Construction method of characteristic spectrum of traditional Chinese medicine Zhikang capsule, its characteristic spectrum and application

Abstract

This invention relates to a method for constructing the characteristic chromatogram of the traditional Chinese medicine Zhikang Capsules, as well as the chromatogram and its application. The method involves gradient elution using high-performance liquid chromatography (HPLC) to determine the contents of catechins, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, thus constructing the characteristic HPLC chromatogram of Zhikang Capsules. Nine characteristic peaks were identified as the characteristic chromatogram, covering major components such as alkaloids, phenols, anthraquinones, and glycosides. A quality evaluation system for Zhikang Capsules was constructed. This method exhibits good specificity, precision, repeatability, and solution stability, and can be used to standardize the production of Zhikang Capsules and provide comprehensive quality control to ensure the uniformity and stability of the product's intrinsic quality.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine preparations, specifically relating to the method for constructing the characteristic spectrum of the traditional Chinese medicine Zhikang capsule, as well as its characteristic spectrum and application. Background Technology

[0002] Zhikang Capsules are an exclusive product manufactured by Xi'an Qianhe Pharmaceutical Co., Ltd. They are composed of 14 medicinal herbs: rhubarb, coptis, notoginseng, angelica dahurica, donkey-hide gelatin, calcined dragon bone, bletilla striata, vinegar-processed myrrh, cuttlebone, madder root, dragon's blood, licorice, pearl, and borneol. They have the effects of clearing heat, cooling blood, stopping bleeding, promoting blood circulation, regenerating tissue, and relieving pain. Clinically, they are mainly used to treat traumatic bleeding, metrorrhagia, hematemesis, and melena. Over the years, Zhikang Capsules have been highly regarded and trusted by medical professionals and patients due to their definite efficacy and high safety. Zhikang Capsules are included in and recommended for clinical use in numerous guidelines, pathways, and textbooks, including the *Guidelines for Clinical Application of Traditional Chinese Medicine*, *International Guidelines for Clinical Practice of Traditional Chinese Medicine*, *Guidelines for the Management of Chronic Peptic Ulcer Disease in the Elderly*, *Guidelines for the Diagnosis and Treatment of Uterine Bleeding Due to Ovulatory Disorders Using Integrated Traditional Chinese and Western Medicine*, *Interpretation of Therapeutic Drugs in Clinical Pathways*, *Modern Colorectal Surgery*, *Colorectal Surgery Nursing*, and *Integrated Traditional Chinese and Western Medicine Obstetrics and Gynecology*. Due to its safety and efficacy, its standards were included in the *Chinese Pharmacopoeia* in 2015, and the current standard is the current edition of the *Chinese Pharmacopoeia*. Although the current quality standards for Zhikang Capsules include microscopic identification of Bletilla striata in the prescription, thin-layer chromatography identification of rhubarb, Coptis chinensis, Panax notoginseng, and Dragon's Blood, limit testing of emodin in rhubarb, content determination of emodin and chrysophanol in rhubarb, and content determination of volatile borneol, making its quality standards higher than those of other traditional Chinese medicines, these quality control indicators cannot fully reflect the overall quality of Zhikang Capsules. In recent years, the method of using fingerprinting or characteristic spectroscopy for overall quality control of traditional Chinese medicine (TCM) has been widely applied, taking into account its multi-component, multi-target, and multi-pathway effects. Using fingerprinting or characteristic spectroscopy allows for effective identification of genuine samples based on their spectral characteristics. Furthermore, by controlling the area, proportion, or retention time of key characteristic peaks, product quality can be effectively controlled, ensuring stability and consistency, thereby further guaranteeing product quality and efficacy. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for constructing a characteristic spectrum of the traditional Chinese medicine Zhikang capsules, along with the characteristic spectrum and its application. The characteristic spectrum established by this method can more comprehensively and effectively control the internal quality of Zhikang capsules.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for establishing the characteristic spectrum of the traditional Chinese medicine Zhikang Capsule, wherein the Zhikang Capsule is composed of rhubarb, coptis, notoginseng, angelica, donkey-hide gelatin, calcined dragon bone, bletilla, vinegar-processed myrrh, cuttlebone, madder root, dragon's blood, licorice, pearl, and borneol.

