Determination method of eight chemical components in Guipi pill and its fingerprint method

Abstract

The application provides a method for determining the content of eight chemical components in Guipi pills. The application also provides a method for detecting the fingerprint of Guipi pills. The application further provides a method for detecting the quality of Guipi pills. The application further provides a method for screening the fingerprint of multiple medicinal materials in Guipi pills. The method for determining the content of eight chemical components in Guipi pills and the fingerprint method provided by the application can quantitatively analyze the eight components in Guipi pills, and the fingerprint of Guipi pills is established, thereby providing reference for scientifically and comprehensively controlling the quality of Guipi pills.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of traditional Chinese medicine components, and relates to a method for determining the contents of 8 chemical components in Guipi Pills and its fingerprint method. Specifically, it relates to a method for determining the contents of 8 chemical components in Guipi Pills: sibiricose A5, sibiricose A6, ferulic acid, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, polygalacin C, costunolide, dehydrocostus lactone, and its fingerprint determined based on 8 components. Background Art

[0002] Guipi Pills originated from the famous ancient prescription Guipi Decoction, which was first recorded in "Jisheng Fang" by the great medical expert Yan Yonghe in the Song Dynasty and is now included in Volume I of "Chinese Pharmacopoeia". It is a honey pill made from 11 traditional Chinese medicines including Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Glycyrrhiza uralensis, Poria cocos, prepared Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan aril, Angelica sinensis, Aucklandia lappa, and large dates (with kernels removed). It has the effects of replenishing qi and strengthening the spleen, nourishing blood and calming the mind, and is used for symptoms such as deficiency of both heart and spleen, shortness of breath and palpitations, insomnia and dreaminess, dizziness and vertigo, limb fatigue and weakness, loss of appetite, metrorrhagia and metrostaxis. In the formula, Astragalus membranaceus and Codonopsis pilosula replenish qi and strengthen the spleen, enabling qi to promote blood production, and are the monarch drugs. Supplementary drugs include Angelica sinensis and longan aril for nourishing blood and tonifying the heart as ministerial drugs; Atractylodes macrocephala replenishes qi and strengthens the spleen, assisting Codonopsis pilosula and Astragalus membranaceus to strengthen the spleen to supply the source of qi and blood production, and is also a ministerial drug. Adjuvant drugs include Ziziphus jujuba var. spinosa, Poria cocos, and Polygala tenuifolia for nourishing blood, calming the mind and soothing the nerves; Aucklandia lappa regulates qi and awakens the spleen to prevent stagnation during supplementation; large dates harmonize the spleen and stomach to assist in qi and blood production. The guiding drug is roasted Glycyrrhiza uralensis to harmonize all the drugs. When these drugs are combined, they exert the effects of replenishing qi and blood, strengthening the spleen and nourishing the heart.

[0003] The quality control index of Guipi Pills preparation is the content of astragaloside IV. Most of the relevant quality control research literature on the preparation analyzes the content of the single index component astragaloside IV in Guipi Pills using ELSD or CAD as detectors, lacking relevant methods for simultaneously detecting and controlling the chemical components and fingerprints of multiple formula herbs in Guipi Pills under the same chromatographic conditions. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a method for determining the contents of 8 chemical components in Guipi Pills and its fingerprint method. By using optimized sample pretreatment and ultra-high performance liquid chromatography methods, a method for determining the contents of 8 components in Guipi Pills: sibiricose A5, sibiricose A6, ferulic acid, 3,6'-di-O-sinapoyl-sucrose, tenuifoliside A, polygalacin C, costunolide, dehydrocostus lactone, and its fingerprint determined based on these 8 index components is established, providing reference for scientifically and comprehensively controlling the quality of Guipi Pills.

[0005] To achieve the above purpose and other related purposes, the first aspect of the present invention provides a method for determining the chemical contents of 8 components in Guipi Pills, including the following steps:

[0006] 1) Preparation of test solution: Dissolve the Guipi Pill sample in solvent, extract by ultrasound, cool, add weight, shake well, filter and take the supernatant, filter again, and take the filtrate to obtain the test solution.

[0007] 2) Preparation of reference solution: Dissolve one or more of the following reference standards in a solvent: Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costus lactone, and dehydrocostus lactone, and then make up to volume to obtain the reference solution.

[0008] 3) Determination: The test solution in step 1) and the reference solution in step 2) were determined by ultra-high performance liquid chromatography (UHPLC), and the contents of one or more of the following components in the test solution were calculated by external standard method: Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinoyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costus lactone, and dehydrocostus lactone.

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

[0010] Preferably, in step 1), the Guipi Pill sample must be accurately weighed before adding the solvent.

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

[0012] Preferably, in step 1), the solvent is weighed after being added.

[0013] Preferably, in step 1), the ratio of the weight (g) of the Guipi Pill 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, and 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, and preferably 30 min.

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

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

[0017] Preferably, in step 1), the weight must be determined before adding the weight.

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

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

[0023] More preferably, the reference stock solution contains 0.2772 mg / mL of Siberian polygalactosyl A5; 0.2348 mg / mL of Siberian polygalactosyl A6; 0.4300 mg / mL of ferulic acid; 0.5724 mg / mL of 3,6'-disinozinc; 0.1724 mg / mL of polygalactosyl A; 0.2992 mg / mL of polygalactosyl C; 0.2576 mg / mL of costunolide; and 0.2876 mg / mL of dehydrocostunolide.

[0024] The reference standard stock solution was stored at 4°C in the dark for later 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%, and more preferably 70%.

[0027] Preferably, in step 2), the CAS number of Siberian polygalactosyl A5 is 107912-97-0, the CAS number of Siberian polygalactosyl A6 is 241125-75-7, the CAS number of ferulic acid is 1135-24-6, the CAS number of 3,6'-disinozinol sucrose is 139891-98-8, the CAS number of polygalactosyl A is 139726-35-5, the CAS number of polygalactosyl C is 139726-37-7, the CAS number of costunolide is 553-21-9, and the CAS number of dehydrocostunolide is 477-43-0.

