Five-flavor precious chrysanthemum tablet fingerprint spectrum, method for establishing same and multi-component content determination method

Abstract

The application provides a fingerprint spectrum of Wawei Zhenju tablets, a method for establishing the fingerprint spectrum and a multi-component content determination method, and belongs to the technical field of analysis.The method comprises the following steps: S1, preparation of a reference substance stock solution and a mixed reference substance solution; S2, preparation of a test sample solution; S3, preparation of a negative sample solution; S4, chromatographic conditions; and S5, determination.The application improves the consistency of the quality of Wawei Zhenju tablets in clinical use and production, improves the standardization, and provides a basis for comprehensively and effectively controlling the quality of the preparation and new drug development.

Description

Technical Field

[0001] This invention relates to the field of analytical technology, specifically to a fingerprint spectrum of Wuwei Zhenju tablets and its establishment method, as well as a method for determining the content of multiple components. Background Technology

[0002] Wuwei Zhenju Tablets are a new traditional Chinese medicine preparation and health food developed by the Guangxi University of Traditional Chinese Medicine Pharmaceutical Factory. It consists of five traditional Chinese medicines: cassia seed, chrysanthemum, kudzu root, wolfberry, and pearl. Cassia seed, listed as a food-medicine homology product by the Ministry of Health in 2018, has high medicinal value in the medical and health field. Domestic and international scholars have found through research that it has effects such as lowering blood pressure, protecting the liver and improving eyesight, antioxidation, and antibacterial properties. It is mainly used to treat eye diseases, constipation, hyperlipidemia, hypertension, and diabetes. Chrysanthemum is a commonly used traditional Chinese medicine in clinical practice, with a cultivation history of over 300 years in my country. It is often used to treat wind-heat colds, headaches, dizziness, red and swollen eyes, and blurred vision. Pharmacological studies have also shown that chrysanthemum mainly contains volatile oils, flavonoids, amino acids, and trace elements, which can promote coronary artery dilation, effectively prevent hypertension, lower hyperlipidemia, have anti-inflammatory and immune-protective effects, protect the liver, have antibacterial and anti-inflammatory effects, antiviral effects, and anti-aging effects. Kudzu root is considered both food and medicine, and can regulate blood sugar, lower blood lipids, reduce inflammation and oxidation, protect the cardiovascular system, improve kidney function, and protect the liver. Its isoflavones, particularly puerarin, have been shown in numerous experiments and clinical studies to have good liver-protective, immune-boosting, and blood pressure-lowering effects. Goji berries are rich in polysaccharides, flavonoids, and polyphenols. Multiple pharmacological experiments have shown that the polysaccharides and flavonoids in goji berries can effectively improve lipid metabolism, enhance the viability of retinal pigment epithelial cells, scavenge free radicals, and inhibit human liver cancer cells, thus playing a role in protecting the liver and kidneys, protecting the eyes, and providing antioxidant and anti-tumor effects. Pearl is one of the more than twenty substances approved by the Ministry of Health for use as a new food resource. It is mainly composed of calcium carbonate and contains various amino acids, such as alanine, aspartic acid, and leucine. It also contains conchiolin, water-soluble proteins, peptides, free amino acids, B vitamins, porphyrins, trace elements, and other functional components. It is commonly used to treat fatigue, improve immunity, combat aging, and prolong life. Clinically, it is often used for eye diseases, skin inflammation, and skin ulcers. When used together, these herbs can calm the mind, clear the liver and improve eyesight, and enhance immunity to regulate the body's metabolic functions, thereby achieving the purpose of protecting the liver. Clinical studies have found that this formula also has significant effects on symptoms such as liver qi stagnation, red and swollen eyes, palpitations, and insomnia.

[0003] Because the components of traditional Chinese medicine (TCM) compound prescriptions are quite complex, it is difficult to quantitatively study the main components that exert their effects and comprehensively reflect the overall quality of TCM preparations. Fingerprint spectroscopy can effectively, comprehensively and accurately reflect chemical component information, and provide an overall description and evaluation of TCM quality. On this basis, combining it with chemical pattern recognition can make the evaluation of TCM quality more reasonable, comprehensive and scientific. Summary of the Invention

[0004] The purpose of this invention is to propose a fingerprint spectrum of Wuwei Zhenju tablets and its establishment method and a method for determining the content of multiple components, thereby improving the consistency of the quality of Wuwei Zhenju tablets in clinical use and production, enhancing its standardization, and providing a basis for comprehensively and effectively controlling the quality of this preparation and for new drug development.

[0005] The technical solution of this invention is implemented as follows:

[0006] This invention provides a method for establishing the fingerprint spectrum of Wuwei Zhenju tablets, comprising the following steps:

[0007] S1. Preparation of reference stock solution and mixed reference solution: Prepare a reference solution from cassia aurantium; separately, accurately weigh appropriate amounts of reference standards neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and betaine; accurately transfer the prepared cassia aurantium reference solution and dilute to volume with methanol solution to prepare a mixed reference stock solution containing neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cassia aurantium, and betaine;

[0008] S2. Preparation of test solution: Take Wuwei Zhenju tablets, grind them into a fine powder, weigh the powder accurately, add methanol solution accurately, weigh, sonicate, cool, weigh and make up the lost weight, filter to obtain the test solution.

[0009] S3. Preparation of negative sample solution: Prepare freeze-dried powders of each negative sample lacking kudzu root, chrysanthemum, cassia seed and wolfberry according to the prescription ratio and preparation process, make tablets, grind finely, take the powder, weigh accurately, add methanol solution accurately, weigh, sonicate, cool, weigh and make up the lost weight, filter to obtain negative sample solution.

