the content according to the above method, determine the best extraction solvent, and the specific results are shown in Table 4:

[0173] Table 5 Influence of different extraction methods on the content of protocatechuic acid in smallleaf fig extract

[0174]

[0175]

[0176] From Table 5, it can be seen that the different extraction methods have no obvious effect on the content of protocatechuic acid in Xiaoganqun extract. Considering the sample processing efficiency, ultrasonic extraction is selected for extraction.

[0177] III. Investigation of different extraction times

[0178] About 0.12 g of Xiaoganqun extract prepared in Example 1 was precisely weighed, and 3 samples were prepared in parallel, each of which was placed in a conical flask with a stopper. Then, 25 mL of 50% (v / v) ethanol was precisely added, the weight was determined, and ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 15 min, 30 min, and 60 min, respectively. After cooling to room temperature, the weight was determined again, and the lost weight was made up with 50% (v / v) ethanol. Then, the mixture was shaken, filtered, and the filtrate was collected. An appropriate amount of the filtrate was passed through a 0.22 μm microporous filter membrane and placed in a liquid sample bottle to obtain each test solution. 2 μl of the control solution and the test solution were precisely taken and injected into the high performance liquid chromatograph for chromatographic analysis according to the chromatographic conditions in 2.1.3. The content of protocatechuic acid was calculated according to the above method to determine the effect of different extraction times on the content. The specific results are shown in Table 6:

[0179] Table 6 Effect of different extraction times on the content of protocatechuic acid in Xiaoganqun extract

[0180]

[0181] From the results in Table 6, it can be seen that different extraction times have no obvious effect on the content of protocatechuic acid in Xiaoganqun extract. Considering the time cost and the content difference, ultrasonic extraction for 15 min is selected.

[0182] The Xiaoganqun extract test solution and the Xiaoganqun decoction piece test solution were prepared using the above optimized conditions, and the method was as follows:

[0183] Preparation of Xiaoganqun extract test solution

[0184] About 0.12 g of Xiaoganqun extract prepared in Example 1 was precisely weighed and placed in a conical flask with a stopper. Then, 25 mL of 50% ethanol was added, the weight was determined, and ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 15 min. After cooling, the weight was determined again, the lost weight was made up with the corresponding solvent, the mixture was shaken, filtered, and the filtrate was collected. An appropriate amount of the filtrate was passed through a 0.22 μm microporous filter membrane and placed in a liquid sample bottle to obtain the Xiaoganqun extract test solution.

[0185] Preparation of Xiaoganqun decoction piece test solution

[0186] Take Xiaoguanzhong decoction pieces (batch number YP2106-1) about 1.0g, accurately weigh, put into a conical flask with a stopper, add 25mL 50% (v / v) ethanol, weigh, ultrasonic treatment (power 500W, frequency 40kHz) for 15min, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take out the filtrate, pass through 0.22μm microporous filter membrane, place in a liquid sample bottle, to obtain Xiaoguanzhong decoction piece test sample solution.

[0187] According to the chromatographic analysis method in 2.1.3 ultra-high performance liquid chromatography analysis, chromatographic analysis was carried out on Xiaoguanzhong extract test sample solution and Xiaoguanzhong decoction piece test sample solution prepared under the above optimized conditions, and the ultra-high performance liquid chromatography spectrum of Xiaoguanzhong extract prepared in Example 1 is shown in Figure 1 According to the mass content calculation formula of protocatechuic acid and the calculation formula of protocatechuic acid transfer rate, the mass content of protocatechuic acid is 3.9mg / g, and the transfer rate of protocatechuic acid is 36.56%.

[0188] 2.2 Investigation of the methodology of the detection method

[0189] 2.2.1 Investigation of specificity

[0190] Take 2μl of Xiaoguanzhong extract test sample solution prepared under the above optimized conditions in 2.1 and 2μl of protocatechuic acid reference solution in 2.1.1 respectively, and determine according to the chromatographic conditions in 2.1.3 above, the results are shown in

[0191] It can be seen from Figure 2 that the analysis method has good specificity for the determination of the content of protocatechuic acid in Xiaoguanzhong extract.

[0192] Figure 2

[0193] 2.2.2 Peak purity

[0194] Take 2μl of Xiaoguanzhong extract test sample solution prepared under the above optimized conditions in 2.1 and 2μl of protocatechuic acid reference solution in 2.1.1 respectively, and determine according to the chromatographic conditions in 2.1.3 above, the results are shown in Figure 3 and Table 7.

[0195] Table 7 Matching values of marker peaks and peak purity

[0196]

[0197] From Figure 3 and Table 7, it can be seen that the purity value of the target component is 1000, which is greater than 996, indicating that the peak purity meets the analysis requirements.

[0198] ​​2.2.3 Linearity test

[0199] A certain amount of protocatechuic acid reference material was precisely weighed, placed in a numbered 20 ml volumetric flask, and 50% (v / v) ethanol was added to prepare a reference solution containing 0.1 mg of protocatechuic acid per 1 ml. The solution was shaken well and stored in the refrigerator for standby.

[0200] The protocatechuic acid reference stock solution was taken and diluted 200, 40, 20, 10, 5, 4, and 2 times to obtain protocatechuic acid control solutions of different concentrations. The solutions were detected by HPLC according to the chromatographic conditions in 2.1.3. The concentration was taken as the abscissa and the peak area value was taken as the ordinate to investigate the linear range of protocatechuic acid. The linear test results are shown in Table 8. Figure 4 and Table 8.

[0201] Table 8 Linear test of protocatechuic acid

[0202] Number Protocatechuic acid concentration (μg / ml) Peak area value (mAU) Protocatechuic acid-1 0.49998 0.36205 Protocatechuic acid-2 2.4999 0.85875 Protocatechuic acid-3 4.9998 1.6056 Protocatechuic acid-4 9.9996 3.3376 Protocatechuic acid-5 19.999 6.6900 Protocatechuic acid-6 24.999 8.37245

[0203] From Figure 4 and Table 8, it can be seen that protocatechuic acid has a good linear relationship with the peak area value in the range of 0.49998 μg / ml to 49.999 μg / ml, and the correlation coefficient r = 0.9998.

[0204] 2.2.4 Precision test

[0205] The Xiaoganzi extract sample solution prepared under the optimized conditions in 2.1 was taken, and 6 parallel samples were prepared, each with 2 injections of 2 μL. The samples were injected into the ultra-high performance liquid chromatograph, and the chromatographic analysis was performed according to the chromatographic conditions in 2.1.3. The content of protocatechuic acid was calculated according to the above method, and the RSD (%) value of the target peak was calculated using the external standard one-point method. The specific results are shown in Table 9.

[0206] Table 9 Precision test results of Xiaoganzi extract content determination method

[0207]

[0208]

[0209] From Table 9, it can be seen that the RSD (%) value of the target peak protocatechuic acid is 0.84, which is less than 2.0%, indicating that the method has good precision.

