Preparation method and detection method of creeping woodbetony root extract and preparation of creeping woodbetony root extract

Abstract

The invention relates to the technical field of traditional Chinese medicines, in particular to a preparation method and a detection method of a creeping woodbetony herb extract and a preparation thereof. The preparation method specifically comprises the following steps: (1) taking a creeper creeper herb decoction piece, adding water, decocting twice, filtering, cooling filtrate to room temperature, and combining the two decoctions to obtain the creeper creeper herb extract; (2) carrying out reduced-pressure low-temperature concentration on the decoction to obtain an extract, and controlling the density of the obtained extract to be 1.03-1.06 g / ml; an extract is obtained; and (3) preparing the extract into a required dosage form. The invention also establishes a specific chromatogram method, adopts ultrahigh-phase liquid chromatography, and has the characteristics of simplicity, convenience, stability, high precision, good reproducibility and the like. The invention also establishes a method for determining and analyzing the content of the creeper belvedere extract and the preparation thereof, and determines the method for determining the content of the creeper belvedere extract and the preparation thereof.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a method for preparing and detecting a Tripterygium wilfordii extract and its preparations. Background Technology

[0002] Triangular vine (Hedera nepalensis K. Kochvar. sinensis (Tobl.) Rehd.) is a plant belonging to the Araliaceae family. It is also known as ivy, and has various common names such as triangular maple, triangular vine, rice horn star, climbing vine, and triangular arrow. Triangular vine is a traditional medicine used by ethnic minorities in Guizhou Province, including the Han, Miao, Yi, Zhuang, Dong, and Tujia peoples. It is pungent, bitter, and cold in nature. It enters the liver, spleen, and lung meridians. It has the effects of dispelling wind and detoxifying, promoting blood circulation and stopping bleeding, reducing swelling and relieving pain. It is used to treat rheumatic pain, paralysis and numbness, hematemesis, hemoptysis, epistaxis, hematochezia, pruritus, eczema, traumatic injuries, and unnamed boils and carbuncles.

[0003] According to the "Quality Standards for Traditional Chinese and Ethnic Medicinal Materials of Guizhou Province" (2003 edition), the vine stem of this product is cylindrical, curved, branched, and varies in length, with a diameter of 0.3-1.2 cm. The surface is light yellowish-brown or grayish-brown, with longitudinal wrinkles, and densely covered with adventitious roots on one side. It is hard and brittle, easily broken; the broken surface has a thin, grayish-green or brown cortex, and a wide, yellowish-white or light brown xylem, with small pores (vascular bundles) visible under a magnifying glass. The pith is small and dot-like. The leaves are simple, alternate, and have long petioles. The leaf blade is leathery, slightly curled, and varies in shape, with entire or three-lobed margins, and is grayish-green or light yellowish-brown. It has a faint odor and a bitter taste.

[0004] Currently, there are few reports on the quality testing of Acer triangularis extracts and their preparations. The evaluation system for the quality standards of Acer triangularis extracts and their preparations is still imperfect, which cannot effectively evaluate their indicators and limits their application and development. Summary of the Invention

[0005] To address the aforementioned technical problems in the prior art, this invention provides a method for preparing and detecting *Tripterygium wilfordii* extract and its formulations, including methods for content analysis, characteristic chromatogram analysis, and thin-layer chromatography identification, as detailed below:

[0006] A method for preparing a triangular herb extract and its formulation includes the following steps:

[0007] (1) Take the slices of Trichoderma, add water and decoct twice, filter, and combine the two decoctions after cooling the filtrate to room temperature, which is the Trichoderma extract.

[0008] (2) The decoction was concentrated under reduced pressure and low temperature to obtain an extract, and the density of the obtained extract was controlled between 1.03 and 1.06 g / ml; the extract was obtained.

[0009] (3) Prepare the extract into the desired dosage form.

[0010] Further, step (1) specifically involves taking the triangular wind slices, adding water and decocting them twice. For the first decoction, add 10 to 14 times the amount of water directly, soak for 20 to 40 minutes, bring to a boil over high heat, then simmer over low heat for 50 to 70 minutes. Filter the decoction while it is still hot through a 200 to 400 mesh sieve, and record the quality of the decoction after the filtrate has cooled to room temperature. For the second decoction, add 8 to 12 times the amount of water, heat to a boil over high heat, then simmer over low heat for 15 to 25 minutes. Filter the decoction while it is still hot through a 200 to 400 mesh sieve, and record the quality of the decoction after the filtrate has cooled to room temperature. Combine the two decoctions.

[0011] Furthermore, the specific conditions for the reduced pressure low-temperature concentration in step (2) are: temperature: 65℃; vacuum degree: -0.080~-0.090MPa.

[0012] Further, step (3) involves freeze-drying the obtained extract to obtain the standard decoction of *Trigonella foenum-graecum*. The freeze-drying parameters are as follows: the pre-freezing temperature of the concentrated *Trigonella foenum-graecum* is -40 to -50°C, the pre-freezing time is 200 to 300 minutes, the sublimation drying temperature is -30°C to 0°C, the sublimation drying time is 800 to 900 minutes, and the vacuum degree is -0.15 to 0.25 mbar; the desorption drying temperature is 5°C to 25°C, the desorption drying time is 400 to 450 minutes, and the vacuum degree is -0.15 to 0.25 mbar.

[0013] Furthermore, step (3) involves adding excipients to the obtained extract, drying it, adding more excipients, mixing it, and granulating it to obtain triangular wind granules.

[0014] Furthermore, the 14,000-16,000g of medicinal slices of the triangular wind granules are made into 1,000g of granules, with each 1g of formula granules equivalent to 14-16g of medicinal slices.

[0015] A method for determining the content of triangular spirulina and its preparations, using high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the stationary phase; 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 0.30 ml / min; column temperature of 30℃; detection wavelength of 326 nm.

[0016]

[0017] Furthermore, the chromatographic column used was 100 mm long, 2.1 mm inner diameter, and 1.8 μm particle size.

[0018] Furthermore, 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 40-60% ethanol accurately, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 40-60% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0019] Furthermore, the ultrasonic treatment has a power of 450-550W and a frequency of 35-45kHz.

[0020] Furthermore, the reference solution is a solution containing 20 μg of chlorogenic acid per 1 ml, prepared using 40-60% ethanol as a solvent.

[0021] A method for detecting the characteristic chromatograms of *Tripterygium wilfordii* extract and its preparations is disclosed, employing high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B, using gradient elution as specified in the table below; the flow rate is 0.30 ml / min; the column temperature is 30℃; and the detection wavelength is 326 nm.

[0022]

[0023] Furthermore, the chromatographic column used was 100 mm long, 2.1 mm inner diameter, and 1.8 μm particle size.

[0024] Furthermore, 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 40-60% ethanol accurately, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 40-60% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0025] Furthermore, the ultrasonic treatment has a power of 450-550W and a frequency of 35-45kHz.

[0026] Furthermore, the reference solution is a solution containing 20 μg of chlorogenic acid per 1 ml, prepared using 40-60% ethanol as a solvent.

[0027] Furthermore, the final standard for the characteristic chromatograms of *Tripterygium wilfordii* and its preparations is determined as follows: the chromatogram of the test sample should show 5 characteristic peaks, corresponding to the retention times of the 5 characteristic peaks in the chromatogram of the reference herb; among them, peak 2 should correspond to the retention time of the chlorogenic acid peak. The peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 1, 3, 4, and 5 with peak S are calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.43 (peak 1), 1.21 (peak 3), 2.09 (peak 4), and 3.17 (peak 5).

