Kadsura longipedunculata extract and preparation method and detection method of kadsura longipedunculata extract preparation

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a kadsura longipedunculata extract and a preparation method and a detection method of a preparation thereof. A standard decoction preparation process comprises the following steps: (1) taking kadsura longipedunculata decoction pieces, adding water to decoct twice, filtering, cooling filtrate to room temperature, and combining the two decoctions; (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 1.03-1.08 g / ml; and (3) preparing the extract into a required dosage form, thereby obtaining the traditional Chinese medicine composition. The invention also establishes a characteristic chromatogram of the kadsura longepedunculata (schisandra sphenanthera), adopts ultrahigh-phase liquid chromatography, and has the characteristics of simplicity, convenience, stability, high precision, good reproducibility and the like. The invention further establishes a method for determining and analyzing the content of the kadsura longepedunculata (schisandra sphenanthera) standard decoction.

Description

Technical Field

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

[0002] Schisandra sphenanthera Rehd. et Wils, a plant in the Schisandraceae family, is the dried stem and root of the vine. Also known as Da Xue Teng, Zi Jin Teng, Zuan Gu Feng, Xiao Xue Teng, Yan Pi Pa, Nei Feng Xiao, and Ye Wu Wei Zi Teng, it is distributed in Hubei, Sichuan, Yunnan, and Tibet. It is warm in nature and pungent and bitter in taste. It enters the spleen, stomach, and liver meridians. It has the effects of regulating qi and relieving pain, dispelling wind and unblocking meridians, promoting blood circulation and reducing swelling. It is used to treat stomach pain, abdominal pain, rheumatic pain, dysmenorrhea, irregular menstruation, postpartum abdominal pain, sore throat, hemorrhoids, unexplained swelling and pain, and injuries from falls. Modern research shows that Schisandra sphenanthera mainly contains lignans, triterpenes, and volatile oils, among which lignans are its main active ingredient, exhibiting pharmacological effects such as anti-oxidation, anti-tumor, anti-HIV, and anti-HBV.

[0003] According to the "Guizhou Province Standard for Quality of Traditional Chinese and Ethnic Medicinal Materials" (2019 edition), this product is cylindrical, irregularly twisted, 2–15 cm long or longer, and 0.2–6 cm in diameter. The surface is brown to dark brown, slightly rough, with fine longitudinal wrinkles and numerous transverse cracks reaching the wood. Deep transverse grooves are present at the twisted areas, and occasionally the cork has peeled off, exposing the brown bark. It is hard and difficult to break, with a coarse fibrous fracture surface. The bark is thick and brown, while the wood is light brown or dark brownish-yellow, densely covered with small pores (vessels). The pith is distinct and dark brown. It has a slightly fragrant and unique odor, and a slightly sweet taste followed by a bitter and pungent aftertaste.

[0004] Experimental studies have shown that *Schizonepeta tenuifolia* contains a high content of catechin-7-O-β-D-glucopyranoside, which possesses antioxidant and anti-inflammatory activities. Currently, there are few reports on the quality determination of *Schizonepeta tenuifolia* extracts and their preparations, and a standardized system is lacking, which is detrimental to the efficacy and safety of these extracts and preparations. Summary of the Invention

[0005] To address the aforementioned technical problems in the existing technology, this invention provides a method for preparing and detecting *Spatholobus suberectus* extract and its preparations, including methods for content and characteristic chromatographic detection, as detailed below:

[0006] A method for preparing a Schisandra chinensis (Chinese Schisandra chinensis) preparation includes the following steps:

[0007] (1) Take the slices of Five-spice Blood Vine, add water and decoct twice, filter, and combine the two decoctions after cooling the filtrate to room temperature, which is the Five-spice Blood Vine 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 1.08 g / ml; the extract was obtained.

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

[0010] Further, in step (1), specifically, take the slices of Five-Spice Blood Vine, add water and decoct twice. For the first decoction, add 8 to 12 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 20 to 40 minutes. Filter the decoction while hot through a 100-300 mesh sieve, and record the quality of the decoction after the filtrate has cooled to room temperature. For the second decoction, add 6 to 10 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 hot through a 100-300 mesh sieve, and record the quality of the decoction after the filtrate has cooled to room temperature. Combine the two decoctions. The preferred method is as follows: Take slices of five-spice blood vine, add water and decoct twice. For the first decoction, add 10 times the amount of water directly, 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, and record the quality of the decoction after cooling to room temperature. For the second decoction, add 8 times the amount of water, heat 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 record the quality of the decoction after cooling to room temperature. Combine the two decoctions.

[0011] Furthermore, the specific conditions for the reduced pressure low-temperature concentration in step (2) are: temperature: 40~70℃ (preferably 65℃); vacuum degree: -0.080~-0.090MPa.

[0012] Further, step (3) involves freeze-drying the obtained extract to obtain the standard decoction of *Spatholobus suberectus*. The freeze-drying parameters are as follows: the pre-freezing temperature of the concentrated *Spatholobus suberectus* extract 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 900 to 950 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 -0.252 mbar. The preferred freeze-drying parameters are as follows: the pre-freezing temperature of the concentrated schizonepeta tenuifolia extract is -45℃, the pre-freezing time is 240 minutes, the sublimation drying temperature is -30℃~0℃, the sublimation drying time is 920 minutes, and the vacuum degree is -0.2mbar; the desorption drying temperature is 5℃~25℃, the desorption drying time is 420 minutes, and the vacuum degree is -0.2mbar.

[0013] Further, step (3) involves adding excipients (maltodextrin) to the obtained extract, mixing and filtering, drying, collecting the powder, mixing, and granulating to obtain the spiced vine granules.

[0014] Furthermore, the spiced blood vine granules are made from 8000-12000g of medicinal slices into 1000g of granules, with each 1g of formula granules equivalent to 8-12g of medicinal slices.

[0015] A method for determining the content of components in Schisandra chinensis (also known as Chinese Schisandra chinensis) and its preparations is disclosed. The method employs high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the stationary phase. Acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B. Gradient elution is performed according to the specifications in the table below. The flow rate is 0.30 ml / min; the column temperature is 30℃; and the detection wavelength is 204 nm.

[0016]

[0017] Furthermore, the chromatographic column used has a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm.

[0018] Furthermore, the test solution is prepared by the following method: Approximately 0.1 g of the sample to be tested is accurately weighed and placed in a stoppered conical flask. 25 ml of 60-80% (preferably 75%) methanol is accurately added, the weight is measured, and the solution is sonicated for 30 min. After cooling, the weight is measured again. The lost weight is replenished with 60-80% (preferably 75%) methanol, the solution is shaken well, filtered, and the filtrate is collected. The sonication treatment is performed at a power of 450-550 W and a frequency of 35-45 kHz; preferably, at a power of 500 W and a frequency of 40 kHz.

[0019] Furthermore, the reference solution is a solution containing 60 μg of catechin-7-O-β-D-glucopyranoside per 1 ml, prepared using 60-80% (preferably 75%) methanol as a solvent.

[0020] A method for detecting the characteristic chromatograms of Schisandra chinensis (Chinese Schisandra chinensis) and its preparations is disclosed, using high-performance liquid chromatography (HPLC) with octadecylsilane-bonded silica gel as the stationary phase; acetonitrile as mobile phase A and water as mobile phase B, with gradient elution as specified in the table below; flow rate of 0.30 ml / min; column temperature of 40℃; and detection wavelength of 204 nm.

[0021]

[0022]

[0023] Furthermore, the final standard for the characteristic chromatograms of Schisandra chinensis (Chinese Schisandra chinensis) and its preparations was determined as follows: the chromatogram of the test sample should show 4 characteristic peaks, and the retention times should correspond to the 4 characteristic peaks in the chromatogram of the reference medicinal material; among them, peak 1 corresponds to the retention time of the characteristic peak of the catechin-7-O-β-D-glucopyranoside reference standard; the peak corresponding to the catechin-7-O-β-D-glucopyranoside reference standard is peak S; the relative retention times of peaks 2, 3, and 4 with peak S should be calculated, and their relative retention times should be within ±10% of the specified values, with specified values ​​of 2.14 (peak 2), 2.54 (peak 3), and 3.45 (peak 4).

