Haemorrhoid-eliminating preparation fingerprint spectrum and construction method thereof

Abstract

The invention discloses a hemorrhoid-eliminating preparation fingerprint spectrum and a construction method thereof, and the construction method comprises the following steps: preparing a test sample solution, and carrying out high performance liquid chromatography detection on the test sample solution, according to the high performance liquid chromatography conditions, an acetonitrile-0. 08%-0.12% phosphoric acid solution is used as a mobile phase, gradient elution is carried out, and the detection wavelength is 200 nm to 280 nm. The fingerprint analysis method established by the invention is strong in specificity and high in sensitivity, can be used for comprehensively monitoring the whole substance component group of the Xiaoling preparation and monitoring the quality of the Xiaoling preparation, and provides a scientific basis for standardized research of the Xiaoling preparation and further improvement of the quality standard of the Xiaoling preparation.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine detection, and in particular, relates to a method for constructing a fingerprint spectrum of a Xiaozhiling preparation, and the fingerprint spectrum constructed thereby. Background Art

[0002] Xiaozhiling tablets (produced by Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd., the standard word of national medicine Z44022857) have the effects of clearing away heat and removing dampness, removing toxic substances and reducing swelling, dispersing stagnation and relieving pain, cooling blood and stopping bleeding, and are composed of Chinese gallnut, Radix Atractylodes, Radix Selaginellae, Radix Sanguisorbae, Flos Sophorae, and cholic acid. Its preparation method and technique are as follows: take Chinese gallnut, Radix Atractylodes, Radix Selaginellae, Radix Sanguisorbae and Flos Sophorae and decoct with water three times, each 2 hours, merge decoction, filter, filtrate is concentrated into appropriate amount, adds ethanol and leaves standstill 24h, filters, reclaims ethanol, concentrating and drying, pulverizes, and is standby. Separately get cholic acid, add auxiliary materials such as sucrose, low-substituted hydroxypropyl cellulose, mix homogeneously with above-mentioned cream powder, add appropriate amount of silicon dioxide and magnesium stearate, be pressed into tablets, bag sugar coating or film coating, to obtain final product.

[0003] Existing research on Xiaozhiling tablets primarily focuses on individual components. Qin Yiqiang et al. determined the gallic acid content in Xiaozhiling tablets using RP-HPLC, while Wang Wei et al. and Ma Lingyun et al. determined the content of monomeric bile acids using RP-HPLC and HPLC-ELSD. However, there are few reports on the identity of the medicinal ingredients in Xiaozhiling preparations, making comprehensive and holistic quality control and evaluation of Xiaozhiling preparations difficult.

[0004] HPLC-DAD fingerprinting technology is a rapid, accurate, and comprehensive quality testing method. Fingerprinting technology enables comprehensive quality control of traditional Chinese medicines, overcoming the limitations of single-ingredient testing. It is one of the core technologies for the modernization and internationalization of traditional Chinese medicine. Currently, fingerprinting technology has been widely used in the field of traditional Chinese medicine. For example, Shan Haitao et al. combined gradient elution (acetonitrile-acetic acid water system) with multiple reference substances (such as epimedium and icariin) to calibrate 13 characteristic peaks of a traditional Chinese medicine compound containing epimedium at a wavelength of 230 nm, clarifying their corresponding relationship with the medicinal material and improving the specificity of quality control. Wang Xianghong et al. used HPLC-DAD detection to construct a fingerprint of Baoji Pills, clarifying the attribution of 22 chemical components.

[0005] On the one hand, current HPLC-DAD detection technology, due to detector sensitivity limitations, has insufficient component coverage, and low-content components (such as trace glycosides and volatile components) may not be detected, resulting in missing chromatographic information. On the other hand, data interpretation of this technology relies on experience, and the attribution of characteristic peaks requires the support of a large number of reference substances. However, many traditional Chinese medicine ingredients lack standards, and the attribution of some peaks can only be inferred by retention time, which limits accuracy. Furthermore, Xiaozhiling tablets contain multiple chemical components such as tannins, flavonoids, and terpenes, with a wide range of polarity, requiring the design of a complex gradient elution program for successful HPLC-DAD detection. Therefore, no HPLC-DAD fingerprint of Xiaozhiling tablets has been reported. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to provide a method for constructing a fingerprint of Xiaozhiling tablets, which can comprehensively and accurately reflect the overall chemical composition of Xiaozhiling tablets, improve the accuracy and comprehensiveness of the quality control of Xiaozhiling tablets, and be more conducive to comprehensive and holistic quality control and evaluation of Xiaozhiling tablets.