[0028] A thin-layer chromatographic detection method for Tripterygium wilfordii extract and its preparations, using Tripterygium wilfordii reference material as a reference, and chloroform-acetone-formic acid (4∶1∶0.1) as the developing solvent, is developed, removed, dried, and examined under ultraviolet light (365nm).

[0029] Compared with the prior art, the technical effects of this invention are reflected in:

[0030] 1. This application selects chlorogenic acid as the indicator component of Tripterygium wilfordii extract and its preparations, and conducts a quantitative determination method of Tripterygium wilfordii extract and its preparations by ultra-high performance liquid chromatography, and formulates quantitative standards for Tripterygium wilfordii extract and its preparations, providing a scientific basis for the establishment of quality standards for Tripterygium wilfordii extract and its preparations.

[0031] 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 Sanjiaofeng medicinal materials was established, and the material basis was studied. The chemical components corresponding to the characteristic peaks were identified and used for the source quality control of Sanjiaofeng formula granules.

[0032] 3. The characteristic spectrum obtained in this application presents 5 characteristic peaks. Peak 2 should correspond to the retention time of the chlorogenic acid peak, and the peak corresponding to the chlorogenic acid reference peak is the S peak. The relative retention times of peaks 1, 3, 4, and 5 with the S peak are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.43 (peak 1), 1.21 (peak 3), 2.09 (peak 4), and 3.17 (peak 5).

[0033] 4. This application establishes an analytical method for determining the content of *Tripterygium wilfordii* extract and its preparations, determines the method for determining the content of *Tripterygium wilfordii* extract and its preparations, and determines the content and transfer rate of 15 batches of *Tripterygium wilfordii* extract and its preparations, thus determining the range of content and transfer rate of *Tripterygium wilfordii* extract and its preparations. Attached Figure Description

[0034] Figure 1 It is a YMW mechanical split-type decoction pot.

[0035] Figure 2 It is a specific investigation map.

[0036] Figure 3 This is a chromatogram of peak purity.

[0037] Figure 4 It is a linear regression equation for chlorogenic acid.

[0038] Figure 5 This study investigates the effect of different chromatographic columns on the content of target components in the standard decoction of Triangle Wind.

[0039] Figure 6 This study examines the influence of different chromatographic instruments on the content of target components in the triangular wind formula granules.

[0040] Figure 7 These are the chromatographic results of the target components in the standard decoction of Triangle Wind at different column temperatures.

[0041] Figure 8 These are the chromatographic results of the target components in the standard decoction of Triangle Wind at different flow rates.

[0042] Figure 9 This is the DAD diagram of the standard decoction for Triangular Wind.

[0043] Figure 10 These are UPLC spectra of different mobile phase systems.

[0044] Figure 11 This is the aqueous phase UPLC spectrum of the characteristic chromatogram of the standard decoction of Triangular Wind.

[0045] Figure 12 This study examines the characteristic chromatograms of the standard decoction of *Tripterygium wilfordii* using different extraction solvents.

[0046] Figure 13 This is a comparative graph of the extraction efficiency of different extraction solvents for the characteristic spectrum of the standard decoction of Triangular Wind.

[0047] Figure 14 It is a common pattern of characteristic chromatogram of the standard decoction of Triangular Wind (R is the reference medicinal material).

[0048] Figure 15 This study examines the specificity of the characteristic chromatograms of the standard decoction for Triangular Wind.

[0049] Figure 16 This is a comprehensive examination of the characteristic spectrum of the standard decoction for Triangular Wind.

[0050] Figure 17 This is a comparison chart of UPLC tests using different chromatographic columns.

[0051] Figure 18 This is a comparison chart of UPLC tests conducted at different column temperatures.

[0052] Figure 19 This is a comparison chart of UPLC under different flow rates.

[0053] Figure 20This is a thin-layer chromatogram for investigating the sample volume of *Triangle Wind* formula granules, where: 1. *Triangle Wind* formula granules 0.5 μl; 2. *Triangle Wind* formula granules 1 μl; 3. *Triangle Wind* formula granules 3 μl; 4. *Triangle Wind* formula granules 5 μl; 5. *Triangle Wind* formula granules 8 μl; 6. *Triangle Wind* reference medicinal material 0.5 μl; 7. *Triangle Wind* reference medicinal material 1 μl; 8. *Triangle Wind* reference medicinal material 3 μl; 9. *Triangle Wind* reference medicinal material 5 μl; 10. *Triangle Wind* reference medicinal material 8 μl.

[0054] Figure 21 The specific chromatogram of the thin-layer chromatography of the Sanjiaofeng formula granules (T: 25℃, RH: 66%; Qingdao Marine prefabricated silica gel thin-layer plate); where 1. negative sample 2μl; 2-4. Sanjiaofeng formula granules (241001-241003) 2μl; 5. Sanjiaofeng reference medicinal material 2μl.

[0055] Figure 22 These are thin-layer chromatograms for identifying the triangular wind formula particles under different temperature conditions. Among them:

[0056] First sheet (T: 40℃, RH: 66%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0057] Second sheet (T: 25℃, RH: 66%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0058] The third sheet (T: 5℃, RH: 66%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0059] Figure 23 These are thin-layer chromatograms for identifying the triangular wind formula particles under different humidity conditions. Among them:

[0060] First sheet (T: 25℃, RH: 88%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0061] Second sheet (T: 25℃, RH: 66%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0062] The third sheet (T: 25℃, RH: 33%; Qingdao Marine prefabricated silica gel thin film): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0063] Figure 24 These are chromatograms obtained from examining silica gel plates from different manufacturers. Among them:

[0064] First sheet (T: 25℃, RH: 66%; Yantai Yinlong Silica Pre-made GF365 plate): 1. Negative sample 2μl; 2-4. Triangular wind formula granules (241001-241003) 2μl; 5. Triangular wind control medicinal material 2μl.

[0065] Second sheet (T: 25℃, RH: 66%; Qingdao Marine Silica G High-Efficiency Plate): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0066] The third sheet (T: 25℃, RH: 66%, Qingdao Marine Silica G plate): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0067] Fourth plate (T: 25℃, RH: 66%; Merck GF365 silica gel plate): 1. Negative sample 2μl; 2-4. Triangle Wind Formula Granules (241001-241003) 2μl; 5. Triangle Wind Control Herbal Material 2μl.

[0068] Figure 25 The results are thin-layer chromatograms of 15 batches of Sanjiaofeng formula granules (T: 25℃, RH: 66%, Qingdao Marine Silica G plate); among them, 1. negative sample 2μl; 2-4. Sanjiaofeng formula granules (241001-241003) 2μl; 5. Sanjiaofeng reference medicinal material 2μl. Detailed Implementation

[0069] 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.

[0070] Example 1

[0071] Basis for proposing process parameters for the standard decoction of Triangle Wind

[0072] 1. Instruments and Materials

[0073] 1.1 Instruments

[0074] 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), 0.01% 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 & Technology Co., Ltd., DLSB-5 / 20B), vacuum freeze dryer (Shanghai Dongfulong Technology Co., Ltd., LYO-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).

[0075] 1.2 Materials

[0076] The following table shows the slices of triangular wind-dispelling herbs:

[0077] Table 1 Information on Triangular Wind Herbs

[0078]

[0079] 2. Methods for determining process optimization indicators

[0080] 2.1 Method for determining solid content

[0081] Method for determining the solid content of triangular wind concentrate

[0082] 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.

[0083] 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%. The yield value of the concentrated liquid of standard decoction is the same as the yield of standard decoction.

