Characteristic spectrum identification method for Xiaoxiao decoction

Abstract

The invention belongs to the field of traditional Chinese medicine detection, and particularly relates to a method for identifying a characteristic spectrum of Xiaoxiao decoction. The method comprises the following steps: a, preparing a reference substance solution; b, preparing a test solution; and c, respectively sucking the reference solution and the test solution and injecting into a liquid chromatograph. Through specific sample solvent and chromatographic conditions, the obtained characteristic chromatogram can identify common peaks of coptis chinensis component group characteristics, common peaks of rhizoma pinelliae preparata component group characteristics and common peaks of trichosanthes kirilowii maxim component group characteristics in the Xiaoxiaoxiao decoction. The common peaks have strong characteristics and can accurately, comprehensively and stably reflect the chemical quality general picture of the classic famous prescription Xiaoxiaoxiao decoction, and accurate identification and comprehensive quality control of the traditional preparation and the modern preparation of the Xiaoxiaoxiao decoction are realized.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine detection, specifically involving a method for identifying the characteristic chromatograms of Xiao Xianxiong Decoction. Background Technology

[0002] Xiao Xianxiong Decoction is the 12th prescription in the "Catalogue of Ancient Classic Prescriptions (Second Batch) - Han Medicine" published by the State Administration of Traditional Chinese Medicine in 2023. It originates from the "Differentiation of Pulse and Symptoms of Taiyang Disease and Treatment" section of the *Shanghan Lun* (Treatise on Cold Damage) by Zhang Zhongjing, a physician of the Han Dynasty. It has the effects of clearing heat and resolving phlegm, relieving chest tightness and dissipating nodules. Its main ingredients include Coptis chinensis, Pinellia ternata, and Trichosanthes kirilowii. Xiao Xianxiong Decoction has various clinical applications: it is mainly used to treat phlegm-heat accumulation and chest pain, primarily for cardiovascular diseases, respiratory diseases, digestive diseases, and other diseases caused by phlegm-heat accumulation, such as cholecystitis and cholelithiasis.

[0003] Xiao Xianxiong Tang is a classic traditional Chinese medicine formula. However, modern formulations, lacking the original appearance of the medicinal materials, cannot effectively control their quality and efficacy. This makes it difficult to prevent counterfeit and substandard products from appearing in modern formulations, and consequently, to ensure their equivalence with ancient or standard formulations, hindering the modernization and international promotion of traditional Chinese medicine. Characteristic chromatograms of traditional Chinese medicines, by analyzing common peaks, can accurately identify the raw material components in traditional Chinese medicine formulas, providing a reliable basis for authenticity identification and quality control. However, there is currently no research on the characteristic chromatogram of Xiao Xianxiong Tang. Summary of the Invention

[0004] To address the above problems, this invention provides a method for identifying the characteristic spectra of Xiaoxianxiong Decoction, comprising the following steps: a. Preparation of reference solutions: Take the reference standards of Coptis chinensis, Trichosanthes kirilowii and Pinellia ternata respectively, dissolve them in methanol to prepare reference solutions; b. Preparation of the test solution: Take the sample to be tested, extract with methanol solution, filter, and take the filtrate to obtain the test solution; c. Inject the reference solution and the test solution into the liquid chromatograph, respectively; Chromatographic conditions for detecting characteristic components of Coptis chinensis: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, and aqueous solution of formic acid and triethylamine as mobile phase B; Gradient elution program as follows: 0~10min, 0→4%A, 10~15min, 4→9%A, 15~35min, 9→13%A, 35~45min, 13→19%A, 45~60min, 19%A; Chromatographic conditions for detecting characteristic components of Trichosanthes kirilowii and Pinellia ternata: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, formic acid solution as mobile phase B; Gradient elution program as follows: 0~3min, 3%A, 3~25min, 3→5%A, 25~55min, 5→20%A, 55~60min, 20→80%A, 60~61min, 80→3%A, 61~71min, 3%A; The reference standards for the Coptis chinensis component are berberine hydrochloride, palmatine hydrochloride, coptisine hydrochloride, magnoflorine, tetrandrine, cypermethrin hydrochloride, and epiberberine; the reference standards for the Trichosanthes kirilowii and Pinellia ternata components are 5-hydroxymethylfurfural, adenosine, uridine, guanosine, and chlorogenic acid. The sample to be tested was a preparation of Xiao Xianxiong Decoction; The Xiaoxianxiong Decoction is composed of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis in a mass ratio of 70-90:30-40:10-20.

[0005] Furthermore, the preparations of Xiaoxianxiong Decoction include Xiaoxianxiong Decoction decoction, Xiaoxianxiong Decoction granules, Xiaoxianxiong Decoction powder, and Xiaoxianxiong Decoction pills.

[0006] Furthermore, the mass ratio of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis is 80:34.5:13.8.

[0007] Further, the reference solution in step a) contains 25-50 µg of reference standard per 1 ml.

[0008] Further, in step b), the mass-to-volume ratio of the sample to be tested to the methanol solution is 0.2 g: 10~20 mL; the extraction is ultrasonic extraction with a power of 250 W, a frequency of 40 kHz, and a time of 30 min.

[0009] Furthermore, when the mass-to-volume ratio of the sample to the methanol solution is 0.2g:20mL, the characteristic spectrum of Coptis chinensis components is detected; when the mass-to-volume ratio of the sample to the methanol solution is 0.2g:10mL, the characteristic spectra of Trichosanthes kirilowii components and Pinellia ternata components are detected.

[0010] Furthermore, the concentration of the methanol solution in step b) is 30%.

[0011] Further, in step c), the chromatographic conditions for detecting the characteristic chromatograms of Coptis chinensis components are as follows: the chromatographic column is Waters ACQUITY UPLC® CSH™, 2.1 mm × 100 mm, 1.7 μm or Waters ACQUITY UPLC® BEH Shield RP18, 2.1 mm × 100 mm, 1.7 μm; mobile phase B is an aqueous solution containing 0.3% formic acid and 1% triethylamine; wavelength is 270 nm; flow rate is 0.3 ml per minute; column temperature is 30 °C; injection volume is 2 μl; and the theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 5000.

[0012] Further, the characteristic spectrum of Coptis chinensis components described in step c) should show 9 characteristic peaks, of which peak 2 is magnoflorine, peak 4 is palmatine hydrochloride, peak 5 is epiberberine, peak 6 is tetrandrine, peak 7 is cypermethrin hydrochloride, peak 8 is berberine hydrochloride, peak 9 is berberine hydrochloride, and the peak corresponding to the magnoflorine reference is peak S1. The relative retention times of the remaining peaks and peak S1 should be calculated, and their relative retention times should be within ±10% of the specified values, which are: peak 1: 0.53, peak 3: 1.64.

[0013] Furthermore, in step c), the chromatographic conditions for detecting the characteristic chromatograms of Trichosanthes kirilowii and Pinellia ternata components were performed using a YMC-Triart C column. 18 5µm250×4.6mm, Shim-pack Gist C 18 5um, 4.6×250mm or Shim-pack Scepter C 18 -120 5um 250×4.6mm; mobile phase B is 0.3% formic acid solution; flow rate is 1.0ml per minute; column temperature is 25℃; detection wavelength for Pinellia ternata is 260nm and for Trichosanthes kirilowii is 300nm; injection volume is 10μl; theoretical plate number calculated based on the 5-hydroxymethylfurfural peak should not be less than 5000.

[0014] Furthermore, the characteristic spectrum of the components of Pinellia ternata should show 8 characteristic peaks, of which peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 5 is 5-hydroxymethylfurfural, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S2 peak. The relative retention times of the remaining peaks and the S2 peak should be calculated, and their relative retention times should be within ±10% of the specified values, which are: peak 4: 0.93, peak 6: 1.03, peak 7: 1.05, peak 8: 1.16; The characteristic spectrum of Trichosanthes kirilowii should show 9 characteristic peaks, of which peak 3 is 5-hydroxymethylfurfural, peak 8 is chlorogenic acid, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S peak. Calculate the relative retention times of the remaining peaks and the S peak. The relative retention times should be within ±10% of the specified values, which are: peak 1: 0.63, peak 2: 0.82, peak 4: 1.35, peak 5: 1.49, peak 6: 2.31, peak 7: 2.43, peak 9: 3.02.