[0006] The method for constructing the characteristic spectrum of the traditional Chinese medicine Zhikang Capsule includes the following steps:

[0007] 1) Chromatographic conditions and system suitability test

[0008] The chromatographic column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of an organic solvent as phase A and a 0.1% (v / v) aqueous solution of phosphoric acid as phase B, with gradient elution; the column temperature was 40℃~50℃; the UV detector was used with a detection wavelength of 215nm~240nm; the flow rate was 0.8ml / min-1.2ml / min; the theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 10000.

[0009] 2) Preparation of reference solution

[0010] Take appropriate amounts of catechin reference standards, magnoflorine reference standards, berberine hydrochloride reference standards, and 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate reference standards, accurately weigh them, and add 50% methanol to prepare a mixed solution containing 50 μg of catechin, 50 μg of magnoflorine, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate per ml, as the reference solution;

[0011] 3) Preparation of the test solution

[0012] Preparation of the test solution: Weigh approximately 0.5–1.5 g of the contents of Zhikang capsules accurately, place them in a stoppered conical flask, add 10–50 ml of 20%–80% methanol accurately, sonicate for 15–45 minutes at a power of 300 W and a frequency of 40 kHz; cool, filter, and collect the filtrate to obtain the test solution.

[0013] 4) Measurement

[0014] Accurately pipette 2-5 μl of the reference solution and the test solution into a high-performance liquid chromatograph (HPLC) to determine the contents of catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, and construct the characteristic HPLC chromatogram of Zhikang capsules.

[0015] Furthermore, the chromatographic column is a 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] Furthermore, gradient elution is performed, and the preferred gradient elution procedure is as follows, wherein all mobile phase proportions are volume percentages:

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

[0018] 15–25 min, 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] The mobile phase was maintained for 55–65 min, with mobile phase A at 20%–2% and mobile phase B at 80%–98%.

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

[0023] This invention further provides the characteristic map of Zhikang capsules obtained by the aforementioned construction method, as shown in the figure below. Figure 15 As shown. This invention also provides the application of the characteristic chromatogram of Zhikang capsules obtained by the described construction method in evaluating the quality and determining the content of Zhikang capsules.

[0024] Furthermore, the characteristic spectrum should exhibit nine characteristic peaks, among which the retention times of peaks 1, 3, 7, and 9 should correspond to the retention times of the reference peaks for catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate. The peak corresponding to the catechin reference peak is peak S1. The relative retention times of peaks 2 and 4 with peak S1 are calculated. The relative retention times of peaks 2 and 4 should be within ±10% of the specified values, which are 1.19 (peak 2) and 2.42 (peak 4). The peak corresponding to the berberine hydrochloride reference peak is peak S2. The relative retention times of peaks 5, 6, and 8 with peak S2 are calculated. Their relative retention times should be within ±10% of the specified values, which are 0.80 (peak 5), 0.86 (peak 6), and 1.05 (peak 8).

[0025] Furthermore, the sources of the nine common characteristic peaks in the HPLC characteristic chromatogram of the drug method are as follows: among the absorption peaks in the characteristic chromatogram, the components derived from rhubarb are peak 1 (catechin) and peak 4 (rhein-8-O-β-D-glucoside); the component derived from Bletilla striata is peak 9 (1,4-bis[4-(glucosoxy)benzyl]-2-isobutyl malate); and the components derived from Coptis chinensis are unknown components peak 2, peak 3 (magnoliine), peak 5 (berberine), peak 6 (herb root alkaloid), peak 7 (berberine hydrochloride), and peak 8 (palmatine).