[0084] 3. Processing of medicinal slices

[0085] Take the raw medicinal materials, remove impurities, wash, cut into sections, and dry.

[0086] Information on the medicinal materials used in this research is shown in Table 2:

[0087] Table 2 Information on Triangular Wind Herbs

[0088]

[0089]

[0090] 4 Extraction process

[0091] 4.1 Selection of cooking utensils

[0092] 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.

[0093] 4.2 Study on extraction process parameters

[0094] (1) Investigation on the dosage of medicinal slices

[0095] 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 *Gnaphalium affine* 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 the *Gnaphalium affine* standard decoction is set at 100g.

[0096] (2) Water addition assessment

[0097] According to the recommendations under "Standard Decoction Preparation" in the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine Formula 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 12 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 10 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 12 times the amount of water for the first decoction and 10 times the amount of water for the second decoction.

[0098] (3) Investigation of soaking time

[0099] According to the "Preparation of Standard Decoctions" section of the "Technical Requirements for Quality Control and Standard Formulation of Traditional Chinese Medicine 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 standard decoction of *Hedyotis diffusa* is determined to be 30 minutes.

[0100] (4) Examination of cooking time

[0101] 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," 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 advisable; 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 *Triangularis* is determined to be decocted twice: the first decoction is boiled for 30 minutes after boiling, and the second decoction is boiled for 20 minutes after boiling.

[0102] (5) Investigation of solid-liquid separation conditions

[0103] 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 decoction of *Triangularis aurea* 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 *Triangularis aurea* standard decoction.

[0104] 4.3 Results of Determination of Extraction Process Parameters

[0105] Take 100g of *Trichoderma triangularis* slices, place them in an electric ceramic kettle, add water, and decoct twice. For the first decoction, add 12 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 60 minutes. Filter the decoction while hot through a 300-mesh sieve, and record the mass of the decoction after cooling to room temperature. For the second decoction, add 10 times the amount of water, heat to a boil over high heat, then simmer over low heat for 40 minutes. Filter the decoction while hot through a 300-mesh sieve, and record the mass of the decoction after cooling to room temperature. Combine the two decoctions.

[0106] 5 Concentration Process

[0107] 5.1 Study on Concentration Process Parameters

[0108] 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.

[0109] The density, extract yield, and concentrated state of the standard decoction of *Trigonella foenum-graecum* were compared and measured at different concentration temperatures. The results showed that the extract 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 a density between 1.03 and 1.06 g / ml. Therefore, a concentration ratio of 1:1 was determined, and other concentration ratios were not considered.

[0110] 5.2 Results of Determination of Concentration Process Parameters

[0111] The decoction was transferred to a 2000ml round-bottom flask and concentrated under reduced pressure at low temperature using a rotary evaporator (temperature: 65℃; vacuum degree: -0.080~-0.090MPa) to 100g of extract.

[0112] 7. Validation of the preparation process

[0113] Three portions (100g each) of *Trichoderma lucidum* slices (batch numbers: P-SJF-240825-QGY-YP01, P-SJF-240825-QGY-YP02, P-SJF-240825-QGY-YP03) 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:

[0114] Table 3. Verification of the preparation method of Triangular Wind Standard Decoction

[0115]

[0116] The experimental results show that the specific gravity, solid content and yield of the three parallel batches of triangular wind standard decoction concentrate are basically the same, indicating that the process is stable and repeatable and can be used as the preparation process of triangular wind standard decoction.

[0117] 6. Drying process

[0118] 6.1 Study on freeze-drying process parameters

[0119] 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 standard decoctions prepared by Triangle Wind.

[0120] Under magnetic stirring, the extract was dispensed into 5ml brown vials, each with a volume of 2ml. 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 Triangle Wind vacuum freeze dryer were -43.6℃, ​​the pre-freezing temperature was -45℃, the sublimation drying temperature was -30℃~0℃, the desorption drying temperature was 5℃~25℃, and the total drying time was 25 hours. The results indicate that under these freeze-drying conditions, the Triangle Wind standard decoction has low moisture content and is easy to preserve.

[0121] 6.2 Confirmation of freeze-drying process parameters

[0122] Results of freeze-drying process parameters: Under magnetic stirring, the product was dispensed into 5ml brown vials, each with a volume of 2ml, half-stopped, and then transferred to a vacuum freeze dryer for freeze-drying. The freeze-drying parameters were as follows: pre-freezing temperature of the triangular wind concentrate was -45℃, pre-freezing time was 240 minutes; sublimation drying temperature was -30℃~0℃, sublimation drying time was 840 minutes, and vacuum degree was -0.2mbar; desorption drying temperature was 5℃~25℃, desorption drying time was 420 minutes, and vacuum degree was -0.2mbar.

[0123] Example 2

[0124] Establishment of a method for content determination and characteristic chromatographic analysis of standard decoction of Triangular Wind.

[0125] 1. Establishment of analytical methods for content determination

[0126] This product is the fresh or dried stem of *Hedera nepalensis* K. Koch var. *sinensis* (Tobl.) Rehd., a plant belonging to the Araliaceae family. Also known as ivy, it is commonly called triangular wind, triangular vine, rice-horn star, climbing vine, and triangular arrow. Triangular wind is a traditional medicine used by ethnic minorities in Guizhou Province, including the Han, Miao, Yi, Zhuang, Dong, and Tujia peoples. It is pungent, bitter, and cold in nature. It enters the liver, spleen, and lung meridians. It has the effects of dispelling wind and detoxifying, promoting blood circulation and stopping bleeding, reducing swelling and relieving pain. It is used to treat rheumatic pain, paralysis and numbness, hematemesis, hemoptysis, epistaxis, hematochezia, pruritus, eczema, traumatic injuries, and unidentified boils and carbuncles. Modern research shows that triangular wind mainly contains sugars, organic acids, alkaloids, saponins, tannins, and phenolic components, possessing antibacterial and antiviral effects. (See *Guizhou Province Traditional Chinese Medicine and Ethnic Medicine Quality Standards*).

[0127] According to the 2003 edition, the vine stem of this product is cylindrical, curved, branched, and varies in length, with a diameter of 0.3-1.2 cm. The surface is pale yellowish-brown or grayish-brown, with longitudinal wrinkles, and densely covered with adventitious roots on one side. It is hard and brittle, easily broken; the broken surface has a thin, grayish-green or brown cortex, and a wide, yellowish-white or pale brown xylem, with small pores (vascular bundles) visible under a magnifying glass. The pith is small and dot-like. The leaves are simple, alternate, and have long petioles. The leaf blade is leathery, slightly curled, and varies in shape, with entire or 3-lobed margins, and is grayish-green or pale yellowish-brown. It has a faint odor and a bitter taste.

[0128] Experimental studies have shown that the triangular wind contains a high amount of chlorogenic acid. Chlorogenic acid is an ester formed by the condensation of the hydroxyl group of quinic acid and the carboxyl group of trans-phenylacrylic acid. It is a natural antioxidant. Studies have found that chlorogenic acid can reduce or prevent oxidative stress by lowering ROS levels and upregulating the activity of antioxidant enzymes.

[0129] 1.1 Instruments and Reagents

[0130] Agilent 1290 Infinity II and Waters Acquity UHPLC systems The following reagents were purchased from Fisher Scientific: H class, 0.01 g electronic balance (model: ml204), electric thermostatic water bath (model: HH-S8), methanol, and acetonitrile were all chromatographic grade, with batch numbers 225331 and F22MB2201, respectively. The water was ultrapure water, and all other reagents were analytical grade. The batch numbers of the 15 batches of Triangle Wind Marker Soup are: D-241001-01, D-241001-02, D-241001-03, D-241001-04, D-241001-05, D-241001-06, D-241001-07, D-241001-08, D-241001-09, D-241001-10, D-241001-11, D-241001-12, D-241001-13, D-241001-14, and D-241001-15 (provided by the laboratory process group).

