Quality control method and application of eyesight-improving and kidney-nourishing tablets

Abstract

The invention discloses a quality control method and application of eyesight-improving and kidney-nourishing tablets, and relates to the technical field of medicines.The control method comprises the steps that firstly, a fingerprint spectrum of the eyesight-improving and kidney-nourishing tablets is established and used for quality detection of the eyesight-improving and kidney-nourishing tablets, and effective components of six medicinal materials in the eyesight-improving and kidney-nourishing tablets can be detected; meanwhile, the content of glucose, salidroside, chlorogenic acid, verbascoside, catalpol, specnuezhenide, rehmannia D, 3, 5-O-dicaffeoylquinic acid, 4, 5-O-dicaffeoylquinic acid, chrysophanol, luteolin, luteoloside, aurantio-obtusin and beta-ecdysterone in a sample can be quantitatively detected. Establishment of the fingerprint spectrum detection method of the eyesight-improving and kidney-nourishing tablets and determination of standard fingerprint spectrum information of the eyesight-improving and kidney-nourishing tablets have important significance on work in the aspects of product quality control, authenticity identification, origin traceability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a quality control method for Mingmu Zishen Tablets and its application. Background Art

[0002] Mingmu Zishen Tablets have the effects of nourishing the liver and kidney, and improving eyesight and benefiting the eyes. They can be used to treat symptoms such as dim eyesight, dizziness and tinnitus, and soreness and weakness of the waist and knees caused by yin deficiency of the liver and kidney. At the same time, it also has a good therapeutic effect on cataracts with both liver and kidney yin deficiency and qi and blood stasis obstruction. The formula of Mingmu Zishen Tablets consists of Chinese wolfberry, glossy privet fruit, rehmannia root, cassia seed, chrysanthemum, and achyranthes bidentata. Among them, Chinese wolfberry has the effects of nourishing the liver and kidney, benefiting essence and blood, and improving eyesight; glossy privet fruit can tonify the liver and kidney, clear away deficiency heat and improve eyesight. This product is tonifying without being greasy and is relatively cool in nature, being an excellent product for mild tonification. Rehmannia root can clear heat and cool blood, and nourish yin and promote the production of body fluid. Chrysanthemum can clear heat, soothe the liver and improve eyesight. When chrysanthemum and Chinese wolfberry are used together, they can clear heat, nourish the liver and improve eyesight. Cassia seed has the effects of clearing the liver and improving eyesight, and loosening the bowels to relieve constipation. It can not only treat constipation due to liver heat but also provide an outlet for heat pathogens. Chrysanthemum and cassia seed together can clear heat and soothe the liver to treat the symptoms of hyperactivity of liver yang. Achyranthes bidentata can remove blood stasis and dredge collaterals, tonify the liver and kidney, strengthen tendons and bones, and promote diuresis and relieve stranguria. At the same time, its diuretic effect can also help with removing blood stasis and dredging collaterals. In view of the complementary effects of each medicinal material in this formula and the combined effect showing its efficacy, the effective detection of its active ingredients has become a key process for controlling product quality. If the Mingmu Zishen Tablet product is to exert the best therapeutic effect, it is necessary to conduct long-term detection of its active ingredients in all aspects to achieve the best state of effectively relieving the pain of patients with this product.

[0003] The formula of Mingmu Zishen Tablets consists of six traditional Chinese medicines, namely Chinese wolfberry, glossy privet fruit, rehmannia root, cassia seed, chrysanthemum, and achyranthes bidentata. The active ingredients are complex. The detection methods in the existing quality control standards only perform quantitative detection of chrysophanol in cassia seed and cannot achieve qualitative and quantitative detection of the active ingredients in all medicinal materials.

[0004] In the current quality control standard of Mingmu Zishen Tablets, only the content of chrysophanol in cassia seed is controlled, and the identification and comparison of traditional Chinese medicines for cassia seed, glossy privet fruit, and Chinese wolfberry are carried out. There is no detection of the active ingredients in all its medicinal materials, and it is impossible to comprehensively and effectively control the quality of the pharmaceutical preparation. There is a lack of a quality detection method for comprehensively monitoring the product quality.

[0005] In view of the fact that the formula of Mingmu Zishen Tablets consists of six traditional Chinese medicines, namely Chinese wolfberry, glossy privet fruit, rehmannia root, cassia seed, chrysanthemum, and achyranthes bidentata, and the components that play a therapeutic role are not single components. Only quantitative control of chrysophanol in cassia seed in the quality standard cannot ensure the uniformity and stability of the quality of each batch of products, nor can it evaluate the stability and quality of the purchased traditional Chinese medicines. There is a lack of a detection method for controlling the active ingredients in the medicinal materials. Summary of the Invention

[0006] The object of the present invention is to solve the above technical problems, and to provide a quality control method for Mingmu Zishen tablets and its application.

[0007] A quality control method for Mingmu Zishen tablets of the present invention is carried out as follows:

[0008] 1) Preparation of the test sample solution: Take Mingmu Zishen tablets, grind them finely, add methanol, perform ultrasonic treatment, filter, and take the

[0009] continued

[0010] filtrate to obtain the solution.

[0011] 2) Preparation of the reference solution: Weigh a proper amount of the reference substance precisely, add methanol to make a solution to obtain the solution.

[0012] 3) Precisely absorb the reference solution and the test sample solution respectively and inject them into a liquid chromatograph for determination to obtain the fingerprint chromatogram of the test sample.

[0013] The chromatographic conditions for the determination by the liquid chromatograph are as follows: Using octadecylsilane chemically bonded silica as the filler, using acetonitrile - 0.2% formic acid as the mobile phase, gradient elution for detection, flow rate 0.9 - 1.1 ml / min, column temperature 25 - 30 °C, detection wavelength 250 - 330 nm, injection volume 5 - 20 μL, running time 130 min 120 - 160 min.

[0014] The gradient elution conditions are as follows:

[0015]

[0016]

[0017] 4) Import the integral signal of the fingerprint chromatogram of the sample to be tested into the software of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints 2012 Edition" of the Chinese Pharmacopoeia Commission, and fit to generate the reference fingerprint chromatogram R of Mingmu Zishen tablets.

[0018] 5) Import the HPLC fingerprint chromatogram data of different test sample solutions into the Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprints 2012 Edition and perform similarity analysis with the reference chromatogram R.

[0019] 6) Use the fingerprint chromatogram established above for the quality control of Mingmu Zishen tablets.

[0020] Furthermore, the fingerprint includes 41 common peaks. Taking acteoside of peak 15 as the reference peak S, their relative retention times are 0.144, 0.204, 0.288, 0.382, 0.404, 0.445, 0.501, 0.535, 0.582, 0.716, 0.782, 0.925, 0.945, 0.958, 1.000, 1.020, 1.035, 1.108 respectively.

