Preparation and detection method of substance standard of Qingjin phlegm-reducing decoction

Abstract

The invention discloses a preparation process of a Qingjin phlegm-reducing decoction material standard, an HPLC content determination method and a fingerprint spectrum establishment method aiming at the material standard, under the content determination method, the peak shapes of chromatographic peaks of all components are symmetrical, the separation effect is remarkable, and excellent stability, specificity, linear response, repeatability, precision and accuracy are shown. According to the present invention, the fingerprint characteristics are accurately recorded, the reversed-phase high performance liquid chromatography technology is adopted to successfully obtain 13 common peaks, the baicalin is adopted as the reference peak, the relative retention time and the relative peak area of the common peaks are calculated, and the reference substance comparison is further performed to identify the 8 common peaks; the established fingerprint spectrum method also has the characteristics of good chromatographic peak shape, high separation degree, stable method, excellent repeatability, high precision and the like. In conclusion, the invention provides a set of comprehensive and reliable Qingjin phlegm-reducing decoction material standard quality control reference system, and a scientific basis is provided for the overall quality evaluation of the Qingjin phlegm-reducing decoction.

Description

Technical Field

[0001] The present invention relates to the field of analysis of traditional Chinese medicine preparations, and in particular to a preparation process of a Qingjin Huatan Decoction substance benchmark, as well as an HPLC (high performance liquid chromatography) content determination method and a fingerprint establishment method for the substance benchmark. Background Art

[0002] Qingjin Huatan Decoction is a classic prescription for treating phlegm-heat congestion in the lungs. It originates from the Ming Dynasty book "Medical General Principles" by Ye Wenling. The original text states: "Qingjin Huatan Decoction is used for those with heat-related symptoms, such as dry and sore throat, flushed face, and hot nasal discharge. The phlegm is difficult to cough up and is thick, yellow, and sometimes blood-tinged or has a fishy odor." The prescription comprises eleven herbs. Scutellaria baicalensis, Gardenia jasminoides, and Anemarrhena rhizome act as the main herbs to clear heat and clear the lungs. Morus alba bark, Fritillaria thunbergii, stir-fried Trichosanthes fruit, and Ophiopogon japonicus act as the auxiliary herbs to clear the lungs, resolve phlegm, and relieve cough. Platycodon grandiflorum is used as an adjuvant to dissipate lung heat and eliminate phlegm; Tangerine peel regulates qi, dries dampness, and resolves phlegm; Poria cocos strengthens the spleen, dissipates dampness, and eliminates phlegm. Licorice root acts as an adjuvant, both resolving phlegm and relieving cough, and harmonizing the other herbs. These herbs work synergistically to clear lung heat, resolve phlegm, and relieve cough. Qingjin Huatan Decoction and its modified prescriptions are often used in modern clinical practice to treat patients with acute exacerbation of chronic obstructive pulmonary disease (phlegm-heat obstructing the lungs syndrome), and also have good therapeutic effects in the treatment of respiratory diseases such as pneumonia, chronic acute bronchitis, and bronchiectasis.

[0003] To promote the research and development of traditional Chinese medicine compound preparations derived from ancient classic formulas and streamline the registration and approval process, the State Administration of Traditional Chinese Medicine and the State Food and Drug Administration published the "Catalogue of Ancient Classic Formulas (First Batch)" in 2018, with Qingjin Huatan Decoction as the 56th formula. Research on traditional Chinese medicine compound preparations managed under the "Catalogue of Ancient Classic Formulas" has been a key initiative in China's recent modernization and internationalization of traditional Chinese medicine. This research aims to discover, preserve, and utilize traditional Chinese medicine classic formulas, integrating modern science and technology to transform them into modern Chinese medicine preparations that meet modern pharmaceutical standards. New drug registration applications for classic formulas generally fall under "Category 3.1" of the Traditional Chinese Medicine registration system. Category 3.1 traditional Chinese medicine compound preparations are those derived from the nationally published "Catalogue of Ancient Classic Formulas" whose formulas, preparation methods, main functions, and usage and dosage are generally consistent with those recorded in ancient medical texts.