[0007] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0008] The first aspect of the present invention provides a method for constructing a fingerprint of a Xiaozhiling preparation, comprising the following steps: preparing a test solution, and then subjecting the test solution to high performance liquid chromatography detection; the high performance liquid chromatography conditions include: mobile phase: mobile phase A is acetonitrile, and mobile phase B is 0.08% to 0.12% phosphoric acid solution; gradient elution, the gradient elution program including: 0 min, 5% mobile phase A; 0 min→0.5 min, 5%→10% mobile phase A; 0.5 min→21 min, 10% mobile phase A; 21 min→61 min, 10%→30% mobile phase A; 61 min→74 min, 30%→55% mobile phase A; 74 min→77 min, 55%→100% mobile phase A; 77 min→79 min, 100% mobile phase A; 79 min→80 min, 100%→5% mobile phase A; detection wavelength: 200 nm to 280 nm.

[0009] The second aspect of the present invention provides a fingerprint of the Xiaozhiling preparation constructed by the above-mentioned construction method.

[0010] In the present invention, 10 batches of Xiaozhiling tablet samples were tested using a DAD detector and a 210nm / 254nm UV detection wavelength switch under a specific mobile phase and elution schedule to obtain HPLC-DAD chromatograms of Xiaozhiling tablets. These chromatograms were compared with those of the raw materials (gallanthus gallnut, ampelopsis radix, selaginella, sanguisorba officinalis, and sophora japonica) to identify 31 common characteristic peaks of the Xiaozhiling preparation. These common characteristic peaks constitute the standard fingerprints of the Xiaozhiling preparation. The fingerprint analysis method established by the present invention has strong specificity and high sensitivity, can be used to comprehensively monitor the overall material composition of the Xiaozhiling preparation, monitor its quality, and provide a scientific basis for the standardization research of the Xiaozhiling preparation and further improvement of the quality standard of the Xiaozhiling preparation.

[0011] Furthermore, by comparing chromatographic peaks with those of reference substances, the present invention identified oleanolic acid in the Xiaozhiling preparation as belonging to Galla chinensis, Bletilla striata, Selaginella truncatula, and Sanguisorba officinalis; gallic acid as belonging to Galla chinensis, Bletilla striata, and Sanguisorba officinalis; rutin as belonging to Sophora japonica and Selaginella truncatula; quercetin as belonging to Sophora japonica and Sanguisorba officinalis; and aconitol as belonging to Selaginella truncatula, as well as a monomeric bile acid component. The present invention, by combining the inference of characteristic peaks from individual medicinal materials and standard substances, facilitates comprehensive quality control and evaluation of Xiaozhiling tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is the comparison graph (top) and fingerprint graph (bottom) of 10 batches of Xiaozhiling tablets under the detection condition of 210nm in Example 1 of the present invention.

[0013] Figure 2 The comparison graph (top) and fingerprint graph (bottom) of 10 batches of Xiaozhiling tablets under the detection condition of 254nm in Example 1 of the present invention.

[0014] Figure 3 This is a partial magnified view (210 nm) of the common peak of Xiaozhiling tablets and single medicinal materials in Example 1 of the present invention; wherein, A: overview view; B: partial magnified view from 2 min to 12 min; C: partial magnified view from 10 min to 70 min.

[0015] Figure 4 The chromatogram (210 nm) of Xiaozhiling Tablets and a mixed reference substance in Example 1 of the present invention is shown; wherein, 1: oleanolic acid, 3: gallic acid, 17: rutin, 24: quercetin, 27: aconitum biflavonoids, and 32: cholic acid.

[0016] Figure 5 This is a partial magnified view (254 nm) of the common peak of Xiaozhiling Tablets and single medicinal materials in Example 1 of the present invention; wherein, A: overview view; B: partial magnified view.

[0017] Figure 6The chromatogram (254 nm) of Xiaozhiling Tablets and a mixed reference substance in Example 1 of the present invention is shown; wherein, 4: gallic acid, 13: rutin, 19: quercetin, and 20: aconitum biflavonoids.