[0210] 2.2.5 Stability test

[0211] Take the extract of small canal crowd prepared in example one, according to the optimized conditions in 2.1 to prepare the test solution of small canal crowd extract, according to 2.1.3 chromatographic conditions for chromatographic analysis, respectively in test solution preparation after 0, 1, 2, 4, 6, 8, 12, 24 hours sample, sample volume 2 μl, according to the above method to calculate the content of protocatechuic acid, calculated by protocatechuic acid, using external standard point method to calculate the RSD (%) value of target peak content, the specific results are shown in table 10.

[0212] Table 10 stability investigation

[0213]

[0214] From table 10, the RSD (%) value of the target peak protocatechuic acid content within 24h is 1.99%, less than 2.0%, indicating that the solution is stable within 24 hours.

[0215] 2.2.6 repeatability investigation

[0216] Take the extract of small canal crowd prepared in example one, according to the optimized conditions in 2.1 to prepare the test solution of small canal crowd extract, according to 2.1.3 chromatographic conditions for chromatographic analysis, respectively in test solution preparation after 0, 1, 2, 4, 6, 8, 12, 24 hours sample, sample volume 2 μl, according to the above method to calculate the content of protocatechuic acid, calculated by protocatechuic acid, using external standard point method to calculate the RSD (%) value of target peak content, the specific results are shown in table 10.

[0217] Table 11 repeatability experiment results

[0218]

[0219] From table 11, the RSD (%) value of the target peak protocatechuic acid content is 1.81%, less than 2.0%, indicating that the method has good repeatability.

[0220] 2.2.7 intermediate precision investigation

[0221] Select 6 experimenters, respectively in two different dates and two different chromatograph (Thermo Vanquish F and Agilent HPLC 1290InfinityⅡ) operation, take the test solution of small canal crowd extract prepared in 2.1 after optimization, according to 2.1.3 chromatographic conditions for chromatographic analysis, according to the above method to calculate the content of protocatechuic acid, calculated by protocatechuic acid in test solution, using external standard point method to calculate the RSD (%) value of target peak content, the specific results are shown in table 12.

[0222] Table 12 determination method intermediate precision experiment results

[0223]

[0224]

[0225] From Table 12, the intermediate precision RSD (%) value of the target peak protocatechuic acid content is 1.61%, which is less than 2.0%, indicating that the method has good intermediate precision.

[0226] 2.2.8 Accuracy test

[0227] Take about 0.06 g of known content of Xuanfuzhong extract (protocatechuic acid content 4 mg / g), precisely weigh a total of 6 portions, and then precisely add 1 mL of protocatechuic acid reference material (concentration 0.238 mg / mL) prepared by 50% (v / v) ethanol, and then precisely add 24 mL of 50% (v / v) ethanol, weigh, ultrasonic treatment (power 500 W, frequency 40 kHz) for 15 min, cool, re-weigh, make up the weight loss with 50% ethanol, shake well, filter, take the filtrate, take an appropriate amount of the filtrate through a 0.22 μm microporous filter membrane, and place it in a liquid sample bottle to obtain the Xuanfuzhong extract test sample solution. Precisely take 2 μL of protocatechuic acid reference material solution and Xuanfuzhong extract test sample solution respectively, inject into the ultra-high performance liquid chromatograph, perform chromatographic analysis according to the chromatographic conditions in 2.1.3, calculate the protocatechuic acid content according to the above method, calculate the content of the target peak by the external standard one-point method, and calculate the recovery rate and RSD (%) value according to the following formula, and the results are shown in Table 13.

[0228]

[0229] Table 13 Results of sample addition recovery experiment of content determination method

[0230]

[0231] From Table 13, the recovery rate of protocatechuic acid in Xuanfuzhong extract is in the range of 92%-105%, and the RSD% (1.32%) is less than 2.0%, indicating that the accuracy of the content determination method is good.

[0232] 2.2.9 Durability investigation

[0233] 2.2.9.1 Investigation of different chromatographic columns

[0234] The effects of Shimadzu, Dima, and Thermo Fisher chromatographic columns on the peak shape and separation degree of protocatechuic acid in Xuanfuzhong extract were compared.

[0235] Take the Xuanfuzhong extract test sample solution prepared under the optimized conditions in 2.1, and determine according to the chromatographic conditions in 2.1.3, record the chromatographic data. The experimental results are shown in Figure 5 and Table 14.

[0236] Table 14 Effects of different chromatographic columns on the chromatographic peak of protocatechuic acid in Xianhuangzong extract

[0237]

[0238] From Figure 5 As can be seen from Table 14, different chromatographic columns have different effects on the resolution, peak shape and purity of protocatechuic acid in Xianhuangzong extract, and the three brands of chromatographic columns can all achieve good separation of the target peak. According to the experimental results, Shimadzu Shim-pack GIST-HP C18-AQ chromatographic column was selected considering the experimental results and the chromatographic column.

[0239] 2.2.9.2 Investigation of different chromatographs

[0240] According to the existing equipment in the laboratory, Thermo Vanquish F ultra-high performance liquid chromatograph and Agilent UPLC 1290 Infinity II ultra-high performance liquid chromatograph were selected to compare the effects of the two chromatographs on the peak shape and resolution of protocatechuic acid in Xianhuangzong extract.

[0241] The Xianhuangzong extract sample solution prepared under the optimized conditions in 2.1 was determined according to the chromatographic conditions in 2.1.3, and the chromatographic data was recorded in terms of protocatechuic acid. The experimental results are shown in Figure 6 and Table 15.

[0242] Table 15 Results of instrument durability investigation

[0243]

[0244]

[0245] From Figure 6 As can be seen from Table 15, the analysis method has good durability on different chromatographs. The variation of the chromatograph can meet the requirements of system adaptability.

[0246] 2.2.9.3 Investigation of different column temperatures

[0247] The Xianhuangzong extract sample solution prepared under the optimized conditions in 2.1 was determined according to the chromatographic conditions in 2.1.3, and the effects of different column temperatures of 30℃, 35℃ and 40℃ on the peak shape and resolution of protocatechuic acid in Xianhuangzong extract were compared. The experimental results are shown in Figure 7 and Table 16.

[0248] Table 16 Detection results of different column temperatures on the content determination method of Xianhuangzong extract

[0249] Column temperature / ℃ Index component Resolution / R Theoretical plate number Purity value 30 Protocatechuic acid 18.76 11983 971 35 Protocatechuic acid 12.54 11884 999 40 Protocatechuic acid 13.18 11839 999

[0250] FromFigure 7 As shown in Table 16, the peak shape and separation effect were good at all three column temperatures. At 30℃, the baseline of the chromatogram showed no drift, and the peak shape was not significantly different compared to the other two temperatures. Considering the column's tolerance and the analysis time required, a column temperature of 40℃ was chosen.

