Establishment of HPLC fingerprint of Qingfuzhiyang granules and identification of its components

Abstract

The application discloses a method for establishing HPLC fingerprint spectrum of Qingfu Zhiyang granules, which comprises the following steps: detecting by high performance liquid chromatography (HPLC), determining the attribution of each peak in the test sample solution according to the retention time of each substance in the mixed control sample solution, and generating the fingerprint spectrum of the test sample solution, i.e. obtaining the fingerprint spectrum of the Qingfu Zhiyang granules; the application establishes a method for identifying salvia miltiorrhiza, smilax glabra, radix scrophulariae, honeysuckle, gardenia and bauhinia by thin layer chromatography and determining the content of five effective components, and establishes the characteristic fingerprint spectrum of the Qingfu Zhiyang granules by HPLC; the experimental method is simple, feasible, specific, accurate, reliable, sensitive, reproducible, and can be used for the quality control of the Qingfu Zhiyang granules, so as to provide a quality standard detection method for the standardized use of the Qingfu Zhiyang granules in clinic.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a method for establishing an HPLC fingerprint of a skin-clearing and antipruritic granule and a method for identifying its components. Background Technology

[0002] Based on the TCM theory of "blood deficiency and wind dryness" and Xia's unique medication experience, Li Fulun et al. summarized and created a dermatological external application formula called "Qingfu Zhiyang Formula" with the principles of clearing heat, cooling blood, detoxifying, and relieving itching. It has been used clinically for many years with definite efficacy. This formula has been developed into a preparation called Qingfu Zhiyang Granules (Chinese Journal of Dermatology and Venereology, 2025, 39(9): 958-68.).

[0003] Patent application CN117244008A discloses the application of a traditional Chinese medicine composition in the preparation of a drug for treating eczema caused by damp-heat accumulation. It is made from the following raw materials in the indicated weight ratios: 20-40 parts of Salvia miltiorrhiza, 20-40 parts of Rehmannia glutinosa, 20-40 parts of Smilax glabra, 20-40 parts of Lonicera japonica, 5-25 parts of Scutellaria baicalensis, 20-40 parts of Rubia cordifolia, 5-25 parts of Rheum palmatum, 20-40 parts of Lonicera japonica, 2-18 parts of Gardenia jasminoides, and 5-25 parts of Dictamnus dasycarpus. Its advantages include: clearing heat and cooling blood, detoxifying and relieving itching; regulating the body's yin-yang balance holistically; simultaneously treating the disease and strengthening the body's resistance to pathogens; addressing both the root cause and symptoms; better improving symptoms such as erythema and itching of eczema; reducing adverse reactions; and clinically proven significant efficacy and safety. It is universally applicable to erythematous and exudative skin diseases, allowing for rapid diagnosis and identification of skin lesions by both doctors and patients, and is easy to promote and apply clinically.

[0004] The formula contains Rehmannia glutinosa, which is sweet and cold in nature, clearing heat from the Yin channels and nourishing Yin to moisturize the skin; Smilax glabra, which expels damp-heat toxins from the meridians and promotes the expulsion of pathogens; and Salvia miltiorrhiza, which cools and disperses blood stasis and also nourishes blood, effectively removing damp-heat stagnation. These three herbs are the principal herbs. Scutellaria baicalensis, Rubia cordifolia, and Lonicera japonica help clear damp-heat from the skin and cool and disperse blood stasis without causing bleeding, and are thus the assistant herbs. Lonicera japonica, Gardenia jasminoides, and Rheum palmatum are added to clear heat and promote diuresis, eliminating residual heat in the blood; and Dictamnus dasycarpus root bark promotes blood circulation, dispels wind, and relieves itching, serving as the guiding herb. The *Orthodox Manual of Surgery* states that damp sores "arise from the interaction of wind-heat, damp-heat, and blood-heat, causing unbearable itching." The combined effects of these herbs in this preparation, with their bitter and cold nature, directly counteract the heat, while their drying properties help clear heat and eliminate dampness, treating both simultaneously and achieving the combined effects of "clearing heat and detoxifying, cooling and dispersing blood stasis, and relieving dampness and itching."

[0005] Qingfu Zhiyang Granules are mainly used clinically for patients with acute eczema caused by damp-heat accumulation in the skin, characterized by red and burning skin lesions, often presenting as papules, papulovesicles, and vesicles, intense itching, and significant erosion and exudation after scratching. The formulation is refined and well-matched, adjusting the body's yin-yang balance holistically, and complemented by traditional therapies. It treats the disease while simultaneously strengthening the body's resistance and eliminating pathogenic factors, addressing both the root cause and symptoms, effectively shortening the acute course of the disease. While improving eczema symptoms, it reduces adverse drug reactions and has good long-term safety. In terms of manufacturing process, while maintaining traditional Chinese medicine preparation techniques, the original decoction for soaking has been changed to granules for soaking.

[0006] Qingfu Zhiyang Granules are formulated with ten traditional Chinese medicines, resulting in a complex chemical composition. This composition includes the characteristic active ingredients of each individual herb, as well as interactions and transformations among multiple components. Current quality standards focus only on the qualitative identification of a few components, lacking both multi-index content determination and fingerprinting, making it difficult to evaluate batch consistency and stability. Therefore, there is an urgent need to establish a quality control system that combines multi-index quantitative analysis and characteristic fingerprinting to provide crucial technical support for uniform formulation quality and controllable efficacy. Summary of the Invention

[0007] The purpose of this invention is to provide a method for establishing the HPLC fingerprint of antipruritic granules.

[0008] Another object of the present invention is to provide a method for identifying the components in the said antipruritic granules.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] In a first aspect, the present invention provides a method for establishing an HPLC fingerprint of a skin-clearing and antipruritic granule, comprising the following steps:

[0011] Mixed reference solution: Dissolve 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B in methanol to prepare mixed reference solutions with concentrations of 0.1~1 mg / mL, 0.1~0.8 mg / mL, 0.05~0.5 mg / mL, 0.1~3 mg / mL, and 0.001~0.1 mg / mL, respectively.

[0012] Test solution: Dissolve the Qingfu Zhiyang granules in water to obtain a solution with a concentration of 0.05~0.8 g / mL (preferably 0.12 g / mL), add an equal volume of water-saturated n-butanol solution to extract 1~3 times (preferably 2 times), let stand (10~50 min, preferably 30 min), combine the extracts and concentrate to dryness, add methanol to make up to volume, and use as the test solution;

[0013] High performance liquid chromatography was used to detect the peaks in the test solution based on the retention time of each substance in the mixed reference solution. The detection results of the test solution were then used to generate a fingerprint spectrum, which is the fingerprint spectrum of Qingfu Zhiyang Granules.

