Method for determining contents of eight chemical components in spleen-invigorating pill and fingerprint spectrum method thereof

Abstract

The invention provides a method for determining the content of eight chemical components in spleen-invigorating pills. The invention also provides a detection method of the fingerprint spectrum of the spleen-invigorating pill. The invention further provides a quality detection method of the spleen-invigorating pill. The invention further provides a screening method of the fingerprint spectrum of the multiple medicinal materials in the spleen-invigorating pill. According to the method for determining the content of the eight chemical components in the spleen-invigorating pill and the fingerprint spectrum method of the eight chemical components, the eight components in the spleen-invigorating pill can be quantitatively analyzed, the fingerprint spectrum of the spleen-invigorating pill is further established, and reference is provided for scientifically and comprehensively controlling the quality of the spleen-invigorating pill.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicine component detection, and relates to a method for determining the contents of 8 chemical components in Guipi Pills and a fingerprint method thereof, and specifically relates to a method for determining the contents of 8 chemical components in Guipi Pills: sibirica polygala sugar A5, sibirica polygala sugar A6, ferulic acid, 3,6'-diesinapoylsucrose, polygala tenuifolia glycoside A, polygala glycoside C, costunolide, and dehydrocostus lactone, and a fingerprint method thereof determined based on the 8 components. Background Art

[0002] Guipi Pills are derived from the famous prescription Guipi Decoction, which was first recorded in the Jisheng Prescription by Yan Yonghe, a great medical expert in the Song Dynasty. It is now included in the Chinese Pharmacopoeia. It is a honey pill made of 11 Chinese medicinal herbs, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan meat, Angelica sinensis, Aucklandia lappa, and jujube (pitted). It has the effects of invigorating Qi and strengthening the spleen, nourishing blood and calming the mind. It is used for the treatment of deficiency of both heart and spleen, shortness of breath and palpitations, insomnia and dreaminess, dizziness, fatigue, loss of appetite, metrorrhagia and bloody stool. Astragalus membranaceus and Codonopsis pilosula in the prescription nourish the spleen and replenish Qi, making Qi prosperous and blood fertilizing, and are the main drugs. Angelica sinensis and longan meat are used to nourish blood and nourish the heart as the assistant drugs; Atractylodes macrocephala nourishes the spleen and replenishes Qi, and helps Astragalus membranaceus and Astragalus membranaceus to nourish the spleen and provide the source of biochemical metabolism as the assistant drugs. It is supplemented with Chinese date seeds, poria cocos, and polygala to nourish blood, soothe the mind and calm the nerves; costus root regulates qi and invigorates the spleen, so that it can replenish without stagnation; jujube harmonizes the spleen and stomach to help metabolism. Roasted licorice is used to harmonize the various medicines, and the various medicines are matched to achieve the effect of replenishing qi and blood, strengthening the spleen and nourishing the heart.

[0003] The quality control indicator of Guipi Pills is the content of astragaloside IV. Most of the relevant quality control research literature on the preparation uses ELSD or CAD as the detector to analyze the content of astragaloside IV, a single indicator component in Guipi Pills. There is a lack of relevant methods for simultaneously detecting and controlling the chemical components and fingerprint spectra of multiple medicinal materials in Guipi Pills under the same chromatographic conditions. Summary of the invention

[0004] In view of the shortcomings of the prior art mentioned above, the object of the present invention is to provide a method for determining the contents of 8 chemical components in Guipi Pills and a fingerprint method thereof, and adopt pretreatment under optimized conditions and ultra-high performance liquid chromatography to establish a method for determining the contents of 8 components in Guipi Pills: Siberian Polygala A5, Siberian Polygala A6, ferulic acid, 3,6'-diesinapoylsucrose, Polygala tenuifolia glycoside A, Polygala glycoside C, costunolide, and dehydrocostus lactone, and a fingerprint spectrum determined based on these 8 indicator components, so as to provide reference and reference for scientific and comprehensive quality control of Guipi Pills.

[0005] To achieve the above-mentioned object and other related objects, the present invention provides a method for determining the chemical contents of 8 ingredients in Guipi Pills in a first aspect, comprising the following steps:

[0006] 1) Preparation of test solution: Dissolve the Guipi Pills sample in solvent, extract by ultrasonic, cool, weigh, shake well, filter, take the supernatant, filter, and take the filtrate to obtain the test solution;

[0007] 2) Preparation of reference substance solution: one or more reference substances selected from Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-dienasinoyl sucrose, Polygala glycoside A, Polygala glycoside C, costunolide, and dehydrocostus lactone are added to a solvent and dissolved, and then fixed to volume to obtain a reference substance solution;

[0008] 3) Determination: The test solution of step 1) and the reference solution of step 2) are respectively determined by ultra high performance liquid chromatography (UHPLC), and the content of one or more components of Siberian polygala sugar A5, Siberian polygala sugar A6, ferulic acid, 3,6'-diesinapoylsucrose, polygala glycoside A, polygala glycoside C, costunolide, and dehydrocostus lactone in the test solution is calculated by the external standard method.

[0009] Preferably, in step 1), the Guipi Pills sample is a powder sample.

[0010] Preferably, in step 1), the Guipi Pills sample is accurately weighed before adding the solvent.

[0011] Preferably, in step 1), the solvent is an aqueous solution containing 55-85% methanol by volume, preferably 65-75%, more preferably 70%.

[0012] Preferably, in step 1), the weight of the solvent is weighed after addition.

[0013] Preferably, in step 1), the ratio of the weight (g) of the Guipi Pills sample added to the volume (mL) of the solvent added is 1:15-25, specifically 1:15-18, 1:18-22, 1:22-25, for example 1:15, 1:16, 1:18, 1:20, 1:22, 1:23, 1:25, preferably 1:20.

[0014] Preferably, in step 1), the ultrasonic extraction time is 20-40 min, specifically 20-25 min, 25-35 min, 35-40 min, for example 20 min, 23 min, 25 min, 27 min, 30 min, 32 min, 35 min, 38 min, 40 min, preferably 30 min.

[0015] Preferably, in step 1), the power of the ultrasonic extraction is 250-350 W, preferably 300 W; the frequency of the ultrasonic extraction is 30-50 kHz, preferably 40 kHz.

[0016] Preferably, in step 1), the cooling is cooling to room temperature. The room temperature is 20-30°C.

[0017] Preferably, in step 1), the weight is measured before adding weight.

[0018] Preferably, in step 1), the solvent used for the weight replenishment is an aqueous solution containing 55-85% methanol by volume, preferably 65-75%, more preferably 70%.

[0019] Preferably, in step 1), the filtration is membrane filtration.

[0020] More preferably, the filter membrane is a 0.22 μm filter membrane.

[0021] The subsequent filtrate is the filtrate collected after the initial filtrate is poured out.

[0022] Preferably, in step 2), the reference substance solution can be directly prepared as a mixed reference substance solution, or can be prepared by first adding a first solvent to prepare a reference substance stock solution and then adding a second solvent to dilute it step by step.

[0023] More preferably, in the reference stock solution, the content of Siberian Polygala sugar A5 is 0.2772 mg / mL; the content of Siberian Polygala sugar A6 is 0.2348 mg / mL; the content of ferulic acid is 0.4300 mg / mL; the content of 3,6'-dianisopylsucrose is 0.5724 mg / mL; the content of Polygala tenuifolia glycoside A is 0.1724 mg / mL; the content of Polygala tenuifolia glycoside C is 0.2992 mg / mL; the content of costunolide is 0.2576 mg / mL; and the content of dehydrocostus lactone is 0.2876 mg / mL.

[0024] The reference substance stock solution was refrigerated at 4°C away from light for future use.

[0025] More preferably, the first solvent is methanol.

[0026] More preferably, the second solvent is an aqueous solution containing 55-85% methanol by volume, preferably 65-75%, more preferably 70%.

