Construction method of specific chromatogram of Chinese patent medicine Zhikang capsule as well as specific chromatogram and application of specific chromatogram

Abstract

The invention relates to a construction method of a specific chromatogram of a Chinese patent medicine Zhikang capsule, and the specific chromatogram and application thereof, and the method comprises the following steps: carrying out gradient elution by using a high performance liquid chromatograph, determining the contents of catechin, magnoflorine, berberine hydrochloride and 1, 4-di [4-(glucose oxygen) benzyl]-2-isobutyl malate, and forming the high performance liquid chromatography specific chromatogram of the Zhikang capsule. Nine characteristic peaks are determined as a characteristic chromatogram, main components such as alkaloids, phenols, anthraquinones and glycosides are covered, and a quality evaluation system of the Chinese patent medicine Zhikang capsule is constructed; the method is good in specificity, precision, repeatability and solution stability, and can be used for standardizing production of the Zhikang capsules and ensuring uniformity and stability of internal quality of products to provide comprehensive quality control.

Description

Technical Field

[0001] The invention belongs to the technical field of quality control of traditional Chinese medicine preparations, and particularly relates to a method for constructing a characteristic spectrum of Zhikang Capsules, a traditional Chinese medicine, and its characteristic spectrum and application. Background Art

[0002] Zhikang Capsule is an exclusive product produced by Xi'an Qianhe Pharmaceutical Co., Ltd. It is composed of 14 medicinal herbs, including rhubarb, coptis root, notoginseng, angelica root, donkey-hide gelatin, dragon bone (calcined), bletilla striata, vinegar myrrh, cuttlebone, madder, dragon's blood, liquorice, pearl and borneol. It has the effects of clearing away heat, cooling blood and stopping bleeding, removing blood stasis, promoting tissue regeneration and relieving pain. It is mainly used clinically to treat various bleeding diseases such as traumatic hemorrhage, metrorrhagia, hematemesis and bloody stool. Zhikang Capsule has been on the market for many years because of its definite efficacy and high safety, and has been recognized and trusted by medical workers and patients. It is included in many guidelines, pathways and textbooks such as "Guidelines for the Clinical Application of Chinese Patent Medicines", "International Guidelines for Clinical Practice of Traditional Chinese Medicine", "Guidelines for the Management of Chronic Diseases of Peptic Ulcers in the Elderly", "Guidelines for the Diagnosis and Treatment of Abnormal Uterine Bleeding with Ovulatory Disorders by Integrated Chinese and Western Medicine", "Interpretation of Clinical Pathway Therapeutic Drugs", "Modern Anorectal Surgery", "Anorectal Surgery Nursing", "Integrated Chinese and Western Medicine Obstetrics and Gynecology" and is recommended for clinical use; because Zhikang Capsules are safe and effective, its standards were included in the "Chinese Pharmacopoeia" in 2015, and the current implementation standard is the current version of the "Chinese Pharmacopoeia". Although the current quality standards of Zhikang Capsules have carried out microscopic identification of Bletilla striata in the prescription, thin layer identification of rhubarb, coptis chinensis, Panax notoginseng, and dragon's blood, limit inspection of rhubarb glycosides in rhubarb, content determination of rhubarb and chrysophanol in rhubarb, and content determination of volatile borneol, its quality standards are higher than those of other Chinese patent medicines, but these quality control indicators cannot fully reflect the overall quality of Zhikang Capsules. In recent years, the method of using fingerprint or characteristic spectrum of traditional Chinese medicine for overall quality control has been widely used in view of the multi-component, multi-target and multi-pathway characteristics of traditional Chinese medicine. The fingerprint or characteristic spectrum can effectively identify the authenticity of the sample through the characteristic of the spectrum, and on the other hand, it can effectively control the quality of the product by controlling the area, proportion or retention time of the main characteristic peak of the spectrum, ensuring the stability and consistency of the product quality, thereby further ensuring the quality and efficacy of the product. Summary of the invention

[0003] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for constructing a characteristic spectrum of the Chinese patent medicine Zhikang Capsule, its characteristic spectrum and application. The characteristic spectrum established by this method can more comprehensively and effectively perform intrinsic quality control on Zhikang Capsule.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] A method for establishing a characteristic spectrum of a Chinese patent medicine Zhikang Capsule, wherein the Zhikang Capsule is composed of rhubarb, coptis root, notoginseng, angelica dahurica, donkey-hide gelatin, dragon bone (calcined), bletilla striata, myrrh, cuttlebone, madder, dragon's blood, liquorice, pearl, and borneol.