[0015] This invention provides a characteristic chromatographic identification method for Xiao Xianxiong Decoction. Through specific sample solvents and chromatographic conditions, the obtained characteristic chromatograms can identify common peaks representing the group characteristics of the Coptis chinensis component, as well as the group characteristics of the Pinellia ternata and Trichosanthes kirilowii components in Xiao Xianxiong Decoction. These common peaks are highly characteristic and can accurately, comprehensively, and stably reflect the overall chemical quality of the classic formula Xiao Xianxiong Decoction. This enables accurate identification and comprehensive quality control of traditional and modern preparations of Xiao Xianxiong Decoction, and has significant potential for widespread application.

[0016] The Xiaoxianxiong Decoction described in this invention is a traditional Chinese medicine formula, originating from Zhang Zhongjing's "Treatise on Febrile Diseases" in the Han Dynasty. Its formula consists of one liang of Coptis chinensis, half a sheng of Pinellia ternata (washed), and one large Trichosanthes kirilowii fruit. The three ingredients are boiled in six sheng of water. First, the Trichosanthes kirilowii fruit is boiled to obtain three sheng of decoction. The dregs are removed, and then the other ingredients are added. The decoction is boiled to obtain two sheng of decoction. The dregs are removed, and the decoction is divided into three warm doses.

[0017] The standard decoction of Xiao Xianxiong Tang used in this invention is prepared based on the preparation method of Xiao Xianxiong Tang recorded in the ancient medical book *Shanghan Lun*. The standard of the medicinal substances in Xiao Xianxiong Tang is obtained by using this method. Except for the molding process, the other preparation methods are basically consistent with those recorded in *Shanghan Lun*. The specific preparation method is as follows: According to the formula of Xiao Xianxiong Decoction, weigh out the raw materials: 80.00g of Trichosanthes kirilowii, 34.50g of Pinellia ternata, and 13.80g of Coptis chinensis. Add 1200ml of water and soak for 30 minutes. First, boil the Trichosanthes kirilowii slices until 600ml remains. Remove the dregs. Then add the Coptis chinensis slices and Pinellia ternata slices and continue to boil until 400ml remains. Remove the dregs. This gives the material basis of Xiao Xianxiong Decoction.

[0018] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0019] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0020] Figure 1 Different wavelengths of the standard decoction for Xiao Xian Xiong Tang; Figure 2 Different mobile phases of the standard decoction for Xiao Xian Xiong Tang; Figure 3 The standard decoction of Xiao Xian Xiong Tang (Minor Decoction for Chest Congestion) was used to explore the gradient elution of the Coptis chinensis portion in steps 1-3. Figure 4 Xiao Xian Xiong Tang standard decoction, Huang Lian partial gradient elution exploration 4 Figure 5 Study on different column temperatures of the standard decoction of Xiaoxianxiong Decoction; Figure 6 Study on different flow rates of Xiaoxianxiong Decoction standard decoction; Figure 7 Delayed study of the standard decoction of Xiaoxianxiong Decoction; Figure 8 An investigation into the standard decoction extraction method of Xiaoxianxiong Decoction; Figure 9 Investigation on the extraction solvent of the standard decoction of Xiaoxianxiong Decoction; Figure 10 Investigation on the extraction time of the standard decoction of Xiaoxianxiong Decoction; Figure 11 Investigation on the amount of solvent added to the standard decoction of Xiaoxianxiong Decoction; Figure 12 Chromatographic peak identification of the standard decoction of Xiaoxianxiong Decoction; Figure 13 Specificity assessment; Figure 14 A schematic diagram showing the common components of Coptis chinensis in 15 batches of Xiaoxianxiong Decoction standard decoctions; Figure 15 Comparative chart of Coptis chinensis components in Xiaoxianxiong Decoction (peak 2: magnoflorine; peak 4: palmatine hydrochloride; peak 5: epiberberine; peak 6: tetrandrine; peak 7: cypermethrin hydrochloride; peak 8: berberine hydrochloride; peak 9: coptisine hydrochloride) Figure 16 Different wavelengths of the standard decoction for Xiao Xian Xiong Tang; Figure 17 Different mobile phases of the standard decoction for Xiao Xian Xiong Tang - 260nm Figure 18 Different mobile phases of Xiaoxianxiong Decoction standard decoction -300nm; Figure 19 The standard decoction of Xiao Xian Xiong Tang (Minor Bupleurum Decoction) includes a gradient elution process for Trichosanthes and Pinellia. Figure 20 The standard decoction of Xiao Xian Xiong Tang (Minor Bupleurum Decoction) includes a gradient elution of Trichosanthes and Pinellia ternata. (2) Figure 21 The standard decoction of Xiao Xian Xiong Tang (Minor Bupleurum Decoction) includes a gradient washout of Trichosanthes and Pinellia, explored in step 3. Figure 22 Study on different column temperatures of the standard decoction of Xiaoxianxiong Decoction; Figure 23 Study on different flow rates of Xiaoxianxiong Decoction standard decoction; Figure 24 Delayed study of the standard decoction of Xiaoxianxiong Decoction; Figure 25 Chromatographic peak identification of the standard decoction of Xiaoxianxiong Decoction; Figure 26 The standard decoction specificity of Xiao Xian Xiong Tang; Figure 27 Diagram showing the common components of Pinellia ternata in 15 batches of Xiaoxianxiong Decoction standard decoction. Figure 28 A schematic diagram showing the common components of Trichosanthes kirilowii in 15 batches of Xiaoxianxiong Decoction standard decoctions; Figure 29 The characteristic spectrum of Pinellia ternata in Xiaoxianxiong Decoction is compared with that at a wavelength of 260nm (peak 1 (S1): uridine; peak 2: adenosine; peak 3: guanosine; peak 5 (S2): 5-hydroxymethylfurfural). Figure 30The characteristic spectrum of Trichosanthes kirilowii components in Xiaoxianxiong Decoction is shown at a wavelength of 300 nm (peak 3 (S): 5-hydroxymethylfurfural; peak 8: chlorogenic acid). Detailed Implementation

[0021] The raw materials, equipment, and reagents used in the specific embodiments of this invention are all known products and were obtained by purchasing commercially available products. The preparation method of the Xiaoxianxiong Decoction standard decoction is as follows: weigh 80.00g of Trichosanthes kirilowii (Gualou) slices, 34.50g of Pinellia ternata slices, and 13.80g of Coptis chinensis slices, add 1200ml of water, and soak for 30 minutes; first boil the Trichosanthes kirilowii (Gualou) slices to 600ml, remove the dregs, then add the Coptis chinensis slices and Pinellia ternata slices, soak for 30 minutes, and continue to boil to 400ml, remove the dregs, and the decoction is obtained.

[0022] Preparation method of standard decoction of Trichosanthes kirilowii: Weigh 80.00g of Trichosanthes kirilowii, add 1200ml of water, soak for 30 minutes, boil down to 600ml, remove the dregs, and the decoction is ready.

[0023] Preparation method of standard decoction of Pinellia ternata: Weigh 34.50g of Pinellia ternata slices, add 600ml of hot water, soak for 30 minutes, boil down to 400ml, remove the dregs, and the decoction is ready.

[0024] Preparation method of standard decoction of Coptis chinensis: Weigh 13.80g of Coptis chinensis slices, add 600ml of hot water, soak for 30 minutes, boil down to 400ml, remove the dregs, and the decoction is ready.

