Fingerprint spectrum of Buzhong Yiqi Pills and method for determining the contents of multiple components

Abstract

The present invention provides a method for determining the contents of seven components in Buzhong Yiqi Pills. The present invention also provides a method for detecting the fingerprint of Buzhong Yiqi Pills. The present invention further provides the use of the above-mentioned method for detecting the fingerprint of Buzhong Yiqi Pills in the quality detection of components in Buzhong Yiqi Pills and a quality detection method thereof. The present invention further provides a method for screening fingerprints of multiple medicinal materials in Buzhong Yiqi Pills. The present invention provides a method for determining the fingerprint of Buzhong Yiqi Pills and the contents of multiple components, which can quantitatively analyze the seven chemical components in Buzhong Yiqi Pills, establish a high-performance liquid fingerprint of Buzhong Yiqi Pills, and confirm the attribution of the chromatographic peaks of each single medicinal material in Buzhong Yiqi Pills, play a role in both qualitative and quantitative quality control, and provide a scientific basis for the quality control of Buzhong Yiqi Pills.

Description

Technical Field

[0001] The present invention belongs to the technical field of detection of components of traditional Chinese medicine, and relates to a fingerprint spectrum of Buzhong Yiqi Pills and a method for determining the contents of multiple components, and specifically relates to a method for determining the contents of seven indicator components in Buzhong Yiqi Pills: calycosin isoflavone glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, and their fingerprint spectrum. Background Art

[0002] Buzhong Yiqi Pills are made by sieving and mixing eight finely ground Chinese medicinal herbs: astragalus, licorice, codonopsis, atractylodes, angelica, tangerine peel, cimicifuga, and bupleurum. The mixture is then coated with refined honey and water, then dried to form water-honeyed pills. They are used to treat diarrhea, anal prolapse, and uterine prolapse caused by spleen and stomach deficiency and qi deficiency. Symptoms include fatigue, loss of appetite, abdominal distension, loose stools, chronic diarrhea, anal prolapse, and uterine prolapse.

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

[0004] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method for determining the fingerprint of Buzhong Yiqi Pills and the content of multiple components. By using optimized pretreatment conditions and high performance liquid chromatography, a method for determining the contents of seven components in Buzhong Yiqi Pills and a fingerprint of Buzhong Yiqi Pills were established, thereby realizing the detection of the chemical components of the entire Buzhong Yiqi Pill formula, reflecting the current status of each component in Buzhong Yiqi Pills in a more comprehensive manner, and providing a reference basis for the overall control and evaluation of the quality of Buzhong Yiqi Pills.

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

[0006] 1) Preparation of test solution: Dissolve the Buzhong Yiqi Pill sample in methanol, extract by ultrasonication, let it stand and cool, filter, and obtain the test solution by taking the filtrate;

[0007] 2) Preparation of reference solution: Dissolve the reference substances of calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin in methanol by ultrasonication and then dilute to volume to prepare the reference solution;

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

[0009] Preferably, in step 1), the Buzhong Yiqi Pills are a Buzhong Yiqi Pills powder sample obtained by crushing and sieving the Buzhong Yiqi Pills.

[0010] More preferably, the screening is through a No. 4 sieve.

[0011] Preferably, in step 1), the ratio of the weight (g) of the Buzhong Yiqi Pills sample added to the volume (mL) of the methanol added is 1:18-22, specifically 1:18-20, 1:20-22, preferably 1:20.

[0012] Preferably, in step 1), the Buzhong Yiqi Pills sample needs to be precisely weighed after adding the solvent.

[0013] Preferably, in step 1), the ultrasonic extraction time is 25-35 min, specifically 25-30 min, 30-35 min, preferably 30 min.

[0014] Preferably, in step 1), the power of the ultrasonic extraction is 80-120 W, specifically 80-100 W, 100-120 W, preferably 100 W; the frequency of the ultrasonic extraction is 35-45 kHz, specifically 35-40 kHz, 40-45 kHz, preferably 40 kHz.

[0015] Preferably, in step 1), the cooling by standing is cooling to room temperature, which is 20-30°C.

[0016] Preferably, in step 1), the mixture needs to be weighed again after standing and cooling, and methanol is used to make up for the weight loss.

[0017] Preferably, in step 1), the filtration is to take the supernatant through a filtration membrane, discard the initial filtrate, and then take the subsequent filtrate.

[0018] More preferably, the filter membrane is a 0.45 μm filter membrane.

[0019] Preferably, in step 2), the reference substance solution is prepared by first adding methanol to prepare a reference substance stock solution, and then adding methanol for stepwise dilution.

[0020] More preferably, the reference substance stock solution needs to be refrigerated and kept away from light for future use.

[0021] Preferably, in step 2), the ultrasonic dissolution time is 25-35 min, specifically 25-30 min, 30-35 min, preferably 30 min.

[0022] Preferably, in step 2), the power of the ultrasonic dissolution is 80-120 W, specifically 80-100 W, 100-120 W, preferably 100 W; the frequency of the ultrasonic dissolution is 35-45 kHz, specifically 35-40 kHz, 40-45 kHz, preferably 40 kHz.

[0023] Preferably, in step 2), the CAS number of the calycosin isoflavone glucoside is 20633-67-4, the CAS number of the liquiritin is 551-15-5, the CAS number of ferulic acid is 1135-24-6, the CAS number of ammonium glycyrrhizate is 53956-04-0, the CAS number of formononetin is 485-72-3, the CAS number of nobiletin is 478-01-3, and the CAS number of tangeretin is 481-53-8.

[0024] Preferably, in step 2), the content of calycosin glucoside in the reference solution is in the range of 4.74 to 75.84 μg / mL, the content of liquiritin is in the range of 183.525 to 978.8 μg / mL, the content of ferulic acid is in the range of 21.045 to 112.24 μg / mL, the content of ammonium glycyrrhizate is in the range of 241.1 to 1261.4 μg / mL, the content of formononetin is in the range of 8.325 to 44.4 μg / mL, the content of nobiletin is in the range of 122.85 to 655.2 μg / mL, and the content of tangeretin is in the range of 81.75 to 436 μg / mL.

[0025] Preferably, in step 3), the chromatographic column used in the HPLC method is a C18 chromatographic column. More preferably, the chromatographic column used in the HPLC method is an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm) with octadecylsilane bonded silica gel as the filler.

[0026] Preferably, in step 3), the detector in the high performance liquid chromatography is a photodiode array detector (DAD).

[0027] Preferably, in step 3), in the high performance liquid chromatography method, the column temperature is 32-38°C, specifically 32-35°C, 35-38°C, preferably 35°C.

[0028] Preferably, in step 3), the injection volume in the HPLC method is 8-12 μL. More preferably, the injection volume in the HPLC method is 10 μL.

[0029] Preferably, in step 3), the high performance liquid chromatography method is analyzed by flow rate switching, wherein:

[0030] When the retention time is 0-17 min, the flow rate is 0.9-1.1 mL / min, specifically 0.9-1.0 mL / min, 1.0-1.1 mL / min, preferably 1.0 mL / min;

[0031] When the retention time is 17-40 min, the flow rate is 0.75-0.85 mL / min, specifically 0.75-0.8 mL / min, 0.8-0.85 mL / min, preferably 0.8 mL / min.

[0032] Preferably, in step 3), the high performance liquid chromatography method is analyzed by wavelength switching, wherein:

[0033] When the retention time is 0-31 min, the detection wavelength is 240-250 nm, specifically 240-245 nm, 245-250 nm, 243-247 nm, preferably 245 nm;

[0034] When the retention time is 31-33 min, the detection wavelength is 270-280 nm, specifically 270-275 nm, 275-280 nm, 273-277 nm, preferably 275 nm;

[0035] When the retention time is 33-40 min, the detection wavelength is 240-250 nm, specifically 240-245 nm, 245-250 nm, 243-247 nm, preferably 245 nm.