[0028] Preferably, in step 2), the content of Siberian polygalactosyl A5 in the reference solution ranges from 1.11 to 277.20 μg / mL; the content of Siberian polygalactosyl A6 ranges from 0.94 to 234.80 μg / mL; the content of ferulic acid ranges from 0.65 to 430.00 μg / mL; the content of 3,6'-disinozylosyl sucrose ranges from 0.86 to 572.40 μg / mL; the content of polygalactosyl A ranges from 0.69 to 172.40 μg / mL; the content of polygalactosyl C ranges from 1.20 to 299.20 μg / mL; the content of costunolide ranges from 2.60 to 257.60 μg / mL; and the content of dehydrocostunolide ranges from 2.90 to 287.60 μg / mL.

[0029] Preferably, in step 3), the chromatographic column in the ultra-high performance liquid chromatography is a C18 column. 18 Chromatographic column. More preferably, the chromatographic column used in the ultra-high performance liquid chromatography is a Waters UPLC Cortecs C1000 HPLC system. 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), the column temperature in the ultra-high performance liquid chromatography is 25-35℃, specifically 25-28℃, 28-32℃, 32-35℃, and preferably 30℃.

[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, or 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, specifically 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, and 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 two of 325-335nm and 215-225nm, preferably 328-332nm and 218-222nm, and more preferably 330nm and 220nm.

[0036] More preferably, the detection wavelength varies with the time of the gradient elution process, specifically as follows: 0-53 min, the detection wavelength is 325-335 nm, preferably 328-332 nm, more preferably 330 nm; 53-85 min, the detection wavelength is 215-225 nm, preferably 218-222 nm, more preferably 220 nm.

[0037] Preferably, in step 3), the mobile phase in the ultra-high performance liquid chromatography is acetonitrile-0.09-0.11% phosphoric acid aqueous solution; in the 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 method, 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), gradient elution is used in the ultra-high performance liquid chromatography method, and the analysis time is 85 min.

[0041] More preferably, the specific procedure for gradient elution is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0055] More preferably, the specific procedure for the gradient elution is shown in Table 1, as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0069] Table 1

[0070]

[0071] Preferably, in step 3), the external standard method refers to: taking a series of different volumes of the reference solution from step 2), preparing a series of solutions with different concentrations, and analyzing them using ultra-high performance liquid chromatography (UHPLC) to obtain the linear relationship between the concentration and peak area of ​​the eight components in the reference solution. Using the peak area of ​​each component to correspond to its corresponding concentration, a standard working curve is plotted, and the regression equation for each standard working curve is calculated. Then, the test solution is detected using UHPLC, and the peak areas of the eight components in the test solution are substituted into the regression equations of each standard working curve to calculate the content of the corresponding component.

[0072] More preferably, in the standard working curve, the peak area of ​​each component is used as the vertical axis, and the concentration of each component in the reference solution is used as the horizontal axis.

[0073] A second aspect of this invention provides a method for detecting the fingerprint spectrum of Guipi Pill, comprising the following steps:

[0074] A) Preparation of the test solution: Same as step 1) of the method for determining the chemical content of the eight components in Guipi Pill;

[0075] B) Preparation of reference solution: Same as step 2) of the method for determining the chemical content of the eight components in Guipi Pill;

[0076] C) Determination: Using the same chromatographic conditions as step 3) of the method for determining the chemical content of the eight components in Guipi Pill, ultra-high performance liquid chromatography (UHPLC) was used to determine the test solution in step A) and the reference solution in step B), respectively. The fingerprint chromatograms of the test solution and the reference solution were obtained. The fingerprint chromatograms of the test solution and the reference solution were compared, and the index components in the fingerprint chromatogram of the test solution were assigned and located, thereby obtaining the fingerprint chromatogram of Guipi Pill.

[0077] Preferably, in step C), the fingerprint spectrum of the test sample solution is compared with the fingerprint spectrum of the reference solution. This is done by identifying the corresponding characteristic peaks in the fingerprint spectrum of the test sample solution based on the known characteristic peaks in the fingerprint spectrum of the reference solution and by using the relative retention time, thereby assigning and locating the indicator components in the fingerprint spectrum of the test sample solution.

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

[0079] Preferably, when comparing the similarity between the obtained fingerprint chromatogram of Guipi Pill and the control fingerprint chromatogram of Guipi Pill, the present invention uses the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission. More preferably, the similarity between the fingerprint chromatogram of Guipi Pill obtained by the present invention and the control fingerprint chromatogram of Guipi Pill is ≥0.981.

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

[0081] Preferably, the control fingerprint spectrum of Guipi Pill is obtained under the same conditions as the aforementioned detection method for the fingerprint spectrum of Guipi Pill. The control fingerprint spectrum of Guipi Pill includes 35 common fingerprint peaks, with peak 11 as the reference peak (S peak, relative retention time of 1.0000). The relative retention times of the other 34 peaks are, in order, peak 1 (0.10±0.0007), peak 2 (0.13±0.0007), peak 3 (0.17±0.0007), peak 4 (0.26±0.0021), and peak 5. Peak 6 (0.37±0.0027), Peak 7 (0.42±0.0057), 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 (0.37±0.0027), Peak 14 (0.42±0.0057), Peak 15 (0.49±0.0006), Peak 16 (0.49±0.0006), Peak 16 (0.49±0.0006), Peak 16 (0.49±0.0006), Peak 17 (0.49±0.0006), Peak 18 (0.56±0.0006), Peak 19 (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 (0.47±0.0177), Peak 16 (0.47±0.0027), Peak 19 (0.42±0.0057), Peak 10 (0.49±0.0006), Peak 12 (0.49±0.0006), Peak 13 (1.17±0.0139), Peak 14 (1.35±0.0242), Peak 15 ( 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 (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 ...162), Peak Peak 1 (2.18±0.0453), Peak 27 (2.37±0.0470), Peak 28 (2.50±0.0496), Peak 29 (2.79±0.0658), Peak 30 (2.81±0.0662), Peak 31 (2.82±0.0663), Peak 32 (2.83±0.0666), Peak 33 (2.89±0.0677), Peak 34 (2.91±0.0679), Peak 35 (2.96±0.0693).