[0004] Material benchmarks and fingerprints are closely related and of great value in quality control and drug analysis in Traditional Chinese Medicine (TCM) research. Fingerprints are used as a crucial tool in the quality evaluation of classic prescriptions, while material benchmarks provide a standard for comparison and verification. A fingerprint is a characteristic chemical fingerprint of a drug or substance generated using analytical techniques such as high-performance liquid chromatography. It can provide a comprehensive picture of all components in a sample, particularly in complex prescriptions. Fingerprints can be used as a tool for substance identification. By comparing the fingerprint of a test sample with that of an existing material benchmark, it is possible to confirm whether it is the intended medicinal material or prescription. This is particularly important in the identification of TCM compound prescriptions. Fingerprints not only focus on the main components but also include trace components. Due to their characteristic properties, they can serve as a "chemical fingerprint" for substances and are used for the identification, comparison, and quality control of compound TCM prescriptions. Summary of the Invention

[0005] The present invention provides a method for preparing a Qingjin Huatan Decoction substance reference, a method for determining the content of the Qingjin Huatan Decoction substance reference, and a method for establishing an HPLC fingerprint of the Qingjin Huatan Decoction substance reference. This method can monitor the quality of the Qingjin Huatan Decoction substance reference and provide a scientific basis for substance reference research.

[0006] A first aspect of the present invention provides a method for preparing a substance reference of Qingjin Huatan Decoction, comprising the following steps:

[0007] The prescribed amount of scutellaria baicalensis, gardenia jasminoides, platycodon grandiflorum, ophiopogon japonicus, mulberry bark, fritillaria thunbergii, anemarrhena asphodeloides, trichosanthes fruit, tangerine peel, poria cocos, and liquorice were weighed, combined, placed in a decoction container, soaked with 5-10 times the amount of deionized water, covered and decocted without covering, first over high heat and then over low heat, filtered, and the filtrate was concentrated under reduced pressure to a fluid extract state, the fluid extract was freeze-dried, and ground evenly to obtain the material standard of Qingjin Huatan Decoction.

[0008] Preferably, the prescription of Qingjin Huatan Decoction is as follows: 5.60g of Scutellaria baicalensis, 5.60g of Gardenia jasminoides, 7.46g of Platycodon grandiflorum, 3.73g of Ophiopogon japonicus, 3.73g of Morus alba bark, 3.73g of Fritillaria thunbergii, 3.73g of Anemarrhena asphodeloides, 3.73g of Trichosanthes kirilowii, 3.73g of Citrus aurantium, 3.73g of Poria cocos, and 1.49g of Licorice.

[0009] More preferably, the soaking time is 15 to 60 minutes, and the decocting time is 30 to 120 minutes.

[0010] A second aspect of the present invention provides an HPLC method for determining the substance standard of Qingjin Huatan Decoction, comprising the following steps:

[0011] (1) Prepare mixed reference solution;

[0012] Accurately weigh the reference substances of gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate, and add 50%-100% by volume of methanol solvent to prepare a mixed reference substance solution;

[0013] (2) Prepare the test solution;

[0014] Accurately weigh the material standard of Qingjin Huatan Decoction, accurately add 50%-100% methanol solvent by volume, plug tightly, sonicate for 20-40 minutes, filter, and take the filtrate to obtain the test solution;

[0015] (3) The components of the test solution were detected by high performance liquid chromatography. The specific detection conditions are as follows:

[0016] The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-40°C, the volume flow rate was 0.8-1.2 mL / min, the injection volume was 5-20 μL, the detection wavelength was 230-285 nm, and the mobile phase was 0.05%-0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution;

[0017] The procedure of the gradient elution is:

[0018] At 0 to 3 minutes, the volume content of the mobile phase A in the mobile phase is 94% to 90%, and the volume content of the mobile phase B in the mobile phase is 6% to 10%;

[0019] In 3 to 15 minutes, the volume content of the mobile phase A in the mobile phase is 90% to 88%, and the volume content of the mobile phase B in the mobile phase is 10% to 12%;

[0020] At 15 to 17 minutes, the volume content of the mobile phase A in the mobile phase is 88% to 85%, and the volume content of the mobile phase B in the mobile phase is 12% to 15%;

[0021] At 17 to 23 minutes, the volume content of the mobile phase A in the mobile phase is 85% to 79%, and the volume content of the mobile phase B in the mobile phase is 15% to 21%;

[0022] At 23 to 73 minutes, the volume content of the mobile phase A in the mobile phase is 79% to 77%, and the volume content of the mobile phase B in the mobile phase is 21% to 23%;

[0023] At 73-74 min, the volume content of the mobile phase A in the mobile phase is 77%-67%, and the volume content of the mobile phase B in the mobile phase is 23%-33%;

[0024] At 74 to 86 minutes, the volume content of the mobile phase A in the mobile phase is 67% to 22%, and the volume content of the mobile phase B in the mobile phase is 33% to 78%;

[0025] (4) Determine the content of the index components in the material standard.

[0026] Preferably, in the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is (0.05-0.8) g: (15-50) mL, more preferably 0.2 g: 25 mL.