[0036] The wavelength switching method is adopted because the Buzhong Yiqi Pills powder contains many compounds and the absorption wavelengths are complex. The commonly used single-wavelength fingerprint spectrum cannot meet the detection requirements, and multiple wavelength detection easily leads to data processing complexity. The spectrum signal intensity and separation at different absorption wavelengths were investigated. According to the multi-wavelength detection results, the multi-wavelength switching method was adopted. This method can improve the sensitivity of the detector and increase the accuracy of the measurement.

[0037] Preferably, in step 3), in the high performance liquid chromatography method, the mobile phase is acetonitrile-0.05-0.2% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.05-0.2% phosphoric acid aqueous solution; the analysis time is 40 min; and gradient elution is used.

[0038] More preferably, in the high performance liquid chromatography method, the mobile phase is acetonitrile-0.1% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.1% phosphoric acid aqueous solution; the analysis time is 40 minutes; and gradient elution is used.

[0039] The 0.05-0.2% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.05-0.2%. The 0.1% phosphoric acid aqueous solution is a phosphoric acid aqueous solution with a volume percentage of 0.1%.

[0040] More preferably, as shown in Table 1, the specific procedure of the gradient elution is:

[0041] 0-10 min, volume ratio of phase A:phase B was 10:90-20:80;

[0042] 10-17 min, the volume ratio of phase A:phase B is 20:80-27:73;

[0043] 17-20 min, the volume ratio of phase A:phase B is 27:73-30:70;

[0044] 20-21 min, the volume ratio of phase A:phase B is 30:70-36:64;

[0045] 21-30 min, the volume ratio of phase A:phase B is 36:64-65:35;

[0046] 30-40min, the volume ratio of phase A:phase B is 65:35-98:2.

[0047] Table 1 Gradient elution program

[0048]

[0049] Preferably, in step 3), the external standard method refers to: respectively taking a series of different volumes of the reference solution of step 2), making a series of solutions of different concentrations, adopting high performance liquid chromatography to analyze the sample, obtaining the linear relationship between the content and peak area of the 7 components in the reference solution, corresponding the chromatographic peak area of each component to its corresponding content, drawing the corresponding standard working curve, and calculating the regression equation of each standard working curve. The test solution is then detected by high performance liquid chromatography, and the chromatographic peak areas of the 7 components in the test solution are respectively substituted into the regression equation of each standard working curve to obtain the content of the corresponding component.

[0050] More preferably, in the standard working curve, the peak area of each component is used as the ordinate, and the content of each component in the reference solution is used as the abscissa.

[0051] A second aspect of the present invention provides a method for detecting the fingerprint of Buzhong Yiqi Pills, comprising the following steps:

[0052] A) Preparation of test solution: Same as step 1) for the determination of the seven components in Buzhong Yiqi Pills;

[0053] B) Preparation of reference solution: Same as step 2) of the method for determining the contents of the seven components in Buzhong Yiqi Pills;

[0054] C) Determination: Using the high performance liquid chromatography (HPLC) method under the same chromatographic conditions as step 3) of the method for determining the contents of the seven components in Buzhong Yiqi Pills, the test solution of step A) and the reference solution of step B) were respectively determined to obtain fingerprints of the test solution and the reference solution. The fingerprints of the test solution and the reference solution were compared, and the index components in the fingerprints of the test solution were attributed and located, thereby obtaining the fingerprint of Buzhong Yiqi Pills.

[0055] 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.

[0056] A third aspect of the present invention provides a method for detecting the fingerprint of Buzhong Yiqi Pills and its use in quality detection of ingredients in Buzhong Yiqi Pills.

[0057] The fourth aspect of the present invention provides a quality detection method for Buzhong Yiqi Pills, comprising obtaining a fingerprint of Buzhong Yiqi Pills using the aforementioned fingerprint detection method for Buzhong Yiqi Pills, and comparing the obtained fingerprint of Buzhong Yiqi Pills with a control fingerprint of Buzhong Yiqi Pills obtained under the same fingerprint detection conditions for similarity.

[0058] Preferably, when comparing the measured fingerprint of Buzhong Yiqi Pills with the reference fingerprint of Buzhong Yiqi Pills for similarity, the comparison is performed using the "Chinese Herbal Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software published by the State Pharmacopoeia Commission. More preferably, the similarity between the measured fingerprint of Buzhong Yiqi Pills and the reference fingerprint of Buzhong Yiqi Pills is greater than 0.990.

[0059] More preferably, when matching the common fingerprint peaks of the Buzhong Yiqi Pills fingerprint spectrum with the control fingerprint spectrum of the Buzhong Yiqi Pills, automatic full spectrum matching is performed with a time window width of 0.10 min, and the fingerprint spectrum and the control fingerprint spectrum are generated using the median method.

[0060] Preferably, the control fingerprint of Buzhong Yiqi Pills is obtained under the same conditions as the detection method of the fingerprint of Buzhong Yiqi Pills, and the control fingerprint of Buzhong Yiqi Pills includes 20 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 19 peaks are peak 1 (0.48±0.0003), peak 2 (0.49±0.0004), peak 3 (0.51±0.0003), peak 4 (0.54±0.0003), peak 5 (0.58±0.0004), peak 6 (0.64±0.0005), peak 7 (0.85 ±0.0004), peak 8 (0.89±0.0003), peak 9 (0.90±0.0002), peak 10 (0.97±0.0001), peak 12 (1.03±0.0002), peak 13 (1.05±0.0002), peak 14 (1.12±0.0001), peak 15 (1.14±0.0002), peak 16 (1.16±0.0002), peak 17 (1.20±0.0002), peak 18 (1.27±0.0000), peak 19 (1.28±0.0002), and peak 20 (1.33±0.0000).

[0061] The specific data of the control fingerprint of the Buzhong Yiqi Pills can be found in Figure 3 .

[0062] More preferably, the control fingerprint of the Buzhong Yiqi Pills is compared with the fingerprint of the reference solution, and peak No. 1 is located and determined to be the fingerprint peak of calycosin glucoside, peak No. 3 is the fingerprint peak of liquiritin, peak No. 4 is the fingerprint peak of ferulic acid, peak No. 11 is the fingerprint peak of ammonium glycyrrhizate, peak No. 13 is the fingerprint peak of formononetin, peak No. 15 is the fingerprint peak of nobiletin, and peak No. 17 is the fingerprint peak of tangeretin.

[0063] A fifth aspect of the present invention provides a method for screening fingerprints of multiple medicinal materials in Buzhong Yiqi Pills, comprising the following steps:

[0064] a) Preparation of single herbal sample solutions: Prepare any one or more of the eight herbal samples of Buzhong Yiqi Pills, including Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga, and Bupleurum, according to step A) of the Buzhong Yiqi Pills fingerprint detection method to obtain at least one single herbal sample solution;

[0065] b) Preparation of negative sample solutions: Eight medicinal material samples of Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga, and Bupleurum were respectively prepared without Tangerine Peel, Licorice, Astragalus, Cimicifuga, and Angelica, according to step A) of the detection method for the fingerprint of Buzhong Yiqi Pills, to obtain negative sample solutions lacking Tangerine Peel, negative sample solutions lacking Licorice, negative sample solutions lacking Astragalus, and negative sample solutions lacking Cimicifuga and Angelica, respectively;

[0066] c) Determination: using high performance liquid chromatography (HPLC) under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Buzhong Yiqi Pills, respectively determining the single medicinal material sample solution in step a), the negative sample solution lacking dried tangerine peel, the negative sample solution lacking licorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica in step b), to obtain fingerprints of the single medicinal material sample solution, the negative sample solution lacking dried tangerine peel, the negative sample solution lacking licorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica, respectively;

[0067] d) Obtaining the control fingerprint: The test solution prepared according to step A) of the detection method for the fingerprint of Buzhong Yiqi Pills is subjected to step C) of the same detection method for the fingerprint of Buzhong Yiqi Pills to obtain the control fingerprint of Buzhong Yiqi Pills;

[0068] e) Quality testing: The fingerprints of the single medicinal material sample solution, the negative sample solution lacking dried tangerine peel, the negative sample solution lacking licorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica are compared with the control fingerprint of Buzhong Yiqi Pills. The corresponding characteristic peaks of the single medicinal material sample solution in the control fingerprint of Buzhong Yiqi Pills are identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single medicinal material sample solution.