[0082] Specific data for the comparative fingerprint spectrum of Guipi Pill can be found in [link to relevant data]. Figure 2 .

[0083] More preferably, the fingerprint spectrum of the Guipi Pill is compared with the fingerprint spectrum of the reference solution, such as... Figure 2 As shown, peak 2 was identified as the fingerprint peak of Siberian polygalactosyl A5; peak 3 as the fingerprint peak of Siberian polygalactosyl A6; peak 4 as the fingerprint peak of ferulic acid; peak 11 as the fingerprint peak of 3,6'-disinozylosyl sucrose; peak 13 as the fingerprint peak of polygalactosyl A; peak 14 as the fingerprint peak of polygalactosyl C; peak 27 as the fingerprint peak of costunolide; and peak 28 as the fingerprint peak of dehydrocostunolide.

[0084] A fourth aspect of this invention provides a method for screening the fingerprint spectra of multiple medicinal materials in Guipi Pill, comprising the following steps:

[0085] a) Preparation of single herbal sample solutions: Any one or more of the 11 herbs in Guipi Pill, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, roasted Astragalus membranaceus, roasted Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube, are prepared according to step A) of the detection method of fingerprint spectrum of Guipi Pill, and at least one single herbal sample solution is obtained respectively.

[0086] b) Preparation of negative sample solutions: Eleven medicinal materials, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube, were prepared according to step A) of the fingerprint spectrum detection method for Guipi Pill, resulting in negative sample solutions for each of the following deficiencies: Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube.

[0087] c) Determination: The fingerprint spectra of the single herb sample solution in step a) and the negative sample solution in step b) were determined by ultra-high performance liquid chromatography (UHPLC) under the same chromatographic conditions as in step C) of the detection method for the fingerprint spectrum of Guipi Pill.

[0088] d) Obtaining the reference fingerprint spectrum: The test solution prepared according to step A) of the detection method of the fingerprint spectrum of Guipi Pill is used to obtain the reference fingerprint spectrum of Guipi Pill by step C) of the same detection method of the fingerprint spectrum of Guipi Pill.

[0089] e) Quality testing: The fingerprint spectra of single herbal sample solutions and negative sample solutions are compared with the control fingerprint spectra of Guipi Pill. By using the relative retention time, the corresponding characteristic peaks of the single herbal sample solutions in the control fingerprint spectra of Guipi Pill are identified, thereby assigning and locating the characteristic peaks in the fingerprint spectra of the single herbal sample solutions.

[0090] Preferably, in step a), the Codonopsis pilosula is the dried root of Codonopsis pilosula, Codonopsis lanceolata, or Codonopsis chuanxiong (all belonging to the Campanulaceae family). The Atractylodes macrocephala in the stir-fried Atractylodes macrocephala is the dried rhizome of Atractylodes macrocephala (Asteraceae family). The Astragalus membranaceus in the prepared Astragalus membranaceus is the dried root of Astragalus membranaceus or Astragalus membranaceus (all belonging to the Fabaceae family). The Poria cocos is the dried sclerotium of the fungus Poria cocos (Polyporaceae family). The Polygala tenuifolia in the prepared Polygala tenuifolia is the dried root of Polygala tenuifolia or Polygala ovoidea (all belonging to the Polygalaceae family). The Ziziphus jujuba var. spinosa in the stir-fried Ziziphus jujuba var. spinosa is the dried mature seed of Ziziphus jujuba (all belonging to the Rhamnaceae family). The Longan pulp is the aril of Longan (all belonging to the Sapindaceae family). The Angelica sinensis is the dried root of Angelica sinensis (Apiaceae family). The Aucklandia lappa is the dried root of Aucklandia lappa (all belonging to the Asteraceae family). The jujube is the mature fruit of Ziziphus jujuba (all belonging to the Rhamnaceae family).

[0091] Preferably, in step e), the present invention assigns and locates the characteristic peaks of the measured fingerprint spectrum of the single herbal sample solution with the control fingerprint spectrum of Guipi Pill. The analysis is performed using the 2012 version of the "Similarity Evaluation System for Chromatographic Fingerprint Spectra of Traditional Chinese Medicine" software published by the National Pharmacopoeia Commission. The assignment of the characteristic peaks of each single herbal ingredient in Guipi Pill is confirmed by the relative retention times of each characteristic peak on the control fingerprint spectrum of Guipi Pill. See the specific results below. Figure 2 , 3 And Table 2.

[0092] Table 2. Attribution of characteristic peaks of each single herb in Guipi Pill.

[0093]

[0094] As shown in Table 2, this fingerprinting method can characterize the characteristic peaks of the five medicinal materials in Guipi Wan (Angelica sinensis, Aucklandia lappa, Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, and Glycyrrhiza uralensis). Since Codonopsis pilosula and Astragalus membranaceus constitute a small proportion of the prescription, almost no corresponding chromatographic peaks appeared under DAD detection. Furthermore, the representative component of Astragalus membranaceus, astragaloside A, and the sugar components of Ziziphus jujuba and Longan pulp were not absorbed under DAD. Therefore, this method failed to characterize the indicator components of Codonopsis pilosula, Astragalus membranaceus, Ziziphus jujuba, and Longan pulp. Additionally, the characteristic components in stir-fried Atractylodes macrocephala and Poria cocos are also present in low concentrations in the medicinal materials, and therefore could not be characterized in the mixed powder of this prescription.

[0095] All water used in this invention is purified water.

[0096] As described above, this invention provides a method for determining the content of eight chemical components in Guipi Pill and its fingerprint chromatographic method. The method employs a UHPLC ultra-high performance liquid chromatograph supplemented with a DAD detector, using a segmented wavelength detection method to determine the content of eight chemical components in the Guipi Pill mixed powder, including ferulic acid, Siberian polygalactosyl A5, Siberian polygalactosyl A6, 3,6'-disinyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costus lactone, and dehydrocostus lactone, and establishes a fingerprint chromatographic representation of Guipi Pill.