[0027] A third aspect of the present invention provides a method for establishing an HPLC fingerprint of a substance benchmark of Qingjin Huatan Decoction, characterized in that it comprises the following steps:

[0028] (1) Preparation of mixed reference solution:

[0029] Accurately weigh five reference substances, namely, gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate, and add 50%-100% by volume of methanol solvent to prepare a mixed reference substance solution;

[0030] (2) Preparation of substance reference test solution:

[0031] Accurately weigh 15 batches of Qingjin Huatan Decoction material standards, accurately add 50%-100% methanol solvent by volume, plug tightly, sonicate for 20-40 minutes, filter, and take the filtrate to obtain the test solution;

[0032] (3) Use high performance liquid chromatography to detect the content of the components of the test solution. The specific detection conditions are as follows:

[0033] The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-40°C, the volume flow rate was 0.8-1.2 mL / min, the injection volume was 5-20 μL, the detection wavelength was 230-285 nm, and the mobile phase was 0.05%-0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution;

[0034] The procedure of the gradient elution is:

[0035] At 0 to 3 minutes, the volume content of the mobile phase A in the mobile phase is 94% to 90%, and the volume content of the mobile phase B in the mobile phase is 6% to 10%;

[0036] In 3 to 15 minutes, the volume content of the mobile phase A in the mobile phase is 90% to 88%, and the volume content of the mobile phase B in the mobile phase is 10% to 12%;

[0037] At 15 to 17 minutes, the volume content of the mobile phase A in the mobile phase is 88% to 85%, and the volume content of the mobile phase B in the mobile phase is 12% to 15%;

[0038] At 17 to 23 minutes, the volume content of the mobile phase A in the mobile phase is 85% to 79%, and the volume content of the mobile phase B in the mobile phase is 15% to 21%;

[0039] At 23 to 73 minutes, the volume content of the mobile phase A in the mobile phase is 79% to 77%, and the volume content of the mobile phase B in the mobile phase is 21% to 23%;

[0040] At 73-74 min, the volume content of the mobile phase A in the mobile phase is 77%-67%, and the volume content of the mobile phase B in the mobile phase is 23%-33%;

[0041] At 74 to 86 minutes, the volume content of the mobile phase A in the mobile phase is 67% to 22%, and the volume content of the mobile phase B in the mobile phase is 33% to 78%;

[0042] At 86-88 min, the volume content of the mobile phase A in the mobile phase is 22%-10%, and the volume content of the mobile phase B in the mobile phase is 78%-90%;

[0043] At 88 to 101 minutes, the volume content of the mobile phase A in the mobile phase is 10% to 10%, and the volume content of the mobile phase B in the mobile phase is 90% to 90%;

[0044] (4) Determine the main peaks of the fingerprint;

[0045] HPLC fingerprints were established for 15 batches of Qingjin Huatan Decoction standard substances. The common characteristic peaks were found through analysis, and a total of 13 common chromatographic peaks were detected. The common peak belonging to Gardenia jasminoides was peak 2, the common peaks belonging to Anemarrhena asphodeloides were peaks 1 and 3, the common peaks belonging to Glycyrrhiza uralensis were peaks 4 and 12, the common peaks belonging to Scutellaria baicalensis were peaks 5, peaks 7-11, and peak 13, and the common peak belonging to Citrus aurantium was peak 6.

[0046] (5) Identification of chromatographic peaks;

[0047] Peak 2 corresponds to gardeniain, peak 3 corresponds to mangiferin, peak 4 corresponds to liquiritin, peak 6 corresponds to hesperidin, peak 11 corresponds to baicalein, peak 12 corresponds to ammonium glycyrrhizate, peak 13 corresponds to wogonin, and peak 7 baicalin is used as the reference peak.

[0048] Preferably, in the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is (0.05-0.8) g: (15-50) mL, more preferably 0.2 g: 25 mL.

[0049] The beneficial effects of the present invention are as follows: the present invention has developed a feasible method for preparing a material benchmark for Qingjin Huatan Decoction, a method for determining the content of the material benchmark for Qingjin Huatan Decoction, and a method for establishing a material benchmark fingerprint spectrum for Qingjin Huatan Decoction. The ingredients of Chinese herbal medicine compounds are complex and come from a wide range of sources. Traditional quality control methods may have certain limitations. Fingerprint technology can establish a standardized method based on the main ingredients and chemical characteristics of the medicine, providing effective support for formulating quality standards for Chinese herbal medicine compounds. The present invention can simultaneously measure the five components of the material benchmark, namely, gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate. At the same time, it has the advantages of strong specificity, good linear relationship, high precision, high stability, good repeatability, and good accuracy. The material benchmark fingerprint spectrum method of the present invention optimizes 13 common characteristic peaks, can simultaneously identify five medicinal materials, and identify 8 common peaks. It has the advantages of high stability, high precision, and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The present invention will be further described below with reference to the accompanying drawings and examples:

[0051] Figure 1 These are specific graphs for the determination of the benchmark content of substances in Qingjin Huatan Decoction; among them, A is a chromatogram comparing the whole prescription with and without Gardenia jasminoides; B is a chromatogram comparing the whole prescription with and without Anemarrhena asphodeloides; C is a chromatogram comparing the whole prescription with and without Citrus aurantium; D is a chromatogram comparing the whole prescription with and without Scutellaria baicalensis; and E is a chromatogram comparing the whole prescription with and without Licorice root.