[0021] Furthermore, among the 41 common peaks, fingerprint peaks 1, 2, 9, 12, 14, 17, 20, 24, 30 are from Rehmanniae Radix; fingerprint peaks 1, 2, 3, 4, 10, 11, 12, 13, 15, 17, 20, 25, 29, 30, 31, 32 are from Glossy Privet Fruit; fingerprint peaks 1, 2, 5, 6, 7, 9, 11 are from Chinese Wolfberry Fruit; fingerprint peaks 1, 8, 13, 14, 16, 17, 18, 20, 22, 28, 29, 31, 33 are from Chrysanthemum Flos; fingerprint peaks 1, 11, 12, 14, 16, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 36, 37, 38, 39, 40, 41 are from Cassiae Semen; fingerprint peaks 1, 2, 9, 13, 29, 37 are from Achyranthis Bidentatae Radix.

[0022] Furthermore, among the 41 common peaks, fingerprint peak 1 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, Chinese Wolfberry Fruit, Chrysanthemum Flos, Cassiae Semen, and Achyranthis Bidentatae Radix; fingerprint peak 2 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, Chinese Wolfberry Fruit, and Achyranthis Bidentatae Radix; fingerprint peak 9 is the common peak of Rehmanniae Radix, Chinese Wolfberry Fruit, and Achyranthis Bidentatae Radix; fingerprint peak 11 is the common peak of Glossy Privet Fruit, Chinese Wolfberry Fruit, and Cassiae Semen; fingerprint peak 12 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, and Cassiae Semen; fingerprint peak 13 is the common peak of Glossy Privet Fruit, Chrysanthemum Flos, and Achyranthis Bidentatae Radix; fingerprint peak 14 is the common peak of Rehmanniae Radix, Chrysanthemum Flos, and Cassiae Semen; fingerprint peak 16 is the common peak of Chrysanthemum Flos and Cassiae Semen; fingerprint peak 17 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, and Chrysanthemum Flos; fingerprint peak 20 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, and Chrysanthemum Flos; fingerprint peak 24 is the common peak of Rehmanniae Radix and Cassiae Semen; fingerprint peak 25 is the common peak of Glossy Privet Fruit and Cassiae Semen; fingerprint peak 28 is the common peak of Chrysanthemum Flos and Cassiae Semen; fingerprint peak 29 is the common peak of Glossy Privet Fruit, Chrysanthemum Flos, Cassiae Semen, and Achyranthis Bidentatae Radix; fingerprint peak 30 is the common peak of Rehmanniae Radix, Glossy Privet Fruit, and Cassiae Semen; fingerprint peak 31 is the common peak of Glossy Privet Fruit and Chrysanthemum Flos; fingerprint peak 32 is the common peak of Glossy Privet Fruit and Cassiae Semen; peak 37 is the common peak of Cassiae Semen and Achyranthis Bidentatae Radix.

[0023] Furthermore, 14 out of the 41 common peaks were identified as fingerprint peaks, namely, peak 2 glucose, peak 4 salidroside, peak 8 chlorogenic acid, peak 12 acteoside, peak 14 catalpol, peak 15 ligustroside, peak 17 rehmannioside D, peak 18 3,5-O-dicaffeoylquinic acid, peak 22 4,5-O-dicaffeoylquinic acid, peak 23 chrysophanol, peak 31 luteolin, peak 33 luteoloside, peak 36 aurantio-obtusin, and peak 37 β-ecdysterone.

[0024] Furthermore, the filler is SHIMADZU Shim-pack Scepter C18 4.6mm×250mm, 5μm.

[0025] Furthermore, the preparation of the test sample solution is as follows:

[0026] Take Mingmu Zishen tablets, remove the film coating, weigh accurately, grind finely, add methanol, and ultrasonically treat for 30 minutes under the conditions of ultrasonic power of 200W and frequency of 40Hz. Let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0027] A fingerprint spectrum of Mingmu Zishen tablets obtained by a method for establishing a fingerprint spectrum of Mingmu Zishen tablets.

[0028] Furthermore, the fingerprint spectrum is used for the quality detection of Mingmu Zishen tablets, and the specific method is as follows:

[0029] 1) Preparation of the test sample solution: Take Mingmu Zishen tablets, grind finely, add methanol, ultrasonically treat, filter, and take the

[0030] subsequent

[0031] filtrate to obtain the solution;

[0032] 2) Accurately pipette the test sample solution and inject it into a liquid chromatograph for determination to obtain the fingerprint spectrum of the test sample;

[0033] The chromatographic conditions for determination by the liquid chromatograph are as follows: Using octadecylsilane chemically bonded silica gel as the filler, using acetonitrile - 0.2% formic acid as the mobile phase, gradient elution for detection, flow rate of 0.9 - 1.1 ml / min, column temperature of 25 - 30°C, detection wavelength of 250 - 330 nm, injection volume of 5 - 20 μL, and running time of 120 - 160 min;

[0034] The gradient elution conditions are as follows:

[0035]

[0036] 2) Establishment of the standard fingerprint spectrum

[0037] Forty-one common peaks were determined, and 14 chromatographic peaks were identified, namely peak No. 2, peak No. 4, peak No. 8, peak No. 12, peak No. 14, peak No. 15, peak No. 17, peak No. 18, peak No. 22, peak No. 23, peak No. 31, peak No. 33, peak No. 36, and peak No. 37. The acteoside peak of Ligustrum quihoui Carr. var. wangii (Lingelsh.) Rehd. at peak No. 15 with good repeatability and resolution was selected as the reference peak S.

[0038] 3) Quality evaluation of samples

[0039] The HPLC fingerprint of the sample was compared with the standard fingerprint. The similarity calculated based on the 41 common peaks should not be less than 0.9.

[0040] Application of a quality control method for Mingmu Zishen Tablets, and the quality control method is used for purposes such as product manufacturer identification and stability investigation of Mingmu Zishen Tablets.

[0041] The present invention discloses a quality control method for Mingmu Zishen Tablets, which can detect the active ingredients of six herbs in Mingmu Zishen Tablets, and at the same time can quantitatively detect the contents of glucose, salidroside, chlorogenic acid, acteoside, catalpol, acteoside, rehmannioside D, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, chrysophanol, luteolin, luteoloside, aurantio-obtusin, and β-ecdysterone in the sample. The fingerprint detection method for the sample of Mingmu Zishen Tablets disclosed by the present invention can simultaneously detect the active components in each herb, improve the inspection efficiency, and can also quantitatively control the contents of 14 active ingredients. Then, using the standard fingerprint information of the sample of Mingmu Zishen Tablets disclosed by the present invention as a reference, the quality of the sample of Mingmu Zishen Tablets can be comprehensively monitored by this fingerprint detection method. The establishment of the fingerprint detection method for Mingmu Zishen Tablets and the determination of the standard fingerprint information are of great significance for product quality control, authenticity identification, origin tracing and other aspects of work. Description of the drawings

[0042] Figure 1 Reference fingerprint of Mingmu Zishen Tablets R; Figure 2 Fingerprint of 3 batches of samples of Mingmu Zishen Tablets; in the figure, R is the reference fingerprint; S1 to S3 respectively represent the samples of Mingmu Zishen Tablets with batch numbers 230601, 230602, and 230603; Figure 3 Detection results of fingerprints of 6 batches of samples of Mingmu Zishen Tablets; in the figure, R is the reference fingerprint; S1 to S6 respectively represent the samples of Mingmu Zishen Tablets with batch numbers 230801, 230901, 231001, 20240101, 20240201, and 20240301; Figure 4 Chromatogram comparison diagram of detection results with different chromatographic columns; in the figure, A: C-154; B: C-118; C: C-028; D: C-056; E: C-147;Figure 5 Chromatogram comparison diagrams of detection results with different mobile phase systems; in the figure, A: acetonitrile - water; B: methanol - water; C: methanol - 0.1% phosphoric acid; D: acetonitrile - 0.1% phosphoric acid; Figure 6 Chromatogram comparison diagrams of detection with different acetonitrile - acid systems;

