Detection method of lipid-lowering traditional Chinese medicine qingzhi huayu recipe and saccharide components contained therein

Abstract

A lipid-lowering traditional Chinese medicine Qingzhi Huayu recipe and a detection method of sugar components contained therein, comprising: water decoction extraction and concentration of traditional Chinese medicine decoction pieces constituting the lipid-lowering traditional Chinese medicine Qingzhi Huayu recipe to obtain water extract dry products, preparation of standard solution from the water extract dry products, liquid chromatography-electrospray detection by high performance liquid chromatography-electrospray detector to obtain standard liquid chromatogram and mark the characteristic peaks therein, then comparison with the liquid chromatogram of the detected decoction / patent medicine obtained by the same method to determine whether the detected decoction / patent medicine belongs to the lipid-lowering traditional Chinese medicine Qingzhi Huayu recipe, and to determine the six sugar substances contained in the detected decoction / patent medicine, and further comparison with the area of the corresponding sugar characteristic peak in the standard liquid chromatogram to determine the content of the corresponding sugar. The detection method and the standard provided by the application can be used as the basis for formulating the quality control standard and the quality detection standard of the lipid-lowering traditional Chinese medicine Qingzhi Huayu recipe decoction / patent medicine.

Description

Technical Field

[0001] This invention relates to a detection method, and more particularly to a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, and a method for detecting the sugar components contained therein, belonging to the technical field of detection and analysis of traditional Chinese medicine formulas and their components. Background Technology

[0002] Many traditional Chinese medicinal herbs contain fructose, sucrose, and other sugars, all of which possess corresponding pharmacological activities. For example, L-rhamnose has a therapeutic effect on obesity; the absorption and metabolism of fructose can activate signaling pathways such as AMPK and Toll-like receptors, thereby triggering metabolic syndromes and dyslipidemia, including non-alcoholic steatohepatitis. Mannitol is a commonly used hypertonic dehydrating agent with cardioprotective effects, and raffinose has a protective effect against ultraviolet stress on the skin. Furthermore, some plant polysaccharides, such as astragalus polysaccharide, platycodon pectin polysaccharide, ganoderma lucidum polysaccharide, and polygonum cuspidatum polysaccharide, possess various pharmacological effects, including immunomodulation, antioxidation, hypoglycemia, and hypolipidemia.

[0003] Carbohydrates are a class of highly polar substances that do not absorb ultraviolet light. Currently, they are mainly detected by methods such as high performance liquid chromatography, gas chromatography, chromatography-mass spectrometry, ion chromatography, capillary electrophoresis, high performance anion exchange chromatography, and pulsed amperometric detection. However, these detection methods generally suffer from problems such as cumbersome pretreatment (e.g., derivatization), complex instrument operation, and high detection costs. Therefore, they are not suitable as routine methods for drug quality testing and control.

[0004] In addition, existing technologies include a method for determining carbohydrate components that combines a Hilic column with an evaporative light scattering detector (ELSD). However, due to the poor repeatability of existing ELSD detectors and the tendency for carbohydrates to remain in the detector, affecting the detection results, although this method is currently used for the quality testing and control of pharmaceuticals, the repeatability and accuracy of carbohydrate detection still need to be improved.

[0005] Dyslipidemia is a risk factor for many cardiovascular diseases such as atherosclerosis, myocardial infarction, and stroke.

[0006] In existing technologies, according to traditional Chinese medicine theory, the lipid-lowering Chinese medicine Qingzhi Huayu Fang, which takes "clearing phlegm and removing blood stasis" as its treatment principle, has the effect of clearing phlegm and removing blood stasis. It is mainly used to treat the syndrome of phlegm and blood stasis in chest pain. It has a good therapeutic effect on patients with coronary heart disease and hyperlipidemia with the syndrome of phlegm and blood stasis. Pharmacological studies have shown that the lipid-lowering Chinese medicine Qingzhi Huayu Fang can reduce the formation of atherosclerotic plaques. Its mechanism may be related to the regulation of the Keap1-Nrf2 / ARE-mediated antioxidant damage mechanism.

[0007] The lipid-lowering Chinese herbal formula for clearing lipids and removing blood stasis consists of six Chinese herbs: Scutellaria baicalensis (fried), Typha pollen, Polygonatum sibiricum, Citrus aurantium fruit, Polygonum cuspidatum, and Curcuma longa.