[0014] High performance liquid chromatography (HPLC) conditions include:

[0015] Chromatographic column: C18 column; mobile phase: phase A is 0.05%~0.15% (v / v) aqueous phosphoric acid solution, phase B is acetonitrile; gradient elution is used with 60%~90% (v / v) phase A and 10%~40% (v / v) phase B; flow rate: 0.8~1.2 mL / min; column temperature: 20~40℃; injection volume: 8~12 µl; detection wavelength: 200~400 nm.

[0016] The preparation method of the skin-clearing and antipruritic granules includes the following steps:

[0017] Weigh the following components according to the following weight proportions: 20-40 parts of Salvia miltiorrhiza, 20-40 parts of Rehmannia glutinosa, 20-40 parts of Smilax glabra, 20-40 parts of Lonicera japonica, 5-25 parts of Scutellaria baicalensis, 20-40 parts of Rubia cordifolia, 5-25 parts of Rheum palmatum, 20-40 parts of Lonicera japonica, 2-18 parts of Gardenia jasminoides, and 5-25 parts of Dictamnus dasycarpus. Crush the Gardenia jasminoides and add 6-10 times (preferably 8 times) of water to the remaining nine herbs. Decoction is carried out once or three times (preferably twice), each time for 0.5-2 hours (preferably 1 hour). Filter the decoction and concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.07-1.12 (60℃). Spray dry the extract to make an extract powder. Add dextrin and mix well. Make granules, granulate, and package to obtain Qingfu Zhiyang Granules.

[0018] The preparation method of the skin-clearing and antipruritic granules includes the following steps:

[0019] Weigh each component according to the following weight proportions: 37.5 parts of Salvia miltiorrhiza, 37.5 parts of Rehmannia glutinosa, 37.5 parts of Smilax glabra, 37.5 parts of Lonicera japonica, 18.75 parts of Scutellaria baicalensis, 37.5 parts of Rubia cordifolia, 18.75 parts of Rheum palmatum, 37.5 parts of Lonicera japonica, 12.5 parts of Gardenia jasminoides, and 18.75 parts of Dictamnus dasycarpus.

[0020] The high-performance liquid chromatography conditions include:

[0021] Chromatographic column: ZORBAX Eclipse SB-C18 column, 250 mm × 4.6 mm, 5 μm;

[0022] Mobile phase A was a 0.1% (v / v) aqueous solution of phosphoric acid, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–20 min, 77% A–90% A; 20–30 min, 77% A–75% A; 30–45 min, 60% A–75% A; 45–50 min, 60% A; 50–55 min, 60% A–90% A; 55–60 min, 90% A.

[0023] The detection wavelength was 280 nm, the flow rate was 1 mL / min, the column temperature was 30 °C, and the injection volume was 10 µl.

[0024] The concentration of the 3,5-O-dicaffeoylquinic acid dissolved in methanol is 0.40 mg / mL.

[0025] The concentration of astilbin dissolved in methanol is 0.24 mg / m³.

[0026] The concentration of the 4,5-O-dicaffeoylquinic acid dissolved in methanol is 0.16 mg / mL.

[0027] The concentration of baicalin dissolved in methanol is 1.32 mg / mL.

[0028] The concentration of salvianolic acid B dissolved in methanol is 0.016 mg / mL.

[0029] The fingerprint spectrum of the skin-clearing and antipruritic granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 18 common peaks were identified.

[0030] Of the 18 common peaks, peak 7 with a retention time of 19.53 min is 3,5-O-dicaffeoylquinic acid, peak 11 with a retention time of 22.22 min is astilbin, peak 12 with a retention time of 24.16 min is 4,5-O-dicaffeoylquinic acid, peak 13 with a retention time of 28.69 min is baicalin, peak 14 with a retention time of 29.97 min is salvianolic acid B, and the remaining common peaks were not identified.

[0031] The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

[0032] A second aspect of the present invention provides a method for establishing a comparative fingerprint spectrum of a skin-clearing and antipruritic granule, comprising the following steps:

[0033] Take n batches of Qingfu Zhiyang Granules and obtain the fingerprint spectrum of each batch of Qingfu Zhiyang Granules according to the method described above; analyze the fingerprint spectrum of the n batches of Qingfu Zhiyang Granules using the software of the Chinese Medicine Chromatography Fingerprint Similarity Evaluation System to obtain the control fingerprint spectrum of Qingfu Zhiyang Granules; wherein, n≥10.

[0034] In a third aspect, the present invention provides a comparative fingerprint chromatogram of the skin-clearing and antipruritic granules obtained by the method described above. The comparative fingerprint chromatogram of the skin-clearing and antipruritic granules is characterized by selecting the chromatographic peaks of the main components with good separation as characteristic peaks, and a total of 18 common peaks are identified.

[0035] Of the 18 common peaks, peak 7 with a retention time of 19.53 min is 3,5-O-dicaffeoylquinic acid, peak 11 with a retention time of 22.22 min is astilbin, peak 12 with a retention time of 24.16 min is 4,5-O-dicaffeoylquinic acid, peak 13 with a retention time of 28.69 min is baicalin, peak 14 with a retention time of 29.97 min is salvianolic acid B, and the remaining common peaks were not identified.

[0036] The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

[0037] A fourth aspect of the present invention provides a quality control method for antipruritic granules, comprising the following steps: taking antipruritic granules to be tested, obtaining a chromatogram of the antipruritic granules to be tested according to the method, and evaluating the similarity between the obtained chromatogram of the antipruritic granules to be tested and the reference fingerprint chromatogram of the antipruritic granules. If the similarity is ≥0.90 (preferably >0.99), the antipruritic granules to be tested are deemed to be of qualified quality.

[0038] A fifth aspect of the present invention provides a method for determining the content of active ingredients in a skin-clearing and antipruritic granule, comprising the following steps:

[0039] The content of active ingredients in Qingfu Zhiyang Granules was determined by high performance liquid chromatography. The active ingredients were selected from 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and tanshinone B.

[0040] The first step is to establish a standard curve for chemical composition:

[0041] 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B were dissolved in methanol to prepare mixed reference solutions with concentrations of 0.1–1 mg / mL, 0.1–0.8 mg / mL, 0.05–0.5 mg / mL, 0.1–3 mg / mL, and 0.001–0.1 mg / mL, respectively. High-performance liquid chromatography (HPLC) was used for analysis. The chromatographic peaks of each active ingredient were determined based on their retention times, and the peak areas of the corresponding active ingredients were obtained. Standard curves for each active ingredient were established, and the linear regression equations and correlation coefficients r for each active ingredient were obtained.