[0018] Figure 7 This is a chromatogram of the bile acid reference substance when different mobile phases (A methanol-0.1% phosphoric acid / B acetonitrile-0.1% phosphoric acid) are used in Example 3 of the present invention.

[0019] Figure 8 This is the HPLC chromatogram of Xiaozhiling tablets under elution procedure 1 in Example 4 of the present invention.

[0020] Figure 9 The HPLC chromatogram of Xiaozhiling tablets under elution procedure 2 in Example 4 of the present invention is shown.

[0021] Figure 10 The HPLC chromatogram of Xiaozhiling tablets under elution procedure 3 in Example 4 of the present invention is shown.

[0022] Figure 11 The number of chromatographic peaks (254 nm) of the Xiaozhiling tablets extracted with different extraction solvents in Example 5 of the present invention.

[0023] Figure 12 This is the chromatogram (254 nm) of Xiaozhiling tablets extracted with different extraction solvents in Example 5 of the present invention. DETAILED DESCRIPTION

[0024] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0026] Unless otherwise specified, all examples were performed according to conventional experimental conditions, such as those in Sambrook et al. Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2013), or the conditions recommended by the manufacturer's instructions.

[0027] In some embodiments of the present invention, a method for establishing a fingerprint of a Xiaozhiling preparation is disclosed, comprising the following steps: preparing a test solution, and then subjecting the test solution to high performance liquid chromatography (HPLC) testing; the HPLC conditions include:

[0028] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.08% to 0.12% phosphoric acid solution;

[0029] Gradient elution, the gradient elution program includes: 0 min, 5% mobile phase A; 0 min → 0.5 min, 5% → 10% mobile phase A; 0.5 min → 21 min, 10% mobile phase A; 21 min → 61 min, 10% → 30% mobile phase A; 61 min → 74 min, 30% → 55% mobile phase A; 74 min → 77 min, 55% → 100% mobile phase A; 77 min → 79 min, 100% mobile phase A; 79 min → 80 min, 100% → 5% mobile phase A;

[0030] Detection wavelength: 200nm~280nm.

[0031] The present invention adopts an acetonitrile-0.08% to 0.12% phosphoric acid solution as a mobile phase, thereby ensuring a stable baseline of the chromatogram, identifying bile acid chromatographic peaks, and achieving good separation. Furthermore, a uniquely designed gradient elution program ensures a reasonable distribution of chromatographic peaks and good separation. Furthermore, a multi-wavelength switching method is adopted for detection, thereby covering different types of chemical components of the Xiaozhiling preparation to the greatest extent.

[0032] In some embodiments, the test solution is prepared using 50% to 90% methanol as the extraction solvent. Using 50% to 90% methanol as the extraction solvent to prepare the test solution yields a greater number of chromatographic peaks, extracts a greater number of components, generates rich characteristic peak information, and has a greater sum of the characteristic peak areas.

[0033] In some embodiments, the concentration of methanol is 65% to 90%.

[0034] In some embodiments, the concentration of methanol is 65% to 75%.

[0035] In some embodiments, the preparation method of the test sample includes the following steps: removing the Zhiling preparation, adding 50% to 90% methanol, ultrasonic treatment, cooling, adding 50% to 90% methanol to make up the weight, shaking well, filtering, and obtaining the product.

[0036] In some embodiments, the ultrasonic treatment has a power of 310W to 330W, a frequency of 30kHz to 40kHz, and a time of 15min to 25min.

[0037] In some embodiments, the dosage ratio of the Xiaozhiling preparation to 50% to 90% methanol is 0.38 g to 0.42 g: 50 mL.

[0038] In some embodiments, the HPLC conditions further include: the chromatographic column is C 18 Chromatographic column; flow rate is 0.8mL / min~1.2mL / min; column temperature is 28℃~32℃.

[0039] In some embodiments, the chromatographic column is XBridge C 18 The size of the chromatographic column is 4.6 mm × 250 mm, 5 μm.

[0040] In some embodiments, the detection wavelength is 210 nm and 254 nm.

