Construction method of fingerprint spectrum of Zhongtongan and standard fingerprint spectrum

Abstract

The present invention relates to a method for constructing a fingerprint of Zhongtongan and a standard fingerprint, and the construction method includes: S1, taking several batches of Zhongtongan capsule samples, dissolving them with 50-100% methanol, and after dissolving, supplementing the weight loss with 50-100% methanol, filtering, and obtaining several test samples; using cimicifugin glycosides, purpurogenol glycosides, 5-O-methylvisaminol glycosides, bergamot glycoside lactones, imperatorin and isoimperatorin as references, and dissolving in methanol to obtain a reference substance; S2, performing high performance liquid chromatography analysis on the test sample and each reference substance, respectively, to obtain the chromatograms of the test sample and each reference substance; S3, determining the common peaks from the chromatogram of the test sample, and comparing and confirming some characteristic peaks with the chromatogram of the reference substance; using the chromatographic peak of purpurogenol glycosides as the reference peak, calculating the relative retention time of each common peak, that is, constructing the fingerprint of Zhongtongan. The present invention can realize the detection and comprehensive evaluation of multiple components of Zhongtongan.

Description

Technical Field

[0001] The invention relates to the technical field of quality control of traditional Chinese medicines, and in particular to a method for constructing a fingerprint spectrum of the traditional Chinese medicine Zhongtongan and a standard fingerprint spectrum. Background Art

[0002] The effects of Zhongtong'an Capsule are to dispel wind and resolve phlegm, promote blood circulation and resolve nodules, reduce swelling and relieve pain. It is used for toothache, sore throat, oral ulcers caused by wind, phlegm and blood stasis, and arthralgia caused by wind, phlegm and blood stasis blocking the collaterals, with symptoms such as swollen and painful joints, cramps of tendons and veins, difficulty in flexion and extension, and as an auxiliary treatment for tetanus.

[0003] Zhongtongan capsules are composed of eight herbs, including Panax notoginseng, Gastrodia elata, Bombyx batryticatus, Rhizoma Aconiti Lateralis (processed), Saposhnikovia divaricata, Rhizoma Notopterygii, Rhizoma Arisaematis (processed), and Angelica dahurica, and have complex chemical compositions. Panax notoginseng in the prescription has the effects of hemostasis, analgesia, detoxification, blood stasis and swelling, and tranquilization, and mainly contains flavonoids, terpenes, alkaloids, phenylpropanoids, and phenolic compounds. Rhizoma Aconiti Lateralis has the effects of dispelling wind and phlegm, calming convulsions, detoxifying and dispersing knots, and relieving pain, and the chemical compositions it contains are mainly lipids, steroids, nitrogen heterocyclics, amino acids, and other compounds. Rhizoma Notopterygii has the effects of clearing heat, resisting rheumatism, and relieving pain, and mainly contains volatile oils, coumarins, phenolic acids, and alkaloid compounds. Rhizoma Arisaematis has the effects of dispersing knots and swelling, drying dampness and resolving phlegm, dispelling wind and calming convulsions, and mainly contains flavonoids, alkaloids, glycosides, esters, lignans, terpenes, sterols, and other chemical compositions. Saposhnikovia divaricata has the effects of dispelling wind, removing dampness, relieving pain and relieving spasm, and its chemical components mainly include volatile oils, chromones and coumarins. Bombyx batryticatus has the effects of calming wind and relieving spasm, dispelling wind and relieving pain, and resolving phlegm and dispersing stagnation, and it mainly contains chemical components such as protein polypeptides, organic acids, flavonoids, and steroids. Angelica dahurica has the effects of dispelling cold, dispelling wind and relieving pain, clearing the nasal cavity, drying dampness and stopping leukorrhea, and reducing swelling and draining pus, and it mainly contains chemical components such as coumarins, volatile oils, polysaccharides, alkaloids, and amino acids. Gastrodia elata has the effects of calming wind and relieving spasm, calming liver yang, and dispelling wind and unblocking collaterals, and it mainly contains barresonides and Gastrodia elata polysaccharide compounds.

[0004] At present, there are many studies on Zhongtong'an Capsules, most of which are clinical studies on Zhongtong'an Capsules combined with other drugs for erosive oral lichen planus, gingivitis, oral ulcers and other diseases. Jiang Yan et al. established an HPLC method to determine the content of five components in Zhongtong'an Capsules, namely, gastrodin, cimicifuga glycoside, 5-O-methylvisaminol glycoside, imperatorin and isoimperatorin; in addition, an HPLC method was established to determine the content of notoginseng saponin R1, ginsenoside Rg1 and ginsenoside Rb1 in Zhongtong'an Capsules. However, the existing technology lacks standards for effective overall control of multiple active ingredients in Zhongtong'an Capsules.