[0042] High performance liquid chromatography (HPLC) conditions include:

[0043] Chromatographic column: C18 column; mobile phase: phase A is 0.05%~0.15% (v / v) aqueous phosphoric acid solution, phase B is acetonitrile; gradient elution is used with 60%~90% (v / v) phase A and 10%~40% (v / v) phase B; flow rate: 0.8~1.2 mL / min; column temperature: 20~40℃; injection volume: 8~12 µl; detection wavelength: 200~400 nm;

[0044] The second step is to obtain the chromatographic peak area of ​​the active ingredient in the antipruritic granules to be tested;

[0045] Dissolve the antipruritic granules in water to obtain a solution with a concentration of 0.05~0.8 g / mL (preferably 0.12 g / mL), add an equal volume of water-saturated n-butanol solution to extract 1~3 times (preferably 2 times), let stand (10~50 min, preferably 30 min), combine the extracts and concentrate to dryness, add methanol to make up to volume, and use as the test solution;

[0046] The test solution was detected under the same high-performance liquid chromatography conditions as in the first step. The chromatographic peaks of each active ingredient were determined based on their retention times, and the peak areas of the corresponding active ingredients were obtained.

[0047] The third step is to determine the content of the active ingredients in the anti-itch granules to be tested;

[0048] Based on the established standard curves of each active ingredient, the chromatographic peak areas of the active ingredients in the Qingfu Zhiyang granules to be tested were substituted into the corresponding linear regression equations. The concentrations of the corresponding ingredients in the test solution were calculated using the external standard method, and the content of each active ingredient in the Qingfu Zhiyang granules to be tested was calculated.

[0049] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0050] This invention establishes a thin-layer chromatography (TLC) identification method for six medicinal materials (Salvia miltiorrhiza, Smilax glabra, Scutellaria baicalensis, Lonicera japonica, Gardenia jasminoides, and Dictamnus dasycarpus) in Qingfu Zhiyang Granules, as well as a method for determining the content of five active ingredients (3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B). Furthermore, a characteristic fingerprint chromatogram of Qingfu Zhiyang Granules is established using HPLC. The experimental method is simple, feasible, specific, accurate, reliable, sensitive, and reproducible, and can be used for the quality control of Qingfu Zhiyang Granules, thus providing a quality standard testing method for the standardized clinical use of Qingfu Zhiyang Granules.

[0051] This invention utilizes the Chinese herbal chromatographic fingerprint similarity evaluation system (2012 version) to compare and analyze 15 batches of Qingfu Zhiyang granules. The results showed that TLC identification and separation were good, with strong specificity and no interference from negative samples. 3,5-O-dicaffeoylquinic acid showed good linearity in the range of 10–120 µg (r=0.9997), with a mean recovery rate of 98.54% and an RSD of 0.93%. Astilbene showed good linearity in the range of 6–72 µg (r=0.9975), with a mean recovery rate of 100.00% and an RSD of 0.57%. 4,5-O-dicaffeoylquinic acid showed good linearity in the range of 4–48 µg (r=0.9994), with a mean recovery rate of 97.86% and an RSD of 0.62%. Baicalin showed good linearity in the range of 33–396 µg (r=0.9995), with a mean recovery rate of 97.31% and an RSD of 1.30%. Tanshinone B showed good linearity in the range of 4–48 µg. The linear relationship was good within the range of r=0.9994, with a mean recovery rate of 97.54% and an RSD of 1.26%. This invention established the fingerprint spectrum of Qingfu Zhiyang Granules and the control fingerprint spectrum, identifying a total of 18 common peaks, and using a mixed reference solution to identify 5 of these common peaks. Attached Figure Description

[0052] Figure 1 This is a schematic diagram of the thin-layer chromatography of Dictamnus dasycarpus root bark.

[0053] Figure 2 This is a schematic diagram of thin-layer chromatography of Salvia miltiorrhiza.

[0054] Figure 3 This is a schematic diagram of the thin-layer chromatography of Scutellaria baicalensis.

[0055] Figure 4 This is a schematic diagram of the thin-layer chromatography of honeysuckle.

[0056] Figure 5 This is a schematic diagram of the thin-layer chromatography of Smilax glabra.

[0057] Figure 6 This is a schematic diagram of the thin-layer chromatography of gardenia.

[0058] Figure 7 This is a schematic diagram of the results of the chromatographic column screening.

[0059] Figure 8 This is a schematic diagram of the mobile phase screening results.

[0060] Figure 9 This is a schematic diagram of the wavelength selection results.

[0061] Figure 10 This is a schematic diagram of the flow rate screening results.

[0062] Figure 11 This is a schematic diagram of the HPLC results for Qingfu Zhiyang Granules.

[0063] Figure 12 This is a schematic diagram of the control fingerprint spectrum results for Qingfu Zhiyang Granules.

[0064] Figure 13 This is a schematic diagram of the fingerprint spectrum results for Qingfu Zhiyang Granules. Detailed Implementation

[0065] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0066] Example 1

[0067] I. Instruments and Reagents

[0068] Instruments: Agilent 1260 high performance liquid chromatograph (Agilent Technologies, USA); BSA822-CW electronic balance (Beijing Sartorius Instrument Systems Co., Ltd.); CQ-250A-DST ultrasonic cleaner (Shanghai Yuejin Medical Instrument Co., Ltd.); SHZ-D(III) circulating water vacuum pump (Zhengzhou Boke Equipment Co., Ltd.); RV-10 rotary evaporator (IKA GmbH, Germany); BD-II multifunctional ultraviolet analyzer (Beijing Qihang Boda Technology Co., Ltd.); HWS28 electric thermostatic water bath (Shanghai Yiheng Scientific Experiment Co., Ltd.).

[0069] Test drugs: Qingfu Zhiyang Granules (Shanghai Caitongdetang Traditional Chinese Medicine Pharmaceutical Factory Co., Ltd., batch numbers 20250203, 20250204, 20250205); 3,5-O-dicaffeoylquinic acid reference standard (product number STC0680120, content ≥98.0%) and chlorogenic acid reference standard (product number ST02150120, content ≥98.0%) were purchased from Shanghai Shidande Standard Technical Service Co., Ltd.; astilbin reference standard (111798-202306), 4,5-O-dicaffeoylquinic acid reference standard (Batch number 111894-202406), baicalin reference standard (110715-202223), salvianolic acid B reference standard (111562-202318), berberine reference standard (111923-202506); morrhubarb reference standard (111700-202405), all purchased from the China National Institutes for Food and Drug Control, silica gel G thin-layer plates (Yantai Huayang New Material Technology Co., Ltd.); silica gel H plates (Yantai Huayang New Material Technology Co., Ltd.), acetonitrile was chromatographic grade, all other reagents were analytical grade, and water was ultrapure water.