[0052] Figure 2 This is the linear relationship diagram for the determination of the standard content of substances in Qingjin Huatan Decoction; among them, A is the standard curve of Gardenia jasminoides; B is the standard curve of mangiferin; C is the standard curve of hesperidin; D is the standard curve of baicalin; E is the standard curve of ammonium glycyrrhizate.

[0053] Figure 3 The chromatograms are obtained under different concentrations of phosphoric acid aqueous solution;

[0054] Figure 4 These are the fingerprint maps of 15 batches of Qingjin Huatan Decoction substance benchmarks. DETAILED DESCRIPTION

[0055] The present invention will be further described in detail below through specific embodiments and corresponding drawings to facilitate understanding of the technical solutions of the present invention, but the embodiments of the present invention are not limited thereto.

[0056] 1. Preparation of mixed reference solution

[0057] Accurately take appropriate amounts of gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate reference substances, respectively, and add methanol to prepare stock solutions containing 167.90 μg of gardeniain, 495.43 μg of mangiferin, 418.35 μg of hesperidin, 582.03 μg of baicalin, and 39.16 μg of ammonium glycyrrhizate per 1 mL. Take appropriate amounts of the stock solutions, mix them, and add methanol to prepare a mixed reference solution containing 147.19 μg of gardeniain, 9.51 μg of mangiferin, 21.75 μg of hesperidin, 291.02 μg of baicalin, and 7.52 μg of ammonium glycyrrhizate per 1 mL.

[0058] 2 Preparation of test solution

[0059] Accurately take 0.2 g of the Qingjin Huatan Decoction standard substance, place it in a stoppered conical flask, accurately add 25 mL of methanol, seal it tightly, sonicate for 30 minutes, shake well, filter through a 0.22 μm nylon membrane, and take the filtrate.

[0060] 3. Screening of material benchmark preparation process

[0061] 3.1 Determination of paste yield

[0062] Record the volume of the material reference decoction after cooling as V, accurately transfer 25 mL of decoction into an evaporating dish with constant weight, place it in a constant temperature water bath at 100°C to evaporate the water, and after complete evaporation, place it in a forced air drying oven at 105°C to dry for 6 hours, take it out, cool it in a desiccator, weigh it as M, and calculate the paste rate according to formula (1).

[0063]

[0064] 3.2 Water quality

[0065] A total of 46.26 g of each decoction piece was accurately weighed and combined in a 2-liter casserole. The heat was adjusted by adjusting the voltage. 400 mL of tap water or deionized water was added and the mixture was soaked for 30 min. The mixture was covered and decocted without wrapping. The mixture was heated to a boil over high heat (220 V) and then reduced to a gentle boil (100 V) until the mixture reached 160 mL. The mixture was filtered through a 200-mesh filter cloth while still hot. After cooling, the cold volume of the decoction was recorded. The decoction was then concentrated under reduced pressure to a fluid extract, pre-frozen in a -80°C refrigerator for 12 h. The pre-frozen sample was freeze-dried for 48 h and ground to obtain the reference sample. The yield of the extract was determined, and the corresponding contents of the index components were calculated (Table 1). The results show that the decoction with deionized water resulted in a slightly higher content of geniposide and a slightly lower content of baicalin than that with tap water. This resulted in a slightly lower yield of the extract. Considering that the quality control study of the material reference involves precise experiments such as component quantitative analysis, deionized water was used uniformly.

[0066] Table 1 Benchmark process water quality investigation (Mean±SD, n=3)

[0067]

[0068] 3.3 Cooking Temperature

[0069] Accurately weigh a total of 46.26 g of each prescription piece, combine them and place them in a 2L casserole, add 400 mL of water, soak for 30 min, cover and decoct without wrapping, and decoct to 160 mL using three different decoction conditions: high heat (220 V) followed by low heat (100 V), high heat (220 V), and low heat (100 V). Filter while hot through a 200-mesh filter cloth, record the cold volume of the decoction after cooling, and decompress the decoction to a fluid extract state, then pre-freeze in a -80°C refrigerator for 12 h, freeze-dry the pre-frozen sample for 48 h, take it out and grind it to obtain the benchmark sample. The yield of paste was determined and the corresponding contents of index components were calculated, as shown in Table 2. The results show that the content of baicalin was significantly higher and more parallel under the conditions of decocting with strong fire first and then gentle fire, and the content of geniposide was significantly higher than that of other groups under the conditions of decocting with strong fire first and then gentle fire. The yield of paste was significantly higher under the conditions of decocting with strong fire first and then gentle fire than that of other groups. Therefore, after comprehensive consideration, according to the general decocting habits, the decocting temperature should be selected to be strong fire first and then gentle fire.

