Fingerprint of a kidney-tonifying and hair-blackening oral liquid, construction method and application thereof, and multi-index component detection method of the kidney-tonifying and hair-blackening oral liquid

Abstract

This invention provides a fingerprint spectrum of Yishen Wufa Oral Liquid, its construction method, and its application, as well as a multi-index component detection method for Yishen Wufa Oral Liquid, relating to the field of pharmaceutical analysis and structural technology. The fingerprint spectrum of Yishen Wufa Oral Liquid constructed by this invention includes 20 characteristic peaks, including ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin. The chromatographic peaks are abundant and attributed to the effective components of psoralen (roasted with black sesame seeds), angelica, poria cocos, wolfberry, fleeceflower root (processed with black bean wine), achyranthes bidentata, and astragalus complanatus. This provides a more comprehensive reflection of the overall chemical composition and quality of Yishen Wufa Oral Liquid, facilitating more effective guidance for feed addition and strict standardization of production operations. It also helps improve the safety and efficacy of Yishen Wufa Oral Liquid in clinical use, possessing high application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis structure technology, specifically to a fingerprint spectrum of Yishen Wufa oral liquid and its construction method and application, and a multi-index component detection method for Yishen Wufa oral liquid. Background Technology

[0002] With the fast pace of life and increasing life pressure, the number of patients suffering from hair loss and gray hair is rising rapidly year by year, but the age of onset is decreasing. Traditional Chinese medicine believes that "hair is the surplus of blood" and "the health of the kidneys is reflected in the hair." Generally, gray hair is related to blood deficiency and kidney weakness. Gray hair in young people is often due to imbalance of qi and blood, resulting in insufficient nourishment of the hair, or inherent weakness and kidney qi deficiency. Gray hair in the elderly is a manifestation of physiological weakness. Therefore, the treatment method in traditional Chinese medicine is to nourish liver blood and kidney qi. Currently, common Chinese patent medicines used to treat hair loss and gray hair include He Shou Wu Wan, Wu Fa Wan, Qi Bao Mei Ran Wan, Ren Shen Shou Wu Jiaonang, Tian Ma Shou Wu Pian, Shen Rong Zhen Bao Pian, and Sheng Fa Pian, etc.

[0003] Yishen Wufa Oral Liquid has the effects of nourishing the liver and kidneys and darkening hair. It is used for hair loss and premature graying caused by deficiency of both liver and kidneys. The formula of Yishen Wufa Oral Liquid is scientifically sound and based on syndrome differentiation. In the formula, Polygonum multiflorum, Psoralea corylifolia, and Lycium barbarum nourish the liver and kidneys, replenish yin and blood; Achyranthes bidentata, Angelica sinensis, and Poria cocos invigorate blood circulation, remove blood stasis, moisten the lungs, eliminate dampness, and calm the mind. It addresses the root cause of hair loss and graying by regulating the liver and kidneys and replenishing essence and blood. This allows essence and blood to flow through the capillaries around the hair follicles, delivering energy for hair growth, activating atrophied and necrotic hair follicles, promoting new hair growth, and ending hair loss. Simultaneously, it promotes melanin production, eliminating gray hair and fundamentally solving various hair problems. Yishen Wufa Oral Liquid is a commonly used medicine among middle-aged and elderly people, with a wide range of applications. No adverse reactions have been reported after use, indicating good application prospects.

[0004] Yishen Wufa Oral Liquid has good clinical efficacy and a large market demand, but its quality control standards are low and its stability is poor. Standard YBZ02192009 determined the content of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Polygonum multiflorum, but this component is unstable to light and heat, which makes the marketed drug prone to product quality problems after long-term storage. Several studies have reported on the content determination of individual components. For example, Zhang Yanfen et al. (Zhang Yanfen, Optimizing the Quality Standard of Yishen Wufa Oral Liquid, Chinese Journal of Drug Evaluation, 2017, Vol. 2, pp. 100-103) studied the determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Yishen Wufa Oral Liquid using high performance liquid chromatography (HPLC); Liu Linhong et al. (Liu Linhong, Discussion on the Quality Standard of Yishen Wufa Oral Liquid, Beijing Journal of Traditional Chinese Medicine, 2011, Vol. 8, pp. 626-630) studied the determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside using HPLC; Zhu Lanlan et al. (Zhu Lanlan, Determination of the Content of Stilbene Glycoside, Emodin, and Emodin Methyl Ether in Yishen Wufa Oral Liquid, Chinese Journal of Drug Vigilance, 2011, Vol. 5, pp. 281-283) studied the determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside using HPLC. The content of emodin, emodin methyl ether, and 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Yishen Wufa Oral Liquid was determined by high performance liquid chromatography (HPLC). Lu Qiang (Lu Qiang, Research on Quality Standards of Yishen Wufa Oral Liquid, China Pharmacy, 2010, No. 31, pp. 2942-2944) studied the determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in Yishen Wufa Oral Liquid using HPLC. Content of D-glucosinolates; Zhang Jing (Zhang Jing, Determination of 2,3,5,4'-Tetrahydroxystilbene-2-O-β-D-glucosinolate content in Yishen Wufa Oral Liquid by HPLC, Anhui Agricultural Sciences, 2010, No. 32, pp. 18146-18147) studied the determination of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucosinolate content in Yishen Wufa Oral Liquid using high performance liquid chromatography. To date, there is no method for quality control of Yishen Wufa Oral Liquid using fingerprinting.