[0005] The ingredients of Zhongtong'an Capsules are complex. It is difficult to fully control the intrinsic quality of drugs by simply qualitative identification and quantitative analysis of index ingredients. Its quality standards should strengthen specific identification and multi-component and overall quality control. Therefore, it is necessary to establish an analytical method for characteristic spectra that can fully reflect the quality of Zhongtong'an Capsules, so as to realize the overall detection of multiple components of Zhongtong'an Capsules, achieve quality control, comprehensive evaluation, and effectively control the clinical efficacy and safety of drugs. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a method for constructing a fingerprint of Zhongtong'an and a standard fingerprint. The characteristic spectrum constructed by the present invention is used to evaluate the quality of Zhongtong'an capsules. It has good durability, precision, stability and repeatability, and can comprehensively and effectively evaluate the quality of Zhongtong'an capsules, solving the technical problems that the chemical composition of Zhongtong'an capsules is complex, the chromatographic peaks of each component are easy to interfere with each other, and it is difficult to comprehensively control the quality of the drug.

[0008] (II) Technical solution

[0009] The present invention provides a method for constructing a fingerprint spectrum of Zhongtongan, which comprises:

[0010] S1. Take appropriate amount of the contents of several batches of Zhongtong'an capsules, dissolve them in 50-100% methanol respectively, make up the weight loss with 50-100% methanol after dissolution, filter and obtain several test samples;

[0011] Take cimicifugin glycoside, purpurogenol, 5-O-methylvisaminol glycoside, bergamot glycoside lactone, imperatorin and isoimperatorin, use 50-100% methanol to dissolve, and prepare the reference substances of cimicifugin glycoside, purpurogenol glycoside, 5-O-methylvisaminol glycoside, bergamot glycoside lactone, imperatorin and isoimperatorin respectively;

[0012] S2. Perform high performance liquid chromatography analysis on all test samples and reference samples to obtain chromatograms of the test samples and reference samples respectively;

[0013] The conditions for the HPLC analysis are as follows: the filler of the chromatographic column is octadecyl bonded silica gel, acetonitrile is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B;

[0014] The elution gradient is as follows: by volume fraction, 0 min, mobile phase A is 6%; 10 min, mobile phase A is 11%; 18 min, mobile phase A is 20%; 28 min, mobile phase A is 35%; 32 min, mobile phase A is 35%; 38 min, mobile phase A is 80%; 42 min, mobile phase A is 80%; 43 min, mobile phase A is 6%; 45 min, mobile phase A is 6%;

[0015] S3. Determine the common peaks from the chromatograms of several test samples, compare the common peaks with the chromatograms of the reference samples, and identify the chemical components of some common peaks; use the chromatographic peak of purpurogenol as the reference peak, calculate the relative retention time of each common peak, and thus construct the fingerprint of Zhongtongan.

[0016] According to a preferred embodiment of the present invention, in S1, 0.4-0.6 g of the content of Zhongtong'an Capsule is taken, accurately weighed, placed in a container, 70% methanol is added, the weight is weighed, ultrasonic treatment is performed for 20-40 min, cooled, 70% methanol is added to make up the weight loss, mixed, filtered, and the result is obtained. The ultrasonic power is 250 W and the frequency is 40 kHz.

[0017] According to a preferred embodiment of the present invention, in S1, the preparation method of the reference substance is: respectively take standard substances of cimicifuga glycosides, bergamot glycosides, 5-O-methylvisaminol glycosides, peucedanum glycosides, imperatorin, and isoimperatorin, accurately weigh them, add methanol to prepare solutions containing 60 μg, 1 μg, 6 μg, 60 μg, 6 μg, and 22 μg per 1 mL, respectively.

[0018] That is, every 1mL of cimicifugin glycosides reference substance contains 60μg of the standard substance; every 1mL of bergamot lactones reference substance contains 1μg of the standard substance; every 1mL of 5-O-methylvisaminol glycosides reference substance contains 6μg of the standard substance; every 1mL of purpurogenol glycosides reference substance contains 60μg of the standard substance; every 1mL of imperatorin reference substance contains 6μg of the standard substance; every 1mL of isoimperatorin reference substance contains 22μg of the standard substance.

[0019] According to a preferred embodiment of the present invention, in S2, the chromatographic column for HPLC analysis is a Waters Symmetry C 18 (4.6×250mm, 5μm), Wondasil C 18 (4.6×250mm, 5μm) and Agilent EclipseXDB-C 18 (4.6×250mm, 5μm); the liquid chromatograph can be any one of Waters e2695, Shimadzu and Agilent 1260II.

[0020] According to a preferred embodiment of the present invention, in S2, during the HPLC analysis, the column temperature is 30-40°C, preferably 30°C, 35°C or 40°C, more preferably 35°C.

[0021] According to a preferred embodiment of the present invention, in S2, during the HPLC analysis, the injection flow rate is 0.95-1.05 mL / min, preferably 0.95 mL / min, 1.05 mL / min or 1.00 mL / min, more preferably 1.0 mL / min; the injection volume is 10 μL.

[0022] According to a preferred embodiment of the present invention, in S2, the detection wavelength is 295nm-305nm, preferably 300nm, 305nm or 295nm, and more preferably 300nm.