[0041] In some embodiments, the detection method further includes the step of performing high performance liquid chromatography on the mixed reference solution, and the preparation method of the mixed reference solution includes the following steps: accurately weighing a bile acid reference, a gallic acid reference, a sedge flavonoid reference, a rutin reference, an oleanolic acid reference, and a quercetin reference, dissolving them in methanol, and making a fixed volume to prepare a mixed reference solution with concentrations of 5 mg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 30 μg / mL, and 30 μg / mL, respectively.

[0042] In some embodiments, the Xiaozhiling preparation is made from raw materials including Galla chinensis, Bletilla striata, Selaginella truncatula, Sanguisorba officinalis, Sophora japonica flower, and cholic acid.

[0043] In some embodiments, the Xiaozhiling preparation is Xiaozhiling tablets.

[0044] In other embodiments of the present invention, the fingerprint of the Xiaozhiling preparation constructed by the above construction method is disclosed.

[0045] In the following examples, the instruments used included an Ultimate 3000 high performance liquid chromatograph (Dionex, USA, LPG-3400SDN quaternary pump, SRD-3600 degasser, WPS-3000SL autosampler, TCC3000-RS column oven, DAD detector, Chromeleon 6.8 data processing software); and a Waters XBridge C18 chromatographic column (4.6 mm × 250 mm, 5 μm).

[0046] In the following examples, the Xiaozhiling tablets used as reagents included 10 batches of finished products, all provided by Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd. Oleanolic acid reference substance (Batch No.: 110709-202109), bile acid reference substance (Batch No.: 100078-202216), gallic acid reference substance (Batch No.: 110831-202407), schizonepeta biflavonoids reference substance (Batch No.: 111902-202304), quercetin reference substance (Batch No.: 100081-201610) and rutin reference substance (Batch No.: 100080-202012) were all purchased from the China Food and Drug Administration.

[0047] In the following examples, acetonitrile used as a liquid chromatography reagent was of chromatographic grade, the other reagents used were of analytical grade, and water was ultrapure water.

[0048] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0049] Example 1 Method for constructing the fingerprint of Xiaozhiling tablets

[0050] The following steps are involved:

[0051] 1. Solution preparation

[0052] (1) Preparation of reference solution

[0053] Take an appropriate amount of bile acid reference substance, accurately weigh it, and add methanol to prepare a 10 mg / mL stock solution; take an appropriate amount of gallic acid, aconitum biflavonoids, and rutin reference substances, accurately weigh them, and each add methanol to prepare a 1 mg / mL stock solution; take an appropriate amount of oleanolic acid and quercetin reference substances, accurately weigh them, and each add methanol to prepare a 0.3 mg / mL stock solution;

[0054] An appropriate amount of the above mother solution was taken to prepare a mixed reference solution containing 5 mg / mL of bile acid, 100 μg / mL of gallic acid, 100 μg / mL of aconitum biflavonoids, 100 μg / mL of rutin, 30 μg / mL of oleanolic acid, and 30 μg / mL of quercetin.

[0055] (2) Preparation of Xiaozhiling Tablets Test Solution

[0056] Remove 10 tablets of Zhiling tablets, remove the coating, grind them into powder, take the weight equivalent to 1 tablet (0.40g), accurately weigh, place in a stoppered conical flask, accurately add 50mL of 70% methanol, stopper, weigh, ultrasonically treat (power 320W, frequency 35kHz) for 20min, let cool, weigh again, make up the lost weight with 70% methanol, shake well, filter, and obtain.

[0057] (3) Preparation of single herbal test solution

[0058] Weigh 5 g of Galla chinensis, 25 g each of Bletilla striata, Selaginella truncatula, Sanguisorba officinalis and Sophora japonica, add water and boil them three times for 2 hours each time, filter and concentrate to about 100 mL, take an appropriate amount of concentrate to a 25 mL volumetric flask, add 70% methanol to make up to volume, cover the bottle with a stopper and shake well, filter with a 0.22 μm microporous membrane, and take the filtrate to obtain the single medicinal material test solution.

[0059] 2. High performance liquid chromatography analysis

[0060] Accurately pipette 10 μL each of the mixed reference solution, Xiaozhiling tablets test solution, and single herbal medicine test solution and inject the sample; obtain a high performance liquid chromatogram.