[0005] Current methods for determining the content of Yishen Wufa oral liquid rely on one or two components to control its intrinsic quality, which is somewhat one-sided. Strictly controlling the quality of Yishen Wufa oral liquid requires more than just characterizing and controlling one or two chemical components; it necessitates controlling the entire composition of its substances. Therefore, establishing and applying a fingerprint spectral detection method for Yishen Wufa oral liquid is crucial, necessitating control over its entire composition to effectively characterize quality standards. Summary of the Invention

[0006] In view of this, the purpose of this invention is to provide a fingerprint spectrum of Yishen Wufa Oral Liquid, its construction method and application, and a multi-index component detection method for Yishen Wufa Oral Liquid. The fingerprint spectrum of Yishen Wufa Oral Liquid constructed by this invention can comprehensively reflect the overall chemical composition of Yishen Wufa Oral Liquid, providing an effective means for the overall quality control and evaluation of Yishen Wufa Oral Liquid, thereby better ensuring the quality stability, consistency and controllability of Yishen Wufa Oral Liquid.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for constructing the fingerprint spectrum of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps:

[0009] The kidney-tonifying and hair-darkening oral liquid was mixed with a low alcohol to obtain the test solution;

[0010] The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain sample HPLC chromatograms and reference HPLC chromatograms, respectively. The fingerprint chromatogram of Yishen Wufa Oral Liquid was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram.

[0011] The reference standards in the mixed control solution include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin;

[0012] The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution of phosphoric acid is 0.05-0.2%; the elution method is gradient elution, and the gradient elution program includes: 0-20 min, mobile phase A volume fraction 5%; 20-30 min, the volume fraction of mobile phase A increases from 5% to 10%; 30-50 min, the volume fraction of mobile phase A increases from 10% to 20%; 50-70 min, the volume fraction of mobile phase A increases from 20% to 35%; 70-85 min, the volume fraction of mobile phase A increases from 35% to 65%; 85-95 min, the volume fraction of mobile phase A increases from 65% to 80%.

[0013] The fingerprint spectrum of the Yishen Wufa Oral Liquid has 20 characteristic peaks, including the characteristic peaks of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin.

[0014] Preferably, the volume ratio of the kidney-tonifying and hair-darkening oral liquid to lower alcohol is 1:1 to 4;

[0015] The lower alcohols include methanol and / or ethanol.

[0016] Preferably, the mixing includes ultrasonic mixing, wherein the ultrasonic mixing power is 50-100W, the frequency is 200-300kHz, and the time is 20-40min.

[0017] Preferably, in the mixed reference solution, the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside is 0.1–0.2 mg / mL, the concentrations of ferulic acid and astragaloside A are independently 0.01–0.02 mg / mL, and the concentrations of psoralen, isopsoralen, and emodin are independently 0.005–0.02 mg / mL;

[0018] The solvent in the mixed reference solution includes lower alcohols.

[0019] Preferably, the conditions for high performance liquid chromatography detection further include: a mobile phase flow rate of 0.8–1.2 mL / min, an ultraviolet detector, a detection wavelength of 230–360 nm, a column temperature of 20–35 °C, and an injection volume of 5–20 μL.

[0020] Preferably, the similarity between the liquid chromatography chromatogram of the sample and the liquid chromatography chromatogram of the reference substance, calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine", is both >0.9.

[0021] The present invention provides a fingerprint spectrum of Yishen Wufa Oral Liquid obtained by the fingerprint spectrum construction method described above, which contains 20 characteristic peaks, including characteristic peaks of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin.

[0022] This invention provides the application of the fingerprint spectrum of Yishen Wufa Oral Liquid described in the above technical solution in the quality control of Yishen Wufa Oral Liquid.

[0023] This invention also provides a method for detecting multiple components of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps:

[0024] The kidney-tonifying and hair-darkening oral liquid to be tested was mixed with a low alcohol to obtain the sample solution to be tested;

[0025] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple index components of Yishen Wufa oral liquid; the multiple index components include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin.

[0026] The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution of phosphoric acid is 0.05-0.2%; the elution method is gradient elution, and the gradient elution program includes: 0-20 min, mobile phase A volume fraction 5%; 20-30 min, the volume fraction of mobile phase A increases from 5% to 10%; 30-50 min, the volume fraction of mobile phase A increases from 10% to 20%; 50-70 min, the volume fraction of mobile phase A increases from 20% to 35%; 70-85 min, the volume fraction of mobile phase A increases from 35% to 65%; 85-95 min, the volume fraction of mobile phase A increases from 65% to 80%.

[0027] Preferably, the volume ratio of the kidney-tonifying and hair-darkening oral liquid to lower alcohol is 1:1 to 4;

[0028] The lower alcohols include methanol and / or ethanol;

[0029] The mixing includes ultrasonic mixing, wherein the power of the ultrasonic mixing is 50-100W, the frequency is 200-300kHz, and the time is 20-40min;

[0030] The detection conditions for the high-performance liquid chromatography (HPLC) detection also include: a mobile phase flow rate of 0.8–1.2 mL / min, a detection wavelength of 230–360 nm, a column temperature of 20–35 °C, and an injection volume of 5–20 μL.

[0031] This invention uses C 18The chromatographic column used acetonitrile as mobile phase A and 0.05–0.2% phosphoric acid aqueous solution as mobile phase B. Gradient elution was employed with controlled gradient elution program. The test solution and mixed reference solution of Yishen Wufa Oral Liquid were separately detected by high performance liquid chromatography (HPLC), and the chromatograms of the two were compared and analyzed. The fingerprint chromatogram of Yishen Wufa Oral Liquid showed a total of 20 characteristic peaks. Among these 20 characteristic peaks were characteristic peaks of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin, which were attributed to the effective components of Psoralea corylifolia (roasted with black sesame seeds), Angelica sinensis, Poria cocos, Lycium barbarum, Polygonum multiflorum (roasted with black bean wine), Achyranthes bidentata, and Astragalus complanatus. The fingerprint spectrum constructed by this invention has abundant chromatographic peaks, providing an effective means to comprehensively reflect, monitor and evaluate the overall chemical composition and overall quality control of Yishen Wufa Oral Liquid. It guides standardized production, thereby better ensuring the quality stability, consistency and controllability of Yishen Wufa Oral Liquid. It also improves the overly simplistic current quality standard for Yishen Wufa Oral Liquid and has high application value.