[0010] S4. Chromatographic conditions: Thermo Fisher Acclaim 120C18 column; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 260-300 nm; flow rate: 0.5-1.5 ml / min; injection volume: 10-20 μl; column temperature: 32-37℃;

[0011] S5. Determination: Accurately pipette the reference stock solution and mixed reference solution prepared in step S1, the test solution prepared in step S2, and the negative sample solution prepared in step S3, respectively, and inject them into the high performance liquid chromatograph. Determine the results according to the chromatographic conditions described in step S3.

[0012] As a further improvement of the present invention, the chromatographic column has a specification of 4.6×250mm and a particle size of 5μm.

[0013] As a further improvement of the present invention, the detection wavelength is 280 nm; the volumetric flow rate is 1 ml / min; the injection volume is 15 μl; and the column temperature is 35 °C.

[0014] As a further improvement of the present invention, the mass concentrations of neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cassia aurantium, and betaine in the mixed reference stock solution in step S1 are 7.5, 18.0, 10.5, 126.5, 31.0, 17.0, 22.0, 57.0, 1.986, and 98.0 μg / mL, respectively.

[0015] As a further improvement of the present invention, the ultrasonic treatment has a power of 60-100W and a frequency of 40-50kHz.

[0016] As a further improvement of the present invention, the gradient elution conditions are as follows:

[0017] Time / min Acetonitrile ratio 0-13 3％-8％ 13-30 8％-10％ 30-55 10％-17％ 55-76 17％-20％ 76-105 20％-40％ 105-113 40％-49％ 113-130 49％-87％

[0018] This invention further protects a fingerprint spectrum of Wuwei Zhenju tablets obtained by the above-mentioned method.

[0019] As a further improvement of the present invention, there are 10 common peaks, numbered sequentially from left to right, including peak 2 (neochlorogenic acid), peak 4 (chlorogenic acid), peak 6 (caffeic acid), peak 7 (puerarin), peak 11 (daidzein), peak 13 (isochlorogenic acid B), peak 14 (isochlorogenic acid A), peak 16 (isochlorogenic acid C), peak 22 (aurocarpine), and peak 26 (betaine).

[0020] This invention further protects a method for determining the content of multiple components, comprising the following steps:

[0021] S1. Preparation of test solution: Take Wuwei Zhenju tablets, grind them into a fine powder, weigh the powder precisely, add methanol solution precisely, weigh, sonicate, cool, weigh and make up the lost weight, filter to obtain the test solution.

[0022] S2. Preparation of mixed reference stock solution: Accurately weigh cassia aurantium, dissolve it in methanol, and dilute to volume to obtain cassia aurantium reference solution. Separately, accurately weigh chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A, dissolve them in an appropriate amount of methanol, and dilute to volume to obtain mixed reference solutions. Then, accurately transfer the above mixed reference solutions of chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A and the cassia aurantium reference solution, add puerarin, add an appropriate amount of methanol, and sonicate to completely dissolve. After cooling, dilute to volume and shake well to obtain a mixed reference stock solution containing chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium.

[0023] S3. Chromatographic conditions: Thermo Fisher Acclaim 120C18 column; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 260-300 nm; flow rate: 0.5-1.5 ml / min; injection volume: 10-20 μl; column temperature: 32-37℃;

[0024] S4. Determination: Accurately pipette the test solution prepared in step S1 and the mixed reference stock solution prepared in step S2, inject them into the high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S3.

[0025] As a further improvement of the present invention, the chromatographic column has specifications of 4.6 × 250 mm and a particle size of 5 μm; the detection wavelength is 280 nm; the flow rate is 1 ml / min; the injection volume is 15 μl; the column temperature is 35 °C; and the gradient elution conditions are as follows:

[0026] Time / min Acetonitrile ratio 0-13 3％-8％ 13-30 8％-10％ 30-55 10％-17％ 55-76 17％-20％ 76-105 20％-40％ 105-113 40％-49％ 113-130 49％-87％

[0027] This invention has the following beneficial effects: It improves the consistency of the quality of Wuwei Zhenju tablets in clinical use and production, and enhances its standardization. Based on the product production information such as the optimal extraction process of Guangxi University of Traditional Chinese Medicine Pharmaceutical Factory in the early stage, a high-performance liquid chromatography (HPLC) fingerprint spectrum is established, and cluster analysis and principal component analysis in chemical pattern recognition are combined for analysis. At the same time, the content of six active ingredients in Wuwei Zhenju tablets, namely chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia seed extract, is determined, aiming to provide a basis for comprehensively and effectively controlling the quality of this preparation and for new drug development. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 Overlay image of fingerprint chromatograms of 10 batches of Wuwei Zhenju tablets and control fingerprint chromatogram;

[0030] Figure 2 This is the chromatogram of the mixed reference solution;

[0031] Figure 3 Fingerprint spectrum of Wuwei Zhenju tablets (a traditional Chinese medicine formula);

[0032] Figure 4 This is the HPLC fingerprint of a negative sample of Wuwei Zhenju tablets.