[0008] Studies on the composition, blood-entry components, and tissue distribution of the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang have revealed that it contains over 143 chemical components, mainly including flavonoids, organic acids, amino acids, nucleotides, terpenes, quinones, and phenols, of which 52 are blood-entry components. However, current technology lacks effective methods for detecting the types and contents of the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang and its sugar components. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, this invention provides a method for detecting the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang and its sugar components. This method enables qualitative detection of the decoction / prepared medicine of Qingzhi Huayu Fang and effectively detects the sugar components in the decoction / prepared medicine. It provides corresponding methods and standards for detecting the sugar components and content of Qingzhi Huayu Fang and its contents, and also provides a basis for quality control of Qingzhi Huayu Fang.

[0010] To achieve the above objectives, the present invention first provides a method for detecting a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, the technical solution of which is as follows:

[0011] A method for detecting a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is used for the qualitative detection of the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. The decoction of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is prepared by decocting the medicinal slices of traditional Chinese medicine including Scutellaria baicalensis (fried), Typha orientalis, Polygonatum sibiricum, Caprifolium arvense, Polygonum cuspidatum and Curcuma longa in water. The prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is prepared by decocting the medicinal slices of traditional Chinese medicine including Scutellaria baicalensis (fried), Typha orientalis, Polygonatum sibiricum, Caprifolium arvense, Polygonum cuspidatum and Curcuma longa in water to obtain an extract, and then adding pharmaceutically acceptable excipients. The method includes the following steps:

[0012] The following traditional Chinese medicine decoction pieces were prepared by decocting and extracting with water to obtain a dried aqueous extract of a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis: Scutellaria baicalensis (fried), Typha pollen, Polygonatum sibiricum, Citrus aurantium fruit, Polygonum cuspidatum, and Curcuma longa. A standard solution of this lipid-lowering traditional Chinese medicine formula was then prepared and analyzed by high-performance liquid chromatography-electrospray ionization (HPLC-ESI) to obtain the standard chromatogram. The characteristic peaks were then marked.

[0013] The high-performance liquid chromatography-electrospray detector has a column packed with aminosilane-bonded silica gel, and the theoretical plate number of the column should be no less than 5000 based on the high-performance liquid chromatogram of sucrose. The liquid chromatography-electrospray detection uses acetonitrile aqueous solution as the mobile phase for elution.

[0014] A water extract of the sample was obtained and dried and a test solution was prepared. The test solution was then tested using the same detection method and conditions as the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the sample.

[0015] The liquid chromatogram of the tested sample is compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. If the liquid chromatogram of the tested sample contains characteristic peaks from the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and the relative retention time of each characteristic peak in the liquid chromatogram of the tested sample is only experimentally permissible, and there is only an experimentally permissible error between the relative retention time of each corresponding characteristic peak in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, then the tested sample is the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula.

[0016] Further:

[0017] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis has at least 14 characteristic peaks, and the relative retention times of the 14 characteristic peaks are as follows:

[0018] 0.37min, 0.43min, 0.54min, 0.62min, 0.65min, 0.73min, 0.78min, 0.94min, 1.00min, 1.13min, 1.23min, 1.27min, 1.38min and 1.40min.

[0019] Furthermore, in the detection method of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, the inner diameter of the chromatographic column is 4.6 mm, the column length is 250 mm, the film thickness is 5 μm, and the column temperature is 35 °C.

[0020] Furthermore, in the detection method of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, the flow rate of the mobile phase is 0.8 mL / min, the elution method is gradient elution, and the elution gradient is:

[0021] Use an acetonitrile aqueous solution containing 82-80% by volume for 0-10 min; 80-75% by volume for 10-20 min; 75-65% by volume for 20-30 min; 65-40% by volume for 30-35 min; 40-82% by volume for 35-36 min; and 82% by volume for 36-45 min.

[0022] Furthermore, the preparation method of the dried product of the water extract of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is as follows:

[0023] Take 120g of Scutellaria baicalensis (fried), 120g of Typha pollen, 100g of Polygonatum sibiricum, 50g of Polygonum cuspidatum, 100g of Curcuma longa, and 90g of Caprifolium arvense fruit. Soak them in 10 times the amount of water for 1 hour, then decoct for 2 hours and collect the decoction. Add 9 times the amount of water and decoct for another 2 hours and collect the decoction. Combine the two decoctions, filter, concentrate under reduced pressure, and vacuum dry to obtain the dried product of the water extract of the lipid-lowering Chinese medicine formula for clearing lipids and removing blood stasis.

[0024] Furthermore, the preparation method of the standard solution of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is as follows:

[0025] Accurately weigh approximately 0.3g of the dried aqueous extract of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, place it in a 25mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20min, let it cool to room temperature, add another 30% by volume methanol aqueous solution to the mark, shake well, then take the supernatant, centrifuge, and filter through a 0.45μm microporous membrane. The filtrate is the standard solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang.