[0032] Furthermore, since fingerprinting is not intended to determine the precise content of a component, but rather to fully reflect the information of the chemical composition, when 254nm is used as the detection wavelength, there are more peaks, the information reflected is more complete, the absorption values ​​of each peak are good, the baseline is stable, and the large absorption of near-ultraviolet impurity peaks is avoided.

[0033] The high-performance liquid chromatography fingerprinting method established in this invention is the first to achieve quality control of the entire formula for nourishing kidney and blackening hair, rather than identifying a single compound or medicinal material. It can more effectively guide the feeding, strictly standardize production operations, and improve the safety and effectiveness of clinical medication.

[0034] This invention establishes a method for simultaneous detection of multiple components in Yishen Wufa Oral Liquid. This method has the advantage of quantitative monitoring of multiple components in the same chromatographic system, and can comprehensively detect the content of six major active ingredients in Yishen Wufa Oral Liquid: ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin. It can be used as a quality control method for Yishen Wufa Oral Liquid, and can completely and accurately evaluate the efficacy, safety, and stability of Yishen Wufa Oral Liquid. It can also control the raw materials of the product more precisely and comprehensively, thus ensuring the quality and efficacy of the product. Attached Figure Description

[0035] Figure 1 The HPLC chromatogram of the mixed reference solution;

[0036] Figure 2 The HPLC chromatogram of the test solution is shown below.

[0037] Figure 3 The fingerprint spectrum overlay of 10 batches of Yishen Wufa Oral Liquid;

[0038] Figure 4 This is a comparison chart of sample fingerprint chromatograms and chromatograms of various medicinal materials.

[0039] Figure 5 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 360nm;

[0040] Figure 6 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 320nm;

[0041] Figure 7 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 280nm;

[0042] Figure 8 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 254nm;

[0043] Figure 9 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 230nm;

[0044] Figure 10 The spectrum of Yishen Wufa Oral Liquid at a wavelength of 210nm;

[0045] Figure 11 The image of Yishen Wufa Oral Liquid at a wavelength of 203nm. Detailed Implementation

[0046] This invention provides a method for constructing the fingerprint spectrum of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps:

[0047] The kidney-tonifying and hair-darkening oral liquid was mixed with a low alcohol to obtain the test solution;

[0048] The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain sample HPLC chromatograms and reference HPLC chromatograms, respectively. The fingerprint chromatogram of Yishen Wufa Oral Liquid was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram.

[0049] The reference standards in the mixed control solution include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin;

[0050] The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphoric acid; the elution method is gradient elution, and the gradient elution program includes: 0–18 min, the volume fraction of mobile phase A increases from 5% to 14%; 18–33 min, the volume fraction of mobile phase A increases from 14% to 28%; 33–50 min, the volume fraction of mobile phase A increases from 28% to 57%; 50–70 min, the volume fraction of mobile phase A is 57%.

[0051] The liquid chromatography chromatogram of the sample is imported into the chromatographic fingerprint similarity evaluation system for traditional Chinese medicine to generate a common pattern of characteristic chromatograms.

[0052] The fingerprint spectrum of the Yishen Wufa Oral Liquid contains 20 characteristic peaks, including those of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, Astragalus complanatus glycoside A, psoralen, isopsoralen, and emodin.

[0053] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0054] This invention involves mixing a kidney-tonifying and hair-darkening oral liquid with a solvent to obtain a test solution.

[0055] In this invention, the kidney-tonifying and hair-darkening oral liquid is prepared from Polygonum multiflorum (processed with black bean wine), Angelica sinensis, Psoralea corylifolia (roasted with black sesame seeds), Lycium barbarum, Astragalus complanatus, Poria cocos, and Achyranthes bidentata. This invention does not specifically limit the source of the kidney-tonifying and hair-darkening oral liquid; it can be prepared using commercially available products well-known to those skilled in the art or using methods well-known to those skilled in the art. In this invention, the commercially available products preferably include the kidney-tonifying and hair-darkening oral liquid produced by Jilin Yinnuoke Pharmaceutical Co., Ltd. In this invention, the preferred method for preparing the kidney-tonifying and hair-darkening oral liquid includes the following steps: Take 250g of Polygonum multiflorum (processed with black bean wine), 62.5g of Angelica sinensis, 31.3g of Psoralea corylifolia (fried with black sesame seeds, 5g), 62.5g of Lycium barbarum, 62.5g of Astragalus complanatus, 62.5g of Poria cocos, and 62.5g of Achyranthes bidentata. Add 8 times the amount of water and decoct twice, the first decoction for 3 hours and the second decoction for 1 hour. Filter, combine the filtrates, let stand at room temperature for 24 hours, take the supernatant, concentrate under reduced pressure to a clear paste with a relative density of 1.10 (50℃), cool, add 250g of refined honey, 150g of syrup and 3g of sodium benzoate, heat to boiling, cool, adjust the total volume to 1000mL, stir well, refrigerate for 24 hours, take the supernatant, fill and seal, sterilize to obtain the kidney-tonifying and hair-darkening oral liquid.

[0056] In this invention, the lower alcohol preferably includes methanol and / or ethanol, more preferably ethanol.

[0057] In this invention, the volume ratio of the kidney-tonifying and hair-darkening oral liquid to lower alcohols (total) is preferably 1:1 to 4, more preferably 1:1.5 to 3, further preferably 1:2 to 2.5, and most preferably 3:7.

[0058] In this invention, the mixing preferably includes ultrasonic mixing, wherein the power of the ultrasonic mixing is preferably 50-100W, more preferably 55-80W, and even more preferably 60-70W; the frequency of the ultrasonic mixing is preferably 200-300kHz, more preferably 240-260kHz, and even more preferably 250kHz; and the time of the ultrasonic mixing is preferably 20-40min, more preferably 25-35min, and even more preferably 30min.

