A method for determining the content and fingerprint of multiple components in Tianwang Buxin Pills

Abstract

The present invention provides a method for determining the content of seven components in Tianwang Buxin Pills. The present invention also provides a method for screening the medicinal materials to which the seven components in Tianwang Buxin Pills belong. The present invention further provides a method for detecting the fingerprint of Tianwang Buxin Pills. The present invention further provides the use of the above-mentioned method for detecting the fingerprint of Tianwang Buxin Pills in the quality detection of components in Tianwang Buxin Pills and the quality detection method thereof. The present invention further provides a method for screening the fingerprint of multiple medicinal materials in Tianwang Buxin Pills. The present invention provides a method for determining the content and fingerprint of multiple components in Tianwang Buxin Pills, which can quantitatively analyze the seven chemical components in Tianwang Buxin Pills and determine the seven medicinal materials to which the seven components belong. A high-performance liquid chromatography fingerprint of Tianwang Buxin Pills is also established, providing 13 index components representing the 12 medicinal materials in Tianwang Buxin Pills, and providing a scientific basis for the quality control of Tianwang Buxin Pills.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of components of traditional Chinese medicine, and relates to a method for determining the content and fingerprint of multiple components in Tianwang Buxin Pills, and specifically relates to a method for determining the content of seven components in Tianwang Buxin Pills: α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrae alcohol A, β-asarone, 3,6'-diesinaroylsucrose and ligustilide, and their fingerprint. Background Art

[0002] Tianwang Buxin Pills are included in the 2020 edition of the Pharmacopoeia of the People's Republic of China. It is composed of sixteen herbs, including Danshen, Acorus gramineus, Poria, Ophiopogon japonicus, Rehmannia root, processed Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds, Platycodon grandiflorus, and Cinnabar. Of the above sixteen herbs, cinnabar is ground into a very fine powder with water, and the remaining fifteen herbs, including Danshen, are ground into a fine powder (raw material powder for Tianwang Buxin Pills). They are then ground with the above powders, sieved, and mixed evenly. For every 100g of powder, add 20-30g of refined honey and an appropriate amount of water to make pills, dry them, and make water-honeyed pills; or add 50-70g of refined honey to make small or large honeyed pills. In the prescription, Rehmannia root is sweet, bitter, and cold, and it nourishes and clears away the body. It is good at nourishing yin and blood, cooling blood and promoting fluid production, and is used to treat the root cause of yin deficiency and internal heat, so it is used as the main medicine. Asparagus cochinchinensis is sweet, moistening, bitter, and cooling, effectively nourishing kidney yin, cooling kidney fire, and moistening the lungs. Ophiopogon japonicus is sweet, slightly bitter, and slightly cold, effectively nourishing yin, clearing the heart, and relieving restlessness. Scrophularia ningpoensis is salty, cold, and bitter, effectively purging, effectively nourishing yin and reducing fire, thereby controlling the rise of yin deficiency fire. Angelica sinensis is sweet, tonifying, and pungent, warming, and effectively nourishing blood, promoting blood circulation, and relieving dryness. Salvia miltiorrhiza is bitter, purging, and cooling, effectively activating blood circulation, cooling the blood, and clearing the heart and calming the mind. Together, these five herbs not only assist the main herb in nourishing yin and blood, but also clear the heart and calm the mind, and moisten dryness and promote bowel movements, thus serving as assistant herbs. Stir-fried Chinese jujube seeds are sweet and sour, nourishing the heart, liver, and gallbladder, while calming the mind. Cypress seeds are sweet and mild, tonifying deficiency, nourishing the heart and kidneys, calming the mind, and also moistening the intestines. Codonopsis pilosula is sweet and mild, not harsh, and is good at replenishing qi, strengthening the spleen, and nourishing blood. Schisandra chinensis is sweet, sour, warming, and astringent, nourishing yin and qi, calming the mind, and tranquilizing the mind. Poria cocos is sweet and mild, and is good at strengthening the spleen, eliminating dampness, and tranquilizing the mind. Processed Polygala tenuifolia is pungent, aphrodisiac, bitter, and purging, and warming, supporting heart yang, benefiting heart qi, connecting the heart and kidneys, and improving intelligence and tranquilizing the mind. Acorus calamus is pungent, dispersing, bitter, and purging, with an aromatic and warming effect, resolving phlegm and dampness, opening the heart, connecting the heart and kidneys, and tranquilizing the mind. Cinnabar is cold and heavy, calming the heart, clearing the mind, and tranquilizing the mind. Together, these eight herbs nourish yin and blood, nourish the source of transformation, moisten the intestines, and tranquilize the mind, thus supporting the efficacy of the ministerial herbs. Therefore, they are collectively considered adjuvants. Platycodon grandiflorum is pungent, dispersing, bitter, and neutral, combining with calamus to regulate qi and nourish without stagnation, while also carrying the herbs upward to the chest and heart. Licorice root is sweet and neutral, tonifying qi and benefiting the heart while also harmonizing the other herbs, making them both guiding herbs. The combined effects of the entire formula nourish, clear, purge, calm, and astringe, tonifying yin and blood, tonifying the heart, and calming the mind, are effective in treating palpitations, forgetfulness, insomnia, and dry stools caused by heart yin deficiency.

[0003] At present, the chemical composition of Tianwang Buxin Pills is complex. In order to better control the quality of Tianwang Buxin Pills, it is necessary to establish a sensitive, efficient, simple and rapid analytical method as the main means of quality control of Tianwang Buxin Pills. Summary of the Invention

[0004] In view of the shortcomings of the prior art mentioned above, the purpose of the present invention is to provide a method for determining the content of multiple components and fingerprints in Tianwang Buxin Pills. By using optimized pretreatment conditions and high-performance liquid chromatography, a method for determining the content of seven components in Tianwang Buxin Pills and a fingerprint of Tianwang Buxin Pills were established, which more comprehensively reflects the current status of each component in Tianwang Buxin Pills and provides a reference basis for controlling and evaluating the quality of Tianwang Buxin Pills.

[0005] To achieve the above-mentioned and other related objects, the present invention provides a method for determining the contents of seven components in Tianwang Buxin Pills in a first aspect, comprising the following steps:

[0006] 1) Preparation of test solution: Dissolve Tianwang Buxin Wan sample in solvent, extract by ultrasonication, cool, and filter the supernatant to obtain the test solution.

[0007] 2) Preparation of reference solution: Dissolve α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-diesinarosyl sucrose, and ligustilide reference substances in a solvent and dilute to volume to prepare a reference solution;

[0008] 3) Determination: The test solution of step 1) and the reference solution of step 2) were respectively determined by ultra performance liquid chromatography (UPLC), and the contents of the seven components in the test solution were calculated by the external standard method.

[0009] Preferably, in step 1), the Tianwang Buxin Pills sample is a powder sample obtained by crushing the Tianwang Buxin Pills.

[0010] Preferably, in step 1) or 2), the solvent is a methanol aqueous solution with a volume percentage of 65-75%, preferably a methanol aqueous solution with a volume percentage of 70%.

[0011] Preferably, in step 1), the ratio of the weight (g) of the Tianwang Buxin Pills sample added to the volume (mL) of the solvent added is 1.5:23-27, specifically 1.5:23-25, 1.5:25-27, preferably 1.5:25.

[0012] Preferably, in step 1), the ultrasonic extraction time is 27-33 min, specifically 27-30 min, 30-33 min, preferably 30 min.

[0013] Preferably, in step 1), the power of the ultrasonic extraction is 200-350W, specifically 200-250W, 250-350W, preferably 250W; the frequency of the ultrasonic extraction is 40-60kHz, specifically 40-50kHz, 50-60kHz, preferably 40kHz.

[0014] Preferably, in step 1), the cooling is to cool the mixture to room temperature, which is 20-30°C.

[0015] Preferably, in step 1), the filtration is membrane filtration.

[0016] More preferably, the filter membrane is a 0.22 μm filter membrane.

[0017] Preferably, in step 2), the reference substance solution is prepared by first adding a solvent to prepare a reference substance stock solution, and then adding a solvent for stepwise dilution.

[0018] More preferably, the reference substance stock solution needs to be stored in a refrigerator at 3-5°C, preferably 4°C, away from light.

[0019] Preferably, in step 2), the reference solution should be shaken and filtered before use to obtain a subsequent filtrate.

[0020] Preferably, in step 2), the CAS number of the α-linolenic acid is 463-40-1, the CAS number of the salvianolic acid B is 121521-90-2, the CAS number of glycyrrhizic acid is 1405-86-3, the CAS number of schisandrae alcohol A is 7432-28-2, the CAS number of β-asarone is 5273-86-9, the CAS number of 3,6'-diesinapoylsucrose is 139891-98-8, and the CAS number of ligustilide is 4431-01-0.

[0021] Preferably, in step 2), the content of α-linolenic acid in the reference solution is in the range of 9.73-389.49 μg / mL, preferably 200 μg / mL; the content of salvianolic acid B is in the range of 23.78-475.52 μg / mL, preferably 100 μg / mL; the content of glycyrrhizic acid is in the range of 9.75-195.03 μg / mL, preferably 40 μg / mL; the content of schisandrin A is in the range of 7. 74-154.73μg / mL, preferably 30μg / mL; the content range of β-asarone is 8.25-165.04μg / mL, preferably 30μg / mL; the content range of 3,6'-diesinapoylsucrose is 4.98-99.66μg / mL, preferably 20μg / mL; the content range of ligustilide is 9.61-192.17μg / mL, preferably 40μg / mL.

[0022] Ultra Performance Liquid Chromatography (UPLC) differs from traditional High Performance Liquid Chromatography (HPLC) by incorporating novel technologies such as small particle size, very low system volume, and rapid detection methods, increasing analytical throughput, sensitivity, and chromatographic peak capacity. Compared to HPLC methods, which suffer from lengthy detection times, complex chromatographic peaks, and reduced resolution, UPLC's detection speed, sensitivity, and resolution are unmatched by HPLC. UPLC's improved speed, sensitivity, and resolution shorten analysis times, reduce solvent usage, and lower analytical costs.

[0023] Preferably, in step 3), the chromatographic column used in the ultra-high performance liquid chromatography is a C18 chromatographic column. More preferably, the chromatographic column used in the high performance liquid chromatography is a Waters Acquity UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm).

[0024] Preferably, in step 3), the detector in the ultra-high performance liquid chromatography is a photodiode array detector (DAD).