[0024] A thin-layer chromatography method for detecting the extract of Schisandra chinensis and its preparations, using Schisandra chinensis (Chinese Schisandra chinensis) as a reference material, and petroleum ether (60-90℃)-ethyl acetate (4:1) as the developing solvent, is developed, removed, air-dried, sprayed with 10% phosphomolybdic acid ethanol solution, and dried at 105℃ until the spots are clearly visible, and then examined under sunlight.

[0025] Furthermore, the specific steps are as follows:

[0026] Solution preparation

[0027] 1. Preparation of the test solution

[0028] Take 2.0g of this product, grind it into a fine powder, add 10ml of petroleum ether (60-90℃), sonicate for 30 minutes, filter, evaporate to dryness, dissolve the residue in 1ml of petroleum ether (60-90℃) to prepare the test solution.

[0029] 2. Preparation of the control herbal solution

[0030] Take 2.0g of Schisandra chinensis (Chinese Schisandra chinensis) as a reference herb, add 50ml of water, heat under reflux for 30 minutes, filter, and extract twice with petroleum ether (60-90℃) by shaking, 50ml each time. Combine the petroleum ether (60-90℃) solutions, evaporate to dryness, and dissolve the residue in 1ml of petroleum ether (60-90℃) to obtain the reference herb solution.

[0031] 3. Preparation of negative sample solution

[0032] Take 2.0g of the negative sample of the formula granules of Schisandra chinensis (Huazhong Schisandra chinensis), grind it into a fine powder, and process it in the same way as the test sample to prepare the negative sample solution.

[0033] Thin-layer plate: Silicone G thin-layer plate

[0034] Developing solvent: petroleum ether (60–90℃) - ethyl acetate (4:1)

[0035] Sampling method: Spray strip sampling

[0036] Deployment method: Deployment is carried out using a double-groove deployment cylinder.

[0037] Inspection: Spray with 10% phosphomolybdic acid ethanol solution, bake at 105°C until the spots are clearly visible, and inspect under sunlight.

[0038] Compared with the prior art, the technical effects of this invention are reflected in:

[0039] 1. The preparation method of the formulation described in this application can provide a standard for the quality control of Schisandra chinensis (Chinese Schisandra) and its preparations, such as standard decoction products, achieving overall quality control and effective supervision of Schisandra chinensis (Chinese Schisandra) and its preparations, such as formulated granules. Furthermore, the process described in this application is stable and repeatable, and can be used as a standard decoction preparation process for Schisandra chinensis (Chinese Schisandra).

[0040] 2. The characteristic chromatographic method established in this application adopts ultra-high performance liquid chromatography, which has the characteristics of being simple, stable, highly precise and reproducible. It has established a characteristic chromatographic spectrum of Schisandra chinensis (Chinese Schisandra chinensis), conducted a material basis study, and identified the chemical components corresponding to the characteristic peaks. It is used for the source quality control of Schisandra chinensis (Chinese Schisandra chinensis) formulation granules.

[0041] 3. This application developed a quantitative determination method for the content of *Schisandra chinensis* (also known as *Schisandra chinensis*) in a standard decoction using ultra-high performance liquid chromatography (UHPLC), and established a quantitative standard for the decoction, providing a scientific basis for establishing a quality standard for *Schisandra chinensis*. An analytical method for determining the content of *Schisandra chinensis* (also known as *Schisandra chinensis*) in the standard decoction was established, and the method for determining the content of *Schisandra chinensis* (also known as *Schisandra chinensis*) in the standard decoction was determined. The content and transfer rate of 15 batches of *Schisandra chinensis* (also known as *Schisandra chinensis*) in the standard decoction were determined, and the range of content and transfer rate of *Schisandra chinensis* (also known as *Schisandra chinensis*) in the standard decoction was determined. Attached Figure Description

[0042] Figure 1 It is a YMW mechanical split-type decoction pot.

[0043] Figure 2 It is a specific investigation map.

[0044] Figure 3 This is a chromatogram of peak purity.

[0045] Figure 4 The linear regression equation for catechin-7-O-β-D-glucopyranoside was investigated.

[0046] Figure 5 The effect of different chromatographic columns on the content of the target components in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0047] Figure 6This study examines the effects of different chromatographic instruments on the content of target components in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0048] Figure 7 These are the chromatographic results of the target components in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) at different column temperatures.

[0049] Figure 8 These are the chromatographic results of the target components in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) at different flow rates.

[0050] Figure 9 This is the DAD diagram of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0051] Figure 10 These are UPLC spectra of different mobile phase systems.

[0052] Figure 11 This is the aqueous phase UPLC spectrum of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0053] Figure 12 This study investigates the characteristic chromatograms of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) using different extraction solvents.

[0054] Figure 13 This is a characteristic chromatogram of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) and a comparison of the extraction efficiency of different extraction solvents.

[0055] Figure 14 It is the common pattern of the characteristic spectrum of the standard decoction of Schisandra chinensis (Wu Xiang Xue Teng (Hua Zhong Wu Wei Zi)) (R is the reference herb).

[0056] Figure 15 This study investigates the specificity of the characteristic chromatograms of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0057] Figure 16 This is a comprehensive study of the characteristic chromatograms of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0058] Figure 17 This is a comparison chart of UPLC tests using different chromatographic columns.

[0059] Figure 18 This is a comparison chart of UPLC tests conducted at different column temperatures.

[0060] Figure 19 This is a comparison chart of UPLC under different flow rates.

[0061] Figure 20 The final characteristic chromatogram of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis). Peak 1 (S): catechin-7-O-β-D-glucopyranoside. Column: ZORBAX SB-C18 (2.1×150mm 1.8μm).

[0062] Figure 21 This is a thin-layer chromatogram for the sampling quantity investigation of the granules of *Schisandra chinensis* (also known as *Schisandra chinensis*). The sample consisted of: 1. 6 μl of *Schisandra chinensis* granules; 2. 8 μl of *Schisandra chinensis* granules; 3. 10 μl of *Schisandra chinensis* granules; 4. 4 μl of *Schisandra chinensis* reference material; 5. 5 μl of *Schisandra chinensis* reference material; and 6. 6 μl of *Schisandra chinensis* reference material.

[0063] Figure 22 This study focuses on the thin-layer chromatography (TLC) specificity of the formula granules of *Schisandra chinensis* (also known as *Schisandra chinensis* from Central China). The samples included (T: 25℃, RH: 66%; Yinlong silica gel G plate); 1. 10 μl of negative sample; 2-4. 10 μl of formula granules of *Schisandra chinensis* (240601-240603); 5. 6 μl of reference material of *Schisandra chinensis* (also known as *Schisandra chinensis* from Central China).

[0064] Figure 23 These are thin-layer chromatograms for identifying the granules of Schisandra chinensis (a type of schisandra chinensis) under different temperature conditions. Among them:

[0065] First sheet (T: 40℃, RH: 66%; Qingdao Marine Silica G plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0066] Second sheet (T: 25℃, RH: 66%; Yinlong silica gel G plate). 1. Negative sample 10μl; 2-4. Five-spice vine (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Five-spice vine (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0067] The third sheet (T: 5℃, RH: 66%; Qingdao Marine Silica G Plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0068] Figure 24 This is a thin-layer chromatogram for identifying the granules of Schisandra chinensis (Chinese Schisandra chinensis) under different humidity conditions.

[0069] in:

[0070] First sheet (T: 25℃, RH: 88%; Qingdao Marine Silica G plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0071] Second sheet (T: 25℃, RH: 66%; Yinlong G plate). 1. Negative sample 10μl; 2-4. Five-spice vine (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Five-spice vine (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0072] Third sheet (T: 25℃, RH: 33%; Qingdao Marine Silica G plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0073] Figure 25 These are chromatograms obtained from examining silica gel plates from different manufacturers. Among them:

[0074] First sheet (T: 25℃, RH: 33%; Qingdao Marine Silica G plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0075] Second sheet (T: 25℃, RH: 66%; Yantai Yinlong Silica G-plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0076] The third sheet (T: 25℃, RH: 66%; Merck silica gel G plate). 1. Negative sample 10μl; 2-4. Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (240601-240603) 10μl; 5. Schisandra chinensis (Huazhong Schisandra chinensis) control medicinal material 6μl.

