Herba chloranthi serrati extract and preparation method and detection method of herba chloranthi serrati extract preparation

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a preparation method and a detection method of a herba chloranthi serrati extract and a preparation thereof. The invention relates to a herba chloranthi serrati extract and a preparation method thereof, which specifically comprises the following steps: (1) decocting herba chloranthi serrati decoction pieces twice with water, filtering, cooling the filtrate to room temperature, combining the two decoctions, namely the herba chloranthi serrati extract, and concentrating the decoctions under reduced pressure to obtain an extract with the extract yield of 4.2-11.2%; and preparing the extract into a required dosage form, thereby obtaining the Sihua Tibet preparation. The invention establishes a quality control method based on a characteristic spectrum, and a content determination analysis method aiming at the herba chloranthi serrati extract and the preparation of the herba chloranthi serrati extract. The established detection method adopts a liquid chromatography technology, has the characteristics of simplicity and convenience in operation, stability, high precision and good reproducibility, and provides a reliable analysis means for comprehensive evaluation of product quality and standard establishment.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and specifically relates to a preparation method and a detection method for an extract of Chloranthus multistachys (H.-M.) Pei and its preparations. Background Art

[0002] Chloranthus multistachys (H.-M.) Pei is the dried rhizome and root of the plant Chloranthus multistachys (H.-M.) Pei of the family Chloranthaceae. It is also known as Four Heavenly Kings, Four Gentle Winds, Four Tiles, Big Four Tiles, Four Pieces of Tiles, Red Four Tiles, Four-leaf Yellow, etc. It has a pungent and bitter taste, a neutral nature, and is toxic. It belongs to the lung and liver meridians. It has the functions of relaxing tendons and activating collaterals, expelling wind and relieving pain, clearing heat and detoxifying, and reducing swelling. It is used for the treatment of pulmonary tuberculosis, unknown swelling toxins, falls and injuries, rheumatic low back and leg pain, etc. Modern research shows that Chloranthus multistachys (H.-M.) Pei mainly contains volatile oils, terpenoids, coumarins and lignans, alkaloids and other components.

[0003] The "Quality Standards for Chinese Medicinal Materials and Ethnic Medicinal Materials in Guizhou Province (2003 Edition)" records that the rhizome of this product is in a clump shape, densely covered with many fibrous roots. The fibrous roots are slender and cylindrical, 10-20 cm long, 0.05-0.15 cm in diameter, with a brown surface and root branch scars. It is brittle, with a flat cross-section. The cortex is yellowish-brown, and the xylem is light yellow. It has a faint smell and a slightly bitter taste.

[0004] The clinical efficacy and pharmacological activity of Chloranthus multistachys (H.-M.) Pei are related to its active ingredients. Current research shows that Chloranthus multistachys (H.-M.) Pei contains chemical components such as volatile oils, terpenoids, coumarins and lignans, alkaloids and other chemical components.

[0005] At present, there are few reports on the content determination of extracts of Chloranthus multistachys (H.-M.) Pei and its preparations. The evaluation system of the quality standards for extracts of Chloranthus multistachys (H.-M.) Pei and its preparations is still not perfect, and it cannot effectively evaluate its indicators, restricting its application and development. Therefore, research needs to be carried out in aspects such as characteristic fingerprints and content determination to establish a complete evaluation system for quality standards. Summary of the Invention

[0006] In order to solve the above technical problems existing in the prior art, the present invention provides a preparation method and a detection method for an extract of Chloranthus multistachys (H.-M.) Pei and its preparations, including a preparation method, content, characteristic fingerprints, and thin-layer chromatography identification detection methods, specifically as follows: A preparation method for an extract of Chloranthus multistachys (H.-M.) Pei and its preparations specifically includes the following steps: (1) Decoct the sliced Chloranthus multistachys (H.-M.) Pei twice with water, filter, and after cooling the filtrate to room temperature, combine the two decoctions to obtain the extract of Chloranthus multistachys (H.-M.) Pei; (2) Concentrate the decoction under reduced pressure to obtain an extract paste, and the paste yield is 4.2% - 11.2%; obtain the extract paste; (3) Prepare the paste into the required dosage form to obtain the product.

[0007] Further, step (1) specifically involves taking four pieces of medicinal slices, adding water and decocting them twice. For the first decoction, add 12 to 16 times the amount of water, soak for 20 to 40 minutes, bring to a boil over high heat, then simmer over low heat for 20 to 40 minutes. Filter the decoction through a 200 to 400 mesh sieve while it is still hot. For the second decoction, add 10 to 14 times the amount of water, bring to a boil over high heat, then simmer over low heat for 15 to 25 minutes. Filter the decoction through a 200 to 400 mesh sieve while it is still hot, and combine the two decoctions.

[0008] Furthermore, the specific conditions for vacuum concentration in step (2) are: temperature: 60~70℃; vacuum degree: -0.080~-0.090MPa.

[0009] Further, step (3) involves freeze-drying the obtained extract to obtain the Four-Piece Standard Decoction. The freeze-drying parameters are as follows: pre-freezing temperature is -45~-55℃, pre-freezing time is 150~200 minutes, sublimation drying temperature is -45℃~0℃, sublimation drying time is 2000~2500 minutes, and vacuum degree is -0.15~0.25mbar; desorption drying temperature is 10℃~30℃, desorption drying time is 300~400 minutes, and vacuum degree is -0.1~0.1mbar.

[0010] Furthermore, step (3) involves adding excipients to the obtained extract, drying it, adding more excipients, mixing it evenly, and granulating it to obtain four-piece tile granules.

[0011] Furthermore, the specifications of the four-piece granule formulation are such that each 1g of the granules is equivalent to 8-12g of medicinal slices.

[0012] A method for determining the content of components in a tetrapod extract and its formulations is disclosed, employing high-performance liquid chromatography (HPLC). The column used is octadecylsilane-bonded silica gel as the packing material, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 µm. Acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, with gradient elution performed according to the specifications in the table below. The flow rate is 0.30 mL / min; the column temperature is 25 °C; and the detection wavelength is 326 nm.

[0013] Furthermore, the test solution is prepared by the following method: Take the sample to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 50% ethanol, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake well, filter it, and collect the filtrate. Further, the sonication treatment is performed with a power of 450~550W, a frequency of 35~45kHz, and a time of 10~30min.

[0014] Furthermore, the reference solution is a solution prepared with methanol as the solvent, containing 20 μg of chlorogenic acid per 1 ml.

[0015] A method for detecting the characteristic chromatograms of a tetrapod extract and its formulations is disclosed, employing high-performance liquid chromatography (HPLC). The column used is octadecylsilane-bonded silica gel as the packing material, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 µm. Acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B, with gradient elution performed according to the specifications in the table below. The flow rate is 0.30 mL / min; the column temperature is 25 °C; and the detection wavelength is 326 nm.

[0016] Furthermore, the test solution is prepared by the following method: Take the sample to be tested, accurately weigh it, place it in a stoppered conical flask, accurately add 50% ethanol, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake well, filter it, and collect the filtrate. Further, the sonication treatment is performed with a power of 450~550W, a frequency of 35~45kHz, and a time of 10~30min.

[0017] Furthermore, the reference solution is a solution prepared with methanol as the solvent, containing 20 μg of chlorogenic acid, 20 μg of isoflavone, and 30 μg of rosmarinic acid per 1 ml.

[0018] Furthermore, the reference solution of the control medicinal material was prepared by the following method: Take four pieces of control medicinal material, weigh them accurately, place them in a stoppered conical flask, add 75% ethanol accurately, weigh them, reflux and extract for 40 minutes, cool them, weigh them again, replenish the lost weight with 75% ethanol, shake well, filter, and take the filtrate to obtain the solution.

[0019] Furthermore, the final standard for the characteristic chromatograms of the four-piece extract and its preparations was determined as follows: the chromatogram of the test sample should show 6 characteristic peaks, corresponding to the retention times of the 6 characteristic peaks in the chromatogram of the reference medicinal material; among them, peaks 2, 4, and 6 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 1, 3, and 5 with peak S are calculated, and their relative retention times should be within ±10% of the specified values, which are 0.64 (peak 1), 1.11 (peak 3), and 2.19 (peak 5).