[0025] Preferably, in step 3), in the ultra-high performance liquid chromatography method, the column temperature is 15-25°C, specifically 15-20°C, 20-25°C, preferably 20°C.

[0026] Preferably, in step 3), the injection volume in the ultra-high performance liquid chromatography method is 0.5-2 μL. More preferably, the injection volume in the high performance liquid chromatography method is 1 μL.

[0027] Preferably, in step 3), the flow rate in the ultra-high performance liquid chromatography method is 0.1-0.5 mL / min, specifically 0.1-0.3 mL / min, 0.3-0.5 mL / min, and preferably 0.3 mL / min.

[0028] Preferably, in step 3), the detection wavelength in the ultra-high performance liquid chromatography is selected from one or more combinations of 203 nm, 250 nm, and 320 nm, preferably 203 nm, 250 nm, and 320 nm.

[0029] When the above detection wavelengths are used for detection, the 203 nm is used to detect a-linolenic acid, the 250 nm is used to detect salvianolic acid B, glycyrrhizic acid, schisandra methanol, and β-asarone, and the 320 nm is used to detect 3,6'-diesinapoylsucrose and ligustilide.

[0030] The multi-wavelength detection method is adopted because Tianwang Buxin Pills contains many compounds and the wavelengths of the main components vary greatly. Single wavelength detection will cause some compounds to have poor absorption and small peak area, which seriously affects the content determination results. The spectral signal intensity and separation conditions at different absorption wavelengths were examined. This method can improve the sensitivity of the detector and increase the accuracy of the determination.

[0031] Preferably, in step 3), in the ultra-performance liquid chromatography method, the mobile phase is acetonitrile-0.08-0.12% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.08-0.12% phosphoric acid aqueous solution; the analysis time is 50 min; and gradient elution is used.

[0032] More preferably, in the ultra-high performance liquid chromatography method, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; the analysis time is 50 minutes; and gradient elution is used.

[0033] The 0.08-0.12% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.08-0.12%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0034] More preferably, as shown in Table 1, the specific procedure of the gradient elution is:

[0035] 0-15 min, the volume ratio of phase A:phase B was 8:92-22:78;

[0036] 15-23 min, the volume ratio of phase A:phase B is 22:78-35:65;

[0037] 23-30 min, the volume ratio of phase A:phase B is 35:65-40:60;

[0038] 30-35 min, the volume ratio of phase A:phase B is 40:60-53:47;

[0039] 35-42 min, the volume ratio of phase A:phase B is 53:47-95:5;

[0040] 42-45 min, the volume ratio of phase A:phase B is 95:5-95:5;

[0041] 45-46 min, the volume ratio of phase A:phase B is 95:5-8:92;

[0042] 46-50min, the volume ratio of phase A:phase B is 8:92-8:92.

[0043] Table 1 Gradient elution program

[0044]

[0045] Preferably, in step 3), the external standard method refers to: respectively taking a series of different volumes of the reference solution of step 2), making a series of solutions of different concentrations, adopting ultra-high performance liquid chromatography to analyze the sample, obtaining the linear relationship between the concentration and peak area of the 7 components in the reference solution, and plotting the corresponding standard working curve with the chromatographic peak area of each component corresponding to its corresponding concentration, and calculating the regression equation of each standard working curve. The test solution is then detected by ultra-high performance liquid chromatography, and the chromatographic peak areas of the 7 components in the test solution are respectively substituted into the regression equation of each standard working curve to calculate the content of the corresponding component.

[0046] More preferably, in the standard working curve, the peak area of each component is used as the ordinate, and the concentration of each component in the reference solution is used as the abscissa.

[0047] A second aspect of the present invention provides a method for screening the medicinal materials of the seven ingredients in Tianwang Buxin Pills, comprising the following steps:

[0048] I) Preparation of single herbal sample solutions: Prepare one or more of the seven herbal samples of Tianwang Buxin Pills (Danshen miltiorrhiza, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Glycyrrhiza uralensis, Angelica sinensis, and Schisandra chinensis) according to step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills to obtain at least one single herbal sample solution;

[0049] II) Preparation of negative sample solutions: Samples of the seven medicinal materials in Tianwang Buxin Pills, namely, Danshen, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, and Schisandra chinensis, were prepared according to step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills to obtain negative sample solutions lacking Danshen, lacking Acorus gramineus, lacking Polygala tenuifolia, lacking Platycladus orientalis, lacking Licorice root, lacking Angelica sinensis, and lacking Schisandra chinensis, respectively;

[0050] III) Determination: The fingerprints of the single herbal medicine sample solution in step I) and the negative sample solution in step II) were determined by ultra-performance liquid chromatography (UPLC) under the same chromatographic conditions as in step 3) of the method for determining the contents of the seven components in Tianwang Buxin Pills;

[0051] IV) Obtaining the control fingerprint: The test solution prepared according to step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills was subjected to step 3) of the same method for determining the contents of the seven components in Tianwang Buxin Pills to obtain the control fingerprint of the seven components in Tianwang Buxin Pills;

[0052] V) Quality testing: The fingerprints of the single herb sample solution and the negative sample solution were compared with the reference fingerprints of the seven ingredients in Tianwang Buxin Pills. The corresponding characteristic peaks of the single herb sample solution in the reference fingerprints of the seven ingredients in Tianwang Buxin Pills were identified by relative retention time, thereby attributing and locating the seven ingredients in Tianwang Buxin Pills.

[0053] Preferably, in step I), the salvia miltiorrhiza is the root and rhizome of Salvia miltiorrhiza Bge., a plant of the Lamiaceae family. The calamus is the rhizome of Acorus tatarinowii Schott., a plant of the Araceae family. The processed polygala is processed polygala, and the polygala is the dried root or root bark of Polygala tenuifolia folia Willd. or Polygala sibirica L., a plant of the Polygalaceae family. The cypress seed is the seed kernel of Platycladus orientails (L.) Franco, a plant of the Cupressaceae family. The licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., a plant of the Leguminosae family. The angelica is the root of Angelica sinensis (Oliv.) Diels., a plant of the Apiaceae family. The schisandra chinensis is the dried mature fruit of Schisandra chinensis (Turcz.) Baill. of the Magnoliaceae family.

[0054] Preferably, in step V), the present invention locates the characteristic peaks of the measured fingerprints of the single medicinal material sample solution and the negative sample solution and the reference fingerprints of the seven components in Tianwang Buxin Pills, and uses the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" issued by the State Pharmacopoeia Commission for analysis and processing, and identifies the corresponding characteristic peaks of the single medicinal material sample solution in the reference fingerprints of the seven components in Tianwang Buxin Pills, thereby attributing and locating the seven components in Tianwang Buxin Pills. Specific results are shown in Figure 1a 、 Figure 1b 、 Figure 1c .

[0055] Preferably, in step V), among the seven ingredients in the Tianwang Buxin Pills, α-linolenic acid is peak 1, coming from cypress seed; salvianolic acid B is peak 2, coming from salvia miltiorrhiza; glycyrrhizic acid is peak 3, coming from liquorice; schisandrae alcohol A is peak 4, coming from schisandra chinensis; β-asarone is peak 5, coming from calamus; 3,6'-diesinarosylsucrose is peak 6, coming from polygala tenuifolia; and ligustilide is peak 7, coming from angelica sinensis.

[0056] A third aspect of the present invention provides a method for detecting the fingerprint of Tianwang Buxin Pills, comprising the following steps:

[0057] A) Preparation of test solution: Same as step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills;

[0058] B) Preparation of reference solution: Dissolve α-linolenic acid, salvianolic acid B, schisandrin A, β-asarone, 3,6'-dieserucyl sucrose, ligustilide, liquiritin, verbascoside, salvianolic acid A, schisandrin B, schisandrin A, schisandrin B, and linoleic acid reference substances in a solvent and calibrate to volume to prepare a reference solution;

[0059] C) Determination: Using ultra performance liquid chromatography (UPLC) under the same chromatographic conditions as in step 3) of the method for determining the contents of the seven components in Tianwang Buxin Pills, the test solution of step A) and the reference solution of step B) were respectively determined to obtain fingerprints of the test solution and the reference solution. The fingerprints of the test solution and the reference solution were compared, and the index components in the fingerprints of the test solution were attributed and located, thereby obtaining a fingerprint of Tianwang Buxin Pills.

[0060] Preferably, in step B), the CAS number of the liquiritin is 551-15-5, the CAS number of the verbascoside is 61276-17-3, the CAS number of salvianolic acid A is 96574-01-5, the CAS number of schisandrin B is 58546-54-6, the CAS number of schisandrin A is 61281-38-7, the CAS number of schisandrin B is 61281-37-6, and the CAS number of linoleic acid is 60-33-3.

[0061] Preferably, in step B), the reference substance solution is prepared by first adding a solvent to prepare a reference substance stock solution, and then adding a solvent for stepwise dilution.

[0062] More preferably, the reference substance stock solution needs to be stored in a refrigerator at 3-5°C, preferably 4°C, away from light.

[0063] Preferably, in step B), the solvent is a methanol aqueous solution with a volume percentage of 65-75%, preferably a methanol aqueous solution with a volume percentage of 70%.

[0064] Preferably, in step B), the content of α-linolenic acid in the reference solution is 200 μg / mL; the content of salvianolic acid B is 100 μg / mL; the content of schisandrae alcohol A is 30 μg / mL; the content of β-asarone is 30 μg / mL; the content of 3,6'-dieserucyl sucrose is 20 μg / mL; the content of ligustilide is 40 μg / mL; the content of liquiritin is 100 μg / mL; the content of verbascoside is 100 μg / mL; the content of salvianolic acid A is 100 μg / mL; the content of schisandrae alcohol B is 100 μg / mL; the content of schisandrin A is 100 μg / mL; the content of schisandrin B is 100 μg / mL; and the content of linoleic acid is 200 μg / mL.

[0065] Preferably, in step C), the detection wavelength in the ultra-high performance liquid chromatography is selected from one or more combinations of 203 nm, 250 nm, and 320 nm, preferably 203 nm.

[0066] Preferably, in step C), the fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peaks in the fingerprint of the test solution are identified by relative retention time based on the known characteristic peaks in the fingerprint of the reference solution, thereby attributing and locating the indicator components in the fingerprint of the test solution.

[0067] A fourth aspect of the present invention provides a method for detecting the fingerprint of Tianwang Buxin Pills and its use in quality detection of ingredients in Tianwang Buxin Pills.