[0070] Preparation of Qingfu Zhiyang Granules: 375 g of Danshen, 375 g of Shengdihuang, 375 g of Tufuling, 375 g of Rendongteng, 187.5 g of Huangqin, 375 g of Qiancao, 187.5 g of Shengdahuang, 375 g of Jinyinhua, 125 g of Zhizi, and 187.5 g of Baixianpi. Crush the Zhizi and add 8 times the amount of water to the other nine herbs. Decoction twice, 1 hour each time. Filter and concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.07~1.12 (60℃). Spray dry to make extract powder. Add dextrin and mix well to make 1000g granules. Granulate, package, and obtain Qingfu Zhiyang Granules.

[0071] II. Methods and Results

[0072] 2.1 Thin-layer chromatography identification

[0073] Identification of Dictamnus dasycarpus root bark: Dissolve 3 g of Qingfu Zhiyang granules in 25 mL of water, extract twice with an equal volume of water-saturated n-butanol solution, let stand for 30 min, combine the extracts, concentrate to dryness under reduced pressure, and dilute to 5 mL with methanol as the test solution; prepare a negative sample without Dictamnus dasycarpus root bark according to the Qingfu Zhiyang granules and process, and prepare a negative control solution according to the test solution preparation method; separately prepare a mixed solution of berberine reference standard with methanol containing 1 mg of each in 1 mL as the reference solution. Perform thin-layer chromatography (General Rule 0502), apply 3 μL of each of the above solutions to the same silica gel G thin-layer plate, develop with petroleum ether (60~90℃)-ethyl formate-acetone (6∶1∶3) as the developing solvent, remove, air dry, spray with 5% vanillin sulfuric acid solution, and heat at 105℃ until the spots are clearly visible. Results are shown in the figure. Figure 1 As shown, Figure 1 This is a schematic diagram of thin-layer chromatography for Dictamnus dasycarpus root bark. Numbers 1-3 are test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is a berberine reference solution; and 5 is a negative control solution. As can be seen from the figure, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram, and the negative control shows no interference. The presence of the target component and the absence of interference from the negative control eliminate the possibility of false positives from excipients or matrices, thus verifying the reliability of the method.

[0074] Preparation of water-saturated n-butanol solution: At room temperature, add n-butanol and distilled water in a separatory funnel at a volume ratio of 1:1, shake thoroughly for 3-5 minutes to saturate the two phases, let stand until complete separation (if necessary, let stand overnight or use sonication to accelerate separation), separate the upper organic phase to obtain water-saturated n-butanol solution.

[0075] Identification of Danshen: The preparation method of the test solution is the same as before; according to the prescription and process of Qingfu Zhiyang Granules, a negative sample without Danshen was prepared, and a negative control solution was prepared according to the preparation method of the test solution; separately, Danshensu B reference standard was prepared by adding methanol to prepare a solution containing 1 mg per 1 mL as the reference solution. According to the thin-layer chromatography method (General Rule 0502), 3 μL of each of the above solutions were applied separately to the same silica gel G thin-layer plate, and developed using dichloromethane-ethyl acetate-formic acid (10:15:2) as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The results are shown below. Figure 2 As shown, Figure 2 This is a schematic diagram of the thin-layer chromatography of Salvia miltiorrhiza. In the diagram, numbers 1-3 represent the test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is the salvianolic acid B reference solution; and 5 is the negative control solution. As can be seen from the diagram, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram, and the negative control shows no interference, indicating the presence of the target component. The absence of interference from the negative control eliminates the possibility of false positives from excipients or matrices, thus verifying the reliability of the method.

[0076] Identification of Scutellaria baicalensis: The preparation method of the test sample solution is the same as before; according to the prescription and process of Qingfu Zhiyang Granules, a negative sample without Scutellaria baicalensis is prepared, and a negative control solution is prepared according to the preparation method of the test sample solution; separately, baicalin reference standard is prepared by adding methanol to prepare a solution containing 1 mg per 1 mL as the reference solution. According to the thin-layer chromatography method (General Rule 0502), 3 μL of each of the above solutions are applied separately to the same silica gel G thin-layer plate. Develop the plate using ethyl acetate-acetone-dimethylformamide-glacial acetic acid-water (15:10:1.1:1.5:5) as the developing solvent. After development, remove the plate, air dry, spray with 5% ferric chloride ethanol solution, and heat at 105℃ until the spots are clearly visible. Results are shown below. Figure 3 As shown, Figure 3 This is a schematic diagram of the thin-layer chromatography of Scutellaria baicalensis. In the diagram, numbers 1-3 represent the test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is the baicalin reference solution; and 5 is the negative control solution. As can be seen from the diagram, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram, and the negative control shows no interference, indicating the presence of the target component. The absence of interference from the negative control eliminates the possibility of false positives from excipients or the matrix, thus verifying the reliability of the method.

[0077] Honeysuckle identification: The preparation method for the test sample solution is the same as before; according to the prescription and process of Qingfu Zhiyang Granules, a negative sample without honeysuckle was prepared, and a negative control solution was prepared according to the preparation method of the test sample solution; chlorogenic acid reference standard was separately prepared with methanol to a concentration of 1 mg per mL as the reference solution. Thin-layer chromatography (General Rule 0502) was performed, and 3 μL of each of the above solutions were applied separately to the same polysiloxane H plate. The upper layer of butyl acetate-formic acid-water (7:2.5:2.5) was used as the developing solvent. After development, the plate was removed, dried, and examined under ultraviolet light (365 nm). The results are shown below. Figure 4 As shown, Figure 4 This is a schematic diagram of the thin-layer chromatography of honeysuckle. In the diagram, numbers 1-3 represent the test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is the chlorogenic acid reference solution; and 5 is the negative control solution. As can be seen from the diagram, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram, and the negative control shows no interference, indicating the presence of the target component. The absence of interference from the negative control eliminates the possibility of false positives from excipients or matrices, thus verifying the reliability of the method.

[0078] Identification of Smilax glabra: The preparation method of the test sample solution is the same as before; according to the prescription and process of Qingfu Zhiyang Granules, a negative sample without Smilax glabra was prepared, and a negative control solution was prepared according to the preparation method of the test sample solution; separately, astilbene reference standard was prepared by adding methanol to a solution containing 1 mg per 1 mL as the reference solution. According to the thin-layer chromatography method (General Rule 0502), 3 μL of each of the above solutions were applied separately to the same silica gel G thin-layer plate, developed with toluene-ethyl acetate-formic acid (13:32:9) as the developing solvent, removed, dried, sprayed with aluminum trichloride test solution, and examined under ultraviolet light (365 nm) after 5 minutes. In the chromatogram of the test sample, fluorescent spots of the same color appeared at the corresponding positions as in the chromatogram of the reference standard. The results are shown below. Figure 5 As shown, Figure 5 This is a schematic diagram of the thin-layer chromatography of Smilax glabra. In the diagram, numbers 1-3 represent the test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is the astilbin reference solution; and 5 is the negative control solution. As can be seen from the diagram, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram, and the negative control shows no interference, indicating the presence of the target component. The absence of interference from the negative control eliminates the possibility of false positives from excipients or the matrix, thus verifying the reliability of the method.