[0025] Example 1: Characteristic chromatogram identification of Xiao Xianxiong Decoction I. Characteristic spectrum of Coptis chinensis components in Xiao Xian Xiong Tang 1) Preparation of reference solution ①: Take berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, magnoflorine, tetrandrine, cypermethrin hydrochloride, and epiberberine reference standards, and add methanol to prepare a reference solution containing 50µg berberine hydrochloride and 25µg each of palmatine hydrochloride, berberine hydrochloride, magnoflorine, tetrandrine, cypermethrin hydrochloride, and epiberberine per ml ①; 2) Preparation of test solution ①: Take 0.2g of Xiaoxianxiong Decoction preparation, place it in an Erlenmeyer flask, add 20ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30 minutes, cool, weigh it, make up the lost weight with 30% methanol, shake well, filter, and the product is obtained. 3) Determination of the characteristic spectrum of components in Coptis chinensis Accurately pipette the reference solution and the test solution into the liquid chromatograph and obtain the chromatograms; The chromatographic conditions are as follows: Chromatographic column: Waters ACQUITY UPLC® CSH™ C18, 2.1 mm × 100 mm, 1.7 µm; acetonitrile as mobile phase A, and 0.3% formic acid solution (1 mL triethylamine per 1000 mL) as mobile phase B for gradient elution; column temperature: 30 °C; flow rate: 0.3 mL / min; detection wavelength: 270 nm; injection volume: 2 µL; theoretical plate number calculated based on the berberine hydrochloride peak should be no less than 5000; gradient elution program: 0~10min, 0→4%A, 10~15min, 4→9%A, 15~35min, 9→13%A, 35~45min, 13→19%A, 45~60min, 19%A; 4) Analyze the characteristic spectrum of Coptis chinensis components. The characteristic spectrum of Coptis chinensis components should show 9 characteristic peaks, of which peak 2 is magnoflorine, peak 4 is palmatine hydrochloride, peak 5 is epiberberine, peak 6 is tetrandrine, peak 7 is cypermethrin hydrochloride, peak 8 is berberine hydrochloride, peak 9 is berberine hydrochloride, and the peak corresponding to the magnoflorine reference is peak S1. Calculate the relative retention times of the remaining peaks with respect to peak S1. The relative retention times should be within ±10% of the specified values, which are: peak 1: 0.53, peak 3: 1.64. II. Characteristic spectrum of Trichosanthes and Pinellia components in Xiao Xian Xiong Tang. 5) Preparation of reference solution ②: Prepare a reference solution ② by adding 5-hydroxymethylfurfural, adenosine, uridine, guanosine, and chlorogenic acid to methanol solution, each containing 25 µg of the reference standard per ml. 6) Preparation of test solution ②: Take 0.2g of Xiaoxianxiong Decoction preparation, accurately weigh it, place it in a stoppered conical flask, add 10ml of 30% methanol, seal tightly, sonicate (600w, 40KHZ) for 30 minutes, cool, weigh it again, make up the lost weight with 30% methanol, shake well, filter, and the product is obtained. 6) Determination of the characteristic chromatogram of components of Trichosanthes kirilowii and Pinellia ternata Accurately pipette the reference solution and the test solution into the liquid chromatograph and obtain the chromatograms; The chromatographic conditions are as follows: Chromatographic column: YMC-Triart C18 250×4.6mm, 5µm; gradient elution using acetonitrile as mobile phase A and 0.3% formic acid solution as mobile phase B; column temperature: 25℃; flow rate: 1.0 ml / min; detection wavelength for Pinellia ternata components: 260 nm; detection wavelength for Trichosanthes kirilowii components: 300 nm; injection volume: 10 μl; theoretical plate number calculated based on the 5-hydroxymethylfurfural peak should be no less than 5000; gradient elution program: 0~3min, 3%A, 3~25min, 3→5%A, 25~55min, 5→20%A, 55~60min, 20→80%A, 60~61min, 80→3%A, 61~71min, 3%A.

[0026] 4) Analysis of the component characteristic spectrum of Trichosanthes kirilowii and Pinellia ternata The characteristic spectrum of Pinellia ternata components at a wavelength of 260 nm should show 8 characteristic peaks, of which peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 5 is 5-hydroxymethylfurfural, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S2 peak. Calculate the relative retention times of the remaining peaks with respect to the S2 peak. The relative retention times should be within ±10% of the specified values, which are: peak 4: 0.93, peak 6: 1.03, peak 7: 1.05, peak 8: 1.16. The characteristic spectrum of Trichosanthes kirilowii components at a wavelength of 300 nm should show 9 characteristic peaks, of which peak 3 is 5-hydroxymethylfurfural, peak 8 is chlorogenic acid, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S peak. Calculate the relative retention times of the remaining peaks and the S peak. The relative retention times should be within ±10% of the specified values, which are: peak 1: 0.63, peak 2: 0.82, peak 4: 1.35, peak 5: 1.49, peak 6: 2.31, peak 7: 2.43, peak 9: 3.02.

[0027] The following experimental examples illustrate the beneficial effects of the present invention.

[0028] Experimental Example 1: Characteristic Graph Study for Differentiating Xiao Xianxiong Decoction I. Characteristic Profile of Coptis chinensis Components in Xiao Xian Xiong Tang 1. Main experimental instruments High-performance liquid chromatographs: Agilent ultra-high performance liquid chromatographs, Waters ultra-high performance liquid chromatographs, Thermo Fisher ultra-high performance liquid chromatographs; Column 1: Waters ACQUITY UPLC® CSH™ C18, 2.1mm × 100mm, 1.7µm; Column 2: Shimadzu Waters ACQUITY UPLC® BEH Shield RP18 2.1mm × 100mm, 1.7μm.

[0029] 2. Main experimental materials Berberine hydrochloride (batch number: 110713-202316, purity: 91%), palmatine hydrochloride (batch number: 110732-201913, purity: 85.7%), berberine hydrochloride (batch number: 110733-202110, purity: 90.3%), berberine hydrochloride (batch number: 112026-201802, purity: 94%), and magnoflorine (batch number: 112090-202201, purity: 100%) were all purchased from the National Institutes for Food and Drug Control; epiberberine (batch number: PS021066, purity: 98%) was purchased from... Biopush; African tetrandrine (batch number: PS020964, purity 99.31%) was purchased from Chengdu Pusi Biotechnology Co., Ltd.; Xiaoxianxiong Decoction standard decoction (batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15), Trichosanthes kirilowii (Gualou) standard decoction, Pinellia ternata standard decoction and Coptis chinensis standard decoction were provided by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.

[0030] 3. Investigation of chromatographic conditions 3.1 Determination of detection wavelength Take the standard decoction of Xiao Xian Xiong Tang, extract it with 30% methanol using ultrasound, and use the filtrate as the test solution. Detect the solution as follows. Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the stationary phase (WatersACQUITY UPLC® CSH™ C18 2.1mm × 100mm, 1.7µm); acetonitrile was used as mobile phase A, and 0.2% formic acid solution (with 1ml of triethylamine per 1000ml) was used as mobile phase B; the flow rate was 0.3ml per minute; the column temperature was 30℃; and the gradient elution program was as follows: A full-band scan was performed using diode array detection, and chromatograms of the test solution were extracted at wavelengths of 230 nm, 260 nm, 270 nm, 280 nm, 300 nm, and 330 nm.

[0031] The results are as follows Figure 1 As shown in the figure, it can be seen that the standard decoction of Xiaoxianxiong Decoction has a large amount of chromatographic peak information and a more stable chromatographic baseline when the detection wavelength is 270nm. Therefore, the detection wavelength is determined to be 270nm.

[0032] 3.2 Investigation of the mobile phase Based on the determined wavelength, the mobile phase in the chromatographic conditions was adjusted, specifically using five different mobile phases for gradient elution: acetonitrile (A)-water (B), acetonitrile (A)-0.2% phosphoric acid (B), acetonitrile (A)-0.1% triethylamine (B), acetonitrile (A)-0.2% phosphoric acid + 0.1% triethylamine (B), and acetonitrile (A)-0.2% formic acid + 0.1% triethylamine (B).