[0077] Figure 26 These are thin-layer chromatograms of three batches of *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, with (T: 25℃, RH: 33%; Qingdao Marine Silica G plate). 1. Negative sample 10 μl; 2-4. *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules (240601-240603) 10 μl; 5. *Schisandra chinensis* (Huazhong Schisandra chinensis) reference material 6 μl. Detailed Implementation

[0078] 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 made.

[0079] Example 1:

[0080] Basis for proposing standard decoction process parameters for Schisandra chinensis (Chinese Schisandra chinensis)

[0081] 1. Instruments and Materials

[0082] 1.1 Instruments

[0083] 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 Industry & Trade 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), guillotine.

[0084] 1.2 Materials

[0085] The following table shows the processed slices of Schisandra chinensis (also known as Chinese Schisandra chinensis):

[0086] Table 1. Information on processed slices of Schisandra chinensis (also known as Chinese Schisandra chinensis).

[0087]

[0088] 2. Methods for determining process optimization indicators

[0089] 2.1 Method for determining solid content

[0090] Method for Determination of Solid Content in Concentrated Solution of Schisandra chinensis (Schisandra chinensis from Central China)

[0091] 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.

[0092] 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.

[0093] 3. Processing of medicinal slices

[0094] Take the raw medicinal material, remove impurities, wash it, soak it thoroughly, cut it into thick slices or sections, and dry it to obtain the final product.

[0095] Information on the medicinal materials used in this research is shown in Table 2:

[0096] Table 2. Information on the medicinal material *Schisandra chinensis* (also known as *Schisandra chinensis* from Central China).

[0097]

[0098]

[0099] 4 Extraction process

[0100] 4.1 Selection of cooking utensils

[0101] 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.

[0102] 4.2 Study on extraction process parameters

[0103] (1) Investigation on the dosage of medicinal slices

[0104] 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 experimental results, 100g of *Schisandra chinensis* (Chinese Schisandra chinensis) 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 standard decoction of *Schisandra chinensis* (Chinese Schisandra chinensis) is set at 100g.

[0105] (2) Water addition assessment

[0106] 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 10 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 8 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 10 times the amount of water for the first decoction and 8 times the amount of water for the second decoction.

[0107] (3) Investigation of soaking time

[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," 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 Schisandra chinensis (Huazhong Schisandra chinensis) is determined to be 30 minutes.

[0109] (4) Examination of cooking time

[0110] According to the recommendations under "Standard Decoction Preparation" 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; 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 of *Schisandra chinensis* (from Central China) 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.

[0111] (5) Investigation of solid-liquid separation conditions

[0112] 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 *Schisandra chinensis* (Huazhong Schisandra chinensis) 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 standard decoction of *Schisandra chinensis* (Huazhong Schisandra chinensis).

[0113] 4.3 Results of Determination of Extraction Process Parameters

[0114] Take 100g of Schisandra chinensis (also known as Chinese Schisandra chinensis) slices, place them in an electric ceramic kettle, add water, and decoct twice. For the first decoction, add 10 times the amount of water, soak for 30 minutes, bring to a boil over high heat (500W), then simmer over low heat (200W) for 30 minutes. Filter the decoction through a 300-mesh sieve while hot, and record the mass of the decoction after cooling to room temperature. For the second decoction, add 8 times the amount of water, heat 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 record the mass of the decoction after cooling to room temperature. Combine the two decoctions.

[0115] 5 Concentration Process

[0116] 5.1 Study on Concentration Process Parameters

[0117] 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.

[0118] The density, extract yield, and concentrated state of the standard decoction of Schisandra chinensis (from Central China) 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 exhibited moderate viscosity, good fluidity, and ease of 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.

[0119] 5.2 Results of Determination of Concentration Process Parameters

[0120] 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 100ml of extract.

[0121] 6. Drying process

[0122] 6.1 Study on freeze-drying process parameters

[0123] 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 selected as the drying method for the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis).

[0124] Under magnetic stirring, the extract was dispensed into 5ml brown vials, each containing 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 product was obtained. The eutectic point of the *Schisandra chinensis* (a type of vine) obtained by vacuum freeze-drying was -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 28 hours. The results indicate that under these freeze-drying conditions, the standard decoction of *Schisandra chinensis* (a type of vine) has low moisture content and is easy to preserve.

[0125] 6.2 Confirmation of freeze-drying process parameters

[0126] 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: the pre-freezing temperature of the concentrated Schisandra chinensis (Huazhong Schisandra chinensis) extract was -45℃, the pre-freezing time was 240 minutes, the sublimation drying temperature was -30℃~0℃, the sublimation drying time was 920 minutes, and the vacuum degree was -0.2mbar; the desorption drying temperature was 5℃~25℃, the desorption drying time was 420 minutes, and the vacuum degree was -0.2mbar.

[0127] 7. Validation of the preparation process

[0128] Three portions (100g each) of *Schisandra chinensis* (a type of vine) slices (batch numbers: YP240613, YP240614, YP240615) 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:

[0129] Table 3. Verification of the preparation method of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0130]

[0131]

[0132] The experimental results show that the specific gravity, solid content and extract yield of the three parallel batches of standard decoction concentrate of Schisandra chinensis (Chinese Schisandra chinensis) are basically the same, indicating that the process is stable and reproducible and can be used as the preparation process of standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0133] Example 2

[0134] Research on the Processing Technology of Schisandra chinensis (Chinese Schisandra chinensis) Granules

[0135] The yield of the standard decoction of *Schisandra chinensis* (from Central China) in Example 1 fluctuated between 5.92% and 9.57%, with a mean of 7.95% and a standard deviation (SD) of 1.08%. The mean yield ranged from 70% to 130% between 5.56% and 10.33%, and the mean yield ±3SD ranged from 4.70% to 11.19%. The yield range of the standard decoction of *Schisandra chinensis* (from Central China) was tentatively determined to be the mean yield ±3SD range of 4.70% to 11.19%, approximately rounded to 4.7% to 11.2%. The yields of multiple batches of the standard decoction were all within this range. Since an appropriate amount of excipients (3% to 6% of the amount of medicinal slices) needs to be added during the drying of the concentrated extract of Schisandra chinensis (Huazhong Schisandra chinensis), and considering the production yield data of different processes and the yield and material balance of each process, it can be seen that the yield of each process meets the requirements of the standard decoction range, and the material balance of the granulation process is ≥97%. At the same time, considering the convenience of rounding and clinical dispensing, based on the granule yield, the equivalent of the Schisandra chinensis (Huazhong Schisandra chinensis) formula granule preparation is confirmed to be: 1g of Schisandra chinensis (Huazhong Schisandra chinensis) formula granules is equivalent to 10.0g of medicinal slices.

[0136] Example 3

[0137] Establishment of a standard decoction method for determining the content of Schisandra chinensis (a type of vine) in Central China

[0138] 1. Establishment of analytical methods for content determination

[0139] According to the "Chinese Materia Medica," *Symplocos buergeriana* is warm in nature and sweet in taste, entering the liver, lung, and stomach meridians. It has the effects of regulating qi and relieving pain, dispelling wind and unblocking meridians, promoting blood circulation and reducing swelling. In traditional Chinese medicine, it is mainly used for stomach pain, abdominal pain, rheumatic pain, dysmenorrhea, irregular menstruation, postpartum abdominal pain, sore throat, hemorrhoids, boils of unknown origin, and injuries from falls. Studies have shown that the alcohol extract of *Symplocos buergeriana* has an inhibitory effect on inflammatory factors in the joints of rats with type II collagen arthritis (CIA), thereby eliminating swelling. *Symplocos buergeriana* combined with the root of *Schisandra chinensis*, washed, dried, and ground into a very fine powder, can be mixed with sesame oil and applied to the affected area to treat burns and scalds. Soaking it in 500ml of wine for one week, then taking 10ml three times a day, can treat injuries from falls and rheumatic back pain. According to literature, *Symplocos buergeriana* mainly contains lignans, triterpenes, and volatile oils.