[0020] A thin-layer chromatography method for detecting the extract of *Symplocos cuspidatum* and its preparations, using *Symplocos cuspidatum* as a reference material, with the following specific parameters: thin-layer plate: silica gel G thin-layer plate; developing solvent: petroleum ether-ethyl acetate (1:1); sample volume: 4 μl; development method: double-groove developing tank, development distance of about 8 cm; inspection: under ultraviolet light at 365 nm. Preparation of test solution: Take the sample to be tested, grind it into a fine powder, add methanol, sonicate, and filter to obtain the test solution; Preparation of the control herbal solution: Take four pieces of control herbal material, add water, boil, filter, evaporate the filtrate to dryness, add methanol to dissolve, filter, and use as the control herbal solution; Preparation of negative sample solution: Take four pieces of tile formula granules as negative sample, grind them finely, and process them in the same way as the test sample to prepare negative sample solution.

[0021] Compared with the prior art, the technical effects of this invention are reflected in: 1. This application selects chlorogenic acid as the indicator component of the extract and its preparations of *Symplocos sipatica*, and conducts a quantitative determination method of ultra-high performance liquid chromatography for the content determination of the extract and its preparations of *Symplocos sipatica*, and formulates quantitative standards for the extract and its preparations of *Symplocos sipatica*, providing a scientific basis for the establishment of quality standards for the extract and its preparations of *Symplocos sipatica*.

[0022] 2. The characteristic chromatographic method established in this application adopts ultra-high performance liquid chromatography, which has the characteristics of simplicity, stability, high precision and good reproducibility. Under the premise of meeting the internal control quality standards of medicinal materials, a characteristic chromatogram of Sikuawa medicinal materials was established, and the material basis was studied. The chemical components corresponding to the characteristic peaks were identified, which is used for the source quality control of Sikuawa extract and its preparations.

[0023] 3. The characteristic chromatographic method established in this application uses ultra-high performance liquid chromatography, which is simple, stable, precise and reproducible. Moreover, the fingerprint spectrum of the obtained extract has many peaks, good peak shape, easy identification and is accurate and reliable.

[0024] 4. The characteristic chromatographic method established in this application is simple, time-saving, and environmentally friendly. The analysis of a batch of preparations takes only 37 minutes, which can greatly shorten the detection and analysis time, improve production efficiency, and enable large-scale production sampling and testing to quickly achieve the purpose of quality control. Attached Figure Description

[0025] Figure 1 It is a YMW mechanical split-type decoction pot.

[0026] Figure 2 It is a specific investigation map.

[0027] Figure 3 This is a chromatogram of peak purity.

[0028] Figure 4 It is a linear regression equation for chlorogenic acid.

[0029] Figure 5 This study investigates the effect of different chromatographic columns on the target components contained in the four standard decoctions.

[0030] Figure 6 This study examines the effects of different chromatographic instruments on the content of the target components in the four-piece tile formulation granules.

[0031] Figure 7 These are the chromatographic results of the target components in four standard decoctions at different column temperatures.

[0032] Figure 8 These are the chromatogram results of the target components in four standard decoctions at different flow rates.

[0033] Figure 9 This is the DAD diagram of the standard decoction of four tiles.

[0034] Figure 10 It is the UPLC spectrum of the aqueous phase of the four-piece standard decoction.

[0035] Figure 11 These are UPLC spectra of different mobile phase systems.

[0036] Figure 12 This study examines the characteristic spectra of the four standard decoctions using different extraction solvents.

[0037] Figure 13 This is a comparison chart of the extraction efficiency of different extraction solvents for the characteristic spectrum of the four standard decoctions.

[0038] Figure 14 It is a common pattern of characteristic spectrum of the four standard decoctions (R is the reference medicinal material).

[0039] Figure 15 This study examines the specificity of the characteristic chromatograms of the four standard decoctions.

[0040] Figure 16 This is a comprehensive examination of the characteristic spectra of the four standard decoctions.

[0041] Figure 17 This is a comparison chart of UPLC tests using different chromatographic columns.

[0042] Figure 18 This is a comparison chart of UPLC tests conducted at different column temperatures.

[0043] Figure 19 This is a comparison chart of UPLC under different flow rates.

[0044] Figure 20 This is a thin-layer chromatogram for the sampling quantity of the Si Kuai Wa formula granules. The sample consisted of: 1. 2 μl of Si Kuai Wa reference medicinal material; 2. 4 μl of Si Kuai Wa reference medicinal material; 3. 8 μl of Si Kuai Wa reference medicinal material; 4. 2 μl of Si Kuai Wa formula granules; 5. 4 μl of Si Kuai Wa formula granules; and 6. 8 μl of Si Kuai Wa formula granules.

[0045] Figure 21This is the thin-layer chromatography specificity of the Sikuawa formula granules. (T: 25℃, RH: 33%; Qingdao Marine Silica G plate) 1. Sikuawa reference medicinal material; 2-4. Sikuawa formula granules (210602-210604) 4 μl; 5. Negative sample.

[0046] Figure 22 These are thin-layer chromatograms of the four-piece tile formulation particles under different temperature conditions.

[0047] The first sheet contains: (T: 5℃, RH: 33%; Qingdao Marine Silica G Plate) 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0048] Second sheet: (T: 25℃, RH: 33%; Qingdao Marine Silica G plate) 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0049] Third sheet: (T: 35℃, RH: 33%; Qingdao Marine Silica G plate) 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0050] Figure 23 These are thin-layer chromatograms of the four tile formulation particles under different humidity conditions for identification.

[0051] The first sheet contains (T: 25 ℃, RH: 33%; Qingdao Marine Silica G Plate): 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0052] Second sheet: (T: 25 ℃, RH: 66%; Qingdao Marine Silica G plate) 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0053] Third sheet: (T: 25 ℃, RH: 88%; Qingdao Marine Silica G plate). 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0054] Figure 24 These are chromatograms of silica gel plates from different manufacturers.

[0055] The first sheet contains: (T: 25℃, RH: 33%; Qingdao Marine Silica G Plate). 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0056] Second sheet: (T: 25℃, RH: 33%; Sinopharm Group silica gel G plate). 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0057] Third sheet: (T: 25℃, RH: 33%; Merck G-plate, Germany). 1. Four-piece control medicinal material; 2-4. Four-piece formula granules (210602-210604) 4 μl; 5. Negative sample.

[0058] Figure 25 These are thin-layer chromatograms of three batches of Sikuawa formula granules. (T: 25℃, RH: 33%; Qingdao Marine Silica G plate) 1. Sikuawa reference medicinal material; 2. Sikuawa reference medicinal material; 3-5. Sikuawa formula granules (210602-210604) 4 μl. Detailed Implementation

[0059] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description.

[0060] Example 1 Basis for proposing standard decoction process parameters for four tiles 1. Instruments and Materials 1.1 Instruments Rotary evaporator (Shanghai Yarong Biochemical Instrument Factory, RE-5205A), ceramic health pot (Huzhou Gangdian Craft Manufacturing Factory), electronic balance (Hangzhou Youheng Weighing Equipment Co., Ltd., HLD-30002), balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd., AL104), circulating water vacuum pump (Gongyi Yuhua Instrument Co., Ltd., SHZ-D(III)), low temperature coolant circulating water pump (Zhengzhou Changcheng Science and Technology Industry and Trade Co., Ltd., DLSB-5 / 20B), vacuum freeze dryer (Dalian Shuangrui Technology Co., Ltd., TRL-0.5), electric thermostatic drying oven (Tianjin Tester Instrument Co., Ltd., 202-2AB), electric blower thermostatic drying oven (Hunan Electric Furnace Drying Oven Factory, 101-4A), electric thermostatic water bath (Beijing Kewei Yongxing Instrument Co., Ltd., HH-S6A), guillotine.