[0070] Table 2 Investigation of boiling temperature in the benchmark process (Mean±SD, n=3)

[0071]

[0072] 3.4 Determination of the Standard Preparation Process for Qingjin Huatan Decoction

[0073] Accurately take the prescription amount of each piece of medicine, 5.6g of Scutellaria baicalensis, 5.6g of Gardenia jasminoides, 7.46g of Platycodon grandiflorum, 3.73g of Ophiopogon japonicus, 3.73g of Morus alba, 3.73g of Fritillaria thunbergii, 3.73g of Anemarrhena asphodeloides, 3.73g of stir-fried Trichosanthes kirilowii, 3.73g of Dried Tangerine peel, 3.73g of Poria cocos, and 1.49g of Licorice root, add 400mL of water after merging and place in a casserole, soak for 30min, cover and do not wrap and decoct, boil over high heat (220V) and then turn to simmering heat (100V) to keep boiling until 160mL is reached, filter through 200 mesh filter cloth while hot, and concentrate under reduced pressure to a fluid extract state. The fluid extract is freeze-dried, ground and mixed to obtain the material standard of Qingjin Huatan Decoction.

[0074] 4. Content determination method

[0075] 4.1 Chromatographic conditions

[0076] Chromatographic column: Agilent Technologies Zorbax SB-C18 (250 mm×4.6 mm, 3.5 μm), column temperature 30°C, volume flow rate 1 mL / min, injection volume 10 μL, UV detection wavelengths 242 nm, 258 nm, and 280 nm, 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, gradient elution 0-3 min, 6-10% B; 3-15 min, 10-12% B; 15-17 min, 12-15% B; 17-23 min, 15-21% B; 23-73 min, 21-23% B; 73-74 min, 23-33% B; 74-86 min, 33-78% B.

[0077] 4.2 Specificity

[0078] Take the mixed reference solution, the Qingjin Huatan Decoction, the test solution without Scutellaria baicalensis, Gardenia jasminoides, Anemarrhena rhizome, Tangerine peel, and Licorice root respectively, and record the chromatogram (see Figure 1 ).

[0079] 4.3 Linearity

[0080] Take the gardeniain reference substance, mangiferin reference substance, hesperidin reference substance, baicalin reference substance, and ammonium glycyrrhizate reference substance respectively, and add methanol to make 1 mL of the stock solution containing 586.64 μg of gardeniain, 619.68 μg of mangiferin, 452.56 μg of hesperidin, 968.11 μg of baicalin, and 632.48 μg of ammonium glycyrrhizate. After taking an appropriate amount of the stock solution and diluting it with methanol solvent, the linear solutions of geniposide containing 200.63μg, 179.51μg, 105.59μg, 63.36μg, and 33.79μg per mL were obtained; the linear solutions of mangiferin containing 35.69μg, 33.71μg, 19.83μg, 11.90μg, and 6.35μg per mL were obtained; the linear solutions of dapoxetine containing 61.10μg, 46.16μg, 27.15μg, and 16.29μg per mL were obtained. g, 8.69 μg of hesperidin linear solution; 1 mL of baicalin linear solution containing 566.35 μg, 314.64 μg, 193.62 μg, 116.17 μg, 61.96 μg; 1 mL of ammonium glycyrrhizate linear solution containing 27.32 μg, 25.81 μg, 15.18 μg, 9.11 μg, 4.86 μg, were injected for detection. The standard curve was plotted with the concentration of the reference solution as the abscissa and the peak area as the ordinate (see Figure 2), the linear regression equation is shown in Table 3. The results show that the r of the standard curve equations of gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate are all greater than 0.999, gardeniain has a good linear relationship in the concentration range of 33.79-200.63 μg / mL, mangiferin has a good linear relationship in the concentration range of 6.35-35.69 μg / mL, hesperidin has a good linear relationship in the concentration range of 8.69-61.10 μg / mL, baicalin has a good linear relationship in the concentration range of 61.96-566.35 μg / mL, and ammonium glycyrrhizate has a good linear relationship in the concentration range of 4.86-27.32 μg / mL.