[0097] The methodological validation results of this content determination method showed that the extraction conditions, linear range, repeatability, precision, stability, and recovery of this method were investigated. The results showed that the method is stable and reliable. The eight active ingredients in Guipi Pill (Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costus lactone, and dehydrocostus lactone) showed good relationships within their respective linear ranges, with average recoveries of 95.72%–102.39% and RSD% of 1.65%–4.67%.

[0098] The fingerprint spectrum of Guipi Pills includes 35 characteristic peaks. Twenty batches of Guipi Pills were analyzed, and their characteristic fingerprint spectra were established and similarity was evaluated. The similarity of the fingerprint spectra of the 20 batches of Guipi Pills was all above 0.981, providing a reference for the scientific and comprehensive control of the quality of Guipi Pills.

[0099] The method for determining the content of eight chemical components in Guipi Pill and its fingerprinting method provided by this invention are stable and reliable, and can comprehensively control the quality of Guipi Pill, providing a scientific basis for its quality evaluation and standard establishment. Attached Figure Description

[0100] Figure 1 The chromatograms shown are those of the reference standard and sample of the eight chemical components in Guipi Pill of the present invention, wherein A: chromatogram of the reference standard; B: chromatogram of the sample; 1: Siberian polygalactosyl A5; 2: Siberian polygalactosyl A6; 3: ferulic acid; 4: 3,6'-disinozylosyl sucrose; 5: Polygalactosyl A; 6: Polygalactosyl C; 7: Costus lactone; 8: Dehydrocostus lactone.

[0101] Figure 2 The UHPLC reference fingerprint of the Guipi Pill sample of the present invention is shown, with 35 characteristic peaks, including: 2: Siberian polygalactose A5; 3: Siberian polygalactose A6; 4: ferulic acid; 11: 3,6'-disinoyl sucrose; 13: polygalactoside A; 14: polygalactoside C; 27: costus lactone; 28: dehydrocostus lactone.

[0102] Figure 3 The chromatogram shown is a fingerprint chromatogram of the Guipi Pill sample of the present invention, showing the characteristic peaks and the peaks belonging to the medicinal materials, wherein: 2: Siberian polygalactosyl A5; 3: Siberian polygalactosyl A6; 4: ferulic acid; 11: 3,6'-disinoyl sucrose; 13: polygalactosyl A; 14: polygalactosyl C; 27: costus lactone; 28: dehydrocostus lactone.

[0103] Figure 4The image shows the UHPLC superimposed fingerprint chromatograms of 20 batches of Guipi Pill samples from the present invention, wherein S1 to S20 are 20 batches of Guipi Pill samples. Detailed Implementation

[0104] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0105] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed 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 standards: Siberian polygalactosyl sucrose A5 reference standard (batch number: 6954, purity ≥98%, Shanghai Shidander Standards & Technical Services Co., Ltd.), Siberian polygalactosyl sucrose A6 reference standard (batch number: 7920, purity ≥98%, Shanghai Shidander Standards & Technical Services Co., Ltd.), ferulic acid (batch number: 110773-200611, China National Institutes for Food and Drug Control), 3,6'-disinoyl sucrose (batch number: 8520, purity ≥97.90%, Shanghai Shidander Standards & Technical Services Co., Ltd.) Polygalactoside A (batch number: 12728, purity ≥98%, Shanghai Shidande Standard Technical Service Co., Ltd.), Polygalactoside C (batch number: 19051525, purity ≥98%, Shanghai Tongtian Biotechnology Co., Ltd.), Costunolide (batch number: 111524-201911, purity ≥99.90%, China National Institutes for Food and Drug Control), and Dehydrocostunolide (batch number: 111524-201911, purity ≥99.50%, China National Institutes for Food and Drug Control).

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

[0110] The medicinal materials are: Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube. These are all commonly used medicinal materials and are provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

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

[0112] 2. Instruments

[0113] Agilent 1290 Infinity II UHPLC system (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 0.001 g electronic balance and XSR205DU / A 0.001 g electronic balance (METTLER TOLEDO Instruments Shanghai Co., Ltd.); DHG-9123A electric drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.); HWS-26 electric thermostatic water bath (Shanghai Yiheng Scientific Instruments Co., Ltd.); KQ-800DE ultrasonic cleaner (Kunshan Ultrasonic Instruments 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 the test solution: Take 1.0 g of powder from batch 230501 of Guipi Pill sample, place it in a 100 mL stoppered conical flask, accurately add 20 mL of 70% methanol, weigh it, and extract it by ultrasonication (power 300 W, frequency 40 kHz) for 30 minutes. Let it stand and cool to room temperature, weigh it again, replenish the lost weight with 70% methanol, shake well, filter, take the supernatant, filter it through a 0.22 μm microporous membrane, and take the filtrate to obtain test solution 1#.

[0117] Preparation of reference solutions: Accurately weigh the reference standards of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl glycoside A, polygalactosyl glycoside C, costus lactone, and dehydrocostus lactone, respectively, and place them in the same 50 mL volumetric flask. Dissolve and dilute to the mark with methanol, shake well, and prepare the reference standard stock solution. The reference stock solution contained the following components: Siberian polygalactosyl A5 (0.2772 mg / mL); Siberian polygalactosyl A6 (0.2348 mg / mL); ferulic acid (0.4300 mg / mL); 3,6'-disinozylosyl sucrose (0.5724 mg / mL); polygalactoside A (0.1724 mg / mL); polygalactoside C (0.2992 mg / mL); costunolide (0.2576 mg / mL); and dehydrocostunolide (0.2876 mg / mL). Store at 4°C protected from light for later use.