[0061] 1.2 Materials The following table shows the four types of medicinal slices: Table 1. Information on Four-Wall Herbal Pieces

[0062] 2. Methods for determining process optimization indicators 2.1 Method for determining solid content Method for determining the solid content of four-piece tile concentrate According to the "Hot Extraction Method" of General Chapter 2201 Extraction Determination Method in Part IV of the 2020 Chinese Pharmacopoeia: accurately weigh 10g of concentrated solution, place it in a pre-weighed evaporating dish, evaporate it to dryness on a water bath, dry it at 105℃ for 3 hours, cool it in a desiccator for 30 minutes, quickly and accurately weigh it, and calculate the solid content of the concentrated solution.

[0063] The formula for calculating the yield of concentrated liquid is: Yield % = weight of concentrated liquid * solid content of concentrated liquid / amount of medicinal slices * 100%.

[0064] The yield of the standard decoction concentrate is the same as the standard decoction yield.

[0065] 3. Processing of medicinal slices Take the raw medicinal materials, remove impurities, soak them thoroughly, cut them into sections, and dry them promptly.

[0066] Information on the medicinal materials used in this research is shown in Table 2: Table 2 Information on Four-Wall Medicinal Herbs

[0067] 4 Extraction process 4.1 Selection of cooking utensils According to the "Management Standards for Traditional Chinese Medicine Decoction Rooms in Medical Institutions" (Document No. 3 of 2009 issued by the State Administration of Traditional Chinese Medicine) regarding decoction containers (decoction containers should preferably be made of ceramic, stainless steel, copper, etc., and containers made of iron or other easily corroded materials are prohibited) and the family's habit of decocting medicines, a 5L automatic ceramic decoction pot was selected as the standard decoction equipment.

[0068] 4.2 Study on extraction process parameters (1) Investigation on the dosage of medicinal slices According to the recommendations under "Preparation of Standard Decoctions" in the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," the amount of medicinal slices used per decoction should generally not be less than 100g, with the amount for flowers, leaves, and other medicinal slices potentially reduced. Based on preliminary test results, 100g of Sikuawa medicinal slices is convenient to handle, and the resulting dry extract is sufficient for experimental research on standard decoctions. Therefore, the amount of medicinal slices used in Sikuawa standard decoctions is set at 100g.

[0069] (2) Water addition assessment According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Granules," the amount of water added should generally be 2-5 cm above the surface of the herbs. For flower and herbaceous herbs or herbs requiring a longer decoction time, the amount of water can be adjusted accordingly. When 14 times the amount of water is added for the first decoction to meet the solvent requirement of "2-5 cm above the surface of the herbs," and 12 times the amount of water is added for the second decoction, the water level exceeds 2 cm. Therefore, the final water addition is determined to be 14 times the amount of water for the first decoction and 12 times the amount of water for the second decoction.

[0070] (3) Investigation of soaking time According to the "Preparation of Standard Decoctions" section of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," the medicinal slices to be decocted should be soaked first, with a soaking time generally not less than 30 minutes. Based on the "Technical Requirements," the soaking time for the Sikuawa standard decoction is determined to be 30 minutes.

[0071] (4) Examination of simmering time According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Granules," each dose of medicine is generally decocted twice. Generally, after boiling, it should be decocted for 30 minutes. For diaphoretic, heat-clearing, and aromatic herbs, prolonged decoction is not recommended; boiling for 20 minutes after boiling is sufficient. For harder herbs, the decoction time can be appropriately extended. For tonifying herbs, first bring to a boil over high heat, then simmer over low heat for about 60 minutes. The second decoction time can be appropriately shortened. Therefore, based on the "Technical Requirements," the standard decoction for Sikuawa is decocted twice: the first decoction is boiled for 30 minutes after boiling, and the second decoction is boiled for 20 minutes after boiling.

[0072] (5) Investigation of solid-liquid separation conditions According to the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," the recommended filter material for solid-liquid separation under the "Standard Decoction Preparation" section should have a mesh size of 100 mesh or higher. To obtain a decoction consistent with traditional decoctions using modern solid-liquid separation methods, the filtration effects of 100-mesh, 200-mesh, and 300-mesh sieves on the Si Kuai Wa decoction were investigated. The results showed that 100-mesh, 200-mesh, and 300-mesh sieves all facilitated solid-liquid separation. However, the filtrates obtained through filtration using 100-mesh and 200-mesh sieves exhibited poor clarity and a higher amount of sediment after settling. The filtrate obtained through filtration using a 300-mesh sieve showed better clarity and a lower amount of sediment after settling. Therefore, a 300-mesh sieve was chosen for solid-liquid separation of the Si Kuai Wa standard decoction.

[0073] 4.3 Results of Determination of Extraction Process Parameters Take 100g of four pieces of medicinal slices, place them in an electric ceramic kettle, add water and decoct twice. For the first decoction, add 14 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter the decoction through a 300-mesh sieve while hot. For the second decoction, add 12 times the amount of water, bring to a boil over high heat, then simmer over low heat for 20 minutes. Filter the decoction through a 300-mesh sieve while hot and combine the two decoctions.

[0074] 5 Concentration Process 5.1 Study on Concentration Process Parameters According to the "Preparation of Standard Decoctions" section of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," it is recommended to use a vacuum concentration method for low-temperature concentration to obtain the specified amount of extract. By comparing different concentration temperatures, a suitable concentration temperature is determined, and a concentration ratio of 1:1 is tentatively set. The concentration state is then observed.

[0075] The density, yield, and state of the concentrated solution of four standard decoctions were compared and measured at different concentration temperatures. The results showed that the yield was basically consistent across different concentration temperatures, with no significant difference between 50℃ and 65℃. Considering all factors, 65℃ was chosen as the optimal concentration temperature. When the concentration ratio was 1:1, the extract had moderate viscosity, good fluidity, and was easy to transfer, with densities ranging from 1.03 to 1.08 g / ml. Therefore, a concentration ratio of 1:1 was determined, and other concentration ratios were not considered.

[0076] 5.2 Results of Determination of Concentration Process Parameters The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure using a rotary evaporator (temperature: 65℃; vacuum degree: -0.080~-0.090MPa) to 100ml of extract.

[0077] 6. Validation of the preparation process Three portions (100g each) of four types of medicinal slices (batch numbers: P-SKW-240719-D-YP01, P-SKW-240719-D-YP01, P-SKW-240719-D-YP01) were taken and prepared as standard decoctions according to the standard decoction preparation process. The yield of the three batches of standard decoctions was determined to evaluate the stability and rationality of the standard decoction preparation process. The results are shown in the table below: Table 3. Verification of the preparation method of the Four-Wall Standard Decoction

[0078] The experimental results show that the specific gravity, solid content and yield of the concentrated solution of the three parallel batches of Sikuawa standard decoction are basically the same, indicating that the process is stable and repeatable and can be used as the preparation process of Sikuawa standard decoction.

[0079] 7. Drying process 7.1 Study on freeze-drying process parameters According to the "Preparation of Standard Decoctions" section of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula Granules," freeze-drying is generally recommended for the preparation of standard decoctions to ensure their quality stability, ease of dissolution, and elimination of excipients. Therefore, freeze-drying was initially chosen as the drying method for the four-piece standard decoction.

[0080] Under magnetic stirring, the extract was dispensed into 10 ml brown vials, each vial containing 1 ml. The vials were partially capped, and after dispensing, they were transferred to a vacuum freeze dryer for freeze-drying. The vials were then removed, capped with aluminum, and the final product was obtained. The eutectic point test results from the four-walled vacuum freeze dryer were -40.9℃, the pre-freezing temperature was -50ºC, the sublimation drying temperature was -45ºC to 0ºC, the desorption drying temperature was 10ºC to 30ºC, and the total drying time was 48 hours. The results indicate that under these freeze-drying conditions, the four-walled standard decoction has low moisture content and is easy to preserve.

[0081] 7.2 Confirmation of freeze-drying process parameters Results of freeze-drying process parameters: Under magnetic stirring, the solution was dispensed into 10 ml brown vials, each with a volume of 2 ml, half-stopped, and then transferred to a vacuum freeze dryer for freeze-drying. The freeze-drying parameters were as follows: the pre-freezing temperature of the four-piece standard decoction concentrate was -50℃, the pre-freezing time was 180 minutes, the sublimation drying temperature was -45℃ to 0℃, the sublimation drying time was 2340 minutes, and the vacuum degree was -0.2 mbar; the desorption drying temperature was 10℃ to 30℃, the desorption drying time was 360 minutes, and the vacuum degree was 0 mbar.