[0251] 2.2.9.4 Investigation of different flow velocities

[0252] The *Dryopteris crassirhizoma* extract test solution prepared under the optimized conditions in section 2.1 was analyzed according to the chromatographic conditions in section 2.1.3. The mobile phase flow rates were selected as 0.25 ml / min, 0.3 ml / min, and 0.35 ml / min, respectively. The effects of different flow rates (0.25 ml / min, 0.3 ml / min, and 0.35 ml / min) on the peak shape and resolution of protocatechuic acid in the *Dryopteris crassirhizoma* extract were compared. The experimental results are as follows: Figure 8 And as shown in Table 17.

[0253] Table 17 Results of the determination of the content of Dryopteris crassirhizoma extract by different flow rates

[0254] Flow rate / (ml / min) Index component Resolution / R Theoretical plate number Purity value 0.25 Protocatechuic acid 6.21 13387 999 0.30 Protocatechuic acid 13.18 11839 999 0.35 Protocatechuic acid 12.88 11513 999

[0255] Depend on Figure 8 As shown in Table 17, the chromatographic peak shape and separation effect were good at all three flow rates. At a flow rate of 0.3 ml / min, the parameters of protocatechuic acid were best, with no baseline drift and no significant difference in peak shape. Therefore, a flow rate of 0.3 ml / min was selected for this experiment.

[0256] 3. Method for constructing the characteristic spectrum of Dryopteris crassirhizoma extract

[0257] 3.1 Optimization of the conditions for the construction method

[0258] 3.1.1 Preparation of reference solution

[0259] Take an appropriate amount of protocatechuic acid reference standard, accurately weigh it, add methanol to prepare a reference solution containing 16 μg of protocatechuic acid per 1 ml, shake well, and the solution is obtained.

[0260] 3.1.2 Preparation of the test solution

[0261] Take about 1g of the Dryopteris crassirhizoma extract prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, add 25ml of solvent, weigh it, sonicate it (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, replenish the lost weight with the corresponding solvent, shake it well, filter it, take out the filtrate, and take an appropriate amount of the filtrate through a 0.22μm microporous membrane and place it in a liquid sample bottle.

[0262] 3.1.3 Ultra-high performance liquid chromatography analysis

[0263] Take 2 μL of the reference solution in 3.1.1 and the test sample solution in 3.1.2 respectively, inject into the ultra-high performance liquid chromatograph for analysis, use octadecylsilane bonded silica gel as the filler; use organic phase as mobile phase A, and water phase as mobile phase B, perform gradient elution according to the provisions in Table 18; perform determination at a certain column temperature and flow rate under a certain detection wavelength.

[0264] Table 18 Gradient elution

[0265] Time (min) Mobile phase A (%) Mobile phase B (%) 0-5 5 95 5-21 5→12 95→88 21-24 12→16 88→84 24-34 16→18 84→82 34-38 18→60 82→40 38-38.2 60→5 40→95 38.2-40 5 95

[0266] 3.1.4 Optimization of the conditions of the construction method

[0267] 3.1.4.1 Determination of the detection wavelength

[0268] Take about 0.12 g of the extract of smallleaf rhododendron prepared in Example One, accurately weigh, place in a conical flask with a stopper, add 100% methanol 25 ml, weigh, ultrasonic treat (power 500 W, frequency 40 kHz) for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent, shake well, filter, take the filtrate, obtain the test sample solution of the extract of smallleaf rhododendron, record the absorption spectrum in the range of 190-400 nm, as shown in Figure 9

[0269] As can be seen from Figure 9 , at the wavelength of 252 nm, the test sample solution of the extract of smallleaf rhododendron can detect more chromatographic peak information and has less baseline noise interference, therefore, 252 nm is selected as the detection wavelength.

[0270] 3.1.4.2 Optimization of the mobile phase

[0271] ① Refer to the method in 3.1.3, the chromatographic column is Shim-pack GIST-HP C18-AQ (150 mm, 2.1 mm, 1.9 μm) of Shimadzu; the column temperature is 40 ℃, the flow rate of the mobile phase is 0.3 ml / min, the test sample solution of the extract of smallleaf rhododendron refers to the preparation method in 3.1.4.1, A is organic phase (100% methanol or 100% acetonitrile), B is 0.1% phosphoric acid aqueous solution, the gradient elution table is shown in Table 19, and the results are shown in Figure 10

[0272] Table 19 Gradient elution table

[0273] Time (min) Mobile phase A (%) Mobile phase B (%) 0～20 5→100 95→0 20～20.1 100→5 0→95 20.1～22 5 95

[0274] As can be seen from Figure 10 ​​It can be seen that the elution ability of acetonitrile-0.1% phosphoric acid solution is stronger than that of methanol-0.1% phosphoric acid solution, so acetonitrile-0.1% phosphoric acid solution is selected for condition exploration, and then various acids used in the mobile phase are investigated.

[0275] 2. Refer to the method in 3.1.3, the chromatographic column is Shim-pack GIST-HP C18-AQ (150 mm, 2.1 mm, 1.9 μm), the column temperature is 40 ℃, the flow rate of the mobile phase is 0.3 ml / min, the Xiaogan Zhong extract sample solution is prepared according to 3.1.4.1, and acetonitrile-water, acetonitrile-0.1% phosphoric acid, acetonitrile-0.1% formic acid and acetonitrile-0.1% acetic acid are used as the mobile phase respectively for chromatographic analysis, and the results are shown in Table 2. Figure 11

[0276] It can be seen from Table 2 that the peak shape is better with acid than without acid, and the peak shape is best when the mobile phase is acetonitrile-0.1% phosphoric acid solution system, so acetonitrile-0.1% phosphoric acid solution is selected for elution. Figure 11

[0277] 3.1.4.3 Investigation of different extraction solvents

[0278] About 0.12 g of Xiaogan Zhong extract prepared in Example 1 was accurately weighed, 2 samples in parallel were placed in a conical flask with a plug, 25 ml of water, 50% (v / v) ethanol, 75% (v / v) ethanol, 95% (v / v) ethanol, 50% (v / v) methanol, 75% (v / v) methanol and 100% methanol were accurately added respectively, the weight was determined, ultrasonic treatment (power 500 W, frequency 40 kHz) was carried out for 30 min, it was cooled and weighed again, the weight loss was made up with the corresponding solvent, shaken uniformly, filtered, and the filtrate was taken as the sample solution. According to the method in 3.1.3, the sample was injected, the chromatographic conditions were as follows: the mobile phase A was 100% acetonitrile, the mobile phase B was 0.1% phosphoric acid solution, the chromatographic column was Shim-pack GIST-HP C18-AQ (150 mm, 2.1 mm, 1.9 μm), the column temperature was 40 ℃, the flow rate was 0.3 ml / min, and the peak area was recorded. The results are shown in Table 20.