[0026] Furthermore, the method for preparing the dried aqueous extract of the sample is as follows:

[0027] The decoction sample to be tested is filtered, then concentrated under reduced pressure, and then dried under vacuum to obtain the dried water extract of the decoction sample.

[0028] The drug sample to be tested is crushed, then 10 times the amount of water is added and decocted for 15 minutes. The decoction is then filtered, concentrated under reduced pressure, and vacuum dried to obtain the dried water extract of the drug sample.

[0029] Furthermore, the method for preparing the test solution is as follows:

[0030] Accurately weigh approximately 0.3 g of the dried aqueous extract of the herbal decoction or the dried aqueous extract of the prepared medicine sample, place it in a 25 mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20 min, let it cool to room temperature, add more methanol aqueous solution with a content of 30% by volume to the mark, shake well, take the supernatant, centrifuge, and then filter through a 0.45 μm microporous membrane. The filtrate is the test solution.

[0031] Secondly, this invention also provides a qualitative detection method for carbohydrate components contained in a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. This method is used for the qualitative detection of carbohydrate components contained in the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. The technical solution includes the following steps:

[0032] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis was obtained according to the detection method of claim 1 above, and the chromatographic peaks with relative retention times of 0.37 min were identified as rhamnose peak, 0.54 min as fructose peak, 0.65 min as mannitol peak, 0.73 min as D-anhydrous glucose peak, 1.00 min as sucrose peak, and 1.27 min as raffinose peak.

[0033] The decoction / prepared medicine was obtained as a dry water extract and the corresponding decoction / prepared medicine test solution was prepared. Then, the decoction / prepared medicine test solution was tested according to the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the decoction / prepared medicine.

[0034] The liquid chromatogram of the tested decoction / prepared medicine was compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis;

[0035] The chromatographic peaks appearing at the positions of the rhamnose peak, fructose peak, mannitol peak, D-anhydrous glucose peak, sucrose peak, and raffinose peak in the liquid chromatogram of the tested decoction / prepared medicine corresponding to the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and the relative retention time of the chromatographic peak is within the experimentally permissible error compared to the relative retention time of the corresponding chromatographic peak in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, are the sugars represented by the chromatographic peak with the corresponding relative retention time in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula.

[0036] Furthermore, this invention also provides a method for quantitatively detecting carbohydrate components in a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. This method is used for the quantitative detection of carbohydrate components in the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. The technical solution includes the following steps:

[0037] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis was obtained according to the detection method of claim 1 above, and the chromatographic peaks with relative retention times of 0.37 min were identified as rhamnose peak, 0.54 min as fructose peak, 0.65 min as mannitol peak, 0.73 min as D-anhydrous glucose peak, 1.00 min as sucrose peak, and 1.27 min as raffinose peak.

[0038] Accurately weigh appropriate amounts of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose reference standards, and add water to prepare reference standard solutions containing 2.7630 mg rhamnose, 3.6314 mg fructose, 1.4169 mg mannitol, 3.0080 mg D-anhydrous glucose, 3.8164 mg sucrose, and 2.4520 mg raffinose per ml, respectively. Then, perform liquid chromatography detection according to the method and conditions for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis as described in claim 1 above to obtain the liquid chromatogram of the reference standard. By comparing the relative retention times, determine the chromatographic peaks of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose on the liquid chromatogram of the reference standard, and record the peak areas corresponding to their respective contents.

[0039] The decoction / prepared medicine was obtained as a dry water extract and the corresponding decoction / prepared medicine test solution was prepared. Then, the decoction / prepared medicine test solution was tested according to the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the decoction / prepared medicine.

[0040] The liquid chromatogram of the tested decoction / prepared medicine is compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. By comparing the relative retention times, the sugars contained in the tested decoction / prepared medicine Qingzhi Huayu formula and their peak areas are determined on the liquid chromatogram of the tested decoction / prepared medicine.

[0041] Then, the peak areas of each sugar in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine are calculated by correspondingly comparing them with the content and peak area of ​​the same sugar recorded in the liquid chromatogram of the reference standard, so as to obtain the content of each sugar in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine.

[0042] Compared with the prior art, the beneficial effects and advancements of the present invention are as follows:

[0043] The present invention provides a method for detecting lipid-lowering traditional Chinese medicine Qingzhi Huayu formula and its sugar components. For the first time, the method uses high performance liquid chromatography-electrospray ionization detector to achieve qualitative detection of the decoction / prepared medicine of Qingzhi Huayu formula and to effectively detect some sugar substances contained in the decoction / prepared medicine of Qingzhi Huayu formula. This provides a method and standard for detecting the decoction / prepared medicine of Qingzhi Huayu formula and its sugar components and content.