[0023] According to a preferred embodiment of the present invention, in S3, 15 batches of Zhongtong'an Capsule samples are selected to prepare 15 test samples according to the test sample preparation conditions in step S1, and high performance liquid chromatography analysis is performed respectively to obtain 15 fingerprint spectra, and the chromatographic peaks with good stability and appropriate response values ​​in these fingerprint spectra are determined as common peaks.

[0024] The present invention provides a standard fingerprint of Zhongtong'an Capsule, which is a high performance liquid chromatography characteristic spectrum, with a total of 15 characteristic peaks, and the chromatographic peak of purpurogenol in Peak 6 is used as a reference peak;

[0025] Among them, the relative retention time of peak 1 is 0.1465; the relative retention time of peak 2 is 0.2228; the relative retention time of peak 3 is 0.2786; the relative retention time of peak 4 is 0.5821; peak 5 is cimicifuga glycoside, with a relative retention time of 0.8451; peak 6 is purpurogenol glycoside, with a relative retention time of 1.0000; peak 7 is 5-O-methylvisaminol glycoside, with a relative retention time of 1.0299; peak 8 is relative retention time of 1.0000. The relative retention time of peak 9 is 1.2354; the relative retention time of peak 9 is 1.2807; peak 10 is bergamotolactone, with a relative retention time of 1.5984; peak 11 is with a relative retention time of 1.6607; peak 12 is with a relative retention time of 1.6874; peak 13 is with a relative retention time of 1.7103; peak 14 is imperatorin, with a relative retention time of 1.7316; peak 15 is isoimperatorin, with a relative retention time of 1.7727;

[0026] When the standard fingerprint is used to evaluate the quality of Zhongtong'an Capsules, the similarity between the test sample fingerprint of qualified Zhongtong'an Capsules and the control fingerprint is not less than 0.90 according to the Chinese medicine chromatographic fingerprint similarity evaluation system.

[0027] In this application, % refers to volume percentage unless otherwise specified; for example, 50% methanol means a mixture of methanol and water in a volume ratio of 1:1.

[0028] (III) Beneficial effects

[0029] (1) The present invention uses 50-100% methanol to prepare the test solution, and the number of chromatographic peaks is greater than that of the test solution prepared using ethanol, which is conducive to obtaining a larger number of chromatographic peaks, so as to achieve the overall detection of multiple components of Zhongtongan Capsules; the number of chromatographic peaks of the liquid chromatography obtained by the elution gradient conditions of the present invention is large and the separation is good. The test sample and the single-flavor decoction piece solution are prepared by the test sample preparation method determined by the present invention, and the chromatographic conditions in the present invention are used to obtain the chromatograms of the test sample and the chromatograms of the single-flavor decoction piece solution, respectively, and the chromatographic peaks of the test sample are attributed to the medicinal flavors, and the 15 chromatographic peaks are respectively attributed to 8 medicinal flavors in Zhongtongan Capsules, namely, Panax notoginseng, Saposhnikovia divaricata, Bombyx batryticatus, Rhizoma Aconiti Lateralis, Arisaema consanguineum, Gastrodia elata, Angelica dahurica, and Notopterygium indica. It can be seen that the fingerprint method can perform comprehensive quality control on Zhongtongan Capsules.

[0030] (2) The fingerprint construction method of the present invention has the following advantages:

[0031] ① Inject the test sample, reference solution and blank solvent (70% methanol) into the high performance liquid chromatograph, measure and record the chromatogram. The results show that the blank solvent has no interference, indicating that the instrument has good specificity. ② Take the same batch of zhongtongan capsule samples, prepare the test solution, and inject 6 times continuously. The relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD of the main common peak is less than 3%, indicating that the stability of the test solution is good. ③ Take the same batch of zhongtongan capsule samples, prepare 6 test solutions, inject and record the fingerprint. The relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD of the main common peak is less than 3%, indicating that the chromatographic conditions of the present invention have good repeatability. ④ Good durability: The fingerprint construction method of the present invention has good peak shape and response of each common peak of the sample fingerprint on different brands of chromatographic columns, and has good durability in the column temperature of 30-40°C, injection speed of 0.95-1.05 mL / min and detection wavelength range of 295-305 nm. In addition, the fingerprint conditions also have good durability on different chromatographic instruments such as Waters e2695, Shimadzu, and Agilent 1260II. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A common pattern diagram was formed for the fingerprints of 15 batches of Zhongtong'an Capsules test samples.

[0033] Figure 2The fingerprint of Zhongtong'an Capsule and the fingerprint of the reference substance were positioned and confirmed, where (a) is the fingerprint of the Zhongtong'an Capsule sample, and (b) is the fingerprint of the reference substance.

[0034] Figure 3 This is the control fingerprint of the Zhongtong'an Capsule constructed in Example 1.