[0006] The characteristic spectrum construction method of the Chinese patent medicine Zhikang Capsule specifically comprises the following steps:

[0007] 1) Chromatographic conditions and system suitability test

[0008] The chromatographic column is filled with octadecylsilane bonded silica gel; the mobile phase is an organic solvent as phase A and a 0.1% phosphoric acid aqueous solution as phase B, and gradient elution is performed; the column temperature is 40°C to 50°C; the ultraviolet detector has a detection wavelength of 215nm to 240nm; the flow rate is 0.8ml / min-1.2ml / min; the theoretical plate number is calculated based on the berberine hydrochloride peak and should not be less than 10000;

[0009] 2) Preparation of reference solution

[0010] Take appropriate amounts of catechin reference substance, magnolia alkaloid reference substance, berberine hydrochloride reference substance, and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate reference substance, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 50 μg of catechin, 50 μg of magnolia alkaloid, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate per 1 ml, as the reference substance solution;

[0011] 3) Preparation of test solution

[0012] Preparation of test solution: take about 0.5-1.5g of Zhikang capsule preparation, weigh accurately, put into a stoppered conical flask, add 10-50ml of 20%-80% methanol accurately, and treat with ultrasound for 15-45 minutes, with an ultrasound power of 300W and a frequency of 40kHz; cool, filter, and take the filtrate to obtain;

[0013] 4) Determination

[0014] Accurately pipette 2-5 μl of the reference solution and the test solution respectively, inject into the HPLC, determine the contents of catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, and construct the HPLC characteristic spectrum of Zhikang Capsules.

[0015] Furthermore, the chromatographic column is Poroshell HPH-C18, 4.6 mm×150 mm, 2.7 μm; the flow rate is 1.0 mL / min; the column temperature is 45° C.; and the detection wavelength is 235 nm.

[0016] Further, gradient elution, the gradient elution program is preferably as follows, wherein the mobile phase ratios are all volume percentages:

[0017] 0-15min, mobile phase A is 2%-10%, mobile phase B is 98%-90%;

[0018] 15-25min, mobile phase A is 10%-15%, mobile phase B is 90%-85%;

[0019] 25-45 min, mobile phase A is 15%-20%, mobile phase B is 85%-80%;

[0020] 45-55 min, mobile phase A is 20%, mobile phase B is 80%;

[0021] 55-65min, mobile phase A is 20%-2%, mobile phase B is 80%-98%.

[0022] Further, in step 3), take about 1.0 g of the preparation of the product, accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of 75% methanol, and ultrasonically treat it for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz; cool it, filter it, and take the filtrate to obtain the product.

[0023] The present invention further provides a characteristic spectrum of Zhikang Capsules obtained by the construction method as follows Fig.15 The present invention also provides the application of the characteristic spectrum of Zhikang Capsules obtained by the construction method in the evaluation of the quality and content determination of Zhikang Capsules.

[0024] Furthermore, the characteristic spectrum should present 9 characteristic peaks, among which the retention times of peaks 1, 3, 7 and 9 should correspond to the retention times of the reference peaks of catechin, magnolia alkaloids, berberine hydrochloride and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate. The peak corresponding to the catechin reference peak is the S1 peak, and the relative retention times of peaks 2, 4 and S1 are calculated. The relative retention times of peaks 2 and 4 should be within ±10% of the specified value, and the specified values ​​are: 1.19 (peak 2) and 2.42 (peak 4); the peak corresponding to the berberine hydrochloride reference peak is the S2 peak, and the relative retention times of peaks 5, 6 and 8 and S2 are calculated. The relative retention times should be within the range of ±10% of the specified value, and the specified values ​​are 0.80 (peak 5), 0.86 (peak 6) and 1.05 (peak 8).

[0025] Furthermore, the sources of the 9 common characteristic peaks of the HPLC characteristic spectrum of the drug method are: among the absorption peaks of the characteristic spectrum, the components derived from rhubarb include peak 1 (catechins) and peak 4 (rhein-8-O-β-D-glucoside); the components derived from Bletilla striata include peak 9 (1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate); the components derived from Coptis chinensis include unknown component peak 2, peak 3 (magnolia pine), peak 5 (coptisine), peak 6 (jatrorrhizine), peak 7 (berberine hydrochloride), and peak 8 (palmatine).

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The method for constructing the characteristic spectrum of Zhikang capsule of the Chinese patent medicine of the present invention adopts high performance liquid chromatography for gradient elution, which can not only determine the content of catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, but also construct the high performance liquid chromatography characteristic spectrum of Zhikang capsule. Nine characteristic peaks are determined as characteristic spectrum, covering the main components such as alkaloids, phenols, anthraquinones and glycosides, and the quality evaluation system of Zhikang capsule of the Chinese patent medicine is constructed; the method has good specificity, precision, repeatability and solution stability, and can be used to standardize the production of Zhikang capsule and ensure the uniformity and stability of the intrinsic quality of the product to provide comprehensive quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below in conjunction with the accompanying drawings:

[0029] Figure 1 It is the liquid chromatogram of different mobile phase gradients;

[0030] Figure 2 It is the liquid chromatogram of different detection wavelengths;

[0031] Figure 3 Examine the chromatograms for different acid regulator concentrations;

[0032] Figure 4 The liquid chromatograms of different chromatographic columns;

[0033] Figure 5 The liquid chromatograms at different column temperatures;