[0279] Table 20 Comparison of extraction efficiency of different extraction methods (peak area / sample weight)

[0280] Characteristic peak Water 95% ethanol 75% ethanol 50% ethanol 100% methanol 75% methanol 50% methanol Peak 1 188 196 196 191 199 194 190 Peak 2 116 104 114 115 113 115 115 Peak 3 39 33 37 37 37 39 37 Peak 4 18 24 24 24 23 24 24

[0281] It can be seen from Table 20 that the extraction efficiency is higher when the extraction solvent is 100% methanol, so 100% methanol is selected as the extraction solvent.

[0282] The above optimized conditions are used to determine the characteristic spectrum of Xiaogan Zhong extract prepared in Example 1, and the method is as follows:​​

[0283] Preparation of test solution

[0284] Take approximately 0.12 g of the Dryopteris crassirhizoma extract prepared in Example 1, accurately weigh it, place it in a stoppered conical flask, add 25 ml of 100% methanol, weigh it, sonicate it (power 500 W, frequency 40 kHz) for 30 min, cool it, weigh it again, replenish the lost weight with the appropriate solvent, shake it well, filter it, and take out the filtrate. Take an appropriate amount of the filtrate and filter it through a 0.22 μm microporous membrane, place it in a liquid sample bottle, and obtain the Dryopteris crassirhizoma extract test solution.

[0285] 10 μL of the reference solution from section 3.1.1 and the above-mentioned test solution were injected into an ultra-high performance liquid chromatograph for analysis. Octadecylsilane-bonded silica gel (Shimadzu GIST-HP C18-AQ) was used as the stationary phase; 100% acetonitrile was used as mobile phase A, and 0.1% phosphoric acid aqueous solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 18; the column temperature was 40℃, and the flow rate was 0.3 mL / min. The results are as follows: Figure 12 As shown.

[0286] Depend on Figure 12 It can be seen that there are 4 characteristic peaks in the characteristic spectrum of the extract of Dryopteris crassirhizoma. The peak corresponding to the reference is the S peak. The relative retention time of each characteristic peak and the S peak is calculated. The relative retention time of peak 1 (S) is 1.00, the relative retention time of peak 2 is 2.15, the relative retention time of peak 3 is 2.53, and the relative retention time of peak 4 is 4.27.

[0287] 3.2 Methodological Examination of the Construction Method

[0288] 3.2.1 Specificity Examination

[0289] One μl each of the *Dryopteris crassirhizoma* extract test solution prepared under the optimized conditions in 3.1 and 100% methanol solvent was injected into the liquid chromatograph, and the determination was performed under the chromatographic conditions optimized in 3.1. The results are as follows: Figure 13 As shown.

[0290] Depend on Figure 13 It can be seen that the solvent does not interfere with the characteristic peaks in the spectrum of the Dryopteris crassirhizoma extract.

[0291] 3.2.2 Holistic Examination

[0292] Take 1 μl of the *Dryopteris crassirhizoma* extract test solution prepared under the optimized conditions in 3.1, and perform the determination under the optimized chromatographic conditions in 3.1. Extend the elution time by one time at the mobile phase ratio at the gradient endpoint. The characteristic chromatogram is shown below. Figure 14 As shown.

[0293] From Figure 14 It can be seen that there is no obvious chromatographic peak after the elution time is doubled, which indicates that the chromatographic condition basically meets the principle of maximum information.

[0294] 3.2.3 Precision examination

[0295] The test sample solution of the Xuanfuzhong extract prepared under the optimized conditions in 3.1 was determined by the chromatographic condition after optimization in 3.1, and the sample was injected for 6 times with the injection volume of 1 μl. The consistency of the relative retention time and relative peak area of the characteristic peaks (4 characteristic peaks were calibrated, and peak 1 was used as the reference peak) was examined. The experimental results are shown in Table 21.

[0296] Table 21 Precision results of the characteristic chromatogram of the Xuanfuzhong extract (relative retention time)

[0297]

[0298] From Table 21, it can be seen that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, which indicates that the precision of the instrument is good.

[0299] 3.2.4 Stability examination

[0300] The Xuanfuzhong extract prepared in Example 1 was taken, and the test sample solution of the Xuanfuzhong extract was prepared according to the conditions after optimization in 3.1. The sample was determined by the chromatographic condition after optimization in 3.1, and the sample was injected at 0, 2, 4, 6, 8, 12 and 24 hours after the preparation of the test sample solution with the injection volume of 1 μl. The consistency of the relative retention time and relative peak area of the characteristic peaks was examined (4 characteristic peaks were calibrated, and peak 1 was used as the reference peak). The experimental results are shown in Table 22.

[0301] Table 22 Stability results of the characteristic chromatogram of the Xuanfuzhong extract (relative retention time)

[0302] Serial number 1 2 3 4 5 6 Average value RSD / % Peak 1 (S) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 Peak 2 2.17 2.17 2.17 2.17 2.17 2.16 2.17 0.19 Peak 3 2.56 2.56 2.56 2.56 2.56 2.55 2.56 0.23 Peak 4 4.32 4.31 4.32 4.32 4.31 4.29 4.31 0.30

[0303] From Table 22, it can be seen that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, which indicates that the sample solution is relatively stable.

[0304] 3.2.5 Reproducibility examination

[0305] The Xuanfuzhong extract prepared in Example 1 was taken, and the test sample solution was prepared according to the conditions after optimization in 3.1. The sample was determined by the chromatographic condition after optimization in 3.1 with the injection volume of 1 μl. The consistency of the relative retention time and relative peak area of the characteristic peaks was examined (4 characteristic peaks were calibrated, and peak 1 was used as the reference peak). The experimental results are shown in Table 23.

[0306] Table 23 Reproducibility results of the characteristic chromatogram of the Xuanfuzhong extract (relative retention time)

[0307] Serial number 1 2 3 4 5 6 Average value RSD % Peak 1 (S) 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.00 Peak 2 2.16 2.16 2.16 2.16 2.16 2.16 2.16 0.04 Peak 3 2.55 2.55 2.55 2.55 2.55 2.55 2.55 0.05 Peak 4 4.30 4.31 4.30 4.31 4.30 4.31 4.31 0.04

[0308] From Table 23, the RSD of each chromatographic peak relative retention time is less than 3.0%, indicating that the method has good repeatability.

[0309] 3.2.6 Durability Investigation

[0310] 3.5.6.1 Investigation of Different Chromatographic Columns

[0311] Take the small canthus extract prepared in Example One, prepare the test sample solution according to the conditions optimized in 3.1, and determine according to the chromatographic conditions optimized in 3.1, with an injection volume of 1 μl, wherein the chromatographic columns are respectively: chromatographic column 1-Shim-pack GIST-HP C18-AQ (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.9 μm); chromatographic column 2-Waters ACQUITY HSS T3 (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.8 μm); and chromatographic column 3-DIKMA Endeavorsil C18 (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.8 μm). Investigate the effects of the three chromatographic columns on the peak elution of the small canthus extract characteristic chromatogram. The results are shown in Figure 15 and Table 24.