[0033] Figure 5 Dendrogram of systematic cluster analysis for 10 batches of Wuwei Zhenju tablets;

[0034] Figure 6 Main component analysis charts for 10 batches of Wuwei Zhenju tablets;

[0035] Figure 7 This is a graph for examining specificity. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The instruments used in this invention are: an Agilent 1260 high-performance liquid chromatograph equipped with a UV detector (Agilent Technologies, USA); an ME204 / E electronic analytical balance (Shanghai Mettler Company); an XM-P06H stepless adjustable power ultrasonic cleaner (Xiaomei Ultrasonic Instruments (Kunshan) Co., Ltd.); an HWS-26 electric thermostatic water bath (Shanghai Qixin Scientific Instruments Co., Ltd.); a Milli-Q ultrapure water preparation system (Guangxi Nanning Bomei Biotechnology Co., Ltd.); and an SHB-III circulating water multi-purpose vacuum pump (Zhengzhou Great Wall Science & Industry Trade Co., Ltd.).

[0038] Reagents and drugs used in this invention

[0039] New chlorogenic acid (batch number: 22040213, purity ≥99.55%), daidzein (batch number: 22022207, purity ≥99.08%), isochlorogenic acid A (batch number: 22110413, purity ≥98.46%), isochlorogenic acid B (batch number: 23012705, purity ≥98.27%), isochlorogenic acid C (batch number: 22081001, purity ≥99.77%), and betaine (batch number: 22072612, purity ≥99.99%) were purchased from Chengdu Manster Biotechnology Co., Ltd.; chlorogenic acid (batch number: 110753-202) was purchased from Chengdu Manster Biotechnology Co., Ltd. 119 (purity ≥96.3%), caffeic acid (batch number: 110885-201703, purity ≥99.7%), puerarin (batch number: 110752-202217, purity ≥96.8%), daidzein (batch number: 111502-202003, purity ≥99.3%), and cassia seed extract (batch number: 111900-201605, purity ≥98.3%) were purchased from the China National Institutes for Food and Drug Control; acetonitrile and methanol (chromatographic grade, Thermo Fisher Scientific (China) Co., Ltd.); phosphoric acid (analytical grade); and ultrapure water (laboratory preparation).

[0040] Ten batches of Wuwei Zhenju tablets (numbered S1 to S10) were produced in the laboratory, with batch numbers 22070901, 22080408, 22080409, 22100709, 22110902, 22120101, 22120804, 23010602, 23020809, and 23030711. The origin information of the cassia seed, chrysanthemum, kudzu root, wolfberry, and Zhenju used in the experiment is shown in Table 1 below. The batch combinations were obtained using a medical statistical random number method, and the extracts were processed using a pharmaceutical factory's optimized process to obtain a dry powder before being tableted.

[0041] Table 1. Sample Information of Five-Flavor Chrysanthemum Tablets

[0042]

[0043]

[0044] Example 1

[0045] S1. Preparation of reference standard stock solutions and mixed reference standard solutions

[0046] Prepare a reference solution with a mass concentration of 0.03972 mg / mL by placing 1.986 mg of cassia aurantium in a 50 mL volumetric flask. Separately, accurately weigh appropriate amounts of the reference standards neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and betaine, and place them in a 25 mL volumetric flask. Accurately transfer 5 mL of the prepared cassia aurantium reference solution into the 25 mL volumetric flask and dilute to volume with 80% methanol to prepare a mixed reference stock solution with mass concentrations of 7.5, 18.0, 10.5, 126.5, 31.0, 17.0, 22.0, 57.0, 1.986, and 98.0 μg / mL, respectively.

[0047] S2. Preparation of the test solution

[0048] Take 20 pieces of Wuwei Zhenju tablets, grind them into a fine powder, take 0.1g of the powder, weigh it accurately, place it in a stoppered conical flask, add 10mL of 80% methanol accurately, weigh it, sonicate (power 80W, frequency 45kHz) for 30min, cool it, weigh it and make up the lost weight, filter it, filter the filtrate through a 0.22μm microporous membrane to obtain the test solution.

[0049] S3. Preparation of negative sample solution

[0050] According to the prescription ratio and preparation process, freeze-dried powders of negative samples lacking kudzu root, chrysanthemum, cassia seed, and wolfberry were prepared respectively. The powders were made into tablets. 20 tablets were taken, ground into a fine powder, and 0.1g of the powder was accurately weighed and placed in a stoppered conical flask. 10mL of 80% methanol was accurately added and weighed. The flask was ultrasonically treated (power 80W, frequency 45kHz) for 30min. After cooling, the flask was weighed and the lost weight was replenished. The flask was filtered and the filtrate was filtered through a 0.22μm microporous membrane to obtain the negative sample solution.

[0051] S4. Chromatographic conditions: Thermo Fisher Acclaim 120C18 column (4.6×250mm, 5μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 260nm; flow rate: 0.5ml / min; injection volume: 10μl; column temperature: 32℃.

[0052] The gradient elution protocol is shown in Table 2.

[0053] Table 2 Gradient elution scheme

[0054]

[0055]

[0056] S5. Determination: Accurately pipette the reference stock solution and mixed reference solution prepared in step S1, the test solution prepared in step S2, and the negative sample solution prepared in step S3, respectively, and inject them into the high performance liquid chromatograph. Determine the results according to the chromatographic conditions described in step S3.

[0057] Example 2

[0058] S1. Preparation of reference standard stock solutions and mixed reference standard solutions

[0059] Prepare a reference solution with a mass concentration of 0.03972 mg / mL by placing 1.986 mg of cassia aurantium in a 50 mL volumetric flask. Separately, accurately weigh appropriate amounts of the reference standards neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and betaine, and place them in a 25 mL volumetric flask. Accurately transfer 5 mL of the prepared cassia aurantium reference solution into the 25 mL volumetric flask and dilute to volume with 80% methanol to prepare a mixed reference stock solution with mass concentrations of 7.5, 18.0, 10.5, 126.5, 31.0, 17.0, 22.0, 57.0, 1.986, and 98.0 μg / mL, respectively.

