HPLC (High Performance Liquid Chromatography) fingerprint spectrum detection method of compound cypress and astragalus pills

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an HPLC fingerprint spectrum detection method of a compound cypress-astragalus pill, which comprises the following steps: S1, extracting a prescription with water; s2, preparing a compound cypress-astragalus pill; s3, preparing a test solution; s4, preparing a reference substance solution; and S5, constructing an HPLC fingerprint spectrum: analyzing the test solution under chromatographic conditions, recording a chromatogram, selecting a reference peak, and determining the relative peak area and the relative retention time of each common peak through analysis and comparison, thereby obtaining the HPLC fingerprint spectrum of the compound cypress-astragalus pill. The extraction process and the established fingerprint spectrum are high in operability and repeatable, and an important method guarantee is provided for effective extraction and quality control of the compound cypress and astragalus pills.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to an HPLC fingerprint detection method for compound Baiqi pills. Background Art

[0002] Fufang Baiqi Wan (Combined Boqi Pills) is an in-hospital formulation adapted from a commonly used prescription for diabetes treatment by renowned Chinese medicine practitioner Shi Jinmo. Its main ingredients are Phellodendron amurense, Astragalus membranaceus, Hawthorn Leaf, and Rehmannia glutinosa. It boasts the effects of clearing heat and dissolving dampness, removing blood stasis and resolving phlegm, and tonifying Qi and Yin. It is used to treat metabolic syndrome, characterized by symptoms such as polydipsia, polyuria, polyphagia, and weight loss. Shi Jinmo believed that metabolic syndrome, characterized by the disturbance of glucose and lipid metabolism, is a syndrome of both underlying deficiency and superficial excess. The underlying deficiency is primarily characterized by Qi and Yin deficiency, while the superficial excess is often characterized by phlegm and dampness, blood stasis, and turbid toxins. Therefore, medication should prioritize and prioritize the three main components of deficiency, heat, and excess. Therefore, treatment primarily focuses on clearing heat, dissolving dampness, tonifying Qi, dissolving blood stasis, and promoting Qi circulation. The main herb in the formula, Phellodendron chinense, is bitter, cold, and has a sinking effect. It is potent in clearing heat and drying dampness, purging fire and eliminating steam, and is "specifically designed to treat symptoms of internal heat caused by yin deficiency," effectively clearing heat and resolving phlegm. The auxiliary herb, Astragalus membranaceus, replenishes qi and promotes diuresis, nourishes yin and produces body fluid, relieves stagnation and relieves numbness, promoting qi and enabling the circulation of yin fluid, which in turn quenches thirst. The adjuvant herb, Rehmannia glutinosa, is a key herb for clearing heat and cooling blood. When combined with Astragalus membranaceus, it enhances its qi-tonifying, yin-nourishing, heat-clearing, and dryness-moistening effects, thereby quenching thirst. The auxiliary herb, Crataegus pinnatifida, activates blood circulation, dissolves stasis, regulates qi and unblocks meridians, and clears turbidity and reduces lipids. Together, these four herbs clear heat and dampness, replenish qi and nourish yin, and simultaneously target blood stasis and phlegm, effectively improving the symptoms of patients with metabolic syndrome and lipid disorders. Modern pharmacological studies have shown that berberine hydrochloride, phellodendron hydrochloride, and hyperoside, the main components of Phellodendron chinense, Rehmannia glutinosa, and Crataegus pinnatifida, can inhibit hepatic fatty acid synthesis and lower blood sugar. It can be seen from this that Fufang Baiqi Pills are effective in treating glucose and lipid metabolism disorders, but there are currently no research reports on their extraction process and quality testing.