[0312] Table 24 Investigation results of chromatographic columns for small canthus extract characteristic chromatogram (relative retention time)

[0313]

[0314] From Figure 15 and Table 24, it can be seen that the chromatographic column has a greater effect on the characteristic chromatogram. The chromatographic peak has a better peak shape and the separation effect is best when using the ultra-high performance liquid chromatographic column of 1 Shim-pack GIST-HP C18-AQ (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.9 μm). Therefore, the liquid chromatographic column with the model number of Shim-pack GIST-HP C18-AQ (column length is 150 mm, inner diameter is 2.1 mm, and particle size is 1.9 μm) is used in this method.

[0315] 3.2.6.2 Investigation of Different Column Temperatures

[0316] Take the small canthus extract prepared in Example One, prepare the test sample solution according to the conditions optimized in 3.1, and determine according to the chromatographic conditions optimized in 3.1, with an injection volume of 1 μl, wherein the column temperatures are respectively 30℃, 35℃, and 40℃. Compare the separation effects of the sample at different column temperatures of 30℃, 35℃, and 40℃, and the results are shown in Figure 16 and Table 25.

[0317] Table 25 Column temperature investigation results of Xiaojianzhong extract characteristic chromatogram (relative retention time)

[0318]

[0319]

[0320] From Figure 16 As can be seen from Table 25, the column temperature has little effect on the peak elution, but the separation effect is best and the peak type is better when the column temperature is 40°C. Therefore, the column temperature of 40°C is selected.

[0321] 3.5.6.3 Investigation of different flow rates

[0322] The Xiaojianzhong extract prepared in Example 1 was taken to prepare the sample solution according to the optimized conditions in 3.1, and was determined according to the optimized chromatographic conditions in 3.1. The injection volume was 1 μl, and the flow rate was 0.2 ml / min, 0.25 ml / min and 0.3 ml / min. The elution under different flow rates (0.2 ml / min, 0.25 ml / min and 0.3 ml / min) was investigated. The results are shown in Table 26 and Figure 17

[0323] Table 26 Investigation results of Xiaojianzhong extract characteristic chromatogram (relative retention time)

[0324] Flow rate Peak 1 (S) Peak 2 Peak 3 Peak 4 0.25 ml / min 1.00 2.01 2.34 3.77 0.30 ml / min 1.00 2.13 2.53 4.23 0.35 ml / min 1.00 2.43 2.73 4.77

[0325] From Figure 17 As can be seen from Table 26, the flow rate has little effect on the peak elution, but the peak type is best when the flow rate is 0.3 ml / min. Therefore, the flow rate of 0.3 ml / min is selected.

[0326] Example 2

[0327] 1. Preparation method of Xiaojianzhong standard decoction

[0328] (1) Xiaojianzhong pieces (YP2106-2) 100 g were taken and placed in an electric ceramic pot. Water was added and decocted twice. The first decoction was directly added to water of 12 times the weight of Xiaojianzhong pieces, soaked for 30 minutes, and then boiled with a strong fire (500 W). After boiling, a weak fire (200 W) was used to maintain a slight boiling for 35 minutes. The decoction was filtered through a 300-mesh screen while hot. The second decoction was added with water of 10 times the weight of Xiaojianzhong pieces. After heating and boiling with a strong fire, a weak fire was used to maintain a slight boiling for 25 minutes. The decoction was filtered through a 300-mesh screen while hot, and the two filtrates were combined.

[0329] ​(2) The filtrate was transferred into a 2000 ml round bottom flask, and concentrated under reduced pressure and low temperature (temperature: 65℃; vacuum degree: -0.080~ -0.090 MPa) to 100 ml of extract, and the density was measured as 1.07 g / ml, and the extract rate was 13.86%; under magnetic stirring, it was divided into 10 ml brown schlenk bottles, with a volume of 1 ml per bottle, half-capped, and then transferred to a vacuum freeze dryer for freeze-drying; pre-freezing was performed at -55℃ for 2 hours, followed by primary drying at -45℃, -35℃, -30℃, -25℃, -20℃, -10℃ and 0℃, respectively, with a vacuum degree of 0.2 mbar, and the drying time was 3 hours, 7 hours, 20 hours, 10 hours, 2 hours, 1 hour and 2 hours, respectively; then secondary drying was performed at 10℃, 20℃ and 30℃, respectively, with a vacuum degree of 0 mbar, and the drying time was 1 hour, 1 hour and 5 hours, respectively; after removal, the aluminum cap was pressed, and the standard decoction of smallleaf galangal was obtained.

[0330] 2. Detection of the mass content and transfer rate of protocatechuic acid

[0331] The mass content of protocatechuic acid in the standard decoction of smallleaf galangal was measured to be 3.22 mg / g by the method in Example 1, and the transfer rate of protocatechuic acid was 40.51%.

[0332] 3. Determination method of the characteristic chromatogram of the standard decoction of smallleaf galangal

[0333] The characteristic chromatogram of the standard decoction of smallleaf galangal was determined according to the detection method shown in Example 1, which contained four peaks, with the relative retention time of peak 1 (S) being 1.00, the relative retention time of peak 2 being 2.15, the relative retention time of peak 3 being 2.53, and the relative retention time of peak 4 being 4.27.

[0334] Determination of common peaks

[0335] The ultra-high performance liquid chromatograms of 15 batches of smallleaf galangal extract samples and smallleaf galangal reference material were determined according to the method for constructing the characteristic chromatogram of smallleaf galangal extract after optimization of the conditions in Example 1, wherein the 15 batches of smallleaf galangal extract were prepared from 15 batches of smallleaf galangal decoction pieces of different sources according to the method in Example 1, and the preparation method of the smallleaf galangal reference material test solution was as follows: about 1.0 g of smallleaf galangal reference material (batch number YP2106-1) was accurately weighed, placed in a conical flask with a plug, 25 ml of 100% methanol was accurately added, the weight was determined, and ultrasonic treatment (power 500 W, frequency 40 kHz) was performed for 30 minutes; after cooling, the weight was determined again, the lost weight was made up with methanol, shaken well, filtered, and the filtrate was taken; an appropriate amount of the filtrate was filtered through a 0.22 μm microporous filter membrane, and placed in a liquid sample bottle to obtain the smallleaf galangal reference material test solution.