[0043] In the figure, A: acetonitrile - 0.1% phosphoric acid; B: acetonitrile - 0.2% phosphoric acid; C: acetonitrile - 0.05% acetic acid; D: acetonitrile - 0.1% acetic acid; E: acetonitrile - 0.1% formic acid; Figure 7 Chromatogram comparison diagrams of detection results with different acetonitrile - formic acid systems; in the figure, A: acetonitrile - 0.1% formic acid; B: acetonitrile - 0.2% formic acid; Figure 8 Chromatogram comparison diagrams of detection results under different gradient elution conditions; in the figure, A: condition 1; B: condition 2; C: condition 3; D: condition 4; E: condition 5; Figure 9 Spectrogram at a detection wavelength of 220 nm;

[0044] Figure 10 Spectrogram at a detection wavelength of 250 nm; Figure 11 Spectrogram at a detection wavelength of 280 nm; Figure 12 Spectrogram at a detection wavelength of 330 nm; Figure 13 Spectrogram at a detection wavelength of 430 nm; Figure 14 Chromatogram comparison diagrams of detection results at different column temperatures; in the figure, A: column temperature 25°C; B: column temperature 30°C; C: column temperature 35°C; Figure 15 Chromatogram of all chromatographic conditions detected; Figure 16 Chromatogram comparison diagrams of detection with different extraction solvents; in the figure, A: extraction solvent 60% methanol; B: extraction solvent 80% methanol; C: extraction solvent methanol;

[0045] Figure 17 Chromatogram comparison diagrams of detection of the test sample with different extraction methods; in the figure, A: ultrasonic extraction; B: reflux extraction; Figure 18 Chromatogram comparison diagrams of detection of the test sample with different extraction solvent dosages; in the figure, A: 2 g → 25 mL; B: 1 g → 25 mL; C: 1 g → 50 mL; D: 1 g → 75 mL; Figure 19 Chromatogram comparison diagrams of detection of the test sample with different extraction times; in the figure, A: extraction time 30 minutes; B: extraction time 40 minutes; C: extraction time 50 minutes; Figure 20 Chromatogram comparison diagram of Rehmannia glutinosa single herb and Mingmu Zishen tablets; in the figure, A: Mingmu Zishen tablets B: Rehmannia glutinosa single herb; Figure 21 Chromatogram comparison diagram of Ligustrum lucidum single herb and Mingmu Zishen tablets; in the figure, A: Mingmu Zishen tablets B: Ligustrum lucidum single herb; Figure 22Comparison Chromatogram of Lycii Fructus Single Herb and Mingmu Zishen Tablets; In the figure, A: Mingmu Zishen Tablets; B: Lycii Fructus Single Herb Figure 23 Comparison Chromatogram of Chrysanthemi Flos Single Herb and Mingmu Zishen Tablets; In the figure, A: Mingmu Zishen Tablets; B: Chrysanthemi Flos Single Herb Figure 24 Comparison Chromatogram of Cassiae Semen Single Herb and Mingmu Zishen Tablets; In the figure, A: Mingmu Zishen Tablets; B: Cassiae Semen Single Herb Figure 25 Comparison Chromatogram of Achyranthis Bidentatae Radix Single Herb and Mingmu Zishen Tablets; In the figure, A: Mingmu Zishen Tablets; B: Achyranthis Bidentatae Radix Single Herb Figure 26 Specificity Chromatogram; In the figure, A is blank excipient and B is the sample of Mingmu Zishen Tablets Specific Embodiment

[0046] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer and more understandable, the spirit of the content disclosed in the present invention will be described in detail below. After any person skilled in the relevant technical field understands the embodiments of the content of the present invention, the techniques taught by the content of the present invention can be changed and modified without departing from the spirit and scope of the content of the present invention

[0047] The illustrative embodiments of the present invention and their descriptions are used to explain the present invention, but not to limit the present invention

[0048] Embodiment 1 A Method for Establishing the Fingerprint of Mingmu Zishen Tablets

[0049] 1. Detection Method: Chromatographic Conditions: Using octadecylsilane-bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6mm×250mm, 5μm), using acetonitrile - 0.2% formic acid as the mobile phase, detecting according to the determined gradient elution conditions (see Table 2 below), with a flow rate of 1.0 mL / min, a column temperature of 30°C, a detection wavelength of 280 nm, an injection volume of 10 μL, and a running time of 130 min

[0050] Table 2 Determined Gradient Elution Conditions

[0051]

[0052] Preparation of Test Solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind finely, take about 2 g, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain

[0053] Preparation of Reference Solution: Weigh accurately an appropriate amount of reference substance, and make a solution containing 1 mg per 1 mL with methanol, that is, obtain

[0054] Assay method: Accurately pipette 10 μL each of the reference substance solution and the test sample solution, inject them into the liquid chromatograph, and determine the results.

[0055] 2. Experimental method: Prepare samples of Mingmu Zishen Tablets in 3 batches (batch numbers: 230601, 230602, 230603) according to the preparation method of the test sample solution, inject and analyze them under the above chromatographic conditions to obtain the fingerprint chromatograms of the 3 batches of Mingmu Zishen Tablets. Then use the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines 2012 Edition" of the Pharmacopoeia Commission of the People's Republic of China to analyze the fingerprint chromatograms of the 3 batches of samples, and fit and generate the reference fingerprint chromatogram R of Mingmu Zishen Tablets. The chromatogram is as Figure 1 shown. The multi-point calibration and automatic matching of the chromatographic peaks of each fingerprint chromatogram were carried out by the average correlation coefficient method. There were 41 common chromatographic peaks in total, and 14 common peaks were identified with reference substances, namely glucose (peak 2), salidroside (peak 4), chlorogenic acid (peak 8), acteoside (peak 12), catalpol (peak 14), ligustroside (peak 15), rehmannioside D (peak 17), 3,5-O-dicaffeoylquinic acid (peak 18), 4,5-O-dicaffeoylquinic acid (peak 22), chrysophanol (peak 23), luteolin (peak 31), cynaroside (peak 33), aurantio-obtusin (peak 36), β-ecdysterone (peak 37). Taking ligustroside (peak 15) as the reference peak S, their relative retention times were 0.144, 0.204, 0.288, 0.382, 0.404, 0.445, 0.501, 0.535, 0.582, 0.716, 0.782, 0.925, 0.945, 0.958, 1.000, 1.020, 1.035, 1.108 respectively. The samples with batch numbers 230601, 230602, and 230603 are represented by S1, S2, and S3 respectively. The detection chromatograms are as Figure 2 shown, and the detection results are shown in Table 3 below.