[0027] Preferably, in step 2), the CAS number of Siberian polygala sugar A5 is 107912-97-0, the CAS number of Siberian polygala sugar A6 is 241125-75-7, the CAS number of ferulic acid is 1135-24-6, the CAS number of 3,6'-dianisinoyl sucrose is 139891-98-8, the CAS number of fine leaf polygala glycoside A is 139726-35-5, the CAS number of polygala glycoside C is 139726-37-7, the CAS number of costusin lactone is 553-21-9, and the CAS number of dehydrocostus lactone is 477-43-0.

[0028] Preferably, in step 2), the content range of Siberian Polygala sugar A5 in the reference solution is 1.11-277.20 μg / mL; the content range of Siberian Polygala sugar A6 is 0.94-234.80 μg / mL; the content range of ferulic acid is 0.65-430.00 μg / mL; the content range of 3,6'-dianisopylsucrose is 0.86-572.40 μg / mL; the content range of fine leaf polygala glycoside A is 0.69-172.40 μg / mL; the content range of polygala glycoside C is 1.20-299.20 μg / mL; the content range of costunolide is 2.60-257.60 μg / mL; the content range of dehydrocostus lactone is 2.90-287.60 μg / mL.

[0029] Preferably, in step 3), the chromatographic column in the ultra-high performance liquid chromatography is C 18 More preferably, the chromatographic column in the ultra-high performance liquid chromatography is a Waters UPLC Cortecs C 18 Chromatographic column (2.1 mm × 100 mm, 1.6 μm).

[0030] Preferably, in step 3), the detector in the ultra-high performance liquid chromatography is a photodiode array detector (DAD).

[0031] Preferably, in step 3), in the ultra-high performance liquid chromatography, the column temperature is 25-35°C, such as 25-28°C, 28-32°C, 32-35°C, and preferably 30°C.

[0032] Preferably, in step 3), the injection volume in the ultra-high performance liquid chromatography is 1-5 μL.

[0033] More preferably, the injection volume in the ultra-high performance liquid chromatography is, for example, 1 μL, 2 μL, 3 μL, 4 μL, 5 μL, and preferably 2 μL.

[0034] Preferably, in step 3), the flow rate in the ultra-high performance liquid chromatography is 0.2-1 mL / min, such as 0.2-0.4 mL / min, 0.4-0.8 mL / min, 0.8-1 mL / min, for example 0.2 mL / min, 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, preferably 0.6 mL / min.

[0035] Preferably, in step 3), the detection wavelength in the ultra-high performance liquid chromatography is selected from one or a combination of 325-335 nm, 215-225 nm, preferably 328-332 nm and 218-222 nm, and more preferably 330 nm and 220 nm.

[0036] More preferably, the detection wavelength changes with the time of the gradient elution program, and the specific changes are: 0-53min, the detection wavelength is 325-335nm, preferably 328-332nm, more preferably 330nm; 53-85min, the detection wavelength is 215-225nm, preferably 218-222nm, more preferably 220nm.

[0037] Preferably, in step 3), in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.09-0.11% phosphoric acid aqueous solution; in acetonitrile-0.09-0.11% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.09-0.11% phosphoric acid aqueous solution.

[0038] More preferably, in the high performance liquid chromatography, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution; in the acetonitrile-0.1% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution.

[0039] The 0.09-0.11% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.09-0.11%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0040] Preferably, in step 3), the ultra-high performance liquid chromatography method uses gradient elution and the analysis time is 85 min.

[0041] More preferably, the specific procedure of the gradient elution is:

[0042] 0-5min, the volume ratio of phase A:phase B is 4-6:94-96-7-9:91-93;

[0043] 5-8min, the volume ratio of phase A:phase B is 7-9:91-93-7-9:91-93;

[0044] 8-9min, the volume ratio of phase A:phase B is 7-9:91-93-10-11:89-90;

[0045] 9-17min, the volume ratio of phase A:phase B is 10-11:89-90-10-11:89-90;

[0046] 17-18min, the volume ratio of phase A:phase B is 10-11:89-90-12-14:86-88;

[0047] 18-30min, the volume ratio of phase A:phase B is 12-14:86-88-12-14:86-88;

[0048] 30-35min, the volume ratio of phase A:phase B is 12-14:86-88-19-21:79-81;

[0049] 35-45min, the volume ratio of phase A:phase B is 19-21:79-81-19-21:79-81;

[0050] 45-50min, the volume ratio of phase A:phase B is 19-21:79-81-28-30:70-72;

[0051] 50-70min, the volume ratio of phase A:phase B is 28-30:70-72-28-30:70-72;

[0052] 70-75min, the volume ratio of phase A:phase B is 28-30:70-72-44-46:54-56;

[0053] 75-80min, the volume ratio of phase A:phase B is 44-46:54-56-79-81:19-21;

[0054] At 80-85min, the volume ratio of phase A:phase B is 79~81:19~21-79~81:19~21.

[0055] Further preferably, the specific procedure of the gradient elution is shown in Table 1, which is:

[0056] 0-5min, the volume ratio of phase A:phase B was 5:95-8:92;

[0057] 5-8min, volume ratio of phase A:phase B is 8:92-8:92;

[0058] 8-9min, the volume ratio of phase A:phase B is 8:92-10:90;

[0059] 9-17min, volume ratio of phase A:phase B is 10:90-10:90;

[0060] 17-18min, the volume ratio of phase A:phase B is 10:90-13:87;

[0061] 18-30min, the volume ratio of phase A:phase B is 13:87-13:87;

[0062] 30-35min, volume ratio of phase A:phase B is 13:87-20:80;

[0063] 35-45min, volume ratio of phase A:phase B is 20:80-20:80;

[0064] 45-50min, the volume ratio of phase A:phase B is 20:80-29:71;

[0065] 50-70min, volume ratio of phase A:phase B is 29:71-29:71;

[0066] 70-75min, the volume ratio of phase A:phase B was 29:71-45:55;

[0067] 75-80min, the volume ratio of phase A:phase B is 45:55-80:20;

[0068] 80-85min, the volume ratio of phase A:phase B is 80:20-80:20.

[0069] Table 1

[0070]

[0071] Preferably, in step 3), the external standard method refers to: respectively taking a series of different volumes of the reference solution of step 2), respectively preparing a series of solutions of different concentrations, using ultra-high performance liquid chromatography for sample analysis, obtaining the linear relationship between the concentration and peak area of ​​the 8 components in the reference solution, and drawing 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. Then the test solution is detected by ultra-high performance liquid chromatography, and the chromatographic peak areas of the 8 components in the test solution are respectively substituted into the regression equation of each standard working curve, and the content of the corresponding component can be calculated.

[0072] 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.

[0073] The second aspect of the present invention provides a method for detecting the fingerprint of Guipi Pills, comprising the following steps:

[0074] A) Preparation of test solution: same as step 1) of the method for determining the chemical contents of the eight components in Guipi Pills;

[0075] B) Preparation of reference solution: same as step 2) of the method for determining the chemical contents of the eight components in Guipi Pills;

[0076] C) Determination: Using ultra high performance liquid chromatography (UHPLC) under the same chromatographic conditions as step 3) of the method for determining the chemical contents of 8 components in Guipi Pills, respectively determine the test solution of step A) and the reference solution of step B) to obtain fingerprints of the test solution and the reference solution, compare the fingerprints of the test solution with those of the reference solution, attribute and locate the index components in the fingerprints of the test solution, and thus obtain the fingerprint of Guipi Pills.

[0077] 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.

[0078] The third aspect of the present invention provides a quality detection method for Guipi Pills, comprising obtaining a fingerprint of Guipi Pills using the aforementioned fingerprint detection method of Guipi Pills, and comparing the obtained fingerprint of Guipi Pills with a control fingerprint of Guipi Pills obtained under the same fingerprint detection conditions for similarity.

[0079] Preferably, when the present invention compares the similarity of the measured fingerprint of Guipi Pills with the control fingerprint of Guipi Pills, the software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version issued by the State Pharmacopoeia Committee is used for comparison. More preferably, the similarity of the fingerprint of Guipi Pills measured by the present invention with the control fingerprint of Guipi Pills is ≥ 0.981.