[0033] The results are as follows Figure 2 As shown, when acetonitrile-aqueous solution and acetonitrile-0.1% triethylamine solution were used as mobile phases, almost all chromatographic peaks in the chromatogram were eluted within the first 10 minutes of elution, but effective separation between peaks was not achieved. When acetonitrile (A)-0.2% phosphoric acid + 0.1% triethylamine solution (B) was used as the mobile phase, the overall peak shape in the chromatogram was poor; peaks 6-8 also showed differences in shape when acetonitrile-0.2% phosphoric acid was used as the mobile phase. These poorly shaped peaks severely affected the matching degree calculation, thus interfering with the accurate identification of common peaks. In contrast, when acetonitrile (A)-0.2% formic acid + 0.1% triethylamine (B) was used as the mobile phase, the chromatogram baseline was more stable, the number of peaks was greater, and the peak shapes showed a normal distribution. Therefore, acetonitrile (A)-0.2% formic acid + 0.1% triethylamine (B) was chosen as the mobile phase for the determination of the characteristic chromatogram of the Xiaoxianxiongtang reference sample.

[0034] 3.3. Examination of the elution procedure Based on the determination of the detection wavelength and mobile phase, adjust the gradient elution program in the chromatographic conditions, specifically as shown in gradient elution exploration 1~4.

[0035] Chromatograms under different gradient elution programs are shown below. Figures 3-4 .from Figure 3 It is evident that increasing the proportion of acetonitrile in the mobile phase at different rates has varying effects on the resolution of peaks in the chromatogram; increasing the proportion of acetonitrile at only one rate throughout the elution process cannot effectively separate the Coptis chinensis components. In the partial gradient elution experiment with Coptis chinensis, increasing the proportion of acetonitrile at different rates at different time points resulted in better peak separation, but the peak shape remained poor within 0-18 minutes, affecting the identification of common peaks. After extensive experimentation, the following gradient elution program was finally determined to effectively separate the most Coptis chinensis components: 0–10 min, 0 → 4% A; 10–15 min, 4 → 9% A; 15–35 min, 9 → 13% A; 35–45 min, 13 → 19% A; 45–60 min, 19% A.

[0036] 3.4 Column Temperature Investigation Based on the determined mobile phase, elution program, and wavelength, the effects were investigated at column temperatures of 25℃, 30℃, and 35℃. Figure 5 As shown. The results of column temperature investigation showed that at a column temperature of 35℃, the separation between peak 1 and peak 3 of Xiaoxianxiong Decoction was poor; at column temperatures of 25℃ and 30℃, the separation of each chromatographic peak was good. Since the elution rate of each chromatographic peak was faster and the separation of the chromatographic peaks was better at a column temperature of 30℃, the column temperature was determined to be 30℃.

[0037] 3.5 Flow velocity investigation Based on the established mobile phase, elution program, wavelength, and column temperature, the effects were investigated at flow rates of 0.25 mL / min, 0.3 mL / min, and 0.35 mL / min. Figure 6 As shown. The flow rate investigation results showed that at a flow rate of 0.25 ml / min, peak 3 had poor resolution; at a flow rate of 0.35 ml / min, peak 1 and its neighboring peaks had poor resolution; and at a flow rate of 0.3 ml / min, all chromatographic peaks had good resolution. Therefore, the flow rate was determined to be 0.3 ml / min.

[0038] 3.6 Delayed Test Based on the established experimental conditions, the chromatogram acquisition time was extended to 90 minutes. For example... Figure 7 As shown. The delay test results indicate that the Xiaoxianxiong Decoction standard decoction sample showed virtually no chromatographic peaks after 60 minutes, therefore the detection time was set at 60 minutes.

[0039] 3.7 Preliminary determination of chromatographic conditions Based on the chromatographic conditions, the chromatographic conditions and system suitability test for the characteristic chromatogram of Xiaoxianxiong Decoction were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.7 μm); acetonitrile as mobile phase A, and 0.2% formic acid solution (1 ml of triethylamine per 1000 ml) as mobile phase B; flow rate 0.3 ml / min; column temperature 30℃; detection wavelength 270 nm; injection volume 2 µL; gradient elution program as shown below. The theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 5000. Gradient elution program: 0–10 min, 0 → 4% A, 10–15 min, 4 → 9% A, 15–35 min, 9 → 13% A, 35–45 min, 13 → 19% A, 45–60 min, 19% A 4. Sample pretreatment investigation 4.1 Examination of Extraction Methods Accurately weigh approximately 0.2g of the standard decoction of Xiaoxianxiong Decoction (batch number: BT-01), place it in a stoppered conical flask, add 20ml of 30% methanol (the extraction solvent), seal tightly, and treat with sonication (600W, 40kHz) for 30min and reflux for 30min respectively. Shake well, filter, and collect the filtrate for analysis according to the final determined chromatographic conditions. The results are as follows: Figure 8 The results of the extraction method investigation showed that changing the extraction method did not significantly affect the number of sample peaks. Since the ultrasonic method is rapid and simple, ultrasonic extraction was determined to be the extraction method for Xiaoxianxiong Decoction.

[0040] 4.2 Investigation of Extraction Solvents Take 0.2g of the standard decoction of Xiaoxianxiong Decoction (BT-01) and place it in separate conical flasks. Test the sample using 20mL each of methanol, 30% methanol, 50% methanol, 70% methanol, water, and ethanol as the extraction solvent. Seal the flasks tightly, weigh them, and sonicate (600W, 40kHz) for 30 minutes. Cool the flasks, collect the filtrate, and analyze it according to the final determined chromatographic conditions. Figure 9 As shown.

[0041] The results of the extraction solvent investigation showed that as the proportion of organic phase decreased, the response of the target component peak 1 showed a significant improvement trend. Pure methanol / ethanol caused the peak shape of peak 1 to deteriorate or even disappear. Peak 1 had almost no response in 70% methanol, and the peak shape was broad and blunt in 50% methanol. However, when the proportion of aqueous phase increased to more than 70%, the peak height of each chromatographic peak increased and the peak shape tended to be sharper. Among them, 30% methanol and water had the best extraction effect, with good peak shape and moderate peak area. However, considering that the sample extracted with pure water had poor stability, 30% methanol was selected as the final extraction solvent after comprehensive consideration.

[0042] 4.3 Examination of extraction time Take 0.2g of the standard decoction of Xiaoxianxiong Decoction (BT-01) and place it in a conical flask. Add 20mL of 30% methanol to each flask and sonicate (600W, 40kHz) for 30, 45, and 60 minutes respectively. After cooling, collect the filtrate and analyze it according to the final determined chromatographic conditions. Figure 10 As shown in the figure. The extraction time results indicate that the peak area of ​​the chromatographic peak at an extraction time of 30 minutes is comparable to that at extraction times of 45 minutes and 60 minutes, indicating that extraction is sufficient at 30 minutes. Therefore, the extraction time for Xiao Xianxiong Decoction was determined to be 30 minutes.

[0043] 4.4 Investigation of Solvent Addition Amount Take 0.2g of the standard decoction of Xiaoxianxiong Decoction (BT-01) and place it in an Erlenmeyer flask. Add 20mL, 50mL, and 100mL of 30% methanol to the flask respectively for analysis. Sonicate the solution (600W, 40kHz) for 30min, cool, collect the filtrate, and analyze it according to the final determined chromatographic conditions. Figure 11 As shown in the figure. The results indicate that when the extraction solvent volume is 20 mL, the baseline fluctuation is small, and the peak shapes and resolutions of each chromatographic peak are good. Therefore, the solvent volume was determined to be 20 mL.

[0044] 4.5 Preliminary determination of the pretreatment method for Xiaoxianxiong Decoction samples Take about 0.2g of Xiaoxianxiong Decoction, place it in an Erlenmeyer flask, add 20ml of 30% methanol, sonicate (power 600W, frequency 40kHz) for 30min, cool, and collect the filtrate to obtain the product.

[0045] 5. Methodological Examination 5.1 Chromatographic peak identification and specificity assessment Preparation of test solution: Take the standard decoction of Xiaoxianxiong Decoction and prepare the test solution of Xiaoxianxiong Decoction standard decoction according to the pre-determined pretreatment method of Xiaoxianxiong Decoction sample.