[0056] Table 3 Detection Results of Fingerprint Chromatograms of 3 Batches of Mingmu Zishen Tablets

[0057] Name S1 S2 S3 Control Fingerprint S1 1.000 0.993 0.975 0.994 S2 0.993 1.000 0.992 1.000 S3 0.975 0.992 1.000 0.994 Control Fingerprint 0.994 1.000 0.994 1.000

[0058] Conclusion: Peak matching was performed on the selected 41 common chromatographic peaks to obtain a common pattern, and this common pattern was used as the standard reference chromatogram for overall similarity evaluation. The results showed that the similarities of the 3 batches of finished products were all greater than 0.900, indicating a relatively high similarity. This shows that the product performs very similarly at multiple characteristic points, contains similar chemical components and has similar biological activities.

[0059] Example 2

[0060] Application of a Detection Method for Fingerprint Chromatogram of Mingmu Zishen Tablets

[0061] 1. Fingerprint detection method: Detection is carried out according to the detection method in Example 1.

[0062] 2. Experimental method: Six batches of Mingmu Zishen tablets (batch numbers: 230801, 230901, 231001, 20240101, 20240201, 20240301) were prepared according to the preparation method of the test solution, and injected and analyzed under the chromatographic conditions of the fingerprint detection method to obtain the fingerprint spectra of the six batches of Mingmu Zishen tablets. The samples with batch numbers 230801, 230901, 231001, 20240101, 20240201, and 20240301 are represented by S1 to S6 respectively. The detection spectra are as Figure 3 shown, and the detection results are shown in Table 4 below.

[0063] Table 4 Detection results of fingerprint spectra of six batches of Mingmu Zishen tablets

[0064] Name S1 S2 S3 S4 S5 S6 Control Fingerprint S1 1.000 0.991 0.966 1.000 0.994 0.978 0.995 S2 0.991 1.000 0.994 0.995 0.997 0.993 0.999 S3 0.966 0.994 1.000 0.980 0.987 1.000 0.993 S4 1.000 0.995 0.980 1.000 0.995 0.982 0.996 S5 0.994 0.997 0.987 0.995 1.000 0.988 0.998 S6 0.978 0.993 1.000 0.982 0.988 1.000 0.994 Control Fingerprint 0.995 0.999 0.993 0.996 0.998 0.994 1.000

[0065] Conclusion: The similarity evaluation was carried out on the fingerprint spectrum information of the six batches of Mingmu Zishen tablets and the standard control spectrum. The results showed that the similarities of the six batches of finished products in different years and batches were all greater than 0.900, and the similarities between batches of products were relatively high. The detection results indicated that the preparation process of Mingmu Zishen tablets was stable and the product quality was reliable. When this detection method was applied to product quality control, it was not only simple in operation but also made the detection results accurate and detailed.

[0066] Experimental Example 1

[0067] Investigation of chromatographic column for fingerprint detection method

[0068] Preparation of test solution: Take 20 tablets of Mingmu Zishen tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place them in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain it.

[0069] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler, five different manufacturers and models of C18 chromatographic columns were investigated respectively. The detailed information is shown in Table 5. Using acetonitrile - 0.1% phosphoric acid as the mobile phase, the gradient elution conditions are shown in Table 6, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the detection wavelength is 280 nm.

[0070] Determination method: Accurately pipette 10 μL of the test solution respectively, inject it into the liquid chromatograph, and determine, that is, obtain it.

[0071] The detection result spectra are as Figure 4 shown.

[0072] Table 5 Chromatographic Column Information

[0073]

[0074] Table 6 Gradient Elution Conditions

[0075] Conclusion: The test solution was detected using the above 5 chromatographic columns respectively. The chromatogram obtained using the SHIMADZU Shim-pack Scepter C18 chromatographic column numbered C-118 had the largest number of chromatographic peaks and good separation effect for each peak. Therefore, this type of chromatographic column was selected for detection.

[0076] Experimental Example 2

[0077] Investigation of the Mobile Phase System for the Fingerprint Detection Method

[0078] Investigation of the Mobile Phase System

[0079] Preparation of the test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain.

[0080] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), different mobile phase systems were investigated respectively. The detailed information is shown in Table 7, and the gradient elution conditions are shown in Table 8. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 280 nm.

[0081] Determination method: Accurately pipette 10 μL of each test solution respectively, inject it into the liquid chromatograph, and determine, that is, obtain.

[0082] The detected result chromatogram is as Figure 5 shown.

[0083] Table 7 Different Mobile Phase Systems

[0084] Serial Number A B 1 Acetonitrile Water 2 Methanol Water 3 Methanol 0.1% Phosphoric Acid 4 Acetonitrile 0.1% Phosphoric Acid

[0085] Table 8 Gradient Elution Conditions

[0086] Time (min) A(％) B(％) 0 5 95 5～50 5→45 95→55 50～60 45→55 55→45 60～65 55 45

[0087] Conclusion: Comparing 4 different mobile phase systems, the chromatogram obtained using the mobile phase of acetonitrile - 0.1% phosphoric acid system had the largest number of chromatographic peaks and good separation effect for each peak. Therefore, the mobile phase system was selected as the acetonitrile - 0.1% phosphoric acid system.

[0088] 2. Investigation of the glycolonitrile system

[0089] Preparation of the test solution: Take 20 Mingmu Zishen tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain it.

[0090] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm × 250 mm, 5 μm), investigate different glycolonitrile systems respectively. The detailed information is shown in Table 9, and the gradient elution conditions are shown in Table 10. The flow rate is 1.0 mL / min, the column temperature is 30 °C, and the detection wavelength is 280 nm.

[0091] Determination method: Accurately pipette 10 μL of each test solution respectively, inject it into the liquid chromatograph, and determine, that is, obtain it.

[0092] The detection result chromatogram is as Figure 6 shown.

[0093] Table 9 Different mobile phase acid systems

[0094] Serial Number A B 1 Acetonitrile 0.1% Phosphoric Acid 2 Acetonitrile 0.2% Phosphoric Acid 3 Acetonitrile 0.05% Acetic Acid 4 Acetonitrile 0.1% Acetic Acid 5 Acetonitrile 0.1% Formic Acid

[0095] Table 10 Gradient elution conditions

[0096] Time (min) A(％) B(％) 0 5 95 5～50 5→45 95→55 50～60 45→55 55→45 60～65 55 45

[0097] Conclusion: Comparing 5 different glycolonitrile systems, the chromatogram obtained by using acetonitrile - 0.1% formic acid as the mobile phase has more chromatographic peaks, and the resolution and peak shape of each peak are better. Therefore, acetonitrile - 0.1% formic acid is selected as the mobile phase for gradient detection.

[0098] 3. Investigation of the acetonitrile - formic acid system

[0099] Preparation of the test solution: Take 20 Mingmu Zishen tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain it.

[0100] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm × 250 mm, 5 μm), different ratios of acetonitrile and formic acid systems were used respectively. The detailed information is shown in Table 11, and the gradient elution conditions are shown in Table 12. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 280 nm.

[0101] Assay method: Precisely pipette 10 μL of each test solution, inject it into the liquid chromatograph, and determine it to obtain the result.

[0102] The detected result chromatogram is as Figure 7 shown.

