A method for constructing a characteristic map of a traditional Chinese medicine and the characteristic map thereof

Abstract

The application discloses a method for constructing a characteristic map of a traditional Chinese medicine and the characteristic map, wherein the method adopts an HPLC method to determine the content of an effective component, imperatorin, in Zhikang capsules, and constructs a high performance liquid chromatography characteristic map of the Zhikang capsules; 10 characteristic peaks are determined as the characteristic map, which are respectively from dragon's blood, rhubarb and Bai Zhi; the components from the dragon's blood are dracorhodin B (peak 2) and pterostilbene (peak 4); the components from the rhubarb are peak 1 (aloe-emodin), peak 3, peak 6 (emodin), peak 9 and peak 10; and the components from the Bai Zhi are imperatorin (peak 5), peak 7 and peak 8; the method covers polyphenols, anthraquinones and furanocoumarins and other main components, and constructs a quality evaluation system of the Zhikang capsules; the method has good specificity, precision, repeatability and solution stability, and can be used for standardizing the production of the Zhikang capsules and guaranteeing the uniformity and stability of the internal quality of products to provide comprehensive quality control.

Description

Technical Field

[0001] This invention belongs to the field of quality control technology for traditional Chinese medicine preparations, specifically relating to a method for constructing a characteristic spectrum of a traditional Chinese medicine preparation and its characteristic spectrum. 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. It has been included in and recommended for clinical use in many guidelines, pathways and textbooks, such as the "Guidelines for Clinical Application of Traditional Chinese Medicine", "International Guidelines for Clinical Practice of Traditional Chinese Medicine", "Guidelines for the Management of Chronic Diseases of Peptic Ulcer in the Elderly", "Guidelines for the Diagnosis and Treatment of Uterine Bleeding Due to Ovulation Disorders by Integrated Traditional Chinese and Western Medicine", "Interpretation of Clinical Pathway Therapeutic Drugs", "Modern Colorectal Surgery", "Colorectal Surgery Nursing", and "Integrated Traditional Chinese and Western Medicine Obstetrics and Gynecology". Because Zhikang Capsules are safe and effective, its standard was included in the "Chinese Pharmacopoeia" in 2015, and the current standard is the current version of the "Chinese Pharmacopoeia".

[0003] 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 medicine preparations, 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, considering its multi-component, multi-target, and multi-pathway effects, has become widely used. Using fingerprinting or characteristic spectroscopy, on the one hand, the authenticity of samples can be effectively identified through the characteristic features of the spectra; on the other hand, by controlling the area, proportion, or retention time of the main characteristic peaks in the spectra, the quality of the product can be effectively controlled, ensuring the stability and consistency of product quality, thereby further guaranteeing the quality and efficacy of the product. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for constructing a characteristic spectrum of the traditional Chinese medicine Zhikang Capsules and the characteristic spectrum thereof. The characteristic spectrum established by this method can more comprehensively and effectively control the internal quality of Zhikang Capsules.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A method for establishing a characteristic spectrum of a traditional Chinese medicine preparation for clearing heat, cooling blood, stopping bleeding, resolving blood stasis, promoting tissue regeneration, and relieving pain, wherein the traditional Chinese medicine preparation is composed of 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.

[0007] This invention provides a method for constructing the characteristic spectrum of the traditional Chinese medicine Zhikang Capsule, specifically including the following steps:

[0008] 1) Chromatographic conditions and system suitability test

[0009] The chromatographic column was packed with octadecylsilane-bonded silica gel; the mobile phase consisted of acetonitrile as phase A and 0.1% (v / v) phosphoric acid aqueous solution as phase B, with gradient elution; the column temperature was 35℃~45℃; the UV detector was used with a detection wavelength of 220nm~280nm; the flow rate was 0.8ml / min~1.2ml / min; the theoretical plate number, calculated based on the imperatorin peak, should not be less than 10000.

[0010] 2) Preparation of reference solution

[0011] Take an appropriate amount of imperatorin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per ml, which is used as the reference solution;

[0012] 3) Preparation of the test solution

[0013] Take approximately 0.5–1.5 g of the contents of the traditional Chinese medicine Zhikang capsules, accurately weigh it, place it in a stoppered conical flask, accurately add 10–30 ml of 70%–80% methanol, 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 product.