[0140] The content of catechin-7-O-β-D-glucopyranoside in *Schizonepeta tenuifolia* is relatively high. Establishing a method for determining the content of catechin-7-O-β-D-glucopyranoside is of important reference value for controlling the quality of *Schizonepeta tenuifolia* decoction pieces. Currently, there are few reports on the determination of the content of *Schizonepeta tenuifolia* in standard decoctions. Therefore, this experiment developed a quantitative determination method of *Schizonepeta tenuifolia* content in standard decoctions using ultra-high performance liquid chromatography (UHPLC), and established a quantitative standard for this standard decoction, providing a scientific basis for establishing the quality standard of *Schizonepeta tenuifolia* standard decoctions.

[0141] 1.1 Instruments and Reagents

[0142] The Agilent 1290 Infinity II ultra-high performance liquid chromatograph, the Shimadzu LC-40D XS ultra-high performance liquid chromatograph, the ML204 electronic balance (model: 0.0001%), the HH-S8 electric thermostatic water bath, 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 Five-Spice Blood Vine Soup are: BT240601, BT240602, BT240603, BT240604, BT240605, BT240606, BT240607, BT240608, BT240609, BT240610, BT240611, BT240612, BT240613, BT240614, and BT240615 (provided by the laboratory process group).

[0143] 1.2 Source and purity test of reference standards

[0144] Catechin-7-O-β-D-glucopyranoside (batch number: 23040307, ​​purity 99.4%) was purchased from Chengdu Glip Biotechnology Co., Ltd. It is for content determination purposes and requires no pretreatment.

[0145] 1.3 Determination of chromatographic conditions

[0146] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 150 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 204 nm. The theoretical plate number, calculated based on the catechin-7-O-β-D-glucopyranoside peak, should be no less than 3000.

[0147]

[0148] 1.4 Preparation of reference solution

[0149] Take an appropriate amount of catechin-7-O-β-D-glucopyranoside reference standard, accurately weigh it, and add 75% methanol to prepare a solution containing 60 μg of catechin-7-O-β-D-glucopyranoside per 1 ml, which will be used as the reference solution.

[0150] 1.5 Preparation of the test solution

[0151] (1) Investigation of different extraction solvents

[0152] Nine portions of the standard decoction of Schisandra chinensis (from Central China) BT240609 were prepared, with two parallel samples per portion. Each portion was placed in a stoppered conical flask, and 25 ml of methanol, 75% methanol, 50% methanol, 25% methanol, anhydrous ethanol, 75% ethanol, 50% ethanol, 25% ethanol, and water were added sequentially. The samples were weighed, sonicated (500W, 40kHz) for 30 minutes, cooled, and weighed again. The lost weight was replenished with the appropriate solvent, shaken well, filtered, and the filtrate was collected to obtain the test solutions. Under the above chromatographic conditions, 1 μl of each of the reference solution and the test solution were precisely injected into an ultra-high performance liquid chromatograph for determination. The content of catechin-7-O-β-D-glucopyranoside was calculated to determine the optimal extraction solvent. The experimental results were calculated using the external standard one-point method. Detailed results are shown in the table below.

[0153] Table 4. Effects of different extraction solvents on the content determination results of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis).

[0154]

[0155] The experimental results show that different solvents have a significant impact on the content of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis). Considering both the content and the peak shape of the chromatogram, 75% methanol was selected as the extraction solvent for further research.

[0156] (2) Examination of extraction methods

[0157] Two portions of the standard decoction of Schisandra chinensis (from Central China) BT240609 were prepared, with two parallel samples per portion. Each portion was placed in a stoppered conical container, and 25 ml of 75% methanol was accurately added. The samples were weighed, and each was ultrasonicated and refluxed for 30 min. After cooling, the samples were weighed again, and the lost weight was replenished with 75% methanol. The mixture was shaken well, filtered, and the filtrate was collected to obtain the test solutions. Under the chromatographic conditions described above, 1 μl of each of the reference solution and the test solution were accurately injected into an ultra-high performance liquid chromatograph for determination. The content of catechin-7-O-β-D-glucopyranoside was calculated to determine the effect of different extraction methods on its content, and the optimal extraction method was determined. The experimental results were calculated using the external standard single-point method. Detailed results are shown in the table below.

[0158] Table 5. Effects of different extraction methods on the content determination results of the standard decoction of Schisandra chinensis (Wuweizi).

[0159]

[0160] The experimental results show that different extraction methods have little effect on the content of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis). Considering the ease of operation and the difference in content, ultrasonic treatment (power 500W, frequency 40kHz) was selected as the extraction method for further research.

[0161] (3) Examination of different extraction times

[0162] Take an appropriate amount of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) BT240609, a total of 4 portions, with 2 parallel samples for each portion. Place each portion in a stoppered conical container, accurately add 25 ml of 75% methanol, weigh, and sonicate (power 500W, frequency 40kHz) for 15, 30, and 60 minutes respectively. After cooling, weigh again, and replenish the lost weight with 75% methanol. Take an appropriate amount of the filtrate and filter it through a 0.22 μm microporous membrane, place it in a high-performance liquid sample bottle, and obtain the test solution for later use. Under the above chromatographic conditions, accurately pipette 1 μl each of the reference solution and the test solution, inject them into the ultra-high performance liquid chromatograph, and determine the content. Calculate the effect of different extraction times on the content of catechin-7-O-β-D-glucopyranoside, and determine the optimal extraction time. The experimental results are calculated using the external standard one-point method. The specific results are shown in the table below:

[0163] Table 6. Effects of different extraction times on the content determination results of the standard decoction of Schisandra chinensis (Chinese schisandra chinensis).

[0164]

[0165] The experimental results show that different extraction times have a significant effect on the content of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis). Considering both time cost and content differences, ultrasonic treatment (500W power, 40kHz frequency) for 30 minutes was selected for further research.

[0166] (4) Investigation of the volume of the extraction solvent

[0167] Take an appropriate amount of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) BT240609, a total of 4 portions, with 2 parallel samples per portion. Place each portion in a stoppered conical container, and accurately add 10 ml, 20 ml, 25 ml, and 50 ml of 75% methanol sequentially, weighing each portion. Sonicate each portion (500W power, 40kHz frequency) for 30 minutes, cool, and weigh again. Make up the lost weight with 75% methanol. Take an appropriate amount of the filtrate, filter it through a 0.22 μm microporous membrane, and place it in a high-performance liquid sample bottle to obtain the test solutions. 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 volumes on the content of catechin-7-O-β-D-glucopyranoside to determine the optimal extraction volume. The experimental results are calculated using the external standard one-point method. Detailed results are shown in the table below.

[0168] Table 7. Effect of different extraction solvent volumes on the content determination results of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis).

[0169]

[0170] The experimental results show that the volume of different extraction solvents has no significant effect on the content of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis). Considering the cost and the difference in content, the extraction solvent volume of 25 ml was selected for further research.

[0171] (5) Determination of the preparation method of the test solution

[0172] Based on the results of the sample pretreatment experiment, the preparation method for the test sample can be determined as follows:

[0173] Take approximately 0.1g of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis), accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, replenish the lost weight with 75% methanol, shake it well, filter it, and collect the filtrate to obtain the final product.

[0174] 2. Validation of content determination method

[0175] (1) Specificity examination

[0176] Accurately pipette 1 μl each of the test solution of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) (BT240609), the reference solution of catechin-7-O-β-D-glucopyranoside, and the negative sample, and perform chromatographic analysis under the conditions described above. Record the chromatographic elution patterns. Results are shown below. Figure 2 .

[0177] The chromatogram results show that this analytical method is specific for determining the content of catechin-7-O-β-D-glucopyranoside in the formula granules of Schisandra chinensis (Chinese Schisandra chinensis).

[0178] (2) Peak purity

[0179] Precisely pipette 1 μl each of the test solution and reference solution of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) (BT240609) 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 below. Figure 3 .

[0180] Table 8 Matching values ​​for target peaks and peak purity

[0181]

[0182] The experimental results show that the peak purity matching value of the components in this index is greater than 960, which indicates that the peak purity meets the analytical requirements.