[0118] The reference standard stock solution was then diluted with 70% methanol to prepare a series of reference standard solutions of different concentrations. In a series of reference solutions of different concentrations, the contents of Siberian polygalactosyl A5 ranged from 1.11 to 277.20 μg / mL; the contents of Siberian polygalactosyl A6 ranged from 0.94 to 234.80 μg / mL; the contents of ferulic acid ranged from 0.65 to 430.00 μg / mL; the contents of 3,6'-disinozinol sucrose ranged from 0.86 to 572.40 μg / mL; the contents of polygalactosyl A ranged from 0.69 to 172.40 μg / mL; the contents of polygalactosyl C ranged from 1.20 to 299.20 μg / mL; the contents of costunolide ranged from 2.60 to 257.60 μg / mL; and the contents of dehydrocostunolide ranged from 2.90 to 287.60 μg / mL.

[0119] 2. Chromatographic conditions

[0120] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) were as follows: the column was a Waters UPLC Cortecs C1000 HPLC system. 18 The chromatographic column was 2.1 mm × 100 mm, 1.6 μm; the detector was a photodiode array detector (DAD); the column temperature was 30 °C; the injection volume was 2 μL; and the flow rate was 0.6 mL / min.

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

[0122] 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 85 min; gradient elution was used.

[0123] The specific procedure for gradient elution is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0137] 3. Measurement

[0138] Using the external standard method, a series of reference solutions of different volumes were transferred to prepare a series of solutions of different concentrations. These solutions were then analyzed using ultra-high performance liquid chromatography (UHPLC) to plot a standard curve. The obtained test solution was then analyzed using UHPLC, and the analytical results were substituted into the standard curve to obtain the content of the eight components in the test solution.

[0139] Specifically, a series of reference solutions of different volumes were transferred and prepared into solutions of different concentrations. These solutions were then analyzed using ultra-high performance liquid chromatography (UHPLC) to obtain the linear relationship between the concentration and peak area of ​​the eight components in the reference solutions. A standard working curve was plotted, with the peak area of ​​each component corresponding to its concentration, and the regression equation for each standard working curve was calculated. The test solution was then analyzed using UHPLC. The peak areas of the eight components in the test solution were substituted into the regression equations of the respective standard working curves to calculate the content of the corresponding components. See the chromatograms below. Figure 1 .

[0140] Example 2

[0141] 1. Sample pretreatment

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

[0143] Preparation of reference solutions: Accurately weigh the reference standards of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl glycoside A, polygalactosyl glycoside C, costunolide, and dehydrocostunolide, and place them in the same 50 mL volumetric flask. Dissolve and dilute to the mark with methanol, shake well, and prepare a reference standard stock solution. Then dilute with 68% methanol to prepare a series of reference standard solutions of different concentrations. The concentration range of the reference standard stock solution and the series of reference standard solutions of different concentrations is the same as in step 1 of Example 1.

[0144] 2. Chromatographic conditions

[0145] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) were as follows: the column was a Waters UPLC Cortecs C1000 HPLC system. 18 The chromatographic column was 2.1 mm × 100 mm, 1.6 μm; the detector was a photodiode array detector (DAD); the column temperature was 32 °C; the injection volume was 1 μL; and the flow rate was 0.4 mL / min.

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

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

[0148] The specific procedure for gradient elution is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0162] 3. Measurement

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

[0164] Example 3

[0165] 1. Sample pretreatment

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

[0167] Preparation of reference solutions: Accurately weigh the reference standards of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl glycoside A, polygalactosyl glycoside C, costunolide, and dehydrocostunolide, and place them in the same 50 mL volumetric flask. Dissolve and dilute to the mark with methanol, shake well, and prepare a reference standard stock solution. Then, dilute again with 72% methanol to prepare a series of reference standard solutions of different concentrations. The concentration range of the reference standard stock solution and the series of reference standard solutions of different concentrations is the same as in step 1 of Example 1.

[0168] 2. Chromatographic conditions

[0169] The chromatographic conditions for ultra-high performance liquid chromatography (UHPLC) were as follows: the column was a Waters UPLC Cortecs C1000 HPLC system. 18 The chromatographic column was 2.1 mm × 100 mm, 1.6 μm; the detector was a photodiode array detector (DAD); the column temperature was 28 °C; the injection volume was 3 μL; and the flow rate was 0.8 mL / min.

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

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

[0172] The specific procedure for gradient elution is as follows:

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

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

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

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

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

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

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

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

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

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

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

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

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

[0186] 3. Measurement

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

[0188] Example 4

[0189] Accurately weigh the reference standards of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosin A, polygalactosin C, costunolide, and dehydrocostunolide, and place them in the same 50 mL volumetric flask. Dissolve and dilute to the mark with methanol, shake well, and prepare a reference standard stock solution. The content of Siberian polygalactosyl A5 in the reference standard stock solution is 0.2772 mg / mL; the content of Siberian polygalactosyl A6 is 0.2348 mg / mL; the content of ferulic acid is 0.4300 mg / mL; the content of 3,6'-disinyl sucrose is 0.5724 mg / mL; the content of polygalactosin A is 0.1724 mg / mL; the content of polygalactosin C is 0.2992 mg / mL; and the content of costunolide is 0.2576 mg / mL. The reference standard stock solution was then diluted with 70% methanol to prepare a series of reference standard solutions of different concentrations.

[0190] In a series of reference solutions of different concentrations, the contents of Siberian polygalactosyl A5 ranged from 1.11 to 277.20 μg / mL; the contents of Siberian polygalactosyl A6 ranged from 0.94 to 234.80 μg / mL; the contents of ferulic acid ranged from 0.65 to 430.00 μg / mL; the contents of 3,6'-disinozinol sucrose ranged from 0.86 to 572.40 μg / mL; the contents of polygalactosyl A ranged from 0.69 to 172.40 μg / mL; the contents of polygalactosyl C ranged from 1.20 to 299.20 μg / mL; the contents of costunolide ranged from 2.60 to 257.60 μg / mL; and the contents of dehydrocostunolide ranged from 2.90 to 287.60 μg / mL.

[0191] The sample was injected at 2 μL under the chromatographic conditions described in step 2 of Example 1. A standard curve was plotted with peak area (Y) on the ordinate and concentration (X) on the abscissa, and regression calculations were performed. The results showed good linearity among the components. The sample was then stepwise diluted with 70% methanol, and 10 μL was taken for determination. The limit of quantitation (LOQ) was set at the concentration of the reference standard with a peak area signal-to-noise ratio of 10 (S / N = 10), and the limit of detection (LOD) was set at the concentration of the reference standard with a peak area signal-to-noise ratio of 3 (S / N = 3). The regression equation and the LQ and LOD data are shown in Table 3.