[0026] The advantages of this invention compared to existing technologies are as follows:

[0027] The method for constructing the characteristic chromatogram of the traditional Chinese medicine Zhikang capsule of this invention uses high-performance liquid chromatography (HPLC) with gradient elution. This method not only determines the content of catechins, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, but also constructs the characteristic HPLC chromatogram of Zhikang capsule. Nine characteristic peaks were identified as characteristic chromatograms, covering major components such as alkaloids, phenols, anthraquinones, and glycosides. A quality evaluation system for Zhikang capsule was constructed. This method exhibits good specificity, precision, repeatability, and solution stability, and can be used to standardize the production of Zhikang capsule and provide comprehensive quality control to ensure the uniformity and stability of the product's intrinsic quality. Attached Figure Description

[0028] The invention will now be further described with reference to the accompanying drawings:

[0029] Figure 1 Liquid chromatograms for different mobile phase gradients;

[0030] Figure 2 Liquid chromatograms at different detection wavelengths;

[0031] Figure 3 Chromatograms were examined for different concentrations of acid regulators;

[0032] Figure 4 Liquid chromatograms for different chromatographic columns;

[0033] Figure 5 Liquid chromatography chromatograms at different column temperatures;

[0034] Figure 6 The liquid chromatograms show different acid modifiers in the mobile phase.

[0035] Figure 7 Chromatograms of blanks, references, and samples for system suitability testing;

[0036] Figure 8 To investigate the specificity of related spectra (dragon's blood, rhubarb);

[0037] Figure 9 For specificity analysis of related spectra (Bletilla striata, donkey-hide gelatin);

[0038] Figure 10 For specificity investigation, relevant spectra (Angelica dahurica, Rubia cordifolia);

[0039] Figure 11 To specifically examine related spectra (pearl, vinegar and myrrh);

[0040] Figure 12 To specifically investigate related maps (cuttlebone, borneol);

[0041] Figure 13 For specificity analysis, relevant atlases (calcined dragon bone, Panax notoginseng) were examined;

[0042] Figure 14 For specificity analysis, relevant spectra (licorice, coptis);

[0043] Figure 15 This is a characteristic spectrum of the test solution of the present invention;

[0044] Figure 16 For multiple batches of spectral measurements;

[0045] In the figure, peak 1: catechin (S1); peak 2: unknown component of Coptis chinensis; peak 3: magnoflorine; peak 4: rhein-8-O-β-D-glucoside; peak 5: berberine; peak 6: berberine; peak 7: berberine hydrochloride (S2); peak 8: palmatine; peak 9: 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate. Detailed Implementation

[0046] The following embodiments can help those skilled in the art to more fully understand the present invention, but should not be construed as limiting the present invention in any way.

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

[0048] Instruments: Agilent 1290 UHPLC (Agilent Technologies), Agilent 1260 HPLC (Agilent Technologies), Waters H-Class UHPLC (Waters Technologies), Agilent Poroshell HPH-C18 (4.6×150mm, 2.7μm) column, 0.001 g balance (Mettler-Toledo, ME104), 0.1 mg balance (Mettler-Toledo, XPR2), CNC ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd., KQ-300DB), electric thermostatic water bath (Shanghai Yiheng Scientific Instrument Co., Ltd., HWS26), cooling liquid circulation pump (Shanghai Dayan Instrument Equipment Co., Ltd., LTC-5 / 30), high-speed benchtop 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 & Beverage Co., Ltd.); Formic acid (mass spectrometry grade, Shanghai Anpu Experimental Technology Co., Ltd.); Phosphoric acid (chromatographic grade, Shanghai Anpu Experimental Technology Co., Ltd.).

[0050] Test drugs: Zhikang capsules (Xi'an Qianhe Pharmaceutical Co., Ltd.), catechin reference standard, magnoflorine reference standard, rhein-8-O-β-D-glucoside reference standard, palmatine reference standard, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate reference standard were all obtained from Shanghai Shidander Standard Technical Service Co., Ltd.

[0051] Example 1. Method for establishing characteristic chromatograms of the contents of Zhikang capsules using HPLC

[0052] Based on extensive experimentation, a preliminary method for determining the content of multiple active ingredients in Zhikang capsules, including catechins, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, was established using high performance liquid chromatography. The content determination method and its characteristic chromatogram for Zhikang capsules were also established.