[0183] (3) Linear

[0184] Accurately weigh an appropriate amount of catechin-7-O-β-D-glucopyranoside reference standard and place it in a numbered 100ml volumetric flask. Add 75% methanol to prepare a solution containing 159.2045 μg of catechin-7-O-β-D-glucopyranoside per ml. Shake well to obtain the catechin-7-O-β-D-glucopyranoside reference standard stock solution, and store it in a refrigerator for later use. Dilute the catechin-7-O-β-D-glucopyranoside reference standard stock solution 1, 2, 4, 8, and 16 times to obtain reference solutions of different concentrations of catechin-7-O-β-D-glucopyranoside. Perform chromatography analysis under the above conditions. Plot concentration on the x-axis and peak area on the y-axis to investigate the linear range of catechin-7-O-β-D-glucopyranoside. The linearity results are shown in the table below. Figure 4 .

[0185] Table 9. Linearity Study of Catechin-7-O-β-D-glucopyranoside

[0186]

[0187] Experimental results showed that catechin-7-O-β-D-glucopyranoside concentration and peak area exhibited a good linear relationship within the concentration range of 9.9503 μg / ml to 159.2045 μg / ml, with a correlation coefficient R0. 2 =0.9999, the linear regression equation is: y = 13.98x - 3.6624.

[0188] (4) Precision test

[0189] 1) Instrument precision test

[0190] Accurately pipette 1 μl each of the test solution of Schisandra chinensis (Batch No.: BT240609) standard decoction and the reference solution of catechin-7-O-β-D-glucopyranoside, inject them into the ultra-high performance liquid chromatograph (UHPLC) for determination. The RSD (%) value of the target peak, calculated using the external standard single-point method, is given by catechin-7-O-β-D-glucopyranoside. Detailed results are shown in the table below.

[0191] Table 10 Precision test results of the method for determining the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction.

[0192]

[0193] The results show that the RSD (%) of the target peak catechin-7-O-β-D-glucopyranoside content is 0.52% < 2%, indicating that the method has good instrument precision.

[0194] 2) Repeatability test

[0195] Take approximately 0.1 g of the same batch of standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) (BT240609), accurately weigh it, and prepare 6 parallel portions according to the above-described method for preparing the test solution. Analyze the solution under the above-described chromatographic conditions. Calculate the RSD (%) of the target peak content using the external standard single-point method, based on catechin-7-O-β-D-glucopyranoside in the test solution. See the table below for detailed results.

[0196] Table 11 Repeatability test results of the method for determining the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction.

[0197]

[0198] The experimental results show that the RSD (%) of the target peak catechin-7-O-β-D-glucopyranoside content is 1.12% < 2%, indicating that the method has good reproducibility.

[0199] 3) Intermediate precision

[0200] Other analysts in this project team operated on different dates and under different chromatographs, taking approximately 0.1g of the same batch of Schisandra chinensis (Huazhong Schisandra chinensis) standard decoction (BT240609), accurately weighed, and prepared the test solution according to the above-mentioned test solution preparation method. The test solution was then analyzed under the above-mentioned chromatographic conditions. The RSD (%) value of the target peak content was calculated using the external standard one-point method, based on catechin-7-O-β-D-glucopyranoside in the test solution. The specific results are detailed in the table below.

[0201] Table 12 Results of intermediate precision test for the determination of the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction.

[0202]

[0203] The experimental results show that the intermediate precision RSD (%) value of the target peak catechin-7-O-β-D-glucopyranoside content is 0.87% < 2%, indicating that the method has good precision.

[0204] (5) Accuracy test

[0205] Six portions of a standard decoction containing 6-spice vine (Schisandra chinensis) of known content (batch number BT240609, catechin-7-O-β-D-glucopyranoside content 6.71 mg / g) were accurately weighed. 25 ml of a catechin-7-O-β-D-glucopyranoside reference solution (concentration 0.0133 mg / ml) prepared in 75% methanol was added to each portion. The test solutions were prepared according to the method described above. The solutions were then analyzed by chromatographic analysis under the conditions described above. 1 μl of each solution was injected. The content of the target peak was calculated using the external standard single-point method, based on the catechin-7-O-β-D-glucopyranoside content. The recovery rate and RSD were calculated using the following formula.

[0206]

[0207] Table 13. Determination of the content of *Schisandra chinensis* (a type of vine) in standard decoction: Results of the recovery experiment of catechin-7-O-β-D-glucopyranoside.

[0208]

[0209] The experimental results show that the recovery rate of catechin-7-O-β-D-glucopyranoside in the standard decoction is within the range of 92%-105%, and the RSD% (1.59%) is less than 2%, indicating that the accuracy of the assay method is good.

[0210] (6) Stability test

[0211] The test solution of the standard decoction of Schisandra chinensis (batch number BT240609) was prepared according to the preparation method of the test solution. The sample was injected at 0, 2, 4, 6, 8, 12 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 one-point external standard method based on catechin-7-O-β-D-glucopyranoside. The specific results are shown in the table below.

[0212] Table 14. Stability test results of the method for determining the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction.

[0213]

[0214]

[0215] Experiments showed that the RSD (%) of the target peak catechin-7-O-β-D-glucopyranoside within 24 hours was 0.69% < 2%, indicating that the solution had good stability within 24 hours.

[0216] (7) Durability test

[0217] 1) Investigation of different chromatographic columns

[0218] Comparison of chromatographic columns ACQUITY The effects of three chromatographic columns—HSS (2.1×150mm, 1.8μm), Agilent ZORBAXSB-C18 (2.1*150mm, 1.8μm), and Thermo Accucore C18 (2.1×150mm, 1.8μm)—on the peak shape and resolution of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0219] Take the test solution from the [Content Determination] section of the standard decoction of Schisandra chinensis (batch number BT240609) and determine its content under the above chromatographic conditions. Calculate the content using catechin-7-O-β-D-glucopyranoside and record the chromatographic data. The experimental results are shown in the table below. Figure 5 As shown.

[0220] Table 15. Effects of different chromatographic columns on the determination results of *Schisandra chinensis* (a type of schisandra fruit) in standard decoction.

[0221]

[0222] Experimental results showed that all three chromatographic columns had good separation effects and could meet the requirements for the determination of catechin-7-O-β-D-glucopyranoside in Schisandra chinensis (a type of schisandra chinensis). However, the resolution and theoretical plate number of the Agilent ZORBAX SB-C18 (2.1*150mm, 1.8μm) column were lower than those of the ACQUITY column. HSS (2.1×150mm, 1.8μm) and ThermoAccucore C18 (2.1×150mm, 1.8μm) columns are superior, therefore the Agilent ZORBAX SB-C18 (2.1*150mm, 1.8μm) column was selected as the preferred column for this experiment.

[0223] 2) Investigation using different chromatographs

[0224] Based on the existing equipment in the laboratory, a Shimadzu ultra-high performance liquid chromatograph (LC-40D XS) and an Agilent high performance liquid chromatograph (Agilent 1290 Infinity II) were selected to compare the effects of the two chromatographs on the peak shape and resolution of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0225] Take the test solution from the [Content Determination] section of the standard decoction of Schisandra chinensis (batch number BT240609) and determine its content under the above chromatographic conditions. Calculate the content using catechin-7-O-β-D-glucopyranoside and record the chromatographic data. The experimental results are shown in the figure below. Figure 6 As shown.

[0226] Table 16 Results of the investigation using different chromatographs

[0227]

[0228] Experimental results show that this analytical method exhibits good durability with different chromatographs. Variations in the chromatograph can meet the system adaptability requirements.

[0229] 3) Investigation at different column temperatures

[0230] The effects of different column temperatures (25℃, 30℃, and 35℃) on the peak shape of catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) were compared.

[0231] Take the test solution from the [Content Determination] section of the standard decoction of Schisandra chinensis (batch number BT240609) and determine its content under the above chromatographic conditions. Calculate the content using catechin-7-O-β-D-glucopyranoside and record the chromatographic data. The experimental results are shown in the table below. Figure 7 As shown.