[0069] Preferably, in step a), the astragalus is the dried root of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge., of the legume family. The licorice is the dried root and rhizome of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., of the legume family. The codonopsis is the dried root of Codonopsis pilosula (Franch.) Nannf., Codonopsis pilosula Nannf. var. modesta (Nannf.) LTShen, or Codonopsis tangshen Oliv., of the Campanulaceae family. The atractylodes is the dried rhizome of Atractylodes macrocephala Koidz., of the Compositae family. The angelica root is the root of Angelica sinensis (Oliv.) Diels., a plant of the Umbelliferae family. The dried tangerine peel (Citri Reticulatae Pericarpium) is the dried mature pericarp of the Rutaceae plant Citrus aurantium and its cultivars. The coix seed is the dried rhizome of Cimici fuga heracleifolia Kom., Cimici fuga dahurica (Turcz) Maxim., or Cimici fuga foetida L., a plant of the Ranunculaceae family. The bupleurum root is the dried root of Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd., a plant of the Umbelliferae family.

[0070] Preferably, in step e), the present invention locates the attribution of characteristic peaks of the measured fingerprint of the single medicinal material sample solution and the control fingerprint of Buzhong Yiqi Pills, and uses the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software issued by the State Pharmacopoeia Commission for analysis and processing, and confirms the attribution of the characteristic peaks of each single medicinal material of Buzhong Yiqi Pills by the relative retention time of each characteristic peak on the control fingerprint of Buzhong Yiqi Pills. Specific results are shown in Figure 5 and Table 2.

[0071] Table 2 The characteristic peaks of each single herbal medicine in Buzhong Yiqi Pills

[0072]

[0073] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Astragalus sample solution includes 5 common fingerprint peaks, and the 5 common fingerprint peaks are Peak 1, Peak 8, Peak 9, Peak 13, and Peak 20.

[0074] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the licorice sample solution includes 7 common fingerprint peaks, and the 7 common fingerprint peaks are peak 2, peak 3, peak 8, peak 10, peak 11, peak 12, and peak 20.

[0075] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Codonopsis pilosula sample solution includes one common fingerprint peak, and the one common fingerprint peak is Peak 20.

[0076] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Atractylodes macrocephala sample solution includes one common fingerprint peak, and the one common fingerprint peak is peak 18.

[0077] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Angelica sinensis sample solution includes two common fingerprint peaks, and the two common fingerprint peaks are Peak 4 and Peak 20.

[0078] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the tangerine peel sample solution includes 4 common fingerprint peaks, and the 4 common fingerprint peaks are peak 6, peak 15, peak 17, and peak 20.

[0079] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Cimicifuga sample solution includes 5 common fingerprint peaks, and the 5 common fingerprint peaks are Peak 4, Peak 5, Peak 7, Peak 10, and Peak 20.

[0080] Preferably, in step e), in the single medicinal material sample solution, the fingerprint of the Bupleurum sample solution includes one common fingerprint peak, and the one common fingerprint peak is peak 20.

[0081] Among the above-mentioned common fingerprint peaks, peak 1 is determined to be the fingerprint peak of calycosin glucoside, peak 3 is the fingerprint peak of liquiritin, peak 4 is the fingerprint peak of ferulic acid, peak 11 is the fingerprint peak of ammonium glycyrrhizate, peak 13 is the fingerprint peak of formononetin, peak 15 is the fingerprint peak of nobiletin, and peak 17 is the fingerprint peak of tangeretin.

[0082] The water used in the present invention is all purified water.

[0083] As described above, the present invention provides a method for determining the fingerprint spectrum and multi-component content of Buzhong Yiqi Pills. The method based on fingerprint spectrum analysis and simultaneous quantification of multiple components will comprehensively analyze the material basis of Buzhong Yiqi Pills from two aspects.

[0084] First, the contents of seven major active ingredients in Buzhong Yiqi Pills (calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin) were determined. A high-performance liquid chromatography (HPLC) wavelength-switching method was established for the simultaneous determination of these ingredients. The seven active ingredients showed good resolution, symmetrical peak shapes, and the method was simple, reliable, and easy to operate, making it suitable for the quantitative determination of multiple components in Buzhong Yiqi Pills. The method was validated for quantitative analysis of the seven ingredients, demonstrating good linearity within their respective ranges, good reproducibility, high accuracy, and high precision. This method meets the requirements for basic research on the substance of Buzhong Yiqi Pills and facilitates quality control of Buzhong Yiqi Pills.

[0085] Secondly, a comparative analysis of the fingerprint similarities of Buzhong Yiqi Pills was conducted, and a fingerprint method for Buzhong Yiqi Pills was established. This method highlights the contribution of different chemical components to the fingerprint system of Buzhong Yiqi Pills from different aspects. After methodological investigation, a total of 20 common peaks were calibrated and the medicinal materials were attributed to these 20 chromatographic peaks. By confirming the attribution of the chromatographic peaks of each single medicinal ingredient in Buzhong Yiqi Pills, the compound peaks of 8 medicinal ingredients were well characterized in the fingerprint, thus achieving effective monitoring of the source of raw materials, strictly controlling the quality of raw medicinal materials, and can be used for quality control in the production process, thereby ensuring the safety and effectiveness of clinical medication.

[0086] In summary, this method is rapid, highly accurate, and reproducible. It can be used to verify the quality stability of different batches of products through fingerprint determination. Furthermore, through quantitative analysis of the main components of Buzhong Yiqi Pills, the content of the main components can be controlled, achieving both qualitative and quantitative quality control, providing a scientific basis for the quality control of Buzhong Yiqi Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 Shown are the chromatograms of the reference solution and the test solution of the Buzhong Yiqi Pills of the present invention, wherein the upper figure is the chromatogram of the test solution, and the lower figure is the fingerprint of the reference solution; 1: calycosin isoflavone glucoside, 2: liquiritin, 3: ferulic acid, 4: ammonium glycyrrhizate, 5: formononetin, 6: nobiletin, 7: tangeretin.

[0088] Figure 2Shown are chromatograms S1, S2, S3, S4, and S5 of the test solution and negative sample solution of the Buzhong Yiqi Pills of the present invention, wherein S1 is the chromatogram of the negative sample lacking tangerine peel, S2 is the chromatogram of the negative sample lacking licorice, S3 is the chromatogram of the negative sample lacking astragalus, S4 is the chromatogram of the negative sample lacking cimicifuga and angelica, and S5 is the chromatogram of the test solution; 1: calycosin isoflavone glucoside, 2: liquiritin, 3: ferulic acid, 4: ammonium glycyrrhizate, 5: formononetin, 6: nobiletin, 7: tangeretin.

[0089] Figure 3 The fingerprint of the Buzhong Yiqi Pills of the present invention is shown, wherein: 1: calycosin isoflavone glucoside; 3: liquiritin; 4: ferulic acid; 11: ammonium glycyrrhizate; 13: formononetin; 15: nobiletin; 17: tangeretin.