[0192] Table 3

[0193]

[0194] Example 5

[0195] 1. Precision

[0196] A sample of Guipi Pills from batch 230501 was taken, and a test solution was prepared according to step 1 in Example 1. The solution was then tested according to step 2 in Example 1. Six consecutive injections were performed for analysis, and the peak areas of each component were recorded. The RSD% of the eight index components was calculated. The results showed that the RSD% of the eight index components were all ≤2.0%, indicating that the instrument precision was good.

[0197] 2. Stability

[0198] A sample of Guipi Pills from batch 230501 was taken, and one test solution was prepared according to step 1 in Example 1. The solution was then tested according to step 2 in Example 1, with injections at 0, 3, 6, 9, 12, 24, and 36 hours. Chromatograms were recorded, peak areas were measured, and the RSD of the peak areas for the eight indicator components was calculated. The results showed that the RSD% of the peak areas for each indicator component was ≤3.0%, indicating that the sample solution was stable within 36 hours.

[0199] 4. Repeatability

[0200] Sample 230501 of Guipi Pills was taken, and six test solutions were prepared according to step 1 in Example 1. Six solutions were accurately weighed and tested according to step 2 in Example 1. Chromatograms were recorded, and the peak areas of each component were recorded. The content (mg / ml) of the eight indicator components was calculated. The results showed that the RSD% of each indicator component was ≤3.0%, indicating good repeatability of the method.

[0201] 2. Recovery rate

[0202] Nine samples of Guipi Pill from batch 230501, each approximately 0.1g, were accurately weighed and added to reference solutions of low, medium, and high concentrations (equivalent to 50%, 100%, and 150% of the original mass fraction, respectively). Three samples of each concentration were taken and the test solution was prepared according to step 1 in Example 1. The test solution was then analyzed according to step 2 in Example 1, and the chromatograms were recorded. The recovery rate and RSD% of each component were calculated based on the measured and added amounts. The results are shown in Table 4. Table 4 shows that the average recoveries of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costunolide, and dehydrocostunolide were 95.72%–102.39%, and the RSD% were 1.65%–4.67%, indicating good accuracy of the method.

[0203] Table 4

[0204]

[0205]

[0206] Example 6

[0207] Twenty batches of Guipi Pill samples (batch numbers: 230501~230520) were taken, and test solutions were prepared according to step 1 in Example 1. The samples were then injected and analyzed according to the chromatographic conditions in step 2 in Example 1. The chromatograms were recorded, and the contents of eight index components in Guipi Pill were calculated using 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 is the same as that in step 1 of Example 1.

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

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

[0215] 3. Determination

[0216] The ultra - performance liquid chromatography with the chromatographic conditions in step 2 above was used to separately determine the test solution and the reference solution in step 1 above, obtaining the fingerprint of the test solution and the fingerprint of the reference solution. Among them, the fingerprint of the test solution should be compared with the fingerprint of the reference solution. According to the known characteristic peaks in the fingerprint of the reference solution, the corresponding characteristic peaks in the fingerprint of the test solution are identified through the relative retention time, so as to locate the target components in the fingerprint of the test solution and obtain the fingerprint of Guipi Pills.

[0217] Example 8

[0218] The detection method of the fingerprint of Guipi Pills established in Example 7 above was used to detect 20 batches of Guipi Pills, obtaining the fingerprint of the test solution and the fingerprint of the reference solution. The fingerprint data of the obtained test samples were imported into the software "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicines" 2012 version released by the Pharmacopoeia Commission of China. Taking sample 230501 (S1) as the reference chromatogram, after multi - point calibration, Mark peak matching was carried out, and a time window width of 0.2 was selected for the matching of common peaks in the fingerprint to generate the fingerprint and the reference fingerprint. The fingerprint of the test sample was compared with the reference fingerprint of Guipi Pills obtained under the same fingerprint detection conditions, and the similarity of the fingerprint of each batch of Guipi Pills was calculated. Taking peak 11 (3,6'-diacylsucrose) as the reference peak S, 35 common peaks were calibrated, as shown in Figure 2 . The results of comparing the similarity between the fingerprints of 20 batches of test samples and the reference fingerprint showed that the similarities of S1 - S20 were all greater than 0.981, as shown in Table 6 specifically, indicating that the overall quality of each batch of samples was relatively stable. The superimposed fingerprint diagram of 20 batches of Guipi Pills is shown in Figure 4 . It shows that the quality of different batches of Guipi Pills is relatively close, and the established fingerprint is representative and can be used for the quality control of Guipi Pills.

[0219] Table 6

[0220]

[0221] Example 9

[0222] The fingerprint spectrum detection method for Guipi Pill established in Example 7 above was used to detect Guipi Pill. Based on the fingerprint spectra of the test solution and the reference solution, 35 common fingerprint peaks were identified. Through comparison with the reference solution, 8 components were identified. Figure 2 As shown, peak 11 has good separation and a moderate peak area, so it is selected as the reference peak (S peak). The relative retention times of the other peaks are calculated. Therefore, using peak 11 as the reference peak (S peak, relative retention time of 1.0000), 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 17: 1.64; Peak 18: 1.70; Peak 19: 1.74; Peak 20: 1.76; Peak 21: 1.78; Peak 22: 1.80; Peak 23: 1.97; Peak 24: 2.05; Peak 25: 2.11; Peak 26: 2.18; Peak 27: 2.37; Peak 28: 2.50; Peak 29: 2.79; Peak 30: 2.81; Peak 31: 2.82; Peak 32: 2.83; Peak 33: 2.89; Peak 34: 2.91; Peak 35: 2.96.