[0080] More preferably, when the fingerprint common peak of the Guipi Pills fingerprint spectrum is matched with the control fingerprint spectrum of Guipi Pills, the time window width is 0.20, and Mark peak matching is performed after multi-point correction to generate the fingerprint spectrum and the control fingerprint spectrum.

[0081] Preferably, the control fingerprint of Guipi Pills is obtained under the same conditions as the detection method of the fingerprint of Guipi Pills, and the control fingerprint of Guipi Pills includes 35 common fingerprint peaks, with peak 11 as the reference peak (S peak, relative retention time is 1.0000), and the relative retention times of the other 34 peaks are peak 1 (0.10±0.0007), peak 2 (0.13±0.0007), peak 3 (0.17±0.0007), peak 4 (0.26±0.0021), peak 5 (0.26±0.0021), peak 6 (0.26±0.0021), peak 7 (0.26±0.0021), peak 8 (0.26±0.0021), peak 9 (0.26±0.0021), peak 10 (0.26±0.0021), peak 11 (0.26±0.0021), peak 12 (0.26±0.0021), peak 13 (0.26±0.0021), peak 14 (0.26±0.0021), peak 15 (0.26±0.0021), peak 16 (0.26±0.0021), peak 17 (0.26±0.0021), peak 18 (0.26±0.0021), peak 19 (0.26±0.0021), peak 20 (0.26±0.0021), peak 21 (0.26±0.0021), peak 22 (0.26±0.0021), peak 23 (0.26±0.0021), peak 24 (0.26±0.0021), peak 25 (0.37±0.0027), peak 6 (0.42±0.0057), peak 7 (0.49±0.0006), peak 8 (0.56±0.0006), peak 9 (0.58±0.0013), peak 10 (0.65±0.0031), peak 12 (1.05±0.0024), peak 13 (1.17±0.0139), peak 14 (1.35±0.0242), peak 15 (1.47±0.0177), peak 16 ( 1.52±0.0162), peak 17 (1.64±0.0110), peak 18 (1.70±0.0280), peak 19 (1.74±0.0333), peak 20 (1.76±0.0363), peak 21 (1.78±0.0367), peak 22 (1.80±0.0383), peak 23 (1.97±0.0420), peak 24 (2.05±0.0379), peak 25 (2.11±0.0444), peak 26 Peak No. (2.18±0.0453), peak No. 27 (2.37±0.0470), peak No. 28 (2.50±0.0496), peak No. 29 (2.79±0.0658), peak No. 30 (2.81±0.0662), peak No. 31 (2.82±0.0663), peak No. 32 (2.83±0.0666), peak No. 33 (2.89±0.0677), peak No. 34 (2.91±0.0679), and peak No. 35 (2.96±0.0693).

[0082] The specific data of the control fingerprint of Guipi Pills can be found in Figure 2 .

[0083] More preferably, the control fingerprint of Guipi Pills is compared with the fingerprint of the reference solution, such as Figure 2 As shown, it was determined that peak 2 was the fingerprint peak of Siberian polygala sugar A5; peak 3 was the fingerprint peak of Siberian polygala sugar A6; peak 4 was the fingerprint peak of ferulic acid; peak 11 was the fingerprint peak of 3,6'-diesinapoylsucrose; peak 13 was the fingerprint peak of fine-leaved polygala glycoside A; peak 14 was the fingerprint peak of polygala glycoside C; peak 27 was the fingerprint peak of costunolide; and peak 28 was the fingerprint peak of dehydrocostus lactone.

[0084] The fourth aspect of the present invention provides a method for screening fingerprints of multiple medicinal materials in Guipi Pills, comprising the following steps:

[0085] a) Preparation of single medicinal material sample solutions: prepare any one or more of the 11 medicinal material samples of Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costusroot, and jujube in Guipi Pills according to step A) of the detection method of the fingerprint of Guipi Pills to obtain at least one single medicinal material sample solution respectively;

[0086] b) Preparation of negative sample solution: 11 medicinal material samples, namely, Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube, are respectively lacking Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube, and are prepared according to step A) of the detection method of Guipi Pill fingerprint, to obtain negative sample solutions lacking Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube;

[0087] c) Determination: using ultra high performance liquid chromatography (UHPLC) under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Guipi Pills to respectively determine the fingerprints of the single medicinal material sample solution in step a) and the negative sample solution in step b);

[0088] d) Obtaining the control fingerprint: The test solution prepared according to step A) of the detection method of the fingerprint of Guipi Pills is subjected to the same step C) as the detection method of the fingerprint of Guipi Pills to obtain the control fingerprint of Guipi Pills;

[0089] e) Quality inspection: Compare the fingerprints of the single medicinal sample solution and the negative sample solution with the control fingerprint of Guipi Pills. By the relative retention time, identify the corresponding characteristic peaks of the single medicinal sample solution in the control fingerprint of Guipi Pills, so as to attribute and locate the characteristic peaks in the fingerprint of the single medicinal sample solution.

[0090] Preferably, in step a), the Codonopsis pilosula is the dried root of Codonopsis pilosula, Codonopsis pilosula or Codonopsis pilosula of Campanulaceae. The Atractylodes macrocephala in the bran-fried Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala of Asteraceae. The Astragalus membranaceus in the roasted Astragalus membranaceus is the dried root of Astragalus mongolica or Astragalus membranaceus of Leguminosae. The Poria cocos is the dried sclerotium of the fungus Poria cocos of Polyporaceae. The Polygala tenuifolia in the processed Polygala tenuifolia is the dried root of Polygala tenuifolia or Polygala ovalifolia of Polygalaceae. The Ziziphus jujuba seeds in the stir-fried Ziziphus jujuba seeds are the dried mature seeds of Ziziphus jujuba of Rhamnaceae. The Longan meat is the aril of Longan of Sapindaceae. The Angelica sinensis is the dried root of Angelica sinensis of Umbelliferae. The Aucklandia lappa is the dried root of Aucklandia lappa of Asteraceae. The Jujube is the mature fruit of Ziziphus jujuba of Rhamnaceae.

[0091] Preferably, in step e), the present invention locates the characteristic peaks of the measured fingerprint of the single medicinal material sample solution and the reference fingerprint of Guipi Pills, uses the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" issued by the State Pharmacopoeia Committee for analysis and processing, and confirms the attribution of the characteristic peaks of each single medicinal material of Guipi Pills by the relative retention time of each characteristic peak on the reference fingerprint of Guipi Pills. Specific results are shown in Figure 2 , 3 and Table 2.

[0092] Table 2 The characteristic peaks of each single herbal medicine in Guipi Pills

[0093]

[0094] As shown in Table 2, this fingerprint method can characterize the characteristic peaks of the five medicinal materials in Guipi Pills (Angelica sinensis, Aucklandia lappa, Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, and Licorice). Since the dosage of Codonopsis pilosula and Astragalus membranaceus is relatively small in the prescription, there is basically no corresponding chromatographic peak under the DAD detection signal, and the representative components of Astragaloside I, jujube, and sugar components of longan pulp of Astragalus membranaceus have no absorption under DAD. Therefore, this method fails to characterize the index components of Codonopsis pilosula, Astragalus membranaceus, jujube, and longan pulp. At the same time, the characteristic components of Atractylodes macrocephala and Poria cocos are also low in the medicinal materials, so they cannot be characterized in the mixed powder of this prescription.

[0095] The water used in the present invention is all purified water.

[0096] As described above, the present invention provides a method for determining the contents of 8 chemical components in Guipi Pills and a fingerprint method thereof, adopting a UHPLC ultra-high performance liquid chromatograph assisted by a DAD detector, and using a segmented wavelength detection method to detect the contents of 8 chemical components in the Guipi Pills mixed powder, including ferulic acid, Siberian Polygala sugar A5, Siberian Polygala sugar A6, 3,6'-dicerinoylsucrose, Polygala tenuifolia glycoside A, Polygala glycoside C, costunolide and dehydrocostus lactone, and establish a fingerprint of Guipi Pills.