[0103] Table 11 Different acetonitrile - formic acid systems

[0104] Serial Number A B 1 Acetonitrile 0.1% Formic Acid 2 Acetonitrile 0.2% Formic Acid

[0105] Table 12 Gradient elution conditions

[0106] Time (min) A(％) B(％) 0 5 95 6～30 5→17 95→83 30～70 17→25 83→75 70～85 25→35 75→65 85～100 35→50 65→50

[0107] Conclusion: Comparing the two different acetonitrile - formic acid systems, the resolution and peak shape of each peak in the chromatogram detected with acetonitrile - 0.2% formic acid as the mobile phase are better. Therefore, acetonitrile - 0.2% formic acid was selected as the mobile phase for gradient detection.

[0108] Experimental Example 3

[0109] Investigation on gradient elution conditions of fingerprint chromatogram detection method

[0110] Preparation of test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of methanol, stopper it, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0111] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm × 250 mm, 5 μm), with acetonitrile - 0.2% formic acid as the mobile phase, gradient elution was carried out according to the conditions in Tables 13, 14, 15, 16, and 17 respectively. The flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelength was 280 nm.

[0112] Assay method: Precisely pipette 10 μL of each test solution, inject it into the liquid chromatograph, and determine it to obtain the result.

[0113] The detected result chromatogram is as Figure 8 shown.

[0114] Table 13 Gradient Elution Conditions 1

[0115]

[0116]

[0117] Table 14 Gradient Elution Conditions 2

[0118] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→26 83→74 70～85 26→30 74→70 85～95 30→50 70→50 95～110 50 50

[0119] Table 15 Gradient Elution Conditions 3

[0120] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→25 83→75 70～85 25→35 75→65 85～100 35→50 65→50 100～110 50 50

[0121] Table 16 Gradient Elution Conditions 4

[0122] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→35 76→65 85～100 35→50 65→50 100～110 50 50

[0123] Table 17 Gradient Elution Conditions 5

[0124] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50

[0125] Conclusion: The samples were detected under the above 5 different gradient elution conditions. The chromatogram obtained under gradient elution condition 5 had a larger number of chromatographic peaks and better resolution for each peak. Therefore, acetonitrile - 0.2% formic acid was used as the mobile phase and the detection was carried out according to gradient elution condition 5.

[0126] Determination of gradient elution conditions: After selecting the gradient elution conditions, the running time was extended by 10 min, that is, the total detection time was 130 min, so that the elution conditions returned to the initial state. The determined chromatographic conditions are shown in Table 18.

[0127] Table 18 Determined Gradient Elution Conditions

[0128] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0129] Experimental Example 4

[0130] Investigation of detection wavelength for fingerprint detection method

[0131] Considering the complex prescription and large polarity differences among the components in the extract, the sample solution of Mingmu Zishen tablets was subjected to DAD full - wavelength spectral scanning. By comprehensively comparing the chromatogram, UV spectral information at multiple wavelengths and literature search, wavelengths of 220 nm, 250 nm, 280 nm, 330 nm, and 430 nm were investigated respectively. It was found that the number of chromatographic peaks at 280 nm was the largest and the resolution of each peak was better. Therefore, the wavelength was set at 280 nm. The detection result chromatogram is as Figures 9 - 13 shown.

[0132] Experimental Example 5

[0133] Investigation on Column Temperature in Fingerprint Detection Method

[0134] Preparation of Test Solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind them finely, take about 1 g, weigh accurately, place it in a stoppered conical flask, accurately add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate, that is, obtain the test solution.

[0135] Chromatographic Conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), using acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 19, the flow rate is 1.0 mL / min, the column temperature is investigated at 25°C, 30°C, and 35°C respectively, and the detection wavelength is 280 nm.

[0136] Determination Method: Accurately pipette 10 μL of the test solution respectively, inject it into the liquid chromatograph for determination, and that is, obtain the results.

[0137] The detected result chromatogram is as Figure 14 shown.

[0138] Table 19 Gradient Elution Conditions

[0139] Time (min) A - Acetonitrile (%) B - 0.1% Phosphoric Acid (%) 0 5 95 5～60 5→60 95→40 60～65 60 40

[0140] Conclusion: The test solution is detected at the above 3 column temperatures respectively. The chromatogram at 30°C has the largest number of peaks, can detect the most components, the peak shapes of each peak are better, and the resolution is good. Therefore, the column temperature is selected as 30°C.

[0141] Experimental Example 6

[0142] Determination of Chromatographic Conditions in Fingerprint Detection Method

[0143] Chromatographic Conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), using acetonitrile - 0.2% formic acid as the mobile phase, detect according to the determined gradient elution conditions (see Table 20), the flow rate is 1.0 mL / min, the column temperature is 30°C, the detection wavelength is 280 nm, the injection volume is 10 μL, and the running time is 130 min.

[0144] The detected result chromatogram is as Figure 15 shown.

[0145] Table 20 Determined Gradient Elution Conditions

[0146] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0147] Experimental Example 7

[0148] Investigation on the sample preparation method

[0149] 1. Investigation on the extraction solvent

[0150] Preparation of the test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind finely, prepare three portions, take about 1 g each, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of 60% methanol, 25 mL of 80% methanol, and 25 mL of methanol respectively, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes respectively, let cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0151] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), using acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 21, flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 280 nm, injection volume 10 μL, running time 130 min.

[0152] Determination method: Accurately pipette 10 μL of each test solution respectively, inject into the liquid chromatograph for determination to obtain the chromatogram.

[0153] The detected result chromatograms are as Figure 16 shown.

[0154] Table 21 Gradient elution conditions

[0155] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0156] Conclusion: By comparing the test solutions prepared with 3 different extraction solvents, it was found that the number of peaks extracted by the 3 different extraction solvents was the same and the extraction effects were consistent. However, the baseline of the methanol solvent was stable without fluctuations. Therefore, methanol was used as the extraction solvent.

[0157] 2. Investigation on the extraction method

[0158] Preparation of the test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh accurately, grind finely, prepare two portions, take about 1 g each, weigh accurately, place in a stoppered conical flask, accurately add 25 mL of methanol respectively, stopper tightly, weigh, ultrasonically treat one portion (power 200 W, frequency 40 Hz) for 40 minutes, and heat the other portion under reflux at 65 °C for 40 minutes (see Table 22), let cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0159] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the packing material (SHIMADZU Shim-pack Scepter C18 4.6 mm × 250 mm, 5 μm), acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 22, flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 280 nm, injection volume 10 μL, running time 130 min.

[0160] Assay method: Precisely pipette 10 μL of each test solution, inject into the liquid chromatograph, determine, and record the chromatogram.

[0161] The detected result chromatogram is as Figure 17 shown.

[0162] Table 22 Gradient elution conditions

[0163] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0164] Conclusion: By comparing the test solutions prepared by two different extraction methods, it was found that the number of peaks extracted by the two different extraction methods was the same and the extraction effects were consistent. Considering the simplicity of the extraction method and energy conservation, ultrasonic extraction was adopted as the extraction method.