[0061] The chromatographic conditions include:

[0062] Chromatographic column: Waters XBridge C18 (4.6 mm × 250 mm, 5 μm);

[0063] Mobile phase: Acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B

[0064] Gradient elution program: 0 min, 5% mobile phase A;

[0065] 0 min → 0.5 min, 5% → 10% mobile phase A;

[0066] 0.5 min → 21 min, 10% mobile phase A;

[0067] 21 min → 61 min, 10% → 30% mobile phase A;

[0068] 61 min → 74 min, 30% → 55% mobile phase A;

[0069] 74 min → 77 min, 55% → 100% mobile phase A;

[0070] 77 min → 79 min, 100% mobile phase A;

[0071] 79 min → 80 min, 100% → 5% mobile phase A;

[0072] Detection wavelength: 210 nm and 254 nm (DAD detector, 210 nm and 254 nm switching detection);

[0073] Flow rate: 1 mL / min;

[0074] Column temperature 30℃.

[0075] 3. Common Peak Identification and Attribution

[0076] The HPLC chromatograms of the 10 batches of Xiaozhiling tablets were compared using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) to determine the common characteristic peaks and obtain the common pattern (reference spectrum). Under the 210nm detection condition, a total of 32 common chromatographic peaks were obtained ( Figure 1 ), of which 27 common peaks have been found to belong to medicinal materials and are considered as characteristic peaks of Xiaozhiling tablets; under the detection condition of 254nm, a total of 20 common peaks were obtained ( Figure 2 ), among which 4 common peaks are newly detected characteristic peaks under the detection condition of this wavelength; the above 31 characteristic peaks (27+4) constitute the fingerprint characteristics of Xiaozhiling tablets, that is, the fingerprint spectrum of Xiaozhiling tablets.

[0077] The overall similarity of the fingerprints of 10 batches of Xiaozhiling tablets was calculated, and the results are shown in Tables 1 and 2.

[0078] Table 110 Similarity evaluation results of fingerprints of batches of Xiaozhiling tablets (210nm)

[0079]

[0080] Table 210 Similarity evaluation results of the fingerprints of batches of Xiaozhiling tablets (254nm)

[0081]

[0082]

[0083] The similarities between the fingerprints of 10 batches of Xiaozhiling tablets and the control fingerprint were all greater than 0.9, indicating that the 10 batches of Xiaozhiling tablets had good correlation and relatively stable quality.

[0084] The specific analysis is as follows:

[0085] (1) The chromatograms of the single herbal medicine sample solution and the Xiaozhiling tablets sample solution (batch number: 8240601) were analyzed under the detection condition of 210 nm. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that under the detection condition of 210 nm, a total of 32 common peaks were obtained.

[0086] The summary results of the comparison between the test solution of Xiaozhiling tablets and the test solution of single medicinal materials are shown in Table 3.

[0087] Table 3 Identification and attribution of common peaks in the chromatogram of Xiaozhiling tablets (210 nm)

[0088]

[0089]

[0090] From the results in Table 3, we can see that 27 of the 32 common peaks in the chromatogram of Xiaozhiling tablets are attributed to Galla chinensis, Bletilla striata, Selaginella tataricus, Sanguisorba officinalis, Sophora japonica and cholic acid, among which Peak 1, Peak 3, Peak 7, Peak 9 and Peak 21 are attributed to Galla chinensis; Peak 1, Peak 2, Peak 6 and Peak 7 are attributed to Bletilla striata; Peak 1, Peak 2, Peak 4, Peak 7, Peak 8, Peak 11, Peak 17, Peak 22, Peak 27 and Peak 28 are attributed to Selaginella tataricus; Peak 1, Peak 3, Peak 4, Peak 6, Peak 7, Peak 12, Peak 15 and Peak 24 are attributed to Sanguisorba officinalis; Peak 8, Peaks 13 to 20 and Peaks 22 to 25 are attributed to Sophora japonica. In addition, peaks 1, 3, 17, 24, 27 and 32 can be identified as oleanolic acid, gallic acid, rutin, quercetin, aconitum biflavonoids and cholic acid, respectively, by comparison with reference substances (see Figure 4 ).

[0091] (2) The chromatograms of the single herbal medicine sample solution and the Xiaozhiling tablets sample solution (batch number: 8240601) were analyzed under the detection condition of 254 nm. The results are as follows: Figure 5 As shown. Figure 5 It can be seen that under the detection condition of 254 nm, a total of 20 common peaks were obtained.