[0035] Figure 4 The response results of the extraction time and chromatographic peak when using 70% methanol to prepare the test sample, where (a) is the chromatographic peak of the test sample prepared by extracting Zhongtong'an capsules with 70% methanol for 20 minutes, (b) is the chromatographic peak of the test sample prepared by extracting Zhongtong'an capsules with 70% methanol for 30 minutes, and (c) is the chromatographic peak of the test sample prepared by extracting Zhongtong'an capsules with 70% methanol for 40 minutes.

[0036] Figure 5 The response results of different gradient elution conditions and chromatographic peaks, where (a) is the chromatographic peak produced by elution under gradient condition 1, (b) is the chromatographic peak produced by elution under gradient condition 2, and (c) is the chromatographic peak produced by elution under gradient condition 3.

[0037] Figure 6 In Example 3, different samples (blank solvent, Zhongtong'an capsule sample, reference substance mixed solution) were injected and tested to investigate the instrument specificity, wherein (a) is the chromatographic peak of the blank solvent, (b) is the chromatographic peak of the reference substance, and (c) is the chromatographic peak of the Zhongtong'an capsule sample.

[0038] Figure 7 In Example 3, the effects of different brands of chromatographic columns on the fingerprint spectrum were investigated, wherein (a) is the chromatographic peak produced by a Waters chromatographic column, (b) is the chromatographic peak produced by a Shimadzu chromatographic column, and (c) is the chromatographic peak produced by an Agilent chromatographic column.

[0039] Figure 8 In order to investigate the influence of different column temperatures on the fingerprint spectrum in Example 3, (a) is the chromatographic peak produced by the detection at a column temperature of 35°C, (b) is the chromatographic peak produced by the detection at a column temperature of 30°C, and (c) is the chromatographic peak produced by the detection at a column temperature of 40°C.

[0040] Fig. 9 In order to investigate the influence of different injection speeds on the fingerprint spectrum in Example 3, (a) is the chromatographic peak generated by the injection speed of 1.00 ml / min, (b) is the chromatographic peak generated by the injection speed of 1.05 ml / min, and (c) is the chromatographic peak generated by the injection speed of 0.95 ml / min.

[0041] Fig.10In order to investigate the influence of different detection wavelengths on the fingerprint spectrum in Example 3, (a) is the chromatographic peak generated at a detection wavelength of 300 nm, (b) is the chromatographic peak generated at a detection wavelength of 295 nm, and (c) is the chromatographic peak generated at a detection wavelength of 305 nm. DETAILED DESCRIPTION

[0042] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific embodiments. The instruments, chromatographic columns, reagents, reference substance information, and Zhongtong'an Capsule sample batch numbers used in the following examples are described as follows:

[0043] Instrument: Waters e2695 high performance liquid chromatography system (Waters Corporation, USA); detector: 2998 PDA Detector; electronic analytical balance: TB-215D, BSA224S-CW (Sartorius, Beijing); KQ-250 ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., power 250 W, frequency 40 kHz). Chromatographic column: Waters Symmetry C18 (4.6×250 mm, 5 μm).

[0044] Reagents: Acetonitrile (chromatographic grade) and phosphoric acid (chromatographic grade) were purchased from Fisher, USA; methanol (analytical grade) was purchased from Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.; laboratory ultrapure water came from UPR-II-20L ultrapure water system (Sichuan Youpu Ultrapure Technology Co., Ltd.).

[0045] Reference substance information: Cimicifugin (PS011304), bergamot lactone (PS010563), and 5-O-methylvisaminol glycoside (PS011303) were all purchased from Chengdu Pusi Biotechnology Co., Ltd.; purpurogenol (97.4%, 111821-201303), imperatorin (99.6%, 110826-201616), and isoimperatorin (99.4%, 110827-201611) were all purchased from China Food and Drug Identification Institute. Sample batch numbers of Zhongtong'an Capsules: 240201, 240202, 240203, 240204, 240205, 240206, 240208, 240209, 240210, 240212, 240213, 240214, 240301, 240302, 240304.

[0046] Example 1

[0047] This embodiment provides a method for constructing a fingerprint spectrum of Zhongtongan, and the steps are as follows:

[0048] (1) Take 15 batches of Zhongtong'an capsule samples, cut the capsules open, take out 0.5 g of the drug content, place in a stoppered conical flask, accurately add 25 mL of 70% methanol, weigh, ultrasonically treat (power 250 W, frequency 40 kHz) for 20 min, cool, make up the lost weight with 70% methanol, mix well, filter, and obtain 15 batches of Zhongtong'an capsule test samples.

[0049] (2) Take appropriate amounts of cimicifuga glycosides, bergamot lactones, 5-O-methylvisaminol glycosides, purpurogenol glycosides, imperatorin, and isoimperatorin reference substances, weigh them accurately, and add methanol to prepare solutions containing 60μg, 1μg, 6μg, 60μg, 6μg, and 22μg per ml, respectively, to obtain reference substances. That is, each 1mL of cimicifuga glycoside reference substance contains 60μg of the standard substance; each 1mL of bergamot lactone reference substance contains 1μg of the standard substance; each 1mL of 5-O-methylvisaminol glycosides contains 6μg of the standard substance; each 1mL of purpurogenol glycosides contains 60μg of the standard substance; each 1mL of imperatorin reference substance contains 6μg of the standard substance; each 1mL of isoimperatorin reference substance contains 22μg of the standard substance.