[0131] 1.2 Source and purity test of reference standards

[0132] Chlorogenic acid (batch number: 110753-202119, purity 96.3%) was purchased from the China National Institutes for Food and Drug Control. It is for content determination purposes and requires no pretreatment.

[0133] 1.3 Determination of chromatographic conditions

[0134] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 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 0.30 mL / min; column temperature 30 °C; detection wavelength 326 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should be no less than 3000.

[0135]

[0136] 1.4 Preparation of reference solution

[0137] Take an appropriate amount of chlorogenic acid reference standard, accurately weigh it, and add 50% ethanol to prepare a solution containing 20 μg of chlorogenic acid per 1 ml, which will be used as the reference solution.

[0138] 1.5 Preparation of the test solution

[0139] (1) Investigation of different extraction solvents

[0140] Take an appropriate amount of the standard decoction D-241001-11 for chlorogenic acid, making 9 portions, with 2 parallel samples per portion. Place each portion in a stoppered conical container, and accurately add 20 ml of methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water sequentially. Weigh the contents, sonicate (500W, 40kHz) for 30 minutes, cool, weigh again, and replenish the lost weight with the appropriate solvent. Shake well, filter, and collect the filtrate to obtain the test solution. Under the above chromatographic conditions, accurately 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.

[0141] Table 4. Effects of different extraction solvents on the content determination results of Triangle Wind Standard Decoction

[0142]

[0143] The experimental results show that different solvents have a significant impact on the chlorogenic acid content in the standard decoction of Triangular Wind. Considering both the content and the peak shape of the chromatogram, 50% ethanol was selected as the extraction solvent for further research.

[0144] (2) Examination of extraction methods

[0145] Take 0.1g of the standard decoction for *Trigonella foenum-graecum* (D-241001-11), in two portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and precisely add 20ml 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 chromatographic conditions described above, precisely 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 methods on the chlorogenic acid content, and 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.

[0146] Table 5. Effects of different extraction methods on the content determination results of the standard decoction of Triangle Wind.

[0147]

[0148] The experimental results show that different extraction methods have a significant impact on the chlorogenic acid content in the standard decoction of Triangular Wind. 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.

[0149] (3) Investigation of the volume of the extraction solvent

[0150] Take 0.1g of the standard decoction for *Trigonella foenum-graecum* (D-241001-11), in two portions, with two parallel samples per portion. Place each portion in a stoppered conical container, and accurately add 10ml, 15ml, 20ml, and 25ml of 50% ethanol sequentially, respectively. 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, and 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.

[0151] Table 6. Effect of different extraction solvent volumes on the content determination results of the Triangle Wind Standard Decoction

[0152]

[0153] The experimental results show that the volume of different extraction solvents has no significant effect on the chlorogenic acid content in the standard decoction of Triangular Wind. Considering the cost and the difference in content, the volume of extraction solvent of 20 ml was selected for further research.

[0154] (4) Examination of different extraction times

[0155] Take 0.1g of the standard decoction for chlorogenic acid (D-241001-11), in two portions, each with two parallel samples. Place each portion in a stoppered conical container, and precisely add 25ml of 50% ethanol sequentially. Weigh the samples, and sonicate them (500W, 40kHz) for 15, 30, 45, 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, precisely 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 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.

[0156] Table 7. Effects of different extraction times on the content determination results of the standard decoction of Triangle Wind.

[0157]

[0158] The experimental results show that different extraction times have no significant effect on the chlorogenic acid content in the standard decoction of Triangular Wind. Considering both time cost and content differences, ultrasonic treatment (power 500W, frequency 40kHz) for 60 minutes was selected for subsequent research.

[0159] (5) Determination of the preparation method of the test solution

[0160] Based on the results of the sample pretreatment experiment, the preparation method of the test sample can be determined as follows: Take about 0.1g of the standard decoction of Triangular Wind, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% ethanol, weigh it, sonicate (power 500W, frequency 40kHz) for 60 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.

[0161] 2. Validation of content determination method

[0162] (1) Specificity examination

[0163] Accurately pipette 1 μl each of the test solution of the triangular wind standard decoction (D-241001-11), the chlorogenic acid reference solution, and the blank solvent, and inject them separately into the ultra-high performance liquid chromatograph. Perform the determination according to the chromatographic conditions under section "1.3", record the chromatograms, and the results are as follows: Figure 2 As shown.

[0164] The chromatogram results show that the analytical method has good specificity for the determination of chlorogenic acid content in the standard decoction of Triangular Wind.

[0165] (2) Peak purity

[0166] Accurately pipette 1 μl each of the test solution and reference solution of the Triangular Wind Standard Decoction (D-241001-11) 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 .

[0167] Table 8 Matching values ​​for target peaks and peak purity

[0168]

[0169] The experimental results show that the peak purity matching value of the indicator component chlorogenic acid is 999.87 > 960, indicating that its peak purity meets the analytical requirements.

[0170] (3) Linear

[0171] Accurately weigh an appropriate amount of chlorogenic acid reference standard and place it in a numbered 100ml volumetric flask. Add methanol to prepare a solution containing 200.4388μg 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 chlorogenic acid reference standard stock solution by 1.25, 1.56, 1.95, 2.44, 3.05, 3.81, 7.63, 15.26, and 30.52 times to obtain reference solutions of different concentrations of chlorogenic acid. Perform chromatographic analysis under the above conditions. Plot concentration on the x-axis and 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 .

[0172] Table 9. Linearity Study of Chlorogenic Acid

[0173]

[0174] Experimental results showed that chlorogenic acid concentration and peak area exhibited a good linear relationship within the concentration range of 6.5680 μg / ml to 200.4388 μg / ml, with a correlation coefficient R0. 2 =0.9999, the linear regression equation is: y = 10.057x - 11.512.

[0175] (4) Precision test

[0176] 1) Instrument precision test

[0177] Accurately pipette 1 μl each of the test solution of the triangular wind standard decoction (D-241001-11) and the reference solution of chlorogenic acid, 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.

[0178] Table 10 Precision Experiment Results of the Method for Determining the Content of Triangular Wind Standard Decoction

[0179]

[0180] The results show that the RSD (%) of the target peak chlorogenic acid content is 0.71% ≤ 2%, indicating that the method has good instrument precision.

[0181] 2) Repeatability test

[0182] Accurately weigh approximately 0.1 g of the same batch of Triangular Wind Standard Decoction (D-241001-11), prepare 6 parallel solutions, and set aside 6 test solutions. 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.

[0183] Table 11 Results of repeatability experiments for the determination of the content of Trichoderma standard decoction

[0184]

[0185] The experimental results show that the RSD (%) of the chlorogenic acid content of the target peak is 0.28% < 2.0%, indicating that the method has good repeatability.

[0186] 3) Intermediate precision

[0187] Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.1 g of the same batch of Triangular Wind Standard Decoction (D-241001-11), accurately weighed, and prepared 6 parallel samples. The test solution was prepared according to the test solution preparation method, and the sample was tested under the above chromatographic 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.

[0188] Table 12 Results of intermediate precision experiments for the determination of content in the standard decoction of Triangular Wind (a type of medica).