[0034] Figure 6 is the liquid chromatogram of different acid regulators in the mobile phase;

[0035] Figure 7 Blank, reference, and sample chromatograms for system suitability testing;

[0036] Figure 8 To investigate the relevant spectra (Dragon Blood Drainage, Rhubarb) for specificity;

[0037] Fig. 9 To investigate the relevant spectra (Bletilla striata, donkey-hide gelatin) for specificity;

[0038] Fig.10 To investigate the relevant spectra (Angelica Dahuricae, Rubia cordifolia) for specificity;

[0039] Fig.11 To investigate the relevant atlases for specificity (pearl, vinegar myrrh);

[0040] Fig.12 To investigate the relevant atlases for specificity (Cuttlebone, Borneol);

[0041] Fig.13 To investigate the relevant spectra for specificity (calcined dragon bone, Panax notoginseng);

[0042] Fig.14 To investigate the relevant spectra (Glycyrrhiza uralensis and Coptis chinensis) for specificity;

[0043] Fig.15 It is the characteristic spectrum of the test solution of the present invention;

[0044] Fig.16 Determine the spectrum for multiple batches;

[0045] In the figure, peak 1: catechin (S1); peak 2: unknown component in Coptis chinensis; peak 3: magnolamine; peak 4: rhein-8-O-β-D-glucoside; peak 5: coptisine; peak 6: jatrorrhizine; peak 7: berberine hydrochloride (S2); peak 8: palmatine; peak 9: 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate. DETAILED DESCRIPTION

[0046] The following embodiments may help those skilled in the art to more fully understand the present invention, but shall not limit the present invention in any way.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified; some of the instruments, reagents and reagents used are as follows:

[0048] Instruments: Agilent 1290 ultra high performance liquid chromatograph (Agilent Technologies, Inc.), Agilent 1260 high performance liquid chromatograph (Agilent Technologies, Inc.), Waters H-Class ultra high performance liquid chromatograph (Waters Technologies, Inc.), Agilent Poroshell HPH-C18 (4.6×150 mm, 2.7 μm) chromatographic column, one-tenth balance (Mettler-Toledo, ME104), one-millionth balance (Mettler-Toledo, XPR2), CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd., KQ-300DB), electric constant temperature water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., HWS26), coolant circulation pump (Shanghai Dayan Instrument Equipment Co., Ltd., LTC-5 / 30), high-speed desktop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., H1650-W).

[0049] Reagents: methanol (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.); acetonitrile (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.); water (distilled water, Guangzhou Watsons Food and Beverage Co., Ltd.); formic acid (mass spectrometry grade, Shanghai Anpu Experiment Technology Co., Ltd.); phosphoric acid (chromatographic grade, Shanghai Anpu Experiment Technology Co., Ltd.).

[0050] Test drugs: Zhikang capsules (Xi'an Qianhe Pharmaceutical Co., Ltd.), catechin reference substance, magnolia alkaloid reference substance, rhein-8-O-β-D-glucoside reference substance, bamipine reference substance, 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate reference substance, all originated from Shanghai Shidande Standard Technology Service Co., Ltd.

[0051] Example 1. Establishment of a characteristic spectrum method of Zhikang capsule contents by HPLC

[0052] Based on a large number of experimental explorations, a high performance liquid chromatography method was preliminarily established to determine the contents of various active ingredients in Zhikang Capsules, including catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, and the content determination and characteristic spectrum of Zhikang Capsules were established.

[0053] Example 1. Establishment of a characteristic spectrum method of Zhikang capsule contents by HPLC

[0054] 1.1 Choice of mobile phase

[0055] 1.1.1 Selection of different gradients in mobile phase:

[0056] Preparation of reference solution: Take 2.5 mg of catechin reference substance, 2.5 mg of magnolia alkaloid reference substance, 12.5 mg of berberine hydrochloride reference substance, and 25 mg of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate reference substance, place them in a 50 ml volumetric flask, add 50% methanol to dissolve and dilute to the scale to obtain a mixed reference solution containing 50 μg of catechins, 50 μg of magnolia alkaloid, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate per 1 ml.

[0057] Preparation of test solution: Take about 1.0 g of the preparation, accurately weigh it, put it into a stoppered conical flask, accurately add 20 ml of 80% methanol, ultrasonically treat for 30 minutes, cool, filter, and take the filtrate for use.

[0058] Chromatographic conditions and system suitability test

[0059] Column: Poroshell HPH-C18, 4.6 mm × 150 mm, 2.7 μm;

[0060] Mobile phase: Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution, flow rate: 1.0mL / min; column temperature: 45℃; detection wavelength: 235nm; theoretical plate number calculated based on berberine hydrochloride peak, should not be less than 10000;

[0061] Determination: Pipette 2-5 μl of the above-mentioned reference solution and Zhikang Capsule test solution and inject them into liquid chromatography for determination.

[0062] In order to investigate the effect of different mobile phase gradients on the characteristic spectrum of Zhikang Capsules, the gradient with better characteristic peak response and better separation effect was selected.