[0046] Preparation of reference solution: Take berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, magnoflorine, cypermethrin hydrochloride, and epiberberine, add 30% methanol solution to prepare a reference solution containing 50µg berberine hydrochloride and 25µg each of berberine hydrochloride, palmatine hydrochloride, berberine hydrochloride, magnoflorine, cypermethrin hydrochloride, and epiberberine per ml.

[0047] Preparation of negative control solutions: Prepare negative control solutions for Qingbanxia decoction, Gualou decoction, and Huanglian decoction using the same method as the pretreatment of Xiaoxianxiong decoction samples.

[0048] The characteristic peaks of the standard Xiaoxianxiong Decoction were located using defined chromatographic conditions. For example... Figure 12 As shown, specificity was examined, and the results are as follows. Figure 13 As shown.

[0049] from Figure 12 As can be seen, peak 2 corresponds to magnoflorine, peak 4 corresponds to palmatine hydrochloride, peak 5 corresponds to epiberberine, peak 6 corresponds to tetrandrine, peak 7 corresponds to cypermethrin hydrochloride, peak 8 corresponds to berberine hydrochloride, and peak 9 corresponds to berberine hydrochloride.

[0050] From Exclusivity Figure 13 It can be seen that peaks 1 to 9 belong to the components of Coptis chinensis. The characteristic spectrum obtained by this method can identify the common peaks of the Coptis chinensis component group characteristics in Xiaoxianxiong Decoction.

[0051] 5.2 Precision Test Accurately weigh the test solution of Xiaoxianxiong Decoction standard decoction (batch number: BT-01), and inject it 6 times consecutively under the determined chromatographic conditions, 2 μL each time. Calculate the retention time and peak area of ​​each characteristic peak. As shown in Tables 1 and 2.

[0052] Table 1. Precision Study of Standard Xiao Xian Xiong Tang Decoction - Relative Retention Time Table 2 Precision Study of Standard Xiaoxianxiong Decoction - Relative Peak Area The results show that the instrument has good precision.

[0053] 5.3 Repeatability Test Six portions of the test solution of Xiaoxianxiong Decoction standard decoction (batch number: BT-01) were accurately weighed and tested under the determined chromatographic conditions. The results are shown in Tables 3 and 4.

[0054] Table 3. Repeatability Study of Standard Xiao Xian Xiong Tang Decoction - Relative Retention Time Table 4. Repeatability Study of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area The results show that the method has good repeatability.

[0055] 5.4 Intermediate Precision Examination 5.4.1 Investigation with different instruments Accurately weigh two portions of the Xiaoxianxiong Decoction standard decoction (batch number: BT-01), prepare the test solution according to the determined sample pretreatment method, and determine the solution using an Agilent, Waters, or Thermo Fisher ultra-high performance liquid chromatograph under the determined chromatographic conditions. See Tables 5 and 6 for details.

[0056] Table 5. Instrument durability test of Xiao Xian Xiong Tang standard decoction - relative retention time Table 6. Instrument durability test of Xiaoxianxiong Decoction standard decoction - relative peak area The results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was less than 5.0%.

[0057] 5.4.2 Investigations by different personnel and at different times Two portions of the Xiaoxianxiong Decoction standard decoction (batch number: BT-01) were accurately weighed by different personnel (A and B) at different times (T1 and T2). The test samples were prepared according to the determined sample pretreatment method, and then determined according to the determined chromatographic conditions. As shown in Tables 7 and 8.

[0058] Table 7. Personnel and Time Observation of Standard Decoction for Minor Chest Congestion - Relative Retention Time Table 8. Personnel and Time Observation of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area The results showed that when different people measured the same sample of the Xiaoxianxiong Decoction standard decoction at different times, the relative retention times (RSD%) of the nine characteristic peaks ranged from 0.00% to 1.08%, indicating that the method had good stability based on the relative retention times.

[0059] 5.5 Durability Test 5.5.1 Column robustness test The chromatographic columns 1 and 2 were investigated according to the established detection methods, as shown in Tables 9 and 10.

[0060] Table 9. Standard decoction of Xiao Xian Xiong Tang - Chromatographic column durability study - Relative retention time Table 10. Standard decoction of Xiao Xian Xiong Tang - Chromatographic column durability study - Relative peak area The results showed that when using two different chromatographic columns to test the robustness of the Xiaoxianxiong Decoction standard, the relative retention time (RSD) of characteristic peak 1 was significantly higher, indicating that it was greatly affected by differences in column efficiency, while the other characteristic peaks remained stable. To ensure method reproducibility and data comparability, the same type of chromatographic column should be used for testing.

[0061] 5.5.2 Stability Test The same Xiaoxianxiong Decoction standard decoction test solution was taken according to the established detection method and measured at 0h, 2h, 4h, 6h, 12h, 16h, and 24h, respectively. As shown in Tables 11 and 12.

[0062] Table 11 Stability and Relative Retention Time of Standard Xiao Xian Xiong Tang Decoction Table 12 Stability of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area The results showed that the RSD of the relative retention time of the characteristic peak of the Xiaoxianxiong Decoction standard decoction was 0.00% to 0.70%, and the RSD% of the relative peak area of ​​the characteristic peak was 0.00% to 6.33%, indicating that the Xiaoxianxiong Decoction sample solution had good stability under the determined chromatographic conditions within 24 hours.

[0063] The above methodological validation results show that this method has strong specificity, good precision, and good robustness. The preliminarily determined sample pretreatment method and chromatographic conditions were used to determine the composition of 15 batches of Xiaoxianxiong Decoction standard decoction. Furthermore, a software system for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine was used to automatically match the chromatographic peaks of the HPLC chromatograms of the 15 batches of Xiaoxianxiong Decoction standard decoction, generating a common pattern diagram. (See the common pattern diagram below.) Figure 14 And establish a comparative atlas (see Figure 15 The spectra all show 9 characteristic peaks. Peak 2, corresponding to the magnoflorine reference, is labeled as peak S1, and peak 5, corresponding to the epiberberine reference, is labeled as peak S2. The relative retention times of peaks 1-3 with peak S1 and peaks 4-9 with peak S2 are calculated. The relative retention times should be within ±10% of the specified values. The specified values ​​are: 0.53 (peak 1), 1.64 (peak 3), 0.93 (peak 4), 1.02 (peak 6), 1.04 (peak 7), 1.15 (peak 8), and 1.19 (peak 9). Among these, peak 4 is palmatine hydrochloride, peak 6 is tetrandrine, peak 7 is berberine hydrochloride, peak 8 is berberine hydrochloride, and peak 9 is berberine hydrochloride. The common peaks in the spectra are sharp, symmetrical, well-separated, and all have a similarity of not less than 0.90.

[0064] II. Spectral Study of Common Peaks in the Trichosanthes and Pinellia Root Components of Xiao Xian Xiong Tang 1. Main experimental instruments High-performance liquid chromatographs (HPLC): Agilent 1260 HPLC, Shimadzu LC-20AD HPLC, Waters e2695 2898 HPLC; Column 1: YMC-Triart C18 250×4.6mm 5μm; Column 2: Shim-pack Gist C18 5um 4.6×250mm 5μm; Column 3: Shim-pack Scepter C18-120 5μm 250×4.6mm.

[0065] 2. Main experimental reagents Uric acid (batch number: 110887-202305, content calculated as 99.6%), guanosine (batch number: 111977-202202, content calculated as 88.6%), adenosine (batch number: 110879-202204, content calculated as 99.4%), chlorogenic acid (batch number: 110753-202119, content calculated as 96.3%), and 5-hydroxymethylfurfural (batch number: 111626-202417, content calculated as 99.8%) were all... Purchased from the China National Institutes for Food and Drug Control; the standard decoctions of Xiaoxianxiong Decoction (batch numbers: BT-01, BT-02, BT-03, BT-04, BT-05, BT-06, BT-07, BT-08, BT-09, BT-10, BT-11, BT-12, BT-13, BT-14, BT-15), Trichosanthes kirilowii (Gualou) standard decoction, Pinellia ternata standard decoction, and Coptis chinensis standard decoction were provided by Sichuan Xinlvse Pharmaceutical Technology Development Co., Ltd.