[0097] The methodological validation results of this content determination method showed that the methodological contents such as extraction conditions, linear range, repeatability, precision, stability and sample recovery of this method were investigated. The results showed that this method was stable and reliable. The eight active ingredients in Guipi Pills (Siberian Polygala A5, Siberian Polygala A6, ferulic acid, 3,6'-diesinapoylsucrose, Polygala glycoside A, Polygala glycoside C, costunolide and dehydrocostus lactone) had a good relationship within their respective linear ranges, with average recoveries of 95.72% to 102.39% and RSD% of 1.65% to 4.67%.

[0098] The fingerprint spectrum of Guipi Pills includes 35 characteristic peaks. 20 batches of Guipi Pills were analyzed, and their characteristic fingerprints were established and similarity evaluation was performed. The similarities of the fingerprints of the 20 batches of Guipi Pills were all above 0.981, which provides reference and reference for the scientific and comprehensive control of the quality of Guipi Pills.

[0099] The method for determining the contents of 8 chemical components in Guipi Pills and the fingerprint method thereof provided by the present invention are stable and reliable, can perform relatively comprehensive quality control on Guipi Pills, and provide a scientific basis for quality evaluation and standard establishment thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1 Shown are chromatograms of the reference substance and samples of 8 chemical components in the Guipi Pills of the present invention, wherein A: chromatogram of the reference substance; B: chromatogram of the sample; 1: Siberian Polygala sugar A5; 2: Siberian Polygala sugar A6; 3: Ferulic acid; 4: 3,6'-dianisinoyl sucrose; 5: Polygala tenuifolia glycoside A; 6: Polygala tenuifolia glycoside C; 7: Costunolide; 8: Dehydrocostus lactone.

[0101] Figure 2 The UHPLC control fingerprint of the Guipi Pill sample of the present invention is shown, which has 35 characteristic peaks, among which 2: Siberian Polygala sugar A5; 3: Siberian Polygala sugar A6; 4: Ferulic acid; 11: 3,6'-dianisinoyl sucrose; 13: Polygala glycoside A; 14: Polygala glycoside C; 27: Costunolide; 28: Dehydrocostus lactone.

[0102] Figure 3 Shown are the characteristic peaks of the fingerprint of the Guipi Pill sample of the present invention and the chromatogram to which the peaks belong, wherein 2: Siberian Polygala sugar A5; 3: Siberian Polygala sugar A6; 4: Ferulic acid; 11: 3,6'-dianisinoyl sucrose; 13: Polygala tenuifolia glycoside A; 14: Polygala glycoside C; 27: Costunolide; 28: Dehydrocostus lactone.

[0103] Figure 4Shown are UHPLC superimposed fingerprints of 20 batches of Guipi Pills samples among the Guipi Pills samples of the present invention, wherein S1 to S20 are 20 batches of Guipi Pills samples. DETAILED DESCRIPTION

[0104] The present invention is further described below in conjunction with 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.

[0105] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0106] The reagents and instruments used in the following examples are as follows:

[0107] 1. Reagents

[0108] Reference substances: Siberian Polygala sugar A5 reference substance (batch number: 6954, purity ≥ 98%, Shanghai Shidande Standard Technology Service Co., Ltd.), Siberian Polygala sugar A6 reference substance (batch number: 7920, purity ≥ 98%, Shanghai Shidande Standard Technology Service Co., Ltd.), ferulic acid (batch number: 110773-200611, China Food and Drug Inspection Institute), 3,6'-bisinacoyl sucrose (batch number: 8520, purity ≥ 97.90%, Shanghai Shidande Standard Technology Service Co., Ltd.) , polygala tenuifolia glycoside A (batch number: 12728, purity ≥98%, Shanghai Stand Standard Technical Service Co., Ltd.), polygala tenuifolia glycoside C (batch number: 19051525, purity ≥98%, Shanghai Tongtian Biotechnology Co., Ltd.), costunolide (batch number: 111524-201911, purity ≥99.90%, China Food and Drug Inspection Institutes) and dehydrocostus lactone (batch number: 111524-201911, purity ≥99.50%, China Food and Drug Inspection Institutes).

[0109] Samples: 20 batches of Guipi Pills samples (230501-230520), all produced and provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0110] Medicinal materials: Codonopsis pilosula, stir-fried Atractylodes macrocephala with bran, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, Costusroot lappa, and jujube are all commonly used medicinal materials and provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0111] Reagents: methanol (analytical grade AR, Sinopharm Chemical Reagent Co., Ltd.), acetonitrile, phosphoric acid (chromatographic grade, Fisher, USA), ultrapure water was prepared by Milli-Q ultrapure water treatment system.

[0112] 2. Instruments

[0113] Agilent 1290 Infinity II UHPLC ultra-high performance liquid chromatograph (including G7120A binary ultra-high speed pump; G7129B standard autosampler; G7116B high capacity column oven; G7117A diode array detector FS; OpenLab CDS2 chromatography workstation software Plus); ME204E 1 / 10,000 electronic balance and XSR205DU / A 1 / 100,000 electronic balance (METTLER TOLEDO Instrument Shanghai Co., Ltd.); DHG-9123A electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); HWS-26 electric constant temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd.); KQ-800DE ultrasonic cleaning machine (Kunshan Ultrasonic Instrument Co., Ltd.); Mill-Q Advantage A10 ultrapure water preparation system (MILLIPORE Shanghai Trading Co., Ltd.).

[0114] Example 1

[0115] 1. Sample pretreatment

[0116] Preparation of test solution: Take the powder of Guipi Pills sample from batch 230501, accurately weigh 1.0 g, place it in a 100-mL conical flask with a stopper, accurately add 20 mL of 70% methanol, weigh the weight, and extract by ultrasonic extraction (power 300 W, frequency 40 kHz) for 30 minutes. Let it stand and cool to room temperature, weigh the weight again, make up the lost weight with 70% methanol, shake well, filter, take the supernatant, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain test solution 1#.

[0117] Preparation of reference solution: Accurately weigh reference substances of Siberian Polygala A5, Siberian Polygala A6, ferulic acid, 3,6'-dienasinoylsucrose, Polygala glycoside A, Polygala glycoside C, costunolide and dehydrocostuslactone, respectively, place them in the same 50mL volumetric flask, dissolve and dilute to the mark with methanol, shake well to prepare the reference stock solution. In the reference stock solution, the content of Siberian Polygala A5 was 0.2772 mg / mL; the content of Siberian Polygala A6 was 0.2348 mg / mL; the content of ferulic acid was 0.4300 mg / mL; the content of 3,6'-biserucyl sucrose was 0.5724 mg / mL; the content of fine leaf polygala glycoside A was 0.1724 mg / mL; the content of polygala glycoside C was 0.2992 mg / mL; the content of costunolide was 0.2576 mg / mL; and the content of dehydrocostus lactone was 0.2876 mg / mL. Refrigerate at 4°C away from light for future use.

[0118] The reference substance stock solution was then diluted with 70% methanol to prepare a series of reference substance solutions of different concentrations. In a series of reference solutions with different concentrations, the content of Siberian Polygala A5 ranged from 1.11 to 277.20 μg / mL; the content of Siberian Polygala A6 ranged from 0.94 to 234.80 μg / mL; the content of ferulic acid ranged from 0.65 to 430.00 μg / mL; the content of 3,6'-dianisopylsucrose ranged from 0.86 to 572.40 μg / mL; the content of fine-leaved polygala glycoside A ranged from 0.69 to 172.40 μg / mL; the content of polygala glycoside C ranged from 1.20 to 299.20 μg / mL; the content of costunolide ranged from 2.60 to 257.60 μg / mL; and the content of dehydrocostus lactone ranged from 2.90 to 287.60 μg / mL.

[0119] 2. Chromatographic conditions

[0120] The chromatographic conditions of ultra-high performance liquid chromatography were as follows: the chromatographic column was Waters UPLC Cortecs C 18 Chromatographic column (2.1mm×100mm, 1.6μm); detector is photodiode array detector (DAD); column temperature is 30℃; injection volume: 2μL; flow rate is 0.6mL / min.