[0063] Method 1: The gradient elution procedure is as follows, wherein the mobile phase ratios are all in volume percentage:

[0064] 0-15min, mobile phase A is 2%-10%, mobile phase B is 98%-90%;

[0065] 15-25min, mobile phase A is 10%-15%, mobile phase B is 90%-85%;

[0066] 25-45 min, mobile phase A is 15%-20%, mobile phase B is 85%-80%;

[0067] 45-55 min, mobile phase A is 20%, mobile phase B is 80%;

[0068] 55-65min, mobile phase A is 20%-2%, mobile phase B is 80%-98%;

[0069] According to the above method 1, except for adjusting the gradient, data were collected according to the following elution gradient. The results are shown in Table 1-2.

[0070] Table 1: Gradient adjustment survey table

[0071]

[0072] Note: Phase A is acetonitrile.

[0073] Table 2: Gradient adjustment investigation results

[0074]

[0075] Note: The two values ​​of separation are the separation between the target peak and its preceding and succeeding peaks. NA means there are no peaks before or after the target peak.

[0076] The results showed that after the method was adjusted, peaks 4 and 7 were split. In addition, the separation of some characteristic peaks was reduced. After comprehensive consideration, method 1 was selected as the elution gradient.

[0077] 1.1.2 Selection of different acid regulators in mobile phase:

[0078] The effects of acid regulators in different mobile phases on the characteristic spectra of Zhikang Capsules were investigated, and the one with better characteristic peak response and better separation effect was selected as the mobile phase acid regulator.

[0079] Except for adjusting the mobile phase acid regulator, the elution gradient of Method 1 was used, and the rest of the data was collected according to the chromatographic conditions and test solution preparation method under "1.1.1". The results are shown in Table 3.

[0080] The results showed that there were obvious differences in chromatographic behaviors under different additives. When formic acid was used as the additive, the baseline drift was more serious. When phosphoric acid was used, the overall chromatographic behavior was better, so phosphoric acid was selected as the additive.

[0081] Table 3: Results of investigation on different acid regulators

[0082]

[0083] The effects of different mobile phase acid regulator concentrations on the characteristic spectrum of Zhikang Capsules were further investigated to select the additive concentration with better characteristic peak response and better separation effect.

[0084] The test solution was prepared in the same manner as in Item “1.1.1”. Except for adjusting the concentration of the mobile phase additive, the data were collected according to the chromatographic conditions in Item “1.1.1”. The results are shown in Table 4.

[0085] Table 4: Results of investigation of different acid regulator concentrations

[0086]

[0087] The results showed that there was no significant difference in chromatographic behavior at different additive concentrations. As the additive concentration decreased, the retention time of alkaloid components shortened. Considering the pH tolerance and service life of the chromatographic column, phosphoric acid with a concentration of 0.1% was selected as the nearest acid regulator.

[0088] 1.2 Selection of detection wavelength

[0089] Data were collected at different detection wavelengths of 220 nm, 235 nm, 254 nm, 280 nm, 300 nm, 330 nm, and 360 nm according to the chromatographic conditions of method 1. The results are shown in Figure 2 .

[0090] The results showed that there were significant differences in chromatographic behaviors at different detection wavelengths, including 220nm, 235nm, 254nm, 280nm, 300nm, 330nm, and 360nm. Except for 220nm and 235nm, the other wavelengths had missing characteristic peaks. At the same time, compared with 220nm, 235nm had less impurity interference around the characteristic peaks, so 235nm was selected as the detection wavelength.

[0091] 1.3 Selection of different chromatographic columns

[0092] The effects of different chromatographic columns on the characteristic spectrum of Zhikang Capsules were investigated. Agilent Poroshell HPH-C18 (4.6×150mm, 2.7μm, number: column 1), Agilent Poroshell 120EC-C18 (4.6×150mm, 2.7μm, number: column 2), and Agilent Poroshell 120AQ-C18 (4.6×150mm, 2.7μm, number: column 3) were selected to prepare the test solution. In addition to adjusting the chromatographic column, the detection wavelength was 235nm, and the rest of the data was collected according to the chromatographic conditions under "1.1". The appropriate chromatographic column was selected by observing the tailing factor and separation of each characteristic peak in the spectrum. The results are shown in Tables 5 and Figure 4 .

[0093] Table 5: Results of investigations on different chromatographic columns

[0094]

[0095] The results showed that different types of filler columns had a certain influence on the characteristic spectra. When column 3 was used, peak 11 did not elute within the effective elution time due to its strong retention. When columns 1 and 2 were used, the characteristic peaks were evenly distributed, the peak shapes were good, and the spectra were more beautiful. In comparison, column 1 had a shorter analysis time and a larger adjustable space than column 2. Considering the subsequent optimization process, Agilent Poroshell HPH-C18 (4.6×150mm, 2.7μm, number: column 1) was selected as the chromatographic column for this method.