[0079] Gardenia identification: The preparation method for the test sample solution is the same as before; prepare a negative sample without gardenia according to the prescription and process of Qingfu Zhiyang Granules, and prepare a negative control solution according to the preparation method of the test sample solution; separately take geniposide reference standard, add methanol to prepare a solution containing 1 mg per 1 mL as the reference solution. Perform the test according to the thin-layer chromatography method (General Rule 0502), take 3 μL of each of the above solutions, and spot them separately on the same silica gel G thin-layer plate. Use ethyl acetate-acetone-anhydrous formic acid-water (5:5:1:1) as the developing solvent, develop, remove, air dry, spray with 10% sulfuric acid ethanol solution, and heat at 105 ℃ until the spots are clearly visible. Results are shown below. Figure 6 As shown, Figure 6 This is a schematic diagram of the thin-layer chromatography of gardenia. In the diagram, numbers 1-3 represent the test solutions, with batch numbers 20250203, 20250204, and 20250205 respectively; 4 is the geniposide reference solution; and 5 is the negative control solution. As can be seen from the diagram, the test solution chromatogram shows spots of the same color at the corresponding positions as the reference solution chromatogram. The spots are clear, and there is no interference from the negative control, indicating the presence of the target component. The absence of interference from the negative control eliminates the possibility of false positives from excipients or the matrix, thus verifying the reliability of the method.

[0080] 2.2 Content Determination

[0081] 2.2.1 Chromatographic conditions

[0082] Chromatographic column: Agilent Zorbax Eclipse SB-C18 column (250 mm × 4.6 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution (A) - acetonitrile (B), elution program as shown in Table 1; flow rate: 1.0 mL / min; column temperature: 30 ℃; detection wavelength: 280 nm; injection volume: 10 μL.

[0083] Table 1 Gradient elution program

[0084]

[0085] 2.2.2 Solution Preparation

[0086] (1) Mixed reference solution: Take appropriate amounts of 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin and salvianolic acid B, accurately weigh them, dissolve them in methanol, and prepare mixed reference solutions with concentrations of 0.40 mg / mL, 0.24 mg / mL, 0.16 mg / mL, 1.32 mg / mL and 0.016 mg / mL respectively.

[0087] (2) Test solution: Take 3 g of Qingfu Zhiyang Granules (batch number 20250203) and dissolve it in 25 mL of water. Add an equal volume of water-saturated n-butanol solution to extract twice. Let stand for 30 min, combine the extracts and concentrate to dryness. Add methanol to make up to 5 mL as the test solution.

[0088] (3) Negative control solution: Prepare negative samples lacking Smilax glabra, Salvia miltiorrhiza, Scutellaria baicalensis and Lonicera japonica according to the prescription and process of Qingfu Zhiyang Granules, and prepare negative control solution according to the test solution preparation method.

[0089] 2.3 Measurement Method:

[0090] Take 10 μL each of the test solution and the mixed reference solution and inject them into the high performance liquid chromatograph. Detect the peaks in the test solution by high performance liquid chromatography. Based on the retention time of each substance in the mixed reference solution, determine the peak assignment of each substance. Generate a fingerprint spectrum from the detection results of the test solution to obtain the fingerprint spectrum of Qingfu Zhiyang Granules.

[0091] The screening test for chromatographic conditions in this invention is as follows:

[0092] Column selection: Due to differences in performance and packing materials among different models and manufacturers of chromatographic columns, three columns were compared: Eclipse Plus-C18 (4.6×250mm, 5μm, Agilent Technologies (China) Co., Ltd.), ZORBAX EclipseXDB-C18 (4.6×250mm, 5μm, Agilent Technologies (China) Co., Ltd.), and ZORBAX Eclipse SB-C18 (4.6×250mm, 5μm, Agilent Technologies (China) Co., Ltd.). The results are as follows: Figure 7 As shown, Figure 7 This is a schematic diagram of the column selection results, where A is an Eclipse Plus-C18 column; B is a ZORBAX Eclipse SB-C18 column; and C is a ZORBAX Eclipse XDB-C18 column. As can be seen from the figure, the Eclipse Plus-C18 and ZORBAX Eclipse SB-C18 have better peak elution performance and more peaks than the ZORBAX Eclipse XDB-C18. However, the resolution of each peak in the ZORBAX Eclipse SB-C18 is slightly better than that in the Eclipse Plus-C18. Therefore, the ZORBAX Eclipse SB-C18 was selected as the column for subsequent research in this invention.

[0093] Screening of mobile phases: Since different mobile phases can significantly affect chromatographic separation, this invention investigated the separation effects of the following two mobile phases: methanol + 0.1% phosphoric acid aqueous solution; acetonitrile + 0.1% phosphoric acid aqueous solution. The results are as follows: Figure 8 As shown, Figure 8 The diagram shows the results of mobile phase screening. In the diagram, A represents the results with acetonitrile + 0.1% phosphoric acid aqueous solution as the mobile phase; B represents the results with methanol + 0.1% phosphoric acid aqueous solution as the mobile phase. It can be seen from the diagram that the separation effect is the best when the mobile phase is acetonitrile + 0.1% phosphoric acid aqueous solution.

[0094] Wavelength selection: This invention investigated the chromatographic results obtainable at wavelengths of 327 nm, 254 nm, 286 nm, and 280 nm, as follows: Figure 9 As shown, Figure 9 The diagram shows the results of wavelength screening. A represents the results at a wavelength of 254 nm; B represents the results at a wavelength of 280 nm; C represents the results at a wavelength of 286 nm; and D represents the results at a wavelength of 327 nm. As can be seen from the diagram, the components in the Qingfu Zhiyang Granules have a good response and good separation effect at 280 nm. Therefore, 280 nm was selected as the detection wavelength.

[0095] Flow rate screening: This invention investigated the effect of flow rate on chromatographic separation, specifically examining the separation effect at flow rates of 0.8–1.2 mL / min. The results are as follows: Figure 10 As shown, Figure 10 This diagram illustrates the separation results at different flow rates. A shows the separation effect at a flow rate of 0.8 mL / min; B shows the separation effect at a flow rate of 1 mL / min; and C shows the separation effect at a flow rate of 1.2 mL / min. The diagram shows that the separation effect is good at a flow rate of 1 mL / min. Therefore, the optimal flow rate is determined to be 1 mL / min, with an allowable fluctuation of ±0.1 mL / min.