[0060] S2. Preparation of the test solution

[0061] Take 20 pieces of Wuwei Zhenju tablets, grind them into a fine powder, take 0.1g of the powder, weigh it accurately, place it in a stoppered conical flask, add 10mL of 80% methanol accurately, weigh it, sonicate (power 80W, frequency 45kHz) for 30min, cool it, weigh it and make up the lost weight, filter it, filter the filtrate through a 0.22μm microporous membrane to obtain the test solution.

[0062] S3. Preparation of negative sample solution

[0063] According to the prescription ratio and preparation process, freeze-dried powders of negative samples lacking kudzu root, chrysanthemum, cassia seed, and wolfberry were prepared respectively. The powders were made into tablets. 20 tablets were taken, ground into a fine powder, and 0.1g of the powder was accurately weighed and placed in a stoppered conical flask. 10mL of 80% methanol was accurately added and weighed. The flask was ultrasonically treated (power 80W, frequency 45kHz) for 30min. After cooling, the flask was weighed and the lost weight was replenished. The flask was filtered and the filtrate was filtered through a 0.22μm microporous membrane to obtain the negative sample solution.

[0064] S4. Chromatographic conditions: Thermo Fisher Acclaim 120C18 column (4.6×250mm, 5μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 300nm; flow rate: 1.5ml / min; injection volume: 20μl; column temperature: 37℃.

[0065] The gradient elution protocol is shown in Table 2.

[0066] Table 2 Gradient elution scheme

[0067] Time / min Acetonitrile ratio 0-13 3％-8％ 13-30 8％-10％ 30-55 10％-17％ 55-76 17％-20％ 76-105 20％-40％ 105-113 40％-49％ 113-130 49％-87％

[0068] S5. Determination: Accurately pipette the reference stock solution and mixed reference solution prepared in step S1, the test solution prepared in step S2, and the negative sample solution prepared in step S3, respectively, and inject them into the high performance liquid chromatograph. Determine the results according to the chromatographic conditions described in step S3.

[0069] Example 3

[0070] S1. Preparation of reference standard stock solutions and mixed reference standard solutions

[0071] Prepare a reference solution with a mass concentration of 0.03972 mg / mL by placing 1.986 mg of cassia aurantium in a 50 mL volumetric flask. Separately, accurately weigh appropriate amounts of the reference standards neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and betaine, and place them in a 25 mL volumetric flask. Accurately transfer 5 mL of the prepared cassia aurantium reference solution into the 25 mL volumetric flask and dilute to volume with 80% methanol to prepare a mixed reference stock solution with mass concentrations of 7.5, 18.0, 10.5, 126.5, 31.0, 17.0, 22.0, 57.0, 1.986, and 98.0 μg / mL, respectively.

[0072] S2. Preparation of the test solution

[0073] Take 20 pieces of Wuwei Zhenju tablets, grind them into a fine powder, take 0.1g of the powder, weigh it accurately, place it in a stoppered conical flask, add 10mL of 80% methanol accurately, weigh it, sonicate (power 80W, frequency 45kHz) for 30min, cool it, weigh it and make up the lost weight, filter it, filter the filtrate through a 0.22μm microporous membrane to obtain the test solution.

[0074] S3. Preparation of negative sample solution

[0075] According to the prescription ratio and preparation process, freeze-dried powders of negative samples lacking kudzu root, chrysanthemum, cassia seed, and wolfberry were prepared respectively. The powders were made into tablets. 20 tablets were taken, ground into a fine powder, and 0.1g of the powder was accurately weighed and placed in a stoppered conical flask. 10mL of 80% methanol was accurately added and weighed. The flask was ultrasonically treated (power 80W, frequency 45kHz) for 30min. After cooling, the flask was weighed and the lost weight was replenished. The flask was filtered and the filtrate was filtered through a 0.22μm microporous membrane to obtain the negative sample solution.

[0076] S4. Chromatographic conditions: Thermo Fisher Acclaim 120C18 column (4.6×250mm, 5μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 280nm; flow rate: 1ml / min; injection volume: 15μl; column temperature: 35℃.

[0077] The gradient elution protocol is shown in Table 2.

[0078] Table 2 Gradient elution scheme

[0079] Time / min Acetonitrile ratio 0-13 3％-8％ 13-30 8％-10％ 30-55 10％-17％ 55-76 17％-20％ 76-105 20％-40％ 105-113 40％-49％ 113-130 49％-87％

[0080] S5. Determination: Accurately pipette the reference stock solution and mixed reference solution prepared in step S1, the test solution prepared in step S2, and the negative sample solution prepared in step S3, respectively, and inject them into the high performance liquid chromatograph. Determine the results according to the chromatographic conditions described in step S3.

[0081] Example 4 Precision Test

[0082] Take the powder of Wuwei Zhenju tablets (S6) and prepare a test solution according to the method in Example 3. Inject the solution six times consecutively under the chromatographic conditions in Example 3 and record the chromatogram. Peak 7 in the obtained chromatogram has a good peak shape and a high response value. It is set as the reference peak. The RSD of the relative retention time of each common peak is calculated to be <0.30%, and the RSD of the relative peak area is <2.27%, indicating that the instrument has good precision.