[0059] The present invention preferably further includes, after mixing, adjusting the volume of the resulting ultrasonic system with lower alcohol and filtering it through a 0.45 μm filter membrane, with the filtrate being the test solution.

[0060] After obtaining the test solution, the present invention performs high performance liquid chromatography on the test solution and the mixed reference solution respectively to obtain the sample liquid chromatogram and the reference liquid chromatogram, and obtains the fingerprint chromatogram of Yishen Wufa Oral Liquid based on the sample liquid chromatogram and the reference liquid chromatogram.

[0061] In this invention, the reference standards in the mixed reference solution include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin. In this invention, the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside in the mixed reference solution is preferably 0.1–0.2 mg / mL, more preferably 0.15–0.16 mg / mL; the concentrations of ferulic acid and astragaloside A in the mixed reference solution are independently preferably 0.01–0.02 mg / mL, more preferably 0.015–0.016 mg / mL; the concentrations of psoralen, isopsoralen, and emodin in the mixed reference solution are independently preferably 0.005–0.02 mg / mL, more preferably 0.01–0.015 mg / mL. In this invention, the solvent in the mixed reference solution preferably includes lower alcohols, more preferably methanol and / or ethanol, and even more preferably methanol.

[0062] In this invention, the preferred method for preparing the mixed reference solution includes the following steps: using lower alcohols as solvents, preparing ferulic acid reference stock solutions, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference stock solutions, astragaloside A reference stock solutions, psoralen reference stock solutions, isopsoralen reference stock solutions, and emodin reference stock solutions respectively; mixing the stock solutions and then adjusting the volume with lower alcohols to obtain the mixed reference solution. In this invention, the concentrations of the 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference stock solution, ferulic acid reference stock solution, and psoralen A reference stock solution are preferably 0.5–1 mg / mL, more preferably 0.75–0.08 mg / mL; and the concentrations of psoralen, isopsoralen, and emodin in the mixed reference solution are preferably 0.25–1 mg / mL, more preferably 0.5–0.75 mg / mL.

[0063] In this invention, the conditions for the high-performance liquid chromatography detection include: the chromatographic column comprising C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile, and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution is 0.05-0.2%, preferably 0.1-0.15%; the mobile phase flow rate is preferably 0.8-1.2 mL / min, more preferably 0.9-1.1 mL / min, and even more preferably 1 mL / min; the detector is preferably an ultraviolet detector, and the detection wavelength is preferably 230-360 nm, more preferably 254-280 nm; the column temperature is preferably 20-35℃, more preferably 25-30℃; The sample volume is preferably 5–20 μL, more preferably 10–15 μL; the elution method is gradient elution, and the gradient elution program includes: 0–20 min, mobile phase A volume fraction is 5%; 20–30 min, mobile phase A volume fraction increases from 5% to 10%; 30–50 min, mobile phase A volume fraction increases from 10% to 20%; 50–70 min, mobile phase A volume fraction increases from 20% to 35%; 70–85 min, mobile phase A volume fraction increases from 35% to 65%; 85–95 min, mobile phase A volume fraction increases from 65% to 80%.

[0064] In this invention, the sample liquid chromatography chromatogram is preferably imported into a similarity evaluation system for chromatographic fingerprints of traditional Chinese medicine to generate a common pattern of characteristic chromatograms, resulting in a fingerprint chromatogram containing 20 characteristic peaks. The fingerprint chromatogram is compared with the retention time of a reference liquid chromatography chromatogram (based on the chromatographic peaks of the reference solution). The fingerprint chromatogram includes characteristic peaks of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin. In this invention, the sample liquid chromatography chromatogram is preferably calculated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software, and the common peaks with a similarity > 0.9 are considered the fingerprint chromatogram of Yishen Wufa Oral Liquid. In this invention, the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software is preferably "Similarity Evaluation System Software for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012A)". This invention preferably uses the median method for similarity analysis, with a preferred time window width of 0.1 min, and identifies 20 common peaks. In an embodiment of this invention, high-performance liquid chromatography (HPLC) analysis was performed on 10 batches of Yishen Wufa oral liquid, and the resulting chromatograms were imported into a traditional Chinese medicine chromatographic fingerprint similarity evaluation system for similarity analysis. The similarity of each batch of Yishen Wufa oral liquid was greater than 0.90.

[0065] In this invention, by comparing the retention times of the fingerprint spectrum with those of the reference liquid chromatography spectrum, it was found that peak 11 in the fingerprint spectrum is ferulic acid, peak 13 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 15 is astragaloside A, peak 17 is psoralen, peak 18 is isopsoralen, and peak 20 is emodin. In this invention, the 20 common peaks are respectively attributed to seven medicinal materials: black sesame-fried psoralea corylifolia, angelica sinensis, poria cocos, wolfberry, black bean wine-processed he shou wu, achyranthes bidentata, and astragalus complanatus. Among them, peak 1 is attributed to black bean wine-processed he shou wu, peak 2 is attributed to angelica sinensis, poria cocos, wolfberry, and achyranthes bidentata, peak 3 is attributed to wolfberry, peak 4 is attributed to wolfberry, peak 5 is attributed to black bean wine-processed he shou wu, peak 6 is attributed to achyranthes bidentata, wolfberry, and angelica sinensis, peak 7 is attributed to black bean wine-processed he shou wu, and peak 8 is attributed to wolfberry and black sesame-fried psoralea corylifolia. Psoralea corylifolia, peak number 9 belongs to black sesame-fried Psoralea corylifolia, peak number 10 belongs to black sesame-fried Psoralea corylifolia, peak number 11 belongs to Angelica sinensis and Astragalus complanatus, peak number 12 belongs to Achyranthes bidentata and Lycium barbarum, peak number 13 belongs to black bean wine-processed Polygonum multiflorum, peak number 14 belongs to sodium benzoate, peak number 15 belongs to Astragalus complanatus, peak number 16 belongs to black bean wine-processed Polygonum multiflorum, peak number 17 belongs to black sesame-fried Psoralea corylifolia, peak number 18 belongs to black sesame-fried Psoralea corylifolia, peak number 19 belongs to Astragalus complanatus, peak number 20 belongs to black bean wine-processed Polygonum multiflorum.