[0232] Table 17 Results of the determination of the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction at different column temperatures

[0233]

[0234] The results showed that the peak shape and separation were good at all three column temperatures. At 30℃, the baseline of the chromatogram showed no drift, and the retention time 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.

[0235] 4) Investigation of different flow velocities

[0236] 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 catechin-7-O-β-D-glucopyranoside in the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0237] Take the test solution from the [Content Determination] section of the standard decoction of Schisandra chinensis (batch number BT240609) and determine its content under the above chromatographic conditions. Calculate the content using catechin-7-O-β-D-glucopyranoside and record the chromatographic data. The experimental results are shown in the table below. Figure 8 As shown.

[0238] Table 18 Results of the determination of the content of Schisandra chinensis (Chinese Schisandra chinensis) in standard decoction at different flow rates

[0239]

[0240] 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.

[0241] Example 4

[0242] Establishment of a characteristic chromatogram analysis of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0243] 1. Instruments and reagents

[0244] Instruments: Agilent 1290 Infinity II ultra-high performance liquid chromatograph; KQ-500DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); ML204 electronic balance (0.0001 g); HH-4 digital display constant temperature water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.).

[0245] Reagents: Acetonitrile was of chromatographic grade; water was ultrapure water; all other reagents were of analytical grade.

[0246] Test drug: Catechin-7-O-β-D-glucopyranoside (batch number: 23040307, ​​purity 99.4%), purchased from Chengdu Glip Biotechnology Co., Ltd. *Schisandra chinensis* reference material (batch number: 2024-090601) purchased from Chengdu Glip Biotechnology Co., Ltd. The batch numbers of 15 batches of *Schisandra chinensis* (Huazhong Schisandra chinensis) standard decoction are: BT240601, BT240602, BT240603, BT240604, BT240605, BT240606, BT240607, BT240608, BT240609, BT240610, BT240611, BT240612, BT240613, BT240614, BT240615 (provided by the laboratory process group).

[0247] 2. Preparation of reference solution

[0248] Accurately weigh approximately 0.4g of *Schisandra chinensis* (a type of vine) reference material, place it in a stoppered conical flask, accurately add 25ml of water, weigh the flask, heat under reflux for 30 minutes, cool, weigh again, replenish the lost weight with water, shake well, filter, and use the filtrate as the reference solution. Accurately weigh an appropriate amount of catechin-7-O-β-D-glucopyranoside reference standard, add 75% methanol to prepare a solution of 60μg of catechin-7-O-β-D-glucopyranoside per 1ml, and use this as the reference solution.

[0249] 3. Determination of chromatographic conditions

[0250] (1) Determination of detection wavelength

[0251] The standard decoction of Schisandra chinensis (from Central China) was injected into the sample for analysis, and the absorption spectrum in the range of 190–400 nm was recorded (see [link to sample analysis]). Figure 9 ).

[0252] Experimental results show that at a wavelength of 204 nm, the standard decoction sample solution of Schisandra chinensis (Huazhong Schisandra chinensis) has more detectable chromatographic peak information and less baseline noise interference. Therefore, 204 nm was selected as the detection wavelength.

[0253] (2) Optimization of the mobile phase

[0254] ① Consider A as the organic phase and B as the aqueous phase. The gradient is as follows:

[0255]

[0256] Experimental results: see details Figure 10Acetonitrile-0.1% phosphoric acid aqueous solution has a stronger elution ability than methanol-0.1% phosphoric acid aqueous solution. Therefore, acetonitrile-aqueous solution was chosen to explore the conditions. The various acids used in the mobile phase will be investigated later.

[0257] ② Examine the cases with and without acid.

[0258] Experimental results: see details Figure 11 The peak shape is better without acid than with acid, and the peak shape is optimal when the mobile phase is an acetonitrile-water solution system. Therefore, acetonitrile-water solution is chosen for elution.

[0259] (3) Determination of chromatographic conditions

[0260] The column was packed with octadecylsilane-bonded silica gel (150 mm column length, 2.1 mm inner diameter, 1.8 μm particle size); acetonitrile was used as mobile phase A, and water 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 40 °C; and the detection wavelength was 204 nm. The theoretical plate number, calculated based on the catechin-7-O-β-D-glucopyranoside peak, should be no less than 5000.

[0261]

[0262] 4. Preparation of the test solution

[0263] This experiment investigated the effects of different extraction solvents on the characteristic chromatograms of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis). 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 different extraction solvents were compared by using the total peak area / sample weight of four tentatively determined chromatographic peaks and the chromatograms.

[0264] Take an appropriate amount of the standard decoction of *Spatholobus suberectus* (BT240609), approximately 0.2 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 30 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 of *Spatholobus suberectus* are shown below. Figure 12-13 .

[0265] Experimental results: The extraction efficiency was higher when the extraction solvent was 75% methanol, therefore 75% methanol was chosen as the extraction solvent.

[0266] Based on the above experimental results, the sample pretreatment method for the characteristic chromatogram of the standard decoction of *Schizonepeta tenuifolia* can be determined as follows:

[0267] Take about 0.2g of this product, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, weigh it, sonicate it (power 500W, frequency 40kHz) for 30min, cool it, weigh it again, make up the lost weight with 75% methanol, shake well, filter it, and take the filtrate to obtain the product.

[0268] 5. Identification and designation of common peaks

[0269] UPLC chromatograms of different batches of standard decoction samples of Schisandra chinensis (Huazhong Schisandra chinensis) were determined, and the results were analyzed using the "Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine (2012 Edition)" recommended by the National Pharmacopoeia Commission, and common peaks were selected.

[0270] Figure 14 The results showed that the characteristic spectrum of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis) had four relatively obvious common peaks.

[0271] 6. Methodological Validation of Feature Map Analysis Method

[0272] (1) Specificity examination

[0273] Accurately pipette 1 μl each of the test solution and blank solvent from the "Characteristic Chromatography" section of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) and inject them into the liquid chromatograph. Determine the chromatographic results under the conditions described above. See results below. Figure 15 .

[0274] The experimental results show that the solvent does not interfere with the characteristic peaks in the standard decoction chromatogram of Schisandra chinensis (Chinese Schisandra chinensis).

[0275] (2) Holistic Examination

[0276] Take the test solution of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) under the [characteristic spectrum] section, 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.

[0277] 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.

[0278] (3) Precision test

[0279] Take the test solution from the [Characteristic Chromatography] section of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis), and inject it 6 times repeatedly, with an injection volume of 1 μl. Temporarily identify 4 characteristic peaks, using peak 1 as the reference peak, and calculate the relative retention time. The experimental results are shown in the table below.

[0280] Table 19 Precision results of the characteristic chromatogram of the standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) (relative retention time)

[0281]

[0282] 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.

[0283] (4) Stability test

[0284] The test solution of the standard decoction of Schisandra chinensis (from Central China) under the [Characteristic Chromatography] section was injected at 0, 2, 4, 8, 12, 16, 20, and 24 hours, with an injection volume of 1 μl. Four characteristic peaks were tentatively identified, and peak 1 was used as the reference peak to calculate the relative retention time. The results are shown in the table below.

[0285] Table 20 Stability results (relative retention time) of the characteristic chromatograms of the standard decoction of Schisandra chinensis (from Central China).

[0286]

[0287] The experimental results show that the RSD of the relative retention time of the chromatographic peak is in the range of 0.17% to 0.47%, and the RSD is <3.0%, indicating that the sample solution is relatively stable.

[0288] (5) Repeated examination

[0289] Take approximately 0.2 g of the same batch of standard decoction of Schisandra chinensis (Huazhong Schisandra chinensis) (BT240609), accurately weigh it, and prepare six parallel portions according to the test solution preparation method under the [Characteristic Chromatography] section. Inject 1 μl into each portion. Temporarily identify four characteristic peaks, using peak 1 as the reference peak, and calculate the relative retention time.

[0290] Table 21 Repeatability results of characteristic chromatograms of the standard decoction of Schisandra chinensis (from Central China) (relative retention time)

[0291]

[0292] Experimental results show that the relative retention time (RSD) of each chromatographic peak is between 0.22% and 0.66%, and the RSD value is <3.0%, indicating that the method has good repeatability.