[0003] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0004] The purpose of the present invention is to provide an HPLC fingerprint detection method for Fufang Baiqi Pills to solve the problems existing in the prior art.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A HPLC fingerprint detection method for Fufang Baiqi Pills comprises the following steps:

[0007] S1. Water Extraction Prescription

[0008] Take two-thirds of the prescribed amount of medicinal materials, add water and reflux extract three times, combine the filtrates, filter, concentrate under reduced pressure, and spray dry to obtain dry paste powder;

[0009] S2. Preparation of Compound Baiqi Pills

[0010] The remaining one-third of the prescribed amount of medicinal materials is taken, dried, crushed, and sieved to obtain a fine powder; the dry paste powder is mixed with the fine powder, wet granulated, made into pills, shaped into pills, dried, and packaged to obtain the finished product of Compound Baiqi Pills;

[0011] S3. Prepare the test solution

[0012] Prepare the test solution from the finished product of Fufang Baiqi Pills;

[0013] S4. Preparation of reference solution

[0014] Accurately weigh rehmannia glutinosa glycoside D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside reference substances respectively to prepare a mixed reference solution;

[0015] S5. Construction of HPLC fingerprint

[0016] 0.5 g of each of 9 batches of Fufang Baiqi Pills were taken to prepare test solutions; the test solutions were analyzed under chromatographic conditions, the chromatograms were recorded, reference peaks were selected, and the relative peak areas and relative retention times of the common peaks were determined by analysis and comparison to obtain the HPLC fingerprint of Fufang Baiqi Pills.

[0017] Furthermore, in step S1, the prescription is as follows, in parts by mass: 10 parts of Phellodendron chinense, 30 parts of Astragalus membranaceus, 15 parts of Rehmannia glutinosa, and 10 parts of Crataegus leaves.

[0018] Furthermore, in step S1, the specific operation of water extraction is: taking two-thirds of the prescribed amount of medicinal materials and adding 12 times the weight of water to boil and extract 3 times, each time for 60 minutes, combining the filtrate, filtering, concentrating under reduced pressure, and spray drying to obtain a dry paste powder.

[0019] Furthermore, in step S2, the specific operation of preparing the Compound Baiqi Pills is as follows: taking the remaining one-third of the prescribed amount of medicinal materials, drying, crushing, and passing through a 100-mesh sieve to obtain a fine powder. The dry paste powder and the fine powder are mixed evenly, 60% ethanol is added, and the mixture is formed into pills using a plastic molding method at a stirring rate of 30 rpm and a pilling speed of 35 rpm. The whole pills are placed in a forced air drying oven at 60°C for 8 hours, and packaged in 6g bags to obtain the finished Compound Baiqi Pills.

[0020] Furthermore, in step S3, the preparation method of the test solution is: take 0.5 g of the finished product of Fufang Baiqi Pills, place it in a 25 mL container, add 25% methanol, ultrasonically treat it for 30 minutes, take it out, cool it, shake it to volume, and filter it with a 0.45 μm microporous filter membrane to obtain the test solution.

[0021] Furthermore, in step S4, the mass concentrations of rehmannia glutinosa D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside in the mixed reference solution are 0.1029 mg / mL, 0.0547 mg / mL, 0.0505 mg / mL and 0.0419 mg / mL, respectively.

[0022] Furthermore, in step S5, the chromatographic conditions are: the chromatographic column is SEVA C TM 18 Opal chromatographic column (250 mm × 4.6 mm, 5 μm), mobile phase was methanol A-0.1% phosphoric acid aqueous solution B, gradient elution, flow rate was 1.0 mL / min, detection wavelength was 210 nm, column temperature was 35 ° C, injection volume was 10 μL.

[0023] Furthermore, the elution program is: 0-10 min, 0%-10% mobile phase A; 10-15 min, 10%-20% mobile phase A; 15-25 min, 20%-22% mobile phase A; 25-35 min, 22%-35% mobile phase A; 35-45 min, 35%-40% mobile phase A; 45-60 min, 40%-40% mobile phase A.