[0096] 2.4 Specificity Test

[0097] Take 10 μL each of the test solution, mixed reference solution, and negative control solution, and inject them according to the chromatographic conditions described in section 2.2.1. The results showed that the theoretical plate number met the requirements, and the baseline separation was good. The negative control solution showed no interfering peaks at the same retention times as the mixed reference solution, indicating good specificity. See the results below. Figure 11 As shown, Figure 11This is a schematic diagram of the HPLC results for Qingfu Zhiyang Granules. In the diagram, 7 represents 3,5-O-dicaffeoylquinic acid; 11 represents astilbin; 12 represents 4,5-O-dicaffeoylquinic acid; 13 represents baicalin; and 14 represents salvianolic acid B. A is the test solution; B is the mixed reference solution; and C is the negative control solution. The relative retention times of the common peaks were 19.53 min, 22.22 min, 24.16 min, 28.69 min, and 29.97 min, respectively. This detection method has good specificity, and Qingfu Zhiyang Granules contain five target components: 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B.

[0098] 2.5 Methodological Examination

[0099] 2.5.1 Examination of Linear Relationships

[0100] The mixed reference solution under section 2.2.2 was diluted at different ratios to prepare six mixed reference solutions of different concentrations. These solutions were then injected and analyzed under the chromatographic conditions described in section 2.2.1, and the peak areas were recorded. A standard curve was plotted with the mixed reference concentration as the abscissa (X) and the peak area as the ordinate (Y) to obtain the linear equations, correlation coefficients, and linear ranges for each component. The results are shown in Table 2. The data in the table show that each component exhibits a good linear relationship within its respective concentration range.

[0101] Table 2. Results of linear relationship examination for the five principal components.

[0102]

[0103] 2.5.2 Intra-day precision test

[0104] Accurately pipette 10 µL of the mixed reference solution under section 2.2.2, and inject it 6 times under the chromatographic conditions under section 2.2.1. Record the peak area and calculate the RSD value. The experimental data are listed in Table 3. The results show that the instrument has good precision.

[0105] Table 3 Precision test results

[0106]

[0107] 2.5.3 Repeatability Test

[0108] Six test solutions were prepared according to the method described in section 2.2.2, and 10 µL of each solution was injected. The peak areas of 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B were recorded, and the RSD values ​​were calculated. The results are shown in Table 4, indicating that the method has good repeatability.

[0109] Table 4 Repeatability Test Results

[0110]

[0111] 2.5.4 Stability Test

[0112] Prepare the test solution according to the method described in section 2.2.2, and incubate it at room temperature for 0, 4, 8, 12, 16, and 24 h. Inject 10 µL of the solution under the chromatographic conditions described in section 2.2.1, determine the peak area, and calculate the RSD value. The experimental data are listed in Table 5, and the results show that the test solution is stable within 24 h.

[0113] Table 5 Stability test results

[0114]

[0115] 2.5.5 Recovery Test

[0116] Accurately weigh 3g of Qingfu Zhiyang Granules (batch number 20250203), making 9 portions. Accurately add reference standards equivalent to 0.8, 1, and 1.2 times the content of 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B, respectively. Prepare the test solution according to the method in section 2.2.2. Accurately measure 1mL of the test solution and determine the content according to the chromatographic conditions in section 2.2.1. The experimental data are listed in Table 6. The results show that the method has good accuracy.

[0117] Table 6 Results of the spiking recovery experiment

[0118]

[0119]

[0120] 2.6 Validation of content determination method

[0121] Three batches of Qingfu Zhiyang Granules (batch numbers 20250203, 20250204, and 20250205) were collected. Test sample solutions were prepared according to the method described in section 2.2.2. The contents of 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B were determined under the chromatographic conditions described in section 2.2.1. The experimental data are listed in Table 7. The results show that the process is accurate, reliable, and feasible, and can be used for industrial production.

[0122] Table 7. Results of content determination of 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin and salvianolic acid B in Qingfu Zhiyang Granules

[0123]

[0124] 3 fingerprint spectrum

[0125] 3.1 Establishment of fingerprint map

[0126] Fifteen batches of prepared Qingfu Zhiyang granules were used to prepare test sample solutions according to the method described in section 2.2.2. The solutions were injected sequentially under the chromatographic conditions described in section 2.2.1 to obtain chromatograms for each batch. The chromatograms of each batch were imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) for analysis to obtain the fingerprint chromatograms of Qingfu Zhiyang granules. The fingerprint chromatogram obtained from the first batch of Qingfu Zhiyang granules was named S1 and used as a reference chromatogram for MARK peak matching to construct a control fingerprint chromatogram. A total of 18 common peaks were identified in the fingerprint chromatograms of the 15 batches of Qingfu Zhiyang granules. By comparing with the control fingerprint chromatogram, peak 7 was identified as 3,5-O-dicaffeoylquinic acid, peak 11 as astilbin, peak 12 as 4,5-O-dicaffeoylquinic acid, peak 13 as baicalin, and peak 14 as salvianolic acid B. The results are as follows: Figure 12 As shown, Figure 12 This is a schematic diagram of the control fingerprint results for Qingfu Zhiyang Granules. Among them, peak 7 is 3,5-O-dicaffeoylquinic acid, peak 11 is astilbin, peak 12 is 4,5-O-dicaffeoylquinic acid, peak 13 is baicalin, and peak 14 is salvianolic acid B.

[0127] The fingerprint spectrum of the obtained Qingfu Zhiyang granules showed 18 common peaks. Among them, peak 7 with a retention time of 19.53 min was 3,5-O-dicaffeoylquinic acid, peak 11 with a retention time of 22.22 min was astilbin, peak 12 with a retention time of 24.16 min was 4,5-O-dicaffeoylquinic acid, peak 13 with a retention time of 28.69 min was baicalin, peak 14 with a retention time of 29.97 min was salvianolic acid B, and the remaining common peaks were not identified.

[0128] The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

[0129] The spectral results for S1 are shown in Figure 13. Figure 13This is a schematic diagram of the fingerprint spectrum results for Qingfu Zhiyang Granules. In the diagram, 7 represents 3,5-O-dicaffeoylquinic acid; 11 represents astilbin; 12 represents 3,5-O-dicaffeoylquinic acid; 13 represents baicalin; and 14 represents salvianolic acid B. A total of 18 common peaks were identified. Peak 7 (retention time 19.53 min) represents 3,5-O-dicaffeoylquinic acid; peak 11 (retention time 22.22 min) represents astilbin; peak 12 (retention time 24.16 min) represents 4,5-O-dicaffeoylquinic acid; peak 13 (retention time 28.69 min) represents baicalin; and peak 14 (retention time 29.97 min) represents salvianolic acid B. The remaining common peaks were not identified.