[0053] Example 1. Method for establishing characteristic chromatograms of the contents of Zhikang capsules using HPLC

[0054] 1.1 Selection of mobile phase

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

[0056] Preparation of reference solution: Take 2.5 mg of catechin reference standard, 2.5 mg of magnoflorine reference standard, 12.5 mg of berberine hydrochloride reference standard, and 25 mg of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate reference standard, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50% methanol to obtain a mixed reference solution containing 50 μg of catechin, 50 μg of magnoflorine, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate per ml.

[0057] Preparation of test solution: Weigh about 1.0 g of the contents of this product preparation accurately, place it in a stoppered conical flask, add 20 ml of 80% methanol accurately, sonicate for 30 minutes, cool, filter, and collect the filtrate for later 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, and mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid. Gradient elution is performed. Flow rate: 1.0 mL / min; Column temperature: 45℃; Detection wavelength: 235 nm; The theoretical plate number, calculated based on the berberine hydrochloride peak, should not be less than 10,000.

[0061] Determination: Inject 2-5 μl each of the above reference solution and the Zhikang capsule test solution into the liquid chromatograph for determination.

[0062] To investigate the effects of different mobile phase gradients on the characteristic spectrum of Zhikang capsules, and to select the gradient with better characteristic peak response and separation effect.

[0063] Method 1: The gradient elution procedure is as follows, where all mobile phase proportions are volume percentages:

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

[0065] 15–25 min, 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] 55–65 min, mobile phase A is 20%–2%, mobile phase B is 80%–98%;

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

[0070] Table 1: Gradient Adjustment Examination Table

[0071]

[0072] Note: Phase A is acetonitrile.

[0073] Table 2: Results of Gradient Adjustment Examination

[0074]

[0075] Note: The two values ​​of separation are the separation between the target peak and its preceding and following peaks, respectively. NA indicates that there are no peaks before or after the target peak.

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

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

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

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

[0080] The results showed that there were significant differences in chromatographic behavior under different additives. When formic acid was used as the additive, baseline drift was more severe. When phosphoric acid was used, the overall chromatographic behavior was better. Therefore, phosphoric acid was chosen as the additive.

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

[0082]

[0083] The effect of different mobile phase acid regulator concentrations on the characteristic chromatograms of Zhikang capsules was further investigated in order to select the additive concentration with better characteristic peak response and separation effect.

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

[0085] Table 4: Results of the study on 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 alkaloids shortened. Considering the pH tolerance and lifespan of the chromatographic column, 0.1% phosphoric acid was selected as the nearest acid adjuster.

[0088] 1.2 Selection of detection wavelength

[0089] Data were acquired at different detection wavelengths (220 nm, 235 nm, 254 nm, 280 nm, 300 nm, 330 nm, and 360 nm) under the chromatographic conditions described in Method 1. See attached results. Figure 2 .

[0090] The results showed that the chromatographic behavior differed significantly at different detection wavelengths, including 220nm, 235nm, 254nm, 280nm, 300nm, 330nm, and 360nm. Except for 220nm and 235nm, the other wavelengths all had missing characteristic peaks. At the same time, compared with 220nm, there was less interference from impurities around the characteristic peak at 235nm. Therefore, 235nm was selected as the detection wavelength.

[0091] 1.3 Selection of Different Chromatographic Columns

[0092] The influence of different chromatographic columns on the characteristic chromatogram of Zhikang capsules was investigated. Agilent Poroshell HPH-C18 (4.6×150mm, 2.7μm, column number: 1), Agilent Poroshell 120EC-C18 (4.6×150mm, 2.7μm, column number: 2), and Agilent Poroshell 120AQ-C18 (4.6×150mm, 2.7μm, column number: 3) were selected for preparing the test solution. Except for column adjustments, the detection wavelength was 235nm, and data acquisition was performed according to the chromatographic conditions described in section "1.1". The appropriate chromatographic column was selected by observing the tailing factor and resolution of each characteristic peak in the chromatogram. The results are detailed in Table 5 and... Figure 4 .