[0024] Furthermore, in step S5, the phellodendrine hydrochloride peak is used as a reference peak.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The orthogonal experiment of the present invention selected rehmannia root representative component rehmannia glutinosa D, berberine hydrochloride and phellodendronine hydrochloride representative components of Phellodendron chinense, and hawthorn leaf representative component hyperoside as index components for content determination. The optimal extraction process was screened out as follows: add 12 times the amount of water to the daily prescription amount of the slices, boil over high heat and then simmer for 60 minutes, filter, decoct three times, and combine the filtrates to obtain the product. Under this extraction condition, the average dry paste yield was 26.89%, and the contents of rehmannia root, phellodendronine hydrochloride, berberine hydrochloride, and hyperoside were 1.0027 mg / g, 0.9863 mg / g, 1.0849 mg / g, and 0.3412 mg / g, respectively, with a comprehensive score of 98.73.

[0027] (2) The present invention established HPLC fingerprints of 9 batches of finished Fufang Baiqi Pills, and their similarities were all greater than 0.936, indicating that the quality of samples from different batches was similar. A total of 13 common peaks were calibrated, of which 1, 2, 3, 4, 6, 7, 8, and 10 were quality markers, including index components such as rehmannia glutinosa glycoside D and phellodendronine hydrochloride. This also shows that the content determination of the index components selected by the orthogonal experiment of the present invention is representative for the optimization of the extraction process of Fufang Baiqi Pills.

[0028] In summary, the preferred extraction process and the established fingerprint of the present invention are highly operable and repeatable, providing an important reference for the effective extraction and quality control of Fufang Baiqi Pills. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A The specific chromatogram of the mixed reference solution;

[0030] Figure 1B This is the chromatogram of the water-extracted sample of Fufang Baiqi Pills;

[0031] Figure 1C The specific chromatogram of the hawthorn leaf negative sample solution is missing;

[0032] Figure 1D The specific chromatogram of the negative sample solution of Rehmannia glutinosa is missing;

[0033] Figure 1E The specific chromatogram of the negative sample solution lacking Phellodendron chinense is shown;

[0034] Figure 2 This is the process flow chart for the preparation of Fufang Baiqi Wan.

[0035] Figure 3 HPLC fingerprints of 9 batches of Fufang Baiqi Pills finished products;

[0036] Figure 4 VIP graph of the common peak fingerprint;

[0037] Figure 5 This is the HCA dendrogram of 9 batches of Fufang Baiqi Pills.

[0038] Description of main reference numerals:

[0039] Figures 1A-1E In the formula, 1--rehmannoside D; 2--phellodendronine hydrochloride; 3--berberine hydrochloride; 4--hyperoside. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts are within the scope of protection of the present invention.

[0041] 1. Materials

[0042] 1.1 Instrument

[0043] Thermo Fisher ULtiMate3000 high performance liquid chromatograph (Thermo Fisher Scientific);

[0044] Electronic balance (1 / 100,000, Sartorius, Germany);

[0045] Ultrapure water machine (Millipore, Germany);

[0046] Electric constant temperature drying oven 202-0A (Tianjin Test Instrument Co., Ltd.);

[0047] Electric constant temperature blast drying oven (Shanghai Jinghong Experimental Equipment Co., Ltd.).

[0048] 1.2 Reagents, medicinal materials and drugs

[0049] Phellodendron amurense (batch number: 20230701), Astragalus membranaceus (batch number: 2024062201), Rehmannia root (batch number: 20231201), and Crataegus pinnatifida leaf (batch number: 2024062202) were purchased from Shaanxi Duoyao Chinese Medicine Pieces Co., Ltd.

[0050] Rehmannia glutinosa D reference substance (lot number: PRF23092521), phellodendrine hydrochloride reference substance (lot number: PRF23031543), berberine hydrochloride reference substance (lot number: PRF23052301), and hyperoside reference substance (lot number: PRF24030741) were purchased from Chengdu Purifa Technology Development Co., Ltd.