[0066] The present invention provides a fingerprint spectrum of Yishen Wufa Oral Liquid obtained by the fingerprint spectrum construction method described above, which contains 20 characteristic peaks, including characteristic peaks of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin.

[0067] This invention also provides a method for detecting multiple components of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps:

[0068] The kidney-tonifying and hair-darkening oral liquid to be tested was mixed with a low alcohol to obtain the sample solution to be tested;

[0069] The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple index components of Yishen Wufa oral liquid; the multiple index components include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin.

[0070] The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution of phosphoric acid is 0.05-0.2%; the elution method is gradient elution, and the gradient elution program includes: 0-20 min, mobile phase A volume fraction 5%; 20-30 min, the volume fraction of mobile phase A increases from 5% to 10%; 30-50 min, the volume fraction of mobile phase A increases from 10% to 20%; 50-70 min, the volume fraction of mobile phase A increases from 20% to 35%; 70-85 min, the volume fraction of mobile phase A increases from 35% to 65%; 85-95 min, the volume fraction of mobile phase A increases from 65% to 80%.

[0071] In this invention, the preparation method of the sample solution to be tested is the same as the preparation method of the test solution; the conditions for high performance liquid chromatography detection are the same as the conditions for high performance liquid chromatography detection in the fingerprint pattern construction process, and will not be described in detail here.

[0072] In this invention, the multi-index component detection results include qualitative detection results and quantitative detection results. Preferably, the qualitative detection results are obtained using fingerprinting.

[0073] In this invention, the quantitative detection results are preferably quantified using the external standard method to calculate the content of each component.

[0074] In this invention, the reference standards in the linear mixed reference solution include ferulic acid reference standard, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside reference standard, astragaloside A reference standard, psoralen reference standard, isopsoralen reference standard, and emodin reference standard; the solvent in the mixed reference solution preferably includes methanol and / or ethanol, more preferably methanol.

[0075] To further illustrate the present invention, the fingerprint spectrum construction method, preparation method and application of Yishen Wufa Oral Liquid, as well as the multi-index component detection method, are described in detail below with reference to the embodiments. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0076] In the following embodiments, the instruments and reagents used are as follows:

[0077] 1.1 Agilent 1220 high performance liquid chromatograph (Agilent Technologies, USA); KQ-250B ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BSA 124S electronic balance (Sartorius); QUINTIX35-1CN (Sartorius);

[0078] 1.2 Purified water (Wahaha), acetonitrile (chromatographic grade), and other reagents were all analytical grade. Ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, Astragalus complanatin A, psoralen, isopsoralen, and emodin reference standards (batch numbers 110773-202316, 110844-202317, 111803-201704, 110739-202319, 110738-202317, and 110756-201913, with purities of 99.3%, 97.9%, 99.1%, 99.7%, 99.8%, and 96.0%, respectively). Kidney-Nourishing and Hair-Darkening Oral Liquid (Batch Nos.: 20221001, 20221002, 20221003, 20230201, 20230202, 20230203, 20230401, 20230402, 20230701, 20230702, Jilin Yinnuoke Pharmaceutical Co., Ltd.)

[0079] 2. HPLC chromatographic conditions: The chromatographic column was C10. 18The chromatographic column (250 mm × 4.6 mm, 5 μm) used acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, with gradient elution at a flow rate of 1.0 mL / min and a column temperature of 30 °C. A VWD detector was used with a detection wavelength of 254 nm, and the injection volume was 10 μL. The gradient elution program was as follows: 0–20 min, mobile phase A volume fraction 5%; 20–30 min, mobile phase A volume fraction increased from 5% to 10%; 30–50 min, mobile phase A volume fraction increased from 10% to 20%; 50–70 min, mobile phase A volume fraction increased from 20% to 35%; 70–85 min, mobile phase A volume fraction increased from 35% to 65%; 85–95 min, mobile phase A volume fraction increased from 65% to 80%.

[0080] Example 1

[0081] Construction of fingerprint spectrum of Yishen Wufa Oral Liquid

[0082] Preparation of test solution: Take 3 mL of Yishen Wufa oral liquid, place it in a 10 mL volumetric flask, add about 6 mL of ethanol, extract by sonication (80 W, 250 kHz) for 30 min, cool to room temperature, dilute to the mark with ethanol, shake well, filter through a 0.45 μm microporous membrane, and take the filtrate as the test solution.

[0083] Preparation of the reference solution:

[0084] Accurately weigh 7.60 mg of ferulic acid into a 10 mL volumetric flask, dissolve and dilute with methanol, shake well to obtain a ferulic acid reference stock solution with a concentration of 0.7547 mg / mL.

[0085] Accurately weigh 7.68 mg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside into a 10 mL volumetric flask, dissolve in methanol and dilute to volume, shake well to obtain a 0.7587 mg / mL stock solution of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside.

[0086] Accurately weigh 7.52 mg of Astragalus complanatus glycoside A and place it in a 10 mL volumetric flask. Dissolve and dilute to volume with methanol, and shake well to obtain a stock solution of Astragalus complanatus glycoside A with a concentration of 0.7452 mg / mL.

[0087] Accurately weigh 5.08 mg of psoralen and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to volume. Shake well to obtain a psoralen reference stock solution with a concentration of 0.5065 mg / mL.