[0090] Figure 4 The superimposed fingerprints of HPLC of 20 batches of Buzhong Yiqi Pill samples in the present invention are shown, wherein S1: 202301-1; S2: 202301-2; S3: 202301-3; S4: 202301-4; S5: 202302-1; S6: 202302-2; S7: 202302-3; S8: 202302-4; S9: 202303-1; S 10: 202303-2; S11: 202303-3; S12: 202303-4; S13: 202304-1; S14: 202304-2; S15: 202 304-3; S16: 202304-4; S17: 202305-1; S18: 202305-2; S19: 202305-3; S20: 202305-4.

[0091] Figure 5 Shown is the HPLC attribution diagram of each characteristic peak of the sample and single medicinal material in the Buzhong Yiqi Pill of the present invention. DETAILED DESCRIPTION

[0092] The present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0093] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0094] The reagents and instruments used in the following examples are as follows:

[0095] 1. Reagents

[0096] Reference substances: calycosin isoflavone glucoside (batch number 111998-201703, mass fraction 97.4%), liquiritin (batch number 111610-201607, mass fraction 93.1%), ammonium glycyrrhizate (batch number 110731-201720, mass fraction 97.7%), ferulic acid (batch number 110773-201614, mass fraction 99.0%), formononetin (batch number 111720-201810, mass fraction 93.5%), nobiletin (batch number 18070602, mass fraction 98%), and tangeretin (batch number 18031501, mass fraction 98%). All of the above reference substances were purchased from the China Food and Drug Inspection Institute.

[0097] Sample: Buzhong Yiqi Pills powder, provided by Shanghai Hutchison Pharmaceutical Co., Ltd., a total of 20 batches (batch numbers are S221101, 202301-1, 202301-2, 202301-3, 202301-4, 202302-1, 202302-2, 202302-3, 202302-4, 202303-1, 202303-2, 202303-3, 202303-4, 202304-1, 202304-2, 202304-3, 202304-4, 202305-1, 202305-2, 202305-3, 202305-4).

[0098] Single medicinal materials: Astragalus, licorice, Codonopsis pilosula, Atractylodes macrocephala, Angelica sinensis, dried tangerine peel, Bupleurum chinense and Cimicifuga foetida were all provided by Shanghai Hutchison Pharmaceuticals Co., Ltd.

[0099] Reagents: methanol (analytical grade AR, Sinopharm Chemical Reagent Co., Ltd.), acetonitrile (chromatographic grade, TEDIA, USA), phosphoric acid (chromatographic grade, TEDIA, USA), and ultrapure water were prepared using a Milli-Q ultrapure water treatment system.

[0100] 2. Instruments

[0101] An Agilent 1260 high-performance liquid chromatograph (Agilent OpenLAB CDS ChemStation workstation, G1311C quaternary pump system, G1329B standard autosampler, G1316A column oven, and G4212B diode array detector, all from Agilent, USA) was used. An AL204 1 / 10,000 electronic balance and an X205BDU 1 / 100,000 electronic balance were purchased from METTLER TOLEDO (Shanghai). A DFY-500 high-speed Chinese medicine pulverizer was purchased from Dade Traditional Chinese Medicine Machinery Co., Ltd. in Wenling, Zhejiang Province. A DHG-9123A electric blast drying oven was purchased from Shanghai Yiheng Scientific Instrument Co., Ltd. An SB-5200 ultrasonic cleaner was purchased from Ningbo Xinzhi Biotechnology Co., Ltd., and a Mill-Q Advantage A10 ultrapure water preparation system was purchased from Millipore Shanghai Trading Co., Ltd.

[0102] Example 1

[0103] 1. Sample pretreatment

[0104] Preparation of test solution: Take the Buzhong Yiqi Pills sample of batch S221101, crush it and pass it through a No. 4 sieve, accurately weigh 1.0 g, place it in a 50 mL conical flask with a stopper, accurately add 20 mL of methanol, stopper it, weigh the mass, and extract it by ultrasonic extraction (power 100 W, frequency 40 kHz) for 30 minutes. Let it stand and cool to room temperature, then weigh the mass again, make up the lost mass with methanol, filter it through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution 1#.

[0105] Preparation of reference solution: Accurately weigh the reference substances of calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, dissolve them in methanol by ultrasonication (power 100 W, frequency 40 kHz) for 30 minutes, then dilute to volume in the same 100 mL volumetric flask, shake well, and prepare the reference stock solution.

[0106] The reference substance stock solution was then precisely measured, diluted stepwise with methanol, and fixed to volume to prepare a series of reference substance solutions of different concentrations. In the series of reference substance solutions of different concentrations, the content of calycosin glucoside ranged from 4.74 to 75.84 μg / mL, the content of liquiritin ranged from 183.525 to 978.8 μg / mL, the content of ferulic acid ranged from 21.045 to 112.24 μg / mL, the content of ammonium glycyrrhizate ranged from 241.1 to 1261.4 μg / mL, the content of formononetin ranged from 8.325 to 44.4 μg / mL, the content of nobiletin ranged from 122.85 to 655.2 μg / mL, and the content of tangeretin ranged from 81.75 to 436 μg / mL.

[0107] 2. Chromatographic conditions

[0108] The chromatographic conditions of the HPLC method are as follows: the chromatographic column is an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); the detector is a photodiode array detector (DAD); the column temperature is 35° C.; and the injection volume is 10 μL.

[0109] The analysis was performed using flow rate switching, where

[0110] When the retention time was 0-17 min, the flow rate was 1.0 mL / min;

[0111] When the retention time was 17-40 min, the flow rate was 0.8 mL / min.

[0112] The analysis is performed using wavelength switching, where:

[0113] When the retention time was 0-31 min, the detection wavelength was 245 nm;

[0114] When the retention time was 31-33 min, the detection wavelength was 275 nm;

[0115] When the retention time was 33-40 min, the detection wavelength was 245 nm.

[0116] 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 40 min; and the elution was gradient.

[0117] As shown in Table 1, the specific procedure of the gradient elution is:

[0118] 0-10 min, volume ratio of phase A:phase B was 10:90-20:80;

[0119] 10-17 min, the volume ratio of phase A:phase B is 20:80-27:73;

[0120] 17-20 min, the volume ratio of phase A:phase B is 27:73-30:70;

[0121] 20-21 min, the volume ratio of phase A:phase B is 30:70-36:64;

[0122] 21-30 min, the volume ratio of phase A:phase B is 36:64-65:35;

[0123] 30-40min, the volume ratio of phase A:phase B is 65:35-98:2.

[0124] 3. Determination

[0125] Using the external standard method, a series of different volumes of reference solution were pipetted to prepare a series of solutions with different concentrations. The samples were analyzed by HPLC and a standard working curve was plotted. The obtained test solution was then injected and analyzed by HPLC, and the analysis results were substituted into the standard working curve to obtain the contents of the seven components in the test solution.

[0126] Specifically, 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 high performance liquid chromatography to obtain the linear relationship between the content and peak area of the seven components in the reference solution. The chromatographic peak area of each component corresponded to its corresponding content, and the corresponding standard working curve was drawn. The regression equation of each standard working curve was calculated. The test solution was then tested by high performance liquid chromatography, and the chromatographic peak areas of the seven components in the test solution were substituted into the regression equation of each standard working curve to obtain the content of the corresponding component. See the table for details. Figure 1 .

[0127] Example 2

[0128] 1. Sample pretreatment

[0129] Preparation of test solution: Take the Buzhong Yiqi Pills sample of batch S221101, crush it and pass it through a No. 4 sieve, accurately weigh 1.0 g, place it in a 50 mL stoppered conical flask, accurately add 18 mL of methanol, stopper it tightly, weigh the mass, and extract it ultrasonically (power 90 W, frequency 35 kHz) for 32 minutes. Let it stand and cool to room temperature, then weigh the mass again, make up the lost mass with methanol, filter it through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution 2#.