[0096] 1.4 Different column temperature selection

[0097] The influence of different column temperatures on the characteristic spectrum of Zhikang Capsules was investigated to select the flow rate with better characteristic peak response and better separation effect. The test solution was prepared according to the same preparation method as "1.1.1". Except for adjusting the column temperature, the rest of the data was collected according to the chromatographic conditions under "1.1.1". The results are shown in Table 6.

[0098] Table 6: Results of investigation at different column temperatures

[0099]

[0100] The results showed that there were certain differences in chromatographic behaviors at different column temperatures. As the column temperature increased, the retention time of each characteristic peak shortened. From an overall perspective, when the column temperature was 45°C, there was less interference from impurities around the characteristic peaks, so the column temperature was selected to be 45°C.

[0101] 1.5 Different flow rate options

[0102] The influence of different flow rates on the characteristic spectrum of Zhikang Capsules was investigated to select the flow rate with better characteristic peak response and better separation effect. The test solution was prepared according to the same preparation method as "1.1.1". Except for adjusting the flow rate, the data were collected according to the chromatographic conditions under "1.1.1". The results are shown in Table 7.

[0103] Table 7: Results of investigation at different flow rates

[0104]

[0105] The results showed that there were certain differences in chromatographic behaviors at different flow rates. With the increase of flow rate, the retention time of each characteristic peak shortened. Peaks 6 and 8 had obvious impurity interference at different flow rates, so the flow rate was selected as 1.0 ml / min.

[0106] Example 2. Investigation of the preparation method of the test solution

[0107] The extraction solvent, extraction method, extraction time and solid-liquid ratio used in the preparation of Zhikang Capsule test solution were investigated to determine the preparation method of Zhikang Capsule test solution.

[0108] 2.1 Investigation of extraction solvent

[0109] The effects of different extraction solvents on the characteristic spectrum of this product were investigated. 25%, 50% and 75% methanol were used as extraction solvents respectively. The peak shape and separation of the characteristic peaks were observed and the "total peak area / sample weight" of the characteristic peaks was calculated to compare the effects of different extraction solvents on the characteristic spectrum of this product and select the best extraction solvent.

[0110] Preparation of test solution: Take the contents of Zhikang Capsules, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 25% methanol, 50% methanol, 75% methanol 20ml, respectively, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, filter, and take the filtrate. Analyze according to the chromatographic conditions under "1.1.1", the results are detailed in Table 8.

[0111] Table 8: Investigation of different extraction solvents

[0112]

[0113] The results showed that as the proportion of methanol increased, the "total peak area / sample weight" increased accordingly. The peak areas of the characteristic peaks, mainly peaks 6 to 10, were greatly affected by the extraction solvent, and there were no obvious differences in the other parameters. Considering that the relevant peaks were the main characteristic components, 75% methanol was selected as the extraction solvent.

[0114] 2.2 Different extraction times

[0115] The effects of different extraction times on the characteristic spectrum of this product were investigated. The extraction times were set as 15 minutes, 30 minutes, and 45 minutes respectively. The peak shape and separation of the characteristic peaks were observed and the “total peak area / sample weight” of the characteristic peaks was calculated to compare the effects of different extraction times on the characteristic spectrum of this product and select the optimal extraction time.

[0116] Preparation of test solution: Take the contents of Zhikang Capsules, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 20ml of 75% methanol, ultrasonically treat (300W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively, cool, filter, and take the filtrate. Analyze according to the chromatographic conditions under "1.1.1". The results are detailed in Table 9.

[0117] The results showed that as the extraction time increased, the "total peak area / sample weight" tended to be stable, and the influence of different extraction times on the extraction effect was not obvious, so a more moderate 30 minutes was selected as the extraction time.

[0118] Table 9: Investigation of different extraction times

[0119]

[0120] 2.3 Investigation of different extraction solid-liquid ratios

[0121] The effects of different extraction material-liquid ratios on the characteristic spectrum of this product were investigated, with ratios of 1:10, 1:20 and 1:50 respectively. The peak shapes and separations of the characteristic peaks were observed, and the “total peak area / sample weight × extraction solution volume” of the characteristic peaks was calculated to compare the effects of different extraction material-liquid ratios on the characteristic spectrum of this product, and the optimal extraction material-liquid ratio was selected.

[0122] Preparation of test solution: Take the contents of Zhikang Capsules, mix well, take about 1.0g, accurately weigh 3 portions, accurately add 75% methanol 10ml, 20ml, 50ml, respectively, ultrasonically treat (300W, 40kHz) for 30 minutes, cool, filter, and take the filtrate. Analyze according to the chromatographic conditions under "1.1.1", and the results are detailed in Table 10.

[0123] Table 10: Investigation of different extraction solid-liquid ratios

[0124]

[0125] The results showed that as the solid-liquid ratio decreased, the "total peak area / sample weight × extraction solution volume" increased and tended to stabilize, indicating that the extraction efficiency increased with the increase in solvent dosage, but the effect was not significant, so 1:20 was selected as the extraction solid-liquid ratio.