[0082] Example 2 Establishment of a method for content determination and characteristic spectral analysis of standard decoction in four-piece tile molds 1. Establishment of analytical methods for content determination This product is *Chloranthus multiflora*, a plant belonging to the Chloranthus family. Chloranthus multistachys The dried rhizome and roots of *H.-M.* Pei. Also known as Four Heavenly Kings, Four Winds, Four Tiles, Big Four Tiles, Four Tiles, Red Four Tiles, Four Yellow Leaves, etc. It has a pungent and bitter taste, and is neutral in nature; it is toxic. It enters the lung and liver meridians. It has the functions of relaxing muscles and tendons, dispelling wind and relieving pain, clearing heat and detoxifying, and reducing swelling. It is used to treat pulmonary tuberculosis, unnamed boils and carbuncles, injuries from falls, and rheumatic lumbago. Modern research shows that *H.-M.* Pei mainly contains volatile oils, terpenes, coumarins and lignans, alkaloids, etc. The 2003 edition of the *Guizhou Province Standards for the Quality of Traditional Chinese Medicine and Ethnic Medicines* records that the rhizome is in a clump-like shape, densely covered with numerous fibrous roots. The fibrous roots are slender and cylindrical, 10–20 cm long and 0.05–0.15 cm in diameter, with a brownish-brown surface and branch root scars. It is brittle, with a smooth cross-section; the bark is yellowish-brown, and the wood is pale yellow. It has a faint odor and a slightly bitter taste.

[0083] Experimental studies have shown that the four tiles contain a high amount of chlorogenic acid, a natural polyphenol compound that is widely found in plants and has antioxidant, anti-inflammatory, metabolic regulating, and antibacterial effects.

[0084] 1.1 Instruments and Reagents The Agilent 1290 Infinity II ultra-high performance liquid chromatograph, the Shimadzu LC40D ultra-high performance liquid chromatograph, the 0.01% electronic balance (model: ml204), the electric thermostatic water bath (model: HH-S8), methanol and acetonitrile were all chromatographic grade, with batch numbers 225331 and F22MB2201 respectively, purchased from Fisher Scientific. The water was ultrapure water, and all other reagents were analytical grade. The batch numbers of the 15 batches of the four-piece tile standard soup are: D240905-01, D240905-02, D240905-03, D240905-04, D240905-05, D240905-06, D240905-07, D240905-08, D240905-09, D240905-10, D240905-11, D240905-12, D240905-13, D240905-14, and D240905-15 (provided by the laboratory process group).

[0085] 1.2 Source and purity test of reference standards Chlorogenic acid (110753-202119, China National Institutes for Food and Drug Control, purity: 96.3%). For content determination, no pretreatment is required.

[0086] 1.3 Determination of chromatographic conditions Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (2.1 mm × 100 mm, 1.8 µm); acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, with gradient elution as specified in the table below; flow rate of 0.20 mL / min; column temperature of 25 °C; and detection wavelength of 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0087] 1.4 Preparation of reference solution Prepare a solution containing 20 μg of chlorogenic acid per 1 ml by adding methanol to chlorogenic acid reference standard and shaking well.

[0088] 1.5 Preparation of the test solution (1) Investigation of different extraction solvents Take an appropriate amount of four standard decoctions (D-240905-01), totaling nine portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and sequentially and precisely add 25 ml of methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water. Weigh the contents, sonicate (500W, 40kHz) for 30 minutes, cool, and weigh again. Make up the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, precisely inject 1 μl each of the reference solution and the test solution into an ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction solvents on the chlorogenic acid content to determine the optimal extraction solvent. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below. Table 4. Effects of different extraction solvents on the content determination results of Sikuawa standard decoction.

[0089] The experimental results show that different solvents have a significant impact on the chlorogenic acid content in the standard decoction of Sikuawa. Considering both the content and the peak shape of the chromatogram, 50% ethanol was selected as the extraction solvent for further research.

[0090] (2) Examination of extraction methods Take 0.2g of four standard decoctions (D-240905-01), making two portions, each with two parallel samples. Place each portion into a stoppered conical container, and precisely add 25 ml of 50% ethanol sequentially. Weigh the samples, and treat them separately by sonication and reflux for 30 minutes. After cooling, weigh them again, and replenish the lost weight with 50% ethanol. Shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, precisely inject 1 μl each of the reference solution and the test solution into an ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction methods on the chlorogenic acid content to determine the optimal extraction method. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below. Table 5. Effects of different extraction methods on the content determination results of Sikuawa standard decoction.

[0091] The experimental results show that different extraction methods have a significant impact on the chlorogenic acid content in the standard decoction of Sikuawa. Considering the ease of operation and the differences in content, ultrasonic treatment (power 500W, frequency 40kHz) was selected as the extraction method for further research.

[0092] (3) Investigation of the volume of extraction solvent Take 0.2g of four standard decoctions (D-240905-01), making two portions, each with two parallel samples. Place each portion in a stoppered conical container, and accurately add 15ml, 25ml, and 50ml of 50% ethanol sequentially. Weigh the samples, sonicate (500W, 40kHz) for 30 minutes, cool, and weigh again. Make up the weight loss with 50% ethanol, shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, accurately inject 1μl each of the reference solution and the test solution into an ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction times on the chlorogenic acid content to determine the optimal extraction time. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below. Table 6. Effect of different extraction solvent volumes on the content determination results of the four-piece standard decoction.

[0093] The experimental results show that the volume of different extraction solvents has no significant effect on the chlorogenic acid content in the four standard decoctions. Considering the cost and the differences in content, an extraction solvent volume of 15 ml was selected for further research.

[0094] (4) Examination of different extraction times Take 0.2g of four standard decoctions (D-240905-01), two portions in total, each with two parallel samples. Place each portion in a stoppered conical container, and accurately add 25 ml of 50% ethanol sequentially. Weigh the samples, and sonicate them (500W power, 40kHz frequency) for 20 minutes, 40 minutes, and 60 minutes respectively. After cooling, weigh the samples again, and replenish the lost weight with 50% ethanol. Shake well, filter, and collect the filtrate to obtain the test solutions. Under the above chromatographic conditions, accurately inject 1 μl each of the reference solution and the test solution into the ultra-high performance liquid chromatograph for determination. Calculate the effect of different extraction solvent volumes on the chlorogenic acid content to determine the optimal extraction time. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below. Table 7. Effect of different extraction times on the content determination results of the Sikuawa standard decoction.

[0095] The experimental results show that different extraction times have no significant effect on the chlorogenic acid content in the standard decoction of Sikuawa. Considering the time cost and the difference in content, ultrasonic treatment (power 500W, frequency 40kHz) for 20 minutes was selected for subsequent research.

[0096] (5) Determination of the preparation method of the test solution Based on the results of the sample pretreatment experiment, the preparation method of the test sample can be determined as follows: Take about 0.2g of the standard decoction of four tiles, weigh it accurately, place it in a stoppered conical flask, add 15ml of 50% ethanol accurately, weigh it, sonicate for 20 minutes, cool it, weigh it again, make up the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test sample.

[0097] 2. Validation of content determination method (1) Specificity examination Accurately pipette 1 μl each of the four standard decoction solutions (D-240905-01), chlorogenic acid reference solution, and blank solvent, and inject them separately into the ultra-high performance liquid chromatograph. Perform the determination under the chromatographic conditions described in section "1.3," and record the chromatograms. The results are as follows: Figure 2 As shown.

[0098] The chromatogram results show that the analytical method has good specificity for the determination of chlorogenic acid content in the Sikua standard decoction.

[0099] (2) Peak purity Precisely pipette 1 μl each of the test solution and reference solution of four standard decoctions (D-240905-01) and inject them into the ultra-high performance liquid chromatograph for determination. The peak purity of the target peak is then obtained. The results are shown in the figure. Figure 3 .