[0165] 3. Investigation on the amount of extraction solvent

[0166] Preparation of test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, precisely weigh, grind finely, prepare four portions, take about 2 g, 1 g, 1 g, 1 g respectively, precisely weigh, place in a stoppered conical flask, precisely add 25 mL, 25 mL, 50 mL, 75 mL of methanol respectively. The detailed amounts of extraction solvent are shown in Table 23 below. Close the stopper, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 40 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0167] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the packing material (SHIMADZU Shim-pack Scepter C18 4.6 mm × 250 mm, 5 μm), acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 24, flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 280 nm, injection volume 10 μL, running time 130 min.

[0168] Assay method: Precisely pipette 10 μL of each test solution, inject into the liquid chromatograph, determine, and record the chromatogram.

[0169] The detected result chromatogram is as Figure 18 shown.

[0170] Table 23 Investigation on the amount of extraction solvent

[0171] Serial Number Dosage of Extraction Solvent 1 2g → 25mL 2 1g → 25mL 3 1g → 50mL 4 1g → 75mL

[0172] Table 24 Gradient elution conditions

[0173]

[0174]

[0175] Conclusion: By comparing the test solution prepared with 4 different dosages of extraction solvent, it was found that the number of peaks extracted with 4 different dosages of extraction solvent was the same, and the extraction effects were consistent. In line with the principles of environmental protection and energy conservation, and with better peak shapes and resolutions for the 2g→25mL ratio, the dosage of extraction solvent was thus adopted as 2g→25mL.

[0176] 4. Investigation of extraction time

[0177] Preparation of test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, accurately weigh, grind them finely, prepare three portions, respectively take about 2g, accurately weigh, place them in a stoppered conical flask, accurately add 25 mL of methanol respectively, tightly stopper, weigh, ultrasonically treat (power 200W, frequency 40Hz) for 30 min, 40 min, and 50 min respectively, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0178] Chromatographic conditions: Using octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6mm×250mm, 5μm), using acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 25, flow rate 1.0 mL / min, column temperature 30°C, detection wavelength 280 nm, injection volume 10 μL, running time 130 min.

[0179] Determination method: Accurately pipette 10 μL of each test solution respectively, inject it into the liquid chromatograph for determination to obtain the results, and record the chromatogram.

[0180] The detection result chromatogram is as Figure 19 shown.

[0181] Table 25 Gradient elution conditions

[0182] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0183] Conclusion: By comparing the test solution prepared with 3 different extraction times, it was found that the number of peaks extracted with 3 different extraction times was the same, and the extraction effects were consistent. Considering energy conservation, the extraction time was thus adopted as 30 minutes.

[0184] Experimental example 8

[0185] Identification of Chromatographic Peaks in Fingerprint and Determination of Reference Peaks

[0186] Preparation of reference substance solution: Weigh an appropriate amount of each reference substance precisely, add methanol to make a solution containing 1 mg per 1 mL, and you will get it.

[0187] Preparation of test solution: Take 20 tablets of Mingmu Zishen Tablets, remove the film coating, weigh precisely, grind them finely, take about 2 g, weigh precisely, place them in a stoppered conical flask, precisely add 25 mL of methanol, stopper tightly, weigh, ultrasonically treat (power 200 W, frequency 40 Hz) for 30 minutes, let it cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the continuous filtrate, and you will get it.

[0188] Chromatographic conditions: Use octadecylsilane chemically bonded silica gel as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), use acetonitrile - 0.2% formic acid as the mobile phase, the gradient elution conditions are shown in Table 26, the flow rate is 1.0 mL / min, the column temperature is 30 °C, the detection wavelength is 280 nm, the injection volume is 10 μL, and the running time is 130 min.

[0189] Determination method: Precisely absorb 10 μL of each reference substance solution and test solution respectively, inject them into the liquid chromatograph for determination, and you will get it.

[0190] Table 26 Gradient Elution Conditions

[0191] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0192] Conclusion: By comparing the retention times of the chromatographic peaks in the chromatograms of each reference substance solution and the test solution of Mingmu Zishen Tablets, a total of 14 characteristic fingerprint peaks were identified. Among them, peak 2 is glucose, peak 4 is salidroside, peak 8 is chlorogenic acid, peak 12 is acteoside, peak 14 is catalpol, peak 15 is specnuezhenide, peak 17 is rehmannioside D, peak 18 is 3,5-O-dicaffeoylquinic acid, peak 22 is 4,5-O-dicaffeoylquinic acid, peak 23 is chrysophanol, peak 31 is luteolin, peak 33 is luteoloside, peak 36 is aurantio-obtusin, and peak 37 is β-ecdysterone. Among them, specnuezhenide has good repeatability, resolution, and appropriate elution time and peak height. Therefore, specnuezhenide was selected as the reference peak (S peak).

[0193] Example 9

[0194] Attribution of Chromatographic Peaks

[0195] Preparation of samples of each single herb:[[]]

[0196] Single herb sample of Rehmannia glutinosa: Take an appropriate amount of Rehmannia glutinosa slices, and prepare the Rehmannia glutinosa herb sample according to the Rehmannia glutinosa process in the production process of Mingmu Zishen Tablets.

[0197] Single-ingredient sample of Ligustrum lucidum: Take appropriate amount of Ligustrum lucidum slices and prepare Rehmannia glutinosa medicinal material sample according to the Ligustrum lucidum process in the production process of Mingmu Zishen tablets.

[0198] Lycium barbarum single-ingredient sample: Take appropriate amount of Lycium barbarum decoction pieces and prepare Rehmannia root medicinal material sample according to the Lycium barbarum process in the production process of Mingmu Zishen Tablets.

[0199] Chrysanthemum single-ingredient sample: Take appropriate amount of chrysanthemum slices and prepare Rehmannia root medicinal material sample according to the chrysanthemum process in the production process of Mingmu Zishen tablets.

[0200] Cassia seed single-ingredient sample: Take appropriate amount of Cassia seed slices and prepare Rehmannia root medicinal material sample according to the Cassia seed process in the production process of Mingmu Zishen tablets.

[0201] Achyranthes bidentata single ingredient sample: Take appropriate amount of Achyranthes bidentata slices and prepare Rehmannia glutinosa medicinal material sample according to the Achyranthes bidentata process in the production process of Mingmu Zishen Tablets.

[0202] Preparation of each test solution: take 20 tablets of Mingmu Zishen Tablets respectively, remove the film coating, weigh accurately, and grind into powder; grind the single medicinal material sample of Rehmannia glutinosa, the single medicinal material sample of Ligustrum lucidum, the single medicinal material sample of Lycium barbarum, the single medicinal material sample of Chrysanthemum, the single medicinal material sample of Cassia seed, and the single medicinal material sample of Achyranthes bidentata, and take about 2g of each, weigh accurately, put in a stoppered conical flask, accurately add 25mL of methanol respectively, stopper, weigh the weight, ultrasonically treat (power 200W, frequency 40Hz) for 30 minutes, cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

[0203] Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler (SHIMADZU Shim-pack Scepter C18 4.6 mm×250 mm, 5 μm), acetonitrile-0.2% formic acid was used as the mobile phase, the gradient elution conditions were shown in Table 27, the flow rate was 1.0 mL / min, the detection wavelength was 280 nm, and the column temperature was 30°C.