[0014] 4) Measurement

[0015] Accurately pipette 2–10 μl of the reference solution and the test solution into a high-performance liquid chromatograph (HPLC) to determine the content of imperatorin and construct the characteristic HPLC chromatogram of Zhikang capsules.

[0016] According to a further description of the present invention: the chromatographic column is a Waters XBridge-C18 (4.6×250mm, 5μm); the flow rate is 1.0mL / min; the column temperature is 40℃; and the detection wavelength is 254nm.

[0017] According to a further description of the present invention: the gradient elution program is as follows, wherein the proportions of the mobile phases are all volume percentages: 0-20 min, mobile phase A is 35%, and mobile phase B is 65%;

[0018] 20–40 min, mobile phase A is 35%–45%, mobile phase B is 65%–55%;

[0019] 40–60 min, mobile phase A is 45%–70%, mobile phase B is 55%–30%;

[0020] The mobile phase was maintained for 60–70 min, with mobile phase A at 70%–35% and mobile phase B at 30%–55%.

[0021] According to a further explanation of the present invention: In step 3), approximately 1.0g of the contents of the Zhikang capsule is accurately weighed, placed in a stoppered conical flask, and 20ml of 75% methanol is accurately added. The mixture is then sonicated for 30 minutes at a power of 300W and a frequency of 40kHz. After cooling, the mixture is filtered, and the filtrate is collected to obtain the final product.

[0022] This invention also provides a characteristic chromatogram of Zhikang capsules obtained by the aforementioned construction method. Furthermore, this invention provides the application of the aforementioned characteristic chromatogram of Zhikang capsules in evaluating the quality and determining the content of Zhikang capsules.

[0023] According to a further explanation of the present invention: the characteristic spectrum should present 10 characteristic peaks, wherein the retention time of peak 5 should correspond to the retention time of the imperatorin reference peak; taking peak 5 corresponding to the imperatorin reference peak as reference peak S, the relative retention times of peaks 1 to 4, peaks 6 to 10 and peak S are calculated: the relative retention times of each characteristic peak should be within ±10% of the specified value, and the specified values ​​are: 0.40 (peak 1), 0.69 (peak 2), 0.83 (peak 3), 0.92 (peak 4), 1.04 (peak 6), 1.12 (peak 7), 1.23 (peak 8), 1.37 (peak 9), 1.50 (peak 10).

[0024] Furthermore, the characteristic spectrum identified the sources of 10 common characteristic peaks, which originated from dragon's blood, rhubarb, and angelica dahurica, respectively. Among them, the components from dragon's blood are dragon's blood extract B (peak 2) and pterostilbene (peak 4); the components from rhubarb are peak 1 (aloe-emodin), peak 3 (unknown component), 6 (rhubarb peak), peak 9 (unknown component), and peak 10 (unknown component); and the components from angelica dahurica are imperatorin (peak 5), peak 7, (unknown component), and peak 8 (unknown component).

[0025] The advantages of this invention compared to existing technologies are as follows:

[0026] The method for constructing the characteristic chromatogram of the traditional Chinese medicine Zhikang Capsules of this invention uses high-performance liquid chromatography (HPLC) with gradient elution. This method not only determines the content of imperatorin but also constructs the HPLC characteristic chromatogram of Zhikang Capsules. Ten characteristic peaks were identified as characteristic chromatograms: Dragon's blood (dragon's blood) components include dragon's blood B (peak 2) and pterostilbene (peak 4); rhubarb (rhubarb) components include peak 1 (aloe-emodin), peak 3, peak 6 (emodin), peak 9, and peak 10; and angelica (angelica dahurica) components include imperatorin (peak 5), peak 7, and peak 8. This method covers major components such as polyphenols, anthraquinones, and furanocoumarins, and constructs a quality evaluation system for Zhikang Capsules. 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. Attached Figure Description

[0027] The invention will now be further described with reference to the accompanying drawings:

[0028] Figure 1 Liquid chromatograms for different mobile phase gradients;

[0029] Figure 2 Chromatograms were examined for different acid modifiers;

[0030] Figure 3 Chromatograms were examined for different concentrations of acid regulators;