[0100] Table 8 Matching values ​​for target peaks and peak purity

[0101] The experimental results show that the peak purity matching value of the indicator component chlorogenic acid is greater than 960, indicating that its peak purity meets the analytical requirements.

[0102] (3) Linear Accurately weigh an appropriate amount of chlorogenic acid reference standard and place it in a numbered 50ml volumetric flask. Add methanol to prepare a solution containing 0.03192mg of chlorogenic acid per ml. Shake well to obtain the chlorogenic acid reference standard stock solution, and store it in a refrigerator for later use. Dilute the stock solution (1ml, 3ml, 5ml, 7ml, and 9ml) to 10ml volumetric flasks and dilute to the mark with methanol to obtain reference solutions of different concentrations of chlorogenic acid. Perform chromatographic analysis under the conditions described above. Plot the concentration on the x-axis and the peak area on the y-axis to investigate the linear range of chlorogenic acid. The linearity results are shown in the table below. Figure 4 .

[0103] Table 9. Linearity Study of Chlorogenic Acid

[0104] Experimental results showed that the concentration of isovitexin and peak area of ​​chlorogenic acid exhibited a good linear relationship within the concentration range of 0.003192 mg / ml to 0.02873 mg / ml, with a correlation coefficient R0. 2 =0.9998, the linear regression equation is: y=5951.3x+1.9907.

[0105] (4) Precision test 1) Instrument precision test Accurately pipette 1 μl each of the test solution of four standard decoctions (D-240905-01) and the chlorogenic acid reference solution, inject them into the ultra-high performance liquid chromatograph, and determine the results. Calculate the RSD (%) value of the target peak using the external standard one-point method, based on chlorogenic acid. The specific results are shown in the table below.

[0106] Table 10 Precision Experiment Results of the Method for Determining the Content of Four-Wall Standard Decoction

[0107] The results show that the RSD (%) values ​​of the target peak chlorogenic acid content are all less than 2%, indicating that the method has good instrument precision.

[0108] 2) Repeatability test Take approximately 0.1 g of the same batch of four standard decoctions (D-240905-01), accurately weigh it, and prepare six parallel solutions to prepare six test solutions for later use. Perform chromatographic analysis under the conditions described above. Calculate the RSD (%) of the target peak content using the external standard single-point method, based on the chlorogenic acid content in the test solution. See the table below for detailed results.

[0109] Table 11 Repeatability test results of the method for determining the content of Sikuawa standard decoction

[0110] The experimental results show that the RSD (%) values ​​of the target peak chlorogenic acid content are all less than 2%, indicating that the method has good repeatability.

[0111] 3) Intermediate precision Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.1g of the same batch of four standard decoctions (D-240905-01), accurately weighed, and prepared in 6 parallel batches. The test solution was prepared according to the test solution preparation method, and the chromatographic analysis was performed under the above conditions. The RSD (%) value of the target peak content was calculated using the external standard one-point method, based on the chlorogenic acid content in the test solution. The specific results are shown in the table below.

[0112] Table 12 Results of intermediate precision experiment for the determination of content in the Four-Wall Standard Decoction

[0113] The experimental results show that the intermediate precision RSD (%) value of the target peak chlorogenic acid content is 142% < 4%, indicating that the intermediate precision of this method is good.

[0114] (5) Accuracy test Take 0.1g of four standard decoctions with known content (batch number: D-240905-01, chlorogenic acid content 3.12mg / g), accurately weigh a total of 6 portions, and accurately add 50ml of chlorogenic acid reference solution prepared with 75% methanol (concentration 0.00626mg / ml) to each portion. Weigh the portions and prepare the test solutions according to the test solution preparation method. Detect the solutions under the above chromatographic conditions, injecting 1µl of each sample. Calculate the content of the target peak using the external standard one-point method, based on chlorogenic acid. Calculate the recovery rate and RSD according to the following formulas. The results are shown in the table below.

[0115] Table 13. Results of the chlorogenic acid recovery experiment in the determination method of the content of the standard decoction of Sikuawa.

[0116] The experimental results show that the recovery rate of chlorogenic acid in the four standard decoctions is within the range of 95%-102%, and the RSD% is less than 2%, indicating that the accuracy of the assay method is good.

[0117] (6) Stability test The test solution of the four-piece standard decoction (D-240905-01) was prepared according to the preparation method of the test solution. The sample was injected at 0, 2, 4, 6, 12 and 24 hours according to the above chromatographic conditions, with an injection volume of 1 μl. The RSD (%) value of the target peak content was calculated using the external standard one-point method based on chlorogenic acid. The specific results are shown in the table below.

[0118] Table 14. Stability test results of the method for determining the content of Sikuawa standard decoction.

[0119] Experiments showed that the RSD (%) of the target peak chlorogenic acid content was less than 2% within 24 hours, indicating that the solution had good stability within 24 hours.

[0120] (7) Durability test 1) Investigation of different chromatographic columns The effects of three chromatographic columns—DiKMA Endsavorsil C18 (2.1*150mm, 1.8μm), JADE-PAK KP-C18 (2.1*100mm, 1.8μm), and Agilent ZORBAX SB-AQ C18 (2.1*150mm, 2.7μm)—on the peak shape and resolution of chlorogenic acid in the Sikua standard decoction were compared. The test solution from the Sikua standard decoction (D240107-01) under the [Content Determination] section was taken and analyzed under the above chromatographic conditions. Chromatographic data were recorded as chlorogenic acid. Experimental Results Figure 5 As shown in the table below.

[0121] Table 15. Effect of different chromatographic columns on the determination results of the content of the standard decoction in Sikuawa.

[0122] Experimental results showed that all three chromatographic columns had good separation performance and could meet the requirements for the determination of four tile contents. However, the resolution and theoretical plate number of the DiKMA Endsavorsil C18 (2.1*150mm, 1.8μm) column were better than those of the JADE-PAKKP-C18 (2.1*100mm, 1.8μm) and Agilent ZORBAX SB-AQ C18 (2.1*150mm, 2.7μm) columns. Therefore, the DiKMA Endsavorsil C18 (2.1*150mm, 1.8μm) column was selected as the preferred column for this experiment.

[0123] 2) Investigation using different chromatographs Based on the existing equipment in the laboratory, an Agilent high performance liquid chromatograph (Agilent 1290 Infinity II) and a Shimadzu ultra-high performance liquid chromatograph (LC40D) were selected to compare the effects of the two chromatographs on the peak shape and resolution of chlorogenic acid in the four-piece tile formulation particles.

[0124] Take four samples of the test solution from the [Content Determination] section of the standard decoction (D-240905-01), and determine the content using the chromatographic conditions described above. Calculate the content as chlorogenic acid and record the chromatographic data. The experimental results are shown in the figure below. Figure 6 As shown.

[0125] Table 16 Results of tests using different chromatographs

[0126] Experimental results show that this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0127] 3) Investigation at different column temperatures The effects of different column temperatures (20℃, 25℃, and 30℃) on the peak shape of chlorogenic acid in the standard decoction of Sikuawa were compared.

[0128] Take four standard decoction samples (D-240905-01) [Content Determination] test solution, and determine the content according to the above chromatographic conditions. Calculate chlorogenic acid and record the chromatographic data. Experimental results are as follows: Figure 7 As shown.

[0129] Table 17 Results of the determination of the content of standard soup in four tiles at different column temperatures

[0130] The results showed that the peak shape and separation were good at different column temperatures. At 25℃, 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 25℃ was chosen.

[0131] 4) Investigation of different flow velocities The effects of different flow rates of 0.28 ml / min, 0.30 ml / min, and 0.32 ml / min on the peak shape and resolution of chlorogenic acid in the standard decoction of Sikuawa were compared.

[0132] Take four standard decoction samples (D-240905-01) [Assay] and determine the content according to the above chromatographic conditions. Calculate chlorogenic acid and record the chromatographic data. Experimental results are as follows: Figure 8 As shown.

[0133] Table 18 Results of different flow rates on the determination of the content of the standard soup in four-piece tile.

[0134] The results showed that the peak shape and separation were good at all three flow rates. The target component showed good separation with no baseline drift at a flow rate of 0.30 ml / min. Therefore, a flow rate of 0.30 ml / min was selected for this experiment.