[0081] Table 3 Linear regression equation

[0082]

[0083] 4.4 Precision

[0084] Reference solutions of 192.94 μg / mL gardeniain, 19.76 μg / mL mangiferin, 134.10 μg / mL hesperidin, 429.23 μg / mL baicalin, and 21.22 μg / mL ammonium glycyrrhizate diluted from the respective stock solutions were injected for testing. Six consecutive injections were performed, and the RSDs of the peak areas of each index component were calculated (see Table 4). The RSDs of the peak areas of gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate were 0.62%, 0.34%, 0.42%, 0.19%, and 0.36%, respectively, all less than 2%, indicating good instrument precision.

[0085] Table 4 Instrument precision

[0086]

[0087] 4.5 Repeatability

[0088] Six parallel sample solutions were prepared and injected for testing, and the RSDs of the index components were calculated, as shown in Table 5. The results show that the RSD values ​​of geniposide, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate in the six parallel samples were 0.78%, 1.43%, 1.08%, 1.40%, and 2.41%, respectively, indicating good reproducibility of the benchmark samples.

[0089] Table 5 Repeatability of content determination

[0090]

[0091] 4.6 Stability

[0092] The test solution was sampled and tested at 0 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, and 48 h, and the RSDs of the index components were calculated, as shown in Table 6. The results show that within 48 h, the RSD values ​​of the contents of geniposide, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate in the reference sample were 0.98%, 2.77%, 1.29%, 0.84%, and 2.38%, respectively, all less than 3%, indicating that at room temperature, the reference sample solution was relatively stable within 48 h after preparation.

[0093] Table 6 Assay stability

[0094]

[0095] 4.7 Accuracy

[0096] Accurately pipette 5 mL of the reproducible sample into a 10 mL volumetric flask, and accurately add approximately equal amounts of gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate reference solution. After diluting to volume with methanol and shaking well, the sample was injected for detection. The results of calculated sample recovery are shown in Table 7. The sample recovery limits of gardeniain and baicalin are in the range of 92%-105%; the sample recovery limits of hesperidin are in the range of 90%-108%; the sample recovery limits of mangiferin and ammonium glycyrrhizate are in the range of 85%-110%, indicating that the accuracy of the reference sample is good.

[0097] Table 7 Accuracy of content determination

[0098]

[0099] Table 7: Content determination accuracy

[0100]

[0101] 4.8 Durability

[0102] The effects of 0.05%, 0.1%, and 0.2% phosphoric acid aqueous solutions on the chromatographic peaks of the test products were investigated. The results are shown in Figure 3 , the sample peaks under the three conditions were all good and the baselines were relatively stable.

[0103] 5 Fingerprint establishment method

[0104] 5.1 Chromatographic conditions

[0105] Chromatographic column: Agilent Technologies Zorbax SB-C18 (250 mm×4.6 mm, 3.5 μm), column temperature 30°C, volume flow rate 1 mL / min, injection volume 10 μL, UV detection wavelength 258 nm, 0.05% phosphoric acid aqueous solution (A)-acetonitrile (B) as the mobile phase, gradient elution 0-3 min, 6-10% B; 3-15 min, 10-12% B; 15-17 min, 12-15% B; 17-23 min, 15-21% B; 23-73 min, 21-23% B; 73-74 min, 23-33% B; 74-86 min, 33-78% B, 86-88 min, 78-90% B; 88-101 min, 90-90% B.

[0106] 5.2 Precision

[0107] The test solution was sampled and tested for 6 consecutive times. The retention time and peak area of ​​the common peak were recorded. The relative retention time RSD and relative peak area RSD of the common peak were calculated with reference to the reference peak. As shown in Table 8, the RSD of the relative retention time of each common peak was ≤1%, and the RSD of the relative peak area was ≤5%, indicating good precision.

[0108] Table 8 Fingerprint precision

[0109]

[0110] 5.3 Repeatability

[0111] Six test solutions were prepared in parallel, sampled and tested, and the retention time and peak area of ​​the common peak were recorded. The relative retention time RSD and relative peak area RSD of the common peak were calculated with reference to the reference peak. As shown in Table 9, the RSD of the relative retention time of each common peak was ≤1%, and the RSD of the relative peak area was ≤5%, indicating good precision.

[0112] Table 9 Fingerprint repeatability

[0113]

[0114] 5.4 Stability

[0115] The test solution was sampled and tested at 0h, 2h, 4h, 8h, 12h, 24h, 36h and 48h, and the retention time and peak area of ​​the common peak were recorded. The relative retention time RSD and relative peak area RSD of the common peak were calculated with reference to the reference peak. It can be seen from Table 10 that the RSD of the relative retention time of each common peak is ≤1%, and the RSD of the relative peak area is ≤5%, indicating that the sample is stable within 48h.