[0189]

[0190] The experimental results show that the intermediate precision RSD (%) of the target chlorogenic acid content is 0.51% < 4%, indicating that the intermediate precision of this method is good.

[0191] (5) Accuracy test

[0192] Take 0.05g of a known concentration of *Trigonella foenum-graecum* standard decoction (batch number D-241001-11, chlorogenic acid content 5.85mg / g), accurately weigh a total of 6 portions, and accurately add 20ml of methanol-prepared chlorogenic acid reference solution (concentration 0.014633mg / ml) to each portion. Prepare the test solution according to the test solution preparation method, and perform chromatographic analysis under the above conditions. Inject 1μl of each sample. Calculate the content of the target peak using the external standard one-point method, based on chlorogenic acid, and calculate the recovery rate and RSD according to the following formula.

[0193]

[0194] Table 13. Results of the chlorogenic acid recovery experiment in the method for determining the content of triangular wind standard decoction.

[0195]

[0196]

[0197] The experimental results show that the recovery rate of chlorogenic acid in the standard decoction of Triangular Wind is within the range of 92%-105%, and the RSD% is less than 2%, indicating that the accuracy of the detection method is good.

[0198] (6) Stability test

[0199] The test solution of the triangular wind standard decoction (D-241001-11) was prepared according to the preparation method of the test solution. The sample was injected at 0, 2, 4, 8, 12, 16, 20 and 24 hours according to the above chromatographic conditions, with an injection volume of 1 μl. The RSD (%) 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.

[0200] Table 14. Stability test results of the method for determining the content of Triangular Wind Standard Decoction

[0201]

[0202] Experiments showed that the RSD (%) of the target peak chlorogenic acid content within 24 hours was 0.48% < 2%, indicating that the solution had good stability within 24 hours.

[0203] (7) Durability test

[0204] 1) Investigation of different chromatographic columns

[0205] The effects of three chromatographic columns—ZORBAX SB-C18 (2.1*100mm, 1.8μm), ACQUITY BEH ShieldRP18 C18 (2.1*100mm, 1.7μm), and Acquity UPLC BEH C18 (2.1*100mm, 1.9μm)—on the peak shape and resolution of chlorogenic acid in *Trichoderma triangularis* were compared. The test solution of the *Trichoderma triangularis* standard decoction (D-241001-11) under the [Content Determination] section was taken and determined according to the above chromatographic conditions. Chromatographic data were recorded as chlorogenic acid. The experimental results are shown in the table below. Figure 5 As shown.

[0206] Table 15. Effects of different chromatographic columns on the determination results of *Trigonella foenum-graecum* in standard decoction.

[0207]

[0208]

[0209] Experimental results showed that all three chromatographic columns had good separation performance and could meet the requirements for the determination of trigonelline content. However, the ZORBAX SB-C18 (2.1*100mm, 1.8μm) column had better resolution and theoretical plate number than the ACQUITY BEHShield RP18 C18 (2.1*100mm, 1.7μm) and Acquity UPLC BEH C18 (2.1*100mm, 1.9μm) columns. Therefore, the ZORBAX SB-C18 (2.1*100mm, 1.8μm) column was selected as the preferred column for this experiment.

[0210] 2) Investigation using different chromatographs

[0211] Based on the existing equipment in the laboratory, Thermo VANQUISH Autosampler and Agilent 1290 Infinity II high performance liquid chromatographs were selected to compare the effects of the two chromatographs on the peak shape and resolution of chlorogenic acid in the triangular wind formulation granules.

[0212] Take the test solution from the [Content Determination] section of the standard decoction for *Triangular Wind* (D-241001-11), and determine its content under 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.

[0213] Table 16 Results of the investigation using different chromatographs

[0214]

[0215] Experimental results show that this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0216] 3) Investigation at different column temperatures

[0217] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape of chlorogenic acid in the standard decoction of Triangle Wind were compared.

[0218] Take the test solution from the [Content Determination] section of the standard decoction for *Triangle Wind* (D-241001-11), and determine its content under the chromatographic conditions described above. Calculate the content as chlorogenic acid and record the chromatographic data. The experimental results are as follows: Figure 7 As shown in the table below.

[0219] Table 17 Results of the Determination of the Content of Triangular Flask Soup by Different Column Temperatures

[0220]

[0221] The results showed that the peak shape and separation were good at different 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 30℃ was chosen.

[0222] 4) Investigation of different flow velocities

[0223] The effects of different flow rates of 0.25 ml / min, 0.30 ml / min, and 0.35 ml / min on the peak shape and resolution of chlorogenic acid in the standard decoction of Triangular Wind were compared.

[0224] Take the test solution from the [Content Determination] section of the standard decoction for *Triangular Wind* (D-241001-11), and determine its content under the chromatographic conditions described above. Calculate the content as chlorogenic acid and record the chromatographic data. The experimental results are as follows: Figure 8 As shown in the table below.

[0225] Table 18 Results of the Determination of Content in Triangular Windvane Soup by Different Flow Rates

[0226]

[0227] 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.

[0228] Establishment of a characteristic chromatogram analysis of the standard decoction for 3-triangle wind

[0229] 3.1 Instruments and Reagents

[0230] 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.).

[0231] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0232] Test drug: chlorogenic acid (China National Institutes for Food and Drug Control, batch number: 110753-202119, purity 96.3%); triangular wind reference material (batch number: 2025-011601, Chengdu Glip Biotechnology Co., Ltd.).

[0233] The batch numbers of the 15 batches of Triangle Wind Marker Soup are: D-241001-01, D-241001-02, D-241001-03, D-241001-04, D-241001-05, D-241001-06, D-241001-07, D-241001-08, D-241001-09, D-241001-10, D-241001-11, D-241001-12, D-241001-13, D-241001-14, and D-241001-15 (provided by the laboratory process group).

[0234] 3.2 Preparation of the reference solution

[0235] Accurately weigh approximately 1.0g of *Hedyotis diffusa* reference material and place it in a stoppered conical flask. Accurately add 100ml of 50% ethanol, weigh the flask, and sonicate (500W, 40kHz) for 60 minutes. Cool the flask, weigh it again, and replenish the lost weight with 50% ethanol. Shake well, filter, and use the filtrate as the reference solution. Use the reference solution from the [Assay] section as the reference solution.

[0236] 3.3 Determination of chromatographic conditions

[0237] (1) Determination of detection wavelength

[0238] Inject the test sample of the standard decoction of *Triangularis tinctoria* into the sample for analysis, and record the absorption spectrum in the range of 190–400 nm (see [link to sample]). Figure 9 ).

[0239] Experimental results show that at a wavelength of 326 nm, the standard decoction sample solution of Triangle Wind has more detectable chromatographic peak information and less baseline noise interference. Therefore, 326 nm was selected as the detection wavelength.

[0240] (2) Optimization of the mobile phase

[0241] ① Consider A as the organic phase and B as the aqueous phase. The gradient is as follows:

[0242]

[0243] Experimental results: see Figure 10 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.

[0244] ② Examine the cases with and without acid.

[0245] Experimental results: see Figure 11 Adding acid resulted in better peak shape than not adding acid. The optimal peak shape was achieved when the mobile phase was an acetonitrile-0.1% phosphoric acid aqueous solution system. Therefore, an acetonitrile-0.1% phosphoric acid aqueous solution was chosen for elution.

[0246] (3) Determination of chromatographic conditions

[0247] The column was packed with octadecylsilane-bonded silica gel (100 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); 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 30 °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.

[0248]

[0249] 3.4 Preparation of the test solution

[0250] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of the standard decoction of Triangular Wind. Methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water were selected as extraction solvents. The characteristic chromatograms of the five tentatively determined chromatographic peaks were compared with the sample weight and the chromatograms.