[0031] Figure 4 Liquid chromatograms at different detection wavelengths;

[0032] Figure 5 Liquid chromatograms for different chromatographic columns;

[0033] Figure 6 These are liquid chromatograms at different column temperatures;

[0034] Figure 7 Chromatograms of liquid phases at different flow rates;

[0035] Figure 8 Chromatograms of liquid phases with different injection volumes;

[0036] Figure 9 Chromatograms of blanks, references, and samples for system suitability testing;

[0037] Figure 10 To investigate the specificity of related spectra (dragon's blood, rhubarb);

[0038] Figure 11 To specifically investigate related spectra (Bletilla striata, donkey-hide gelatin);

[0039] Figure 12 For specificity investigation, relevant spectra (Angelica dahurica, Rubia cordifolia) were examined;

[0040] Figure 13 To investigate the specificity of related spectra (pearl, vinegar and myrrh);

[0041] Figure 14 To specifically investigate related maps (cuttlebone, borneol);

[0042] Figure 15 For specificity analysis, relevant atlases (calcined dragon bone, Panax notoginseng) were examined;

[0043] Figure 16 To investigate the specificity of related spectra (licorice, coptis);

[0044] Figure 17 This is a characteristic spectrum of the test solution of the present invention;

[0045] Figure 18 For multiple batches of spectral measurements;

[0046] In the figure, peak 1: aloe-emodin; peak 2: dracoside B; peak 4: pterostilbene; peak 5: imperatorin (S); peak 6: emodin. Detailed Implementation

[0047] 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.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the instruments, reagents, and reagents used are as follows:

[0049] Instruments: Agilent 1260 HPLC system (Agilent Technologies), Waters 2695 HPLC system (Waters Technologies), AB Sciex Triple 4600 high-resolution mass spectrometry (SCIEX), Waters XBridge-C18 (4.6×250mm, 5μm) column, Agilent Eclipse XDB-C18 (4.6×250mm, 5μm) column, Waters Atlantis TM T3 (4.6×250mm, 5μm) chromatographic column, 0.001 g / mL balance (Mettler-Toledo, ME104), 0.1 mg / mL 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), coolant circulation pump (Shanghai Dayan Instrument Equipment Co., Ltd., LTC-5 / 30), high-speed benchtop centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd., H1650-W).

[0050] 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.).

[0051] Test drugs: Zhikang capsules (Xi'an Qianhe Pharmaceutical Co., Ltd.), rhein reference solution, aloe-rhein reference solution, imperatorin reference solution, pterostilbene reference solution, dracosanol A reference solution, and dracosanol B reference solution were all from Shanghai Shidander Standard Technical Service Co., Ltd.

[0052] Example 1: Method for establishing characteristic chromatograms of the contents of Zhikang capsules using HPLC

[0053] Based on extensive experimentation, a preliminary method for determining the content of imperatorin in Zhikang capsules using high performance liquid chromatography was established, and a characteristic chromatogram for the content determination of Zhikang capsules was also developed.

[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 12.5 mg of imperatorin reference standard, place it in a 25 ml volumetric flask, add methanol to prepare a solution containing 0.5 mg per ml, which is used as the reference solution.

[0057] Preparation of the test solution: Weigh approximately 1.0 g of the contents of Zhikang capsules 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 collect the filtrate to obtain the test solution.

[0058] Chromatographic conditions and system suitability test:

[0059] Chromatographic column: Octadecylsilane-bonded silica gel as the packing material, the chromatographic column is: Waters XBridge-C18 (4.6×250mm, 5μm);

[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: 40℃; Detection wavelength: 254 nm; The theoretical plate number, calculated based on the eugenol peak, should not be less than 10,000.

[0061] Determination: Inject 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 chromatograms of Zhikang capsules, and to select the gradient with the best characteristic peak response and separation effect, the following method was used: Method 1: The gradient elution program is as follows, where all mobile phase proportions are volume percentages:

[0063] From 0 to 20 minutes, mobile phase A was 35% and mobile phase B was 65%.

[0064] 20–40 min, mobile phase A is 35%–45%, mobile phase B is 65%–55%;

[0065] 40–60 min, mobile phase A is 45%–70%, mobile phase B is 55%–30%;

[0066] The mobile phase was maintained for 60–70 min, with mobile phase A at 70%–35% and mobile phase B at 30%–55%.