[0130] Preparation of reference solution: Accurately weigh the reference substances of calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, dissolve them in methanol by ultrasonication (power 90 W, frequency 35 kHz) for 32 minutes, then dilute to volume in the same 100 mL volumetric flask, shake well, and prepare the reference stock solution.

[0131] The reference substance stock solution was then accurately measured, diluted stepwise with methanol, and fixed to volume to prepare a series of reference substance solutions of different concentrations. The concentration range of the series of reference substance solutions of different concentrations was the same as that of step 1 in Example 1.

[0132] 2. Chromatographic conditions

[0133] The chromatographic conditions of the HPLC method are as follows: the chromatographic column is an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); the detector is a photodiode array detector (DAD); the column temperature is 34° C.; and the injection volume is 8 μL.

[0134] The analysis was performed using flow rate switching, where

[0135] When the retention time was 0-17 min, the flow rate was 0.9 mL / min;

[0136] When the retention time was 17-40 min, the flow rate was 0.75 mL / min.

[0137] The analysis is performed using wavelength switching, where:

[0138] When the retention time was 0-31 min, the detection wavelength was 243 nm;

[0139] When the retention time was 31-33 min, the detection wavelength was 273 nm;

[0140] When the retention time was 33-40 min, the detection wavelength was 243 nm.

[0141] The mobile phase was acetonitrile-0.08% phosphoric acid aqueous solution, wherein phase A was acetonitrile and phase B was 0.08% phosphoric acid aqueous solution; the analysis time was 40 min; and the elution was gradient.

[0142] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0143] 3. Determination

[0144] The specific measurement process is the same as step 3 in Example 1.

[0145] Example 3

[0146] 1. Sample pretreatment

[0147] Preparation of test solution: Take the Buzhong Yiqi Pills sample of batch S221101, crush it and pass it through a No. 4 sieve, accurately weigh 1.0 g, place it in a 50 mL stoppered conical flask, accurately add 22 mL of methanol, stopper it, weigh the mass, and extract it ultrasonically (power 110 W, frequency 45 kHz) for 28 minutes. Let it stand and cool to room temperature, then weigh the mass again, make up the lost mass with methanol, filter it through a 0.45 μm microporous filter membrane, and take the filtrate to obtain the test solution 3#.

[0148] Preparation of reference solution: Accurately weigh the reference substances of calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, dissolve them in methanol by ultrasonication (power 110 W, frequency 45 kHz) for 28 minutes, then dilute to volume in the same 100 mL volumetric flask, shake well, and prepare the reference stock solution.

[0149] The reference substance stock solution was then accurately measured, diluted stepwise with methanol, and fixed to volume to prepare a series of reference substance solutions of different concentrations. The concentration range of the series of reference substance solutions of different concentrations was the same as that of step 1 in Example 1.

[0150] 2. Chromatographic conditions

[0151] The chromatographic conditions of the HPLC method are as follows: the chromatographic column is an Agilent TC-C18 chromatographic column (4.6 mm×250 mm, 5 μm); the detector is a photodiode array detector (DAD); the column temperature is 36° C.; and the injection volume is 12 μL.

[0152] The analysis was performed using flow rate switching, where

[0153] When the retention time was 0-17 min, the flow rate was 1.1 mL / min;

[0154] When the retention time was 17-40 min, the flow rate was 0.85 mL / min.

[0155] The analysis is performed using wavelength switching, where:

[0156] When the retention time was 0-31 min, the detection wavelength was 247 nm;

[0157] When the retention time was 31-33 min, the detection wavelength was 277 nm;

[0158] When the retention time was 33-40 min, the detection wavelength was 247 nm.

[0159] The mobile phase was acetonitrile-0.12% phosphoric acid aqueous solution, wherein phase A was acetonitrile and phase B was 0.12% phosphoric acid aqueous solution; the analysis time was 40 min; and the elution was gradient.

[0160] The specific procedure of gradient elution is the same as step 2 in Example 1.

[0161] 3. Determination

[0162] The specific measurement process is the same as step 3 in Example 1.

[0163] Example 4

[0164] Prepare a series of solutions of different concentrations of the above 7 reference substances, accurately draw the reference substance stock solutions, respectively, and perform high performance liquid chromatography analysis using the HPLC detection conditions of Example 1. Record the chromatogram, plot the standard curve with the concentration of each reference substance (X, μg / mL) as the abscissa and the peak area (Y) as the ordinate, and perform linear regression calculation to obtain the regression equation, correlation coefficient, and linear range. Dilute the concentration of each mixed reference substance so that the limit of quantification S / N ≥ 10 and the limit of detection S / N ≥ 3. The specific results are shown in Table 3.

[0165] As shown in Table 3, the regression equation has a good linear relationship when the sample is injected within the corresponding concentration range, and the correlation coefficient r 2 Not less than 0.9993.

[0166] Table 3 Linear relationships among 7 active ingredients (n=6)

[0167]

[0168]

[0169] Example 5

[0170] 1. Precision

[0171] Any reference solution prepared in Example 1 was taken and tested according to the method in Example 1. The sample was continuously injected 6 times for analysis and the peak area was recorded. The results showed that the RSDs of the peak areas of the seven components, including calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin and tangeretin, were all less than 3%, indicating that the instrument had good precision.

[0172] 2. Repeatability

[0173] Six samples of Buzhong Yiqi Pills from the same batch (batch number S221101) were taken and six test samples were prepared in parallel according to the method in Example 1. The peak areas were recorded and the results showed that the RSDs of the peak areas of the seven components, including calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, were all less than 3%, indicating that the method had good repeatability and high accuracy.

[0174] 3. Stability

[0175] Take one portion of the Buzhong Yiqi Pills sample from the same batch (batch number S221101) and prepare one portion of the test solution according to the method in Example 1 above. The samples were then placed for 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h for detection, and the peak areas were recorded. The results showed that the RSDs of the peak areas of the seven components, including calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin, were all less than 2.87%, indicating that the test solution had good stability within 48 h.

[0176] 4. Sample recovery rate

[0177] Nine samples of Buzhong Yiqi Pills with batch number S221101 were taken and accurately weighed. Calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin reference substances were added at low, medium, and high mass concentration levels (equivalent to 80%, 100%, and 120% of the original mass fraction, respectively). Three parts were taken for each mass concentration. The test solution was prepared according to step 1 in Example 1, and the solution was analyzed according to the chromatographic conditions of step 2 in Example 1. The sample recoveries and RSD% of the seven components at different addition ratios were calculated. The results are shown in Table 4. As shown in Table 4, the recoveries of the seven components, including calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin and tangeretin, ranged from 98.13% to 100.92%, with average recoveries of 98.76%, 100.56%, 99.7%, 100.92%, 100.61%, 98.13% and 100.50%, respectively. The RSDs (n=3) were 2.78%, 2.51%, 2.20%, 2.55%, 1.62%, 1.82% and 2.06%, respectively, indicating that the method had good accuracy.

[0178] Table 4 Sample recovery test results (n=9)

[0179]

[0180]

[0181] Example 6

[0182] Take 20 batches of Buzhong Yiqi Pills samples (202301-1, 202301-2, 202301-3, 202301-4, 202302-1, 202302-2, 202302-3, 202302-4, 202303-1, 202303-2, 202303-3, 202303-4, 202304-1, 202304-2, 202304-3, 2023 04-4, 202305-1, 202305-2, 202305-3, 202305-4), the test solution was prepared according to step 1 in Example 1, and the samples were injected and analyzed according to the chromatographic conditions of step 2 in Example 1, and the chromatograms were recorded. The contents of calycosin isoflavone glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin and tangeretin in the Buzhong Yiqi Pills sample were calculated by external standard method. The results are shown in Table 5.