[0135] Establishment of a characteristic chromatogram analysis of the four-piece standard decoction 3.1 Instruments and Reagents Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph; KQ-500DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 0.01% electronic balance (model: ml204); HH-4 digital display constant temperature water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.).

[0136] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0137] Test reagents: Chlorogenic acid (110753-202119, National Institutes for Food and Drug Control, purity: 96.3%), rosmarinic acid (111871-202408, National Institutes for Food and Drug Control, purity: 99.6%), and isoflavone (DST241024-010, Lemeitian Pharmaceutical Desite, purity: 98%). Four-piece control medicinal material (batch number: 25061, Zhuhai Anzhe Biotechnology Co., Ltd.).

[0138] The batch numbers of the 15 batches of the four-piece tile standard soup are: D240107-01, D240107-02, D240107-03, D240107-04, D240107-05, D240107-06, D240107-07, D240107-08, D240107-09, D240107-10, D240107-11, D240107-12, D240107-13, D240107-14, and D240107-15 (provided by the laboratory process group).

[0139] 3.2 Preparation of the reference solution Accurately weigh approximately 2.0 g of four pieces of reference medicinal material and place them in a stoppered conical flask. Accurately add 50 ml of 75% methanol, weigh the flask, and sonicate (500 W, 40 kHz) for 40 minutes. Cool the flask, weigh it again, and replenish the lost weight with 75% methanol. Shake well, filter, and collect the filtrate as the reference solution for the reference medicinal material. Take the reference solution from the [Assay] section as the reference solution for the reference substance.

[0140] 3.3 Determination of chromatographic conditions (1) Determination of detection wavelength Four standard decoction samples were injected for analysis, and the absorption spectra in the range of 190–400 nm were recorded (see...). Figure 9 ).

[0141] Experimental results show that at a wavelength of 326 nm, the four standard decoction sample solution can detect more chromatographic peak information and has less baseline noise interference. Therefore, 326 nm was chosen as the detection wavelength.

[0142] (2) Optimization of the mobile phase ① The effects of adding and not adding acid were investigated. A was the organic phase, and B was the aqueous phase. The gradient is as follows:

[0143] Experimental results: see details Figure 10The peak shape was better with the addition of acid than without acid, and the peak shape was best with a methanol-0.1% phosphoric acid aqueous solution system. Therefore, a methanol-0.1% phosphoric acid aqueous solution was selected for investigation.

[0144] ② Investigate different mobile phase systems.

[0145] Experimental results: see details Figure 11 Acetonitrile-0.1% phosphoric acid aqueous solution has a stronger elution ability than methanol-0.1% phosphoric acid aqueous solution. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution was chosen to explore the conditions. Later, the various acids used in the mobile phase will be investigated.

[0146] (3) Determination of chromatographic conditions The column was packed with octadecylsilane-bonded silica gel (2.1 mm × 100 mm, 1.8 µm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in the table below; the flow rate was 0.30 mL / min; the column temperature was 25 °C; and the detection wavelength was 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0147] 3.4 Preparation of the test solution This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of the Sikua standard decoction. Methanol, 75% methanol, 50% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, and water were selected as extraction solvents. The characteristic chromatograms of different extraction solvents were compared by using the total peak area / sample weight of six tentatively determined chromatographic peaks and the chromatograms.

[0148] Take an appropriate amount of the four-piece standard decoction (D240107-01), approximately 0.2g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 25ml each of methanol, 75% methanol, 50% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, and water, respectively. Weigh the flask, sonicate (500W) for 30 minutes, cool, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Inject the sample according to the determined chromatographic conditions and record the chromatogram. The results of the investigation of the characteristic chromatogram of the four-piece standard decoction with different extraction solvents are shown below. Figure 12 , Figure 13 .

[0149] Experimental results: The extraction efficiency was high when 50% ethanol was used as the extraction solvent, therefore 50% ethanol was chosen as the extraction solvent.

[0150] Based on the above experimental results, the sample pretreatment method for the characteristic chromatogram of the four-piece standard decoction can be determined as follows: Take about 0.2g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 50ml of 50% ethanol, weigh it, sonicate it (power 500W) for 20 minutes, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the product.

[0151] 3.5 Determination and Identification of Common Peaks UPLC spectra of different batches of Sikua standard decoction samples were determined, and the results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and common peaks were selected.

[0152] Figure 14 The results showed that there were 6 relatively obvious common peaks in the characteristic spectrum of the four standard decoctions.

[0153] 3.6 Methodological Validation of Feature Map Analysis Method (1) Specificity examination Accurately pipette 1 μl each of the test solution and blank solvent from the "Four-Piece Standard Decoction [Characteristic Chromatography]" section and inject them into the liquid chromatograph. Determine the chromatographic conditions as described above. Results are shown below. Figure 15 .

[0154] The experimental results show that the solvent did not interfere with the characteristic peaks in the spectrum of the four standard decoctions.

[0155] (2) Holistic examination Take the test solution of the standard decoction [characteristic spectrum] from four tiles, inject it into the liquid chromatograph, extend the elution time by one time at the mobile phase ratio of the gradient endpoint, and analyze the characteristic spectrum.

[0156] Figure 16 The results showed that no obvious chromatographic peaks were observed after doubling the elution time under these chromatographic conditions, indicating that the chromatographic conditions basically met the principle of maximizing information content.

[0157] (3) Precision test Take the test solution from the "[Characteristic Spectrum]" section of the four-piece standard decoction and inject it six times, with an injection volume of 1 μl. Temporarily identify six characteristic peaks, among which peaks 2, 4, and 6 should correspond to the retention times of the corresponding reference peaks. Using peak 2 as the reference peak, calculate the relative retention time. The experimental results are shown in the table below.

[0158] Table 19 Precision results of the characteristic spectrum of the Four-Piece Standard Decoction (relative retention time)

[0159] Experimental results show that the RSD of the relative retention time of each chromatographic peak is less than 3.0%, indicating that the instrument has good precision.

[0160] (4) Stability test Take four standard decoction samples from the [Characteristic Spectrum] section and inject them at 0, 2, 4, 8, 12, and 24 hours, with an injection volume of 1 μl. Temporarily identify six characteristic peaks, where peaks 2, 4, and 6 should correspond to the retention times of their respective reference peaks. Using peak 2 as the reference peak, calculate the relative retention time. The results are shown in the table below.

[0161] Table 20 Stability results of the characteristic spectra of the Four-Piece Standard Decoction (relative retention time)

[0162] The experimental results show that the RSD of the relative retention time of the chromatographic peak is less than 3.0%, indicating that the sample solution is relatively stable.

[0163] (5) Repeated examination Accurately weigh approximately 0.2 g of the same batch of four standard decoction pieces (D-240107-01), prepare six parallel aliquots, and prepare test solutions according to the method for preparing test solution under the [Characteristic Spectrum] section. Inject 1 μl into each solution. Temporarily identify six characteristic peaks, among which peaks 2, 4, and 6 should correspond to the retention times of the corresponding reference peaks. Using peak 2 as the reference peak, calculate the relative retention time.

[0164] Table 21 Repeatability results of characteristic spectra of the Four-Piece Standard Decoction (relative retention time)

[0165] Experimental results show that the relative retention time (RSD) of each chromatographic peak is less than 3.0%, indicating that the method has good repeatability.

[0166] (6) Durability test ① Column analysis Three chromatographic columns were investigated: DiKMA Endsavorsil C18 (2.1 * 100 mm, 1.8 µm); JADE-PAK K C18 (2.1 * 100 mm, 1.8 µm); and Agilent ZORBAX SB-AqC18 (2.1 * 100 mm, 1.8 µm). The effects of the three columns on the peak characteristics of the four-piece standard decoction were examined.

[0167] Table 22. Chromatographic column analysis results of the characteristic chromatograms of the four-piece standard soup (relative retention time).

[0168] Experimental results show that: (See details below) Figure 17 Table 22. Different chromatographic columns have a certain impact on the peak elution. Elution using a DiKMA Endsavorsil C18 (2.1 * 100 mm, 1.8 µm) ultra-high performance liquid chromatography column resulted in better peak shapes and the best separation effect. Therefore, it is recommended to use an ultra-high performance liquid chromatography column of model DiKMA Endsavorsil C18 (2.1 * 100 mm, 1.8 µm) for this method.