[0088] Accurately weigh 5.12 mg of isopsoralen and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to volume. Shake well to obtain a stock solution of isopsoralen reference standard with a concentration of 0.5110 mg / mL.

[0089] Accurately weigh 5.25 mg of emodin and place it in a 10 mL volumetric flask. Dissolve it in methanol and dilute to volume. Shake well to obtain a stock solution of emodin reference standard with a concentration of 0.5040 mg / mL.

[0090] Accurately pipette 0.2 mL of each of the single reference stock solutions of ferulic acid, astragaloside A, psoralen, isopsoralen, and emodin, and accurately pipette 2 mL of the reference stock solution of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside. Place them in a 10 mL volumetric flask, dilute to volume with methanol, shake well, filter through a 0.45 μm microporous membrane, and use the filtrate as the mixed reference solution.

[0091] Accurately pipette 10 μL each of the mixed reference solution and the test solution into the high-performance liquid chromatograph (HPLC) for detection, and record the HPLC chromatograms within 95 min to obtain the HPLC chromatograms of the reference solution. Figure 1 ) and the liquid chromatogram of the test solution ( Figure 2 The liquid chromatogram of the sample was imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012A) for similarity analysis to obtain the fingerprint chromatogram of Yishen Wufa Oral Liquid. The similarity analysis was performed using the median method, with a preferred time window width of 0.1 min. A total of 20 common peaks were identified. Peaks at the same retention time as the reference standards were identified. The results showed that peaks 11, 13, 15, 17, 18, and 20 were identified as ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, Astragalus complanatin A, psoralen, isopsoralen, and emodin, respectively. According to standard fingerprint spectral conditions, the 20 common peaks were assigned components to seven medicinal herbs: Psoralea corylifolia (roasted with black sesame seeds), Angelica sinensis, Poria cocos, Lycium barbarum, Polygonum multiflorum (processed with black beans and wine), Achyranthes bidentata, and Astragalus complanatus. Specifically, peak 1 was assigned to Polygonum multiflorum (processed with black beans and wine), peak 2 to Angelica sinensis, Poria cocos, Lycium barbarum, and Achyranthes bidentata, peak 3 to Lycium barbarum, peak 4 to Lycium barbarum, peak 5 to Polygonum multiflorum (processed with black beans and wine), peak 6 to Achyranthes bidentata, Lycium barbarum, and Angelica sinensis, peak 7 to Polygonum multiflorum (processed with black beans and wine), and peak 8 to Lycium barbarum. Peak 9 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 10 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 11 belongs to Angelica sinensis and Astragalus complanatus, Peak 12 belongs to Achyranthes bidentata and Lycium barbarum, Peak 13 belongs to Polygonum multiflorum (processed with black beans and wine), Peak 14 belongs to sodium benzoate, Peak 15 belongs to Astragalus complanatus, Peak 16 belongs to Polygonum multiflorum (processed with black beans and wine), Peak 17 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 18 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 19 belongs to Astragalus complanatus, Peak 20 belongs to Polygonum multiflorum (processed with black beans and wine).

[0092] Example 2

[0093] Fingerprint analysis of 10 batches of Yishen Wufa oral liquid

[0094] 1. Fingerprint similarity analysis

[0095] Ten batches of Yishen Wufa Oral Liquid were prepared according to the test sample preparation method, and analyzed under chromatographic conditions. The fingerprint chromatograms of Yishen Wufa Oral Liquid were recorded. The fingerprint chromatograms of the ten batches of Yishen Wufa Oral Liquid were analyzed using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (Version 2012A)" issued by the National Pharmacopoeia Commission. S1 was set as the reference chromatogram. Using the median method, the time window width was set to 0.1 min, and a total of 20 common peaks were identified. The similarity of the ten batches of Yishen Wufa Oral Liquid was greater than 0.90. The similarity evaluation results are shown in Table 1. The relative retention times of the 20 common peaks were basically consistent, while the relative peak areas showed significant differences. The standard fingerprint chromatogram data are shown in Table 2. The overlay diagram of the fingerprint chromatograms of the ten batches of Yishen Wufa Oral Liquid is shown in Table 2. Figure 3 , Figure 3 S1-S10 correspond to batch numbers 20221001, 20221002, 20221003, 20230201, 20230202, 20230203, 20230401, 20230402, 20230701, and 20230702, respectively.

[0096] Table 1. Similarity Evaluation Results of 110 Batches of Yishen Wufa Oral Liquid Samples

[0097]

[0098] Table 2 Standard fingerprint data

[0099]

[0100]

[0101] 2. Common peak identification: The HPLC chromatogram obtained from the mixed reference standards ( Figure 1 Peak 11 is ferulic acid, peak 13 is 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, peak 15 is astragaloside A, peak 17 is psoralen, peak 18 isopsoralen, and peak 20 is emodin) and the sample fingerprint spectrum ( Figure 3 The chromatographic peaks at the same retention time as the reference standards were identified. The results showed that peaks 11, 13, 15, 17, 18, and 20 among the 20 common peaks were identified as ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin, respectively.

[0102] 3. Preparation of test solutions for each control extract

[0103] Weigh 75g of Polygonum multiflorum (processed with black bean wine), add 8 times the amount of water and decoct twice, the first decoction for 3 hours and the second decoction for 1 hour. Filter, combine the filtrates, let stand at room temperature for 24 hours, take the supernatant, concentrate, and dry to constant weight to obtain Polygonum multiflorum extract; weigh 1% of the Polygonum multiflorum extract and place it in a 10mL volumetric flask, add 9mL of 70v / v% ethanol aqueous solution, extract by sonication (80W, 250kHz) for 30min, cool to room temperature, dilute to the mark with 70v / v% ethanol aqueous solution, shake well, filter through a 0.45μm microporous membrane, the filtrate is the test solution of Polygonum multiflorum reference extract.