[0121] The analysis was performed using a multi-wavelength method, with detection wavelengths of 330 nm and 220 nm. Specifically, from 0 to 53 min, the detection wavelength was 330 nm; from 53 to 85 min, the detection wavelength was 220 nm.

[0122] 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 85 min; and the elution is gradient elution.

[0123] The specific procedure of gradient elution is:

[0124] 0-5min, the volume ratio of phase A:phase B was 5:95-8:92;

[0125] 5-8min, volume ratio of phase A:phase B is 8:92-8:92;

[0126] 8-9min, the volume ratio of phase A:phase B is 8:92-10:90;

[0127] 9-17min, volume ratio of phase A:phase B is 10:90-10:90;

[0128] 17-18min, the volume ratio of phase A:phase B is 10:90-13:87;

[0129] 18-30min, the volume ratio of phase A:phase B is 13:87-13:87;

[0130] 30-35min, volume ratio of phase A:phase B is 13:87-20:80;

[0131] 35-45min, volume ratio of phase A:phase B is 20:80-20:80;

[0132] 45-50min, the volume ratio of phase A:phase B is 20:80-29:71;

[0133] 50-70min, volume ratio of phase A:phase B is 29:71-29:71;

[0134] 70-75min, the volume ratio of phase A:phase B was 29:71-45:55;

[0135] 75-80min, the volume ratio of phase A:phase B is 45:55-80:20;

[0136] 80-85min, the volume ratio of phase A:phase B is 80:20-80:20.

[0137] 3. Determination

[0138] Using the external standard method, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations, and the samples were analyzed by ultra-high performance liquid chromatography to draw a standard working curve. The obtained test solution was then analyzed by ultra-high performance liquid chromatography, and the analysis results were substituted into the standard working curve to obtain the contents of the 8 components in the test solution.

[0139] Specifically, a series of different volumes of reference solution are pipetted respectively, and a series of solutions of different concentrations are prepared respectively. The samples are analyzed by ultra-high performance liquid chromatography to obtain the linear relationship between the concentration and peak area of ​​the eight components in the reference solution. The corresponding standard working curve is drawn with the chromatographic peak area of ​​each component corresponding to its corresponding concentration, and the regression equation of each standard working curve is calculated. The test solution is then detected by ultra-high performance liquid chromatography, and the chromatographic peak areas of the eight 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. See the specific chromatogram for details. Figure 1 .

[0140] Example 2

[0141] 1. Sample pretreatment

[0142] Preparation of test solution: Take the powder of Guipi Pills sample from batch 230502, accurately weigh 1.0 g, place it in a 100-mL conical flask with a stopper, accurately add 22 mL of 68% methanol, weigh the weight, and perform ultrasonic extraction (power 330 W, frequency 45 kHz) for 32 minutes. Let it stand and cool to room temperature, weigh the weight again, make up the lost weight with 68% methanol, shake well, filter, take the supernatant, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain test solution 2#.

[0143] Preparation of reference solution: accurately weigh reference substances of Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-biserucyl sucrose, Polygala glycoside A, Polygala glycoside C, costusinolide, and dehydrocostusinolide, respectively, place in the same 50mL volumetric flask, dissolve and dilute to volume with methanol, shake well, prepare reference substance stock solution, and then dilute with 68% methanol to prepare a series of reference substance solutions of different concentrations. The concentration range of the reference substance stock solution and a series of reference substance solutions of different concentrations is the same as step 1 in Example 1.

[0144] 2. Chromatographic conditions

[0145] The chromatographic conditions of ultra-high performance liquid chromatography were as follows: the chromatographic column was Waters UPLC Cortecs C 18 Chromatographic column (2.1mm×100mm, 1.6μm); detector is photodiode array detector (DAD); column temperature is 32℃; injection volume: 1μL; flow rate is 0.4mL / min.

[0146] The analysis was performed using a multi-wavelength method, with detection wavelengths of 328 nm and 222 nm. Specifically, from 0 to 53 min, the detection wavelength was 328 nm; from 53 to 85 min, the detection wavelength was 222 nm.

[0147] The mobile phase is acetonitrile-0.11% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.11% phosphoric acid aqueous solution; the analysis time is 85 min; and the elution is gradient elution.

[0148] The specific procedure of gradient elution is:

[0149] 0-5min, the volume ratio of phase A:phase B was 4:96-9:91;

[0150] 5-8min, volume ratio of phase A:phase B is 9:91-9:91;

[0151] 8-9min, the volume ratio of phase A:phase B is 9:91-11:89;

[0152] 9-17min, the volume ratio of phase A:phase B is 11:89-11:89;

[0153] 17-18min, the volume ratio of phase A:phase B is 11:89-14:86;

[0154] 18-30min, the volume ratio of phase A:phase B is 14:86-14:86;

[0155] 30-35min, the volume ratio of phase A:phase B is 14:86-21:79;

[0156] 35-45min, volume ratio of phase A:phase B is 21:79-21:79;

[0157] 45-50min, the volume ratio of phase A:phase B is 21:79-28:72;

[0158] 50-70min, the volume ratio of phase A:phase B is 28:72-28:72;

[0159] 70-75min, the volume ratio of phase A:phase B was 28:72-46:54;

[0160] 75-80min, the volume ratio of phase A:phase B is 46:54-81:19;

[0161] At 80-85min, the volume ratio of phase A:phase B is 81:19-81:19.

[0162] 3. Determination

[0163] The specific determination process is the same as step 3 in Example 1.

[0164] Example 3

[0165] 1. Sample pretreatment

[0166] Preparation of test solution: Take the powder of Guipi Pills sample from batch 230503, accurately weigh 1.0 g, place it in a 100-mL conical flask with a stopper, accurately add 18 mL of 72% methanol, weigh the weight, and perform ultrasonic extraction (power 280 W, frequency 35 kHz) for 28 minutes. Let it stand and cool to room temperature, weigh the weight again, make up the lost weight with 72% methanol, shake well, filter, take the supernatant, filter through a 0.22 μm microporous filter membrane, and take the filtrate to obtain test solution 3#.

[0167] Preparation of reference solution: accurately weigh reference substances of Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-biserucyl sucrose, Polygala glycoside A, Polygala glycoside C, costus lactone, and dehydrocostus lactone, respectively, place in the same 50mL volumetric flask, dissolve with methanol and dilute to volume, shake well, prepare reference substance stock solution, and then dilute with 72% methanol to prepare a series of reference substance solutions of different concentrations. The concentration range of the reference substance stock solution and a series of reference substance solutions of different concentrations is the same as step 1 in Example 1.

[0168] 2. Chromatographic conditions

[0169] The chromatographic conditions of ultra-high performance liquid chromatography were as follows: the chromatographic column was Waters UPLC Cortecs C 18 Chromatographic column (2.1mm×100mm, 1.6μm); detector is photodiode array detector (DAD); column temperature is 28℃; injection volume: 3μL; flow rate is 0.8mL / min.

[0170] The analysis was performed using a multi-wavelength method, with detection wavelengths of 332 nm and 218 nm. Specifically, from 0 to 53 min, the detection wavelength was 332 nm; from 53 to 85 min, the detection wavelength was 218 nm.

[0171] The mobile phase is acetonitrile-0.09% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.09% phosphoric acid aqueous solution; the analysis time is 85 min; and the elution is gradient.

[0172] The specific procedure of gradient elution is:

[0173] 0-5min, the volume ratio of phase A:phase B was 6:94-7:93;

[0174] 5-8min, the volume ratio of phase A:phase B is 7:93-7:93;

[0175] 8-9min, the volume ratio of phase A:phase B is 7:93-10:90;

[0176] 9-17min, volume ratio of phase A:phase B is 10:90-10:90;

[0177] 17-18min, the volume ratio of phase A:phase B is 10:90-12:88;

[0178] 18-30min, volume ratio of phase A:phase B is 12:88-12:88;

[0179] 30-35min, volume ratio of phase A:phase B is 12:88-19:81;

[0180] 35-45min, volume ratio of phase A:phase B is 19:81-19:81;

[0181] 45-50min, the volume ratio of phase A:phase B is 19:81-30:70;

[0182] 50-70min, volume ratio of phase A:phase B is 30:70-30:70;

[0183] 70-75min, the volume ratio of phase A:phase B is 30:70-44:56;

[0184] 75-80min, the volume ratio of phase A:phase B is 44:56-79:21;

[0185] At 80-85min, the volume ratio of phase A:phase B is 79:21-79:21.