[0044] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula established by the detection method provided by this invention is accurate and reliable. Moreover, the detection method of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine and its six sugar components has high sensitivity, good repeatability, and wide linear range. The accuracy meets the analytical method validation guidelines of the 2020 edition of the Chinese Pharmacopoeia, Part IV. It can provide a basis for the quality control and quality standard formulation of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine. Attached Figure Description

[0045] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the embodiments of the present invention will be briefly described below.

[0046] It is obvious:

[0047] The accompanying drawings described below are only some of the drawings of embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort, but these other drawings are also within the scope of drawings required for the embodiments of the present invention.

[0048] Figure 1 This is a high-performance liquid chromatography-electrospray chromatogram comparing a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis with six sugar reference standards and a blank reagent, as provided in an embodiment of the present invention.

[0049] Figure 2 The liquid chromatogram obtained by high performance liquid chromatography-electrospray spectroscopy comparison of a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, six sugar reference standards, single traditional Chinese medicine test sample and blank reagent provided in the embodiments of the present invention;

[0050] Figure 3 This is a high-performance liquid chromatography-electrospray ionization (HPLC-ESI) comparison diagram of multiple batches of lipid-lowering traditional Chinese medicine decoction for clearing lipids and removing blood stasis, provided as an embodiment of the present invention.

[0051] In the picture:

[0052] B – Chromatogram of high performance liquid chromatography-electrospray ionization detection of blank reagent;

[0053] T-Chromatograms of six carbohydrate reference standards detected by high performance liquid chromatography-electrospray ionization;

[0054] D1~D6 – High performance liquid chromatography-electrospray chromatograms of the water extracts of the single herbs in the lipid-lowering and lipid-clearing formula, wherein D1 is Scutellaria baicalensis (fried), D2 is Caprifolium arvense fruit, D3 is Polygonum cuspidatum, D4 is Polygonatum sibiricum, D5 is Typha pollen, and D6 is Curcuma longa extract.

[0055] S1~S15- are the high performance liquid chromatography-electrospray chromatograms of 15 batches of lipid-lowering traditional Chinese medicine decoctions for clearing lipids and removing blood stasis.

[0056] R - Standard liquid chromatogram of lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. Detailed Implementation

[0057] To make the objectives, technical solutions, beneficial effects and significant progress of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings provided for the embodiments of the present invention. Obviously, all the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0058] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0059] It should be noted that:

[0060] The terms "firstly," "secondly," etc., used in the specification, claims, and accompanying drawings of the embodiments of this invention are merely for distinguishing different objects and are not for describing a specific order.

[0061] Furthermore, the term "comprising" and any variations thereof are intended to cover non-exclusive inclusion, such as a process, method, system, product, or apparatus that includes a series of steps or units that is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0062] What needs to be understood is:

[0063] In the description of the embodiments of this invention, some basic operational terms commonly used in the art are used, such as "heating", "stirring", "mixing", "dissolving", "weighing", "filtering" and "drying". These terms should be interpreted broadly, and can refer to routine operations performed using various conventional equipment and instruments in the art, as well as operations performed using the latest equipment, such as programmed operations and unmanned automatic operations. Unless otherwise explicitly limited, those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances and adopt specific operating methods to achieve their operational objectives.

[0064] It should also be noted that:

[0065] The following specific embodiments can be combined with each other, and the same or similar concepts or processes may not be described again in some embodiments;

[0066] Furthermore, the raw materials, auxiliary materials, and reaction equipment and facilities involved in the following specific embodiments are all commercially available.

[0067] The technical solution of the present invention will now be described in detail with reference to specific embodiments.

[0068] Example 1

[0069] This embodiment provides a detection method for the qualitative analysis of lipid-lowering traditional Chinese medicine decoctions / prepared medicines.

[0070] A method for detecting a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is provided for the qualitative detection of the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. In this embodiment, the decoction of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is prepared by decocting the medicinal slices including Scutellaria baicalensis (fried), Typha orientalis, Polygonatum sibiricum, Caprifolium spp., Polygonum cuspidatum, and Curcuma longa in water. The prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is prepared by decocting the medicinal slices including Scutellaria baicalensis (fried), Typha orientalis, Polygonatum sibiricum, Caprifolium spp., Polygonum cuspidatum, and Curcuma longa in water to obtain an extract, and then adding pharmaceutically acceptable excipients. The detection method includes the following steps:

[0071] The following traditional Chinese medicine decoction pieces were prepared by decocting and extracting with water to obtain the dried aqueous extract of the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang. Then, a standard solution of Qingzhi Huayu Fang was prepared from the dried aqueous extract and analyzed by high-performance liquid chromatography-electrospray ionization (HPLC-ESI) to obtain the standard chromatogram of Qingzhi Huayu Fang and mark its characteristic peaks.