[0050] The peak areas of chromatographic peaks of cimicifugin, purpurogenol, 5-O-methylvisaminol, bergamotol lactone, imperatorin and isoimperatorin account for a large proportion and are relatively stable, so these 6 compounds were selected as references to prepare control substances.

[0051] (3) Accurately pipette 10 μL of each of the reference and test solutions, inject into the liquid chromatograph, and measure to obtain the HPLC chromatograms of the test sample and each reference solution.

[0052] The conditions for HPLC analysis are as follows: Column: Waters Symmetry C 18 (4.6×250mm, 5μm); acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, gradient elution as specified in the following table; flow rate 1.0ml / min; detection wavelength 300nm; column temperature 35℃. The elution gradient system is shown in Table 1:

[0053] Table 1: Elution gradient system

[0054]

[0055] (4) Calibration of common peaks and establishment of reference spectra

[0056] The chromatograms of the 15 batches of test samples were analyzed, and the chromatographic peaks with good stability and appropriate response values ​​in the fingerprints of the 15 batches of Zhongtong'an capsule samples were selected as common peaks. The 2012 edition of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation Software System" issued by the National Pharmacopoeia Committee was used to automatically match the chromatographic peaks of the HPLC spectra of the 15 batches of Zhongtong'an capsule samples to form a common pattern diagram (see Figure 1 ), and a total of 15 common peaks were identified.

[0057] like Figure 2 As shown in FIG. 1 , the chromatogram of the test sample (the chromatogram of the Zhongtong'an capsule sample) was compared with the chromatogram of the reference sample (the chromatogram of the 6 reference sample chromatogram peaks were combined in one spectrum), and the corresponding positions were made according to the retention time of each reference sample chromatogram peak. Six of the 15 common peaks on the chromatogram of the Zhongtong'an capsule sample were identified, and the following was obtained: Figure 3 The fingerprint is shown. In the fingerprint, the six peaks identified are: Peak 5: Cimicifugin glycoside, Peak 6: Purpurogenol glycoside, Peak 7: 5-O-methylvisaminol glycoside, Peak 10: bergamotol lactone, Peak 14: imperatorin, Peak 15: isoimperatorin.

[0058] (5) Calculation of similarity

[0059] Since the retention time of the chromatographic peak of purpurogenol in the six reference substances is moderate and the response value is high, purpurogenol was selected as the reference peak of the reference substance, marked as peak S (peak 6), and its retention time and peak area were defined as 1. The relative retention time and relative peak area of ​​the 15 common peaks of the chromatograms of 15 batches of Zhongtongan Capsules were calculated respectively, as shown in Table 2-3. According to the relative retention time of each peak, the position of each peak on the horizontal axis was adjusted, and the reference fingerprint of Zhongtongan was constructed. Furthermore, the similarity evaluation software system of the chromatographic fingerprint of traditional Chinese medicine was used to calculate the similarity of the 15 batches of Zhongtongan Capsules samples and the reference fingerprint with the common peaks, and the results are shown in Table 4.

[0060] Table 2: Relative retention time, mean and RSD of 15 common peaks in 15 batches of Zhongtong'an capsules

[0061]

[0062] Table 3: Relative retention area, mean and RSD of 15 common peaks in 15 batches of Zhongtong'an capsules

[0063]

[0064] Table 4: Similarity evaluation results of fingerprint spectra of 15 batches of Zhongtong'an capsule samples

[0065]

[0066] It can be seen from Table 2-4 that the similarity between the 15 batches of Zhongtongan Capsule samples and the control fingerprint spectrum is greater than 0.958. The similarity between the Zhongtongan Capsule samples of different batches is good, so the control fingerprint spectrum ( Figure 3 As shown in the figure, the fingerprint of the test sample should show 15 common peaks, and the retention time should be consistent with that of the reference chromatographic peak. According to the similarity evaluation system of Chinese medicine chromatographic fingerprint, the similarity between the test sample fingerprint and the reference fingerprint should not be less than 0.90 based on the common peaks.

[0067] Comparative Example 1

[0068] Under the same chromatographic detection conditions as in Example 1, the preparation method of the test sample was changed, and the extraction effects of 50% methanol, 70% methanol, pure methanol, 50% ethanol, 70% ethanol, and pure ethanol on the Zhongtong'an capsule samples were compared. The results showed that when 50% ethanol, 70% ethanol, and pure ethanol were used as extraction solvents, the number of chromatographic peaks was less than that of methanol of different concentrations; while when 50% methanol, 70% methanol, and pure methanol were used as extraction solvents, there was no significant difference in the number of chromatographic peaks, among which the response was higher when 70% methanol was used as the extraction solvent. Therefore, 70% methanol was selected as the extraction solvent for the fingerprint of Zhongtong'an capsule samples.