[0068] The fifth aspect of the present invention provides a quality detection method for Tianwang Buxin Pills, comprising obtaining a fingerprint of Tianwang Buxin Pills using the aforementioned fingerprint detection method for Tianwang Buxin Pills, and comparing the obtained fingerprint of Tianwang Buxin Pills with a control fingerprint of Tianwang Buxin Pills obtained under the same fingerprint detection conditions for similarity.

[0069] Preferably, when comparing the measured fingerprint of Tianwang Buxin Pills with the reference fingerprint of Tianwang Buxin Pills for similarity, the comparison is performed using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software published by the State Pharmacopoeia Commission. More preferably, the similarity between the measured fingerprint of Tianwang Buxin Pills and the reference fingerprint of Tianwang Buxin Pills is greater than 0.96.

[0070] More preferably, when matching the common fingerprint peaks of the Tianwang Buxin Pills fingerprint spectrum with the control fingerprint spectrum of Tianwang Buxin Pills, automatic full spectrum matching is performed with a time window width of 0.2 min, and the fingerprint spectrum and the control fingerprint spectrum are generated using the average method.

[0071] Preferably, the control fingerprint of Tianwang Buxin Pills is obtained under the same conditions as the detection method of the fingerprint of Tianwang Buxin Pills. The control fingerprint of Tianwang Buxin Pills includes 25 common fingerprint peaks, with peak 17 as the reference peak (S peak, relative retention time is 1.000), and the relative retention times of the other 24 peaks are peak 1 (0.108±0.007), peak 2 (0.124±0.001), peak 3 (0.201±0.012), peak 4 (0.249±0.013), peak 5 (0.300±0.001), peak 6 (0.314±0.045), peak 7 (0.320±0.036), peak 8 (0.366±0.011), peak 9 (0.416±0.030), peak 10 (0.444±0.031), peak 11 (0.434±0.033), peak 12 (0.466±0.011), peak 13 (0.466±0.031), peak 14 (0.466±0.033), peak 15 (0.466±0.033), peak 16 (0.466±0.033), peak 17 (0.466±0.011), peak 18 (0.466±0.033), peak 19 (0.466±0.033), peak 20 (0.466±0.0 Peak (0.541±0.013), Peak 11 (0.573±0.001), Peak 12 (0.588±0.001), Peak 13 (0.821±0.053), Peak 14 (0.835±0.008), Peak 15 (0.861±0.001), Peak 16 (0.927±0.047), Peak 18 (1.176 ±0.012), peak 19 (1.198±0.012), peak 20 (1.204±0.016), peak 21 (1.242±0.021), peak 22 (1.261±0.021), peak 23 (1.274±0.026), peak 24 (1.289±0.026), and peak 25 (1.308±0.035).

[0072] The specific data of the control fingerprint of Tianwang Buxin Pills can be found in Figure 3 .

[0073] More preferably, the control fingerprint of the Tianwang Buxin Pills is compared with the fingerprint of the reference solution, such as Figure 2 As shown, the positioning determined that peak 6 is the fingerprint peak of liquiritin, peak 8 is the fingerprint peak of verbascoside, peak 9 is the fingerprint peak of 3,6'-dieserinoylsucrose, peak 10 is the fingerprint peak of salvianolic acid B, peak 11 is the fingerprint peak of salvianolic acid A, peak 13 is the fingerprint peak of schisandrae alcohol A, peak 14 is the fingerprint peak of β-asarone, peak 16 is the fingerprint peak of schisandrae alcohol B, peak 17 is the fingerprint peak of ligustilide, peak 18 is the fingerprint peak of schisandrin A, peak 20 is the fingerprint peak of schisandrin B, peak 21 is the fingerprint peak of α-linolenic acid, and peak 23 is the fingerprint peak of linoleic acid.

[0074] A sixth aspect of the present invention provides a method for screening fingerprints of multiple medicinal materials in Tianwang Buxin Pills, comprising the following steps:

[0075] a) Preparation of single medicinal material sample solutions: preparing any one or more of the 16 medicinal material samples in Tianwang Buxin Pills, namely, Danshen, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia root, Polygala tenuifolia root, Platycladi seed, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seed, Platycodon grandiflorum, and Cinnabaris, according to step A) of the Tianwang Buxin Pills fingerprint detection method, to obtain at least one single medicinal material sample solution;

[0076] b) Preparation of negative sample solution: 16 medicinal material samples including Salvia miltiorrhiza, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds, Platycodon grandiflorus and Cinnabar were prepared according to step A) of the detection method of Tianwang Buxin Pill fingerprint, and the samples were respectively lacking Salvia miltiorrhiza, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds, Platycodon grandiflorus and Cinnabar. The negative sample solution lacking Salvia miltiorrhiza, the negative sample solution lacking Acorus gramineus, the negative sample solution lacking Poria cocos, the negative sample solution lacking Ophiopogon japonicus, the negative sample solution lacking Rehmannia glutinosa, the negative sample solution lacking Polygala tenuifolia, the negative sample solution lacking Platycladi seed, the negative sample solution lacking Licorice root, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Codonopsis pilosula, the negative sample solution lacking Schisandra chinensis, the negative sample solution lacking Asparagus cochinchinensis, the negative sample solution lacking Scrophularia ningpoensis, the negative sample solution lacking stir-fried Ziziphus jujuba seeds, the negative sample solution lacking Platycodon grandiflorum, and the negative sample solution lacking Cinnabar;

[0077] c) Determination: The fingerprints of the single herbal medicine sample solution in step a) and the negative sample solution in step b) are respectively determined by ultra performance liquid chromatography (UPLC) under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Tianwang Buxin Pills;

[0078] d) Obtaining the control fingerprint: The test solution prepared according to step A) of the detection method for the fingerprint of Tianwang Buxin Pills is subjected to step C) of the same detection method for the fingerprint of Tianwang Buxin Pills to obtain the control fingerprint of Tianwang Buxin Pills;

[0079] e) Quality testing: The fingerprints of the single medicinal material sample solution and the negative sample solution are compared with the control fingerprint of Tianwang Buxin Pills. The corresponding characteristic peaks of the single medicinal material sample solution in the control fingerprint of Tianwang Buxin Pills are identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single medicinal material sample solution.

[0080] Preferably, in step a), the Poria cocos is the sclerotium of the fungus Poria cocos (Schw.) Wolf. of the Polyporaceae family. The Ophiopogon japonicus is the tuberous root of the plant Ophiopogon japonicus (Thunb.) ker-Gawl. of the Liliaceae family. The Rehmannia glutinosa is the tuberous root of the plant Rehmannia glutinosa Libosch. of the plant Scrophulariaceae family. The Codonopsis pilosula is the dried root of the plant Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen or Codonopsis tangshen Oliv. of the plant Campanulaceae family. The Asparagus cochinchinensis is the tuberous root of the plant Asparagus cochinchinensis (Lour.) Merr. of the Liliaceae family. The Scrophulariaceae is the dried root of Scrophularia ningpoensis Hemsl. or Scrophularia buergeriana Miq. (S.oldhami Oliv.), both of the Scrophulariaceae family. The stir-fried Chinese jujube kernel is stir-fried Chinese jujube kernel, which is the mature seed of Ziziphus jujuba Mill.var.spinosa (Bunge) Hu ex H.F.Chou, both of the Rhamnaceae family. The Platycodon grandiflorum is the dried root of Platycodon grandiflorum (Jacq.) A.DC., both of the Campanulaceae family. The cinnabar is the ore of Cinnabar, a sulfide mineral.

[0081] Preferably, in step e), the present invention locates the attribution of characteristic peaks of the measured fingerprint of the single medicinal material sample solution and the reference fingerprint of Tianwang Buxin Pills, and uses the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" issued by the State Pharmacopoeia Commission for analysis and processing, and confirms the attribution of the characteristic peaks of each single medicinal material of Tianwang Buxin Pills by the relative retention time of each characteristic peak on the reference fingerprint of Tianwang Buxin Pills. Specific results are shown in Figure 3 and Table 2.

[0082] Table 2 The characteristic peaks of each single medicinal ingredient in Tianwang Buxin Pills

[0083]

[0084] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Salvia miltiorrhiza sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are peak 10 and peak 11.

[0085] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Acorus tatarinowii sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are peak 14 and peak 23.

[0086] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Rehmannia glutinosa sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak 4, peak 5, peak 8, and peak 23.

[0087] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the processed Polygala tenuifolia sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are peak 9 and peak 15.

[0088] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Platycladi seed sample solution includes 5 common fingerprint peaks, and the 2 common fingerprint peaks are peak 21, peak 22, peak 23, peak 24, and peak 25.

[0089] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the licorice sample solution includes three common fingerprint peaks, and the three common fingerprint peaks are peak 2, peak 6, and peak 7.

[0090] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Angelica sinensis sample solution includes three common fingerprint peaks, and the three common fingerprint peaks are Peak 1, Peak 17, and Peak 23.

[0091] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Schisandra chinensis sample solution includes 6 common fingerprint peaks, and the 6 common fingerprint peaks are peak 13, peak 16, peak 18, peak 19, peak 20, and peak 23.

[0092] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Asparagus cochinchinensis sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are Peak 1 and Peak 2.

[0093] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Scrophularia ningpoensis sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are peak 8 and peak 12.

[0094] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the stir-fried spinach seed sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak 3, peak 7, peak 23, and peak 25.

[0095] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Platycodon grandiflorum sample solution includes one common fingerprint peak, and the one common fingerprint peak is peak 1.

[0096] Preferably, in step e), among the single medicinal material sample solutions, the fingerprints of the Poria cocos, Ophiopogon japonicus, Codonopsis pilosula, and Cinnabar bark sample solutions have no common fingerprint peaks.

[0097] Among the above-mentioned common fingerprint peaks, peak 6 is determined to be the fingerprint peak of liquiritin, peak 8 is the fingerprint peak of verbascoside, peak 9 is the fingerprint peak of 3,6'-dieserinoylsucrose, peak 10 is the fingerprint peak of salvianolic acid B, peak 11 is the fingerprint peak of salvianolic acid A, peak 13 is the fingerprint peak of schisandra alcohol A, peak 14 is the fingerprint peak of β-asarone, peak 16 is the fingerprint peak of schisandra alcohol B, peak 17 is the fingerprint peak of ligustilide, peak 18 is the fingerprint peak of schisandrin A, peak 20 is the fingerprint peak of schisandrin B, peak 21 is the fingerprint peak of α-linolenic acid, and peak 23 is the fingerprint peak of linoleic acid.

[0098] The water used in the present invention is all purified water.