[0067] Following method 1 above, except for gradient adjustment, data were collected according to the elution gradient in Table 1. The experimental results are shown in Table 2. Figure 1 .

[0068] Table 1: Gradient Adjustment Examination Table

[0069]

[0070] Note: Phase A is acetonitrile.

[0071] Table 2: Results of Gradient Adjustment Examination

[0072]

[0073] 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.

[0074] The results showed that some characteristic peaks in Method 4 had early retention times, making them difficult to distinguish. In Methods 2, 3 and 5, peak 11 had a retention time around 60 min, which was close to the end of the effective elution time. Considering that different instruments have different dead volumes, the retention time of this peak may exceed the effective elution time. Therefore, taking all factors into consideration, the gradient elution condition of Method 1 was selected as the final condition.

[0075] 1.1.2 Selection of different acid regulators in the mobile phase:

[0076] The effects of different mobile phase acid adjusters on the characteristic chromatograms of Zhikang capsules were investigated. An acid adjuster with superior characteristic peak response and better separation effect was selected as the mobile phase additive. The test solution was prepared according to the same preparation method as described in "Example 1, 1.1.1". Gradient elution conditions were selected as method 1. Except for adjustments to the mobile phase acid adjuster, data acquisition was performed according to the chromatographic conditions described in "Example 1, 1.1.1". The experimental results are shown in Table 3 and... Figure 2 .

[0077] Table 3: Results of the investigation of different acid regulators

[0078]

[0079] The results showed that the chromatographic behavior differed significantly under different acid regulators. When formic acid was used as the acid regulator, peak 4 was missing. Therefore, phosphoric acid was chosen as the acid regulator.

[0080] The effect of different mobile phase acid regulator phosphoric acid concentrations on the characteristic chromatograms of Zhikang capsules was further investigated in order to select the concentration of phosphoric acid regulator with better characteristic peak response and separation effect. The experimental results are shown in Table 4 and below. Figure 3 .

[0081] Table 4: Results of the study on different acid regulator concentrations

[0082]

[0083] The results showed that peak 4 was missing when phosphoric acid was not added. The chromatographic behavior did not change significantly as the concentration of the additive decreased. Considering the pH tolerance and lifespan of the chromatographic column, the concentration of the phosphoric acid additive was selected as 0.1%, that is, a 0.1% phosphoric acid aqueous solution was used as the mobile phase B.

[0084] In summary, the optimal mobile phase and gradient system conditions are as follows: Acetonitrile is used as phase A, and a 0.1% (v / v) aqueous solution of phosphoric acid is used as phase B for gradient elution. The gradient elution program is as follows, where all mobile phase proportions are volume percentages: 0–20 min, mobile phase A is 35%, mobile phase B is 65%; 20–40 min, mobile phase A is 35%–45%, mobile phase B is 65%–55%; 40–60 min, mobile phase A is 45%–70%, mobile phase B is 55%–30%; 60–70 min, mobile phase A is 70%–35%, mobile phase B is 30%–55%.

[0085] 1.2 Selection of detection wavelength

[0086] The test solution was prepared according to the same preparation method as described in "Example 1, 1.1.1". Except for adjusting the detection wavelength, data acquisition was performed under the chromatographic conditions described in "Example 1, 1.1.1". The test results are shown in [Figure Number]. Figure 4The results showed that the responses of each characteristic peak differed significantly at different detection wavelengths, including 220nm, 254nm, 280nm, 300nm, 330nm, and 360nm. Considering the responses of each characteristic peak, interference from impurities around the characteristic peaks, and baseline flatness, 254nm was selected as the detection wavelength.

[0087] 1.3 Selection of Different Chromatographic Columns

[0088] The effects of different chromatographic columns on the characteristic chromatograms of Zhikang capsules were investigated. Waters XBridge-C18 (4.6×250mm, 5μm, column number: Column 1), Agilent Eclipse XDB-C18 (4.6×250mm, 5μm, column number: Column 2), and Waters Atlantis were selected. TM T3 (4.6×250mm, 5μm, column number: 3) was used to prepare the test solution according to the same preparation method as described in "Example 1, 1.1.1". Except for column adjustments, data acquisition was performed using gradient elution under the chromatographic conditions described in "Example 1, 1.1.1" and Method 1. A suitable column was selected by observing the tailing factor and resolution of each characteristic peak in the chromatogram. The experimental results are shown in Table 5. Figure 5 .