[0083] Example 5 Stability Test

[0084] Take the powder of Wuwei Zhenju tablets (S6) and prepare the test solution according to the method in Example 3. After being placed at room temperature for 0, 3, 6, 9, 12 and 24 h respectively, the sample was injected and measured according to the chromatographic conditions in Example 3. The chromatogram was recorded. With peak No. 7 as the reference peak, the RSD of the relative retention time of each common peak was calculated to be <0.75% and the RSD of the relative peak area was <2.22%, indicating that the test solution has good stability within 24 h.

[0085] Example 6 Repeatability Test

[0086] Take the powder of Wuwei Zhenju tablets (S6) and prepare 6 test solutions according to the method in Example 3. Determine the solution under the chromatographic conditions in Example 3, record the chromatogram, take peak No. 7 as the reference peak, calculate the RSD of the relative retention time of each common peak (<0.35%) and the RSD of the relative peak area (<2.69%), indicating that the method has good repeatability.

[0087] Example 7: Establishment of HPLC fingerprint chromatogram, identification of common peaks, and evaluation of similarity.

[0088] 1. Establishment of fingerprint spectrum

[0089] Following the method described in Example 3, batches S1-S10 of Wuwei Zhenju tablets were prepared into corresponding test solutions and injected for detection, with chromatograms recorded. The chromatographic image data of the 10 batches of Wuwei Zhenju tablets were saved in "AIA" format and sequentially imported into the "2012 Edition Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for analysis. Peak 7 had a higher peak area and better resolution, therefore it was selected as the reference peak. Through comparison with the reference standard, it was identified as puerarin. Using the mean method (time window length = 0.10), the full spectrum peaks were corrected at multiple points and then matched to obtain the fingerprint chromatograms of the 10 batches of Wuwei Zhenju tablets. Figure 1 (S6) and a reference fingerprint spectrum, from which 26 common peaks were identified.

[0090] 2. Identification and Attribution of Common Peaks

[0091] The mixed reference solution from Example 3 was injected and analyzed under the chromatographic conditions described in Example 3 to obtain the chromatogram of the mixed reference solution. Figure 2 Based on the HPLC fingerprint chromatograms of 10 batches of Wuwei Zhenju tablets ( Figure 1 ) and the fingerprint spectrum of Wuwei Zhenju tablets () Figure 3 The relative retention times of each chromatographic peak and the chromatogram of the mixed reference solution. Figure 2 By comparison, 10 common peaks were identified, namely, peak 2 (neochlorogenic acid), peak 4 (chlorogenic acid), peak 6 (caffeic acid), peak 7 (puerarin), peak 11 (daidzein), peak 13 (isochlorogenic acid B), peak 14 (isochlorogenic acid A), peak 16 (isochlorogenic acid C), peak 22 (aurocarin), and peak 26 (betaine).

[0092] The negative sample solutions prepared in Example 3 were injected for testing, and chromatograms of each negative sample solution were obtained. The obtained negative sample chromatograms were simultaneously compared and assigned common peaks with the chromatograms of the mixed reference standard and the fingerprint chromatogram of Wuwei Zhenju tablets. Figure 4 The results showed that samples 3, 5, 7-9, and 11 came from kudzu root; sample 26 came from wolfberry; samples 2, 4, 6, 10, 12-16, and 20 came from chrysanthemum; and samples 17-19 and 21-25 came from cassia seed.

[0093] Example 8 Similarity Evaluation

[0094] The similarity of the fingerprint chromatograms of 10 batches of Wuwei Zhenju tablets was evaluated using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012 Edition)". The results showed that the similarity between the fingerprint chromatograms of the 10 batches of Wuwei Zhenju tablets and the control fingerprint chromatograms were 0.997, 0.998, 0.990, 0.986, 0.994, 0.998, 0.996, 0.998, 0.995, and 0.985, respectively, all above 0.98 (Table 3). This indicates that the components among the batches are basically similar, the overall quality is relatively stable, and the average RSD value of the relative retention time of each common peak is less than 1.0%.

[0095] Table 3. Similarity Evaluation of 10 Batches of Wuwei Zhenju Tablets

[0096]

[0097] Example 9 Chemical Pattern Recognition

[0098] 1. Cluster Analysis (CA)

[0099] The peak area data of 26 common peaks from 10 batches of Wuwei Zhenju tablets were imported into IBM SPSS Statistics 26.0 software for cluster analysis. Hierarchical clustering was used, with each common peak as a variable. The mean linkage method and squared Euclidean distance were used as the metric. The clustering results are shown below. Figure 5 According to the dendrogram, when the Euclidean distance is 10, these 10 batches of Wuwei Zhenju slices can be divided into 4 categories: S1, S3, S4, S8, and S9 form one category; S10 forms a separate category; S2, S6, and S7 form another category; and S5 forms a separate category. This indicates that there are certain differences in composition between batches, which is preliminarily speculated to be related to the origin of the original medicinal slices.