[0069] Under the same chromatographic detection conditions as in Example 1, 70% methanol was selected as the extraction solvent for the fingerprint of the Zhongtong'an capsule sample, and the extraction effects of three extraction times, namely 20 min, 30 min, and 40 min, on the sample were compared ( Figure 4 ). The results showed that there was no significant difference in the response and uniformity of the chromatographic peaks under the three extraction times. Therefore, the extraction time of Zhongtong'an Capsule was determined to be 20 minutes.

[0070] Comparative Example 2

[0071] Under the same chromatographic detection conditions and chromatographic columns as in Example 1, the separation effects of the three different gradient conditions on the chromatographic peaks in the test sample were compared ( Figure 5 ). The results show that under the gradient 2 condition, the number and separation of chromatographic peaks are significantly better than the other two gradient conditions. The three different gradient conditions have mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid / water (aqueous solution of phosphoric acid with a volume ratio concentration of 0.1%). The three different gradient conditions are as follows:

[0072] Gradient conditions 1: 0-15 min, 10%→45%A, 15-22 min, 45%→60%A, 22-27 min, 60%A, 27-30min, 60%→90%A, 30-34 min, 90%→10%A, 34-40 min, 10%A;

[0073] Gradient condition 2: 0-10 min, 6%→11%A, 10-18 min, 11%→20%A, 18-28 min, 20%→35%A, 28-32 min, 35%A, 32-38 min, 35%→80%A; 38-42 min, 80%A, 42-43 min, 80%→6%A, 43-45min, 6%A;

[0074] Gradient condition 3: 0-5 min, 10%→20%A, 5-15 min, 20%→35%A, 15-30 min, 35%→60%A, 30-35min, 60%→80%A, 35-40min, 80%A, 40-41min, 80%→10%A; 41-45 min, 10%A.

[0075] Example 2

[0076] According to the sample preparation method of Example 1, the sample and 8 single-flavor medicine (Sanqi, Fangfeng, Bombyx Batryticatus, Rhizoma Aconiti Lateralis, Arisaema, Gastrodia, Angelica Dahurica, Notopterygium) decoction solutions were prepared, and the chromatographic conditions determined in Example 1 were used for high performance liquid chromatography analysis to obtain the fingerprints of the 8 single-flavor medicines. The 15 common peaks of the fingerprint of the Zhongtongan capsule sample calibrated in Example 1 were compared and analyzed with the fingerprints of the 8 single-flavor medicines, and the source medicinal flavors of the 15 chromatographic peaks in the fingerprint of the Zhongtongan capsule sample were attributed, and the attribution results are shown in Table 5.

[0077] Table 5: The medicinal properties of the 15 peaks in the chromatogram of the test product

[0078]

[0079] It can be seen from Table 5 that the 15 common peaks of the Zhongtong'an capsule samples are attributed to the eight medicinal ingredients in Zhongtong'an capsules, namely, Panax notoginseng, Saposhnikovia divaricata, Bombyx batryticatus, Rhizoma Aconitum, Arisaema, Gastrodia elata, Angelica dahurica and Rhizoma Notopterygii. This shows that the fingerprint method can perform comprehensive quality control on Zhongtong'an capsules.

[0080] Example 3

[0081] This example is a methodological investigation of the construction method of the fingerprint spectrum of Zhongtong'an Capsule determined in Example 1, including investigations on specificity, precision, stability, repeatability, durability, etc. The specific investigation methods and conclusions are as follows:

[0082] 1. Exclusivity

[0083] To investigate whether the blank solvent has interference, the test solution was prepared according to the method of Example 1 and injected according to the chromatographic conditions of Example 1; the test sample, reference solution (mixed by 6 kinds of standard substances dissolved in methanol) and blank solvent (70% methanol) were injected into the high performance liquid chromatograph, measured, and the chromatogram was recorded (such as Figure 6 The results show that the blank solvent has no interference, indicating that the instrument has good specificity.

[0084] 2. Precision

[0085] Take the same batch (batch number: 240203) of Zhongtong'an capsule samples, prepare the test solution according to the method of Example 1, and inject the sample continuously for 6 times according to the chromatographic conditions of Example 1, record the fingerprint spectrum, and calculate the relative retention time and relative peak area of ​​each common peak, see Table 6-7.

[0086] Table 6: Relative retention time of common peaks in 6 injections of the same batch of Zhongtong'an capsule samples

[0087]

[0088] Table 7: Relative peak areas of common peaks in 6 injections of the same batch of Zhongtong'an capsule samples

[0089]

[0090] It can be seen from Table 6-7 that the same batch of Zhongtong'an Capsules samples were injected 6 times, and the RSD of the relative retention time of each common peak was less than 1%, and the RSD of the relative peak area of ​​the main common peak was less than 3%, indicating good precision.