[0104] Following the preparation method for the reference extract solution of Polygonum multiflorum, reference extract solutions of Angelica sinensis, Psoralea corylifolia (roasted with black sesame seeds), Lycium barbarum, Astragalus complanatus, Poria cocos, and Achyranthes bidentata were prepared. The amounts of each herb were weighed according to the prescription ratio of Yishen Wufa Oral Liquid: Angelica sinensis 18.75g, Psoralea corylifolia (roasted with black sesame seeds 5g) 9.39g, Lycium barbarum 18.75g, Astragalus complanatus 18.75g, Poria cocos 18.75g, and Achyranthes bidentata 18.75g. The reference extract solutions were analyzed by high-performance liquid chromatography (HPLC). The chromatograms are shown below. Figure 4 As shown.

[0105] Medicinal herb attribution: Chromatographic peaks were assigned according to standard fingerprint chromatographic conditions, see [link to relevant documentation]. Figure 4 , Figure 4 S1 is the test sample (Yishen Wufa Oral Liquid), and S2-S8 are Psoralea corylifolia (roasted with black sesame seeds), Angelica sinensis, Poria cocos, Lycium barbarum, Polygonum multiflorum (processed with black beans and wine), Achyranthes bidentata, and Astragalus complanatus, respectively. The 20 common peaks belong to seven medicinal materials: Psoralea corylifolia (roasted with black sesame seeds), Angelica sinensis, Poria cocos, Lycium barbarum, Polygonum multiflorum (processed with black beans and wine), Achyranthes bidentata, and Astragalus complanatus. Specifically, peak 1 belongs to Polygonum multiflorum (processed with black beans and wine), peak 2 belongs to Angelica sinensis, Poria cocos, Lycium barbarum, and Achyranthes bidentata, peak 3 belongs to Lycium barbarum, peak 4 belongs to Lycium barbarum, peak 5 belongs to Polygonum multiflorum (processed with black beans and wine), peak 6 belongs to Achyranthes bidentata, Lycium barbarum, and Angelica sinensis, peak 7 belongs to Polygonum multiflorum (processed with black beans and wine), peak 8 belongs to Lycium barbarum and Psoralea corylifolia (roasted with black sesame seeds), peak 9 belongs to... Peak 10 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 11 belongs to Angelica sinensis and Astragalus complanatus, Peak 12 belongs to Achyranthes bidentata and Lycium barbarum, Peak 13 belongs to Polygonum multiflorum (processed with black beans and wine), Peak 14 belongs to sodium benzoate, Peak 15 belongs to Astragalus complanatus, Peak 16 belongs to Polygonum multiflorum (processed with black beans and wine), Peak 17 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 18 belongs to Psoralea corylifolia (roasted with black sesame seeds), Peak 19 belongs to Astragalus complanatus, and Peak 20 belongs to Polygonum multiflorum (processed with black beans and wine).

[0106] This invention established HPLC fingerprints for 10 batches of Yishen Wufa Oral Liquid, with similarity greater than 0.90. Through systematic component identification and attribution of single medicinal materials with common peaks in different batches of the drug, it comprehensively reflects the chemical information contained in Yishen Wufa Oral Liquid and the current status of each component. It can more comprehensively reflect the quality of Yishen Wufa Oral Liquid and provide a reference for the quality control of Yishen Wufa Oral Liquid.

[0107] Example 3

[0108] Selection of detection wavelength

[0109] Sample batch 20221001 was used for testing. The overall effect of the chromatograms at different wavelengths (360nm, 320nm, 280nm, 254nm, 230nm, 210nm, and 203nm) was examined. Other chromatographic conditions were the same as in Example 1. The liquid chromatograms are shown below. Figure 5 , Figure 6 , Figure 7 , Figure 8 , Figure 9 , Figure 10 and Figure 11 .Depend on Figure 5 It can be seen that the number of peaks is very small at a wavelength of 360nm. From Figure 6 It can be seen that at a wavelength of 320nm, the response value is low and the number of peaks is relatively small. From Figure 7 It can be seen that at a wavelength of 280 nm, the chromatogram baseline is relatively flat, the peak shape is better, the number of peaks is greater, and the response value is the highest; however, the chromatographic peaks cannot reflect all the characteristics of medicinal materials. Figure 8 It can be seen that at a wavelength of 254 nm, the chromatogram baseline is relatively flat, the peak shape is better, the number of peaks is greater, and the response value is the highest, which is the optimal condition. Figure 9 It can be seen that at a wavelength of 230 nm, the chromatogram baseline is not flat, the baseline is not in a standard position, and the number of peaks is relatively small. From... Figure 10 It can be seen that at a wavelength of 210 nm, the chromatogram baseline is uneven, and the chromatographic peaks fluctuate significantly. From... Figure 11 It is known that the chromatogram baseline is unstable at a wavelength of 203 nm. Considering all factors, the fingerprint spectrum obtained with a detection wavelength of 254 nm best reflects the composition of the formulation and has the best peak shape; therefore, 254 nm is the optimal detection wavelength.

[0110] Example 4

[0111] Methodological investigation

[0112] The chromatographic conditions and preparation method of the test solution were the same as in Example 1. Batch 20221001 was used for testing.

[0113] (1) Precision test

[0114] The test solution was prepared using Yishen Wufa Oral Liquid, and injected six times consecutively. The retention times and areas of 20 major chromatographic peaks were recorded. Using 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as the reference peak, the relative peak area RSD < 3.0% (n = 6) and the relative retention time RSD < 1.0% (n = 6) demonstrated good instrument precision. The experimental results are shown in Table 3.

[0115] (2) Stability test

[0116] The test solution was prepared using Yishen Wufa Oral Liquid, and tests were conducted at 0h, 2h, 4h, 8h, 12h, and 24h. The retention times and areas of 20 major chromatographic peaks were recorded. Using 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as the reference peak, the relative peak area RSD < 3.0% (n = 6) and the relative retention time RSD < 1.0% (n = 6) demonstrated good stability of the sample within 24h. The experimental results are shown in Table 3.