[0100] 2. Principal Component Analysis (PCA)

[0101] The peak areas of 26 common peaks of 10 batches of Wuwei Zhenju tablets were imported into SPSS26.0 software for principal component factor analysis

[20] . The principal component eigenvalues ​​and contribution rates were obtained, as shown in Table 4. As can be seen from the table, when the principal component is 5, the cumulative contribution rate can reach 96.877% and the eigenvalues ​​are all greater than 1, which is 1.284. This suggests that principal component factors 1 to 5 can represent most of the information data of the 26 common peaks in the fingerprint spectrum of Wuwei Zhenju tablets. The contribution values ​​of each peak to the main components of 10 batches of Zhenju chewable tablets can be reflected by the principal component loading matrix

[21] . As shown in Table 5, in the main component 1, the absolute values ​​of the loadings of peaks 4 (chlorogenic acid), 6 (caffeic acid), 12, 13 (isochlorogenic acid B), 14 (isochlorogenic acid A), and 16 (isochlorogenic acid C) are relatively large, and the factor loading coefficients are 0.971, 0.968, 0.962, 0.916, 0.908, and 0.886, respectively, indicating that they have a greater impact on the quality of the preparation. Peak 24 is the main factor affecting the main component 2, and the factor loading coefficient is 0.934. Peak 5 is the feature vector that has a greater impact on the main component 3, and the factor loading coefficient is 0.698. The main component 4 mainly reflects the information of peak 1, and the factor loading coefficient is 0.838.

[0102] To better and more objectively reflect the differences between batches of Wuwei Zhenju tablets, the peak area results of the common peaks of the 10 batches of Wuwei Zhenju tablets samples were imported into SIMCA 14.1 statistical software. After standardizing the 26 common peaks, principal component analysis was performed using the common peak area as a variable. The results showed that the 10 batches of Wuwei Zhenju tablets samples were clustered into 4 categories: the first category consisted of S1, S3, S4, S8, and S9, with S10 forming a separate category; the third category consisted of S2, S6, and S7; and S5 formed the fourth category. Figure 6 The principal component analysis results are basically consistent with the cluster analysis results above.

[0103] Table 4. Principal Component Eigenvalues ​​and Contribution Rates

[0104] principal component Eigenvalues Contribution rate / % Cumulative contribution rate / % 1 13.578 52.222 52.222 2 4.737 18.218 70.440 3 3.525 13.557 83.997 4 2.065 7.940 91.938 5 1.284 4.940 96.877

[0105] Table 5 Principal Component Loading Matrix

[0106]

[0107] Peak 10 0.744 0.052 -0.212 0.239 0.539 Peak 7 0.729 0.280 0.570 -0.246 -0.048 Peak 8 0.705 0.248 0.527 -0.392 0.075 Peak 15 0.685 -0.320 -0.316 0.039 -0.499 Peak 24 0.254 0.934 -0.156 0.125 -0.080 Peak 22 0.356 0.864 -0.283 0.144 0.033 Peak 26 0.260 -0.861 0.046 -0.081 0.193 Peak 23 0.351 0.784 -0.216 0.176 0.305 Peak 25 0.109 0.776 -0.124 0.431 -0.423 Peak 5 0.497 0.399 0.698 -0.068 -0.293 Peak 18 0.490 -0.007 -0.677 -0.535 0.039 Peak 1 0.059 -0.211 0.441 0.838 0.196

[0108] Example 10

[0109] This embodiment provides a method for determining the content of multiple components, including the following steps:

[0110] S1. Preparation of test solution: Take 20 pieces of Wuwei Zhenju tablets, grind them into a fine powder, take 0.1g of the powder, weigh it accurately, place it in a stoppered conical flask, add 10mL of 80% methanol accurately, weigh it, sonicate (power 80W, frequency 45kHz) for 30min, cool it, weigh it and make up the lost weight, filter it, filter the filtrate through a 0.22μm microporous membrane to obtain the test solution.

[0111] S2. Preparation of mixed reference solution

[0112] Accurately weigh cassia aurantium into a 25 mL volumetric flask, dissolve it in an appropriate amount of methanol, and dilute to volume to obtain a 0.356 mg / mL cassia aurantium reference solution. Separately, accurately weigh chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A into the same 5 mL volumetric flask, dissolve them in an appropriate amount of methanol, and dilute to volume to obtain mixed reference solutions of 1.086, 1.046, and 1.028 mg / mL, respectively. Separately, accurately transfer 2 mL of the mixed reference solution of chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A, and 1 mL of the reference solution of cassia aurantium to the same 25 mL volumetric flask. Weigh 12.24 mg of puerarin into the same 25 mL volumetric flask, add an appropriate amount of methanol, and sonicate until completely dissolved. After cooling, dilute to volume and shake well to obtain a mixed reference stock solution with mass concentrations of 0.0869, 0.4898, 0.1408, 0.0838, 0.0822, and 0.0142 mg / mL for chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium, respectively.

[0113] S3. Chromatographic conditions: Thermo Fisher Acclaim 120V18 column (4.6×250mm, 5μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 280nm; flow rate: 1ml / min; injection volume: 15μl; column temperature: 35℃.

[0114] The gradient elution protocol is shown in Table 2.

[0115] Example 11 Specificity Investigation

[0116] Take the above-mentioned mixed reference solution, the test solution prepared in Example 10, and the negative control solution prepared in Example 3, and analyze them separately under the chromatographic conditions in Example 10 to obtain HPLC chromatograms, as shown below. Figure 7 The results showed that the theoretical plate numbers of chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium were all no less than 25,000, and the resolution with adjacent peaks was greater than 1.5, indicating that the resolution with adjacent peaks and the theoretical plate number of each peak met the requirements.