[0186] 3. Determination

[0187] The specific determination process is the same as step 3 in Example 1.

[0188] Example 4

[0189] The reference substances of Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-bisinuoyl sucrose, Polygala glycoside A, Polygala glycoside C, costunolide, and dehydrocostus lactone were accurately weighed respectively, placed in the same 50 mL volumetric flask, dissolved with methanol and diluted to the scale, shaken well, and prepared into reference substance stock solution. In the reference substance stock solution, the content of Siberian Polygala sugar A5 was 0.2772 mg / mL; the content of Siberian Polygala sugar A6 was 0.2348 mg / mL; the content of ferulic acid was 0.4300 mg / mL; the content of 3,6'-bisinuoyl sucrose was 0.5724 mg / mL; the content of Polygala glycoside A was 0.1724 mg / mL; the content of Polygala glycoside C was 0.2992 mg / mL; and the content of costunolide was 0.2576 mg / mL. The reference substance stock solution was then diluted with 70% methanol to prepare a series of reference substance solutions of different concentrations.

[0190] In a series of reference solutions with different concentrations, the content of Siberian Polygala A5 ranged from 1.11 to 277.20 μg / mL; the content of Siberian Polygala A6 ranged from 0.94 to 234.80 μg / mL; the content of ferulic acid ranged from 0.65 to 430.00 μg / mL; the content of 3,6'-dianisopylsucrose ranged from 0.86 to 572.40 μg / mL; the content of fine-leaved polygala glycoside A ranged from 0.69 to 172.40 μg / mL; the content of polygala glycoside C ranged from 1.20 to 299.20 μg / mL; the content of costunolide ranged from 2.60 to 257.60 μg / mL; and the content of dehydrocostus lactone ranged from 2.90 to 287.60 μg / mL.

[0191] According to the chromatographic conditions of step 2 in Example 1, 2 μL was injected for determination. With the peak area (Y) as the ordinate and the concentration (X) as the abscissa, a standard curve was drawn and regression calculation was performed, and the results showed that the linear relationship of each component was good. It was gradually diluted with 70% methanol, and 10 μL was taken for determination. The concentration of the reference substance when the signal-to-noise ratio of the peak area was 10 times (S / N=10) was taken as the limit of quantification (LOQ), and the concentration of the reference substance when the signal-to-noise ratio of the peak area was 3 times (S / N=3) was taken as the limit of detection (LOD). The regression equation and the data of the limit of quantification and the limit of detection are shown in Table 3.

[0192] Table 3

[0193]

[0194] Example 5

[0195] 1. Precision

[0196] Take 230501 batches of Guipi Pills sample, prepare 1 portion of test solution according to step 1 in Example 1, detect according to step 2 in the above Example 1, inject 6 injections continuously for analysis, record the chromatographic peak area of ​​each component, and calculate the RSD% of the 8 index components. The results show that the RSD% of the 8 index components are all ≤2.0%, indicating that the instrument precision is good.

[0197] 2. Stability

[0198] Take 230501 batches of Guipi Pills sample, prepare 1 portion of the test solution according to step 1 in Example 1, and perform the test according to step 2 in Example 1, inject samples at 0, 3, 6, 9, 12, 24 and 36 hours, record the chromatogram, measure the peak area, and calculate the RSD of the peak area of ​​the 8 index components. The results show that the RSD% of the peak area of ​​each index component is ≤3.0%, indicating that the sample solution is stable within 36 hours.

[0199] 4. Repeatability

[0200] Take 230501 batches of Guipi Pills sample, prepare 6 portions of test solution according to step 1 in Example 1, accurately weigh 6 portions, detect according to step 2 in the above Example 1, record the chromatogram, record the chromatographic peak area of ​​each component, and calculate the content (mg / ml) of 8 index components. The results show that the RSD% of the content of each index component is ≤3.0%, indicating that the method has good repeatability.

[0201] 2. Sample recovery rate

[0202] Take 9 portions of Guipi Pills sample from batch 230501, each portion is about 0.1g, accurately weighed, and add three mass concentrations of reference solution of low, medium and high (equivalent to 50%, 100% and 150% of the original mass fraction, respectively), take 3 portions of each mass concentration, prepare the test solution according to step 1 in Example 1, detect according to step 2 in the above Example 1, record the chromatogram, calculate the sample recovery rate and RSD% of each component according to the measured amount and the added amount, and the results are shown in Table 4. As can be seen from Table 4, the average recovery rates of Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-bisinacoyl sucrose, Polygala tenuifolia glycoside A, Polygala tenuifolia glycoside C, costunolide and dehydrocostus lactone are 95.72% to 102.39%, and the RSD% is 1.65% to 4.67%, indicating that the accuracy of the method is good.

[0203] Table 4

[0204]

[0205]

[0206] Example 6

[0207] Take 20 batches of Guipi Pills samples (batch numbers: 230501-230520), prepare the test solution according to step 1 in Example 1, and analyze the samples according to the chromatographic conditions of step 2 in Example 1, record the chromatograms, and calculate the contents of the 8 index components in Guipi Pills by the external standard method. The results are shown in Table 5.

[0208] Table 5

[0209]

[0210] Example 7

[0211] 1. Sample pretreatment

[0212] Preparation of the test solution: The preparation process of the test solution is the same as that in step 1 of Example 1.

[0213] Preparation of reference solution: The preparation process of reference solution is the same as that in step 1 of Example 1.

[0214] 2. Chromatographic conditions: The chromatographic conditions of the ultra-high performance liquid chromatography are the same as those of the ultra-high performance liquid chromatography in step 2 of Example 1.

[0215] 3. Determination

[0216] The ultra-high performance liquid chromatography method with the chromatographic conditions in the above step 2 is used to respectively measure the test solution and the reference solution in the above step 1 to obtain the fingerprint of the test solution and the 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, so as to attribute and locate the index components in the fingerprint of the test solution and obtain the fingerprint of Guipi Pills.

[0217] Example 8

[0218] The detection method of the Guipi Pills fingerprint established in the above-mentioned Example 7 was used to detect 20 batches of Guipi Pills, and the fingerprint of the test solution and the fingerprint of the reference solution were obtained. The fingerprint data of the test sample was imported into the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" issued by the State Pharmacopoeia Committee. Sample 230501 (S1) was used as the reference spectrum, and Mark peak matching was performed after multi-point correction. 0.2 was selected as the time window width for matching the common peaks of the fingerprints to generate fingerprints and reference fingerprints. The fingerprint of the test sample was compared with the reference fingerprint of the Guipi Pills obtained under the same fingerprint detection conditions for similarity, and the similarity of the fingerprint of each batch of Guipi Pills was calculated. Peak 11 (3,6'-diacyl sucrose) was used as the reference peak S, and 35 common peaks were calibrated. See details. Figure 2 The results of comparing the similarity between the fingerprints of the 20 batches of test products and the control fingerprints show that the similarities of S1 to S20 are all greater than 0.981, as shown in Table 6, indicating that the overall quality of each batch of samples is relatively stable. Figure 4 The results showed that the quality of different batches of Guipi Pills was similar, and the established fingerprint was representative and could be used for the quality control of Guipi Pills.