[0099] As described above, the present invention provides a method for determining the content and fingerprint of multiple components in Tianwang Buxin Pills, and comprehensively controls the quality of Tianwang Buxin Pills by combining fingerprints with multi-component quantification.

[0100] First, an ultra-performance liquid chromatography (UPLC) method was established to determine the contents of the seven main active ingredients in Tianwang Buxin Pills (α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-dieserucyl sucrose, and ligustilide). The method demonstrated good separation of the seven active ingredients, with symmetrical peak shapes. The simple, reliable, and easy-to-use method is suitable for multi-component content determination in Tianwang Buxin Pills. The method was validated for quantitative analysis of the seven ingredients, demonstrating good linearity within their respective ranges, good reproducibility, high accuracy, and high precision. This method meets the requirements of material basis research for Tianwang Buxin Pills and facilitates quality control of the pills. Furthermore, the method was able to screen the medicinal materials to which the seven ingredients in Tianwang Buxin Pills belong, identifying the seven medicinal materials to which they belong (Salvia miltiorrhiza, Acorus gramineus, Polygala tenuifolia, Platycladi seed, Glycyrrhiza uralensis, Angelica sinensis, and Schisandra chinensis).

[0101] Secondly, the similarity of the fingerprints of Tianwang Buxin Pills was compared and analyzed, and a fingerprint method for Tianwang Buxin Pills was established. Thirteen index components (a-linolenic acid, salvianolic acid B, schisandrin A, β-asarone, 3,6'-dieserucyl sucrose, ligustilide, liquiritin, verbascoside, salvianolic acid A, schisandrin B, schisandrin A, schisandrin B and linoleic acid) were identified. The contributions of different categories of chemical components to the fingerprint system of Tianwang Buxin Pills were highlighted from different aspects. After methodological investigation, a total of 25 common peaks were calibrated and the 25 chromatographic peaks were attributed to the medicinal materials. By identifying and confirming the chromatographic peaks of each single medicinal ingredient in Tianwang Buxin Pills, the compound peaks of 12 medicinal ingredients (Danshen, Acorus gramineus, Rehmannia root, processed Polygala tenuifolia, Platycladus chinensis seeds, Licorice root, Angelica sinensis, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds and Platycodon grandiflorum) can be well characterized in the fingerprint, thereby achieving effective monitoring of the source of raw materials, strictly controlling the quality of the original medicinal materials, and can be used for quality control of the production process, thereby ensuring the safety and effectiveness of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG1 shows the specificity investigation of the 7 ingredients in Tianwang Buxin Pills of the present invention Figure 1a 、 1b , 1c, where Figure 1a This is a map of the specificity of the lack of cypress seeds. Figure 1b This is a specific investigation chart for the lack of Schisandra chinensis, Licorice root, Acorus calamus, and Salvia miltiorrhiza. Figure 1c This is a chart showing the specificity of Angelica sinensis and processed Polygala tenuifolia. 1: a-linolenic acid, 2: salvianolic acid B, 3: glycyrrhizic acid, 4: schisandrae alcohol A, 5: β-asarone, 6: 3,6'-diesinaroylsucrose, 7: ligustilide.

[0103] Figure 2 The fingerprint of Tianwang Buxin Pills of the present invention is shown, wherein: 6: glycyrrhizin; 8: verbascoside; 9: 3,6'-dieserucyl sucrose; 10: salvianolic acid B; 11: salvianolic acid A; 13: schisandra alcohol A; 14: β-asarone; 16: schisandra alcohol B; 17: ligustilide; 18: schisandrin A; 20: schisandrin B; 21: a-linolenic acid; 23: linoleic acid.

[0104] Figure 3 Shown is the UPLC attribution diagram of the characteristic peaks of the Tianwang Buxin Pill sample and single medicinal materials in the present invention.

[0105] Figure 4Shown are the superimposed fingerprints of UPLC of 15 batches of Tianwang Buxin Pills samples in the present invention, wherein S1: 20230201; S2: 20230202; S3: 20230203; S4: 20230204; S5: 20230205; S6: 20230309; S7: 20230315; S8: 20230322; S9: 20230407; S10: 20230418; S11: 20230509; S12: 20230517; S13: 20230529; S14: 20230605; S15: 20230612; R: control fingerprint of Tianwang Buxin Pills sample. DETAILED DESCRIPTION

[0106] The present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0107] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0108] The reagents and instruments used in the following examples are as follows:

[0109] 1. Reagents

[0110] Reference substances: a-linolenic acid (batch number 111631-202006, purity 98%), schisandra chinensis alcohol (batch number 110857-201815, purity 99.7%), β-asarone (batch number 112018-201802, purity 99.3%), 3,6'-diesinapoylsucrose (batch number 111848-202006, purity 97.9%), liquiritin (Batch number 111610-201908, purity 95.0%), verbascoside (Batch number 111520-201713, purity 92.5%), schisandra chinensis (Batch number 110764-201915, purity 99.5%), and linoleic acid (Batch number 111622-202105, purity 99.6%). All the above reference substances were purchased from the China Food and Drug Inspection Institute. Salvianolic acid B (batch number 5323, purity 95.2%), glycyrrhizic acid (batch number 5520, purity 96.5%), ligustilide (batch number 8155, purity 97.4%), salvianolic acid A (batch number 13483, purity ≥98.0%), schisandrin B (batch number RS02881120, purity ≥98.0%) and schisandrin B (batch number 7852, purity ≥98.0%). All the above reference substances were purchased from Shanghai Shidande Standard Technology Service Co., Ltd.

[0111] Sample: Tianwang Buxin Pills raw material powder, provided by Shanghai Hutchison Pharmaceutical Co., Ltd., a total of 15 batches (batch numbers are S1-20230201; S2-20230202; S3-20230203; S4-20230204; S5-20230205; S6-20230309; S7-20230315; S8-20230322; S9-20230407; S10-20230418; S11-20230509; S12-20230517; S13-20230529; S14-20230605; S15-20230612).

[0112] Single medicinal materials: Salvia miltiorrhiza, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Polygala tenuifolia, Platycladi seed, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seed, Platycodon grandiflorum and Cinnabar were all provided by Shanghai Hutchison Pharmaceutical Co., Ltd.

[0113] Reagents: anhydrous methanol (analytical grade AR, Sinopharm Chemical Reagent Co., Ltd.), acetonitrile (chromatographic grade, TEDIA, USA), phosphoric acid (chromatographic grade, TEDIA, USA), and ultrapure water were prepared by a Milli-Q ultrapure water treatment system.

[0114] 2. Instruments

[0115] A Waters Acquity UPLC H-Class ultra-high performance liquid chromatograph (equipped with an Empower 3 chromatography workstation, a quaternary ultrahigh pressure solvent manager, an automatic sample manager, a column oven, and a PDAeλ detector, all from Waters, USA) was used; an SB-5200DTD ultrasonic cleaner was used (Ningbo Xinzhi Biotechnology Co., Ltd.); AL204 and XS205 analytical electronic balances were used (METTLER TOLEDO Instruments Shanghai Co., Ltd.); and a Milli-Q Advantage A10 ultrapure water system was used (Merck Millipore, Germany).

[0116] Example 1

[0117] 1. Sample pretreatment

[0118] Preparation of test solution: Take the powder sample of Tianwang Buxin Pills batch 20230201, accurately weigh 1.5 g, place it in a 50 mL centrifuge tube, accurately add 25 mL of 70% methanol, and ultrasonically extract (power 250 W, frequency 40 kHz) for 30 minutes. Let it stand and cool to room temperature, take the supernatant, and filter it through a 0.22 μm microporous filter membrane to obtain test solution 1#.

[0119] Preparation of reference solution: Accurately weigh α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandra chinensis alcohol A, β-asarone, 3,6'-diesinarosyl sucrose and ligustilide reference substances respectively, dissolve them in 70% methanol in a 100 mL volumetric flask, make up to volume, prepare the reference stock solution, and store in a refrigerator at 4°C away from light.

[0120] The reference substance stock solution was then precisely measured and serially diluted with 70% methanol to the desired volume to prepare a series of reference substance solutions of varying concentrations. In these reference substance solutions, the content of α-linolenic acid ranged from 9.73 to 389.49 μg / mL, the content of salvianolic acid B ranged from 23.78 to 475.52 μg / mL, the content of glycyrrhizic acid ranged from 9.75 to 195.03 μg / mL, the content of schisandrin A ranged from 7.74 to 154.73 μg / mL, the content of β-asarone ranged from 8.25 to 165.04 μg / mL, the content of 3,6'-dieserucyl sucrose ranged from 4.98 to 99.66 μg / mL, and the content of ligustilide ranged from 9.61 to 192.17 μg / mL. The reference substance solutions were shaken well and filtered before use, and the filtrate was collected.

[0121] 2. Chromatographic conditions

[0122] The chromatographic conditions of the ultra-high performance liquid chromatography method are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm); the detector is a photodiode array detector (DAD); the column temperature is 20° C.; the injection volume is 1 μL; and the flow rate is 0.3 mL / min.

[0123] The analysis was performed using a multi-wavelength method, wherein a detection wavelength of 203 nm was used to detect a-linolenic acid, a detection wavelength of 250 nm was used to detect salvianolic acid B, glycyrrhizic acid, schisandra methanol, and β-asarone, and a detection wavelength of 320 nm was used to detect 3,6'-diesinapoylsucrose and ligustilide.

[0124] The mobile phase was acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase A was acetonitrile and phase B was 0.1% phosphoric acid aqueous solution; the analysis time was 50 min; and the elution was gradient.

[0125] As shown in Table 1, the specific procedure of the gradient elution is:

[0126] 0-15 min, the volume ratio of phase A:phase B was 8:92-22:78;

[0127] 15-23 min, the volume ratio of phase A:phase B is 22:78-35:65;

[0128] 23-30 min, the volume ratio of phase A:phase B is 35:65-40:60;

[0129] 30-35 min, the volume ratio of phase A:phase B is 40:60-53:47;

[0130] 35-42 min, the volume ratio of phase A:phase B is 53:47-95:5;

[0131] 42-45 min, the volume ratio of phase A:phase B is 95:5-95:5;

[0132] 45-46 min, the volume ratio of phase A:phase B is 95:5-8:92;

[0133] 46-50min, the volume ratio of phase A:phase B is 8:92-8:92.