[0091] 3. Stability

[0092] Take the same batch of Zhongtong'an capsule samples, prepare the test solution according to the method of Example 1, inject into the liquid chromatograph (chromatographic conditions refer to Example 1) for analysis at 0h, 8h, 12h, 16h, 20h and 24h respectively, record the fingerprint, and calculate the relative retention time and relative peak area of ​​each common peak, see Table 8-9.

[0093] Table 8: Relative retention time of common peaks after the test solution was placed for different time periods

[0094]

[0095] Table 9: Relative peak areas of common peaks of test solution at different time

[0096]

[0097] It can be seen from Tables 8-9 that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD of the main common peak is less than 3%, indicating that the test solution has good stability within 24 hours.

[0098] 4. Repeatability

[0099] Take the same batch (batch number: 240203) of Zhongtong'an capsule samples, prepare 6 test solutions according to the method of Example 1, inject and detect according to the chromatographic conditions of Example 1, record the fingerprint, and calculate the relative retention time and relative peak area of ​​each common peak, see Table 10-11.

[0100] Table 10: Relative retention time of common peaks in 6 test solutions prepared from the same batch of Zhongtong'an capsule samples

[0101]

[0102] Table 11: Relative peak areas of common peaks in 6 test solutions prepared from the same batch of Zhongtong'an capsule samples

[0103]

[0104] It can be seen from Tables 10-11 that the relative retention time RSD of each common peak is less than 1%, and the relative peak area RSD of the main common peak is less than 3%, indicating that the fingerprint spectrum construction method of the present invention has good repeatability.

[0105] 5. Durability

[0106] The durability test includes the durability of different brands of chromatographic columns, different column temperatures, different flow rates, different detection wavelengths and different high performance liquid chromatographs for this chromatographic condition:

[0107] (1) Influence of different brands of chromatographic columns

[0108] Take the same batch of Zhongtong'an capsule samples, prepare the test samples and perform sample injection testing in accordance with the test sample preparation method and chromatographic detection conditions in Example 1, and compare the results of Waters Symmetry C 18 (4.6×250mm, 5μm), Shimadzu Wondasil C 18 (4.6×250mm, 5μm), Agilent Eclipse XDB-C 18 Separation effect of three chromatographic columns of different brands (4.6×250mm, 5μm) on the chromatographic peaks of the same sample (see Figure 7 ). The results show that the peak shapes and responses of the common peaks of the sample fingerprints on different brands of chromatographic columns are good. In summary, the fingerprint construction conditions of the present invention have good durability on different brands of chromatographic columns.

[0109] (2) Effect of different column temperatures

[0110] On the same chromatographic column, samples of Zhongtong'an capsules of the same batch were prepared and tested in sequence according to the sample preparation method and chromatographic detection conditions in Example 1, and the separation effects of three column temperatures, namely 30°C, 35°C and 40°C, on the chromatographic peaks in the same sample were compared (see Figure 8 ). The results show that there is no significant difference in the separation effect of the samples at different column temperatures. In summary, the fingerprint spectrum construction conditions of the present invention have good durability within the column temperature range of 30-40°C.

[0111] (3) Take samples of Zhongtong'an Capsules of the same batch number, prepare the test samples and perform sample injection testing in accordance with the test sample preparation method and chromatographic detection conditions in Example 1, and compare the separation effects of three flow rate conditions, namely 0.95 mL / min, 1.05 mL / min, and 1.00 mL / min, on the chromatographic peaks in the same test sample (see Fig. 9 ). The results show that under the flow rate condition of 0.95-1.05 mL / min, the peak shape and separation degree of each common peak of the fingerprint spectrum are good. In summary, the fingerprint spectrum construction conditions of the present invention have good durability within the flow rate range of 0.95-1.05 mL / min.

[0112] (4) Influence of different detection wavelengths

[0113] Take the same batch of Zhongtong'an capsule samples on the same chromatographic column, prepare the test samples and inject the samples according to the test sample preparation method and chromatographic detection conditions in Example 1, and investigate the separation effect of 300nm, 305nm, 295nm and other three detection wavelengths on the chromatographic peaks in the same test sample (see Fig.10 ). The results show that the peak shape and response value of the sample chromatogram are good at different detection wavelengths. In summary, the fingerprint construction conditions of the present invention have good durability within the detection wavelength range of 295-305nm.