[0251] Take an appropriate amount of the standard decoction for *Tripterygium wilfordii* (D-241001-11), approximately 0.1 g, accurately weigh it, and place it in a stoppered conical flask. Accurately add 20 ml each of methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water. Weigh the flask, sonicate (500 W, 40 kHz) for 60 minutes, cool, replenish the lost weight with the appropriate solvent, shake well, filter, and collect the filtrate. Inject the sample under the determined chromatographic conditions and record the chromatogram. The results of the investigation of different extraction solvents for the characteristic chromatogram of the standard decoction for *Tripterygium wilfordii* are shown below. Figure 12 , 13 .

[0252] Experimental results: The extraction efficiency was higher when 50% ethanol was used as the extraction solvent, so 50% ethanol was chosen as the extraction solvent.

[0253] Based on the above experimental results, the sample pretreatment method for the characteristic chromatogram of the triangular wind standard decoction can be determined as follows: Take about 0.1g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 20ml of 50% ethanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 60 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 product.

[0254] 3.5 Determination and Identification of Common Peaks

[0255] UPLC chromatograms of different batches of *Trichoderma lucidum* standard decoction samples were determined. The results were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission. Common peaks were selected. Specific results are shown in [link to results]. Figure 14 .

[0256] The results showed that there were 5 relatively obvious common peaks in the characteristic spectrum of the standard decoction of Triangular Wind.

[0257] 3.6 Methodological Validation of Feature Map Analysis Method

[0258] (1) Specificity examination

[0259] Accurately pipette 1 μl each of the test solution and blank solvent from the "Characteristic Chromatography" section of the Triangular Wind Standard Decoction, and inject them into the liquid chromatograph. Determine the chromatographic results under the conditions described above. See results below. Figure 15 .

[0260] The experimental results show that the solvent does not interfere with the characteristic peaks in the spectrum of the standard decoction of Triangular Wind.

[0261] (2) Holistic Examination

[0262] Take the test solution from the [Characteristic Chromatography] section of the Triangular Wind Standard Decoction, 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 chromatogram. See details below. Figure 16 .

[0263] 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.

[0264] (3) Precision test

[0265] Take the test solution from the [Characteristic Spectrum] section of the Triangular Wind Standard Decoction, and inject it 6 times repeatedly, with an injection volume of 1 μl. Temporarily identify 5 characteristic peaks, using peak 2 as the reference peak, and calculate the relative retention time. The experimental results are shown in the table below.

[0266] Table 19 Precision results of the characteristic chromatograms of the standard decoction for Triangular Wind (relative retention time)

[0267]

[0268] 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.

[0269] (4) Stability test

[0270] Take the test solution of the standard decoction for *Trichoderma triangularis* under the [characteristic spectrum] section and inject it at 0, 2, 4, 8, 12, 16, 20, and 24 hours, with an injection volume of 1 μl. Tentatively identify 5 characteristic peaks, using peak 2 as the reference peak, and calculate the relative retention time. The results are shown in the table below.

[0271] Table 20 Stability results of the characteristic spectra of the standard decoction of Triangular Wind (relative retention time)

[0272]

[0273] 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.

[0274] (5) Repeated examination

[0275] Accurately weigh approximately 0.1 g of the same batch of Triangular Wind Standard Decoction (D-241001-12), prepare six parallel portions, and prepare test solutions according to the test solution preparation method under the [Characteristic Spectrum] section. Inject 1 μl into each sample. Temporarily identify five characteristic peaks, using peak 2 as the reference peak, and calculate the relative retention time.

[0276] Table 21. Repeatability results of characteristic chromatograms of the standard decoction for Triangular Wind (relative retention time)

[0277]

[0278] 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.

[0279] (6) Durability test

[0280] ① Column analysis

[0281] Three chromatographic columns were investigated: ZORBAX SB-C18 (2.1*100mm, 1.8μm), ACQUITYBEH Shield RP18 C18 (2.1*100mm, 1.7μm), and Acquity UPLC BEH C18 (2.1*100mm, 1.9μm). The effects of the three columns on the peak characteristics of the triangular wind standard decoction were examined.

[0282] Table 22. Chromatographic column analysis results of the characteristic chromatogram of the triangular wind vane soup (relative retention time).

[0283]

[0284] Experimental results show that, for details, please refer to the table above and... Figure 17 Different chromatographic columns have a certain impact on the peak elution. Elution using a ZORBAX SB-C18 (2.1*100mm, 1.8μm) ultra-high performance liquid chromatography column yielded better peak shapes and the best separation effect. Therefore, it is recommended to use a ZORBAX SB-C18 (2.1*100mm, 1.8μm) ultra-high performance liquid chromatography column for this method.

[0285] ② Column temperature investigation

[0286] The elution performance at different column temperatures (25℃, 30℃, 35℃) was investigated.

[0287] Table 23. Results of column temperature analysis of the characteristic spectra of the triangular wind vane soup (relative retention time)

[0288]

[0289]

[0290] Experimental results show: (See) Figure 18 See the table above. Column temperature has some influence on peak elution, but the separation effect is best and the peak shape is better when the column temperature is 30℃. Therefore, it is recommended to use a column temperature of 30℃ for the determination.

[0291] ③ Flow rate test: The elution effect at different flow rates (0.25 ml / min, 0.30 ml / min, 0.35 ml / min) was investigated.

[0292] Table 24 Results of flow velocity investigation in the characteristic spectrum of the triangular wind vane soup (relative retention time)

[0293]

[0294] Experimental results show: (See) Figure 19See the table above. Flow rate has some influence on peak formation, but the peak shape is optimal at a flow rate of 0.30 ml / min. Therefore, it is recommended to use 0.30 ml / min as the measurement flow rate.

[0295] Example 3

[0296] Research on the process of triangular wind granules

[0297] 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.

[0298] The yield was determined to be 6.7%. The average yield of the 15 batches of standard decoctions, ranging from -3SD to +3SD, should be between 3.4% and 11.9%. At the same time, in order to strictly control the yield of the triangular wind formula granules in large-scale production and ensure the uniformity and stability of the quality of the triangular wind formula granules, the yield range of the dry extract of the formula granules was determined to be 3.4% to 6.7%.

[0299] In summary, the yield of this product is determined to be 6.7%, that is, 15,000g of medicinal slices are made into 1,000g of granules, and 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%.

[0300] Example 4

[0301] Triangular wind thin-layer chromatography identification

[0302] 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 *Trichoderma triangularis*. Considering the specificity of TLC identification for *Trichoderma triangularis*, a reference material of *Trichoderma triangularis* was used as a control. The developing solvent was chloroform-acetone-formic acid (4:1:0.1). After development, the sample was removed, dried, and examined under ultraviolet light (365 nm). In the chromatogram of the test sample, spots of the same color appeared at the corresponding positions as in the chromatogram of the reference material. The results showed that spots corresponding to those in the reference material could be detected in the sample, and the spots were clear and well-separated. This method is highly operable and reproducible; therefore, it was included in the main text of the quality standard for *Trichoderma triangularis* formula granules.

[0303] 1. Instruments, reagents and reagents

[0304] Instruments: Automated thin-layer imaging system (TLC VISUALIZER2, CAMAG, Switzerland), dual-groove developing tank, fully automated thin-layer sampling system (AUTOMATIC TLC SAMP1ER4, CAMAG, Switzerland), AL-104 electronic balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.], KQ-500DA ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.), silica gel G thin-layer plates (10cm×20cm, Qingdao Ocean Chemical Co., Ltd., batch number: 20240809), silica gel GF... 365 Thin-layer laminate (10cm x 10cm, Merck AG & Limited), silicone GF 365 Thin-layer plate (10cm×10cm, Yantai Huayang New Material Technology Co., Ltd., batch number: 20231204).