[0134] 3. Determination

[0135] Using the external standard method, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by ultra-high performance liquid chromatography (UPLC) and a standard working curve was plotted. The obtained test solution was then injected and analyzed by ultra-high performance liquid chromatography (UPLC). The analysis results were substituted into the standard working curve to obtain the contents of the seven components in the test solution.

[0136] Specifically, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations. The samples were analyzed using an ultra-high performance liquid chromatograph to obtain a linear relationship between the content and peak area of the seven components in the reference solution. The corresponding standard working curve was drawn by corresponding the chromatographic peak area of each component to its corresponding content, and the regression equation of each standard working curve was calculated. The test solution was then tested using an ultra-high performance liquid chromatograph, and the chromatographic peak areas of the seven components in the test solution were substituted into the regression equation of each standard working curve to obtain the content of the corresponding component.

[0137] Example 2

[0138] 1. Sample pretreatment

[0139] Preparation of test solution: Take the powder sample of Tianwang Buxin Pills batch 20230201, accurately weigh 1.5 g, place it in a 50 mL centrifuge tube, accurately add 25 mL of 68% methanol, and ultrasonically extract (power 250 W, frequency 40 kHz) for 32 minutes. Let it stand and cool to room temperature, take the supernatant, and filter it through a 0.22 μm microporous filter membrane to obtain test solution 2#.

[0140] Preparation of reference solution: Accurately weigh α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-diesinarosylsucrose and ligustilide reference substances respectively, dissolve them in 100 mL volumetric flask with 68% methanol, make up to volume, prepare reference stock solution, and store in a refrigerator at 3°C away from light.

[0141] Then, the reference substance stock solution was precisely measured, diluted stepwise with 68% methanol and fixed to volume to prepare a series of reference substance solutions with different concentrations. The concentration range of the series of reference substance solutions with different concentrations was the same as that of step 1 in Example 1.

[0142] 2. Chromatographic conditions

[0143] The chromatographic conditions of the ultra-high performance liquid chromatography method are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm); the detector is a photodiode array detector (DAD); the column temperature is 22° C.; the injection volume is 1.5 μL; and the flow rate is 0.2 mL / min.

[0144] The analysis was performed using a multi-wavelength method, wherein a detection wavelength of 203 nm was used to detect a-linolenic acid, a detection wavelength of 250 nm was used to detect salvianolic acid B, glycyrrhizic acid, schisandra methanol, and β-asarone, and a detection wavelength of 320 nm was used to detect 3,6'-diesinapoylsucrose and ligustilide.

[0145] The mobile phase was acetonitrile-0.12% phosphoric acid aqueous solution, wherein phase A was acetonitrile and phase B was 0.12% phosphoric acid aqueous solution; the analysis time was 50 min; and the elution was gradient.

[0146] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0147] 3. Determination

[0148] The specific measurement process is the same as step 3 in Example 1.

[0149] Example 3

[0150] 1. Sample pretreatment

[0151] Preparation of test solution: Take the powder sample of Tianwang Buxin Pills batch 20230201, accurately weigh 1.5 g, place it in a 50 mL centrifuge tube, accurately add 25 mL of 72% methanol, and ultrasonically extract (power 250 W, frequency 40 kHz) for 28 minutes. Let it stand and cool to room temperature, take the supernatant, and filter it through a 0.22 μm microporous filter membrane to obtain test solution 3#.

[0152] Preparation of reference solution: Accurately weigh α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-diesinarosylsucrose and ligustilide reference substances respectively, dissolve them in a 100 mL volumetric flask with 72% methanol, make up to volume, prepare the reference stock solution, and store in a refrigerator at 5°C away from light.

[0153] The reference substance stock solution was then accurately measured, diluted stepwise with 72% methanol, and fixed to volume to prepare a series of reference substance solutions of different concentrations. The concentration range of the series of reference substance solutions of different concentrations was the same as that of step 1 in Example 1.

[0154] 2. Chromatographic conditions

[0155] The chromatographic conditions of the ultra-high performance liquid chromatography method are as follows: the chromatographic column is a Waters Acquity UPLC BEH C18 chromatographic column (2.1 mm×100 mm, 1.7 μm); the detector is a photodiode array detector (DAD); the column temperature is 18° C.; the injection volume is 0.8 μL; and the flow rate is 0.4 mL / min.

[0156] The analysis was performed using a multi-wavelength method, wherein a detection wavelength of 203 nm was used to detect a-linolenic acid, a detection wavelength of 250 nm was used to detect salvianolic acid B, glycyrrhizic acid, schisandra methanol, and β-asarone, and a detection wavelength of 320 nm was used to detect 3,6'-diesinapoylsucrose and ligustilide.

[0157] The mobile phase was acetonitrile-0.08% phosphoric acid aqueous solution, wherein phase A was acetonitrile and phase B was 0.08% phosphoric acid aqueous solution; the analysis time was 50 min; and the elution was gradient.

[0158] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0159] 3. Determination

[0160] The specific measurement process is the same as step 3 in Example 1.

[0161] Example 4

[0162] A series of solutions of different concentrations of the above seven reference substances were prepared, and the reference substance stock solutions were accurately aspirated. Ultra-high performance liquid chromatography analysis was performed using the UPLC detection conditions of step 2 of Example 1. The chromatogram was recorded. The standard curve was plotted with the concentration of each reference substance (x, μg / mL) as the abscissa and the peak area (y) as the ordinate. Linear regression calculation was performed to obtain the regression equation, correlation coefficient, and linear range. The concentration of each mixed reference substance was diluted so that the limit of quantification S / N ≥ 10 and the limit of detection S / N ≥ 3. The specific results are shown in Table 3.

[0163] As shown in Table 3, the regression equation has a good linear relationship when the sample is injected within the corresponding concentration range, and the correlation coefficient R 2 Not less than 0.9996.

[0164] Table 3 Linear relationships among seven components (n=6)

[0165]

[0166] Example 5

[0167] 1. Precision

[0168] Any reference solution prepared in Example 1 was taken and tested according to the method in Example 1. The sample was continuously injected 6 times for analysis and the peak area was recorded. The results showed that the RSDs of the peak areas of the seven components, including α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin A, β-asarone, 3,6'-diesinarosylsucrose and ligustilide, were all less than 0.31%, indicating that the instrument had good precision.

[0169] 2. Repeatability

[0170] Six samples of Tianwang Buxin Pills from the same batch (batch number 20230201) were taken and six test samples were prepared in parallel according to the method in Example 1 above. The peak areas were recorded and the results showed that the RSDs of the peak areas of seven components, including α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrae alcohol A, β-asarone, 3,6'-diesinarosyl sucrose and ligustilide, were all less than 0.99%, indicating that the method had good repeatability and high accuracy.

[0171] 3. Stability

[0172] Take one portion of Tianwang Buxin Pills sample from the same batch (batch number 20230201) and prepare one portion of the test solution according to the method in Example 1 above. The solution was then placed for 1 h, 3 h, 7 h, 12 h, 24 h, and 36 h for detection, and the peak areas were recorded. The results showed that the RSDs of the peak areas of the seven components, including α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandra chinensis alcohol A, β-asarone, 3,6'-diesinarosyl sucrose, and ligustilide, were all less than 1.44%, indicating that the test solution had good stability within 36 h.

[0173] 4. Sample recovery rate

[0174] Nine samples of Tianwang Buxin Pills with batch number 20230201 were taken and accurately weighed. α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrae alcohol A, β-asarone, 3,6'-diesinarosyl sucrose and ligustilide reference solution were added at low, medium and high mass concentration levels (equivalent to 50%, 100% and 150% of the original mass fraction, respectively). Three parts were taken for each mass concentration. The test solution was prepared according to step 1 in Example 1, and the solution was analyzed according to the chromatographic conditions of step 2 in Example 1. The sample recoveries and RSD% of the seven components at different addition ratios were calculated. The results are shown in Table 4. As shown in Table 4, the average recoveries of the seven components, including α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-diesinarosylsucrose and ligustilide, were 93.72% to 99.05%, with RSDs (n=9) ranging from 0.78% to 2.91%, indicating that the method had good accuracy.

[0175] Table 4 Sample recovery test results (n=9)

[0176]

[0177]

[0178] Example 6

[0179] Fifteen batches of Tianwang Buxin Pills samples were taken, and the test solution was prepared according to step 1 in Example 1. The samples were respectively injected and analyzed according to the chromatographic conditions of step 2 in Example 1, and the chromatograms were recorded. The contents of α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrae alcohol A, β-asarone, 3,6'-diesinarosoylsucrose and ligustilide in the Tianwang Buxin Pills samples were calculated by the external standard method. The results are shown in Table 5.

[0180] As shown in Table 5, the established content detection method has good separation of the 7 compounds in Tianwang Buxin Pills, symmetrical peak shape, simple and reliable method, easy to operate, good reproducibility and accuracy, and can be used to accurately determine the content of the 7 compounds in Tianwang Buxin Pills.

[0181] Table 5 Content determination results of 15 batches of Tianwang Buxin Pills (mg / g, n=2)

[0182]

[0183] Example 7

[0184] The sample pretreatment step in step 1 of the above embodiment 1 was used to prepare the test solution.

[0185] Seven medicinal material samples, namely, Salvia miltiorrhiza, Acorus gramineus, Polygala tenuifolia (processed), Platycladus chinensis seeds, Licorice root, Angelica sinensis, and Schisandra chinensis, were taken respectively, and the sample pretreatment steps in step 1 of the above embodiment 1 were used to prepare seven single medicinal material sample solutions.

[0186] Seven medicinal material samples, namely, Salvia miltiorrhiza, Acorus gramineus, processed Polygala tenuifolia, Platycladi seed, Licorice, Angelica sinensis, and Schisandra chinensis, were respectively lacking Salvia miltiorrhiza, Acorus gramineus, processed Polygala tenuifolia, Platycladi seed, Licorice, Angelica sinensis, and Schisandra chinensis. The sample pretreatment steps in step 1 of the above embodiment 1 were adopted to obtain negative sample solutions lacking Salvia miltiorrhiza, negative sample solutions lacking Acorus gramineus, negative sample solutions lacking processed Polygala tenuifolia, negative sample solutions lacking Platycladi seed, negative sample solutions lacking Licorice, negative sample solutions lacking Angelica sinensis, and negative sample solutions lacking Schisandra chinensis, respectively.