[0051] Methanol (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0052] Phosphoric acid (chromatographic grade, Tianjin Damao Chemical Reagent Factory);

[0053] Ultrapure water was homemade;

[0054] The 9 batches of finished products of Fufang Baiqi Pills were homemade samples, with batch numbers S1, S2, S3, S4, S5, S6, S7, S8, and S9.

[0055] 2. Methods and Results

[0056] 2.1 Optimization of the extraction process of Fufang Baiqi Pills

[0057] 2.1.1 Solution preparation

[0058] Preparation of test solution: Take 130 g of the prescribed amount of Fufang Baiqi Wan decoction pieces (20 g of Phellodendron amurense, 60 g of Astragalus membranaceus, 30 g of Rehmannia glutinosa, and 20 g of Hawthorn leaves), add water and reflux for extraction. Filter, combine the filtrates, and concentrate under reduced pressure to a concentrate with a relative density of 1:1.12-1.15. Vacuum dry to obtain a dry powder. Accurately take 0.5 g of the dry powder and place it in a 25 mL volumetric flask. Add an appropriate amount of 25% methanol and ultrasonically treat for 30 min. Remove, shake well, and dilute to the mark with 25% methanol. Filter through a 0.45 μm microporous membrane to obtain the test solution.

[0059] Preparation of mixed reference solution: Accurately weigh the reference substances rehmannia glutinosa D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside and place them in 10 mL volumetric flasks respectively, add 25% methanol to dissolve, ultrasonicate for 30 min, dilute to the scale, and shake well to obtain reference substance stock solutions with concentrations of 0.5145 mg / mL, 0.5468 mg / mL, 0.5047 mg / mL and 0.5243 mg / mL for rehmannia glutinosa D, 0.5468 mg / mL, 0.5047 mg / mL and 0.5243 mg / mL, respectively.

[0060] Accurately pipette appropriate amounts of rehmannia glutinosa glycoside D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside stock solutions, place them in a 10 mL volumetric flask, dilute to the mark with 25% methanol, and shake well to obtain a mixed reference solution with concentrations of 0.1029 mg / mL, 0.0547 mg / mL, 0.0505 mg / mL and 0.0419 mg / mL for rehmannia glutinosa glycoside D, 0.0547 mg / mL, 0.0505 mg / mL and 0.0419 mg / mL for hyperoside.

[0061] Preparation of negative sample solution: prepare negative samples lacking Rehmannia root, Phellodendron chinense and Crataegus leaves according to the prescription process, and prepare negative sample solution according to the preparation method of the test solution.

[0062] 2.1.2 Chromatographic conditions

[0063] SEVAC TM Opal 18 column (250 mm × 4.6 mm, 5 μm); gradient elution with methanol (A) and 0.1% aqueous phosphoric acid (B); flow rate 1.0 mL / min; column temperature 35°C; detection wavelength 210 nm; injection volume 10 μL. The elution program was as follows: 0%-10% mobile phase A for 0-10 min; 10%-20% mobile phase A for 10-15 min; 20%-22% mobile phase A for 15-25 min; 22%-35% mobile phase A for 25-35 min; 35%-40% mobile phase A for 35-45 min; and 40%-40% mobile phase A for 45-60 min.

[0064] 2.1.3 Methodological Investigation

[0065] (1) Specificity test

[0066] Accurately pipette the mixed reference solution and test solution, measure according to the above chromatographic conditions, inject into the chromatograph, and the results are shown in Figures 1A-1E .

[0067] Depend on Figures 1A-1E It can be seen that the test solution and the mixed reference solution peak at the same time, and have good separation from other chromatographic peaks. The negative sample solution has no interfering peak here, and methanol has no interference with the reference solution chromatographic peak, indicating that the method of the present invention has good specificity.