[0092] The summary results of the comparison between the test solution of Xiaozhiling tablets and the test solution of single medicinal materials are shown in Table 4.

[0093] Table 4 Identification and attribution of common peaks of chromatographic peaks (254 nm) of Xiaozhiling tablets

[0094]

[0095] Note: * indicates a newly detected chromatographic peak at 254 nm that can be attributed to the raw medicinal materials.

[0096] As shown in Table 4, excluding peaks 3 and 6, the remaining 18 shared peaks can be identified as medicinal herbs. Peaks 1, 4, and 12 are assigned to Galla chinensis; peaks 2, 4, and 12 to Bletilla striata; peaks 2, 5, 8, 13, and 20 to Selaginella; peaks 4, 7, and 19 to Sanguisorba officinalis; and peaks 1, 5, 9–11, and 13–19 to Sophora japonica. Further analysis revealed that six peaks, namely peaks 1, 2, 3, 6, 8, 12, and 14, were newly detected shared peaks at 254 nm. Four peaks, namely peaks 1, 8, 12, and 14, can be identified as medicinal herbs and are characteristic peaks newly detected at 254 nm for Xiaozhiling tablets. In addition, peaks 4, 13, 19 and 20 can be identified as gallic acid, rutin, quercetin and aconitum biflavonoids respectively by comparison with reference substances (see Figure 6 ).

[0097] Example 2 Methodology Verification

[0098] 1. Precision test

[0099] The same Xiaozhiling tablet test solution was injected 6 times continuously, and the chromatogram was recorded. The chromatogram was evaluated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition). The results are shown in Table 5.

[0100] Table 5

[0101] Precision 1 Precision 2 Precision 3 Precision 4 Precision 5 Precision 6 Precision 1 1.000 1.000 1.000 1.000 1.000 1.000 Precision 2 1.000 1.000 1.000 1.000 1.000 1.000 Precision 3 1.000 1.000 1.000 1.000 1.000 1.000 Precision 4 1.000 1.000 1.000 1.000 1.000 1.000 Precision 5 1.000 1.000 1.000 1.000 1.000 1.000 Precision 6 1.000 1.000 1.000 1.000 1.000 1.000

[0102] As can be seen from Table 5, the similarities are all greater than 0.99, which shows that the precision of the instrument is good.

[0103] 2. Stability inspection

[0104] The same Xiaozhiling tablet test solution was taken and sampled and analyzed at 0, 2, 4, 6, 8, 12, 24, and 48 hours, respectively. The chromatograms were recorded and evaluated using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition). The results are shown in Table 6.

[0105] Table 6

[0106]

[0107] As can be seen from Table 6, the similarities are all greater than 0.99, indicating that the test solution is stable within 48 hours.

[0108] 3. Repeatability test

[0109] Take 6 portions of the same batch of Xiaozhiling tablets, prepare them according to the test solution preparation method of Example 1, inject and analyze, record the chromatogram, and evaluate them using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition). The results are shown in Table 7.

[0110] Table 7

[0111]

[0112]

[0113] As shown in Table 7, the similarities are all greater than 0.99, indicating that the method has good repeatability.

[0114] Example 3 Comparison of the effects of different mobile phases on fingerprint construction

[0115] A 10 mg / mL bile acid reference stock solution was diluted two-fold with 70% methanol to obtain a 5 mg / mL bile acid reference solution. The bile acid reference solution was injected and tested using either methanol-0.1% phosphoric acid solution or acetonitrile-0.1% phosphoric acid solution as the mobile phases, respectively, at a wavelength of 192 nm (the wavelength of maximum bile acid absorption). Due to the varying elution capacities of methanol and acetonitrile, a commonly used elution procedure was employed for each mobile phase.

[0116] When the mobile phase is methanol (A)-0.1% phosphoric acid solution (B), the elution program is: 0 min, 5% mobile phase A; 0 min→2.5 min, 5%→9% mobile phase A; 2.5 min→21 min, 9%→35% mobile phase A; 21 min→45 min, 35%→48% mobile phase A; 45 min→50 min, 48%→70% mobile phase A; 50 min→57 min, 70%→100% mobile phase A; 57 min→58 min, 100%→5% mobile phase A; 58 min→60 min, 5% mobile phase A.