[0336] The results were analyzed by using the "Chinese medicine chromatographic fingerprint similarity evaluation system (2012 version)" recommended by the National Pharmacopoeia Committee, and the common peaks were selected, as shown in Table 27. Figure 18

[0337] Table 27 Sample determination results

[0338]

[0339]

[0340] From Table 27, it can be seen that the test sample characteristic spectrum of Xuanfuzhong extract presents 4 characteristic peaks, which correspond to the retention time of 4 characteristic peaks in the test sample chromatogram of the control medicinal material. Taking peak 1 as the reference peak, the relative retention time RSD values of the remaining 3 characteristic peaks of the characteristic spectrum of 15 batches of Xuanfuzhong extract are 0.24%-0.25%, all less than 1.0%, which meets the standard requirements of the characteristic spectrum of Xuanfuzhong extract. Figure 18 Example Four

[0341] 1. Preparation method of Xuanfuzhong formula granules

[0342] (1) Take 100 g of Xuanfuzhong decoction pieces (YP2106-1), add water and decoct twice, soak for 0.5 hours before decoction, add 6 times water for the first time, heat to boiling, keep micro-boiling for 1.0 hour, filter the decoction through a 200 mesh screen. Add 5 times water for the second time, heat to boiling, keep micro-boiling for 0.5 hour, filter the decoction through a 200 mesh screen, and combine the two filtrates.

[0343] (2) Transfer the filtrate to a 2000 ml round bottom flask, use a rotary evaporator to concentrate under reduced pressure and low temperature (temperature: 65°C; vacuum degree: -0.04~-0.08 MPa) to 100 ml of extract, measure its density as 1.05 g / ml, after the extract is fully dissolved, add 10 g of malt dextrin, mix well, pass through a No. 6 sieve (100 mesh), and then spray dry, wherein the material temperature is (80°C), the inlet air temperature of spray drying is (155°C), and the outlet air temperature is (100°C); add appropriate amount of silicon dioxide and magnesium stearate to pass through a No. 5 sieve (80 mesh), mix the dry powder with the ingredients, and sieve (upper layer 10 mesh, lower layer 40 mesh);

[0344] (3) Take 10 g of dry extract powder obtained in step (3), crush and pass through an 80 mesh sieve, add 0.01 g of silicon dioxide and 0.01 g of magnesium stearate, mix well, and sieve (upper layer 10 mesh, lower layer 40 mesh) to obtain Xuanfuzhong formula granules-1.

[0345] 2. Detection of characteristic spectrum of Xuanfuzhong formula granules

[0346]

[0347] ​​2.1 Preparation of reference solution

[0348] Take an appropriate amount of protocatechuic acid reference standard, accurately weigh it, add 100% methanol to prepare a reference solution containing 16 μg of protocatechuic acid per 1 ml, shake well, and the solution is obtained.

[0349] 2.2 Preparation of the test solution

[0350] Take approximately 0.15g of the prepared small-grained ginseng granules-1, accurately weigh it, place it in a stoppered conical flask, add 25ml of 100% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, replenish the lost weight with 100% methanol, shake it well, filter it, take out the filtrate, and take an appropriate amount of the filtrate through a 0.22μm microporous membrane and place it in a liquid sample bottle.

[0351] 2.3 Ultra-high performance liquid chromatography analysis

[0352] 2 μL of the reference solution from 2.1 and the test solution from 2.2 were injected into the ultra-high performance liquid chromatograph for analysis. Octadecylsilane-bonded silica gel was used as the stationary phase. 100% acetonitrile was used as the mobile phase A and 0.1% phosphoric acid solution was used as the mobile phase B. Gradient elution was performed according to the specifications in Table 18. The flow rate was 0.3 mL / min, the column temperature was 40 °C, and the determination was performed at a wavelength of 252 nm.

[0353] The characteristic spectrum of Xiaoguanzhong formula granules measured according to the above method is as follows: Figure 19 As shown, it contains four peaks: peak 1 (S) has a relative retention time of 1.00, peak 2 has a relative retention time of 2.15, peak 3 has a relative retention time of 2.53, and peak 4 has a relative retention time of 4.27.

[0354] Example 5

[0355] 1. Thin-layer chromatography identification method for Xiaoguanzhong formula granules

[0356] 1.1 Preparation of the test solution

[0357] Different batches of *Dryopteris crassirhizoma* (batch numbers YP2106-2 and YP2106-3) were selected, and *Dryopteris crassirhizoma* formula granules-2 and granules-3 were prepared according to the method in Example 4. 0.2g of each of the three formula granules were weighed, ground into a fine powder, dissolved in 5ml of 100% methanol, sonicated for 10 minutes, filtered, and used as the test solution.

[0358] 1.2 Dryopteris crassirhizoma reference solution

[0359] Take 1 g of Xiaoganzi decoction pieces (YP2106-1), add 20 mL of water, heat reflux for 30 min, filter, evaporate the filtrate to collect the residue, add 2 mL of 100% methanol to dissolve as Xiaoganzi reference solution.

[0360] 1.3 Xiaoganzi dispensing granules negative sample solution

[0361] Respectively, take 0.2 g of malt dextrin, finely grind, add 5 ml of 100% methanol, ultrasonic treatment for 10 minutes, filter, as test solution.

[0362] 1.4 Thin layer chromatography conditions

[0363] The thin layer plate is a silica gel G thin layer plate;

[0364] The developing agent is toluene-ethyl acetate-formic acid-water (volume ratio is 1:12:2.5:3);

[0365] Developing method: using double tank developing cylinder, developing distance is 8 cm, developing twice;

[0366] Inspection: spray with 2% aluminum chloride ethanol solution, under UV light 365 nm.

[0367] 2. Investigation of sample size

[0368] Take 1.1 Xiaoganzi dispensing granules-1 test solution, 1.2 Xiaoganzi reference solution, investigate the sample size (2, 4, 8 μL) in Xiaoganzi dispensing granules identification method, develop according to the above thin layer chromatography conditions, take out, dry, spray with 2% aluminum chloride ethanol solution, under UV light 365 nm. The thin layer chromatogram is as shown in Figure 20 .

[0369] From Figure 20 It can be seen that when the sample size is 4 μl, the main spot in the test sample corresponding to the control medicinal material is clear, and there is no other interference, so the subsequent identification selects the sample size of 4 μl.

[0370] 3. Xiaoganzi dispensing granules thin layer chromatography identification method specificity investigation

[0371] Respectively, take 1.1 Xiaoganzi dispensing granules test solution, 1.2 Xiaoganzi reference solution and 1.3 Xiaoganzi dispensing granules negative sample 4 μl respectively, point on the same silica gel G thin layer plate, develop according to the above thin layer chromatography conditions, take out, dry, spray with 2% aluminum chloride ethanol solution, under UV light 365 nm. The thin layer chromatogram is as shown in Figure 21 .

[0372] From Figure 21It can be seen that the same color spots appeared on the corresponding positions of Xiaogan Zhong dispensing granules and Xiaogan Zhong reference material chromatogram, and the negative sample had no interference. It showed that the thin layer method had good specificity.