[0068] (2) Linear relationship investigation

[0069] Using the mixed reference solution prepared in "2.1" as the linear solution, dilute to volume with methanol and dilute sequentially at a 1 / 2 ratio to produce six different concentrations of mixed reference solution. Shake well, filter through a 0.45 μm microporous membrane, and inject 10 μL of each solution. Measure according to the chromatographic conditions and record the peak area. Perform a linear regression of peak area (A) against concentration (C). The results are shown in Table 1.

[0070] Table 1 Standard curves of 4 components

[0071]

[0072] As shown in Table 1, the four components, namely, rehmannia glutinosa glycoside D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside, have a good linear relationship within their respective linear ranges.

[0073] (3) Precision test

[0074] The same mixed reference solution was accurately aspirated and sampled continuously for 6 times according to the above chromatographic conditions. The RSDs of the peak areas of rehmannia glutinosa, phellodendronine hydrochloride, berberine hydrochloride, and hyperoside were calculated, and the results were 0.47%, 0.15%, 1.18%, and 0.56%, respectively, indicating that the instrument had good precision.

[0075] (4) Stability test

[0076] The test solution was injected and the peak area was determined according to the above chromatographic conditions at 0, 2, 4, 8 and 12 hours after preparation. The sample was injected and analyzed at each time point, and the peak area was recorded. The RSDs of the peak areas of rehmannia glutinosa, phellodendronine hydrochloride, berberine hydrochloride and hyperoside were calculated to be 1.63%, 1.39%, 1.49% and 1.81%, respectively, indicating that the solution had good stability within 12 hours.

[0077] (5) Repeatability test

[0078] Take orthogonal sample 2, accurately weigh 6 portions, prepare the test solution, and inject the sample continuously 6 times according to the proposed chromatographic conditions. Record the peak area and calculate the RSDs of the peak areas of rehmannia glutinosa D, phellodendronine hydrochloride, berberine hydrochloride, and hyperoside, which are 1.90%, 1.84%, 1.37%, and 1.34%, respectively, indicating that the method has good repeatability.

[0079] (6) Sample recovery test

[0080] Weigh half the sample powder of known content (6 portions, 0.25 g each) and place them in 25 mL volumetric flasks. Accurately add 0.6 mL of the reference stock solution of rehmannia glutinosa D, 0.31 mL of phellodendronine hydrochloride, 0.48 mL of berberine hydrochloride, and 0.135 mL of hyperoside. Add 25% methanol and sonicate for 30 minutes. Remove, calibrate to volume, and shake well. Inject and measure according to the above chromatographic conditions. Calculate the recoveries of the four components. The results are shown in Table 2.

[0081] Recovery rate = (measured value of spiked sample - measured value of sample) ÷ spiked amount × 100%.

[0082] Table 2 Sample recovery test results

[0083]

[0084] As can be seen from Table 2, the method of the present invention has good accuracy.

[0085] 2.1.4 Dry paste yield test

[0086] Take half of 200 mL of concentrated medicinal solution, place it in a constant weight evaporating dish, evaporate to dryness in a water bath, dry in a 105℃ oven for more than 3 hours, transfer to a desiccator and cool for 30 minutes, accurately weigh the mass, dry in a 105℃ oven for more than 1 hour, weigh the mass after cooling, and calculate the yield of dry paste until the difference between the two results is no more than 5 mg.

[0087] Dry extract yield = dry extract weight / medicinal material weight × 2 × 100%.

[0088] 2.1.5 Orthogonal Experimental Design and Results

[0089] Use L9(3 4 An orthogonal test was conducted, with water addition (A), extraction time (B), and number of extractions (C) as influencing factors, and dry extract yield and the contents of rehmannia glutinosa D, berberine hydrochloride, phellodendronine hydrochloride, and hyperoside as evaluation indicators. A comprehensive score was calculated, with each indicator weighted at 20%.

[0090] Comprehensive score = dry extract yield / maximum dry extract yield × 100 × 20% + rehmannia glutinosa D content / maximum rehmannia glutinosa D content × 100 × 20% + phellodendron hydrochloride content / maximum phellodendron hydrochloride content × 100 × 20% + berberine hydrochloride content / maximum berberine hydrochloride content × 100 × 20% + hyperoside content / maximum hyperoside content × 100 × 20%.