[0072] The high-performance liquid chromatography-electrospray detector uses an aminosilane-bonded silica gel column as the packing material, and the theoretical plate number of the column should be no less than 5000 based on the high-performance liquid chromatogram of sucrose. The liquid chromatography-electrospray detection uses acetonitrile aqueous solution as the mobile phase for elution.

[0073] The sample water extract was dried and prepared into a test solution. The test solution was then tested using the same detection method and conditions as the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula to obtain the liquid chromatogram of the test sample.

[0074] The liquid chromatogram of the sample to be tested is compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang. If the liquid chromatogram of the sample to be tested contains the characteristic peaks of the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, and the relative retention time of each characteristic peak in the liquid chromatogram of the sample to be tested is only within the experimentally permissible error range between the relative retention time of each corresponding characteristic peak in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, then the sample to be tested is the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang.

[0075] Furthermore, the standard liquid chromatogram of the above-mentioned lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis has at least 14 characteristic peaks, and the relative retention times of the 14 characteristic peaks are as follows:

[0076] 0.37min, 0.43min, 0.54min, 0.62min, 0.65min, 0.73min, 0.78min, 0.94min, 1.00min, 1.13min, 1.23min, 1.27min, 1.38min and 1.40min.

[0077] Furthermore, in the above-mentioned detection method for the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, the chromatographic column used has an inner diameter of 4.6 mm, a column length of 250 mm, a film thickness of 5 μm, and a column temperature of 35 °C.

[0078] Furthermore, in the above-mentioned detection method for the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, the flow rate of the mobile phase is 0.8 mL / min, the elution method adopts gradient elution, and the elution gradient is:

[0079] Use an acetonitrile aqueous solution containing 82-80% by volume for 0-10 min; 80-75% by volume for 10-20 min; 75-65% by volume for 20-30 min; 65-40% by volume for 30-35 min; 40-82% by volume for 35-36 min; and 82% by volume for 36-45 min.

[0080] Furthermore, in the above-mentioned detection method, the preparation method of the dried water extract of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang is as follows:

[0081] Take 120g of Scutellaria baicalensis (fried), 120g of Typha pollen, 100g of Polygonatum sibiricum, 50g of Polygonum cuspidatum, 100g of Curcuma longa, and 90g of Caprifolium arvense fruit. Soak them in 10 times the amount of water for 1 hour, then decoct for 2 hours and collect the decoction. Add 9 times the amount of water and decoct for another 2 hours and collect the decoction. Combine the two decoctions, filter, concentrate under reduced pressure, and vacuum dry to obtain the dried product of the water extract of the lipid-lowering Chinese medicine Qingzhi Huayu Fang.

[0082] Furthermore, in the above detection method, the preparation method of the standard solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula is as follows:

[0083] Accurately weigh approximately 0.3g of the dried water extract of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, place it in a 25mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20min, let it cool to room temperature, add another 30% methanol aqueous solution to the mark, shake well, then take the supernatant, centrifuge, and filter through a 0.45μm microporous membrane. The filtrate is the standard solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang.

[0084] Furthermore, in the above detection method, the preparation method of the dried water extract of the sample is as follows:

[0085] The decoction sample to be tested is filtered, then concentrated under reduced pressure, and then vacuum dried to obtain the dried water extract of the decoction sample; or

[0086] The drug sample to be tested is crushed, then 10 times the amount of water is added and decocted for 15 minutes. The decoction is then filtered, concentrated under reduced pressure, and vacuum dried to obtain the dried water extract of the drug sample.

[0087] Furthermore, in the above detection method, the preparation method of the test solution is as follows:

[0088] Accurately weigh approximately 0.3 g of the dried water extract of the decoction sample or the dried water extract of the prepared medicine sample, place it in a 25 mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20 min, let it cool to room temperature, add another 30% methanol aqueous solution to the mark, shake well, take the supernatant, centrifuge, and then filter through a 0.45 μm microporous membrane. The filtrate is the test solution.

[0089] Verification Example

[0090] 1. Instruments and Materials

[0091] 1.1 Instruments

[0092] The Thermo U3000 high performance liquid chromatograph was purchased from Thermo Fisher Scientific (China) Co., Ltd., and the chromatographic column Dikma Polyamino HILIC (4.6mm×250mm, film thickness 5μm) was purchased from Shanghai Dima Technology Co., Ltd.

[0093] BSA224S 0.01 g / 10,000 balance, purchased from Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0094] XP-105 1 / 100,000 balance, purchased from Mettler Toledo Technologies (China) Co., Ltd.