[0223] Through with Figure 2 By comparison, peak 2 was identified as the fingerprint peak of Siberian polygalactosyl A5; peak 3 as the fingerprint peak of Siberian polygalactosyl A6; peak 4 as the fingerprint peak of ferulic acid; peak 11 as the fingerprint peak of 3,6'-disinozylosyl sucrose; peak 13 as the fingerprint peak of polygalactosyl A; peak 14 as the fingerprint peak of polygalactosyl C; peak 27 as the fingerprint peak of costunolide; and peak 28 as the fingerprint peak of dehydrocostunolide.

[0224] Example 10

[0225] 1. Precision

[0226] Take the powder of Guipi Pill sample from batch 230501, accurately weigh it, and prepare one part of the test solution according to step 1 in Example 7. Perform the test according to step 2 in Example 7 above, inject the sample 6 times consecutively, record the chromatogram, and use peak 11 (3'6-diacylsucrose) as the reference peak. The relative retention time RSD% of the 35 common peaks is less than 0.56%, and the relative peak area RSD is less than 2.49%, indicating that the instrument has good precision.

[0227] 2. Repeatability

[0228] Sample 230501 of Guipi Pills was taken, and six test solutions were prepared in parallel according to step 1 in Example 7. The solutions were then tested according to step 2 in Example 7, and the chromatograms were recorded. Peak 11 (3'6-diacylsucrose) was used as the reference peak. The relative retention time RSD% of the 35 common peaks was less than 0.29%, and the relative peak area RSD was less than 2.60%. The results showed that the method had good repeatability.

[0229] 3. Stability

[0230] Take 230501 batch of Guipi Pills samples, prepare one test solution according to step 1 in Example 7, and perform detection according to step 2 in Example 7. Inject at 0, 3, 6, 9, 12, and 24 h, and record the chromatogram. Using peak 11 (3'6-diacylsucrose) as the reference peak, calculate the relative retention time RSD of 35 common peaks. All RSD of the relative peak area is less than 2.55%, and all RSD of the relative peak area is less than 3.01%, indicating that the test solution has good stability within 36 h.

[0231] Example 11

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

[0233] Eleven medicinal materials were taken as powder, namely Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube. The powders were then used in step 1 of Example 7 above to prepare 11 single-herb sample solutions.

[0234] Following step 2 of Example 7, the test solution and the 11 single-herb sample solutions were measured respectively, yielding the test solution and the 11 single-herb sample solutions. The attribution of the chromatographic peaks of each single-herb sample solution was confirmed by comparing them with the fingerprint chromatographic peaks of Guipi Pill, as detailed below. Figure 3 Table 2.

[0235] Depend on Figure 2 Table 2 shows that four common peaks (peaks 1, 12, 27, and 28) originate from *Saussurea costus*, while 17 common peaks (peaks 2, 3, 5-11, 12, 13, 14, and 18-22) originate from *Polygala tenuifolia*. Two common peaks (peaks 4 and 23) originate from *Angelica sinensis*. Peaks 10, 32, and 34 may originate from stir-fried *Ziziphus jujuba* seeds, and peaks 23, 25, and 26 may originate from *Glycyrrhiza uralensis*. This fingerprinting method can characterize the characteristic peaks of five medicinal materials in the prescription (*Angelica sinensis*, *Saussurea costus*, *Polygala tenuifolia*, stir-fried *Ziziphus jujuba* seeds, and *Glycyrrhiza uralensis*). In other words, the characteristic peaks of five out of the eleven medicinal materials are well represented in this fingerprint and their attribution is confirmed.

[0236] In summary, the present invention provides a method for determining the content of eight chemical components in Guipi Pills and its fingerprint spectroscopy method. This method enables quantitative analysis of the eight components in Guipi Pills and establishes a fingerprint spectroscopy for Guipi Pills, providing a reference for the scientific and comprehensive control of Guipi Pills quality. Therefore, the present invention overcomes the various shortcomings of existing technologies and has high industrial application value.

[0237] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A method for determining the chemical content of eight components in Guipi Pill, comprising the following steps: 1) Preparation of test solution: Dissolve the Guipi Pill sample in solvent, extract by ultrasound, cool, add weight, shake well, filter and take the supernatant, filter again, and take the filtrate to obtain the test solution. 2) Preparation of reference solution: Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl a, polygalactosyl glycoside C, costus lactone and dehydrocostus lactone reference standards are dissolved in solvent and then diluted to volume to obtain the reference solution. 3) Determination: The test solution in step 1) and the reference solution in step 2) were determined by ultra-high performance liquid chromatography, and the contents of Siberian polygalactosyl A5, Siberian polygalactosyl A6, ferulic acid, 3,6'-disinyl sucrose, polygalactosyl A, polygalactosyl C, costus lactone and dehydrocostus lactone in the test solution were calculated by external standard method. In step 1), the solvent is an aqueous solution containing 55-85% methanol by volume. In step 3), the detector is a photodiode array detector; the column temperature is 25-35℃; the injection volume is 1-5 μL; the flow rate is 0.2-1 mL / min; mobile phase A is acetonitrile, and mobile phase B is 0.09-0.11% phosphoric acid aqueous solution; the chromatographic column is a WatersUPLC Cortecs C1000. 18 The chromatographic column is 2.1 mm × 100 mm with a diameter of 1.6 μm. The detection wavelength varies with the time of the gradient elution program, specifically: 325-335 nm for 0-53 min and 215-225 nm for 53-85 min. In the ultra-high performance liquid chromatography method, gradient elution is used, and the analysis time is 85 min. The specific procedure for gradient elution is as follows: 0-5 min, the volume ratio of phase A: phase B is 4~6:94~96-7~9:91~93; 5-8 min, the volume ratio of phase A to phase B is 7~9:91~93-7~9:91~93; 8-9 min, the volume ratio of phase A: phase B is 7~9:91~93-10~11:89~90; 9-17 min, the volume ratio of phase A to phase B is 10~11:89~90-10~11:89~90; 17-18 min, the volume ratio of phase A to phase B is 10~11:89~90 -12~14:86~88; 18-30 min, the volume ratio of phase A to phase B is 12~14:86~88 - 12~14:86~88; 30-35 min, the volume ratio of phase A to phase B is 12~14:86~88-19~21:79~81; 35-45 min, the volume ratio of phase A to phase B is 19~21:79~81-19~21:79~81; 45-50 min, the volume ratio of phase A to phase B is 19~21:79~81-28~30:70~72; 50-70 min, A phase:B phase volume ratio is 28~30:70~72; 70-75 min, the volume ratio of phase A to phase B is 28-30:70-72-44-46:54-56; 75-80 min, the volume ratio of phase A to phase B is 44~46:54~56-79~81:19~21; 80-85 min, the volume ratio of phase A to phase B is 79~81:19~21-79~81:19~21.