[0091] The factor levels are shown in Table 3, and the orthogonal test results and variance analysis results are shown in Tables 4 and 5.

[0092] Table 3 Extraction process factor levels

[0093]

[0094] Table 4 Orthogonal experiment results

[0095]

[0096]

[0097] Table 5 Results of variance analysis of orthogonal test

[0098]

[0099] The R values in Table 4 indicate that the order of influence of various factors on the extraction process is C > B > A, that is, number of extractions > extraction time > amount of water added. ANOVA results showed that the number of extractions had a significant effect on the extraction process (P ≤ 0.05), while the amount of water added and extraction time had no significant effect (P > 0.05), as shown in Table 5. By visually analyzing the optimal levels of each factor, combining orthogonal experiments and ANOVA results, and considering energy conservation and high efficiency, the optimal extraction scheme for Fufang Baiqi Wan was determined to be A3B2C3: adding 12 times the amount of water, extracting for 60 minutes each time, and performing three extractions.

[0100] 2.1.6 Verification test

[0101] Verification was carried out according to the optimized process A3B2C3 under "2.1.5", i.e., 12 times the amount of water was added, the decoction time was 60 min, and the number of decoctions was 3. Three batches of samples were prepared, and the content of each indicator and the dry paste yield were determined. The results are shown in Table 7.

[0102] Table 7 Verification experimental results

[0103]

[0104] As shown in Table 7, the RSD values of various indicators were all less than 3%, and the comprehensive scores were 98.74, 99.38, and 98.08 points, respectively, indicating that the extraction process of the present invention was stable and reliable.

[0105] 2.2 HPLC fingerprint establishment

[0106] 2.2.1 Preparation of Compound Baiqi Pills

[0107] Weigh two-thirds of 31 times, 310 times, and 620 times the prescribed amount of medicinal materials respectively, add 12 times the mass of water and boil and extract three times, each time for 60 minutes, combine the filtrates, filter, and concentrate under reduced pressure to a relative density of 1:1.12-1.15, spray dry, and obtain dry paste powder. Take the remaining one-third of the prescribed amount of medicinal materials, dry, grind, and pass through a 100-mesh sieve to obtain fine powder. Mix the dry paste powder with the fine powder, add 60% ethanol, and make pills by plastic molding at a stirring rate of 30 rpm and a pill-making rate of 35 rpm. The whole pills are placed in a blast drying oven at 60°C and dried for 8 hours, and packaged, 6g per bag, to obtain the finished product of Compound Baiqi Pills. Compound Baiqi Pills Preparation Process Flow Figure 2 .

[0108] 2.2.2 Preparation of test solution

[0109] Grind 0.5 g of the finished product of Fufang Baiqi Pills into powder, place in a 25 mL container, and add 25% methanol for ultrasonic extraction. The specific preparation method is the same as "2.1 Solution Preparation".

[0110] 2.2.3 Chromatographic conditions

[0111] Same as “2.1 Chromatographic Conditions”.

[0112] 2.2.4 Methodological Investigation

[0113] Taking peak 2 as a reference, the relative peak area RSD of the determination precision was 0.15%, the relative peak area RSD of the repeatability was 1.84%, and the relative peak area RSD of the stability within 12 hours was less than 1.39%, indicating that the instrument precision, method repeatability and stability within 12 hours of the present invention are good.

[0114] 2.2.5 Graph production and similarity evaluation

[0115] The chromatograms of 9 batches of samples were recorded and imported into the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 version). Using S1 as the reference spectrum, the time window width was set to 0.10 min, and multi-point calibration, Mark peak matching, and median method were selected to generate superimposed fingerprints and reference fingerprints. As a result, 13 common peaks were identified, see Figure 3 .