[0130] The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

[0131] 3.2 Analysis of Feature Fingerprint Result

[0132] Chemical fingerprint data of 15 batches of Qingfu Zhiyang granules were processed in AIA format and entered into similarity evaluation software for comparison with control fingerprints to determine the degree of similarity between granules. The results showed that the similarity between the fingerprints of the 15 batches of Qingfu Zhiyang granules and the generated control fingerprints was >0.99, indicating high similarity and uniform quality among different batches of granules. This also proves the maturity and stability of the initial process. Analysis showed that the retention time RSD of all common peaks in the common patterns of the 15 batches of samples was less than 2%; the peak area RSD of the 18 common peaks ranged from 0.50% to 6.03%, indicating that the quality of Qingfu Zhiyang granules was relatively stable. The results are shown in Tables 8 and 9.

[0133] Table 8. Retention Time of Fingerprint Spectrum of Qingfu Zhiyang Granules

[0134]

[0135] Table 9. Peak area of ​​fingerprint spectrum of Qingfu Zhiyang Granules

[0136]

[0137] 4 Discussion

[0138] This invention relates to thin-layer chromatography (TLC) identification of six medicinal materials in Qingfu Zhiyang Granules: Danshen, Tufuling, Huangqin, Jinyinhua, Zhizi, and Baixianpi, referring to the method in the 2020 edition of the Pharmacopoeia of the People's Republic of China (Part I) (China Medical Science and Technology Press, 2020). The main components of the corresponding medicinal materials listed in the Pharmacopoeia, astilbin, chlorogenic acid, baicalin, berberine, morrhuin, geniposide, and salvianolic acid B, were selected for identification (Chinese Agricultural Science Bulletin, 2023, 39(33): 140-6). The above identification method is highly operable, has good repeatability, produces clear and easily identifiable spots, and has no interference from the negative control.

[0139] To determine the content of the main components in the formula, this invention employs high-performance liquid chromatography (HPLC) (Chinese Journal of Integrated Traditional and Western Medicine in Dermatology and Venereology, 2017, 16(3): 238-40. Chinese Journal of Traditional and Folk Medicine, 2024, 33(22): 21-6. Chemical Analysis and Metrology, 2025, 34(7): 87-95.), using a DAD detector for content determination. The results showed that the resolution of the main components was greater than 1.5, the theoretical plate number exceeded 5000, and the similarity was greater than 0.9. Compared with the pharmacopoeia method, this method is simpler and easier to implement, and more suitable for the separation and identification of the main components in the formula. Through experiments, DAD detectors were used at 254, 280, 286, and 327 nm, respectively. It was found that the five main standard substances could be determined at 280 nm. Therefore, this invention uses a single wavelength to simultaneously detect the five main substances, thus simplifying the content determination method.

[0140] The Qingfu Zhiyang Granules were analyzed by HPLC. A characteristic fingerprint spectrum of the Qingfu Zhiyang Granules was established, and 18 common peaks were identified. Five common peaks were identified by reference standards, namely 3,5-O-dicaffeoylquinic acid, astilbin, 4,5-O-dicaffeoylquinic acid, baicalin and tanshinone B.

[0141] This invention establishes a thin-layer chromatography method for the identification of six medicinal materials (Salvia miltiorrhiza, Smilax glabra, Scutellaria baicalensis, Lonicera japonica, Gardenia jasminoides, and Dictamnus dasycarpus) in Qingfu Zhiyang Granules, as well as a method for the determination of the content of five active ingredients (3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B). The experimental method is simple, feasible, highly sensitive, and reproducible, and can be used for the quality control of Qingfu Zhiyang Granules, thus providing a quality standard testing method for the standardized clinical use of Qingfu Zhiyang Granules.

[0142] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for establishing the HPLC fingerprint of a skin clearing and itching relieving granule, characterized in that, Includes the following steps: Mixed reference solution: Dissolve 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B in methanol to prepare mixed reference solutions with concentrations of 0.1~1 mg / mL, 0.1~0.8 mg / mL, 0.05~0.5 mg / mL, 0.1~3 mg / mL, and 0.001~0.1 mg / mL, respectively. Test solution: Dissolve the Qingfu Zhiyang granules in water to obtain a solution with a concentration of 0.05~0.8 g / mL, add an equal volume of water-saturated n-butanol solution to extract 1~3 times, let stand, combine the extracts and concentrate to dryness, add methanol to make up to volume, and use as the test solution; High performance liquid chromatography was used to detect the peaks in the test solution based on the retention time of each substance in the mixed reference solution. The detection results of the test solution were then used to generate a fingerprint spectrum, which is the fingerprint spectrum of Qingfu Zhiyang Granules. High performance liquid chromatography (HPLC) conditions include: Chromatographic column: C18 column; Mobile phase: Phase A is a 0.05%~0.15% (v / v) aqueous solution of phosphoric acid, and Phase B is acetonitrile; gradient elution is used with 60%~90% (v / v) Phase A and 10%~40% (v / v) Phase B; flow rate is 0.8~1.2 mL / min; column temperature is 20~40℃; injection volume is 8~12 µl; detection wavelength is 200~400 nm.

2. The method for establishing the HPLC fingerprint of Qingshen Zhiyang Granules according to claim 1, characterized in that, The preparation method of the skin-clearing and antipruritic granules includes the following steps: Weigh the following components according to the following weight proportions: 20-40 parts of Salvia miltiorrhiza, 20-40 parts of Rehmannia glutinosa, 20-40 parts of Smilax glabra, 20-40 parts of Lonicera japonica, 5-25 parts of Scutellaria baicalensis, 20-40 parts of Rubia cordifolia, 5-25 parts of Rheum palmatum, 20-40 parts of Lonicera japonica, 2-18 parts of Gardenia jasminoides, and 5-25 parts of Dictamnus dasycarpus. Crush the Gardenia jasminoides and add 6-10 times the amount of water to the remaining nine herbs. Decoction is carried out once or three times, each time for 0.5-2 hours. Filter the decoction and concentrate the filtrate under reduced pressure to a clear extract with a relative density of 1.07-1.

12. Spray dry the extract to make an extract powder. Add dextrin and mix well. Make granules, granulate, and package to obtain Qingfu Zhiyang Granules.