[0116] Table 10 Fingerprint stability

[0117]

[0118] 6 Fingerprint establishment and characteristic peak calibration

[0119] 6.1 Determination and attribution of common peaks

[0120] The test solution, reference solution, and each individual herb solution were sampled, tested, and chromatograms recorded. Common peaks were assigned to their respective medicinal properties, and reference peaks for the fingerprints were selected. Of the 13 common peaks selected, eight components were known and specific. Peak 7 (baicalin), which exhibited high stability and good resolution, was selected as the reference peak. The remaining components were unknown (see Table 11 for details).

[0121] Table 11 Characteristic peaks of reference samples

[0122]

[0123] 6.2 Similarity Evaluation

[0124] Fifteen batches of standard substances were sampled and tested, and the chromatograms were recorded. The chromatograms were imported into the 2012 version of the Chinese medicine chromatographic fingerprint similarity evaluation system software. The median method was used with the time window set to 0.1 to perform multi-point chromatographic peak correction. Figure 4 , calculated the similarities of the fifteen batches, see Table 12, the similarities are all greater than 0.99, indicating that the chromatographic peaks of the 15 batches of fingerprints are basically consistent as a whole.

[0125]

[0126] In summary, the Qingjin Huatan Decoction substance benchmark preparation method, content determination method, and fingerprint establishment method established in the present invention are stable and reliable. A 15-batch Qingjin Huatan Decoction substance benchmark HPLC fingerprint was established using baicalin as the reference peak, and 13 common peaks were calibrated. Using the standard comparison method, peak 2 corresponds to gardeniain, peak 3 corresponds to mangiferin, peak 4 corresponds to liquiritin, peak 6 corresponds to hesperidin, peak 11 corresponds to baicalein, peak 12 corresponds to ammonium glycyrrhizate, and peak 13 corresponds to wogonin, with peak 7, baicalin, as the reference peak.

[0127] The above examples are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A method for preparing a material reference of Qingjin Huatan Decoction, characterized in that: The method comprises the following steps: weighing a prescribed amount of scutellaria baicalensis, gardenia jasminoides, platycodon grandiflorum, ophiopogon japonicus, mulberry bark, thunbergia thunbergii, anemarrhena asphodeloides, stir-fried trichosanthes fruit, dried orange peel, poria cocos, and liquorice, combining the mixture and placing the mixture in a decoction container, adding 5-10 times the amount of deionized water for soaking, covering the container but not covering it, decocting over high heat first and then over low heat, filtering, decompressing the filtrate to a fluid extract state, freeze-drying the fluid extract, and grinding the mixture evenly to obtain the material standard of Qingjin Huatan Decoction.

2. The method according to claim 1, characterized in that The prescription of Qingjin Huatan Decoction is as follows: 5.60g of Scutellaria baicalensis, 5.60g of Gardenia jasminoides, 7.46g of Platycodon grandiflorum, 3.73g of Ophiopogon japonicus, 3.73g of Morus alba bark, 3.73g of Fritillaria thunbergii, 3.73g of Anemarrhena asphodeloides, 3.73g of stir-fried Trichosanthes kirilowii seeds, 3.73g of Citrus aurantium, 3.73g of Poria cocos, and 1.49g of Licorice root.

3. The method according to claim 1 or 2, characterized in that The soaking time is 15 to 60 minutes, and the decocting time is 30 to 120 minutes.

4. A method for determining the content of the substance reference of Qingjin Huatan Decoction, characterized in that: The following steps are involved: (1) Preparation of mixed reference solution: Accurately weigh five reference substances, namely, gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate, and add 50%-100% by volume of methanol solvent to prepare a mixed reference substance solution; (2) Prepare the test solution: Accurately weigh the material standard of Qingjin Huatan Decoction, accurately add 50%-100% methanol solvent by volume, plug tightly, sonicate for 20-40 minutes, filter, and take the filtrate to obtain the test solution; (3) Use high performance liquid chromatography to detect the content of the components of the test solution. The specific detection conditions are as follows: The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-40°C, the volume flow rate was 0.8-1.2 mL / min, the injection volume was 5-20 μL, the detection wavelength was 230-285 nm, and the mobile phase was 0.05%-0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution; The procedure of the gradient elution is: At 0 to 3 minutes, the volume content of the mobile phase A in the mobile phase is 94% to 90%, and the volume content of the mobile phase B in the mobile phase is 6% to 10%; In 3 to 15 minutes, the volume content of the mobile phase A in the mobile phase is 90% to 88%, and the volume content of the mobile phase B in the mobile phase is 10% to 12%; At 15 to 17 minutes, the volume content of the mobile phase A in the mobile phase is 88% to 85%, and the volume content of the mobile phase B in the mobile phase is 12% to 15%; At 17 to 23 minutes, the volume content of the mobile phase A in the mobile phase is 85% to 79%, and the volume content of the mobile phase B in the mobile phase is 15% to 21%; At 23 to 73 minutes, the volume content of the mobile phase A in the mobile phase is 79% to 77%, and the volume content of the mobile phase B in the mobile phase is 21% to 23%; At 73-74 min, the volume content of the mobile phase A in the mobile phase is 77%-67%, and the volume content of the mobile phase B in the mobile phase is 23%-33%; At 74 to 86 minutes, the volume content of the mobile phase A in the mobile phase is 67% to 22%, and the volume content of the mobile phase B in the mobile phase is 33% to 78%; (4) Determine the content of the index components in the material standard.