[0373] 4. Investigation on the durability of Xiaogan Zhong dispensing granules thin layer chromatography identification method

[0374] 4.1 Investigation on different temperatures

[0375] Respectively, 4 μl of Xiaogan Zhong dispensing granules test solution, Xiaogan Zhong reference solution and Xiaogan Zhong dispensing granules negative sample were spotted on the same silica gel G thin layer plate, and developed under different temperature conditions (5 ℃, 25 ℃, 35 ℃) according to the above thin layer chromatography conditions, taken out, air dried, sprayed with 2% aluminum chloride ethanol solution, and observed under ultraviolet light 365 nm. The thin layer chromatogram is shown in Figure 22-1 to Figure 22-3 .

[0376] From Figure 22-1 to Figure 22-3 It can be seen that under different temperature conditions, the same color fluorescent spots appeared on the corresponding positions of Xiaogan Zhong dispensing granules test chromatogram and Xiaogan Zhong reference thin layer chromatogram, with clear color development, good separation degree, no tailing phenomenon, and no background interference. It showed that temperature had no obvious effect on Xiaogan Zhong dispensing granules thin layer identification, and the thin layer identification method had good durability to humidity.

[0377] 4.2 Investigation on different humidity

[0378] Respectively, 4 μl of Xiaogan Zhong dispensing granules test solution, Xiaogan Zhong reference solution and Xiaogan Zhong dispensing granules negative sample were spotted on the same silica gel G thin layer plate, and developed under different humidity conditions (RH: 33%, RH: 66%, RH: 88%) according to the above thin layer chromatography conditions, taken out, air dried, sprayed with 2% aluminum chloride ethanol solution, and observed under ultraviolet light 365 nm. The thin layer chromatogram is shown in Figure 23-1 to Figure 23-3 .

[0379] From Figure 23-1 to Figure 23-3 It can be seen that under different humidity conditions, the same color fluorescent spots appeared on the corresponding positions of Xiaogan Zhong dispensing granules test chromatogram and Xiaogan Zhong reference thin layer chromatogram, and the main spots were clear in color development, with good separation degree, no tailing phenomenon, and no background interference. It showed that humidity had no obvious effect on Xiaogan Zhong dispensing granules thin layer identification, and the thin layer identification method had good durability to humidity.

[0380] 4.3 Investigation on thin layer plates from different manufacturers

[0381] Four microliters each of the Xiaoguanzhong formula granule test solution, Xiaoguanzhong reference solution, and Xiaoguanzhong formula granule negative sample were spotted onto thin-layer plates from different manufacturers (Qingdao Ocean Silica G plate, Sinopharm Chemical Reagent Silica G plate, and Merck plate from Germany). The plates were developed under the same temperature and humidity conditions as described above. After development, the plates were removed, air-dried, sprayed with 2% aluminum trichloride ethanol solution, and examined under ultraviolet light at 365 nm. The thin-layer chromatograms are shown below. Figure 24-1 to Figure 24-3 .

[0382] Depend on Figure 24-1 to Figure 24-3 It is evident that, using silica gel thin-layer plates from different manufacturers (Qingdao Ocean Silica G plate, Sinopharm Chemical Reagent Silica G plate, and German Merck plate), the main spots of the chromatograms of the Xiaoguanzhong formula granules test sample and the reference standard correspond to each other without significant influence, indicating that this thin-layer chromatography identification method has good durability with silica gel G plates from different manufacturers.

[0383] 5. Thin-layer chromatographic identification of Xiaoguanzhong formula granules

[0384] 4 μl of each of the following granule solutions were separately spotted onto the same silica gel G thin-layer plate: * **Xiao Guan Zhong Formula Granules-1**, * **Xiao Guan Zhong Formula Granules-2**, * **Xiao Guan Zhong Formula Granules-3**, * **Xiao Guan Zhong Reference Solution**, and a negative sample of *Xiao Guan Zhong Formula Granules**. The plate was developed under the conditions described above. The plate was then removed, air-dried, sprayed with 2% aluminum trichloride ethanol solution, and examined under UV light at 365 nm. The thin-layer chromatogram is shown below. Figure 25 As shown.

[0385] Depend on Figure 25 It is evident that the chromatograms of the test samples in the three batches of Xiaoguanzhong formula granules showed the same color spots at the corresponding positions as the chromatograms of Xiaoguanzhong reference standard, indicating that the thin-layer identification of the three batches of Xiaoguanzhong formula granules met the requirements.

[0386] In summary, based on the chromatographic spots, the separation effect of the *Dryopteris crassirhizoma* formula granules is good, and the corresponding positions of the chromatograms of the *Dryopteris crassirhizoma* formula granules and the *Dryopteris crassirhizoma* reference standard show fluorescent spots of the same color. This method can effectively identify *Dryopteris crassirhizoma* formula granules that have lost their medicinal slice characteristics. Through thin-layer chromatography methodology evaluation, this method demonstrates good specificity and robustness, and is suitable for the thin-layer chromatographic identification of *Dryopteris crassirhizoma* formula granules.

[0387] Experimental Example 1

[0388] Accelerated stability test and long-term stability test were conducted on the small-root formula granules-1 prepared in Example 4 and the small-root formula granules-2 and small-root formula granules-3 prepared in Example 5, according to the following methods.

[0389] 1. Accelerated stability test

[0390] Take Xiaogan Zong Formula Granules-1, Xiaogan Zong Formula Granules-2 and Xiaogan Zong Formula Granules-3 respectively, and pack them with aluminum foil composite film. Place them at a temperature of 40℃±2℃ and a relative humidity of 75%±5% for 6 months. Take samples at the end of the first month, the second month, the third month and the sixth month during the test period, and test the shape, particle size, moisture, solubility, thin layer chromatography identification, microbial freshness, characteristic spectrum, extract and protocatechuic acid content of the formula granules. The results are shown in Table 28.

[0391] 2. Long-term stability test

[0392] Take Xiaogan Zong Formula Granules-1, Xiaogan Zong Formula Granules-2 and Xiaogan Zong Formula Granules-3 respectively, and pack them with aluminum foil composite film. Place them at a temperature of 30℃±2℃ and a relative humidity of 65%±5% for 12 months. Take samples every 3 months, and take samples at the end of the 0th month, the 3rd month, the 6th month, the 9th month and the 12th month. Test the shape, particle size, moisture, solubility, thin layer chromatography identification, microbial freshness, characteristic spectrum, extract and protocatechuic acid content of the formula granules. The results are shown in Table 29.

[0393] Table 28 Results of accelerated stability test

[0394]

[0395]

[0396] Table 29 Results of long-term stability test

[0397]

[0398]

[0399] From Table 28 and Table 29, it can be seen that the results of the accelerated stability test and the long-term stability test show that Xiaogan Zong Formula Granules containing Xiaogan Zong extract prepared by the method of the present application has stable moisture content, is resistant to moisture absorption, and has stable extract and protocatechuic acid content. The characteristic spectrum is detected according to the detection method of the characteristic spectrum of Xiaogan Zong Formula Granules in Example Four. The results show that the characteristic spectrum of Xiaogan Zong Formula Granules before and after the stability test also meets the requirements, indicating that the Xiaogan Zong extract prepared by the present application has good stability and can be used for quality control of Xiaogan Zong extract and its preparations.