[0116] by Figure 3 Peak No. 5, phellodendrine hydrochloride, was used as the reference peak to calculate the relative peak areas and relative retention times of the common peaks. The results are shown in Table 8.

[0117] Table 8 Relative peak areas and relative retention times of the common peaks

[0118]

[0119] The similarity results of the control fingerprints of 9 batches of Fu Fang Baiqi Wan are shown in Table 9.

[0120] Table 99 batches of compound Baiqi pills fingerprint similarity

[0121]

[0122]

[0123] As shown in Table 9, the similarities of the nine batches of samples are all greater than 0.936, indicating that the similarities between different batches of samples are relatively high.

[0124] 2.7 Chemical Pattern Recognition Research

[0125] 2.7.1 Partial Least Squares Discriminant Analysis

[0126] SIMCA software was used to perform partial least squares discriminant analysis (PLS-DA). The variable importance in projection (VIP) value > 1 was used as the standard to screen the components with the largest contribution to the difference. The results are shown in Figure 4 .

[0127] Depend on Figure 4 It can be seen that there are 8 peaks with VIP values greater than 1, namely chromatographic peaks 1, 2, 3, 4, 6, 7, 8, and 10, which can be used as quality markers of Fufang Baiqi Pills.

[0128] 2.7.2 Cluster Analysis

[0129] All peak data in the fingerprints of 9 batches of Fufang Baiqi Wan samples were homogenized. The homogenized results were used as variables and analyzed using SPSS21.0 software. Intergroup linkage and squared Euclidean distance were used as clustering methods and measurement intervals. The results are shown in Figure 5 .

[0130] Depend on Figure 5 It can be seen that when the Euclidean square distance is 15, the 9 batches of samples are clustered into 3 categories. The first category is S1, S4-S6, and S8, the second category is S2-S3 and S7, and the third category is S9, indicating that there are subtle differences between different batches of samples and there is an overlap phenomenon.

[0131] In summary, the orthogonal experiment of the present invention selected rehmannia root representative component rehmannia glutinosa D, berberine hydrochloride and phellodendronine hydrochloride representative components of Phellodendron chinense, and hawthorn leaf representative component hyperoside as indicator components for content determination. The optimal extraction process was screened and obtained as follows: adding 12 times the amount of water to the daily prescription amount of decoction pieces, reflux extraction for 60 minutes, filtering, extracting three times, and combining the filtrate. Extraction under these conditions resulted in an average dry paste yield of 26.89%, with rehmannia root glutinosa D, phellodendronine hydrochloride, berberine hydrochloride, and hyperoside contents of 1.0027 mg / g, 0.9863 mg / g, 1.0849 mg / g, and 0.3412 mg / g, respectively, and a comprehensive score of 98.73.

[0132] The present invention established HPLC fingerprints for nine batches of Fufang Baiqi Pills, with similarities greater than 0.936, indicating that the quality of samples from different batches of Fufang Baiqi Pills was similar. A total of 13 common peaks were identified, of which 1, 2, 3, 4, 6, 7, 8, and 10 were quality markers, including index components such as rehmannia glutinosa glycoside D and phellodendronine hydrochloride. This also demonstrates that the content determination of the index components selected by the orthogonal experiment in the present invention is representative for optimizing the extraction process.

[0133] The preferred extraction process and the established fingerprint spectrum of the present invention are highly operable and repeatable, and provide an important reference basis for the effective extraction and quality control of Fufang Baiqi Pills.