[0183] As shown in Table 5, the contents of the seven compounds in each batch of Buzhong Yiqi Pills powder showed little difference (RSD < 2.8%). The established content detection method can be used to accurately determine the contents of the seven compounds in Buzhong Yiqi Pills.

[0184] Table 5 Content determination results of 20 batches of Buzhong Yiqi Pills

[0185]

[0186]

[0187] Example 7

[0188] 1. Sample pretreatment

[0189] Preparation of the test solution: The preparation process of the test solution is the same as that in step 1 of Example 1.

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

[0191] 2. Chromatographic conditions

[0192] The chromatographic conditions of the HPLC method are the same as those of the HPLC method in step 2 of Example 1.

[0193] 3. Determination

[0194] The test solution and the reference solution in step 1 are respectively measured by high performance liquid chromatography using the chromatographic conditions in step 2 to obtain a fingerprint of the test solution and a fingerprint of the reference solution. The fingerprint of the test solution is compared with the fingerprint of the reference solution, and the corresponding characteristic peaks in the fingerprint of the test solution are identified by relative retention time based on the known characteristic peaks in the fingerprint of the reference solution, thereby attributing and locating the index components in the fingerprint of the test solution to obtain the fingerprint of Buzhong Yiqi Pills.

[0195] Example 8

[0196] The fingerprint detection method of Buzhong Yiqi Pills established in Example 7 was used to detect 20 batches of Buzhong Yiqi Pills to obtain fingerprints of the test solution and the reference solution. The fingerprint data of the test sample were imported into the 2012 version of the software "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" issued by the State Pharmacopoeia Commission. Sample 202201-1 was used as a reference spectrum, and automatic full spectrum matching (time window width of 0.1 min) was used to generate fingerprints and reference fingerprints using the median method. The fingerprints of the test samples were compared with the control fingerprints of Buzhong Yiqi Pills obtained under the same fingerprint detection conditions, and the similarity of the fingerprints of each batch of Buzhong Yiqi Pills was calculated. The similarities between the fingerprints of the 20 batches of test samples and the control fingerprints were 0.998, 0.998, 0.999, 0.997, 0.998, 0.998, 0.995, 0.998, 0.993, and 0.9 96, 0.993, 0.998, 0.994, 0.996, 0.992, 0.993, 0.991, 0.998, 0.996, 0.998. The similarities of the fingerprints of the 20 batches of Buzhong Yiqi Pills were all greater than 0.990. There was no increase or loss of the number of chromatographic peaks in each batch of samples, indicating that the fingerprints of the 20 batches of samples had good similarity and a relatively stable overall quality. For specific similarity, see Figure 4 .

[0197] Example 9

[0198] The Buzhong Yiqi Pills were tested using the fingerprint detection method of the Buzhong Yiqi Pills established in Example 7. Based on the fingerprints of the test solution and the reference solution, 20 common characteristic peaks were identified.

[0199] The specific fingerprint of Buzhong Yiqi Pills can be found in Figure 3 ,Depend on Figure 3It can be seen that according to the relative retention time of each chromatographic peak in the chromatogram, the common peaks were determined, and 20 common peaks were selected as characteristic fingerprint peaks. The specific data are shown in Table 6. Among them, peak 11 has a moderate elution time, good separation, and a large peak area. Therefore, peak 11 is used as the reference peak (S peak, relative retention time is 1.0000). The relative retention times of the other 19 peaks are peak 1 (0.48±0.0003), peak 2 (0.49±0.0004), peak 3 (0.51±0.0003), peak 4 (0.54±0.0003), peak 5 (0.58±0.0004), peak 6 (0.64±0.0005), peak 7 (0.85±0.0004), peak 8 (0.89±0.0 003), peak 9 (0.90±0.0002), peak 10 (0.97±0.0001), peak 12 (1.03±0.0002), peak 13 (1.05±0.0002), peak 14 (1.12±0.0001), peak 15 (1.14±0.0002), peak 16 (1.16±0.0002), peak 17 (1.20±0.0002), peak 18 (1.27±0.0000), peak 19 (1.28±0.0002), and peak 20 (1.33±0.0000).

[0200] Table 6 Relative retention time of common peaks in fingerprint

[0201]

[0202] Note: Peak 11 is the positioning peak (S peak)

[0203] The control fingerprint of the Buzhong Yiqi Pills was compared with the fingerprint of the reference solution. Figure 1 The known characteristic peaks in the fingerprint of the reference solution in the test sample were identified by relative retention time, and peak 1 was located as the fingerprint peak of calycosin glucoside, peak 3 was the fingerprint peak of liquiritin, peak 4 was the fingerprint peak of ferulic acid, peak 11 was the fingerprint peak of ammonium glycyrrhizate, peak 13 was the fingerprint peak of formononetin, peak 15 was the fingerprint peak of nobiletin, and peak 17 was the fingerprint peak of tangeretin.

[0204] Example 10

[0205] The method for detecting the fingerprint of Buzhong Yiqi Pills in the present invention was methodologically verified, and the performance index results are as follows.

[0206] 1. Precision

[0207] One sample of Buzhong Yiqi Pills from the same batch (batch number 221101) was prepared and tested according to the detection method of the fingerprint of Buzhong Yiqi Pills in Example 7. The sample was injected six times continuously and the chromatogram was recorded. Peak 11 (ammonium glycyrrhizate) was used as the reference peak. The relative retention time RSDs of the 20 common peaks were calculated to be less than 0.10%, and the relative peak area RSDs were less than 2.20%, indicating that the instrument had good precision.

[0208] 2. Repeatability

[0209] Six samples of Buzhong Yiqi Pills from the same batch (batch number 221101) were prepared and tested according to the detection method of the Buzhong Yiqi Pills fingerprint in Example 7. The chromatograms were recorded. Peak 11 (ammonium glycyrrhizate) was used as the reference peak. The relative retention time RSDs of the 20 common peaks were calculated to be less than 0.07%, and the relative peak area RSDs were less than 2.20%. The results showed that the method had good repeatability.

[0210] 3. Stability

[0211] Take one sample of Buzhong Yiqi Pills from the same batch (batch number 221101) and prepare and test it according to the detection method of the fingerprint of Buzhong Yiqi Pills in Example 7 above. After preparing the test solution, place it for 0h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h for detection, record the chromatogram, and use peak 11 (ammonium glycyrrhizate) as the reference peak. The relative retention time RSD of the 20 common peaks is calculated to be less than 0.13%, and the relative peak area RSD is less than 2.80%. The results show that the test solution has good stability within 48h.

[0212] Example 11

[0213] The sample pretreatment steps in Example 7 were used to prepare the test solution.

[0214] Eight single herb sample solutions were prepared by respectively taking powders of eight medicinal materials, namely, astragalus, liquorice, codonopsis, atractylodes, angelica, tangerine peel, cimicifuga and bupleurum. The sample pretreatment steps in Example 7 were used.

[0215] Eight medicinal material samples of Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga, and Bupleurum were respectively lacking Tangerine Peel, lacking Licorice, lacking Astragalus, lacking Cimicifuga, and Angelica, and the sample pretreatment steps in Example 7 were used to obtain negative sample solutions lacking Tangerine Peel, negative sample solutions lacking Licorice, negative sample solutions lacking Astragalus, and negative sample solutions lacking Cimicifuga and Angelica, respectively.