[0400] To sum up, the application adopts decocting Xiaoganzhong with water, filtering to obtain filtrate, and concentrating and drying the filtrate to obtain Xiaoganzhong extract, the extract has an extract yield of 6.3-16.8%, and the prepared Xiaoganzhong extract is used for detecting the mass content and transfer rate of protocatechuic acid by ultra-high performance liquid chromatography analysis, the results are as follows: the mass content of protocatechuic acid is 1.82-5.00 mg / g, and the transfer rate of protocatechuic acid is 20.14-56.56%. The detection method has high precision, good repeatability, and good stability of the test solution within 24 hours.

[0401] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A method for detecting the mass content of proto-catechuic acid and / or the proto-catechuic acid transfer rate in a water extract of small-leaf galangal, characterized in that, It comprises the following steps: (1) Preparation of reference solution Take protocatechuic acid control sample, add solvent to make solution; (2) Preparation of test solution Take small channel crowd water extract, add solvent for extraction; (3) Ultra-high performance liquid chromatography analysis Take octadecylsilane bonded silica as filler, take organic solvent as mobile phase A, and take 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution, take reference solution and test solution and inject into ultra-high performance liquid chromatograph for analysis; The small Pteridium is the Pteridium aquilinum of the Pteridaceae plant Cyrtomium fortunei J. Sm. dried rhizome and petiole base; The solvent in steps (1) and (2) is 50% ethanol; The organic solvent as mobile phase A in step (3) is acetonitrile, The gradient elution conditions are as follows: within 0-5 min, mobile phase A is 5%, and mobile phase B is 95%, Within 5-15 min, mobile phase A increases from 5% to 9%, and mobile phase B decreases from 95% to 91%, Within 15-15.1 min, mobile phase A decreases from 9% to 5%, and mobile phase B increases from 91% to 95%, Within 15.1-20 min, mobile phase A is 5%, and mobile phase B is 95%.

2. The detection method according to claim 1, wherein, The extraction in step (2) is one of reflux extraction, shaking extraction or ultrasonic extraction.

3. The detection method according to claim 1, wherein, The extraction in step (2) is ultrasonic extraction.

4. The assay of any one of claims 1-3, wherein, The extraction time in step (2) is 15-60 min.

5. The detection method according to claim 4, wherein, The extraction time in step (2) is 15 min.

6. A method for constructing a characteristic chromatogram of a water extract of Pyrrhocephalus speciosus, characterized by, It comprises the following steps: (1) Preparation of reference solution Take protocatechuic acid control sample, add solvent to make solution; (2) Preparation of test solution Take small channel crowd water extract, add solvent for extraction; (3) Ultra-high performance liquid chromatography analysis Take octadecylsilane bonded silica as filler, take organic solvent as mobile phase A, and take 0.1% phosphoric acid aqueous solution as mobile phase B for gradient elution, take reference solution and test solution and inject into ultra-high performance liquid chromatograph for analysis; The small Pteridium is the Pteridium aquilinum of the Pteridaceae plant Cyrtomium fortunei J. Sm. dried rhizome and petiole base The solvent in steps (1) and (2) is 100% methanol; The organic solvent as mobile phase A in step (3) is acetonitrile, The aqueous phase as mobile phase B is 0.1% phosphoric acid solution, The gradient elution conditions are as follows: within 0-5 min, mobile phase A is 5%, and mobile phase B is 95%, Within 5-21 min, mobile phase A increases from 5% to 12%, and mobile phase B decreases from 95% to 88%, Within 21-24 min, mobile phase A increases from 12% to 16%, and mobile phase B decreases from 88% to 84%, Within 24-34 min, mobile phase A increases from 16% to 18%, and mobile phase B decreases from 84% to 82%, Within 34-38 min, mobile phase A increases from 18% to 60%, and mobile phase B decreases from 82% to 40%, Within 38-38.2 min, mobile phase A decreases from 60% to 5%, and mobile phase B increases from 40% to 95%, Within 38.2-40 min, mobile phase A is 5%, and mobile phase B is 95%.

7. The method for constructing the characteristic pattern of the extract of Xanthium sibiricum according to claim 6, wherein, The extraction in step (2) is one of reflux extraction, shaking extraction or ultrasonic extraction.

8. The method for constructing the characteristic pattern of the extract of Xanthium sibiricum according to claim 6, wherein, The extraction in step (2) is ultrasonic extraction.

9. The method for constructing the characteristic pattern of the extract of Smilax china L. according to any one of claims 6-8, wherein, The extraction time in step (2) is 15-60 min.

10. The method for constructing the characteristic pattern of the extract of Xanthium sibiricum according to claim 9, wherein, The extraction time in step (2) is 30 min.

11. A method for identifying a water extract of Pyrrho cumulus, characterized by, The thin layer chromatography comprises the following steps: a. preparing a test solution of the water extract of smallleaf galangal and a control solution of smallleaf galangal; b. spotting the test solution and the control solution on the same silica gel G thin layer plate, developing with a developing agent, taking out and drying, spraying with a color developing agent to develop the spots, and detecting, to obtain the characteristic chromatogram of the water extract of smallleaf galangal, The developing agent is toluene-ethyl acetate-formic acid-water, and the volume ratio of toluene, ethyl acetate, formic acid and water is 1:12:2.5:

3.

12. The method of claim 11, wherein the extract of Saururus chinensis is identified by the following steps of: The test solution of the water extract of smallleaf galangal is prepared by the following steps: taking 0.1-1 g of the water extract of smallleaf galangal, dissolving in methanol, and filtering after ultrasonic treatment.

13. The method of claim 11, wherein the extract of Saururus chinensis is identified by the following steps of: The control solution of smallleaf galangal is prepared by the following steps: taking 1-3 g of smallleaf galangal, adding water and heating to reflux, filtering, evaporating the filtrate to dryness, and dissolving the residue in methanol.

14. The application of the method for detecting the mass content of protocatechuic acid and / or the protocatechuic acid transfer rate in the water extract of smallleaf galangal according to any one of claims 1-3 or the method for constructing the characteristic chromatogram of the water extract of smallleaf galangal according to any one of claims 6-8 in the water extract of smallleaf galangal and its preparation.

15. The application of the method for detecting the mass content of protocatechuic acid and / or the protocatechuic acid transfer rate in the water extract of smallleaf galangal according to claim 4 in the water extract of smallleaf galangal and its preparation.

16. The application of the method for constructing the characteristic chromatogram of the water extract of smallleaf galangal according to claim 9 in the water extract of smallleaf galangal and its preparation.

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