[0117] Example 12: Examination of Linear Relationships

[0118] The mixed reference stock solution prepared in Example 10 was precisely measured and serially diluted with methanol to prepare mixed reference standards of six series concentrations. These were then analyzed under the chromatographic conditions described above, and the chromatograms were recorded. Regression equations were obtained by plotting the mass concentration (X, mg / μL) of each reference standard on the x-axis and the peak area (A) on the y-axis, as detailed in Table 6. The results showed that the mass concentrations of chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium exhibited good linear relationships with the peak area within the ranges of 0.00850-0.0869, 0.0452-0.4898, 0.0146-0.1408, 0.00826-0.0838, 0.00779-0.0822, and 0.00144-0.0142 mg / μL, respectively, with correlation coefficients not less than 0.9990.

[0119] Table 6. Linear equations, correlation coefficients, and linear ranges of six indicator components in Wuwei Zhenju tablets.

[0120] Element Regression equation Correlation coefficient r Linear range / mg / μL chlorogenic acid Y = 22639.9755X - 9.8258 0.99968 0.00850-0.0869 Puerarin Y = 22337.8506X + 95.1022 0.99982 0.0452-0.4898 daidzein Y = 23794.6833X + 9.6820 0.99972 0.0146-0.1408 Isochlorogenic acid B Y = 27886.2793X - 20.0430 0.99902 0.00826-0.0838 Isochlorogenic acid A Y = 27488.6431X - 8.4670 0.99976 0.00779-0.0822 Orange-yellow cassia seed extract Y = 86547.7432X + 7.6197 0.99991 0.00144-0.0142

[0121] Example 13 Precision Test

[0122] Take the same batch of Wuwei Zhenju tablets (S6), prepare the test solution according to the preparation method of Example 10, and inject the sample 6 times consecutively under the chromatographic conditions of Example 10 to obtain the chromatogram. Record the peak area of ​​the chromatogram and calculate the peak area RSD values ​​of the 6 mixed standards, which are 1.09%, 0.40%, 0.47%, 0.49%, 0.51%, and 0.39%, respectively, indicating that the instrument has good precision.

[0123] Example 14 Stability Test

[0124] Take the same batch of Wuwei Zhenju tablets (S6), prepare the test solution according to the preparation method of Example 10, and after standing at room temperature for 0h, 3h, 6h, 9h, 12h and 24h, inject the sample according to the chromatographic conditions of Example 10, and measure the chromatograms. Record the peak areas of the chromatograms, and calculate the peak area RSD values ​​of the 6 mixed standards. The values ​​are 1.78%, 0.57%, 0.78%, 1.61%, 1.06% and 0.63%, respectively, indicating that the sample has good stability within 24h.

[0125] Example 15 Repeatability Test

[0126] Six test solutions were prepared from the same batch of Wuwei Zhenju tablets (S6) according to the preparation method in Example 10. Chromatographic analysis was performed under the chromatographic conditions of Example 10, and the peak areas were recorded. The RSD values ​​of the peak areas of the six mixed standards were calculated to be 2.64%, 2.07%, 1.92%, 2.31%, 2.67%, and 2.54%, respectively, indicating good repeatability of the method. The average contents of the six components were also measured to be 5.053, 43.438, 9.259, 4.829, 5.035, and 0.688 mg / g, respectively.

[0127] Example 16: Recovery Test

[0128] Six parallel portions of the same batch of Wuwei Zhenju tablets (S6), each approximately 0.05 g, were accurately weighed and placed in stoppered conical flasks. 10 mL of a mixed reference solution (chlorogenic acid 0.235 mg / mL, puerarin 2.0967 mg / mL, daidzein 0.4367 mg / mL, isochlorogenic acid B 0.2583 mg / mL, isochlorogenic acid A 0.2483 mg / mL, and cassia seed extract 0.0331 mg / mL) were accurately added at a 1:1 ratio to each flask. The test solution was prepared according to the method described in Example 10. Chromatographic analysis was performed under the conditions of Example 10, and chromatograms were obtained. Peak areas were recorded, and the recovery rates of each component were calculated. The results showed that the average recoveries of chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium were 101.73%, 95.73%, 97.42%, 103.22%, 96.99%, and 97.66%, respectively, indicating that the recovery rate of this method met the requirements (see Table 7).

[0129] Table 7. Results of the recovery rate test for six components in Wuwei Zhenju tablets.

[0130]

[0131]

[0132] Example 17: Determination of Sample Content

[0133] Ten batches of Wuwei Zhenju tablets were taken, and test solutions were prepared according to the method in Example 10. The chromatographic conditions of Example 10 were used for determination, and HPLC chromatograms were obtained. The mass fractions of chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia seed extract in the ten batches of samples were calculated by peak area using the external standard one-point method, as shown in Table 8. The contents were 2.902-5.819, 3.693-49.680, 1.449-10.654, 2.768-7.724, 2.941-6.343, and 0.071-0.089 mg / g, respectively.

[0134] Table 8. Results of content determination of six chemical components in 10 batches of Wuwei Zhenju tablets (n=10)

[0135]

[0136] This invention establishes the fingerprint spectrum of Wuwei Zhenju tablets by first performing a full-wavelength scan. Based on the number of peaks and the maximum absorption wavelength of each peak, a wavelength of 280 nm was ultimately selected for the entire process, as this wavelength exhibits strong chromatographic peak characteristics. Subsequently, the separation effects on each component were investigated using different mobile phase compositions, including methanol-water, methanol-0.1% phosphoric acid, acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, acetonitrile-0.1% formic acid, and acetonitrile-0.1% glacial acetic acid. The results showed that acetonitrile-0.1% phosphoric acid provided the best separation effect with more peaks. Therefore, acetonitrile-0.1% phosphoric acid was used as the mobile phase composition for further exploration. For sample preparation, the sample was first extracted with methanol and anhydrous ethanol via ultrasonic extraction. Based on the number and area of ​​peaks, methanol extraction was selected. Further ultrasonic extraction with 30%, 50%, 80%, and 100% methanol was then performed. It was found that 80% methanol extraction yielded the highest number of peaks and the highest content.