[0089] Table 5: Results of experiments with different chromatographic columns

[0090]

[0091] The results showed that different types of packed columns had a certain impact on the characteristic chromatograms. By comparing the peak area, peak height, and the spectra of each chromatographic peak to locate the characteristic peaks, it was found that when column 2 was used, peak 4 was missing; when column 3 was used, peaks 1 to 3 were all missing; when column 1 was used, the characteristic peaks were evenly distributed, the peak shapes were good, and the chromatograms were more aesthetically pleasing. Considering the subsequent optimization process, Waters X-Bridge C18 (4.6×250mm, 5μm, column number: column 1) was selected as the chromatographic column for this method.

[0092] 1.4 Selection of different column temperatures

[0093] 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 described in "Example 1, 1.1.1". Except for adjusting the column temperature, data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in "Example 1, 1.1.1". The experimental results are shown in Table 6. Figure 6 .

[0094] Table 6: Results of experiments at different column temperatures

[0095]

[0096] 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 40℃, there was less interference from impurities around the characteristic peaks. Therefore, the column temperature of 40℃ was selected.

[0097] 1.5 Selection of different flow rates

[0098] The effect of different flow rates on the characteristic chromatogram of Zhikang capsules was investigated in order to select the flow rate with better characteristic peak response and better separation effect.

[0099] The test solution was prepared using the same method as in "Example 1". Except for adjusting the flow rate, data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in section 1.1.1 of "Example 1". The experimental results are shown in Table 7. Figure 7 .

[0100] Table 7: Results of investigation at different flow velocities

[0101]

[0102] 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. When the flow rate was 0.8 ml / min, peaks 4 and 5 showed significant interference. When the flow rate was 1.2 ml / min, peak 1 showed some interference. Therefore, a flow rate of 1.0 ml / min was selected.

[0103] 1.6 Selection of different injection volumes

[0104] The effect of different injection volumes on the characteristic chromatograms of Zhikang capsules was investigated to select the injection volume with better characteristic peak response and separation effect. The test solution was prepared according to the same preparation method as described in "Example 1, 1.1.1". Except for adjusting the injection volume, data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in "Example 1, 1.1.1". The experimental results are shown in Table 8. Figure 8 .

[0105] Table 8: Results of the study with different injection volumes

[0106]

[0107] The results showed that there was no significant difference in chromatographic behavior with different injection volumes, and an injection volume of 5 μl was selected for further investigation.

[0108] Example 2: Investigation of the preparation method of the test solution

[0109] The extraction solvent, extraction method, extraction time, and solid-liquid ratio used in the preparation of the test solution of Zhikang Capsules were investigated to determine the optimal method for preparing the test solution of Zhikang Capsules.

[0110] 2.1 Investigation of extraction solvent

[0111] 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.

[0112] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 3 portions, and accurately add 20ml of 25% methanol, 50% methanol, and 75% methanol respectively. Sonicate (300W, 40kHz) for 30 minutes, cool, filter, and collect the filtrate to obtain the final product. Data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in section 1.1.1 of Example 1; the experimental results are shown in Table 9.

[0113] Table 9: Investigation of different extraction solvents

[0114]

[0115] The results showed that as the proportion of methanol increased, the "total peak area / sample weight" also increased, and the extraction effect of each characteristic peak was significantly improved. Therefore, 75% methanol was chosen as the extraction solvent.

[0116] 2.2 Different extraction times

[0117] 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.

[0118] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh 3 portions, add 20ml of 75% methanol to each portion, and sonicate (300W, 40kHz) for 15 minutes, 30 minutes, and 45 minutes respectively. Cool, filter, and collect the filtrate to obtain the final product. Data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in section 1.1.1 of Example 1; the experimental results are shown in Table 10.

[0119] Table 10: Examination of Different Extraction Times

[0120]

[0121] 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.

[0122] 2.3 Examination of different extraction methods

[0123] 2.3.1 Examination of different extraction methods

[0124] 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.