[0216] Adopting step c and d in the detection method of the fingerprint of Buzhong Yiqi Pills of Example 7, respectively measure the test solution, 8 kinds of single medicinal material sample solutions, the negative sample solution lacking dried orange peel, the negative sample solution lacking liquorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica, and obtain the fingerprints of the test solution, 8 kinds of single medicinal material sample solutions, the negative sample solution lacking dried orange peel, the negative sample solution lacking liquorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica. The fingerprints of the obtained test solution, 8 kinds of single medicinal material sample solutions, the negative sample solution lacking dried orange peel, the negative sample solution lacking liquorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica are imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" 2012 version software issued by the State Pharmacopoeia Commission for analysis and processing, and obtain the control fingerprint of Buzhong Yiqi Pills obtained by simultaneously adopting the detection method of the fingerprint of Buzhong Yiqi Pills of Example 7. The fingerprints of the test solution, 8 single herbal sample solutions, negative sample solution lacking dried tangerine peel, negative sample solution lacking licorice, negative sample solution lacking astragalus, and negative sample solution lacking cimicifuga and angelica were compared with the control fingerprint of Buzhong Yiqi Pills, and the corresponding characteristic peaks of the 8 single herbal sample solutions in the control fingerprint of Buzhong Yiqi Pills were identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprints of the 8 single herbal sample solutions. The specific results are shown in Figure 2 、 5 .

[0217] Depend on Figure 2 、 5 It can be seen that among the single medicinal material sample solutions, the fingerprint peaks in common in the fingerprint spectra of the sample solutions of eight medicinal materials including Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga and Bupleurum are shown in Table 2. Specifically, it was determined that Peak 1 (calycosin isoflavone glucoside), Peak 8, Peak 9, Peak 13 (formononetin) and Peak 20 all came from Astragalus membranaceus; Peak 2, Peak 3 (liquiritin), Peak 8, Peak 10, Peak 11 (ammonium glycyrrhizate), Peak 12 and Peak 20 all came from Glycyrrhiza uralensis; Peak 20 came from Codonopsis pilosula; Peak 18 came from Atractylodes macrocephala; Peak 4 (ferulic acid) and Peak 20 all came from Angelica sinensis; Peak 6, Peak 15 (nobiletin), Peak 17 (tangeretin) and Peak 20 all came from Citrus reticulatae; Peak 4 (ferulic acid), Peak 5, Peak 7, Peak 10 and Peak 20 all came from Cimicifuga foetida; Peak 20 came from Bupleurum chinense.

[0218] It can be seen that the chemical characteristic peaks of the eight medicinal materials in Buzhong Yiqi Pills are well reflected in the fingerprint spectrum and their attribution is confirmed.

[0219] In summary, the present invention provides a fingerprint spectrum and multi-component content determination method for Buzhong Yiqi Pills, which can quantitatively analyze the seven chemical components in Buzhong Yiqi Pills, establish a high-performance liquid chromatography fingerprint spectrum for Buzhong Yiqi Pills, and confirm the identity of the chromatographic peaks of each single medicinal ingredient in Buzhong Yiqi Pills. This method plays a dual role in qualitative and quantitative quality control and provides a scientific basis for the quality control of Buzhong Yiqi Pills. Therefore, the present invention overcomes various shortcomings of the prior art and has high industrial application value.

[0220] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by one of ordinary skill in the art without departing from the spirit and technical principles disclosed herein are intended to be covered by the claims of the present invention.

Claims

1. A method for determining the contents of seven ingredients in Buzhong Yiqi Pills, comprising the following steps: 1) Preparation of test solution: Dissolve the Buzhong Yiqi Pill sample in methanol, extract by ultrasonication, allow to cool, filter, and obtain the test solution by taking the filtrate. 2) Preparation of reference solution: Dissolve the reference substances of calycosin glucoside, liquiritin, ferulic acid, ammonium glycyrrhizate, formononetin, nobiletin, and tangeretin in methanol by ultrasonication and then dilute to volume to prepare the reference solution. 3) Determination: Determine the test solution from step 1) and the reference solution from step 2) by high performance liquid chromatography, and calculate the contents of the seven components in the test solution by the external standard method; In step 3), the chromatographic column used in the high performance liquid chromatography method is an Agilent TC-C18 chromatographic column; In step 3), the flow rate switching method is used for analysis, wherein, When the retention time is 0-17 min, the flow rate is 0.9-1.1 mL / min; When the retention time is 17-40 min, the flow rate is 0.75-0.85 mL / min; In step 3), the analysis is performed by wavelength switching, wherein: When the retention time was 0-31 min, the detection wavelength was 240-250 nm; When the retention time is 31-33 min, the detection wavelength is 270-280 nm; When the retention time is 33-40 min, the detection wavelength is 240-250 nm; In step 3), the mobile phase is acetonitrile-0.05-0.2% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.05-0.2% phosphoric acid aqueous solution; the analysis time is 40 minutes; gradient elution; The specific procedure of the gradient elution is: 0-10 min, the volume ratio of phase A:phase B was 10:90-20:80; 10-17 min, the volume ratio of phase A:phase B is 20:80-27:73; 17-20 min, the volume ratio of phase A:phase B is 27:73-30:70; 20-21 min, the volume ratio of phase A:phase B is 30:70-36:64; 21-30 min, the volume ratio of phase A:phase B is 36:64-65:35; 30-40min, the volume ratio of phase A:phase B is 65:35-98:

2.

2. The method for determining the contents of seven components in Buzhong Yiqi Pills according to claim 1, characterized in that: In step 1) or 2), any one or more of the following conditions are included: A1) In step 1), the Buzhong Yiqi Pills are crushed and then sieved to obtain a Buzhong Yiqi Pills powder sample; the sieving is performed through a No. 4 sieve; A2) In step 1), the ratio of the weight of the Buzhong Yiqi Pill sample added to the volume of the methanol added is 1:18-22, g / mL; A3) In step 1), the ultrasonic extraction time is 25-35 minutes; A4) In step 1), the power of the ultrasonic extraction is 80-120W; the frequency of the ultrasonic extraction is 35-45kHz; A5) In step 1), the filtration is performed by filtering the supernatant through a membrane, discarding the primary filtrate, and then obtaining the subsequent filtrate; the membrane is a 0.45 μm filter membrane; A6) In step 2), the ultrasonic dissolution time is 25-35 minutes; A7) In step 2), the power of the ultrasonic dissolution is 80-120W; the frequency of the ultrasonic dissolution is 35-45kHz; A8) In step 2), the content of calycosin glucoside in the reference solution ranges from 4.74 to 75.84 μg / mL, the content of liquiritin ranges from 183.525 to 978.8 μg / mL, the content of ferulic acid ranges from 21.045 to 112.24 μg / mL, the content of ammonium glycyrrhizate ranges from 241.1 to 1261.4 μg / mL, the content of formononetin ranges from 8.325 to 44.4 μg / mL, the content of nobiletin ranges from 122.85 to 655.2 μg / mL, and the content of tangeretin ranges from 81.75 to 436 μg / mL.

3. The method for determining the contents of seven components in Buzhong Yiqi Pills according to claim 1, characterized in that: In step 3), the chromatographic conditions of the high performance liquid chromatography method include any one or more of the following conditions: B1) The detector is a photodiode array detector; B2) Column temperature is 32-38°C; B3) The injection volume is 8-12 μL; B4) Analyze using flow rate switching, where: When the retention time is 0-17 min, the flow rate is 0.9-1.0 mL / min or 1.0-1.1 mL / min; When the retention time is 17-40 min, the flow rate is 0.75-0.8 mL / min or 0.8-0.85 mL / min; B5) Use wavelength switching to perform analysis, where: When the retention time is 0-31 min, the detection wavelength is 240-245 nm, 245-250 nm, or 243-247 nm; When the retention time is 31-33 min, the detection wavelength is 270-275 nm, 275-280 nm, or 273-277 nm; When the retention time is 33-40 min, the detection wavelength is 240-245 nm, 245-250 nm or 243-247 nm.