3. The method for establishing the HPLC fingerprint of Qingshen Zhiyang Granules according to claim 1, characterized in that, The high-performance liquid chromatography conditions include: Chromatographic column: ZORBAX Eclipse SB-C18 column, 250 mm × 4.6 mm, 5 μm; Mobile phase A was a 0.1% (v / v) aqueous solution of phosphoric acid, and mobile phase B was acetonitrile. The gradient elution program was as follows: 0–20 min, 77% A–90% A; 20–30 min, 77% A–75% A; 30–45 min, 60% A–75% A; 45–50 min, 60% A; 50–55 min, 60% A–90% A; 55–60 min, 90% A. The detection wavelength was 280 nm, the flow rate was 1 mL / min, the column temperature was 30 °C, and the injection volume was 10 µl.

4. The method for establishing the HPLC fingerprint of Qingshen Zhiyang Granules according to claim 1, characterized in that, The concentration of 3,5-O-dicaffeoylquinic acid soluble in methanol is 0.40 mg / mL; Alternatively, the concentration of astilbin dissolved in methanol is 0.24 mg / m³. Alternatively, the concentration of the 4,5-O-dicaffeoylquinic acid dissolved in methanol is 0.16 mg / mL; Alternatively, the concentration of baicalin dissolved in methanol is 1.32 mg / mL; Alternatively, the concentration of salvianolic acid B dissolved in methanol is 0.016 mg / mL.

5. The method for establishing the HPLC fingerprint of Qingshen Zhiyang Granules according to claim 1, characterized in that, The fingerprint spectrum of the skin-clearing and antipruritic granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 18 common peaks were identified.

6. The method for establishing the HPLC fingerprint of Qingshen Zhiyang Granules according to claim 5, characterized in that, Of the 18 common peaks, peak 7 with a retention time of 19.53 min is 3,5-O-dicaffeoylquinic acid, peak 11 with a retention time of 22.22 min is astilbin, peak 12 with a retention time of 24.16 min is 4,5-O-dicaffeoylquinic acid, peak 13 with a retention time of 28.69 min is baicalin, peak 14 with a retention time of 29.97 min is salvianolic acid B, and the remaining common peaks were not identified. The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

7. A method for establishing a control fingerprint of Qingshen Zhiyang granules, characterized in that, Includes the following steps: Take n batches of Qingfu Zhiyang Granules and obtain the fingerprint spectrum of each batch of Qingfu Zhiyang Granules according to the method of any one of claims 1 to 6; analyze the fingerprint spectrum of the n batches of Qingfu Zhiyang Granules using the software of the Chinese Medicine Chromatography Fingerprint Similarity Evaluation System to obtain the control fingerprint spectrum of Qingfu Zhiyang Granules; wherein, n≥10.

8. The control fingerprint spectrum of Qingshen Zhiyang granules obtained by the method of claim 7. The comparative fingerprint spectrum of the Qingfu Zhiyang granules: the chromatographic peaks of the main components with good separation were selected as characteristic peaks, and a total of 18 common peaks were identified. Of the 18 common peaks, peak 7 with a retention time of 19.53 min is 3,5-O-dicaffeoylquinic acid, peak 11 with a retention time of 22.22 min is astilbin, peak 12 with a retention time of 24.16 min is 4,5-O-dicaffeoylquinic acid, peak 13 with a retention time of 28.69 min is baicalin, peak 14 with a retention time of 29.97 min is salvianolic acid B, and the remaining common peaks were not identified. The retention times of the other common peaks are as follows: Peak 1 with a retention time of 3.74 min, Peak 2 with a retention time of 4.09 min, Peak 3 with a retention time of 5.11 min, Peak 4 with a retention time of 8.42 min, Peak 5 with a retention time of 17.43 min, Peak 6 with a retention time of 18.62 min, Peak 8 with a retention time of 20.47 min, Peak 9 with a retention time of 21.10 min, Peak 10 with a retention time of 21.70 min, Peak 15 with a retention time of 30.72 min, Peak 16 with a retention time of 38.32 min, Peak 17 with a retention time of 41.74 min, and Peak 18 with a retention time of 44.56 min.

9. A method for quality control of a skin clearing and itching relieving granule, characterized by, Includes the following steps: Take the Qingfu Zhiyang Granules to be tested. The method according to claim 1 obtains a chromatogram of the Qingfu Zhiyang granules to be tested. The chromatogram of the obtained Qingfu Zhiyang granules to be tested and the control fingerprint chromatogram of the Qingfu Zhiyang granules are evaluated for similarity. If the similarity is ≥0.90, the quality of the Qingfu Zhiyang granules to be tested is determined to be qualified.

10. A method for determining the content of effective ingredients in a skin clearing and itching relieving granule, characterized by, Includes the following steps: The content of active ingredients in Qingfu Zhiyang Granules was determined by high performance liquid chromatography. The active ingredients were selected from 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and tanshinone B. The first step is to establish a standard curve for chemical composition: 3,5-O-dicaffeoylquinic acid, astilbene, 4,5-O-dicaffeoylquinic acid, baicalin, and salvianolic acid B were dissolved in methanol to prepare mixed reference solutions with concentrations of 0.1–1 mg / mL, 0.1–0.8 mg / mL, 0.05–0.5 mg / mL, 0.1–3 mg / mL, and 0.001–0.1 mg / mL, respectively. High-performance liquid chromatography (HPLC) was used for analysis. The chromatographic peaks of each active ingredient were determined based on their retention times, and the peak areas of the corresponding active ingredients were obtained. Standard curves for each active ingredient were established, and the linear regression equations and correlation coefficients r for each active ingredient were obtained. High performance liquid chromatography (HPLC) conditions include: Chromatographic column: C18 column; Mobile phase: Phase A is a 0.05%~0.15% (v / v) aqueous solution of phosphoric acid, and Phase B is acetonitrile; gradient elution is used with 60%~90% (v / v) Phase A and 10%~40% (v / v) Phase B; flow rate is 0.8~1.2 mL / min; column temperature is 20~40℃; injection volume is 8~12 µl; detection wavelength is 200~400 nm. The second step is to obtain the chromatographic peak area of ​​the active ingredient in the antipruritic granules to be tested; Dissolve the Qingfu Zhiyang granules in water to obtain a solution with a concentration of 0.05~0.8 g / mL. Add an equal volume of water-saturated n-butanol solution and extract 1~3 times. Let stand, combine the extracts and concentrate to dryness. Add methanol to make up to volume to obtain the test solution. The test solution was detected under the same high-performance liquid chromatography conditions as in the first step. The chromatographic peaks of each active ingredient were determined based on their retention times, and the peak areas of the corresponding active ingredients were obtained. The third step is to determine the content of the active ingredients in the antipruritic granules to be tested; Based on the established standard curves of each active ingredient, the chromatographic peak areas of the active ingredients in the Qingfu Zhiyang granules to be tested were substituted into the corresponding linear regression equations. The concentrations of the corresponding ingredients in the test solution were calculated using the external standard method, and the content of each active ingredient in the Qingfu Zhiyang granules to be tested was calculated.

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