[0169] ② Column temperature investigation The elution performance at different column temperatures (20℃, 25℃, 30℃) was investigated.

[0170] Table 23 Column Temperature Study Results of Four Standard Broth Samples (Relative Retention Time)

[0171] Experimental results show: See details below. Figure 18 Table 23. Column temperature has a certain impact on peak elution, but the separation effect is best and the peak shape is better when the column temperature is 25℃. Therefore, it is recommended to use a column temperature of 25℃ for the determination.

[0172] ③ Flow rate study: The elution effect at different flow rates (0.28 ml / min, 0.30 ml / min, 0.32 ml / min) was investigated.

[0173] Table 24 Results of flow velocity investigation (relative retention time) of characteristic spectra of the four-piece standard soup.

[0174] Experimental results show: See details below. Figure 19 Table 24. Flow rate has some influence on peak formation, but the peak shape is optimal when the flow rate is 0.3 ml / min. Therefore, it is recommended to use 0.3 ml / min as the measurement flow rate.

[0175] Example 3 Research on the process of four-piece tile granules Based on the yield of the extract in Example 1, in order to strictly control the yield of extract in large-scale production, and in accordance with the principle of minimizing the amount of excipients, the final yield of this product was determined to be 6.7%, that is, 15,000g of medicinal slices were made into 1,000g of granules, with each 1.0g of formula granules equivalent to 15.0g of medicinal slices.

[0176] The yield was determined to be 8.7%. The average yield of the 15 batches of standard decoctions, ranging from -3SD to +3SD, should be between 3.8% and 7.0%. At the same time, in order to strictly control the yield of the four-piece tile formula granules in large-scale production and ensure the uniformity and stability of the quality of the four-piece tile formula granules, the yield range of the dry extract of the formula granules was determined to be 3.4% to 6.7%.

[0177] In summary, the yield of this product is determined to be 6.7%, meaning 15,000g of medicinal slices will be made into 1,000g of granules. The specification is that each 1.0g of formula granules is equivalent to 15.0g of medicinal slices, and the dry extract yield ranges from 3.4% to 6.7%. Example 4 Thin-layer identification of four tiles The 2003 edition of the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials of Guizhou Province" did not include a thin-layer chromatography (TLC) method for identifying *Si Kuai Wa* (a type of medicinal herb). Considering the specificity of TLC identification for *Si Kuai Wa*, a reference herb of *Si Kuai Wa* was used as a control. Petroleum ether-ethyl acetate (1:1) was used as the developing solvent. The samples were removed, dried, and examined under ultraviolet light (365 nm). The results showed that fluorescent spots corresponding to the reference herb could be detected in all samples, and the spots were clear and well-separated. This method is simple to operate and has good reproducibility; therefore, it was included in the main text of the quality standard for *Si Kuai Wa* formula granules.

[0178] 1. Instruments, reagents and reagents Instruments: Automatic thin-layer imaging system (TLC VISUALIZER2, CAMAG, Switzerland), dual-groove developing tank, fully automatic thin-layer sampling system (AUTOMATIC TLC SAMPIER4, CAMAG, Switzerland), AL-104 electronic balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.], KQ-500DA ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), silicone G thin-layer plate (10cm×10cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20231106), silicone G thin-layer plate (10cm×10cm, Yantai Huayang New Material Technology Co., Ltd., batch number: 20231204), silicone G thin-layer plate (10cm×10cm, Merck AG).

[0179] Reagents: Methanol (batch number: 20230301) and ethyl acetate (batch number: 20231001) were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd.

[0180] Trial drugs: 3 batches of Sikuawa formula granules (batch numbers: 240901, 240902, 240903); source: Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd.; Sikuawa control medicinal material (batch number: 25061, Zhuhai Anzhe Biotechnology Co., Ltd.).

[0181] 2. Preparation of solution 2.1 Preparation of the test solution Take 0.2 g of the four-piece tile formula granules, grind them finely, add 5 ml of methanol, sonicate for 10 minutes, filter, and use as the test solution.

[0182] 2.2 Preparation of control herbal solution Take 1 g of the reference medicinal material from four tiles, add 20 ml of water, boil for 30 minutes, filter, evaporate the filtrate to dryness, add 2 ml of methanol to dissolve, filter, and use as the reference medicinal material solution.

[0183] 2.3 Preparation of negative sample solution Take 1.0 g of negative sample of four tile formula granules, grind it into a fine powder, and process it in the same way as the test sample to prepare a negative sample solution.

[0184] 3 Thin-layer chromatography conditions Thin-layer plate: Silicone G thin-layer plate Developing solvent: petroleum ether-ethyl acetate (1:1) Sample volume: 4 μl Unfolding method: A double-groove unfolding cylinder is used, with an unfolding distance of approximately 8 cm.

[0185] Inspection: Examine under ultraviolet light at 365nm.

[0186] 4. Sample Size Investigation Four samples of the test solution of the formula granules and four samples of the reference medicinal material were spotted onto the same silica gel G thin-layer plate. The plate was developed under the thin-layer chromatographic conditions described above. The plate was then removed, dried, and examined under ultraviolet light at 365 nm. The thin-layer chromatogram is shown below. Figure 20 .

[0187] Depend on Figure 20 It can be seen that when the sample volume is 4 μl, the main spot in the test sample at the corresponding position of the control herb is clear and there is no other interference. Therefore, the sample volume of 4 μl was selected.

[0188] 5. Specificity of the four-piece tile formula granules Two μL each of the four-piece granule test solution, the four-piece granule herbal solution, and the four-piece granule negative sample were spotted onto the same silica gel G thin-layer plate. The plate was developed under the above-described thin-layer chromatographic conditions. The plate was then removed, air-dried, and examined under UV light at 365 nm. The thin-layer chromatogram is shown below. Figure 21 .

[0189] Depend on Figure 21 As can be seen, the chromatogram of the four-piece tile formula granule test sample showed spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, and there was no interference from the negative sample. This indicates that the thin-layer chromatography method has good specificity.

[0190] 6. Investigation at different temperatures Four μL of each of the four-piece granule test solution, the four-piece granule reference herbal solution, and the four-piece granule negative sample were spotted onto the same silica gel G thin-layer plate. The plates were developed under the aforementioned thin-layer chromatographic conditions at different temperatures. The plates were then removed, air-dried, and examined under UV light at 365 nm. The thin-layer chromatograms are shown below. Figure 22 .

[0191] Depend on Figure 22 It can be seen that under different temperature conditions, the main spots of the same color appear at the corresponding positions of the chromatograms of the test sample of the Sikuawa formula granules and the chromatograms of the reference medicinal materials. The color development is clear, the separation is good, there is no tailing phenomenon, and there is no background interference. Only with the increase of temperature, the position of the corresponding spots does not change significantly, indicating that temperature has no significant effect on the thin-layer identification of the Sikuawa formula granules, indicating that the thin-layer identification method has good temperature durability.

[0192] 7. Investigation of different humidity levels Four μL each of the test solution of the Sikua granules formula, the reference herbal solution of Sikua granules, and the negative sample of Sikua granules formula were spotted onto the same silica gel G thin-layer plate. The plates were developed under different humidity conditions according to the thin-layer chromatographic conditions described above. The plates were then removed, air-dried, and examined under UV light at 365 nm. The thin-layer chromatograms are shown in the figure. Figure 23 .

[0193] Depend on Figure 23 It can be seen that under different humidity conditions, the test sample chromatogram and the reference medicinal material chromatogram of the Sikuawa formula granules show spots of the same color at the corresponding positions, and the main spots are clearly visible with good separation and no tailing phenomenon. This indicates that the change of humidity has no significant effect on the thin-layer identification of the Sikuawa formula granules, and that the thin-layer identification method is durable to humidity.