[0095] SK7200LHC ultrasonic cleaner, purchased from Shanghai Kedao Ultrasonic Instrument Co., Ltd.

[0096] The Smart-due ultrapure water system was purchased from Shanghai Hetai Instrument Co., Ltd.

[0097] 1.2 Reagents and Solvents

[0098] Acetonitrile (batch number: R142428, grade: LC-MS) and methanol (batch number: R142432, grade: LC-MS) were purchased from Shanghai Dima Technology Co., Ltd., and ultrapure water was prepared by a Smart-due water purifier.

[0099] 1.3 Carbohydrate Reference Standards

[0100] Rhamnose (batch number: 111683-201502), fructose (batch number: 100231-202008, purity 99.9%), mannitol (batch number: 100533-202006, purity 99.5%), D-anhydrous glucose (batch number: 110833-202109, purity 99.9%), and sucrose (batch number: 11107-202105, purity 99.8%) were all purchased from the China National Institutes for Food and Drug Control.

[0101] Raffinose (batch number: 10680, purity 98.0%) was purchased from Shanghai Shidander Standard Technical Service Co., Ltd.

[0102] 1.4 Traditional Chinese Medicine Decoction Pieces

[0103] Scutellaria baicalensis Georgi (fried), Typha angustifolia L., Polygonum cuspidatum Sieb. et Zucc., Curcuma kwangsiensis SGLee et C.F. Liang, and Polygonatum sibiricum Red. were all provided by Shanghai Kangqiao Pharmaceutical Co., Ltd., and met the testing requirements of the 2020 edition of the Chinese Pharmacopoeia.

[0104] The Capparis spinosa L. fruit was provided by Anhui Zhihetang Pharmaceutical Co., Ltd., and met the testing requirements of the 2020 edition of the "Medicinal Materials Standards of Xinjiang Uygur Autonomous Region Drug Administration", with all samples accompanied by test reports.

[0105] 1.5 Sample of lipid-lowering traditional Chinese medicine decoction for clearing lipids and removing blood stasis

[0106] Prepared by Shanghai Huanghai Pharmaceutical Co., Ltd., batch number / number: 210820~210834 / S1~S15.

[0107] 2. Preparation of test solution

[0108] 2.1 Preparation of lipid-lowering traditional Chinese medicine decoctions / prepared medicines for clearing lipids and removing blood stasis

[0109] Take 120g of Scutellaria baicalensis (fried), 120g of Typha pollen, 100g of Polygonatum sibiricum, 50g of Polygonum cuspidatum, 100g of Curcuma longa, and 90g of Caprifolium arvense fruit. Soak them in 10 times the amount of water for 1 hour, then decoct for 2 hours and take the decoction. Then add 9 times the amount of water and decoct for another 2 hours and take the decoction. Combine the two decoctions and filter them to obtain the lipid-lowering Chinese herbal formula for clearing lipids and removing blood stasis.

[0110] The above-mentioned lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang decoction is concentrated to obtain an extract, and then pharmaceutically acceptable excipients are added for processing to obtain the finished drug Qingzhi Huayu Fang.

[0111] 2.2 Preparation of the dried product of water extract of lipid-lowering traditional Chinese medicine Qingzhi Huayu formula

[0112] The lipid-lowering traditional Chinese medicine decoction prepared according to the method described in section 2.1 above was concentrated under reduced pressure and then vacuum dried to obtain the dried water extract of the lipid-lowering traditional Chinese medicine decoction.

[0113] The above-mentioned lipid-lowering Chinese medicine Qingzhi Huayu Fang is crushed, then 10 times the amount of water is added and decocted for 15 minutes. The decoction is then filtered, concentrated under reduced pressure, and vacuum dried to obtain the dried water extract of the lipid-lowering Chinese medicine Qingzhi Huayu Fang.

[0114] It should be noted that, for the convenience of the experiment, this verification example uses the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang decoction with batch number 210831 (S12) prepared by Shanghai Huanghai Pharmaceutical Co., Ltd. as the precursor decoction of the water extract of Qingzhi Huayu Fang (the same below).

[0115] 2.3 Preparation of dried water extracts of single-herb components of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis

[0116] Weigh out 100g each of Scutellaria baicalensis (fried), Typha pollen, Polygonatum sibiricum, Polygonum cuspidatum, Curcuma longa, and Caprifolium arvense fruit. Soak each herb in 10 times the amount of water for 1 hour, decoct for 2 hours, and collect the decoction. Then add 9 times the amount of water and decoct for another 2 hours. Combine the two decoctions of each herb, filter them separately, concentrate them under reduced pressure, and vacuum dry them to obtain the dried water extracts of each herb in the lipid-lowering and blood-activating formula.