[0293] (6) Durability test

[0294] ① Column analysis

[0295] Three chromatographic columns were examined: column 1 ACQUITY The effects of three chromatographic columns on the peak characteristics of the standard decoction of *Schizonepeta tenuifolia* were investigated: HSS T3 (2.1×150mm, 1.8μm); column 2 ZORBAX SB-C18 (2.1×150mm, 1.8μm); column 3 VANQUISH C18 (2.1*150mm, 2.2μm).

[0296] Table 22. Chromatographic column analysis results of the characteristic chromatogram of the decoction of Schisandra chinensis (from Central China) (relative retention time).

[0297]

[0298] Experimental results show that ( Figure 17 Different chromatographic columns have a certain impact on the peak elution. Using a ZORBAX SB-C18 (2.1×150mm, 1.8μm) ultra-high performance liquid chromatography column for elution resulted in better peak shapes and the best separation effect. Therefore, it is recommended to use a ZORBAX SB-C18 (2.1×150mm, 1.8μm) ultra-high performance liquid chromatography column for this method.

[0299] ② Column temperature investigation

[0300] The elution performance at different column temperatures (30℃, 35℃, 40℃) was investigated.

[0301] Table 23. Column temperature study results (relative retention time) of the characteristic chromatogram of the Five-Spice Blood Vine (Schisandra chinensis from Central China) decoction.

[0302]

[0303]

[0304] Experimental results show that ( Figure 18 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 40℃. Therefore, it is recommended to use a column temperature of 40℃ for the determination.

[0305] 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.

[0306] Table 24. Results of flow rate investigation (relative retention time) of the characteristic chromatogram of Schisandra chinensis (Wu Xiang Xue Teng) (Hua Zhong Wu Wei Zi) decoction.

[0307]

[0308] Experimental results show that ( Figure 19Flow rate has some influence on peak formation, but the peak shape is optimal when the flow rate is 0.30 ml / min. Therefore, it is recommended to use 0.30 ml / min as the measurement flow rate.

[0309] The final characteristic chromatogram of the standard decoction of Schisandra chinensis (from Central China) is shown below. Figure 20 The peak corresponding to the reference standard catechin-7-O-β-D-glucopyranoside is peak S. Calculate the relative retention times of peaks 2, 3, and 4 with peak S. The relative retention times should be within ±10% of the specified values, which are 2.14 (peak 2), 2.54 (peak 3), and 3.45 (peak 4).

[0310] Example 5

[0311] Establishment of a characteristic chromatogram analysis of the standard decoction of Schisandra chinensis (Chinese Schisandra chinensis).

[0312] 1. Instruments, reagents and reagents

[0313] 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), ML-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 Haiyang 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), silicone G high-efficiency plate (10cm×10cm, Qingdao Haiyang Chemical Co., Ltd., batch number: 20230315).

[0314] Reagents: Ethyl acetate (batch number: 20231101) and petroleum ether (60-90℃) (batch number: 20231001) were purchased from Chongqing Chuandong Chemical (Group) Co., Ltd.

[0315] Test drugs: Three batches of Schisandra chinensis (Huazhong Schisandra chinensis) formula granules (batch numbers: 240601, 240602, 240603; source: Sinopharm Tongjitang (Guizhou) Pharmaceutical Co., Ltd.); Schisandra chinensis (Huazhong Schisandra chinensis) reference material (batch number: 2024-090601) were purchased from Chengdu Glippe Biotechnology Co., Ltd.

[0316] 2. Preparation of the solution

[0317] 2.1 Preparation of the test solution

[0318] Take 2.0g of this product, grind it into a fine powder, add 10ml of petroleum ether (60-90℃), sonicate for 30 minutes, filter, evaporate to dryness, dissolve the residue in 1ml of petroleum ether (60-90℃) to prepare the test solution.

[0319] 2.2 Preparation of control herbal solution

[0320] Take 2.0g of Schisandra chinensis (Chinese Schisandra chinensis) as a reference herb, add 50ml of water, heat under reflux for 30 minutes, filter, and extract twice with petroleum ether (60-90℃) by shaking, 50ml each time. Combine the petroleum ether (60-90℃) solutions, evaporate to dryness, and dissolve the residue in 1ml of petroleum ether (60-90℃) to obtain the reference herb solution.

[0321] 2.3 Preparation of negative sample solution

[0322] Take 2.0g of the negative sample of the formula granules of Schisandra chinensis (Huazhong Schisandra chinensis), grind it into a fine powder, and process it in the same way as the test sample to prepare the negative sample solution.

[0323] Thin-layer plate: Silicone G thin-layer plate

[0324] Developing solvent: petroleum ether (60–90℃) - ethyl acetate (4:1)

[0325] Sampling method: Spray strip sampling

[0326] Deployment method: Deployment is carried out using a double-groove deployment cylinder.

[0327] Inspection: Spray with 10% phosphomolybdic acid ethanol solution, bake at 105°C until the spots are clearly visible, and inspect under sunlight.

[0328] 4-point sample size investigation

[0329] The test solution of *Schisandra chinensis* (a type of vine) formula granules and the reference herb solution were spotted separately onto the same silica gel G thin-layer plate. Petroleum ether (60–90℃)-ethyl acetate (4:1) was used as the developing solvent. The plates were then removed, air-dried, sprayed with 10% phosphomolybdic acid ethanol solution, and dried at 105℃ until the spots were clearly visible. The results were examined under sunlight. (See attached image). Figure 21 .

[0330] Depend on Figure 21 It can be seen that when the sample volume of the test sample is 10 μl, the key spots at the corresponding positions of the test sample and the control medicinal material are relatively clear and exhibit good spot characteristics; when the sample volume of the control medicinal material is 6 μl, the key spots at the corresponding positions of the test sample and the control medicinal material are relatively clear and exhibit good spot characteristics. Therefore, the sample volume is selected as 10 μl for the test sample solution and 6 μl for the control medicinal material solution.

[0331] 5. Specificity of the formulation granules of Schisandra chinensis (Chinese Schisandra chinensis)

[0332] Take 10 μl each of the test solution and negative sample solution of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, and 6 μl of the control herbal material solution, and spot them onto the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (4:1) as the developing solvent. Remove the plate, air dry, spray with 10% phosphomolybdic acid ethanol solution, and bake at 105℃ until the spots are clearly visible. Examine the plate under sunlight. The experimental results are shown below. Figure 22 .

[0333] 6. Investigation at different temperatures

[0334] 10 μl each of the test solution and negative sample solution of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, and 6 μl of the control herbal material solution were spotted onto the same silica gel G thin-layer plate. Petroleum ether (60–90℃)-ethyl acetate (4:1) was used as the developing solvent, and the plates were developed at different temperatures (5℃, 25℃, 40℃). The plates were then removed, air-dried, sprayed with 10% phosphomolybdic acid ethanol solution, and baked at 105℃ until the spots were clearly visible. After further drying, the spots were examined under sunlight. The experimental results are shown below. Figure 23 .

[0335] Depend on Figure 23 It is evident that, under different temperature conditions, the test sample chromatogram of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules showed spots of the same color at corresponding positions on the chromatograms of the reference medicinal materials. The color development was clear, the separation was good, there was no tailing phenomenon, and there was no background interference. Only with the increase of temperature, the position of the corresponding spots did not change significantly, indicating that temperature had no significant effect on the thin-layer identification of *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, demonstrating that the thin-layer identification method has good temperature robustness.

[0336] 7. Investigation of different humidity levels

[0337] 10 μl each of the test solution and negative sample solution of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, and 6 μl of the control herbal material solution were spotted onto the same silica gel G thin-layer plate. The plates were developed using petroleum ether (60–90℃)-ethyl acetate (4:1) as the developing solvent at different relative humidity levels (33%, 66%, 88%). The plates were then removed, air-dried, sprayed with 10% phosphomolybdic acid ethanol solution, and dried at 105℃ until the spots were clearly visible. The spots were then examined under sunlight. The experimental results are shown below. Figure 24 .