[0219] Table 6

[0220]

[0221] Example 9

[0222] The detection method of Guipi Pills fingerprint established in Example 7 was used to detect Guipi Pills. Based on the fingerprints of the test solution and the reference solution, 35 common fingerprint peaks were identified. Eight of them were identified by comparing with the reference solution. Figure 2 As shown, peak 11 has good separation and moderate peak area, so peak 11 is selected as the reference peak (S peak), and the relative retention time of the other peaks is calculated. Therefore, peak 11 is taken as the reference peak (S peak, relative retention time is 1.0000), and the relative retention times of the other 34 peaks are as follows: peak 1 0.10, peak 2 0.13, peak 3 0.17, peak 4 0.26, peak 5 0.37, peak 6 0.42, peak 7 0.49, peak 8 0.56, peak 9 0.58, peak 10 0.65, peak 12 1.05, peak 13 1.17, peak 14 1.35, peak 15 1.47, peak 16 1.52, Peak No. 17 is 1.64, Peak No. 18 is 1.70, Peak No. 19 is 1.74, Peak No. 20 is 1.76, Peak No. 21 is 1.78, Peak No. 22 is 1.80, Peak No. 23 is 1.97, Peak No. 24 is 2.05, Peak No. 25 is 2.11, Peak No. 26 is 2.18, Peak No. 27 is 2.37, Peak No. 28 is 2.50, Peak No. 29 is 2.79, Peak No. 30 is 2.81, Peak No. 31 is 2.82, Peak No. 32 is 2.83, Peak No. 33 is 2.89, Peak No. 34 is 2.91, and Peak No. 35 is 2.96.

[0223] Through Figure 2 By comparison, it was determined that Peak 2 was the fingerprint peak of Siberian Polygala sugar A5; Peak 3 was the fingerprint peak of Siberian Polygala sugar A6; Peak 4 was the fingerprint peak of ferulic acid; Peak 11 was the fingerprint peak of 3,6'-dianisopylsucrose; Peak 13 was the fingerprint peak of Polygala tenuifolia glycoside A; Peak 14 was the fingerprint peak of Polygala glycoside C; Peak 27 was the fingerprint peak of costunolide; and Peak 28 was the fingerprint peak of dehydrocostus lactone.

[0224] Example 10

[0225] 1. Precision

[0226] Take 230501 batches of Guipi Pills sample powder, accurately weigh it, prepare 1 portion of the test solution according to step 1 in Example 7, detect it according to step 2 in the above Example 7, inject it 6 times continuously, record the chromatogram, take peak 11 (3'6-diacyl sucrose) as the reference peak, calculate the relative retention time RSD% of 35 common peaks, all of which are less than 0.56%, and the relative peak area RSD% are all less than 2.49%, indicating that the instrument has good precision.

[0227] 2. Repeatability

[0228] Take 230501 batches of Guipi Pills sample, prepare 6 test solutions in parallel according to step 1 in Example 7, detect according to step 2 in the above Example 7, record the chromatogram, take peak 11 (3'6-diacyl sucrose) as the reference peak, calculate the relative retention time RSD% of 35 common peaks, all of which are less than 0.29%, and the relative peak area RSD% are all less than 2.60%. The results show that the method has good repeatability.

[0229] 3. Stability

[0230] Take 230501 batches of Guipi Pills sample, prepare 1 portion of the test solution according to step 1 in Example 7, and detect according to step 2 in the above Example 7, inject samples at 0, 3, 6, 9, 12, and 24 h, record the chromatogram, take peak 11 (3'6-diacyl sucrose) as the reference peak, calculate the relative retention time RSD of 35 common peaks, all of which are less than 2.55%, and the relative peak area RSD is less than 3.01%, indicating that the test solution has good stability within 36 h.

[0231] Embodiment 11

[0232] The test solution was prepared by using step 1 in Example 7 above.

[0233] Eleven kinds of medicinal material powders were taken respectively, and 11 kinds of single medicinal material sample solutions were prepared by using step 1 in the above embodiment 7, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Semen Ziziphi spinulosae, Longan pulp, Angelica sinensis, Aucklandia lappa and Ziziphus jujuba.

[0234] The test solution and 11 single herbal sample solutions were measured by step 2 in Example 7 to obtain the test solution and 11 single herbal sample solutions. The attribution was confirmed by the retention time of the chromatographic peak of each single herbal sample solution and the chromatographic peak of the fingerprint of Guipi Pills. Figure 3 , Table 2.

[0235] Depend on Figure 2 As shown in Table 2, 4 common peaks, including peaks 1, 12, 27 and 28, are derived from costusroot, 17 common peaks, including peaks 2, 3, 5-11, 12, 13, 14, 18-22, are derived from polygala root; 2 common peaks, including peaks 4 and 23, are derived from angelica root; peaks 10, 32 and 34 may be derived from stir-fried spiny jujube seeds; peaks 23, 25 and 26 may be derived from liquorice root. This fingerprint method can characterize the characteristic peaks of 5 medicinal materials in the prescription (angelica, costusroot, polygala root, stir-fried spiny jujube seeds, liquorice), that is, the characteristic peaks of 5 medicinal materials out of 11 medicinal materials are well represented in the fingerprint and the attribution is confirmed.

[0236] In summary, the present invention provides a method for determining the contents of 8 chemical components in Guipi Pills and a fingerprint method thereof, which can quantitatively analyze the 8 components in Guipi Pills, and also establish a fingerprint of Guipi Pills, providing a reference for scientific and comprehensive control of the quality of Guipi Pills. Therefore, the present invention overcomes various shortcomings in the prior art and has a high industrial utilization value.

[0237] 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 familiar with 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 a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for determining the chemical contents of 8 ingredients in Guipi Pills, comprising the following steps: 1) Preparation of test solution: Dissolve the Guipi Pills sample in solvent, extract by ultrasonic, cool, weigh, shake well, filter, take the supernatant, filter, and take the filtrate to obtain the test solution; 2) Preparation of reference substance solution: one or more reference substances selected from Siberian Polygala sugar A5, Siberian Polygala sugar A6, ferulic acid, 3,6'-dienasinoyl sucrose, Polygala glycoside A, Polygala glycoside C, costunolide, and dehydrocostus lactone are added to a solvent and dissolved, and then fixed to volume to obtain a reference substance solution; 3) Determination: The test solution of step 1) and the reference solution of step 2) are respectively determined by ultra performance liquid chromatography, and the content of one or more components of Siberian polygala sugar A5, Siberian polygala sugar A6, ferulic acid, 3,6'-dicerinoylsucrose, polygala glycoside A, polygala glycoside C, costunolide, and dehydrocostus lactone in the test solution is calculated by external standard method.

2. The method for determining the chemical contents of 8 ingredients in Guipi Pills according to claim 1, characterized in that: In step 1), any one or more of the following conditions are included: A1) The Guipi Pills sample is a powder sample; A2) the solvent is an aqueous solution containing 55-85% methanol by volume; A3) the ratio of the weight of the Guipi Pill sample added to the volume of the solvent added is 1:15-25, g / mL; A4) The ultrasonic extraction time is 20-40 min; A5) the power of the ultrasonic extraction is 250-350W; the frequency of the ultrasonic extraction is 30-50kHz; A6) the solvent used for the weight replenishment is an aqueous solution containing 55-85% methanol by volume; A7) The filtration is membrane filtration; the membrane is a 0.22 μm membrane.

3. The method for determining the chemical contents of 8 ingredients in Guipi Pills according to claim 1, characterized in that: In step 2), the reference substance solution can be directly prepared into a mixed reference substance solution, or can be prepared by first adding a first solvent to prepare a reference substance stock solution and then adding a second solvent to dilute it step by step.

4. The method for determining the chemical contents of 8 ingredients in Guipi Pills according to claim 3, characterized in that: Includes any one or more of the following conditions: B1) the first solvent is methanol; B2) the second solvent is an aqueous solution containing 55-85% methanol by volume; B3) The content range of Siberian Polygala sugar A5 in the reference solution is 1.11-277.20 μg / mL; the content range of Siberian Polygala sugar A6 is 0.94-234.80 μg / mL; the content range of ferulic acid is 0.65-430.00 μg / mL; the content range of 3,6'-dianisopylsucrose is 0.86-572.40 μg / mL; the content range of fine leaf polygala glycoside A is 0.69-172.40 μg / mL; the content range of polygala glycoside C is 1.20-299.20 μg / mL; the content range of costunolide is 2.60-257.60 μg / mL; the content range of dehydrocostus lactone is 2.90-287.60 μg / mL.