[0093] Table 5: Results of experiments with different chromatographic columns

[0094]

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

[0096] 1.4 Selection of different column temperatures

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

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

[0099]

[0100] The results showed that the chromatographic behavior differed 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℃, there was less interference from impurities around the characteristic peaks, so the column temperature of 45℃ was selected.

[0101] 1.5 Selection of different flow rates

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

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

[0104]

[0105] The results showed that the chromatographic behavior differed at different flow rates. As the flow rate increased, the retention time of each characteristic peak shortened. Peaks 6 and 8 showed significant impurity interference at different flow rates. Therefore, a flow rate of 1.0 ml / min was selected.

[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 the Zhikang capsule test solution were investigated to determine the preparation method of the Zhikang capsule test solution.

[0108] 2.1 Investigation of Extraction Solvents

[0109] The effects of different extraction solvents on the characteristic chromatograms of this product were investigated. 25%, 50%, and 75% methanol were used as extraction solvents, respectively. The effects of different extraction solvents on the characteristic chromatograms of this product were compared by observing the peak shape and resolution of the characteristic peaks and calculating the "total peak area / sample weight" of the characteristic peaks, and the optimal extraction solvent was selected.

[0110] Preparation of the test solution: Take the contents of Zhikang capsules, mix well, and accurately weigh three portions (approximately 1.0 g each). Add 20 ml of 25% methanol, 50% methanol, and 75% methanol to each portion, respectively. Sonicate (300 W, 40 kHz) for 30 minutes, cool, filter, and collect the filtrate. Analyze according to the chromatographic conditions described in section 1.1.1. See Table 8 for the results.

[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" also increased. Observing the peak areas of each characteristic peak, it was found that peaks 6 to 10 were significantly affected by the extraction solvent, while the other parameters showed no significant differences. Considering that the relevant peaks are 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 chromatogram of this product were investigated. Extraction times of 15 minutes, 30 minutes, and 45 minutes were used. The peak shape and resolution of the characteristic peaks were observed, and the "total peak area / sample weight" of the characteristic peaks were calculated to compare the effects of different extraction times on the characteristic chromatogram of this product, and the optimal extraction time was selected.

[0116] Preparation of the test solution: Take the contents of Zhikang capsules, mix well, and accurately weigh three portions (approximately 1.0 g each). Accurately add 20 ml of 75% methanol to each portion, and sonicate (300 W, 40 kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, filter, and collect the filtrate. Analyze according to the chromatographic conditions under section "1.1.1". See Table 9 for the results.

[0117] The results showed that as the extraction time increased, the "total peak area / sample weight" tended to stabilize, and different extraction times had no significant effect on the extraction effect. Therefore, 30 minutes was selected as a more suitable extraction time.

[0118] Table 9: Examination of Different Extraction Times

[0119]

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

[0121] The effect of different extraction-liquid ratios on the characteristic chromatogram of this product was investigated. Extraction-liquid ratios of 1:10, 1:20, and 1:50 were used respectively. The peak shape and resolution of the characteristic peaks were observed, and the effect of different extraction-liquid ratios on the characteristic chromatogram of this product was compared by calculating the "total peak area / sample weight × extraction solution volume" of the characteristic peaks, and the optimal extraction-liquid ratio was selected.

[0122] Preparation of the test solution: Take the contents of Zhikang capsules, mix well, and accurately weigh three portions (approximately 1.0 g each). Add 10 ml, 20 ml, and 50 ml of 75% methanol to each portion, respectively. Sonicate (300 W, 40 kHz) for 30 minutes, cool, filter, and collect the filtrate. Analyze according to the chromatographic conditions described in section "1.1.1". Results are detailed in Table 10.

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

[0124]

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

[0126] 2.4 Examination of Different Extraction Methods

[0127] The effects of different extraction methods on the characteristic chromatogram of this product were investigated. Ultrasonic extraction and reflux extraction were used as extraction methods, respectively. The effects of different extraction methods on the characteristic chromatogram of this product were compared by observing the peak shape and resolution of characteristic peaks and calculating the "total peak area / sample weight" of characteristic peaks, and the optimal extraction method was selected.