[0134] The foregoing descriptions of specific exemplary embodiments of the present invention are for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise forms disclosed, and it is apparent that many variations and modifications are possible in light of the foregoing teachings. The exemplary embodiments have been selected and described for the purpose of explaining the specific principles of the invention and their practical application, thereby enabling those skilled in the art to realize and utilize a variety of exemplary embodiments of the invention and various options and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A HPLC fingerprint detection method for Fufang Baiqi Pills, characterized in that: The following steps are involved: S1. Water Extraction Prescription Take two-thirds of the prescribed amount of medicinal materials, add water and reflux extract three times, combine the filtrates, filter, concentrate under reduced pressure, and spray dry to obtain dry paste powder; S2. Preparation of Compound Baiqi Pills One third of the prescribed amount of medicinal materials is taken, dried, crushed, and sieved to obtain fine powder; the dry paste powder is mixed with the fine powder, wet granulated, made into pills, shaped into pills, dried, and packaged to obtain the finished compound Baiqi pills; S3. Prepare the test solution Prepare the test solution from the finished product of Fufang Baiqi Pills; S4. Prepare reference solution Accurately weigh rehmannia glutinosa glycoside D, phellodendronine hydrochloride, berberine hydrochloride and hyperoside reference substances respectively to prepare a mixed reference solution; S5. Construction of HPLC fingerprint The test solution was analyzed under chromatographic conditions, the chromatogram was recorded, the reference peak was selected, and the relative peak area and relative retention time of each common peak were determined by analysis and comparison to obtain the HPLC fingerprint of Fufang Baiqi Pills.

2. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S1, the prescription is as follows, in parts by mass: 10 parts of Phellodendron chinense, 30 parts of Astragalus membranaceus, 15 parts of Rehmannia glutinosa, and 10 parts of Crataegus pinnatifida leaves.

3. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S1, the specific operation of water extraction is: take two-thirds of the prescribed amount of medicinal materials, add 12 times the weight of water, and boil and extract three times, each time for 60 minutes, combine the filtrate, filter, concentrate under reduced pressure, and spray dry to obtain a dry paste powder.

4. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S2, the preparation method of the compound Baiqi is as follows: take the remaining one-third of the prescribed amount of medicinal materials, dry them at 50°C, crush them, and pass them through a 100-mesh sieve to obtain a fine powder. The dry paste powder obtained above is mixed with the fine powder, 60% ethanol is added, and the mixture is formed into pills using a plastic method at a stirring rate of 30 rpm and a pilling speed of 35 rpm. The whole pills are placed in a forced air drying oven at 60°C for 8 hours, and packaged in bags of 6g to obtain the finished compound Baiqi pills.

5. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S3, the preparation method of the test solution is as follows: take 0.5 g of the finished product of Fufang Baiqi Pills, grind it into powder, place it in a 25 mL container, add 25% methanol, ultrasonically treat it for 30 minutes, take it out, cool it, shake it to volume, and filter it with a 0.45 μm microporous filter membrane to obtain the test solution.

6. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S4, the mass concentrations of rehmannia glutinosa D, phellodendronine hydrochloride, berberine hydrochloride, and hyperoside in the mixed reference solution are 0.1029 mg / mL, 0.0547 mg / mL, 0.0505 mg / mL, and 0.0419 mg / mL, respectively.

7. The HPLC fingerprint detection method of the compound Baiqi pill according to claim 1, characterized in that: In step S5, the chromatographic conditions are: the chromatographic column is SEVAC TM 18 Opal chromatographic column (250 mm × 4.6 mm, 5 μm), mobile phase was methanol A-0.1% phosphoric acid aqueous solution B, gradient elution, flow rate was 1.0 mL / min, detection wavelength was 210 nm, column temperature was 35 ° C, injection volume was 10 μL.

8. The HPLC fingerprint detection method of the compound Baiqi pills according to claim 1, characterized in that: The elution program was as follows: 0-10 min, 0%-10% mobile phase A; 10-15 min, 10%-20% mobile phase A; 15-25 min, 20%-22% mobile phase A; 25-35 min, 22%-35% mobile phase A; 35-45 min, 35%-40% mobile phase A; 45-60 min, 40%-40% mobile phase A.

9. The HPLC fingerprint detection method of Fufang Baiqi Pills according to claim 1, characterized in that: In step S5, the phellodendrine hydrochloride peak is used as a reference peak.

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