[0114] (5) Impact of different instruments

[0115] Take the same batch of Zhongtong'an capsule samples on the same chromatographic column, prepare the sample and sample injection test in turn according to the sample preparation method and chromatographic detection conditions in Example 1, and investigate the separation effect of chromatographic peaks in the same sample by three different instruments, including Waters e2695, Shimadzu, and Agilent 1260II. The results show that the peak shape and response value of the chromatogram of the sample are good when tested on different instruments. In summary, the fingerprint construction conditions of the present invention have good durability on different instruments.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a fingerprint of Zhongtongan, characterized in that: The steps include: S1. Take appropriate amount of the contents of several batches of Zhongtong'an capsules, dissolve them in 50-100% methanol respectively, make up the weight loss with 50-100% methanol after dissolution, filter and obtain several test samples; Take cimicifugin glycoside, purpurogenol, 5-O-methylvisaminol glycoside, bergamot glycoside lactone, imperatorin and isoimperatorin, use 50-100% methanol to dissolve, and prepare the reference substances of cimicifugin glycoside, purpurogenol glycoside, 5-O-methylvisaminol glycoside, bergamot glycoside lactone, imperatorin and isoimperatorin respectively; S2. Perform high performance liquid chromatography analysis on all test samples and reference samples to obtain chromatograms of the test samples and reference samples respectively; The conditions for the HPLC analysis are as follows: the filler of the chromatographic column is octadecyl bonded silica gel, acetonitrile is used as mobile phase A, and 0.1% phosphoric acid aqueous solution is used as mobile phase B; The elution gradient is as follows: by volume fraction, 0 min, mobile phase A is 6%; 10 min, mobile phase A is 11%; 18 min, mobile phase A is 20%; 28 min, mobile phase A is 35%; 32 min, mobile phase A is 35%; 38 min, mobile phase A is 80%; 42 min, mobile phase A is 80%; 43 min, mobile phase A is 6%; 45 min, mobile phase A is 6%; S3. Determine the common peaks from the chromatograms of several test samples, compare the common peaks with the chromatograms of the reference samples, and identify the chemical components of some common peaks; use the chromatographic peak of purpurogenol as the reference peak, calculate the relative retention time of each common peak, and thus construct the fingerprint of Zhongtongan.

2. The construction method according to claim 1, characterized in that: In S1, the test sample preparation method is as follows: take 0.4-0.6g of the content of Zhongtong'an Capsule, accurately weigh it, put it in a container, add 70% methanol, weigh it, ultrasonically treat it for 20-40min, cool it, make up the lost weight with 70% methanol, mix it well, filter it, and obtain it.

3. The construction method according to claim 1, characterized in that: In S1, the preparation method of the reference substance is: take the standard substances of cimicifuga glycosides, bergamot glycosides, 5-O-methylvisaminol glycosides, peucedanum rutaecarpon glycosides, imperatorin and isoimperatorin respectively, weigh them accurately, add methanol to make solutions containing 60μg, 1μg, 6μg, 60μg, 6μg and 22μg per 1mL respectively, to obtain; that is, each 1mL of cimicifuga glycosides reference substance contains 60μg of the standard substance; each 1mL of bergamot glycosides reference substance contains 1μg of the standard substance; each 1mL of 5-O-methylvisaminol glycosides reference substance contains 6μg of the standard substance; each 1mL of peucedanum rutaecarpon glycosides reference substance contains 60μg of the standard substance; each 1mL of imperatorin reference substance contains 6μg of the standard substance; each 1mL of isoimperatorin reference substance contains 22μg of the standard substance.

4. The construction method according to claim 1, characterized in that: In S2, during the HPLC analysis, the column temperature is 30-40°C.

5. The construction method according to claim 1, characterized in that: In S2, during the high performance liquid chromatography analysis, the injection flow rate is 0.95-1.05 mL / min; the injection volume is 10 μL.

6. The construction method according to claim 1, characterized in that: In S2, the detection wavelength is 295nm-305nm.

7. The construction method according to claim 1, characterized in that: In S3, 15 batches of Zhongtong'an capsule samples are selected to prepare 15 test samples according to the test sample preparation conditions in step S1, and high performance liquid chromatography analysis is performed respectively to obtain 15 fingerprint spectra, and the chromatographic peaks with good stability and appropriate response values ​​in these fingerprint spectra are determined as common peaks.

8. The construction method according to claim 1, characterized in that: In S3, the fingerprint of Zhongtongan constructed is a high performance liquid chromatography characteristic spectrum, and the fingerprint includes 15 characteristic peaks in total, with the chromatographic peak of peak 6 of purpurogenol as the reference peak; Among them, the relative retention time of peak 1 is 0.1465; the relative retention time of peak 2 is 0.2228; the relative retention time of peak 3 is 0.2786; the relative retention time of peak 4 is 0.5821; peak 5 is cimicifuga glycoside, with a relative retention time of 0.8451; peak 6 is purpurogenol glycoside, with a relative retention time of 1.0000; peak 7 is 5-O-methylvisaminol glycoside, with a relative retention time of 1.0299; peak 8 is relative retention time of 1.0000. The relative retention time of peak 9 is 1.2354; the relative retention time of peak 9 is 1.2807; peak 10 is bergamotolactone, with a relative retention time of 1.5984; peak 11 is with a relative retention time of 1.6607; peak 12 is with a relative retention time of 1.6874; peak 13 is with a relative retention time of 1.7103; peak 14 is imperatorin, with a relative retention time of 1.7316; peak 15 is isoimperatorin, with a relative retention time of 1.7727; When the fingerprint is used to evaluate the quality of Zhongtong'an Capsules, according to the Chinese medicine chromatographic fingerprint similarity evaluation system, the similarity between the test sample fingerprint of qualified Zhongtong'an Capsules and the control fingerprint is not less than 0.90.

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