[0126] 2.4 Investigation of different extraction methods

[0127] The effects of different extraction methods on the characteristic spectrum of this product were investigated. Ultrasound and reflux were used as the extraction methods respectively. The peak shape and separation degree of the characteristic peaks were observed and the “total peak area / sample weight” of the characteristic peaks was calculated to compare the effects of different extraction methods on the characteristic spectrum of this product and select the best extraction method.

[0128] Preparation of test solution: Take the contents of Zhikang Capsules, mix well, take about 1.0g, accurately weigh 2 portions, accurately add 20ml of 75% methanol, respectively, ultrasonically treat (300W, 40kHz), reflux for 30 minutes, cool, filter, and take the filtrate. Analyze according to the chromatographic conditions under "1.1.1". The results are detailed in Table 11.

[0129] Table 11: Investigation of different extraction methods

[0130]

[0131] The results showed that different extraction methods had little effect on the extraction efficiency of each component, and the chromatographic behavior had no significant change. For ease of operation, ultrasonic extraction was selected.

[0132] Example 3. System suitability test and specificity investigation

[0133] As can be seen from Examples 1-2, the optimal HPLC method for determining the characteristic spectrum of Zhikang Capsules specifically comprises the following steps:

[0134] 1) Preparation of reference solution: Take 2.5 mg of catechin reference substance, 2.5 mg of magnolia alkaloid reference substance, 12.5 mg of berberine hydrochloride reference substance, and 25 mg of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate reference substance, place them in a 50 ml volumetric flask, add 50% methanol to dissolve and dilute to the scale to obtain a mixed reference solution containing 50 μg of catechins, 50 μg of magnolia alkaloid, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate per 1 ml.

[0135] 2) Preparation of test solution: Take about 1.0 g of the preparation, accurately weigh, place in a stoppered conical flask, accurately add 20 ml of 75% methanol, ultrasonically treat (300 W, 40 kHz) for 30 minutes, cool, filter, and take the filtrate for use.

[0136] 3) Chromatographic conditions and system suitability test

[0137] Column: Poroshell HPH-C18, 4.6 mm × 150 mm, 2.7 μm;

[0138] Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a 0.1% by volume phosphoric acid aqueous solution, and gradient elution is performed. The gradient elution procedure is as follows, wherein the mobile phase ratios are all volume percentages:

[0139] 0-15min, mobile phase A is 2%-10%, mobile phase B is 98%-90%;

[0140] 15-25min, mobile phase A is 10%-15%, mobile phase B is 90%-85%;

[0141] 25-45 min, mobile phase A is 15%-20%, mobile phase B is 85%-80%;

[0142] 45-55 min, mobile phase A is 20%, mobile phase B is 80%;

[0143] 55-65min, mobile phase A is 20%-2%, mobile phase B is 80%-98%;

[0144] Flow rate: 1.0 mL / min; column temperature: 45°C; detection wavelength: 235 nm; the theoretical plate number is calculated based on the berberine hydrochloride peak and should not be less than 10,000;

[0145] 4) Determination: 2-5 μl of the above-mentioned reference solution and Zhikang Capsule test solution were injected into the liquid chromatograph, and the HPLC standard characteristic spectrum of Zhikang Capsule and the contents of catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate were obtained according to the high performance liquid chromatography method.

[0146] 3.1 System suitability test

[0147] The test solution and reference solution were prepared according to the same preparation method as in Example 3, and analyzed under the same chromatographic conditions as in Example 1 to examine the chromatographic conditions and system suitability of the characteristic spectrum of Zhikang Capsules, and to examine whether the blank solvent would cause interference. The results showed that the blank solvent had no interference with the sample, and the retention time of the reference substance in the reference solution was consistent with the retention time of the corresponding component in the test sample. See the relevant spectrum below Figure 6 .

[0148] 3.2 Exclusivity Investigation

[0149] The test solution and reference solution were prepared according to the same preparation method as in Example 3, and the analysis was performed under the same chromatographic conditions as in Example 1 to verify the sources of the characteristic peaks in the Zhikang Capsule test solution. Figure 7 .

[0150] According to the results, the 9 characteristic peaks displayed in the characteristic spectrum are from Coptis chinensis, Rheum officinale, and Bletilla striata. Among them, the components from Rheum officinale are catechins (peak 1) and rhein-8-O-β-D-glucoside (peak 4); the components from Bletilla striata are 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate (peak 9); Fig.14 It can be seen from the relevant spectrum of the investigation of the specificity of Coptis chinensis that Peak 2 is an unknown component in Coptis chinensis; that is, it is determined to be from Coptis chinensis through the specificity experiment, but it is an unknown component that has not been studied or reported yet. Therefore, the components derived from Coptis chinensis include Peak 2 (unknown component in Coptis chinensis), magnolamine (peak 3), coptisine (peak 5), jatrorrhizine (peak 6), berberine hydrochloride (peak 7), and palmatine (peak 8).