[0187] Using the chromatographic conditions in step 2 of Example 1 above, the test solution, 7 single medicinal material sample solutions, the negative sample solution lacking Salvia miltiorrhiza, the negative sample solution lacking Acorus gramineus, the negative sample solution lacking processed Polygala tenuifolia, the negative sample solution lacking Platycladi seed, the negative sample solution lacking Licorice, the negative sample solution lacking Angelica sinensis, and the negative sample solution lacking Schisandra chinensis were respectively measured to obtain the fingerprints of the test solution, 7 single medicinal material sample solutions, and 7 negative sample solutions, respectively. The fingerprints of the test solution, 7 single herbal medicine sample solutions, and 7 negative sample solutions were imported into the 2012 version of the software "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" issued by the State Pharmacopoeia Commission for analysis and processing. The fingerprints of the single herbal medicine sample solutions and the negative sample solutions were compared with the reference fingerprints of the 7 ingredients in Tianwang Buxin Pills. The corresponding characteristic peaks of the single herbal medicine sample solutions in the reference fingerprints of the 7 ingredients in Tianwang Buxin Pills were identified by relative retention time, thereby attributing and locating the 7 ingredients in Tianwang Buxin Pills. The specific results are shown in Figure 1a 、 Figure 1b 、 Figure 1cThe results showed that the chromatographic peaks of the seven components had no negative interference, and the method had good specificity. Among them, α-linolenic acid was peak 1, which came from Platycladus orientalis; salvianolic acid B was peak 2, which came from Salvia miltiorrhiza; glycyrrhizic acid was peak 3, which came from Glycyrrhiza uralensis; schisandrin A was peak 4, which came from Schisandra chinensis; β-asarone was peak 5, which came from Acorus tatarinowii; 3,6'-diesinarosylsucrose was peak 6, which came from Polygala tenuifolia; and ligustilide was peak 7, which came from Angelica sinensis.

[0188] Example 8

[0189] 1. Sample pretreatment

[0190] Preparation of the test solution: The preparation process of the test solution is the same as that in step 1 of Example 1.

[0191] Preparation of reference solution: Accurately weigh α-linolenic acid, salvianolic acid B, schisandrin A, β-asarone, 3,6'-dieserinoylsucrose, ligustilide, liquiritin, verbascoside, salvianolic acid A, schisandrin B, schisandrin A, schisandrin B and linoleic acid reference substances, accurately weigh them, dissolve them in 70% methanol in a 100 mL volumetric flask, make up to volume, prepare the reference substance stock solution, and store in a 4°C refrigerator away from light.

[0192] Then accurately measure the reference substance stock solution, dilute it step by step with 70% methanol and make up to volume to prepare a series of reference substance solutions with different concentrations. In a series of reference solutions with different concentrations, the content of α-linolenic acid was 200 μg / mL; the content of salvianolic acid B was 100 μg / mL; the content of schisandrae alcohol A was 30 μg / mL; the content of β-asarone was 30 μg / mL; the content of 3,6'-dieserucylsucrose was 20 μg / mL; the content of ligustilide was 40 μg / mL; the content of liquiritin was 100 μg / mL; the content of verbascoside was 100 μg / mL; the content of salvianolic acid A was 100 μg / mL; the content of schisandrae alcohol B was 100 μg / mL; the content of schisandrae B was 100 μg / mL; and the content of linoleic acid was 200 μg / mL.

[0193] 2. Chromatographic conditions

[0194] The chromatographic conditions of the ultra-high performance liquid chromatography method are the same as those of the ultra-high performance liquid chromatography method in step 2 of Example 1, except that the detection wavelength is 203 nm.

[0195] 3. Determination

[0196] The test solution and the reference solution in step 1 are respectively measured by ultra-high performance liquid chromatography under the chromatographic conditions in step 2 to obtain a fingerprint of the test solution and a fingerprint of the reference solution. The fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peaks in the fingerprint of the test solution are identified by relative retention time based on the known characteristic peaks in the fingerprint of the reference solution, thereby attributing and locating the index components in the fingerprint of the test solution to obtain the fingerprint of Tianwang Buxin Wan.

[0197] Example 9

[0198] 15 batches of Tianwang Buxin Pills were tested using the fingerprint detection method of Tianwang Buxin Pills established in Example 8 above, and the fingerprints of the test solution and the reference solution were obtained. The fingerprint data of the test sample obtained were imported into the 2012 version of the software "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" issued by the State Pharmacopoeia Commission, and automatic full spectrum matching (time window width of 0.2 min) was used to generate fingerprints and reference fingerprints using the average method. The fingerprints of the test sample were compared with the reference fingerprints of Tianwang Buxin Pills obtained under the same fingerprint detection conditions for similarity, and the similarity of the fingerprints of each batch of Tianwang Buxin Pills was calculated. The results of the similarity comparison between the fingerprints of 15 batches of test samples and the reference fingerprints are shown in Table 6 below. The similarity of the fingerprints of the 15 batches of Tianwang Buxin Pills was greater than 0.96, indicating that the fingerprints of the 15 batches of samples had good similarity and a relatively stable overall quality. The specific similarity is shown in Table 6. Figure 4 .

[0199] Table 6 Similarity results of Tianwang Buxin Pill fingerprints

[0200] batch number Similarity 20230201 0.970 20230202 0.992 20230203 0.996 20230204 0.998 20230205 0.991 20230309 0.999 20230315 0.961 20230322 0.963 20230407 0.997 20230418 0.996 20230509 0.998 20230517 0.991 20230529 0.999 20230605 0.989 20230612 0.989

[0201] Example 10

[0202] The Tianwang Buxin Pills were tested using the fingerprint detection method established in Example 8. According to the fingerprints of the test solution and the reference solution, 25 common fingerprint peaks were identified. Among them, peak 17 had a moderate elution time, good separation, and a large peak area. Therefore, peak 17 was used as the reference peak (S peak, relative retention time of 1.000). The relative retention times of the other 24 peaks were ranked in this order. Peak 1 (0.108±0.007), peak 2 (0.124±0.001), peak 3 (0.201±0.012), peak 4 (0.249±0.013), peak 5 (0.300±0.001), peak 6 (0.314±0.045), peak 7 (0.320±0.036), peak 8 (0.366±0.011), peak 9 (0.4 16±0.030), peak 10 (0.541±0.013), peak 11 (0.573±0.001), peak 12 (0.588±0.001), peak 13 (0.821±0.053), peak 14 (0.835±0.008), peak 15 (0.861±0.001), peak 16 (0.927±0.047), peak 18 (1. 176±0.012), peak 19 (1.198±0.012), peak 20 (1.204±0.016), peak 21 (1.242±0.021), peak 22 (1.261±0.021), peak 23 (1.274±0.026), peak 24 (1.289±0.026), and peak 25 (1.308±0.035), see Table 7 below for details.

[0203] Table 7 Relative retention time of common peaks in fingerprint

[0204]

[0205] Note: Peak 17 is the positioning peak (S peak)

[0206] The fingerprint of the control Tianwang Buxin Pills was compared with that of the reference solution, and the relative retention time was used to determine the fingerprint of the control Tianwang Buxin Pills. Figure 2 、 3, identified the corresponding characteristic peaks in the control fingerprint of Tianwang Buxin Pills, and located and determined that peak 6 was the fingerprint peak of liquiritin, peak 8 was the fingerprint peak of verbascoside, peak 9 was the fingerprint peak of 3,6'-dieserucyl sucrose, peak 10 was the fingerprint peak of salvianolic acid B, peak 11 was the fingerprint peak of salvianolic acid A, peak 13 was the fingerprint peak of schisandra alcohol A, peak 14 was the fingerprint peak of β-asarone, peak 16 was the fingerprint peak of schisandra alcohol B, peak 17 was the fingerprint peak of ligustilide, peak 18 was the fingerprint peak of schisandrin A, peak 20 was the fingerprint peak of schisandrin B, peak 21 was the fingerprint peak of a-linolenic acid, and peak 23 was the fingerprint peak of linoleic acid.

[0207] Example 11

[0208] The fingerprint detection method of Tianwang Buxin Pills in the present invention was methodologically verified, and the performance index results are as follows.

[0209] 1. Precision

[0210] One sample of Tianwang Buxin Pills from the same batch (batch number 20230201) was taken and tested after preparation according to the detection method of the Tianwang Buxin Pills fingerprint in Example 8 above. The sample was injected continuously for 6 times, and the chromatogram was recorded. Peak 17 (ligustilide) was used as the reference peak. The relative retention time RSDs of the 25 common peaks were calculated to be less than 0.51%, and the relative peak area RSDs were less than 2.87%, indicating that the instrument had good precision.

[0211] 2. Repeatability

[0212] Six samples of Tianwang Buxin Pills from the same batch (batch number 20230201) were prepared and tested according to the detection method of the Tianwang Buxin Pills fingerprint in Example 8. The chromatograms were recorded. Peak 17 (ligustilide) was used as the reference peak. The relative retention time RSDs of the 25 common peaks were calculated to be less than 0.02%, and the relative peak area RSDs were less than 2.87%. The results showed that the method had good repeatability.

[0213] 3. Stability

[0214] One sample of Tianwang Buxin Pills from the same batch (batch number 20230201) was taken and tested after preparation according to the detection method of the Tianwang Buxin Pills fingerprint in Example 8 above. After preparing the test solution, it was placed for 1h, 3h, 5h, 12h, 24h, and 36h for detection, and the chromatogram was recorded. Peak 17 (ligustilide) was used as the reference peak. The relative retention time RSD of the 25 common peaks was calculated to be less than 0.39%, and the RSD of the relative peak area was less than 2.99%. The results showed that the test solution had good stability within 36h.

[0215] Example 12

[0216] The sample pretreatment steps in Example 8 were used to prepare the test solution.

[0217] Sixteen medicinal material powders, including salvia miltiorrhiza, calamus tatarinowii, poria, ophiopogon japonicus, rehmannia root, processed polygala tenuifolia, cypress seed, liquorice, angelica sinensis, codonopsis pilosula, schisandra chinensis, asparagus cochinchinensis, scrophularia ningpoensis, stir-fried jujube seed, platycodon grandiflorum, and cinnabar, were respectively taken and the sample pretreatment steps in the above Example 8 were used to prepare 16 single medicinal material sample solutions.