[0066] 3. Investigation of chromatographic conditions 3.1 Determination of detection wavelength The wavelength determination was performed using the same method as in "I. Characteristic chromatographic study of the Coptis chinensis component group in Xiaoxianxiong Decoction", with the difference being the chromatographic conditions: chromatographic column: YMC-Triart C18 250×4.6mm 5μm; mobile phase: acetonitrile (A) ~ 0.3% formic acid (B); wavelengths: 260nm, 280nm, 300nm, 330nm; flow rate: 1.0ml / min; column temperature: 25℃; gradient elution program: The results are as follows Figure 16 As shown in the figure, the standard decoction of Xiao Xian Xiong Tang exhibits high peak information content at detection wavelengths of 260 and 300 nm. Considering both Trichosanthes kirilowii and Pinellia ternata separately, the Pinellia ternata component group shows high peak information content at a detection wavelength of 260 nm, while the Trichosanthes kirilowii component group shows relatively high peak information content at a detection wavelength of 300 nm. Therefore, the detection wavelength is determined to be 300 nm for Trichosanthes kirilowii components in Xiao Xian Xiong Tang and 260 nm for Pinellia ternata components.

[0067] 3.2 Investigation of the mobile phase Based on the wavelength determination above, the mobile phase in the chromatographic conditions was adjusted, specifically using five different mobile phases for gradient elution: acetonitrile-water, acetonitrile-0.3% phosphoric acid, acetonitrile-0.3% formic acid, and methanol-0.3% phosphoric acid.

[0068] The results are as follows Figures 17-18As shown, when methanol-0.3% phosphoric acid and acetonitrile-water were used as mobile phases, the peak separation was poor and the peak information content was low. When acetonitrile-0.3% formic acid and acetonitrile-0.3% phosphoric acid were used as mobile phases, the peak separation was improved. However, when acetonitrile-0.3% formic acid was used as the mobile phase, two peaks within 6-7 minutes in the chromatogram at 260 nm showed better separation and better peak shape than when acetonitrile-0.3% phosphoric acid was used as the mobile phase. In the chromatogram at 300 nm, the peaks within 20-23 minutes and 38-40 minutes showed better separation and better peak shape than when acetonitrile-0.3% phosphoric acid was used as the mobile phase. Therefore, acetonitrile-0.3% formic acid was used as the mobile phase in the experiment.

[0069] 3.3 Examination of gradient elution procedures Based on the determined wavelength and mobile phase, adjust the gradient elution program in the chromatographic conditions, specifically as in gradient elution exploration 1~3.

[0070] Gradient elution experiment 1: 0–25 min, 2% → 10% A; 25–32 min, 10% → 15% A; 32–45 min, 15% → 25% A; 45–60 min, 25% → 54% A; 60–61 min, 54% → 1% A; 61–71 min, 1% A; Gradient elution experiment 2: 0–22 min, 1% → 7% A; 22–35 min, 7% → 15% A; 35–48 min, 15% → 25% A; 48–60 min, 25% → 54% A; 60–61 min, 54% → 1% A; 61–71 min, 1% A; Gradient elution experiment 3: 0-3 min, 3% A; 3-25 min, 3% → 5% A; 25-55 min, 5% → 20% A; 55-60 min, 20% → 80% A; 60-61 min, 80% → 3% A; 61-71 min, 3% A; Chromatograms under different gradient elution programs are shown below. Figures 19-21 .from Figure 19 It is evident that when eluting according to gradient elution experiment 1, the peak separation is incomplete within the 0–15 min period for 260 nm detection; and the peak elution is delayed within the 12–20 min period for 300 nm detection, resulting in an excessively long blank at the front end, poor overall separation efficiency, and unreasonable peak position distribution.

[0071] from Figure 20 It is evident that, following the gradient elution experiment 2, the peak separation at 260 nm wavelength from 12 to 16 min was poor, while the peak clusters at 300 nm wavelength from 16 to 18 min were severely overlapping, and a blank area appeared from 0 to 14 min. Overall, there were problems with unsatisfactory separation and uneven peak distribution.

[0072] from Figure 21 As can be seen, by following the gradient elution method 3, the peaks of Xiaoxianxiong Decoction are well separated and have good peak shapes. Therefore, the gradient elution program can be used to identify the characteristic chromatograms of the Pinellia and Trichosanthes parts of Xiaoxianxiong Decoction.

[0073] 3.4 Column Temperature Investigation Based on the determined mobile phase, elution program, and wavelength, the effects were investigated at column temperatures of 20℃, 25℃, 30℃, and 35℃. Figure 22 As shown. The results of column temperature investigation show that at column temperatures of 20℃, 30℃ and 35℃, peaks 4 and 5 at 300nm wavelength and peak 7 at 260nm wavelength have some overlap, and the separation effect is not good; at a column temperature of 25℃, the separation of each peak of the sample is better at both wavelengths; therefore, the column temperature can be determined to be 25℃ based on the two wavelengths.

[0074] 3.5 Flow velocity investigation Based on the determined mobile phase, wavelength, and flow rate, investigations were conducted at flow rates of 0.8 mL / min, 1.0 mL / min, and 1.2 mL / min. Figure 23 As shown. The flow rate investigation results showed that the peak separation was good at a flow rate of 1.0 ml / min; at a flow rate of 0.8 ml / min, the separation of peaks 6 and 7 at 300 nm and peak 4 at 260 nm was poor; at a flow rate of 1.2 ml / min, the separation of peaks 8 and 9 at 300 nm and peaks 1 and 2 at 260 nm was poor. Although the slightly poor separation would not affect the judgment of common peaks, considering that it would affect the precision and stability of the methodology, the flow rate was finally determined to be 1.0 ml / min.

[0075] 3.6 Delayed Test Based on the established experimental conditions, the chromatogram acquisition time was extended to 90 minutes. For example... Figure 24 As shown. The delay test results indicate that the Xiaoxianxiong Decoction standard decoction sample showed virtually no chromatographic peaks after 60 minutes, therefore the detection time was set at 60 minutes.

[0076] 3.7 Final chromatographic conditions Based on the chromatographic conditions, the chromatographic conditions and system suitability test for the characteristic chromatogram of Xiaoxianxiong Decoction were determined as follows: Octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.6 mm, particle size 5 μm); acetonitrile as mobile phase A, and 0.3% formic acid as mobile phase B; flow rate 1.0 mL / min; column temperature 25℃; detection wavelength of Pinellia ternata 260 nm and Trichosanthes kirilowii 300 nm; gradient elution program as shown below. The theoretical plate number, calculated based on the 5-hydroxymethylfurfural peak, should not be less than 5000.

[0077] 2. Sample pretreatment investigation Using the same method as described in "I. Characteristic Chromatographic Study of Coptis chinensis Components in Xiaoxianxiong Decoction", the extraction method, solvent, time, and amount of solvent added were systematically investigated. Under the finally determined characteristic chromatographic conditions of the Trichosanthes kirilowii-Pinellia ternata component group, only the amount of solvent added showed a slight difference: when the sample was extracted with 30% methanol at a mass-volume ratio of 0.2 g: 10 mL, the relative peak areas of each chromatographic peak were the most ideal, while the other parameters remained unchanged.

[0078] 3. Methodological Examination 3.1 Specificity Examination Preparation of test solution: Take the standard decoction of Xiaoxianxiong Decoction and prepare the test solution of Xiaoxianxiong Decoction standard decoction according to the determined pretreatment method of Xiaoxianxiong Decoction sample.