[0338] Depend on Figure 24 It is evident that under different humidity conditions, the test sample chromatogram of the Schisandra chinensis (Huazhong Schisandra chinensis) formula granules showed spots of the same color at corresponding positions on the chromatogram of the reference medicinal material. Moreover, the main spots were clearly visible, with good separation and no tailing phenomenon. This indicates that changes in humidity have no significant impact on the thin-layer identification of the Schisandra chinensis (Huazhong Schisandra chinensis) formula granules, demonstrating that the thin-layer identification method is durable to humidity.

[0339] Investigation of Thin-Layer Boards from 8 Different Manufacturers

[0340] 10 μl each of the test solution and negative sample solution of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, and 6 μl of the control herbal material solution were spotted onto silica gel G thin-layer plates from different manufacturers (marine silica gel G plate, Yantai Yinlong pre-made silica gel G plate, and Merck silica gel G plate). Petroleum ether (60–90℃)-ethyl acetate (4:1) was used as the developing solvent. After development, the plates were removed, air-dried, sprayed with 10% phosphomolybdic acid ethanol solution, and dried at 105℃ until the spots were clearly visible. The results were then examined under sunlight. The experimental results are shown below. Figure 25 .

[0341] Depend on Figure 25 It is evident that when using silica gel thin-layer plates from different manufacturers (Qingdao Ocean Silica G plate, Yantai Yinlong Silica Pre-made Silica G plate, and German Merck plate), the main spots of the test sample chromatogram and the reference medicinal material chromatogram of the Wuxiang Xue Teng (Huazhong Wuweizi) formula granules 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.

[0342] 9. Thin-layer chromatogram for identifying granules of Schisandra chinensis (Chinese Schisandra chinensis)

[0343] Take 10 μl each of the test solution and negative sample solution of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules, and 6 μl of the control herbal material solution, and spot them onto the same silica gel G thin-layer plate. Develop the plate using petroleum ether (60–90℃)-ethyl acetate (4:1) as the developing solvent. Remove the plate, air dry, spray with 10% phosphomolybdic acid ethanol solution, and bake at 105℃ until the spots are clearly visible. Examine the plate under sunlight. The experimental results are shown below. Figure 26 .

[0344] Depend on Figure 26 It is evident that the chromatograms of the test samples in the three batches of Wuxiang Xueteng (Huazhong Wuweizi) formula granules showed fluorescent spots of the same color at the corresponding positions as the chromatograms of the reference medicinal materials, indicating that the thin-layer identification of the three batches of Wuxiang Xueteng (Huazhong Wuweizi) formula granules met the requirements.

[0345] 10 Summary

[0346] From the chromatographic spots, the fluorescent spots of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules showed good separation. Furthermore, the chromatograms of the *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules and the chromatograms of the *Schisandra chinensis* (Huazhong Schisandra chinensis) reference material showed spots of the same color at corresponding positions, and these spots corresponded one-to-one. This method can effectively identify *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules that have lost their processed form. Through thin-layer chromatography methodology evaluation, this method demonstrated good specificity and robustness, making it suitable for the thin-layer chromatographic identification of *Schisandra chinensis* (Huazhong Schisandra chinensis) formula granules.

[0347] 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 preparation method of an extract of five-flavor blood vine and a preparation method of a preparation thereof, characterized in that, Specifically, the steps include the following: (1) Take the slices of Five-spice Blood Vine, add water and decoct twice, filter, and combine the two decoctions after cooling the filtrate to room temperature, which is the Five-spice Blood Vine extract. (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 1.08 g / ml; the extract was obtained. (3) Prepare the extract into the desired dosage form.

2. The preparation method of the extract of *Schizonepeta tenuifolia* and its preparations according to claim 1, characterized in that, The specific step (1) involves taking slices of five-spice vine and decocting them twice with water. For the first decoction, add 8 to 12 times the amount of water and soak for 20 to 40 minutes. After bringing it to a boil over high heat, simmer over low heat for 20 to 40 minutes. Filter the decoction while it is still hot through a 100-300 mesh sieve. Record the quality of the decoction after the filtrate has cooled to room temperature. For the second decoction, add 6 to 10 times the amount of water, heat it to a boil over high heat, and then simmer over low heat for 15 to 25 minutes. Filter the decoction while it is still hot through a 100-300 mesh sieve. Record the quality of the decoction after the filtrate has cooled to room temperature. Combine the two decoctions.

3. The method of claim 1, wherein the five-flavored-merremia is prepared by the steps of: The specific conditions for the reduced pressure and low temperature concentration in step (2) are: temperature: 40~70℃; vacuum degree: -0.080~-0.090MPa. ​ 4. The method of preparing the five-flavored Radix Tylophori and its preparations according to claim 1, characterized in that, Step (3) involves freeze-drying the obtained extract to obtain the standard decoction of *Spatholobus suberectus*. The freeze-drying parameters are as follows: the pre-freezing temperature of the concentrated *Spatholobus suberectus* extract 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 900 to 950 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 -0.252 mbar.

5. The method of preparing the five-flavored Radix Tylophori and its preparations according to claim 1, characterized in that, Step (3) involves adding excipients to the obtained extract, mixing and filtering, drying, collecting the powder, mixing, and granulating to obtain the Five-Spice Blood Vine Granules.

6. The Malus sieboldii leaf extract and the preparation method of the preparation thereof according to claim 5, characterized in that, The Five-Spice Blood Vine Granules are made from 8000-12000g of medicinal slices into 1000g of granules, with each 1g of formula granules equivalent to 8-12g of medicinal slices.

7. A method for determining the content of components of *Schizonepeta tenuifolia* and its preparations, characterized in that, The determination was performed 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, using gradient elution as specified in the table below; the flow rate was 0.30 mL / min; the column temperature was 30℃; and the detection wavelength was 204 nm. 8.The method for determining the content of various components of the five-flavored Radix Tylophori and its preparation according to claim 7, characterized in that, The chromatographic column used was 150 mm long, 2.1 mm inner diameter, and 1.8 μm particle size. 9.The method for determining the content of multiple components in the five-flavor Radix Tylophori and its preparation according to claim 8, 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 60-80% methanol accurately, weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 60-80% methanol, shake it well, filter it, and take the filtrate to obtain the test solution.

10. The method for determining the content of multiple components in *Schizonepeta tenuifolia* and its preparations according to claim 9, characterized in that, The ultrasonic treatment has a power of 450-550W and a frequency of 35-45kHz.

11. The method for determining the content of multiple components in *Schizonepeta tenuifolia* and its preparations according to claim 9, characterized in that, The reference solution is a solution containing 60 μg of catechin-7-O-β-D-glucopyranoside per 1 ml, prepared using 60-80% methanol as a solvent.

12. A method for detecting the fingerprint of an extract of Radix Taxilli and its preparation, characterized in that, Determination was carried out by high performance liquid chromatography with octadecylsilane-bonded silica gel as the filling agent; acetonitrile as mobile phase A and water as mobile phase B, gradient elution was carried out according to the following table; the flow rate was 0.30 ml per minute; the column temperature was 40 ℃; the detection wavelength was 204 nm:

13. The method for detecting the characteristic chromatogram of the extract of the five-flavored Radix Tylophori and its preparation according to claim 12, characterized in that, The final determination of the characteristic spectrum standard of Wuxiang Houteng and its preparation is that four characteristic peaks should be presented in the test sample chromatogram, and correspond to the retention time of the four characteristic peaks in the reference chromatogram of the control medicinal material; peak 1 corresponds to the retention time of the reference characteristic peak of the catechin-7-O-beta-D-glucopyranoside control; the peak corresponding to the catechin-7-O-beta-D-glucopyranoside control is the S peak; calculate the relative retention time of peaks 2, 3, and 4 to the S peak, which should be within ±10% of the specified value, the specified value is 2.14 (peak 2), 2.54 (peak 3), and 3.45 (peak 4).

14. A method for detecting an extract of Radix Taxilli and its preparation by thin layer chromatography, characterized in that, Take the Wuxiang Houteng control medicinal material as the control, use petroleum ether (60-90 ℃)-ethyl acetate (4:1) as the developing agent, develop, take out, air dry, spray with 10% phosphomolybdic acid ethanol solution, and bake at 105 ℃ until the spots are clearly colored, and then observe under daylight.

Images