[0137] Establishing a fingerprint chromatogram for Wuwei Zhenju tablets using high-performance liquid chromatography (HPLC) and determining the content of its components can effectively control the quality of Wuwei Zhenju tablets. This study found that the established method is relatively stable, controllable, and reproducible, with all indicative components present in the fingerprint chromatogram. Therefore, this research is beneficial for evaluating the quality of Wuwei Zhenju tablets, providing a basis for quality control of this tablet, and offering insights for new drug development and the utilization of medicinal resources.

[0138] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for establishing a fingerprint spectrum of Wuwei Zhenju tablets, characterized in that, Includes the following steps: S1. Preparation of reference stock solution and mixed reference solution: Prepare a reference solution of cassia aurantium by taking cassia aurantium; separately take appropriate amounts of reference standards neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and betaine, accurately weigh them, accurately transfer the above-prepared cassia aurantium reference solution, and dilute to volume with methanol solution to prepare a mixed reference stock solution containing neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cassia aurantium, and betaine; S2. Preparation of test solution: Take Wuwei Zhenju tablets, grind them into a fine powder, weigh the powder accurately, add methanol solution accurately, weigh, sonicate, cool, weigh and make up the lost weight, filter to obtain the test solution. S3. Preparation of negative sample solution: Prepare freeze-dried powders of each negative sample lacking kudzu root, chrysanthemum, cassia seed and wolfberry according to the prescription ratio and preparation process, make them into tablets, grind them into fine powder, take the powder, weigh it accurately, add methanol solution accurately, weigh it, sonicate it, cool it, weigh it and make up the lost weight, filter it to obtain the negative sample solution. S4. Chromatographic conditions: Thermo Fisher Acclaim 120 C18 column; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 260-300 nm; flow rate: 0.5-1.5 ml / min; injection volume: 10-20 μl; column temperature: 32-37℃. The gradient elution conditions are as follows: ； S5. Determination: Accurately pipette the reference stock solution and mixed reference solution prepared in step S1, the test solution prepared in step S2, and the negative sample solution prepared in step S3, respectively, and inject them into the high performance liquid chromatograph. Determine the results according to the chromatographic conditions described in step S3.

2. The method for establishing the fingerprint spectrum of Wuwei Zhenju tablets according to claim 1, characterized in that, The chromatographic column has a size of 4.6 × 250 mm and a particle size of 5 μm.

3. The method for establishing the fingerprint spectrum of Wuwei Zhenju tablets according to claim 1, characterized in that, The detection wavelength is 280 nm; the volumetric flow rate is 1 ml / min; the injection volume is 15 μl; and the column temperature is 35 °C.

4. The method for establishing the fingerprint spectrum of Wuwei Zhenju tablets according to claim 1, characterized in that, In step S1, the mass concentrations of neochlorogenic acid, chlorogenic acid, caffeic acid, puerarin, daidzein, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, cassia aurantium, and betaine in the mixed reference stock solution were 7.5, 18.0, 10.5, 126.5, 31.0, 17.0, 22.0, 57.0, 1.986, and 98.0 μg / mL, respectively.

5. The method for establishing the fingerprint spectrum of Wuwei Zhenju tablets according to claim 1, characterized in that, The ultrasonic treatment has a power of 60-100W and a frequency of 40-50kHz.

6. A method for determining the content of multiple components in Wuwei Zhenju tablets, characterized in that, The multiple components are chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia seed extract, and include the following steps: S1. Preparation of test solution: Take Wuwei Zhenju tablets, grind them into a fine powder, weigh the powder precisely, add methanol solution precisely, weigh, sonicate, cool, weigh and make up the lost weight, filter to obtain the test solution. S2. Preparation of mixed reference stock solution: Accurately weigh cassia aurantium, dissolve it in methanol, and dilute to volume to obtain cassia aurantium reference solution. Separately, accurately weigh chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A, dissolve them in an appropriate amount of methanol, and dilute to volume to obtain mixed reference solution. Separately, accurately transfer the above mixed reference solution of chlorogenic acid, isochlorogenic acid B, and isochlorogenic acid A and the cassia aurantium reference solution, add puerarin, add an appropriate amount of methanol, sonicate to completely dissolve, cool, dilute to volume, and shake well to obtain a mixed reference stock solution containing chlorogenic acid, puerarin, daidzein, isochlorogenic acid B, isochlorogenic acid A, and cassia aurantium. S3. Chromatographic conditions: Thermo Fisher Acclaim 120 C18 column; mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution, gradient elution; detection wavelength: 260-300 nm; flow rate: 0.5-1.5 ml / min; injection volume: 10-20 μl; column temperature: 32-37℃; The gradient elution conditions are as follows: ； S4. Determination: Accurately pipette the test solution prepared in step S1 and the mixed reference stock solution prepared in step S2, inject them into the high performance liquid chromatograph, and determine them according to the chromatographic conditions described in step S3.

7. The method for determining the content of multiple components according to claim 6, characterized in that, The chromatographic column has a specification of 4.6×250mm and a particle size of 5μm. The detection wavelength is 280nm; the volumetric flow rate is 1ml / min; the injection volume is 15μl; and the column temperature is 35℃.

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