[0125] Take this product, remove the capsule shell, mix well, take about 1.0g, accurately weigh two portions, accurately add 20ml of 75% methanol to each, sonicate (300W, 40kHz) and reflux for 30 minutes respectively, cool, filter, and collect the filtrate to obtain the final product. For the remaining samples, data acquisition was performed using the chromatographic conditions of Method 1 with gradient elution as described in section 1.1.1 of Example 1; the experimental results are shown in Table 11.

[0126] Table 11: Examination of Different Extraction Methods

[0127]

[0128] 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.

[0129] 2.3.2 Investigation of different extraction material-to-liquid ratios

[0130] 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.

[0131] Take this product, remove the capsule shell, mix well, and accurately weigh approximately 1.0 g into three portions. 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. For the remaining samples, perform gradient elution under the chromatographic conditions of Method 1 according to section 1.1.1 of Example 1; the experimental results are shown in Table 12.

[0132] Table 12: Investigation of different extraction material-liquid ratios

[0133]

[0134] The results showed that as the material-to-liquid ratio decreased, the ratio of "total peak area / sample weight × extraction solution volume" remained relatively stable, indicating that the material-to-liquid ratio did not have a significant impact on the extraction efficiency. Therefore, a material-to-liquid ratio of 1:20 was selected as the optimal ratio.

[0135] Example 3: System Suitability Test and Specificity Assessment

[0136] As shown in Examples 1-2, the optimal HPLC method for determining the characteristic chromatograms of Zhikang capsules specifically includes the following steps:

[0137] 1) Chromatographic conditions and system suitability test

[0138] The chromatographic column was a Waters XBridge-C18 (4.6×250mm, 5μm); the column temperature was 40℃; the UV detector was used with a detection wavelength of 254nm; the flow rate was 1.0ml / min; the theoretical plate number, calculated based on the eugenol peak, should not be less than 10000.

[0139] 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:

[0140] From 0 to 20 minutes, mobile phase A was 35% and mobile phase B was 65%.

[0141] 20–40 min, mobile phase A is 35%–45%, mobile phase B is 65%–55%;

[0142] 40–60 min, mobile phase A is 45%–70%, mobile phase B is 55%–30%;

[0143] 60–70 min, mobile phase A is 70%–35%, mobile phase B is 30%–55%;

[0144] 2) Preparation of reference solution

[0145] Take an appropriate amount of imperatorin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per ml, which is used as the reference solution;

[0146] 3) Preparation of the test solution

[0147] Weigh approximately 1.0g of the contents of Zhikang capsules accurately, place them in a stoppered conical flask, add 20ml of 75% methanol accurately, and sonicate for 30 minutes at a power of 300W and a frequency of 40kHz; cool, filter, and collect the filtrate to obtain the product.

[0148] 4) Measurement

[0149] Accurately pipette 2–10 μl of the reference solution and the test solution into a high-performance liquid chromatograph (HPLC) to determine the content of imperatorin and construct the characteristic HPLC chromatogram of Zhikang capsules.

[0150] 3.1 System Suitability Test

[0151] The test solution and reference solution were prepared in accordance with the law and analyzed according to the chromatographic conditions under “Example 3” to examine the chromatographic conditions and system suitability of the characteristic chromatogram of Zhikang Capsules, and to examine whether the blank solvent would cause interference.

[0152] The results showed that the blank solvent had no interference with the sample, and the retention times of the reference standard in the reference solution were consistent with those of the corresponding components in the test sample. Relevant chromatograms are shown below. Figure 9 .

[0153] 3.2 Specificity Examination

[0154] The test solution, reference solution, medicinal herb slices, and negative control solution were prepared according to law, and analyzed under the chromatographic conditions described in "Example 3" to verify the origin of each characteristic peak in the Zhikang capsule test solution. Relevant chromatograms are shown below. Figure 10-16 According to the results, the 10 characteristic peaks shown in the collected feature maps were derived from dragon's blood, rhubarb, and angelica. Among them, the components derived from dragon's blood were dracosanol B (peak 2) and pterostilbene (peak 4); the components derived from rhubarb were aloe-emodin (peak 1), peak 3, emodin (peak 6), peak 9, and peak 10; and the components derived from angelica were imperatorin (peak 5), peak 7, and peak 8.