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

3. The method for determining the chemical content of eight components in Guipi Pill according to claim 1, characterized in that, In step 2), the reference solution can be prepared directly as a mixed reference solution, or it can be prepared by first adding a first solvent to prepare a reference stock solution and then adding a second solvent for stepwise dilution.

4. The method for determining the chemical content of eight components in Guipi Pill according to claim 3, characterized in that, Includes 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 of Siberian polygalactosyl A5 in the reference solution ranges from 1.11 to 277.20 μg / mL; the content of Siberian polygalactosyl A6 ranges from 0.94 to 234.80 μg / mL; the content of ferulic acid ranges from 0.65 to 430.00 μg / mL; the content of 3,6'-disinozylosyl sucrose ranges from 0.86 to 572.40 μg / mL; the content of polygalactosyl A ranges from 0.69 to 172.40 μg / mL; the content of polygalactosyl C ranges from 1.20 to 299.20 μg / mL; the content of costunolide ranges from 2.60 to 257.60 μg / mL; and the content of dehydrocostunolide ranges from 2.90 to 287.60 μg / mL.

5. A method for detecting the fingerprint spectrum of Guipi Pill, comprising the following steps: A) Preparation of the test solution: Same as step 1) of the method for determining the chemical content of the eight components in Guipi Pill according to any one of claims 1-4; B) Preparation of reference solution: Same as step 2) of the method for determining the chemical content of the eight components in Guipi Pill according to any one of claims 1-4; C) Determination: Using the same chromatographic conditions as step 3) of the method for determining the chemical content of the eight components in Guipi Pill according to any one of claims 1-4, the test solution in step A) and the reference solution in step B) are determined by ultra-high performance liquid chromatography, respectively, to obtain the fingerprint spectrum of the test solution and the fingerprint spectrum of the reference solution. The fingerprint spectrum of the test solution is compared with the fingerprint spectrum of the reference solution, and the index components in the fingerprint spectrum of the test solution are assigned and located to obtain the fingerprint spectrum of Guipi Pill.

6. A quality testing method for Guipi Pills, comprising obtaining a fingerprint spectrum of Guipi Pills using the fingerprint spectrum detection method of Guipi Pills according to claim 5, and comparing the similarity of the obtained fingerprint spectrum of Guipi Pills with a control fingerprint spectrum of Guipi Pills obtained under the same fingerprint spectrum detection conditions.

7. The quality testing method for Guipi Pills according to claim 6, characterized in that, A control fingerprint spectrum of Guipi Pill was obtained under the same conditions as the detection method for the fingerprint spectrum of Guipi Pill according to claim 5. The control fingerprint spectrum of Guipi Pill includes 35 common fingerprint peaks, with peak 11 as the reference peak S, and a relative retention time of 1.0000. The relative retention times of the other 34 peaks are as follows: 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±0.0007, peak 6±0.0007, peak 7±0.0007, peak 8±0.0007, peak 9±0.0007, peak 10±0.0007, peak 11±0.0007, peak 12±0.0007, peak 13±0.0007, peak 14±0.0007, peak 15±0.0007, peak 16±0.0007, peak 17±0.0007, peak 18±0.0007, peak 19 ... Peak 5: 0.0021; 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: 2.18±0.0453; Peak 27: 2.37±0.0470; Peak 28: 2.50±0.0496; Peak 29: 2.79±0.0658; Peak 30: 2.81±0.0662; Peak 31: 2.82±0.0663; Peak 32: 2.83±0.0666; Peak 33: 2.89±0.0677; Peak 34: 2.91±0.0679; Peak 35: 2.96±0.0693.

8. A method for screening the fingerprint spectra of multiple medicinal materials in Guipi Pill, comprising the following steps: a) Preparation of single-herb sample solutions: Take one or more of the 11 herbs in Guipi Pill, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube, and prepare them according to step A) of the detection method of fingerprint spectrum of Guipi Pill as described in claim 5, and obtain at least one single-herb sample solution respectively. b) Preparation of negative sample solutions: Eleven medicinal materials, including Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube, were prepared according to step A) of the fingerprint spectrum detection method for Guipi Pills as described in claim 5, to obtain negative sample solutions for each of the following deficiencies: Codonopsis pilosula, stir-fried Atractylodes macrocephala, processed Astragalus membranaceus, processed Glycyrrhiza uralensis, Poria cocos, processed Polygala tenuifolia, stir-fried Ziziphus jujuba var. spinosa, longan pulp, Angelica sinensis, Aucklandia lappa, and jujube. c) Determination: The fingerprint spectra of the single herb sample solution in step a) and the negative sample solution in step b) were determined by ultra-high performance liquid chromatography under the same chromatographic conditions as in step c) of the detection method of Guipi Pill fingerprint spectrum according to claim 5. d) Obtaining the control fingerprint spectrum: The test solution prepared according to step A) of the detection method of the fingerprint spectrum of Guipi Pill according to claim 5 is used to obtain the control fingerprint spectrum of Guipi Pill by the same step C) as the detection method of the fingerprint spectrum of Guipi Pill according to claim 5. e) Quality testing: The fingerprint spectra of single herbal sample solutions and negative sample solutions are compared with the control fingerprint spectra of Guipi Pill. By using the relative retention time, the corresponding characteristic peaks of the single herbal sample solutions in the control fingerprint spectra of Guipi Pill are identified, thereby assigning and locating the characteristic peaks in the fingerprint spectra of the single herbal sample solutions.

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