[0218] Sixteen medicinal material samples of Salvia miltiorrhiza, Acorus calamus, Poria cocos, Radix Ophiopogonis, Rehmannia root, processed Polygala tenuifolia, Platycladus chinensis, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds, Platycodon grandiflorus and Cinnabar were respectively lacking Salvia miltiorrhiza, Acorus calamus, Poria cocos, Radix Ophiopogonis, Rehmannia root, processed Polygala tenuifolia, Platycladus chinensis, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds, Platycodon grandiflorus and Cinnabar, and the sample pretreatment steps in Example 8 were adopted to obtain negative sample solutions lacking Salvia miltiorrhiza. , the negative sample solution lacking Acorus calamus, the negative sample solution lacking Poria cocos, the negative sample solution lacking Ophiopogon japonicus, the negative sample solution lacking Rehmannia glutinosa, the negative sample solution lacking processed Polygala tenuifolia, the negative sample solution lacking Platycladi seed, the negative sample solution lacking Licorice root, the negative sample solution lacking Angelica sinensis, the negative sample solution lacking Codonopsis pilosula, the negative sample solution lacking Schisandra chinensis, the negative sample solution lacking Asparagus cochinchinensis, the negative sample solution lacking Scrophularia ningpoensis, the negative sample solution lacking stir-fried Ziziphus jujuba seed, the negative sample solution lacking Platycodon grandiflorum and the negative sample solution lacking Cinnabar.

[0219] Steps 2 and 3 of the fingerprint detection method for Tianwang Buxin Pills in Example 8 were used to measure the test sample solution, 16 single medicinal material sample solutions, and 16 negative sample solutions, respectively, to obtain fingerprints of the test sample solution, 16 single medicinal material sample solutions, and 16 negative sample solutions, respectively. The fingerprints of the test sample solution, 16 single medicinal material sample solutions, and 16 negative sample solutions were imported into the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software published by the State Pharmacopoeia Commission for analysis and processing, to obtain a control fingerprint of Tianwang Buxin Pills obtained by simultaneously using the fingerprint detection method for Tianwang Buxin Pills in Example 8. The fingerprints of the test solution, 16 single herbal sample solutions, and 16 negative sample solutions were compared with the control fingerprint of Tianwang Buxin Pills. The corresponding characteristic peaks of the 16 single herbal sample solutions in the control fingerprint of Tianwang Buxin Pills were identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprints of the 16 single herbal sample solutions. The specific results are shown in Figure 2 、 3 .

[0220] Depend on Figure 2 、 3It can be seen that in the single medicinal material sample solutions, the fingerprint peaks in common in the fingerprint spectra of the sample solutions of 16 medicinal materials including Salvia miltiorrhiza, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia glutinosa, Polygala tenuifolia, Platycladi seed, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seed, Platycodon grandiflorum and Cinnabar are shown in Table 2. Specifically, it was determined that peak 1 originated from Angelica sinensis, Asparagus cochinchinensis and Platycodon grandiflorum; peak 2 originated from Licorice and Asparagus cochinchinensis; peak 3 originated from stir-fried Ziziphus jujuba seeds, peaks 4 and 5 originated from Rehmannia glutinosa, and peak 6 originated from Licorice; peak 7 originated from Licorice and stir-fried Ziziphus jujuba seeds; peak 8 originated from Rehmannia glutinosa and Scrophularia ningpoensis; peaks 9 and 15 originated from processed Polygala tenuifolia; peaks 10 and 11 originated from Salvia miltiorrhiza; peak 12 originated from Scrophularia ningpoensis; peak 14 originated from Acorus tatarinowii; peaks 13, 16, 18-20 originated from Schisandra chinensis; peaks 21, 22 and 24 originated from Platycladus chinensis seeds; peak 23 originated from Acorus tatarinowii, Rehmannia glutinosa, Platycladus chinensis seeds, Angelica sinensis, Schisandra chinensis and stir-fried Ziziphus jujuba seeds; peak 25 originated from Platycladus chinensis seeds and stir-fried Ziziphus jujuba seeds.

[0221] It can be seen that 13 index components are provided that can reflect the 12 medicinal materials in the prescription (Danshen, Acorus gramineus, Rehmannia root, processed Polygala tenuifolia, Platycladus chinensis seeds, Licorice root, Angelica sinensis, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seeds and Platycodon grandiflorum).

[0222] It can be seen that the chemical characteristic peaks of the 15 medicinal materials in Tianwang Buxin Pills are well reflected in the fingerprint spectrum and their attribution is confirmed.

[0223] In summary, the present invention provides a method for determining the content and fingerprint of multiple components in Tianwang Buxin Pills, which can quantitatively analyze the seven chemical components in Tianwang Buxin Pills and determine the seven medicinal materials to which the seven components belong. Furthermore, a high-performance liquid chromatography fingerprint of Tianwang Buxin Pills is established, providing 13 index components representing the 12 medicinal materials in Tianwang Buxin Pills, thus providing a scientific basis for quality control of Tianwang Buxin Pills. Therefore, the present invention overcomes various shortcomings of the prior art and has high industrial application value.

[0224] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for determining the contents of seven ingredients in Tianwang Buxin Pills, comprising the following steps: 1) Preparation of test solution: Dissolve Tianwang Buxin Wan sample in solvent, extract by ultrasonication, cool, and filter the supernatant to obtain the test solution. 2) Preparation of reference solution: Dissolve α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin, β-asarone, 3,6'-diesinarosylsucrose, and ligustilide reference substances in solvent and dilute to volume to prepare the reference solution. 3) Determination: Determine the test solution from step 1) and the reference solution from step 2) by ultra-performance liquid chromatography, and calculate the contents of the seven components in the test solution by the external standard method; In steps 1) and 2), the solvent is a methanol aqueous solution with a volume percentage of 65-75%; In step 3), the chromatographic column used in the ultra-high performance liquid chromatography method is a Waters Acquity UPLC BEH C18 chromatographic column; In step 3), a multi-wavelength method is used for analysis, wherein a detection wavelength of 203 nm is used to detect α-linolenic acid, a detection wavelength of 250 nm is used to detect salvianolic acid B, glycyrrhizic acid, schisandra methanol, and β-asarone, and a detection wavelength of 320 nm is used to detect 3,6'-diesinapoylsucrose and ligustilide; In step 3), the ultra-high performance liquid chromatography method comprises the mobile phase of acetonitrile-0.08-0.12% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.08-0.12% phosphoric acid aqueous solution; the analysis time is 50 min; and gradient elution is used; The specific procedure of the gradient elution is: 0-15 min, the volume ratio of phase A:phase B was 8:92-22:78; 15-23 min, the volume ratio of phase A:phase B is 22:78-35:65; 23-30 min, the volume ratio of phase A:phase B is 35:65-40:60; 30-35 min, the volume ratio of phase A:phase B is 40:60-53:47; 35-42 min, the volume ratio of phase A:phase B is 53:47-95:5; 42-45 min, the volume ratio of phase A:phase B is 95:5-95:5; 45-46 min, the volume ratio of phase A:phase B is 95:5-8:92; 46-50min, the volume ratio of phase A:phase B is 8:92-8:

92.

2. The method for determining the contents of the seven components in Tianwang Buxin Pills according to claim 1, characterized in that: In step 1), 2), or 3), any one or more of the following conditions must be included: A1) In step 1), the Tianwang Buxin Pills sample is a powder sample obtained by crushing the Tianwang Buxin Pills; A2) In step 1), the ratio of the weight of the Tianwang Buxin Pill sample added to the volume of the solvent added is 1.5:23-27, g / mL; A3) In step 1), the ultrasonic extraction time is 27-33 minutes; A4) In step 1), the power of the ultrasonic extraction is 200-350W, and the frequency of the ultrasonic extraction is 40-60kHz; A5) In step 1), the filtration is performed by membrane filtration; the membrane is a 0.22 μm membrane; A6) In step 2), the content of α-linolenic acid in the reference solution ranges from 9.73 to 389.49 μg / mL, the content of salvianolic acid B ranges from 23.78 to 475.52 μg / mL, the content of glycyrrhizic acid ranges from 9.75 to 195.03 μg / mL, the content of schisandrin A ranges from 7.74 to 154.73 μg / mL, the content of β-asarone ranges from 8.25 to 165.04 μg / mL, the content of 3,6'-diesinapoylsucrose ranges from 4.98 to 99.66 μg / mL, and the content of ligustilide ranges from 9.61 to 192.17 μg / mL; A7) In step 3), the detector in the ultra-high performance liquid chromatography method is a photodiode array detector; A8) In step 3), the column temperature in the ultra-high performance liquid chromatography method is 15-25°C; A9) In step 3), the injection volume in the ultra-high performance liquid chromatography method is 0.5-2 μL; A10) In step 3), the flow rate of the ultra-high performance liquid chromatography is 0.1-0.5 mL / min.

3. A method for screening the medicinal materials of the seven ingredients in Tianwang Buxin Pills, comprising the following steps: I) Preparation of single herbal sample solutions: preparing any one or more of the seven herbal samples of Tianwang Buxin Pills, namely, Danshen, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, and Schisandra chinensis, according to step 1) of the method for determining the contents of seven components in Tianwang Buxin Pills according to any one of claims 1-2, to obtain at least one single herbal sample solution; II) Preparation of negative sample solutions: Samples of the seven medicinal materials in Tianwang Buxin Pills, namely, Danshen, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, and Schisandra chinensis, are prepared according to step 1) of the method for determining the contents of the seven ingredients in Tianwang Buxin Pills according to any one of claims 1-2, respectively, with Danshen, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, and Schisandra chinensis lacking, to obtain negative sample solutions lacking Danshen, Acorus gramineus, Polygala tenuifolia, Platycladus orientalis, Licorice root, Angelica sinensis, and Schisandra chinensis, respectively; III) Determination: using ultra-high performance liquid chromatography under the same chromatographic conditions as in step 3) of the method for determining the contents of the seven components in Tianwang Buxin Pills according to any one of claims 1-2, respectively determining the fingerprints of the single herbal medicine sample solution in step I) and the negative sample solution in step II); IV) Obtaining a reference fingerprint: applying the test solution prepared in step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills according to any one of claims 1-2 to step 3) of the method for determining the contents of the seven components in Tianwang Buxin Pills according to any one of claims 1-2 to obtain reference fingerprints of the seven components in Tianwang Buxin Pills; V) Quality testing: The fingerprints of the single herb sample solution and the negative sample solution were compared with the reference fingerprints of the seven ingredients in Tianwang Buxin Pills. The corresponding characteristic peaks of the single herb sample solution in the reference fingerprints of the seven ingredients in Tianwang Buxin Pills were identified by relative retention time, thereby identifying and locating the seven ingredients in Tianwang Buxin Pills: α-linolenic acid, salvianolic acid B, glycyrrhizic acid, schisandrin A, β-asarone, 3,6'-diesinarosoylsucrose and ligustilide.