4. A method for detecting the fingerprint of Buzhong Yiqi Pills, comprising the following steps: A) Preparation of the test solution: the same as step 1) of the method for determining the contents of the seven components in Buzhong Yiqi Pills according to any one of claims 1 to 3; B) Preparation of reference solution: the same as step 2) of the method for determining the contents of the seven components in Buzhong Yiqi Pills according to any one of claims 1 to 3; C) Determination: Using high performance liquid chromatography, 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 the fingerprints of the reference solution, attribute and locate the index components in the fingerprints of the test solution, and thus obtain a fingerprint of Buzhong Yiqi Pills; the fingerprint of Buzhong Yiqi Pills includes 20 common fingerprint peaks, and peak 1 is located and determined to be the fingerprint peak of calycosin isoflavone glucoside, peak 3 is the fingerprint peak of liquiritin, peak 4 is the fingerprint peak of ferulic acid, peak 11 is the fingerprint peak of ammonium glycyrrhizate, peak 13 is the fingerprint peak of formononetin, peak 15 is the fingerprint peak of nobiletin, and peak 17 is the fingerprint peak of tangeretin; In step C), the chromatographic column used in the high performance liquid chromatography method is an Agilent TC-C18 chromatographic column; In step C), the analysis is performed by switching the flow rate, wherein: When the retention time is 0-17 min, the flow rate is 0.9-1.1 mL / min; When the retention time is 17-40 min, the flow rate is 0.75-0.85 mL / min; In step C), the analysis is performed by wavelength switching, wherein: When the retention time was 0-31 min, the detection wavelength was 245 nm; When the retention time was 31-33 min, the detection wavelength was 275 nm; When the retention time was 33-40 min, the detection wavelength was 245 nm; In step C), the mobile phase is acetonitrile-0.05-0.2% phosphoric acid aqueous solution, wherein phase A is acetonitrile and phase B is 0.05-0.2% phosphoric acid aqueous solution; the analysis time is 40 minutes; gradient elution; The specific procedure of the gradient elution is: 0-10 min, the volume ratio of phase A:phase B was 10:90-20:80; 10-17 min, the volume ratio of phase A:phase B is 20:80-27:73; 17-20 min, the volume ratio of phase A:phase B is 27:73-30:70; 20-21 min, the volume ratio of phase A:phase B is 30:70-36:64; 21-30 min, the volume ratio of phase A:phase B is 36:64-65:35; 30-40min, the volume ratio of phase A:phase B is 65:35-98:

2.

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

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

7. The quality inspection method of Buzhong Yiqi Pills according to claim 6, characterized in that: The control fingerprint of Buzhong Yiqi Pills is obtained under the same conditions as the detection method of the fingerprint of Buzhong Yiqi Pills according to claim 4, wherein the control fingerprint of Buzhong Yiqi Pills includes 20 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 19 peaks are as follows: peak 1 0.48±0.0003, peak 2 0.49±0.0004, peak 3 0.51±0.0003, peak 4 0.54±0.0003, peak 5 0.58±0.0004, peak 6 0.64±0.0005, peak 7 Peak No. 0.85±0.0004, peak No. 8 0.89±0.0003, peak No. 9 0.90±0.0002, peak No. 10 0.97±0.0001, peak No. 12 1.03±0.0002, peak No. 13 1.05±0.0002, peak No. 14 1.12±0.0001, peak No. 15 1.14±0.0002, peak No. 16 1.16±0.0002, peak No. 17 1.20±0.0002, peak No. 18 1.27±0.0000, peak No. 19 1.28±0.0002, peak No. 20 1.33±0.0000.

8. A method for screening fingerprints of multiple medicinal materials in Buzhong Yiqi Pills, comprising the following steps: a) Preparation of single herbal sample solutions: preparing any one or more of the eight herbal samples of Buzhong Yiqi Pills, including Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga, and Bupleurum, according to step A) of the method for detecting the fingerprint of Buzhong Yiqi Pills according to claim 4, to obtain at least one single herbal sample solution; b) Preparation of negative sample solutions: 8 medicinal material samples of Astragalus, Licorice, Codonopsis, Atractylodes, Angelica, Tangerine Peel, Cimicifuga, and Bupleurum were prepared according to step A) of the fingerprint detection method of Buzhong Yiqi Pills according to claim 4, respectively, with Tangerine Peel, Licorice, Astragalus, Cimicifuga, and Angelica missing, to obtain negative sample solutions missing Tangerine Peel, Licorice, Astragalus, Cimicifuga, and Angelica missing, respectively; c) Determination: using high performance liquid chromatography under the same chromatographic conditions as in step C) of the method for detecting the fingerprint of Buzhong Yiqi Pills according to claim 4, respectively determining the single medicinal material sample solution in step a), the negative sample solution lacking dried tangerine peel, the negative sample solution lacking licorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica in step b), to obtain fingerprints of the single medicinal material sample solution, the negative sample solution lacking dried tangerine peel, the negative sample solution lacking licorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica, respectively; d) Obtaining a control fingerprint: applying the test solution prepared in step A) of the method for detecting the fingerprint of Buzhong Yiqi Pills according to claim 4 to step C) of the method for detecting the fingerprint of Buzhong Yiqi Pills according to claim 4 to obtain a control fingerprint of Buzhong Yiqi Pills; e) Quality testing: The fingerprints of the single medicinal material sample solution, the negative sample solution lacking dried tangerine peel, the negative sample solution lacking liquorice, the negative sample solution lacking astragalus, and the negative sample solution lacking cimicifuga and angelica were compared with the control fingerprint of Buzhong Yiqi Pills. The corresponding characteristic peaks of the single medicinal material sample solution in the control fingerprint of Buzhong Yiqi Pills were identified by relative retention time, thereby attributing and locating the characteristic peaks in the fingerprint of the single medicinal material sample solution; the control fingerprint of Buzhong Yiqi Pills included 20 common fingerprint peaks, and peak 1 was identified as the fingerprint peak of calycosin isoflavone glucoside, peak 3 as the fingerprint peak of liquiritin, peak 4 as the fingerprint peak of ferulic acid, peak 11 as the fingerprint peak of ammonium glycyrrhizate, peak 13 as the fingerprint peak of formononetin, peak 15 as the fingerprint peak of nobiletin, and peak 17 as the fingerprint peak of tangeretin.

9. The method for screening fingerprints of multiple medicinal materials in Buzhong Yiqi Pills according to claim 8, characterized in that: In step e), any one or more of the following conditions are included: C1) The fingerprint of the Astragalus sample solution includes 5 common fingerprint peaks, which are peak 1, peak 8, peak 9, peak 13, and peak 20; C2) The fingerprint of the licorice sample solution includes 7 common fingerprint peaks, which are peak 2, peak 3, peak 8, peak 10, peak 11, peak 12, and peak 20; C3) The fingerprint of the Codonopsis pilosula sample solution includes one common fingerprint peak, which is peak 20; C4) The fingerprint of the Atractylodes macrocephala sample solution includes one common fingerprint peak, which is peak 18; C5) The fingerprint of the Angelica sinensis sample solution includes two common fingerprint peaks, which are Peak 4 and Peak 20; C6) The fingerprint of the tangerine peel sample solution includes four common fingerprint peaks, wherein the four common fingerprint peaks are peak 6, peak 15, peak 17, and peak 20; C7) The fingerprint of the Cimicifuga sample solution includes 5 common fingerprint peaks, wherein the 5 common fingerprint peaks are peak 4, peak 5, peak 7, peak 10, and peak 20; C8) The fingerprint of the Bupleurum sample solution includes one common fingerprint peak, which is Peak 20.

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