[0079] Preparation of reference solution: Take appropriate amounts of 5-hydroxymethylfurfural, adenosine, uridine, guanosine, chlorogenic acid, and neochlorogenic acid, and add 30% methanol solution to prepare a reference solution containing 25µg each of 5-hydroxymethylfurfural, chlorogenic acid, neochlorogenic acid, adenosine, uridine, and guanosine per ml.

[0080] Preparation of negative control solutions: Prepare negative control solutions for Qingbanxia decoction, Gualou decoction, and Huanglian decoction using the same method as the pretreatment of Xiaoxianxiong decoction samples.

[0081] The characteristic peaks of the standard Xiaoxianxiong Decoction were located using defined chromatographic conditions. For example... Figure 25 As shown, specificity was examined, and the results are as follows. Figure 26 As shown.

[0082] from Figure 25 It can be seen that at 260 nm, Xiaoxianxiong decoction identified peak 1 as uridine, peak 2 as adenosine, peak 3 as guanosine, and peak 5 as 5-hydroxymethylfurfural; at 300 nm, Xiaoxianxiong decoction identified peak 3 as 5-hydroxymethylfurfural and peak 8 as chlorogenic acid.

[0083] From Exclusivity Figure 26 It can be seen that in the characteristic spectrum of Xiaoxianxiong Decoction at 260nm, peaks 1-2 and 5-8 are common peaks of Pinellia ternata and Trichosanthes kirilowii, peak 3 is a unique peak of Pinellia ternata, and peak 4 is a unique peak of Trichosanthes kirilowii. In the characteristic spectrum of Xiaoxianxiong Decoction at 300nm, peaks 1 and 3 are common peaks of Pinellia ternata and Trichosanthes kirilowii, and peaks 2, 4, 5, 6, 7, and 9 are unique peaks of Trichosanthes kirilowii. The characteristic spectrum obtained by this method can identify the common peaks of the Trichosanthes kirilowii component group characteristics and the common peaks of the Pinellia ternata component group characteristics in Xiaoxianxiong Decoction.

[0084] 3.2 Precision Test Accurately weigh the test solution of Xiaoxianxiong Decoction standard decoction (batch number: BT-01), and inject it 6 times consecutively under the determined sample pretreatment method and chromatographic conditions, 10 μL each time. Calculate the retention time and peak area of ​​each characteristic peak. As shown in Tables 13 and 16.

[0085] Table 13 Precision Study of Standard Xiao Xian Xiong Tang Decoction - Relative Retention Time Table 14 Precision Study of Standard Xiao Xian Xiong Tang Decoction - Relative Peak Area Table 15 Precision Study of Xiao Xian Xiong Tang Standard Decoction - Relative Retention Time (300nm) Table 16 Precision Study of Xiao Xianxiong Decoction Standard Preparation - Relative Peak Area (300nm) The results show that the instrument has good precision.

[0086] 3.3 Repeatability Test Six portions of the test solution of Xiaoxianxiong Decoction standard decoction (batch number: BT-01) were accurately weighed and tested according to the determined sample pretreatment method and chromatographic conditions. The results are shown in Tables 17-20.

[0087] Table 17 Repeatability Study of Xiao Xianxiong Decoction Standard Preparation - Relative Retention Time (260nm) Table 18 Repeatability Study of Xiao Xianxiong Decoction Standard Preparation - Relative Peak Area (260nm) Table 19 Repeatability Study of Xiao Xianxiong Decoction Standard Preparation - Relative Retention Time (300nm) Table 20 Repeatability Study of Xiao Xianxiong Decoction Standard Preparation - Relative Peak Area (300nm) The results show that the method has good repeatability.

[0088] 3.4 Intermediate Precision Examination 3.4.1 Investigation with different instruments Accurately weigh two portions of the Xiaoxianxiong Decoction standard decoction (batch number: BT-01), prepare the test solution according to the determined sample pretreatment method, and determine the chromatographic conditions using an Agilent instrument (instrument 1), a Waters instrument (instrument 2), and a Thermo Fisher instrument (instrument 3). See Tables 21-24 for details.

[0089] Table 21 Instrument durability test of Xiao Xian Xiong Tang standard decoction - relative retention time - 260nm Table 22 Instrument Durability Test of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area Table 23 Instrument Durability Study of Xiao Xian Xiong Tang Standard Decoction - Relative Retention Time Table 24 Instrument Durability Test of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area The results showed that when the test samples were detected using the above three instruments, the RSD of the relative retention time of each characteristic peak was less than 5.0%.

[0090] 3.4.2 Investigations by different personnel and at different times Two portions of the Xiaoxianxiong Decoction standard decoction (batch number: BT-01) were accurately weighed by different personnel (A and B) at different times (T1 and T2). The test samples were prepared according to the determined sample pretreatment method, and then determined according to the determined chromatographic conditions. As shown in Table 25-28.

[0091] Table 25. Personnel and Time Observation of Standard Decoction for Minor Chest Congestion - Relative Retention Time - 260nm Table 26. Personnel and Time Observation of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area - 260nm Table 27. Personnel and Time Observation of Standard Decoction for Minor Chest Congestion - Relative Retention Time - 300 nm Table 28. Personnel and Time-Based Investigation of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area - 300 nm The results showed that when different personnel measured the same sample of the Xiaoxianxiong Decoction standard decoction at different times, the relative retention time (RSD%) of the characteristic peak was between 0.00% and 1.08%, indicating good method stability.

[0092] 3.5 Durability Test 3.5.1 Column robustness test The chromatographic columns 1, 2, and 3 were investigated according to the established detection methods, as shown in Tables 29-32.

[0093] Table 29 Standard decoction of Xiao Xian Xiong Tang - Chromatographic column robustness study - Relative retention time - 260 nm Table 30 Standard decoction of Xiao Xian Xiong Tang - Chromatographic column durability study - Relative peak area - 260 nm Table 31 Standard decoction of Xiao Xian Xiong Tang - Chromatographic column durability study - Relative retention time - 300 nm Table 32 Standard decoction of Xiao Xian Xiong Tang - Chromatographic column robustness study - Relative peak area - 300 nm The results showed that when the samples were detected using the above three chromatographic columns, the RSD values ​​of the relative retention times of each characteristic peak were all <3.0, indicating that each characteristic peak had good robustness with different chromatographic columns.

[0094] 3.5.2 Stability Assessment According to the established detection method, the same Xiaoxianxiong Decoction standard decoction test solution was taken and measured at 0h, 2h, 4h, 6h, 12h, 16h, and 24h, respectively. As shown in Tables 33-36.

[0095] Table 33 Stability of Xiao Xian Xiong Tang Standard Decoction - Relative Retention Time - 260nm Table 34. Standard decoction of Xiao Xian Xiong Tang - Stability - Relative peak area ratio - 260nm Table 35 Stability of Xiao Xian Xiong Tang Standard Decoction - Relative Retention Time - 300 nm Table 36 Stability of Xiao Xian Xiong Tang Standard Decoction - Relative Peak Area - 300 nm The results showed that the RSD of the relative retention time of the characteristic peak of the Xiaoxianxiong Decoction standard decoction was 0.00% to 0.67%, and the RSD% of the relative peak area of ​​the characteristic peak was 0.00% to 8.45%, indicating that the sample solution was relatively stable within 24 hours. The above methodological validation results show that this method has strong specificity, good precision, and good robustness. The preliminarily determined sample pretreatment method and chromatographic conditions were used to determine the composition of 15 batches of Xiaoxianxiong Decoction standard decoction. Furthermore, a software system for evaluating the similarity of chromatographic fingerprints of traditional Chinese medicine was used to automatically match the chromatographic peaks of the HPLC chromatograms of the 15 batches of Xiaoxianxiong Decoction standard decoction, forming a common pattern diagram. Figure 27-28 ), and establish a comparative atlas ( Figures 29-30 ).