[0305] Reagents: n-Butanol (batch number: 20240401), methanol (batch number: 20230301), chloroform (batch number: 20240601), and acetone (batch number: 20240501) were all purchased from Chongqing Chuandong Chemical (Group) Co., Ltd., and formic acid (batch number: 20220802) was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0306] Trial drugs: 3 batches of Sanjiaofeng formula granules (batch numbers: 241001, 241002, 241003; source: Sinopharm Group Tongjitang (Guizhou) Pharmaceutical Co., Ltd.); Sanjiaofeng control medicinal material (batch number: 2025-011601, Chengdu Glip Biotechnology Co., Ltd.).

[0307] 2. Preparation of the solution

[0308] 2.1 Preparation of the test solution

[0309] Take 1.0g of this product, grind it into a fine powder, add 20ml of water-saturated n-butanol, sonicate for 60 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve, filter through a microporous membrane, and use as the test solution.

[0310] 2.2 Preparation of control herbal solution

[0311] Take 2.0g of the reference herb *Trichoderma triangularis*, add 50ml of water, heat under reflux for 60 minutes, filter, evaporate the filtrate to dryness, dissolve the residue in 20ml of water-saturated n-butanol, sonicate for 60 minutes, filter, evaporate the filtrate to dryness, add 2ml of methanol to dissolve, filter through a microporous membrane, and use as the reference herb solution.

[0312] 2.3 Preparation of negative sample solution

[0313] Take 1.0g of the negative sample of the triangular wind formula granules, grind it into a fine powder, and process it in the same way as the test sample to prepare the negative sample solution.

[0314] 3 Thin-layer chromatography conditions

[0315] Thin-layer plate: Silicone G thin-layer plate

[0316] Developing solvent: chloroform-acetone-formic acid (4:1:0.1)

[0317] Spotting method: round dot sample

[0318] Deployment method: Deployment is carried out using a double-groove deployment cylinder.

[0319] Inspection: Inspection under ultraviolet light (365nm)

[0320] 4-point sample size investigation

[0321] Two μL each of the test solution of the Sanjiaofeng formula granules and the reference herbal solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using chloroform-acetone-formic acid (4:1:0.1) as the developing solvent. The plate was then removed, air-dried, and examined under ultraviolet light (365 nm). The results are shown in the figure. Figure 20 .

[0322] As shown in the figure above, when the amount of the test sample solution is 1-8 μl and the amount of the control medicinal material solution is 1-8 μl, the key spots at the corresponding positions of the test sample and the control medicinal material are clearer and have better spot characteristics. Therefore, the sampling amount is selected as 2 μl for the test sample solution and 2 μl for the control medicinal material solution.

[0323] 5. Specificity of Triangle Wind Formula Granules

[0324] Two μL each of the test solution of the Sanjiaofeng formula granules and the control herbal solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using chloroform-acetone-formic acid (4:1:0.1) as the developing solvent. The plate was then removed, air-dried, and examined under ultraviolet light (365 nm). The experimental results are shown below. Figure 21 .

[0325] As shown in the figure above, the chromatogram of the Triangle Wind Formula Granules test sample shows spots of the same color at the corresponding positions as the chromatogram of the reference medicinal material, and there is no interference from the negative sample. This indicates that the thin-layer chromatography method has good specificity.

[0326] 6. Investigation at different temperatures

[0327] Two μL each of the test solution of the Sanjiaofeng formula granules and the reference herbal solution were spotted onto the same silica gel G thin-layer plate. The plate was developed using chloroform-acetone-formic acid (4:1:0.1) as the developing solvent at different temperatures (5℃, 25℃, 40℃). The plates were then removed, air-dried, and examined under ultraviolet light (365nm). The experimental results are shown below. Figure 22 .

[0328] As shown in the figure above, under different temperature conditions, the test sample chromatogram and the reference medicinal material chromatogram of the triangular wind formula granules show the same main spot of the same color at the corresponding positions. The color development is clear, the separation is good, there is no tailing phenomenon, and there is no background interference. As the temperature increases, the position of the corresponding spot does not change significantly, indicating that the temperature has no significant effect on the thin-layer identification of the triangular wind formula granules, which shows that the thin-layer identification method has good temperature durability.

[0329] 7. Investigation of different humidity levels Two μL each of the *Triangle Wind* formula granule test solution and the *Triangle Wind* control herbal material solution were spotted onto the same silica gel G thin-layer plate. Trichloromethane-acetone-formic acid (4:1:0.1) was used as the developing solvent. The plates were developed at different relative humidities (33%, 66%, and 88%), removed, air-dried, and examined under ultraviolet light (365 nm). The experimental results are shown below. Figure 23 .

[0331] As shown in the figure above, under different humidity conditions, the test sample chromatogram and the reference medicinal material chromatogram of the Triangle Wind Formula Granules showed spots of the same color at the corresponding positions, and the main spots were 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 Triangle Wind Formula Granules, and that the thin-layer identification method is durable to humidity.

[0332] Investigation of Thin-Layer Boards from 8 Different Manufacturers

[0333] Two μL samples of the Triangle Wind Formula Granules test solution and the Triangle Wind Control Herbal Material Solution were spotted onto silica gel G thin-layer plates from different manufacturers (Yantai Yinlong, Qingdao Haiyang, and Merck). The plates were developed using chloroform-acetone-formic acid (4:1:0.1) as the developing solvent. The plates were then removed, dried, and examined under ultraviolet light (365 nm). The experimental results are shown below. Figure 24 .

[0334] Depend on Figure 24 It can be seen that using silica gel thin-layer plates from different manufacturers (Yantai Yinlong pre-made silica gel G plate, Qingdao Haiyang silica gel G high-efficiency plate, Qingdao Haiyang silica gel G plate, and German Merck plate), the main spots of the chromatograms of the Triangle Wind Formula Granules test sample and the reference medicinal material can correspond without significant influence, but their Rf values ​​will be affected to varying degrees. Qingdao Haiyang silica gel G plate is recommended.

[0335] 9. Thin-layer chromatogram for identification of triangular wind formula granules

[0336] Three batches of the Triangle Wind formula granule test solution were each sampled in 2 μl and spotted onto the same silica gel G thin-layer plate. A chloroform-acetone-formic acid (4:1:0.1) solution was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The experimental results are shown below. Figure 25 .

[0337] Depend on Figure 25 It is evident that the corresponding positions of the chromatograms of the three batches of formula granules test samples and the chromatograms of the reference medicinal materials show fluorescent spots of the same color, indicating that the thin-layer identification of the three batches of Sanjiaofeng formula granules all meet the requirements.

[0338] 10 Summary

[0339] From the chromatographic spots, the fluorescent spots of the Sanjiaofeng formula granules showed good separation, and the corresponding positions of the Sanjiaofeng formula granules and the Sanjiaofeng reference herb chromatogram showed fluorescent main spots of the same color, with a one-to-one correspondence. This method can effectively identify Sanjiaofeng formula granules that have lost their processed medicinal characteristics. Through thin-layer chromatography methodology evaluation, this method demonstrated good specificity and robustness, and is suitable for the chromatographic identification of Sanjiaofeng formula granules.