[0117] When the mobile phase is acetonitrile (A)-0.1% phosphoric acid solution (B), the elution program is: 0 min→7 min, 10% mobile phase A; 7 min→15 min, 10%→21% mobile phase A; 15 min→33 min, 21%→29% mobile phase A; 33 min→36 min, 29%-40% mobile phase A; 36 min→45 min, 40%→45% mobile phase A; 45 min→55 min, 45%→100% mobile phase A; 55 min→56 min, 100%→5% mobile phase A; 56 min→60 min, 5% mobile phase A.

[0118] Chromatograms under different mobile phases Figure 7 The results showed that the bile acid chromatographic peak could not be identified even under the optimal elution procedure using a mobile phase of methanol-0.1% phosphoric acid, and the baseline was not flat ( Figure 7 A), using acetonitrile-0.1% phosphoric acid as the mobile phase, the bile acid chromatographic peak can be identified with good separation and a relatively stable baseline ( Figure 7 In B), we analyzed that this may be due to the strong UV absorption of methanol at low wavelengths, which leads to increased baseline noise, possibly masking the signal of the target compound and reducing the detection sensitivity. Therefore, methanol-0.1% phosphoric acid is not suitable as a mobile phase for the detection of bile acid.

[0119] In addition, when acetonitrile-0.1% phosphoric acid was used as the mobile phase, although the response of bile acid was high and the baseline was relatively stable, the baseline was still high. In subsequent experiments, we increased the detection wavelength to 210 nm (and combined it with other chromatographic conditions). Although the response of bile acid was slightly lower, the baseline became more stable (e.g. Figure 1 ).

[0120] Example 4 Comparison of the effects of different elution procedures on fingerprint construction

[0121] The same Xiaozhiling tablet test solution was taken and sampled for detection. Except for the different elution procedures (3 in total), other chromatographic conditions (detection wavelength 254 nm) were the same as in Example 1.

[0122] 1. Elution procedure 1 is shown in Table 8, and the chromatogram is shown in Figure 8 shown.

[0123] Table 8 Elution procedure 1

[0124] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 2.5 9 91 5.5 11 89 21 35 65 45 48 52 50 70 30 55 95 5 57 100 0 58 5 95 60 5 95

[0125] from Figure 8 It can be seen that under the conditions of elution procedure 1, the obtained chromatographic peaks eluted too early, most of the chromatographic peaks were concentrated before 30 minutes, the overall chromatographic peak distribution was unreasonable, and the separation was poor.

[0126] 2. Elution procedure 2 is shown in Table 9, and the chromatogram is shown in Figure 9 shown.

[0127] Table 9 Elution procedure 2

[0128]

[0129]

[0130] from Figure 9 It can be seen that under the conditions of elution procedure 2, the obtained chromatographic peak is between 15 and 20 minutes, the chromatographic peak is too concentrated, the distribution is unreasonable, and the separation is poor.

[0131] 3. Elution procedure 3 is shown in Table 10, and the chromatogram is shown in Figure 10 shown.

[0132] Table 10 Elution program 3

[0133] Time (min) Mobile phase A (%) Mobile phase B (%) 0 5 95 0.5 10 90 21 10 90 61 30 70 74 55 45 77 100 0 79 100 0 80 5 95

[0134] The results are as follows Figure 10 As shown, under elution procedure 3 (i.e., the elution procedure of Example 1), the chromatographic peaks are reasonably distributed and the separation of the chromatographic peaks is good.

[0135] Example 5 Comparison of the effects of different extraction solvents on fingerprint construction

[0136] Remove 10 tablets of Zhiling tablets, remove the coating, grind them into powder, take the weight equivalent to 1 tablet (0.40 g), accurately weigh, place in a stoppered conical flask, accurately add 50 mL of different solvents (water, 50% methanol, 70% methanol and 90% methanol), stopper, weigh, ultrasonically treat (power 320 W, frequency 35 kHz) for 20 min, cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and obtain the test solution.

[0137] The sample was injected and detected under the same chromatographic conditions as in Example 1 (detection wavelength 254 nm).