[0128] Preparation of the test solution: Take the contents of Zhikang capsules, mix well, and accurately weigh two portions of approximately 1.0 g each. Add 20 ml of 75% methanol to each portion, and sonicate (300 W, 40 kHz) and reflux for 30 minutes respectively. Cool, filter, and collect the filtrate. Analyze according to the chromatographic conditions in section "1.1.1". The results are detailed in Table 11.

[0129] Table 11: Examination of Different Extraction Methods

[0130]

[0131] The results showed that different extraction methods had no significant effect on the extraction efficiency of each component, and the chromatographic behavior was not significantly changed. For ease of operation, the ultrasonic extraction method was selected.

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

[0133] As shown in Examples 1-2, the optimal HPLC method for determining the characteristic chromatograms of Zhikang capsules specifically includes the following steps:

[0134] 1) Preparation of reference solution: Take 2.5 mg of catechin reference standard, 2.5 mg of magnoflorine reference standard, 12.5 mg of berberine hydrochloride reference standard, and 25 mg of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate reference standard, place them in a 50 ml volumetric flask, dissolve and dilute to the mark with 50% methanol to obtain a mixed reference solution containing 50 μg of catechin, 50 μg of magnoflorine, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate per ml.

[0135] 2) Preparation of test solution: Weigh about 1.0 g of the contents of this product preparation accurately, place it in a stoppered conical flask, add 20 ml of 75% methanol accurately, sonicate (300 W, 40 kHz) for 30 minutes, cool, filter, and keep the filtrate for later 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% (v / v) aqueous solution of phosphoric acid. Gradient elution is performed, and the gradient elution program is as follows, where all mobile phase proportions are volume percentages:

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

[0140] 15–25 min, 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] 55–65 min, mobile phase A is 20%–2%, mobile phase B is 80%–98%;

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

[0145] 4) Determination: Inject 2-5 μl of the above reference solution and the test solution of Zhikang capsules into the liquid chromatograph, and determine the contents of Zhikang capsules, catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate by high performance liquid chromatography.

[0146] 3.1 System Suitability Test

[0147] The test solution and reference solution were prepared using the same 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 chromatograms of Zhikang capsules, and to investigate whether the blank solvent would cause interference. The results showed that the blank solvent did not interfere with the sample, and the retention time of the reference standard in the reference solution was consistent with the retention time of the corresponding component in the test sample. Relevant chromatograms are shown below. Figure 6 .

[0148] 3.2 Specificity Examination

[0149] The test solution and reference solution were prepared using the same preparation method as in "Example 3", and analyzed under the same chromatographic conditions as in "Example 1" to verify the origin of each characteristic peak in the Zhikang capsule test solution. Relevant chromatograms are shown below. Figure 7 .

[0150] According to the results, the nine characteristic peaks shown in the characteristic spectrum originated from Coptis chinensis, rhubarb, and Bletilla striata. Among them, the components derived from rhubarb include catechin (peak 1) and rhein-8-O-β-D-glucoside (peak 4); the component derived from Bletilla striata is 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate (peak 9); and the components derived from Bletilla striata are... Figure 14 The specificity analysis of Coptis chinensis revealed that peak 2 is an unknown component in Coptis chinensis; that is, the specificity experiment confirmed that it originated from Coptis chinensis, but it is an unknown component that has not been studied or reported to date. Therefore, the components derived from Coptis chinensis include peak 2 (an unknown component in Coptis chinensis), magnoflorine (peak 3), berberine (peak 5), berberine (peak 6), berberine hydrochloride (peak 7), and palmatine (peak 8).

[0151] 4. Methodological Validation

[0152] 4.1 Repeatability

[0153] Six parallel test solutions were prepared from the same batch of Zhikang capsules according to the test solution preparation method described in section "1.1.1". Data were collected under 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 values ​​of the relative retention time of each characteristic peak were all less than 2%, and the RSD values ​​of the relative peak areas were all less than 5%, indicating that the method has good repeatability. Detailed results are shown in Table 12-15 below.