[0194] Investigation of Thin-Layer Boards from 8 Different Manufacturers Four μl samples of the test solution, reference herbal solution, and negative sample of the four-piece formula granules were spotted onto thin-layer plates from different manufacturers (Qingdao Ocean Silica G plate, Sinopharm Chemical Reagent Silica G plate, and Merck plate). The plates were developed under the same temperature and humidity conditions as described above. After development, the plates were removed, air-dried, and examined under UV light at 365 nm. The thin-layer chromatograms are shown in the figure. Figure 24 .

[0195] Depend on Figure 24 It is evident that when using silica gel thin-layer plates from different manufacturers (Qingdao Marine Silica G plate, Sinopharm Chemical Reagent Silica G plate, and German Merck plate), the main spots of the four-panel formula granule test sample chromatogram and the four-panel reference medicinal material chromatogram correspond to each other without significant influence, indicating that this thin-layer identification method has good durability with silica gel G plates from different manufacturers.

[0196] 9. Thin-layer chromatogram of the four-piece tile formula particles for identification Four μl samples of different batches of Sikua granules (test solution), Sikua reference herbal solution, and negative sample of Sikua granules were spotted onto the same silica gel G thin-layer plate. The plate was developed under the above-described thin-layer chromatographic conditions. The plate was then removed, air-dried, and examined under UV light at 365 nm. The thin-layer chromatogram is shown in the figure. Figure 25 .

[0197] Depend on Figure 25 It is evident that the test samples in the three batches of Sikuawa formula granules showed fluorescent spots of the same color at the corresponding positions as the reference medicinal materials, indicating that the thin-layer identification of the three batches of Sikuawa formula granules met the requirements.

[0198] 10 Summary The chromatograms showed good separation of fluorescent spots from the Si Kuai Wa formula granules. Furthermore, the corresponding positions on the chromatograms of the Si Kuai Wa formula granules and the Si Kuai Wa reference medicinal material showed the same color of main fluorescent spot, with a one-to-one correspondence. This method can effectively identify Si Kuai Wa formula granules that have lost their processed medicinal characteristics. Through thin-layer chromatography methodology evaluation, this method demonstrated good specificity and robustness, making it suitable for the chromatographic identification of Si Kuai Wa formula granules.

[0199] Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments, and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in this invention should be considered within the scope of protection of this invention.

Claims

1. A method for preparing a four-piece tile extract and its formulation, characterized in that, Specifically, the steps include the following: (1) Four pieces of medicinal slices, add water and decoct twice, filter, cool the filtrate to room temperature and combine the two decoctions, that is, the four pieces of medicinal slices extract; (2) The decoction was concentrated under reduced pressure to obtain an extract, with a yield of 4.2% to 11.2%; the extract was obtained. (3) Prepare the extract into the desired dosage form.

2. The method for preparing the four-piece tile extract and its formulation according to claim 1, characterized in that, The specific step (1) involves taking four pieces of medicinal slices, adding water and decocting them twice. For the first decoction, add 12 to 16 times the amount of water, soak for 20 to 40 minutes, bring to a boil over high heat, and then simmer over low heat for 20 to 40 minutes. Filter the decoction through a 200 to 400 mesh sieve while it is still hot. For the second decoction, add 10 to 14 times the amount of water, bring to a boil over high heat, and then simmer over low heat for 15 to 25 minutes. Filter the decoction through a 200 to 400 mesh sieve while it is still hot, and combine the two decoctions.

3. The method for preparing the four-piece tile extract and its formulation according to claim 1, characterized in that, The specific conditions for vacuum concentration in step (2) are: temperature: 60~70℃; vacuum degree: -0.080~-0.090MPa.

4. The method for preparing the four-piece tile extract and its formulation according to claim 1, characterized in that, Step (3) involves freeze-drying the obtained extract to obtain the Four-Piece Standard Decoction. The freeze-drying parameters are as follows: pre-freezing temperature is -45~-55℃, pre-freezing time is 150~200 minutes, sublimation drying temperature is -45℃~0℃, sublimation drying time is 2000~2500 minutes, and vacuum degree is -0.15~0.25mbar; desorption drying temperature is 10℃~30℃, desorption drying time is 300~400 minutes, and vacuum degree is -0.1~0.1mbar.

5. The method for preparing the four-piece tile extract and its formulation according to claim 1, characterized in that, Step (3) involves adding excipients to the obtained extract, drying it, adding more excipients, mixing it evenly, and granulating it to obtain four-piece tile granules.

6. The method for preparing the four-piece tile extract and its formulation according to claim 5, characterized in that, The specifications of the four-piece tile granules are such that each 1g of the formula granules is equivalent to 8-12g of medicinal slices.

7. A method for determining the content of components in a four-piece tile extract and its preparations, characterized in that, The determination was performed using high-performance liquid chromatography (HPLC). The column was packed with octadecylsilane-bonded silica gel, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 µm. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below. The flow rate was 0.30 mL / min; the column temperature was 25 °C; and the detection wavelength was 326 nm. 。 8. The method for determining the content of components in the extract of four tiles and its preparations according to claim 7, characterized in that, The test solution is prepared by the following method: take the sample to be tested, weigh it accurately, place it in a stoppered conical flask, add 50% ethanol accurately, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

9. The method for determining the content of components in the extract of four tiles and its preparations according to claim 7, characterized in that, The reference solution is a solution containing 20 μg of chlorogenic acid per 1 ml, prepared using methanol as the solvent.

10. A method for detecting the characteristic chromatograms of a four-piece tile extract and its formulation, characterized in that, The determination was performed using high-performance liquid chromatography (HPLC). The column was packed with octadecylsilane-bonded silica gel, with a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 µm. Acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution as specified in the table below. The flow rate was 0.30 mL / min; the column temperature was 25 °C; and the detection wavelength was 326 nm. 。 11. The method for detecting the characteristic chromatograms of the four-piece tile extract and its preparation according to claim 10, characterized in that, The test solution is prepared by the following method: take the sample to be tested, weigh it accurately, place it in a stoppered conical flask, add 50% ethanol accurately, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

12. The method for detecting the characteristic chromatograms of the four-piece tile extract and its preparations according to claim 10, characterized in that, The reference solution is a solution prepared with methanol as the solvent, containing 20 μg of chlorogenic acid, 20 μg of isoflavone, and 30 μg of rosmarinic acid per 1 ml.

13. The method for detecting the characteristic chromatograms of the four-piece tile extract and its preparations according to claim 10, characterized in that, The reference solution of the control medicinal material was prepared by the following method: Take four pieces of control medicinal material, weigh them accurately, place them in a stoppered conical flask, add 75% ethanol accurately, weigh them, reflux and extract for 40 minutes, cool them, weigh them again, replenish the lost weight with 75% ethanol, shake well, filter, and take the filtrate to obtain the solution.

14. The method for detecting the characteristic chromatograms of the four-piece tile extract and its preparations according to claim 10, characterized in that, The final standard for the characteristic chromatograms of the extract and its preparations of the four-piece tile was determined as follows: the chromatogram of the test sample should show 6 characteristic peaks, and the retention times should correspond to the 6 characteristic peaks in the chromatogram of the reference medicinal material; among them, peaks 2, 4, and 6 should correspond to the retention times of the corresponding reference peaks. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 1, 3, and 5 with peak S are calculated, and their relative retention times should be within ±10% of the specified values, which are 0.64 (peak 1), 1.11 (peak 3), and 2.19 (peak 5).

15. A thin-layer chromatographic detection method for a four-piece tile extract and its preparations, characterized in that, Four pieces of control medicinal materials were used as a reference. The specific parameters are as follows: thin-layer plate: silica gel G thin-layer plate; developing solvent: petroleum ether-ethyl acetate (1:1); sample volume: 4 μl; development method: double-groove developing tank, development distance is about 8 cm. Inspection: Examine under 365nm ultraviolet light; Preparation of test solution: Take the sample to be tested, grind it into a fine powder, add methanol, sonicate, and filter to obtain the test solution; Preparation of the control herbal solution: Take four pieces of control herbal material, add water, boil, filter, evaporate the filtrate to dryness, add methanol to dissolve, filter, and use as the control herbal solution; Preparation of negative sample solution: Take four pieces of tile formula granules as negative sample, grind them finely, and process them in the same way as the test sample to prepare negative sample solution.

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