4. A method for detecting the fingerprint of Tianwang Buxin Pills, comprising the following steps: A) Preparation of the test solution: the same as step 1) of the method for determining the contents of the seven components in Tianwang Buxin Pills according to any one of claims 1-2; B) Preparation of reference solution: Dissolve α-linolenic acid, salvianolic acid B, schisandrin A, β-asarone, 3,6'-dieserucyl sucrose, ligustilide, liquiritin, verbascoside, salvianolic acid A, schisandrin B, schisandrin A, schisandrin B, and linoleic acid reference substances in a solvent and saturate to volume to prepare a reference solution; C) Determination: The test solution of step A) and the reference solution of step B) are respectively determined by ultra-high performance liquid chromatography to obtain fingerprints of the test solution and the reference solution. The fingerprints of the test solution and the reference solution are compared to identify and locate the index components in the fingerprints of the test solution, thereby obtaining a fingerprint of Tianwang Buxin Pills. The fingerprint of Tianwang Buxin Pills includes 25 common fingerprint peaks. Peak 6 is located to be the fingerprint peak of liquiritin, and peak 8 is located to be the fingerprint peak of calycin. The fingerprint peaks of anthosides are: peak 9 is the fingerprint peak of 3,6'-dieserinoylsucrose, peak 10 is the fingerprint peak of salvianolic acid B, peak 11 is the fingerprint peak of salvianolic acid A, peak 13 is the fingerprint peak of schisandrin A, peak 14 is the fingerprint peak of β-asarone, peak 16 is the fingerprint peak of schisandrin B, peak 17 is the fingerprint peak of ligustilide, peak 18 is the fingerprint peak of schisandrin A, peak 20 is the fingerprint peak of schisandrin B, peak 21 is the fingerprint peak of α-linolenic acid, and peak 23 is the fingerprint peak of linoleic acid; In step B), the solvent is a methanol aqueous solution with a volume percentage of 65-75%; In step C), the chromatographic column used in the ultra-high performance liquid chromatography method is a Waters Acquity UPLC BEH C18 chromatographic column; In step C), the detection wavelength in the ultra-high performance liquid chromatography is 203 nm; In step C), the ultra-high performance liquid chromatography method comprises the mobile phase of acetonitrile-0.08-0.12% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.08-0.12% phosphoric acid aqueous solution; the analysis time is 50 min; and gradient elution is used; The specific procedure of the gradient elution is: 0-15 min, the volume ratio of phase A:phase B was 8:92-22:78; 15-23 min, the volume ratio of phase A:phase B is 22:78-35:65; 23-30 min, the volume ratio of phase A:phase B is 35:65-40:60; 30-35 min, the volume ratio of phase A:phase B is 40:60-53:47; 35-42 min, the volume ratio of phase A:phase B is 53:47-95:5; 42-45 min, the volume ratio of phase A:phase B is 95:5-95:5; 45-46 min, the volume ratio of phase A:phase B is 95:5-8:92; 46-50min, the volume ratio of phase A:phase B is 8:92-8:

92.

5. Use of the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4 in the quality detection of ingredients in Tianwang Buxin Pills.

6. A method for detecting the quality of Tianwang Buxin Pills, comprising obtaining a fingerprint of Tianwang Buxin Pills using the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, and comparing the obtained fingerprint of Tianwang Buxin Pills with a control fingerprint of Tianwang Buxin Pills obtained under the same fingerprint detection conditions for similarity.

7. The quality inspection method of Tianwang Buxin Pills according to claim 6, characterized in that: The control fingerprint of Tianwang Buxin Pills was obtained under the same conditions as the detection method of the fingerprint of Tianwang Buxin Pills according to claim 4, and the control fingerprint of Tianwang Buxin Pills included 25 common fingerprint peaks, with peak 17 as the reference peak S peak, and the relative retention time was 1.000; the relative retention times of the other 24 peaks were as follows: peak 1 0.108±0.007, peak 2 0.124±0.001, peak 3 0.201±0.012, peak 4 0.249±0.013, peak 5 0.300±0.001, peak 6 0.314±0.045, peak 7 0.320±0.036, peak 8 0.366±0.011, peak 9 0.416±0.0 30, peak 10 0.541±0.013, peak 11 0.573±0.001, peak 12 0.588±0.001, peak 13 0.821±0.053, peak 14 0.835±0.008, peak 15 0.861±0.001, peak 16 0.927±0.047, peak 18 1.176±0.012, peak 19 1.198±0.012, peak 20 1.204±0.016, peak 21 1.242±0.021, peak 22 1.261±0.021, peak 23 1.274±0.026, peak 24 1.289±0.026, peak 25 1.308±0.

035.

8. A method for screening fingerprints of multiple medicinal materials in Tianwang Buxin Pills, comprising the following steps: a) Preparation of single medicinal material sample solutions: preparing any one or more of the 16 medicinal material samples of Tianwang Buxin Pills, namely, Danshen, Acorus gramineus, Poria cocos, Ophiopogon japonicus, Rehmannia root, Polygala tenuifolia root, Platycladi seed, Licorice root, Angelica sinensis, Codonopsis pilosula, Schisandra chinensis, Asparagus cochinchinensis, Scrophularia ningpoensis, stir-fried Ziziphus jujuba seed, Platycodon grandiflorum, and Cinnabaris, according to step A) of the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, to obtain at least one single medicinal material sample solution; b) Preparation of negative sample solution: 16 medicinal material samples including salvia miltiorrhiza, calamus tatarinowii, poria, ophiopogon japonicus, rehmannia root, processed polygala tenuifolia, cypress seed, liquorice, angelica sinensis, codonopsis pilosula, schisandra chinensis, asparagus cochinchinensis, figwort root, stir-fried jujube seed, platycodon grandiflorum, and cinnabar were prepared according to step A) of the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, with salvia miltiorrhiza, calamus tatarinowii, poria, ophiopogon japonicus, rehmannia root, processed polygala tenuifolia, cypress seed, liquorice, angelica sinensis, codonopsis pilosula, schisandra chinensis, asparagus cochinchinensis, figwort root, stir-fried jujube seed, platycodon grandiflorum, and cinnabar being omitted respectively. The negative sample solutions lacking Danshen, the negative sample solutions lacking Acorus gramineus, the negative sample solutions lacking Poria cocos, the negative sample solutions lacking Ophiopogon japonicus, the negative sample solutions lacking Rehmannia glutinosa, the negative sample solutions lacking Polygala tenuifolia, the negative sample solutions lacking Platycladi seed, the negative sample solutions lacking Licorice root, the negative sample solutions lacking Angelica sinensis, the negative sample solutions lacking Codonopsis pilosula, the negative sample solutions lacking Schisandra chinensis, the negative sample solutions lacking Asparagus cochinchinensis, the negative sample solutions lacking Scrophularia ningpoensis, the negative sample solutions lacking stir-fried Ziziphus jujuba seeds, the negative sample solutions lacking Platycodon grandiflorum and the negative sample solutions lacking Cinnabar were obtained respectively; c) Determination: using ultra-high performance liquid chromatography (UPLC) under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, respectively determining the fingerprints of the single medicinal material sample solution in step a) and the negative sample solution in step b); d) Obtaining a control fingerprint: using the test solution prepared in step A) of the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, and performing step C) identical to the method for detecting the fingerprint of Tianwang Buxin Pills according to claim 4, to obtain a control fingerprint of Tianwang Buxin Pills; e) Quality testing: The fingerprints of the single herb sample solution and the negative sample solution were compared with the control fingerprint of Tianwang Buxin Pills. The corresponding characteristic peaks of the single herb sample solution in the control fingerprint of Tianwang Buxin Pills were identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single herb sample solution; the control fingerprint of Tianwang Buxin Pills includes 25 common fingerprint peaks, and peak 6 was located as the fingerprint peak of liquiritin and peak 8 as the fingerprint peak of verbascoside. Peak 9 is the fingerprint peak of 3,6'-dieserucylsucrose, peak 10 is the fingerprint peak of salvianolic acid B, peak 11 is the fingerprint peak of salvianolic acid A, peak 13 is the fingerprint peak of schisandrin A, peak 14 is the fingerprint peak of β-asarone, peak 16 is the fingerprint peak of schisandrin B, peak 17 is the fingerprint peak of ligustilide, peak 18 is the fingerprint peak of schisandrin A, peak 20 is the fingerprint peak of schisandrin B, peak 21 is the fingerprint peak of α-linolenic acid, and peak 23 is the fingerprint peak of linoleic acid.

9. The method for screening fingerprints of multiple medicinal materials in Tianwang Buxin Pills according to claim 8, characterized in that: In step e), any one or more of the following conditions are included: B1) The fingerprint of the Danshen sample solution includes two common fingerprint peaks, which are peak 10 and peak 11; B2) The fingerprint of the Acorus tatarinowii sample solution includes two common fingerprint peaks, namely peak 14 and peak 23; B3) The fingerprint of the Rehmannia root sample solution includes four common fingerprint peaks, namely peak 4, peak 5, peak 8, and peak 23; B4) The fingerprint of the prepared Polygala tenuifolia sample solution includes two common fingerprint peaks, namely Peak 9 and Peak 15; B5) The fingerprint of the Semen Platycladi sample solution includes five common fingerprint peaks, wherein the two common fingerprint peaks are Peak 21, Peak 22, Peak 23, Peak 24, and Peak 25; B6) The fingerprint of the licorice sample solution includes three common fingerprint peaks, which are peak 2, peak 6, and peak 7; B7) The fingerprint of the Angelica sinensis sample solution includes three common fingerprint peaks, namely Peak 1, Peak 17, and Peak 23; B8) The fingerprint of the Schisandra chinensis sample solution includes 6 common fingerprint peaks, which are peak 13, peak 16, peak 18, peak 19, peak 20, and peak 23; B9) The fingerprint of the Asparagus cochinchinensis sample solution includes two common fingerprint peaks, which are Peak 1 and Peak 2; B10) The fingerprint of the Scrophularia ningpoensis sample solution includes two common fingerprint peaks, which are peak 8 and peak 12; B11) The fingerprint of the stir-fried spinach seed sample solution includes four common fingerprint peaks, namely peak 3, peak 7, peak 23, and peak 25; B12) The fingerprint of the Platycodon grandiflorum sample solution includes one common fingerprint peak, which is Peak 1; B13) The fingerprints of Poria cocos, Ophiopogon japonicus, Codonopsis pilosula and Cinnabar bark sample solutions have no common fingerprint peaks.

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