[0151] 4. Methodological Validation

[0152] 4.1 Repeatability

[0153] Take the same batch of Zhikang capsule samples, prepare 6 test solutions in parallel according to the test solution preparation method under "1.1.1", and collect data according to the specified chromatographic conditions, calculate the relative retention time and relative peak area of ​​each characteristic peak, and the results show that the RSD value of the relative retention time of each characteristic peak is less than 2%, and the RSD value of the relative peak area is less than 5%, indicating that the method has good repeatability. Detailed results are shown in Tables 12-15 below.

[0154] Table 12: Results of repeatability study on retention time of each characteristic peak

[0155]

[0156] Table 13: Results of repeatability test on peak areas of characteristic peaks

[0157]

[0158] Table 14: Results of the relative retention time of each characteristic peak in the repeatability study

[0159]

[0160] Table 15: Results of the relative peak areas of each characteristic peak in the repeatability study

[0161]

[0162] 4.2 Investigation of the intermediate precision of different personnel

[0163] Two experimenters, A and B, each took samples of Zhikang Capsules from the same batch, and prepared 6 test solutions in parallel according to the test solution preparation method under "Example 1". Data were collected on the same instrument according to the specified chromatographic conditions, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. The results showed that the RSD value of the relative retention time of each characteristic peak was less than 2%, and the RSD value of each relative peak area was less than 5%, indicating that the method had good intermediate precision among different personnel. Detailed results are shown in Tables 16-21. (S1~S6 test solutions were prepared by experimenter A, and S7~S12 test solutions were prepared by experimenter B)

[0164] Table 16: Results of the retention time of each characteristic peak in the intermediate precision study of different personnel

[0165]

[0166] Note: This table is the relevant results of person B. The relevant results of person A can be found in the relevant table under the repeatability test, the same below.

[0167] Table 17: Results of the peak areas of each characteristic peak in the intermediate precision study of different personnel

[0168]

[0169]

[0170] Table 18: Results of relative retention time of characteristic peaks in the intermediate precision study by different personnel

[0171]

[0172] Table 19: Results of relative peak areas of characteristic peaks in the intermediate precision study of different personnel

[0173]

[0174] Table 20: Results of relative retention time of characteristic peaks in the intermediate precision study by different personnel (12 samples)

[0175]

[0176] Table 21: Results of relative peak areas of characteristic peaks investigated by different personnel for intermediate precision (12 samples)

[0177]

[0178] Example 5. Multiple batch determination

[0179] 17 batches of Zhikang capsules were prepared into test solutions according to the method of Example 3, injected into a high performance liquid chromatograph, and measured according to the chromatographic conditions of Example 3. The results are shown in Tables 22-26 below.

[0180] Table 22: Multiple batches of retention time results

[0181]

[0182] Table 23: Peak area results of multiple batches

[0183]

[0184] Table 24: Relative retention time results of multiple batches

[0185]

[0186] Table 25: Relative peak area results of multiple batches

[0187]

[0188] Through the detection of multiple batches of Zhikang capsules, 9 characteristic peaks were found in the chromatograms of each batch of test samples, among which the retention times of peaks 1, 3, 7 and 9 corresponded to the retention times of the reference peaks of catechins, magnolia alkaloids, berberine hydrochloride and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutylmalate. The peak corresponding to the catechin reference peak is the S1 peak, and the relative retention times of peaks 2 and 4 with the S1 peak are calculated. The relative retention times are within the range of ±10% of the specified values, and the specified values ​​are: 1.19 (peak 2) and 2.42 (peak 4); the peak corresponding to the berberine hydrochloride reference peak is the S2 peak, and the relative retention times of peaks 5, 6 and 8 with the S2 peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values ​​are: 0.80 (peak 5), 0.86 (peak 6) and 1.05 (peak 8); From Tables 22-25 and Fig.16 It can be seen that there are 9 relatively obvious common peaks in the overlay diagram of the characteristic spectrum of Zhikang Capsules, and the relative retention time of the characteristic peaks in the above 17 batches of samples is very stable, with RSD less than 0.5%, far lower than the 3% specified in the standard; the relative peak area is also relatively stable, with RSD less than 4.0%, lower than the 5% specified in the standard.

[0189] Example 6. Accelerated stability study

[0190] The same batch (batch 20240101) of Zhikang capsules was taken for accelerated stability study at a temperature of 40°C ± 2°C and a relative humidity of 75 ± 5%. During the test, samples were taken at the 0th month, 3rd month, and 6th month, respectively. According to the same preparation method and chromatographic conditions as in Example 3, the relative retention time of each characteristic peak and the content of its characteristic components, catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate, were calculated. The results showed that the relative retention time of each characteristic peak was less than 2%. In the 0th month, each capsule contained 0.265 mg of catechins, 0.165 mg of magnolia alkaloids, 2.297 mg of berberine hydrochloride, and 1.251 mg of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate. The results were consistent after removing water at 3 and 6 months. The specific spectra and results are as follows. The experiment shows that this method is suitable for the quality control of Zhikang capsules.