[0204] Determination method: Accurately aspirate 10 μL of each single medicinal material solution and test sample solution, inject into liquid chromatograph, and determine.

[0205] The test result graph is as follows Figures 20 - 25 shown.

[0206] Table 27 Gradient elution conditions

[0207] Time (min) A - Acetonitrile (%) B - 0.2% Formic Acid (%) 0～6 5 95 6～30 5→17 95→83 30～70 17→24 83→76 70～85 24→30 76→70 85～100 30→50 70→50 100～120 50 50 120～130 50→5 50→95

[0208] Conclusion: By comparing the retention times of the chromatographic peaks in the chromatograms of the solutions of individual crude drugs and the test solution of Mingmu Zishen Tablets, peaks 1, 2, 9, 12, 14, 17, 20, 24, and 30 are derived from Rehmanniae Radix; peaks 1, 2, 3, 4, 10, 11, 12, 13, 15, 17, 20, 25, 29, 30, 31, and 32 are derived from Ligustri Lucidi Fructus; peaks 1, 2, 5, 6, 7, 9, and 11 are derived from Lycii Fructus; peaks 1, 8, 13, 14, 16, 17, 18, 20, 22, 28, 29, 31, and 33 are derived from Chrysanthemi Flos; peaks 1, 11, 12, 14, 16, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 36, 37, 38, 39, 40, and 41 are derived from Cassiae Semen; peaks 1, 2, 9, 13, 29, and 37 are derived from Achyranthis Bidentatae Radix.

[0209] Among them, peak 1 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, Lycii Fructus, Chrysanthemi Flos, Cassiae Semen, and Achyranthis Bidentatae Radix; peak 2 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, Lycii Fructus, and Achyranthis Bidentatae Radix; peak 9 is the common peak of Rehmanniae Radix, Lycii Fructus, and Achyranthis Bidentatae Radix; peak 11 is the common peak of Ligustri Lucidi Fructus, Lycii Fructus, and Cassiae Semen; peak 12 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, and Cassiae Semen; peak 13 is the common peak of Ligustri Lucidi Fructus, Chrysanthemi Flos, and Achyranthis Bidentatae Radix; peak 14 is the common peak of Rehmanniae Radix, Chrysanthemi Flos, and Cassiae Semen; peak 16 is the common peak of Chrysanthemi Flos and Cassiae Semen; peak 17 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, and Chrysanthemi Flos; peak 20 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, and Chrysanthemi Flos; peak 24 is the common peak of Rehmanniae Radix and Cassiae Semen; peak 25 is the common peak of Ligustri Lucidi Fructus and Cassiae Semen; peak 28 is the common peak of Chrysanthemi Flos and Cassiae Semen; peak 29 is the common peak of Ligustri Lucidi Fructus, Chrysanthemi Flos, Cassiae Semen, and Achyranthis Bidentatae Radix; peak 30 is the common peak of Rehmanniae Radix, Ligustri Lucidi Fructus, and Cassiae Semen; peak 31 is the common peak of Ligustri Lucidi Fructus and Chrysanthemi Flos; peak 32 is the common peak of Ligustri Lucidi Fructus and Cassiae Semen; peak 37 is the common peak of Cassiae Semen and Achyranthis Bidentatae Radix.

[0210] Experimental Example 10

[0211] Specificity Experiment

[0212] Prepare a negative control preparation according to the proportion of the prescription excipients and the production process of Mingmu Zishen Tablets, and then prepare a negative control solution according to the preparation method of the test solution.

[0213] Take another sample of Mingmu Zishen Tablets, prepare it according to the preparation method of the test solution, inject the negative control solution and the test solution for detection respectively, and record the chromatograms respectively.

[0214] The detection result chromatogram is as Figure 26 shown.

[0215] Conclusion: In this detection method, the excipients have no influence on the detection of the active ingredients in the product, which can ensure the specificity in the fingerprint chromatogram detection process and the accuracy of the detection data.

[0216] Experimental Example 11

[0217] Precision Experiment

[0218] Take the Mingmu Zishen Tablets sample, prepare it according to the preparation method of the test solution, and inject it continuously for 6 times for detection. The serial numbers are 1 - 6 in sequence. Take the 15th peak, acteoside, as the reference peak S, and record and calculate the relative retention time and relative peak area of each characteristic peak.

[0219] The calculation results of the RSD values of the relative retention time and relative peak area are shown in Tables 28 and 29 as follows.

[0220] Table 28 RSD Values of Relative Retention Time in the Precision Experiment of Common Peaks

[0221]

[0222]

[0223] Table 29 RSD Values of Relative Peak Area in the Precision Experiment of Common Peaks

[0224]

[0225]

[0226] Result: The RSD of the relative retention time of 41 common peaks is all < 2.0%, and the RSD of the relative peak area of 41 common peaks is all < 3.0%, indicating that the precision of this method is good.

[0227] Experimental Example 12

[0228] Repeatability Experiment

[0229] Take 6 portions of the Mingmu Zishen Tablets sample, prepare it according to the preparation method of the test solution, and inject each portion for 1 time for detection. The serial numbers are 1 - 6 in sequence. Take the 15th peak, acteoside, as the reference peak S, and record and calculate the relative retention time and relative peak area of each main chromatographic peak.

[0230] The calculation results of the RSD values of the relative retention time and relative peak area are shown in Tables 30 and 31 as follows.

[0231] Table 30 RSD Values of Relative Retention Time in the Repeatability Experiment of Common Peaks

[0232]

[0233]

[0234] Table 31 RSD Values of Relative Peak Area in the Repeatability Experiment of Common Peaks

[0235]

[0236]

[0237] Result: The RSD of the relative retention times of 41 common peaks was all < 2.0%, and the RSD of the relative peak areas of 41 common peaks was all < 3.0%, indicating that the method had good repeatability.

[0238] Experimental Example 13

[0239] Stability experiment

[0240] Take the Mingmu Zishen Tablets sample, prepare it according to the preparation method of the test solution, and inject 1 injection for detection at 0, 4, 8, 12, 16, 20, 24, 32, 40, and 48 h respectively. Take the 15th peak, acteoside, as the reference peak S, and record and calculate the relative retention times and relative peak areas of each main chromatographic peak.

[0241] The calculation results of the RSD values of the relative retention times and relative peak areas are shown in Tables 32 and 33 below.

[0242] Table 32 RSD values of relative retention times in the stability experiment of common peaks

[0243]

[0244]

[0245] Table 33 RSD values of relative peak areas in the stability experiment of common peaks

[0246]

[0247]

[0248] Result: The RSD of the relative retention times of 41 common peaks was all < 2.0%, and the RSD of the relative peak areas of 41 common peaks was all < 3.0%. The results showed that the test solution was stable within 48 h.