[0155] Example 4: Methodological Validation

[0156] 4.1 Repeatability

[0157] Six test solutions were prepared in parallel using the same batch of Zhikang capsules according to the test solution preparation method described in Example 3. 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 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 that the method has good repeatability. Detailed results are shown in Tables 13-16.

[0158] Table 13: Results of Retention Times of Characteristic Peaks in Repeatability Testing

[0159]

[0160] Table 14: Results of Peak Area of ​​Each Characteristic Peak in Repeatability Test

[0161]

[0162] Table 15: Results of Relative Retention Times of Characteristic Peaks in Repeatability Testing

[0163]

[0164] Table 16: Results of Relative Peak Areas of Characteristic Peaks in Repeatability Testing

[0165]

[0166] 4.2 Intermediate Precision Assessment Among Different Personnel

[0167] Two experimenters, A and B, each took samples of the same batch of Zhikang capsules and prepared six parallel test solutions according to the test solution preparation method in Example 3. Data were collected on the same instrument under the specified chromatographic conditions, and the relative retention time and relative peak area of ​​each characteristic peak were calculated. Detailed results are shown in Tables 17-22.

[0168] Table 17: Results of Retention Times of Characteristic Peaks in Intermediate Precision Testing for Different Personnel

[0169]

[0170] 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.

[0171] Table 18: Results of Peak Area of ​​Various Characteristic Peaks in Intermediate Precision Examination for Different Personnel

[0172]

[0173] Table 19: Results of Relative Retention Times of Characteristic Peaks in Intermediate Precision Testing by Different Personnel

[0174]

[0175] Table 20: Results of Relative Peak Areas of Characteristic Peaks in Intermediate Precision Testing by Different Personnel

[0176]

[0177] Table 21: Results of Relative Retention Times of Characteristic Peaks in Intermediate Precision Testing for Different Personnel (12 Samples)

[0178]

[0179] Table 22: Results of Relative Peak Areas of Characteristic Peaks in Intermediate Precision Testing by Different Personnel (12 Samples)

[0180]

[0181] The results show that the RSD values ​​of the relative retention times of each characteristic peak are all less than 2%, and the RSD values ​​of the relative peak areas are all less than 5%, indicating that the method has good intermediate precision among different personnel.

[0182] Example 5: Multiple batch determinations

[0183] 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 23-26 below. Figure 18 .

[0184] Table 23: Retention Time Results of Multiple Batch Determination Tests

[0185]

[0186] Table 24: Peak Area Results of Multiple Batch Determination Tests

[0187]

[0188] Table 25: Relative Retention Time Results of Multiple Batch Determination Tests

[0189]

[0190] Table 26: Results of relative peak area in multiple batch determination experiments

[0191]

[0192] Multiple batches of Zhikang capsules were tested, and each batch of test samples showed 10 characteristic peaks in the chromatogram. The retention time of peak 5 corresponds to the retention time of the imperatorin reference peak. The peak corresponding to the imperatorin reference peak is peak S. The relative retention times of peaks 1-4 and peaks 6-10 with peak S were calculated. The relative retention times of each characteristic peak were within ±10% of the specified values, which are: 0.40 (peak 1), 0.69 (peak 2), 0.83 (peak 3), 0.92 (peak 4), 1.04 (peak 6), 1.12 (peak 7), 1.23 (peak 8), 1.37 (peak 9), and 1.50 (peak 10). (See Tables 23-26 and...) Figure 18 It can be seen that the superimposed characteristic chromatograms of Zhikang capsules all show 10 relatively obvious common peaks. Furthermore, 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 below 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%. While there are no significant differences in the relative retention times of the characteristic peaks, the relative peak areas of some characteristic peaks vary considerably. Therefore, retention times can be incorporated into the standard for control.

[0193] Example 6: Accelerated Stability Assessment

[0194] Accelerated stability studies were conducted on the same batch (batch 20240101) of Zhikang capsules under the conditions of temperature: 40℃±2℃ and relative humidity: 75±5%. Samples were taken at month 0, month 3, and month 6 during the experiment, and the relative retention time of each characteristic peak and the content of its characteristic component, imperatorin, were calculated according to the same preparation method and chromatographic conditions as in "Example 3". The experimental results are shown in Tables 27-28.