[0191] Table 26: Accelerated stability test spectrum results

[0192]

[0193] Table 27: Summary of test results

[0194]

[0195] The above embodiments are preferred examples for implementing the present invention, and the present invention is not limited to the above embodiments. Any non-essential additions and substitutions made by those skilled in the art based on the technical features of the technical solution of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, characterized in that: The specific steps include: 1) Chromatographic conditions and system suitability test The chromatographic column is filled with octadecylsilane bonded silica gel; the mobile phase is an organic solvent as phase A and a 0.1% phosphoric acid aqueous solution as phase B, and gradient elution is performed; the column temperature is 40°C to 50°C; the ultraviolet detector has a detection wavelength of 215nm to 240nm; the flow rate is 0.8ml / min-1.2ml / min; the theoretical plate number is calculated based on the berberine hydrochloride peak and should not be less than 10000; 2) Preparation of reference solution Take appropriate amounts of catechin reference substance, magnolia alkaloid reference substance, berberine hydrochloride reference substance, and 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate reference substance, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 50 μg of catechin, 50 μg of magnolia alkaloid, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-di[4-(glucose oxygen) benzyl]-2-isobutyl malate per 1 ml, as the reference substance solution; 3) Preparation of test solution Preparation of test solution: take about 0.5-1.5g of Zhikang capsule preparation, weigh accurately, put into a stoppered conical flask, add 10-50ml of 20%-80% methanol accurately, and treat with ultrasound for 15-45 minutes, with an ultrasound power of 300W and a frequency of 40kHz; cool, filter, and take the filtrate to obtain; 4) Determination Accurately pipette 2-5 μl of the reference solution and the test solution respectively, inject into the HPLC, determine the contents of catechins, magnolia alkaloids, berberine hydrochloride, and 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate, and construct the HPLC characteristic spectrum of Zhikang Capsules.

2. The method for constructing the characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, according to claim 1, is characterized in that: The chromatographic column is Poroshell HPH-C18, 4.6 mm×150 mm, 2.7 μm; the flow rate is 1.0 mL / min; the column temperature is 45° C.; and the detection wavelength is 235 nm.

3. The method for constructing the characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, according to claim 2, is characterized in that: Gradient elution, the gradient elution program is preferably as follows, wherein the mobile phase ratios are all volume percentages: 0-15min, mobile phase A is 2%-10%, mobile phase B is 98%-90%; 15-25min, mobile phase A is 10%-15%, mobile phase B is 90%-85%; 25-45 min, mobile phase A is 15%-20%, mobile phase B is 85%-80%; 45-55 min, mobile phase A is 20%, mobile phase B is 80%; 55-65min, mobile phase A is 20%-2%, mobile phase B is 80%-98%.

4. The method for constructing the characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, according to claim 1, is characterized in that: In step 3), take about 1.0 g of the preparation of the product, accurately weigh it, put it in a stoppered conical flask, accurately add 20 ml of 75% methanol, and ultrasonically treat it for 30 minutes at an ultrasonic power of 300 W and a frequency of 40 kHz; cool it, filter it, and take the filtrate to obtain the product.

5. The method for constructing the characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, according to claim 1, is characterized in that: The characteristic spectrum should present 9 characteristic peaks, among which the retention times of peak 1, peak 3, peak 7 and peak 9 should correspond to the retention times of the reference peaks of catechin, magnolia alkaloids, berberine hydrochloride and 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate; the peak corresponding to the catechin reference peak is taken as reference peak S1, and the relative retention times of peak 2, peak 4 and S1 are calculated: the relative retention time of peak 2 is 1.19±10%, and the relative retention time of peak 4 is 2.42±10%; the peak corresponding to the berberine hydrochloride reference peak is taken as reference peak S2, and the relative retention times of peak 5, peak 6, peak 8 and S2 are calculated: the relative retention time of peak 5 is 0.80±10%, the relative retention time of peak 6 is 0.86±10%, and the relative retention time of peak 8 is 1.05±10%.

6. The method for constructing the characteristic spectrum of Zhikang Capsule, a Chinese patent medicine, according to claim 1, is characterized in that: The sources of 9 common characteristic peaks of the HPLC characteristic spectrum of the drug method are as follows: among the absorption peaks of the characteristic spectrum, the components derived from rhubarb include catechins in peak 1 and rhein-8-O-β-D-glucoside in peak 4; the components derived from bletilla striata include 1,4-di[4-(glucose oxygen)benzyl]-2-isobutylmalate in peak 9; the components derived from coptis chinensis include an unknown component in peak 2, magnolamine in peak 3, coptisine in peak 5, jatrorrhizine in peak 6, berberine hydrochloride in peak 7, and bamipine in peak 8.

7. The characteristic spectrum of Zhikang Capsules obtained according to the construction method described in any one of claims 1-6.

8. The characteristic spectrum of Zhikang Capsules obtained by the construction method according to claim 7 is used in evaluating the quality and content determination of Zhikang Capsules.

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