[0117] (3) Repeatability test

[0118] Six test solutions were prepared from Yishen Wufa Oral Liquid, and the retention times and areas of 20 major chromatographic peaks were recorded. Using 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside as the reference peak, the relative peak area RSD < 3.0% (n = 6) and the relative retention time RSD < 1.0% (n = 6) demonstrated good stability. The experimental results are shown in Table 3.

[0119] Table 3. Methodological Investigation Experiment Results

[0120]

[0121] In summary, the fingerprint spectrum of the Yishen Wufa Oral Liquid constructed in this invention includes 20 characteristic peaks, including ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen, and emodin. The chromatographic peaks are abundant and attributed to the effective components of psoralen (roasted with black sesame seeds), angelica, poria, wolfberry, fleeceflower root (processed with black bean wine), achyranthes, and astragalus. This fingerprint spectrum more comprehensively reflects the overall chemical composition and quality of the Yishen Wufa Oral Liquid, facilitating more effective guidance on feed addition and strict standardization of production operations. Furthermore, it helps improve the safety and efficacy of the Yishen Wufa Oral Liquid in clinical use, demonstrating high application value.

[0122] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on the present invention without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing the fingerprint spectrum of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps: The kidney-tonifying and hair-darkening oral liquid was mixed with a low alcohol to obtain the test solution; The test solution and the mixed reference solution were subjected to high performance liquid chromatography (HPLC) to obtain sample HPLC chromatograms and reference HPLC chromatograms, respectively. The fingerprint chromatogram of Yishen Wufa Oral Liquid was obtained based on the sample HPLC chromatogram and the reference HPLC chromatogram. The reference standards in the mixed reference solution include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin; The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution of phosphoric acid is 0.05~0.2%; the elution method is gradient elution, and the gradient elution program includes: 0~20 min, mobile phase A volume fraction 5%; 20~30 min, mobile phase A volume fraction increases from 5% to 10%; 30~50 min, mobile phase A volume fraction increases from 10% to 20%; 50~70 min, mobile phase A volume fraction increases from 20% to 35%; 70~85 min, mobile phase A volume fraction increases from 35% to 65%; 85~95 min, mobile phase A volume fraction increases from 65% to 80%; the detection wavelength is 254~280 nm; The fingerprint spectrum of the Yishen Wufa Oral Liquid contains 20 characteristic peaks, including those of ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, Astragalus complanatus glycoside A, psoralen, isopsoralen, and emodin.

2. The fingerprint map construction method according to claim 1, characterized in that, The volume ratio of the kidney-tonifying and hair-darkening oral liquid to lower alcohols is 1:1~4; The lower alcohols include methanol and / or ethanol.

3. The fingerprint map construction method according to claim 1 or 2, characterized in that, The mixing includes ultrasonic mixing, wherein the ultrasonic mixing power is 50~100W, the frequency is 200~300kHz, and the time is 20~40min.

4. The fingerprint map construction method according to claim 1, characterized in that, In the mixed reference solution, the concentration of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside was 0.1~0.2 mg / mL, the concentrations of ferulic acid and astragaloside A were independently 0.01~0.02 mg / mL, and the concentrations of psoralen, isopsoralen and emodin were independently 0.005~0.02 mg / mL. The solvent in the mixed reference solution includes lower alcohols.

5. The fingerprint map construction method according to claim 1, characterized in that, The conditions for high-performance liquid chromatography detection also include: a mobile phase flow rate of 0.8~1.2 mL / min, an ultraviolet detector, a column temperature of 20~35℃, and an injection volume of 5~20 μL.

6. The application of the fingerprint spectrum of Yishen Wufa Oral Liquid constructed according to any one of claims 1 to 5 in the quality control of Yishen Wufa Oral Liquid.

7. A method for detecting multiple components of a kidney-tonifying and hair-darkening oral liquid, comprising the following steps: The kidney-tonifying and hair-darkening oral liquid to be tested was mixed with a low alcohol to obtain the sample solution to be tested; The sample solution to be tested was subjected to high performance liquid chromatography to obtain the detection results of multiple index components of Yishen Wufa oral liquid; the multiple index components include ferulic acid, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, astragaloside A, psoralen, isopsoralen and emodin. The conditions for the high-performance liquid chromatography detection include: the chromatographic column includes C... 18 The chromatographic column; the mobile phase system includes mobile phase A and mobile phase B, wherein mobile phase A is acetonitrile and mobile phase B is an aqueous solution of phosphoric acid, wherein the volume fraction of phosphoric acid in the aqueous solution of phosphoric acid is 0.05~0.2%; the elution method is gradient elution, and the gradient elution program includes: 0~20 min, mobile phase A volume fraction 5%; 20~30 min, the volume fraction of mobile phase A increases from 5% to 10%; 30~50 min, the volume fraction of mobile phase A increases from 10% to 20%; 50~70 min, the volume fraction of mobile phase A increases from 20% to 35%; 70~85 min, the volume fraction of mobile phase A increases from 35% to 65%; 85~95 min, the volume fraction of mobile phase A increases from 65% to 80%; the detection wavelength is 254~280 nm.

8. The multi-index component detection method according to claim 7, characterized in that, The volume ratio of the kidney-tonifying and hair-darkening oral liquid to lower alcohols is 1:1~4; The lower alcohols include methanol and / or ethanol; The mixing includes ultrasonic mixing, wherein the power of the ultrasonic mixing is 50~100W, the frequency is 200~300kHz, and the time is 20~40min; The detection conditions for the high-performance liquid chromatography (HPLC) detection also include: a mobile phase flow rate of 0.8~1.2 mL / min, a column temperature of 20~35℃, and an injection volume of 5~20 μL.

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