[0195] Table 27: Analysis of Peak Results During the Study Period

[0196]

[0197] Table 28: Summary Table of Experimental Results

[0198] Inspection period Imperatorin content results (μg / capsule) October 2.75 Accelerate March 2.76 Accelerate June 2.72

[0199] The results showed that the relative retention times of each characteristic peak were all less than 2%. Each capsule contained 2.75 μg of imperatorin at 0 months, 2.76 μg at 3 months (accelerated), and 2.72 μg at 6 months (accelerated), with consistent detection results. Specific spectra and results are shown below. The experiment demonstrates that this method is suitable for the quality control of Zhikang capsules.

[0200] 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 traditional Chinese medicine preparations, characterized in that, The traditional Chinese medicine mentioned is Zhikang Capsules, which specifically includes the following steps: 1) Chromatographic conditions and system suitability test The chromatographic column is packed with octadecylsilane-bonded silica gel; the column is a Waters XBridge-C. 18 4.6 × 250 mm, 5 μm; column temperature 40℃; The mobile phase consisted of acetonitrile as phase A and a 0.1% (v / v) aqueous solution of phosphoric acid as phase B, and gradient elution was performed. The gradient elution procedure is as follows, where all mobile phase proportions are volume percentages: From 0 to 20 minutes, mobile phase A was 35% and mobile phase B was 65%. 20–40 min, mobile phase A is 35%–45%, mobile phase B is 65%–55%; 40–60 min, mobile phase A is 45%–70%, mobile phase B is 55%–30%; 60–70 min, mobile phase A is 70%–35%, mobile phase B is 30%–65%; The UV detector has a flow rate of 1.0 mL / min and a detection wavelength of 254 nm. The theoretical plate number, calculated based on the eugenol peak, should be no less than 10,000. 2) Preparation of reference solution Take an appropriate amount of imperatorin reference standard, accurately weigh it, and add methanol to prepare a solution containing 0.5 mg per ml, which is used as the reference solution; 3) Preparation of the test solution Weigh approximately 1.0g of the contents of Zhikang capsules (a traditional Chinese medicine), place it in a stoppered conical flask, add 20ml of 75% methanol, and sonicate for 30 minutes at a power of 300W and a frequency of 40kHz. Cool, filter, and collect the filtrate to obtain the final product. 4) Measurement Accurately pipette 2-10 μl of the reference solution and the test solution into a high-performance liquid chromatograph to determine the content of imperatorin and construct the characteristic high-performance liquid chromatogram of Zhikang capsules. The characteristic spectrum identified the sources of 10 common characteristic peaks, which originated from dragon's blood, rhubarb, and angelica dahurica, respectively. Among them, the components from dragon's blood were dragon's blood B at peak 2 and pterostilbene at peak 4; the components from rhubarb were aloe-emodin at peak 1, an unknown component at peak 3, emodin at peak 6, an unknown component at peak 9, and an unknown component at peak 10; and the components from angelica dahurica were imperatorin at peak 5, an unknown component at peak 7, and an unknown component at peak 8.

2. The method for constructing the characteristic spectrum of traditional Chinese medicine preparations according to claim 1, characterized in that: The characteristic spectrum should present 10 characteristic peaks, among which the retention time of peak 5 should correspond to the retention time of the imperatorin reference peak; using peak 5 corresponding to the imperatorin reference peak as reference peak S, the relative retention times of peaks 1-4, peaks 6-10 and peak S are calculated: the relative retention time of peak 1 is 0.40±10%, the relative retention time of peak 2 is 0.69±10%, the relative retention time of peak 3 is 0.83±10%, the relative retention time of peak 4 is 0.92±10%, the relative retention time of peak 6 is 1.04±10%, the relative retention time of peak 7 is 1.12±10%, the relative retention time of peak 8 is 1.23±10%, the relative retention time of peak 9 is 1.37±10%, and the relative retention time of peak 10 is 1.50±10%.

3. The characteristic chromatogram of Zhikang capsules obtained by the construction method according to any one of claims 1-2 is used in the evaluation of the quality and content determination of Zhikang capsules.

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