[0117] 2.4 Preparation of blank reagent solution

[0118] Take a 30% (v / v) methanol aqueous solution and filter it through a 0.45 μm microporous membrane. Collect the filtrate, which is the blank reagent solution.

[0119] 2.5 Preparation of a mixed solution of six sugar reference standards

[0120] Take appropriate amounts of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose reference standards, respectively, accurately weigh them, add water to prepare solutions containing 2.7630, 3.6314, 1.4169, 3.0080, 3.8164, and 2.4520 mg per ml, respectively. Filter the solutions through a 0.45 μm microporous membrane, collect the filtrate, and obtain a mixed solution of the six sugar reference standards. Store at 2–8℃ for later use.

[0121] 2.6 Preparation of the test solution of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis

[0122] Accurately weigh approximately 0.3g of the dried aqueous extract of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, place it in a 25mL volumetric flask, add an appropriate amount of 30% methanol, sonicate (500W, 53kHz) for 20min, let it cool to room temperature, add 30% methanol aqueous solution to the mark, shake well, take the supernatant, centrifuge (12000r / min) for 5min, filter through a 0.45μm microporous membrane, and take the filtrate, which is the test solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang.

[0123] 2.7 Preparation of single-herb test solutions

[0124] Accurately weigh approximately 0.3g of the dried aqueous extracts of each of the single herbs in the lipid-lowering and blood-activating formula, and place them in separate 25mL volumetric flasks. Add an appropriate amount of 30% (v / v) methanol aqueous solution to each flask, sonicate (500W, 53kHz) for 20min, allow to cool to room temperature, and then add 30% (v / v) methanol aqueous solution to the mark. Shake well, take the supernatant, centrifuge (12000r / min) for 5min, and filter through a 0.45μm microporous membrane. Take the filtrate from each flask to obtain the test solution of each single herb.

[0125] 3. Detection and Results

[0126] 2.1 Chromatographic conditions

[0127] The chromatographic system used was a Thermo U3000 high-performance liquid chromatograph with an aminosilane-bonded silica column (Dikma Polyamino HILIC, 4.6 mm × 250 mm, 5 μm) and a column temperature of 35 °C, equipped with an electrospray ionization detector (CAD detector).

[0128] The injection volume was 3 μL;

[0129] The theoretical plate number, calculated based on the sucrose peak, should be no less than 5000.

[0130] Gradient elution was performed using acetonitrile aqueous solution as the mobile phase at a flow rate of 0.8 mL / min and a mobile phase gradient of:

[0131] Use an acetonitrile aqueous solution containing 82-80% by volume for 0-10 minutes;

[0132] Use an acetonitrile aqueous solution containing 80-75% by volume for 10-20 minutes;

[0133] Use an acetonitrile aqueous solution containing 75-65% by volume for 20-30 minutes;

[0134] Use an acetonitrile aqueous solution containing 65-40% by volume for 30-35 minutes;

[0135] Use an acetonitrile aqueous solution containing 40-82% by volume for 35-36 minutes;

[0136] Use an acetonitrile aqueous solution containing 82% acetonitrile by volume for 36–45 minutes.

[0137] 2.2 System Applicability Results

[0138] Accurately pipette 3 μl each of the blank reagent solution, the mixed solution of six sugar reference standards, and the test solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and inject them into the above-mentioned high-performance liquid chromatograph-electrospray detector. Perform the determination under the chromatographic conditions specified in section "2.1" above. The detection results are shown in [Figure 1]. Figure 1 The image shows a comparison of high-performance liquid chromatography-electrospray ionization (HPLC-ESI) results obtained by analyzing a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, six sugar reference standards, and a blank reagent, as provided in an embodiment of the present invention.

[0139] from Figure 1 It can be seen from this:

[0140] The HPLC chromatogram of the blank reagent solution was stable, with no other detection peaks except for the acetonitrile solvent detection peak.

[0141] The high-performance liquid chromatography-electrospray chromatograms of the mixed solution of six sugar reference standards and the test solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula showed that the theoretical plate number of the detection system in this experiment was greater than 5000 based on the sucrose peak, and the resolution of the chromatographic peaks of the six sugar reference standards was greater than 1.5, and the peak area and relative retention time RSD were less than 2.0%, indicating that the system suitability met the relevant detection requirements.

[0142] through Figure 1 The comparison between the high-performance liquid chromatography-electrospray ionization (HPLC-ESI) chromatogram S12 (the chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang) and the HPLC-ESI chromatogram T (the chromatogram of the six carbohydrate reference standards) clearly identifies the six carbohydrate components contained in the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang.