[0340] 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 of preparing a trigonella foenum-graecum extract and a preparation thereof, characterized by, Specifically comprising the following steps: (1) Take the Sanjiaofeng decoction pieces, decoct twice with water, filter, and combine the two decoctions after the filtrate is cooled to room temperature, i.e. Sanjiaofeng extract; (2) Concentrate the decoction under reduced pressure and low temperature to obtain extract extract, control the density of the obtained extract extract between 1.03-1.06g / ml; obtain extract extract; (3) Prepare the extract extract into the required dosage form, i.e. Sanjiaofeng granules.

2. The method of preparing the extract of G. triquetrum and its formulations as claimed in claim 1, wherein, The step (1) is specifically to take the Sanjiaofeng decoction pieces, decoct twice with water, directly add 10-14 times the amount of water in the first decoction, soak for 20-40 minutes, boil with strong fire and then keep the fire small for 50-70 minutes, filter the decoction with a 200-400 mesh screen while hot, and record the mass of the filtrate after it is cooled to room temperature; add 8-12 times the amount of water in the second decoction, heat and boil with strong fire and then keep the fire small for 15-25 minutes, filter the decoction while hot with a 200-400 mesh screen, and record the mass of the filtrate after it is cooled to room temperature, and combine the two decoctions.

3. The method of preparing the extract of G. triquetrum and its formulations as claimed in claim 1, wherein, The step (2) is specifically to concentrate under reduced pressure and low temperature, and the conditions are: temperature: 60-70℃; vacuum degree: -0.080--0.090MPa.

4. The method for preparing Trichoderma and its formulations according to claim 1, characterized in that, The step (3) is to freeze dry the obtained extract extract, i.e. Sanjiaofeng standard decoction; the freeze drying parameters are: the pre-freezing temperature of Sanjiaofeng concentrated liquid is -40--50℃, the pre-freezing time is 200-300 minutes, the sublimation drying temperature is -30℃-0℃, the sublimation drying time is 800-900 minutes, and the vacuum degree is -0.15--0.25mbar; the analytical drying temperature is 5℃-25℃, the analytical drying time is 400-450 minutes, and the vacuum degree is -0.15--0.25mbar.

5. The method for preparing Trichoderma and its preparations according to claim 1, characterized in that, The step (3) is to add excipients to the obtained extract extract, dry, add excipients again, mix uniformly, and granulate, i.e. Sanjiaofeng granules.

6. The method of preparing the extract of G. triquetrum and its formulations as claimed in claim 5, wherein, The Sanjiaofeng granules 14000-16000g decoction pieces are prepared into 1000g granules, and the specification is that 1g of the formula granules is equivalent to 14-16g of decoction pieces.

7. A method for determining the content of ingredients of a triangular wind and its preparation, characterized by, Determination is performed by high performance liquid chromatography, octadecylsilyl bonded silica is used as the filler, acetonitrile is used as the mobile phase A, and 0.1% phosphoric acid solution is used as the mobile phase B, gradient elution is performed according to the following table, the flow rate is 0.30ml per minute, the column temperature is 30℃, and the detection wavelength is 326nm 8. The method according to claim 7, wherein the method is a method for determining the contents of the components of the triangular wind and its preparation. The chromatographic column used has a column length of 100mm, an inner diameter of 2.1mm, and a particle size of 1.8μm.

9. The method according to claim 8, wherein the amount of the plurality of components in the triangular wind and the preparation thereof is determined. The test sample is precisely weighed, placed in a conical flask with a plug, 50% ethanol is precisely added, the weight is determined, ultrasonic treatment is performed, it is cooled, the weight is determined again, 50% ethanol is added to make up for the weight loss, it is shaken uniformly, filtered, and the filtrate is obtained, i.e. the test solution.

10. The method according to claim 9, wherein the amount of the components in the triangular wind and the preparation thereof is determined. The ultrasonic treatment has a power of 450-550W and a frequency of 35-45kHz.

11. The method according to claim 9, wherein the amount of the plurality of components in the triangular wind and preparation thereof is determined. The control solution is a solution containing 20μg of chlorogenic acid per 1ml prepared with 50% ethanol as the solvent.

12. A method for detecting the extract of Ginkgo biloba L. and its preparation characteristic map, characterized in that, Determination is carried out by high performance liquid chromatography, octadecylsilane-bonded silica gel is used as filler, acetonitrile is used as mobile phase A, 0.1% phosphoric acid solution is used as mobile phase B, gradient elution is carried out according to the following table, flow rate is 0.30 ml per minute, column temperature is 30 DEG C, and detection wavelength is 326 nm:

13. The method for detecting the characteristic pattern of the extract of Ginkgo biloba L. and its preparation according to claim 12, wherein, The chromatographic column used has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

14. The method according to claim 12, wherein the amount of the plurality of components in the triangular wind and the preparation thereof is determined. The test sample is precisely weighed, placed in a conical flask with a plug, 50% ethanol is precisely added, the weight is determined, ultrasonic treatment is carried out, the sample is cooled, the weight is determined again, 50% ethanol is added to make up for the weight loss, the sample is shaken and filtered, and the filtrate is obtained.

15. The method according to claim 14, wherein the amount of the plurality of components in the triangular wind and the preparation thereof is determined. The ultrasonic treatment is carried out at a power of 450-550 W and a frequency of 35-45 kHz.

16. The method according to claim 12, wherein the amount of the plurality of components in the triangular wind and preparation thereof is determined. The control sample solution is prepared by dissolving 20 μg of chlorogenic acid in 50% ethanol to obtain a solution containing 20 μg of chlorogenic acid per 1 ml.

17. The method for detecting the profile of the extract of Ginkgo biloba L. and its preparation according to claim 12, characterized in that, The final determination of the characteristic chromatogram of the Triangular Wind and its preparation is that five characteristic peaks should appear in the test sample chromatogram, and the retention time of the five characteristic peaks in the reference chromatogram of the control medicinal material should correspond; peak 2 should correspond to the retention time of the chlorogenic acid peak. The peak corresponding to the chlorogenic acid reference peak is the S peak, the relative retention time of peaks 1, 3, 4, 5 and the S peak is calculated, and the relative retention time should be within ±10% of the specified value, which is: 0.43 (peak 1), 1.21 (peak 3), 2.09 (peak 4), and 3.17 (peak 5).

18. A thin-layer chromatographic detection method for a triangular herb extract and its preparations, characterized in that, The specific steps are as follows: the control medicinal material of Triangular Wind is used as the control, chloroform-acetone-formic acid (4:1:0.1) is used as the developing agent, the sample is developed, dried, and observed under ultraviolet light (365 nm); the thin layer plate is a silica gel G thin layer plate; the sample is spotted in a circular manner; and the sample is developed in a double-tank developing tank; The test sample is precisely weighed, placed in a conical flask with a plug, 50% ethanol is precisely added, the weight is determined, ultrasonic treatment is carried out, the sample is cooled, the weight is determined again, 50% ethanol is added to make up for the weight loss, the sample is shaken and filtered, and the filtrate is obtained. The test sample is precisely weighed, placed in a conical flask with a plug, 50% ethanol is precisely added, the weight is determined, ultrasonic treatment is carried out, the sample is cooled, the weight is determined again, 50% ethanol is added to make up for the weight loss, the sample is shaken and filtered, and the filtrate is obtained. The test sample is precisely weighed, placed in a conical flask with a plug, 50% ethanol is precisely added, the weight is determined, ultrasonic treatment is carried out, the sample is cooled, the weight is determined again, 50% ethanol is added to make up for the weight loss, the sample is shaken and filtered, and the filtrate is obtained.

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