[0138] The number of chromatographic peaks extracted by the four solvents is as follows Figure 11 The specific chromatogram is shown in Figure 12 When water was used for extraction, 45 chromatographic peaks were obtained; when 50% methanol was used for extraction, 53 chromatographic peaks were obtained; when 70% methanol was used for extraction, 61 chromatographic peaks were obtained; when 90% methanol was used for extraction, 59 chromatographic peaks were obtained ( Figure 11 ); It can be seen that when 70% methanol is used as the extraction solvent, the number of chromatographic peaks obtained is the largest, indicating that the extracted components are the largest and the sum of the areas of all characteristic peaks is the largest. Therefore, 70% methanol is the best extraction solvent and the characteristic peak information obtained is the richest.

[0139] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0140] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A method for constructing a fingerprint of a Xiaozhiling preparation, characterized in that: The following steps are involved: Prepare a test solution, and then perform high performance liquid chromatography on the test solution; the high performance liquid chromatography conditions include: Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.08% to 0.12% phosphoric acid solution; Gradient elution, the gradient elution program includes: 0 min, 5% mobile phase A; 0 min → 0.5 min, 5% → 10% mobile phase A; 0.5 min → 21 min, 10% mobile phase A; 21 min → 61 min, 10% → 30% mobile phase A; 61 min → 74 min, 30% → 55% mobile phase A; 74 min → 77 min, 55% → 100% mobile phase A; 77 min → 79 min, 100% mobile phase A; 79 min → 80 min, 100% → 5% mobile phase A; Detection wavelength: 200nm~280nm.

2. The method for constructing the fingerprint of the Xiaozhiling preparation according to claim 1, wherein: The test solution is prepared by using 50% to 90% methanol as an extraction solvent.

3. The method for constructing the fingerprint of the Xiaozhiling preparation according to claim 2, wherein: The concentration of the methanol is 65% to 90%, preferably 65% to 75%.

4. The method for constructing the fingerprint of the Xiaozhiling preparation according to claim 1, wherein: The preparation method of the test sample comprises the following steps: removing the Zhiling preparation, adding 50% to 90% methanol, ultrasonically treating, cooling, adding 50% to 90% methanol to make up the weight, shaking well, filtering, and obtaining the test sample.

5. The method for constructing the fingerprint of the Xiaozhiling preparation according to claim 4, wherein: The dosage ratio of the Xiaozhiling preparation to 50% to 90% methanol is 0.38g to 0.42g:50mL.

6. The method for constructing the fingerprint of the Xiaozhiling preparation according to claim 4, characterized in that: The ultrasonic treatment has a power of 310W to 330W, a frequency of 30kHz to 40kHz, and a time of 15min to 25min.

7. The method for constructing the fingerprint of Xiaozhiling preparation according to claim 1, characterized in that: The high performance liquid chromatography conditions also include: the chromatographic column is C 18 Chromatographic column; flow rate of 0.8mL / min~1.2mL / min; column temperature 28℃~32℃; preferably, the chromatographic column is XBridge C 18 , the size of the chromatographic column is 4.6 mm × 250 mm, 5 μm; And / or, the detection wavelength is 210 nm and 254 nm.

8. The method for constructing the fingerprint of the Xiaozhiling preparation according to any one of claims 1 to 7, characterized in that: The detection method also includes the step of performing high performance liquid chromatography detection on a mixed reference solution. The preparation method of the mixed reference solution includes the following steps: accurately weighing a bile acid reference, a gallic acid reference, a schizonepeta biflavonoid reference, a rutin reference, an oleanolic acid reference, and a quercetin reference, dissolving them in methanol, and constant volume to prepare mixed reference solutions with concentrations of 5 mg / mL, 100 μg / mL, 100 μg / mL, 100 μg / mL, 30 μg / mL, and 30 μg / mL, respectively.

9. The method for constructing the fingerprint of the Xiaozhiling preparation according to any one of claims 1 to 7, characterized in that: The Xiaozhiling preparation is made from raw materials including Galla chinensis, Ampelopsis pilosula, Selaginella chinensis, Sanguisorba officinalis, Sophora japonica flower, and cholic acid; preferably, the Xiaozhiling preparation is Xiaozhiling tablets.

10. The fingerprint of the Xiaozhiling preparation constructed by the construction method according to any one of claims 1 to 9.

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