5. The method for determining the chemical contents of 8 ingredients in Guipi Pills according to claim 1, characterized in that: In step 3), the ultra-high performance liquid chromatography method includes any one or more of the following conditions: C1) The chromatographic column is C 18 Chromatographic columns; C2) the detector is a photodiode array detector; C3) the column temperature is 25-35°C; C4) the injection volume is 1-5 μL; C5) the flow rate is 0.2-1 mL / min; C6) the detection wavelength is selected from one or a combination of 325-335nm, 215-225nm; C7) the mobile phase is acetonitrile-0.09-0.11% phosphoric acid aqueous solution; in acetonitrile-0.09-0.11% phosphoric acid aqueous solution, phase A is acetonitrile and phase B is 0.09-0.11% phosphoric acid aqueous solution; C8) In the ultra-high performance liquid chromatography, gradient elution is adopted and the analysis time is 85 min.

6. The method for determining the chemical contents of 8 ingredients in Guipi Pills according to claim 5, characterized in that: Also includes any one or more of the following conditions: C11) In item C1), the chromatographic column is a Waters UPLC Cortecs C 18 Chromatographic column, 2.1 mm × 100 mm, 1.6 μm; C61) In item C6), the detection wavelength changes with the time of the gradient elution program, and the specific changes are: 0-53 min, the detection wavelength is 325-335 nm; 53-85 min, the detection wavelength is 215-225 nm; C81) In item C8), the specific procedure of the gradient elution is: 0-5min, the volume ratio of phase A:phase B is 4-6:94-96-7-9:91-93; 5-8min, the volume ratio of phase A:phase B is 7-9:91-93-7-9:91-93; 8-9min, the volume ratio of phase A:phase B is 7-9:91-93-10-11:89-90; 9-17min, the volume ratio of phase A:phase B is 10-11:89-90-10-11:89-90; 17-18min, the volume ratio of phase A:phase B is 10-11:89-90-12-14:86-88; 18-30min, the volume ratio of phase A:phase B is 12-14:86-88-12-14:86-88; 30-35min, the volume ratio of phase A:phase B is 12-14:86-88-19-21:79-81; 35-45min, the volume ratio of phase A:phase B is 19-21:79-81-19-21:79-81; 45-50min, the volume ratio of phase A:phase B is 19-21:79-81-28-30:70-72; 50-70min, the volume ratio of phase A:phase B is 28-30:70-72-28-30:70-72; 70-75min, the volume ratio of phase A:phase B is 28-30:70-72-44-46:54-56; 75-80min, the volume ratio of phase A:phase B is 44-46:54-56-79-81:19-21; At 80-85min, the volume ratio of phase A:phase B is 79~81:19~21-79~81:19~21.

7. A method for detecting the fingerprint of Guipi Pills, comprising the following steps: A) Preparation of the test solution: the same as step 1) of the method for determining the chemical contents of 8 ingredients in Guipi Pills according to any one of claims 1 to 6; B) Preparation of reference solution: the same as step 2) of the method for determining the chemical contents of 8 ingredients in Guipi Pills according to any one of claims 1 to 6; C) Determination: respectively determine the test solution of step A) and the reference solution of step B) by ultra-high performance liquid chromatography under the same chromatographic conditions as step 3) of the method for determining the chemical contents of eight components in Guipi Pills according to any one of claims 1 to 6, to obtain a fingerprint of the test solution and a fingerprint of the reference solution, compare the fingerprint of the test solution with the fingerprint of the reference solution, attribute and locate the index components in the fingerprint of the test solution, and thereby obtain a fingerprint of Guipi Pills.

8. A quality detection method for Guipi Pills, comprising obtaining a fingerprint of Guipi Pills using the detection method for the fingerprint of Guipi Pills according to claim 7, and comparing the obtained fingerprint of Guipi Pills with a control fingerprint of Guipi Pills obtained under the same fingerprint detection conditions for similarity.

9. The quality detection method of Guipi Pills according to claim 8, characterized in that: The control fingerprint of Guipi Pills was obtained under the same conditions as the detection method of the fingerprint of Guipi Pills according to claim 7, wherein the control fingerprint of Guipi Pills includes 35 common fingerprint peaks, with peak 11 as the reference peak S peak, and the relative retention time is 1.0000; the relative retention times of the other 34 peaks are peak 1 0.10±0.0007, peak 2 0.13±0.0007, peak 3 0.17±0.0007, peak 4 0.26±0.0007, peak 5 0.26±0.0007, peak 6 0.26±0.0007, peak 7 0.26±0.0007, peak 8 0.26±0.0007, peak 9 0.26±0.0007, peak 10 0.26±0.0007, peak 11 0.26±0.0007, peak 11 0.26±0.0007, peak 12 0.26±0.0007, peak 13 0.26±0.0007, peak 14 0.26±0.0007, peak 15 0.26±0.0007, peak 16 0.26±0.0007, peak 17 0.26±0.0007, peak 18 0.26±0.0007, peak 19 0.26±0.0007, peak 20 0.26±0.0007, peak 21 0.26±0.0007, peak 22 0.26±0.0007, peak 23 0.26±0.0007, peak 24 0.0021, peak 5 0.37±0.0027, peak 6 0.42±0.0057, peak 7 0.49±0.0006, peak 8 0.56±0.0006, peak 9 0.58±0.0013, peak 10 0.65±0.0031, peak 12 1.05±0.0024, peak 13 1.17±0.0139, peak 14 1.35±0.0242, peak 15 1.47±0.0177 , peak 16 1.52±0.0162, peak 17 1.64±0.0110, peak 18 1.70±0.0280, peak 19 1.74±0.0333, peak 20 1.76±0.0363, peak 21 1.78±0.0367, peak 22 1.80±0.0383, peak 23 1.97±0.0420, peak 24 2.05±0.0379, peak 25 2.11±0.0444 , peak No. 26 2.18±0.0453, peak No. 27 2.37±0.0470, peak No. 28 2.50±0.0496, peak No. 29 2.79±0.0658, peak No. 30 2.81±0.0662, peak No. 31 2.82±0.0663, peak No. 32 2.83±0.0666, peak No. 33 2.89±0.0677, peak No. 34 2.91±0.0679, peak No. 35 2.96±0.0693.

10. A method for screening fingerprints of multiple medicinal materials in Guipi Pills, comprising the following steps: a) Preparation of single medicinal material sample solutions: prepare any one or more of the 11 medicinal material samples of Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costusroot, and jujube in Guipi Pills according to step A) of the detection method of the fingerprint of Guipi Pills according to claim 7, and obtain at least one single medicinal material sample solution respectively; b) Preparation of negative sample solution: 11 medicinal material samples, namely, Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube, are respectively lacking Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube, and are prepared according to step A) of the detection method of Guipi Pill fingerprint according to claim 7, to obtain negative sample solutions lacking Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Licorice root, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba seeds, longan pulp, Angelica sinensis, costus root, and jujube; c) Determination: using ultra-high performance liquid chromatography under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Guipi Pills according to claim 7 to respectively determine the fingerprints of the single medicinal material sample solution in step a) and the negative sample solution in step b); d) Obtaining the control fingerprint: using the test solution prepared according to step A) of the method for detecting the fingerprint of Guipi Pills according to claim 7, and adopting the same step C) as the method for detecting the fingerprint of Guipi Pills according to claim 7 to obtain the control fingerprint of Guipi Pills; e) Quality inspection: Compare the fingerprints of the single medicinal sample solution and the negative sample solution with the control fingerprint of Guipi Pills. By the relative retention time, identify the corresponding characteristic peaks of the single medicinal sample solution in the control fingerprint of Guipi Pills, so as to attribute and locate the characteristic peaks in the fingerprint of the single medicinal sample solution.

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