Claims

1. A quality control method for Mingmu Zishen Tablets, characterized in that It is done as follows: 1) Preparation of test sample solution: Take Mingmu Zishen Tablets, grind them into powder, add methanol, treat with ultrasound, filter, and take the filtrate to obtain the solution; 2) Preparation of reference solution: Accurately weigh an appropriate amount of reference substance and add methanol to make a solution. That's it; 3) Accurately pipette the reference solution and the test solution into the liquid chromatograph for determination to obtain the fingerprint of the test sample; The chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane bonded silica gel as filler, acetonitrile-0.2% formic acid as mobile phase, gradient elution detection, flow rate 0.9-1.1 ml / min, column temperature 25-30°C, detection wavelength 250-330 nm, injection volume 5-20 μL, running time 130 min 120-160 min; The gradient elution conditions are: 4) The fingerprint integral signal of the sample to be tested is imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software of the Chinese Pharmacopoeia Committee to generate the reference fingerprint R of Mingmu Zishen Tablets by fitting; 5) Import the HPLC fingerprint data of different test sample solutions into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition and perform similarity analysis with the reference spectrum R; 6) The fingerprint established above was used for quality control of Mingmu Zishen Tablets.

2. The quality control method of Mingmu Zishen Tablets according to claim 1 is characterized in that The fingerprint spectrum includes 41 common peaks, with peak No. 15 special privet glycoside as the reference peak S, and the relative retention times are 0.144, 0.204, 0.288, 0.382, 0.404, 0.445, 0.501, 0.535, 0.582, 0.716, 0.782, 0.925, 0.945, 0.958, 1.000, 1.020, 1.035, and 1.108, respectively.

3. The quality control method of Mingmu Zishen Tablets according to claim 1 or 2, characterized in that Among the 41 common peaks, fingerprint peaks 1, 2, 9, 12, 14, 17, 20, 24, 30 are derived from Rehmannia root; fingerprint peaks 1, 2, 3, 4, 10, 11, 12, 13, 15, 17, 20, 25, 29, 30, 31, 32 are derived from Ligustrum lucidum; fingerprint peaks 1, 2, 5, 6, 7, 9, 11 are derived from Lycium barbarum; fingerprint peaks 1, 8, 13, 14, 1 6, 17, 18, 20, 22, 28, 29, 31, and 33 are derived from chrysanthemum; fingerprint peaks 1, 11, 12, 14, 16, 19, 21, 23, 24, 25, 26, 27, 28, 29, 30, 32, 34, 35, 36, 37, 38, 39, 40, and 41 are derived from Cassia seed; fingerprint peaks 1, 2, 9, 13, 29, and 37 are derived from Achyranthes bidentata.

4. The quality control method of Mingmu Zishen Tablets according to claim 1, 2 or 3, characterized in that Among the 41 common peaks, fingerprint peak 1 is the common peak of Rehmannia root, Ligustrum lucidum, Lycium barbarum, Chrysanthemum, Cassia seed, and Achyranthes bidentata; fingerprint peak 2 is the common peak of Rehmannia root, Ligustrum lucidum, Lycium barbarum, and Achyranthes bidentata; fingerprint peak 9 is the common peak of Rehmannia root, Lycium barbarum, and Achyranthes bidentata; fingerprint peak 11 is the common peak of Ligustrum lucidum, Lycium barbarum, and Cassia seed; fingerprint peak 12 is the common peak of Rehmannia root, Ligustrum lucidum, and Cassia seed; fingerprint peak 13 is the common peak of Ligustrum lucidum, Chrysanthemum, and Achyranthes bidentata; fingerprint peak 14 is the common peak of Rehmannia root, Chrysanthemum, and Cassia seed; fingerprint peak 16 is the common peak of Chrysanthemum and Cassia seed; fingerprint peak Peak 17 is a common peak of Rehmannia root, Ligustrum lucidum and Chrysanthemum; fingerprint peak 20 is a common peak of Rehmannia root, Ligustrum lucidum and Chrysanthemum; fingerprint peak 24 is a common peak of Rehmannia root and Cassia seed; fingerprint peak 25 is a common peak of Ligustrum lucidum and Cassia seed; fingerprint peak 28 is a common peak of Chrysanthemum and Cassia seed; fingerprint peak 29 is a common peak of Ligustrum lucidum, Chrysanthemum, Cassia seed and Achyranthes bidentata; fingerprint peak 30 is a common peak of Rehmannia root, Ligustrum lucidum and Cassia seed; fingerprint peak 31 is a common peak of Ligustrum lucidum and Chrysanthemum; fingerprint peak 32 is a common peak of Ligustrum lucidum and Cassia seed; peak 37 is a common peak of Cassia seed and Achyranthes bidentata.

5. The quality control method of Mingmu Zishen Tablets according to claim 2 or 3 is characterized in that Among the 41 common peaks, 14 fingerprint peaks were identified, namely, peak 2 glucose, peak 4 salidroside, peak 8 chlorogenic acid, peak 12 verbascoside, peak 14 catalpol, peak 15 privet glycoside, peak 17 rehmannia glutinosa glycoside D, peak 18 3,5-O-dicaffeoylquinic acid, peak 22 4,5-O-dicaffeoylquinic acid, peak 23 chrysophanol, peak 31 luteolin, peak 33 luteolin glycoside, peak 36 aurantium dulcisin, and peak 37 β-ecdysterone.

6. The quality control method of Mingmu Zishen Tablets according to claim 1 is characterized in that The filler is SHIMADZUShim-packScepterC184.6mm×250mm, 5μm.

7. The quality control method of Mingmu Zishen Tablets according to claim 1 is characterized in that Preparation of the test sample solution: Take Mingmu Zishen Tablets, remove the film coating, accurately weigh, grind, add methanol, and ultrasonically treat for 30 minutes at an ultrasonic power of 200 W and a frequency of 40 Hz. Cool, weigh again, make up the lost weight with methanol, shake well, filter, and take the filtrate to obtain.

8. A fingerprint of Mingmu Zishen Tablets obtained by the quality control method of Mingmu Zishen Tablets as described in any one of claims 1 to 7.

9. The quality control method of Mingmu Zishen Tablets according to claim 1, characterized in that: The fingerprint spectrum is used for quality inspection of Mingmu Zishen Tablets, and the specific method is as follows: 1) Preparation of test sample solution: Take Mingmu Zishen Tablets, grind them into powder, add methanol, treat with ultrasound, filter, and take the filtrate to obtain the solution; 2) Accurately pipette the test sample solution and inject it into the liquid chromatograph for determination to obtain the fingerprint of the test sample; The chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane bonded silica gel as filler, acetonitrile-0.2% formic acid as mobile phase, gradient elution detection, flow rate 0.9-1.1 ml / min, column temperature 25-30° C., detection wavelength 250-330 nm, injection volume 5-20 μL, running time 120-160 min; The gradient elution conditions are: 3) Establishment of standard fingerprint 41 common peaks were determined, and 14 chromatographic peaks were identified, namely peak 2, peak 4, peak 8, peak 12, peak 14, peak 15, peak 17, peak 18, peak 22, peak 23, peak 31, peak 33, peak 36, and peak 37. The 15th special ligustrin peak with good repeatability and excellent separation was selected as the reference peak S. 4) Sample quality evaluation Compare the HPLC fingerprint of the sample with the standard fingerprint, and the similarity calculated based on 41 common peaks should not be less than 0.

9.

10. The use of the quality control method of Mingmu Zishen Tablets as claimed in claim 1, characterized in that The quality control method is used for the identification and stability investigation of Mingmu Zishen Tablets.

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