[0096] The spectra at a wavelength of 260 nm all exhibit eight characteristic peaks. The peak corresponding to the uridine reference is designated as peak 1 (S1 peak), and the peak corresponding to the 5-hydroxymethylfurfural reference is designated as peak 5 (S2 peak). The relative retention times of characteristic peaks 2-3 with peak S1, and the relative retention times of characteristic peaks 4, 6-8 with peak S2, were calculated. These relative retention times should be within ±10% of the specified values. The specified values ​​are: 1.24 (peak 2), 1.62 (peak 3), 0.93 (peak 4), 1.03 (peak 6), 1.05 (peak 7), and 1.16 (peak 8). Among these characteristic peaks, peak 2 represents adenosine, and peak 3 represents guanosine. All common peaks in the spectra are sharp, symmetrical, well-separated, and have a similarity of no less than 0.90.

[0097] The spectra at 300 nm all exhibit nine characteristic peaks. Peak 3, corresponding to the 5-hydroxymethylfurfural reference, is designated as peak S. The relative retention times of the remaining peaks and peak S are calculated, and these relative retention times should be within ±10% of the specified values, which are: 0.63 (peak 1), 0.82 (peak 2), 1.35 (peak 4), 1.49 (peak 5), 2.31 (peak 6), 2.43 (peak 7), 2.95 (peak 8), and 3.02 (peak 9). Peak 8 is chlorogenic acid. All common peaks in the spectra are sharp, symmetrical, well-separated, and have a similarity of no less than 0.90.

[0098] In summary, by using specific sample solvents and chromatographic conditions, the obtained characteristic chromatograms can identify the common peaks of the Coptis chinensis component, as well as the common peaks of the Pinellia ternata and Trichosanthes kirilowii components in Xiao Xianxiong Decoction. These common peaks are highly characteristic and can accurately, comprehensively, and stably reflect the overall chemical quality of the classic formula Xiao Xianxiong Decoction, enabling accurate identification and comprehensive quality control of traditional and modern preparations of Xiao Xianxiong Decoction.

Claims

1. A method for identifying the characteristic chromatograms of Xiaoxianxiong Decoction, characterized in that: Includes the following steps: a. Preparation of reference solutions: Take the reference standards of Coptis chinensis, Trichosanthes kirilowii and Pinellia ternata respectively, dissolve them in methanol to prepare reference solutions; b. Preparation of the test solution: Take the sample to be tested, extract with methanol solution, filter, and take the filtrate to obtain the test solution; c. Inject the reference solution and the test solution into the liquid chromatograph, respectively; Chromatographic conditions for detecting characteristic components of Coptis chinensis: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, and aqueous solution of formic acid and triethylamine as mobile phase B; Gradient elution program as follows: 0~10min, 0→4%A, 10~15min, 4→9%A, 15~35min, 9→13%A, 35~45min, 13→19%A, 45~60min, 19%A; Chromatographic conditions for detecting characteristic components of Trichosanthes kirilowii and Pinellia ternata: Column: octadecylsilane-bonded silica gel; Mobile phase: acetonitrile as mobile phase A, formic acid solution as mobile phase B; Gradient elution program as follows: 0~3min, 3%A, 3~25min, 3→5%A, 25~55min, 5→20%A, 55~60min, 20→80%A, 60~61min, 80→3%A, 61~71min, 3%A; The reference standards for the Coptis chinensis component are berberine hydrochloride, palmatine hydrochloride, coptisine hydrochloride, magnoflorine, tetrandrine, cypermethrin hydrochloride, and epiberberine; the reference standards for the Trichosanthes kirilowii and Pinellia ternata components are 5-hydroxymethylfurfural, adenosine, uridine, guanosine, and chlorogenic acid. The sample to be tested was a preparation of Xiao Xianxiong Decoction; The Xiaoxianxiong Decoction is composed of Trichosanthes kirilowii, Pinellia ternata, and Coptis chinensis in a mass ratio of 70-90:30-40:10-20.

2. The identification method according to claim 1, characterized in that: The preparations of Xiaoxianxiong Decoction include Xiaoxianxiong Decoction decoction, Xiaoxianxiong Decoction granules, Xiaoxianxiong Decoction powder, and Xiaoxianxiong Decoction pills.

3. The identification method according to claim 1, characterized in that: Step a) The reference solution contains 25-50 µg of reference standard per 1 ml.

4. The identification method according to claim 1, characterized in that: In step b), the mass-to-volume ratio of the sample to the methanol solution is 0.2 g: 10~20 mL; the extraction is ultrasonic extraction with a power of 250 ohms, a frequency of 40 kHz, and a time of 30 min.

5. The identification method according to claim 1 or 4, characterized in that: The mass-to-volume ratio of the sample to the methanol solution was 0.2 g: 20 mL, and the characteristic chromatogram of Coptis chinensis components was detected; the mass-to-volume ratio of the sample to the methanol solution was 0.2 g: 10 mL, and the characteristic chromatograms of Trichosanthes kirilowii components and Pinellia ternata components were detected.

6. The identification method according to claim 1 or 4-5, characterized in that: The concentration of the methanol solution in step b) is 30%.

7. The identification method according to claim 1, characterized in that: Step c) Chromatographic conditions for detecting the characteristic chromatogram of Coptis chinensis components: The chromatographic column is a Waters ACQUITY UPLC® CSH™, 2.1 mm × 100 mm, 1.7 μm or a Waters ACQUITY UPLC® BEH Shield RP18, 2.1 mm × 100 mm, 1.7 μm; mobile phase B is an aqueous solution containing 0.3% formic acid and 1% triethylamine; wavelength is 270 nm; flow rate is 0.3 ml per minute; column temperature is 30 °C; injection volume is 2 μl; the theoretical plate number calculated based on the berberine hydrochloride peak should not be less than 5000.

8. The identification method according to claim 1, characterized in that: The characteristic spectrum of Coptis chinensis components should show 9 characteristic peaks, of which peak 2 is magnoflorine, peak 4 is palmatine hydrochloride, peak 5 is epiberberine, peak 6 is tetrandrine, peak 7 is cypermethrin hydrochloride, peak 8 is berberine hydrochloride, peak 9 is berberine hydrochloride, and the peak corresponding to the magnoflorine reference is peak S1. The relative retention times of the remaining peaks and peak S1 should be calculated, and their relative retention times should be within ±10% of the specified values, which are: peak 1: 0.53, peak 3: 1.

64.

9. The identification method according to claim 1, characterized in that: Step c) The chromatographic conditions for detecting the characteristic chromatograms of Trichosanthes kirilowii and Pinellia ternata components were performed using a YMC-Triart C column. 18 5µm250×4.6mm, Shim-pack Gist C 18 5um, 4.6×250mm or Shim-pack Scepter C 18 -120 5um 250×4.6mm; mobile phase B is 0.3% formic acid solution; flow rate is 1.0ml per minute; column temperature is 25℃; detection wavelength for Pinellia ternata is 260nm, and detection wavelength for Trichosanthes kirilowii is 300nm; injection volume is 10μl; theoretical plate number calculated based on the 5-hydroxymethylfurfural peak should not be less than 5000.

10. The detection method according to claim 1, characterized in that: The characteristic spectrum of the components of the prepared Pinellia ternata should show 8 characteristic peaks, of which peak 1 is uridine, peak 2 is adenosine, peak 3 is guanosine, peak 5 is 5-hydroxymethylfurfural, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S2 peak. The relative retention times of the remaining peaks with respect to the S2 peak should be calculated, and their relative retention times should be within ±10% of the specified values. The specified values ​​are: peak 4: 0.93, peak 6: 1.03, peak 7: 1.05, Peak 8: 1.

16. The characteristic spectrum of Trichosanthes kirilowii should show 9 characteristic peaks, of which peak 3 is 5-hydroxymethylfurfural, peak 8 is chlorogenic acid, and the peak corresponding to the 5-hydroxymethylfurfural reference is the S peak. Calculate the relative retention times of the remaining peaks with respect to the S peak. The relative retention times should be within ±10% of the specified values, which are: peak 1: 0.63, peak 2: 0.82, peak 4: 1.35, Peak 5: 1.49, Peak 6: 2.31, Peak 7: 2.43, Peak 9: 3.02.

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