5. The method according to claim 4, characterized in that In the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is (0.05-0.8) g: (15-50) mL.

6. The method according to claim 5, characterized in that In the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is 0.2 g: 25 mL.

7. A method for establishing a fingerprint of the substance benchmark of Qingjin Huatan Decoction, characterized in that: The following steps are involved: (1) Preparation of mixed reference solution: Accurately weigh five reference substances, namely, gardeniain, mangiferin, hesperidin, baicalin, and ammonium glycyrrhizate, and add 50%-100% by volume of methanol solvent to prepare a mixed reference substance solution; (2) Prepare 15 batches of substance standard test solution: Accurately weigh 15 batches of Qingjin Huatan Decoction material standards, accurately add 50%-100% methanol solvent by volume, plug tightly, sonicate for 20-40 minutes, filter, and take the filtrate to obtain the test solution; (3) Use high performance liquid chromatography to detect the content of the components of the test solution. The specific detection conditions are as follows: The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-40°C, the volume flow rate was 0.8-1.2 mL / min, the injection volume was 5-20 μL, the detection wavelength was 230-285 nm, and the mobile phase was 0.05%-0.2% phosphoric acid aqueous solution (A)-acetonitrile (B) with gradient elution; The procedure of the gradient elution is: At 0 to 3 minutes, the volume content of the mobile phase A in the mobile phase is 94% to 90%, and the volume content of the mobile phase B in the mobile phase is 6% to 10%; In 3 to 15 minutes, the volume content of the mobile phase A in the mobile phase is 90% to 88%, and the volume content of the mobile phase B in the mobile phase is 10% to 12%; At 15 to 17 minutes, the volume content of the mobile phase A in the mobile phase is 88% to 85%, and the volume content of the mobile phase B in the mobile phase is 12% to 15%; At 17 to 23 minutes, the volume content of the mobile phase A in the mobile phase is 85% to 79%, and the volume content of the mobile phase B in the mobile phase is 15% to 21%; At 23 to 73 minutes, the volume content of the mobile phase A in the mobile phase is 79% to 77%, and the volume content of the mobile phase B in the mobile phase is 21% to 23%; At 73-74 min, the volume content of the mobile phase A in the mobile phase is 77%-67%, and the volume content of the mobile phase B in the mobile phase is 23%-33%; At 74 to 86 minutes, the volume content of the mobile phase A in the mobile phase is 67% to 22%, and the volume content of the mobile phase B in the mobile phase is 33% to 78%; At 86-88 min, the volume content of the mobile phase A in the mobile phase is 22%-10%, and the volume content of the mobile phase B in the mobile phase is 78%-90%; At 88 to 101 minutes, the volume content of the mobile phase A in the mobile phase is 10% to 10%, and the volume content of the mobile phase B in the mobile phase is 90% to 90%; (4) Determine the main peaks of the fingerprint: By comparing 15 batches of Qingjin Huatan Decoction substance benchmarks, an HPLC fingerprint was established, and a total of 13 common chromatographic peaks were detected. The common peak attributed to Gardenia jasminoides was peak 2, the common peaks attributed to Anemarrhena asphodeloides were peaks 1 and 3, the common peaks attributed to Glycyrrhiza uralensis were peaks 4 and 12, the common peaks attributed to Scutellaria baicalensis were peaks 5, peaks 7-11, and peak 13, and the common peak attributed to Citrus aurantium was peak 6. (5) Identification of chromatographic peaks: Peak 2 corresponds to gardeniain, peak 3 corresponds to mangiferin, peak 4 corresponds to liquiritin, peak 6 corresponds to hesperidin, peak 11 corresponds to baicalein, peak 12 corresponds to ammonium glycyrrhizate, peak 13 corresponds to wogonin, and peak 7 baicalin is used as the reference peak.

8. The method according to claim 7, characterized in that In the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is (0.05-0.8) g: (15-50) mL.

9. The method according to claim 8, characterized in that In the test solution, the mass volume ratio of the Qingjin Huatan Decoction substance standard to the methanol solvent is 0.2 g: 25 mL.

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