[0154] Table 12: Results of Retention Times of Characteristic Peaks in Repeatability Testing

[0155]

[0156] Table 13: Results of Peak Area of ​​Each Characteristic Peak in Repeatability Study

[0157]

[0158] Table 14: Results of Relative Retention Times of Characteristic Peaks in Repeatability Testing

[0159]

[0160] Table 15: Results of Relative Peak Areas of Characteristic Peaks in Repeatability Testing

[0161]

[0162] 4.2 Intermediate Precision Assessment Among Different Personnel

[0163] Two researchers, A and B, each took samples of the same batch of Zhikang capsules and prepared six parallel test solutions according to the method described in "Example 1". Data were collected on the same instrument under the specified chromatographic conditions, and the relative retention times and relative peak areas of each characteristic peak were calculated. The results showed that the RSD values ​​of the relative retention times of each characteristic peak were all less than 2%, and the RSD values ​​of the relative peak areas were all less than 5%, indicating good intermediate precision of the method among different researchers. Detailed results are shown in Table 16-21. (Test solutions S1-S6 were prepared by researcher A, and test solutions S7-S12 were prepared by researcher B.)

[0164] Table 16: Results of Retention Times of Characteristic Peaks in Intermediate Precision Testing for Different Personnel

[0165]

[0166] Note: This table shows the results for Person B. The results for Person A can be found in the relevant table under the repeatability test. The same applies below.

[0167] Table 17: Results of Peak Area of ​​Various Characteristic Peaks in Intermediate Precision Examination for Different Personnel

[0168]

[0169]

[0170] Table 18: Results of Relative Retention Times of Characteristic Peaks in Intermediate Precision Testing for Different Personnel

[0171]

[0172] Table 19: Results of Relative Peak Areas of Characteristic Peaks in Intermediate Precision Testing by Different Personnel

[0173]

[0174] Table 20: Results of Relative Retention Times of Characteristic Peaks in Intermediate Precision Study for Different Personnel (12 Samples)

[0175]

[0176] Table 21: Results of Relative Peak Areas of Characteristic Peaks in Intermediate Precision Testing by Different Personnel (12 Samples)

[0177]

[0178] Example 5. Multiple batch determinations

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

[0180] Table 22: Results of Retention Time Measurements for Multiple Batches

[0181]

[0182] Table 23: Results of Peak Area Measurements in Multiple Batches

[0183]

[0184] Table 24: Results of Relative Retention Time Measurements for Multiple Batches

[0185]

[0186] Table 25: Results of Relative Peak Area Measurements in Multiple Batches

[0187]

[0188] Multiple batches of Zhikang capsules were tested, and each batch of test samples showed 9 characteristic peaks in the chromatogram. The retention times of peaks 1, 3, 7, and 9 corresponded to the retention times of the reference peaks of catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutylmalate. The peak corresponding to the catechin reference peak was peak S1. The relative retention times of peaks 2 and 4 with peak S1 were calculated, and their relative retention times were within ±10% of the specified values, which were 1.19 (peak 2) and 2.42 (peak 4). The peak corresponding to the berberine hydrochloride reference peak was peak S2. The relative retention times of peaks 5, 6, and 8 with peak S2 were calculated, and their relative retention times were within ±10% of the specified values, which were 0.80 (peak 5), 0.86 (peak 6), and 1.05 (peak 8). (See Tables 22-25 and...) Figure 16 It can be seen that the superimposed characteristic chromatograms of Zhikang capsules all show 9 relatively obvious common peaks, and the relative retention times of the characteristic peaks in the above 17 batches of samples are very stable, with RSDs all less than 0.5%, far lower than the standard requirement of 3%; the relative peak areas are also relatively stable, with RSDs all less than 4.0%, lower than the standard requirement of 5%.

[0189] Example 6. Accelerated Stability Assessment

[0190] Accelerated stability studies were conducted on the same batch (batch 20240101) of Zhikang capsules under the conditions of temperature: 40℃±2℃; relative humidity: 75±5%. During the experiment, samples were taken at 0 months, 3 months, and 6 months, using the same preparation method and chromatographic conditions as in "Example 3". The relative retention times of each characteristic peak and the contents of its characteristic components—catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate—were calculated. The results showed that the relative retention times of each characteristic peak were all less than 2%. At 0 months, each capsule contained 0.265 mg of catechin, 0.165 mg of magnoflorine, 2.297 mg of berberine hydrochloride, and 1.251 mg of 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate. The results after removing moisture at 3 and 6 months were consistent. Specific chromatograms and results are shown below. The experiment demonstrates that this method is suitable for the quality control of Zhikang capsules.

[0191] Table 26: Results of Accelerated Stability Experiment

[0192]

[0193] Table 27: Summary Table of Experimental Results

[0194]

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

Claims

1. A method for constructing a characteristic spectrum of the traditional Chinese medicine Zhikang capsule, characterized in that, Specifically, it includes the following steps: 1) Chromatographic conditions and system suitability test The chromatographic column was Poroshell HPH-C. 18 , 4.6 mm × 150 mm, 2.7 μm; Mobile phase: Mobile phase A is acetonitrile, and mobile phase B is a 0.1% (v / v) aqueous solution of phosphoric acid. Gradient elution is performed, and the gradient elution program is as follows, where all mobile phase proportions are volume percentages: 0–15 min, mobile phase A is 2%–10%, mobile phase B is 98%–90%; 15–25 min, 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%; 55–65 min, mobile phase A is 20%–2%, mobile phase B is 80%–98%; The flow rate was 1.0 mL / min; the column temperature was 45℃; and the detection wavelength was 235 nm. The theoretical plate number, calculated based on the berberine hydrochloride peak, should be no less than 10,000. 2) Preparation of reference solution Accurately weigh appropriate amounts of catechin reference standards, magnoflorine reference standards, berberine hydrochloride reference standards, and 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate reference standards, and add 50% methanol to prepare a mixed solution containing 50 μg of catechin, 50 μg of magnoflorine, 0.25 mg of berberine hydrochloride, and 0.5 mg of 1,4-bis[4-(glucosoxy)benzyl]-2-isobutylmalate per ml, as the reference solution; 3) Preparation of the test solution Preparation of the test solution: Weigh 1.0 g of the contents of Zhikang capsules accurately, place them in a stoppered conical flask, add 20 ml of 75% methanol accurately, sonicate for 30 minutes at a power of 300 W and a frequency of 40 kHz; cool, filter, and collect the filtrate to obtain the test solution. 4) Measurement Accurately pipette 2-5 μl of the reference solution and the test solution into a high-performance liquid chromatograph (HPLC) to determine the contents of catechin, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate, and construct the characteristic HPLC chromatogram of Zhikang capsules.

2. The method for constructing the characteristic spectrum of the traditional Chinese medicine Zhikang capsule according to claim 1, characterized in that: 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, magnoflorine, berberine hydrochloride, and 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate. Taking the peak corresponding to the catechin reference peak as reference peak S1, the relative retention times of peaks 2 and 4 with peak 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%. Taking the peak corresponding to the berberine hydrochloride reference peak as reference peak S2, the relative retention times of peaks 5, 6, and 8 with peak 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%.

3. The method for constructing the characteristic spectrum of the traditional Chinese medicine Zhikang capsule according to claim 1, characterized in that: The sources of the nine common characteristic peaks in the HPLC characteristic chromatogram of the method are as follows: among the absorption peaks of the characteristic chromatogram, the components derived from rhubarb are catechin at peak 1 and rhein-8-O-β-D-glucoside at peak 4; the components derived from Bletilla striata are 1,4-bis[4-(glucoseoxy)benzyl]-2-isobutyl malate at peak 9; and the components derived from Coptis chinensis are an unknown component at peak 2, magnoflorine at peak 3, coptisine at peak 5, berberine at peak 6, berberine hydrochloride at peak 7, and palmatine at peak 8.

4. The characteristic chromatogram of Zhikang capsules obtained by the construction method according to any one of claims 1-3 is used in the evaluation of the quality and content determination of Zhikang capsules.

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