[0143] T1 peak is rhamnose, T2 peak is fructose, T3 peak is mannitol, T4 peak is D-anhydrous glucose, T5 peak is sucrose, and T6 peak is raffinose.

[0144] 2.3 Characteristic Peak Identification and Specificity Results

[0145] Accurately pipette 3 μl each of the blank reagent solution, the mixed solution of six sugar reference standards, the test solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and the test solutions of each of the six single traditional Chinese medicines, and inject them into the above-mentioned high-performance liquid chromatograph-electrospray detector. Perform the determination under the chromatographic conditions specified in section "2.1" above to obtain the following results. Figure 2The detection results are shown in the high-performance liquid chromatography-electrospray ionization comparison diagram obtained by performing high-performance liquid chromatography-electrospray ionization on a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis provided in this embodiment of the invention, six sugar reference standards, single traditional Chinese medicine test samples, and blank reagents.

[0146] Through the Figure 2 Analysis and comparison of various liquid chromatograms revealed 14 common characteristic chromatographic peaks on the obtained high-performance liquid chromatography-electrospray ionization (HPLC-ESI) chromatogram S12 of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. These peaks include rhamnose (peak 1), fructose (peak 3), mannitol (peak 5), D-anhydrous glucose (peak 6), sucrose (peak 9), and raffinose (peak 12).

[0147] Peak 1 mainly comes from Scutellaria baicalensis and Caprifolium arvense fruit; Peak 3 comes from all six herbs in the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang; Peak 5 mainly comes from Caprifolium arvense, Typha pollen, and Curcuma longa; Peak 6 mainly comes from the other five herbs except for Scutellaria baicalensis (fried); Peak 9 also comes from all six herbs in the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang; Peak 12 mainly comes from herbs except for Typha pollen; Peaks 4, 10, 13, and 14 are unknown components unique to Typha pollen, Polygonum cuspidatum, Polygonatum sibiricum, and Scutellaria baicalensis (fried), respectively, and require further research.

[0148] Table 1 shows the relevant data and specificity of 14 characteristic chromatographic peaks, as well as the results of tracing the main sources of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula.

[0149] In Table 1, D1 is Scutellaria baicalensis (fried), D2 is Caprifolium arvense fruit, D3 is Polygonum cuspidatum, D4 is Polygonatum sibiricum, D5 is Typha pollen, and D6 is Curcuma longa extract.

[0150] Table 1

[0151]

[0152] 2.4 Precision Examination

[0153] Methods: Take the same lipid-lowering traditional Chinese medicine Qingzhi Huayu formula test solution (S12), inject it continuously 6 times under the chromatographic conditions specified in "2.1", and record the peak area and relative retention time of each of the 14 characteristic chromatographic peaks on each liquid chromatogram. Taking the peak with the larger and more stable peak area of ​​peak 9 (sucrose) as the reference peak S, calculate the peak area and relative retention time of each of the 14 common characteristic chromatographic peaks.

[0154] Results: The RSD of the peak area of ​​the 14 common characteristic chromatographic peaks on each of the six detections were all in the range of 0.9–12.7%, and the RSD of the relative retention time was all in the range of 0.0–0.9%. Among them, the RSD of the peak area of ​​the six sugars were all in the range of 0.9–2.4%, and the RSD of the relative retention time was all in the range of 0.0–0.9%, indicating that the high performance liquid chromatography-electrospray ionization detection system used in this validation example has good precision.

[0155] 2.5 Stability Test

[0156] The same lipid-lowering traditional Chinese medicine Qingzhi Huayu formula test solution (S12) was taken and injected under the chromatographic conditions specified in section "2.1" after ultrasonic treatment for 0, 3, 6, 9, 12, and 24 hours. The peak area and relative retention time of 14 characteristic chromatographic peaks on each liquid chromatogram were recorded. Peak No. 9 (sucrose), which has a larger and more stable peak area, was used as the reference peak S. The peak area and relative retention time of the 14 common characteristic chromatographic peaks were calculated. The results showed that the RSD of the peak area of ​​the 14 common characteristic chromatographic peaks were all in the range of 0.2-13.3%, and the RSD of the relative retention time were all in the range of 0.0-1.2%. Among them, the RSD of the peak area of ​​the 6 sugars were all in the range of 0.2-2.3%, and the RSD of the relative retention time were all in the range of 0.0-1.2%. The results indicate that the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula test solution has good stability within 24 hours.

[0157] 2.6 Examination of Linear Relationships

[0158] Six sugar reference solutions of varying concentrations were prepared and injected into the samples. A standard curve was plotted with concentration as the x-axis (X) and peak area of ​​the reference solution as the y-axis (Y). A regression equation was established for peak area versus concentration. The results showed that the six sugar components exhibited good linearity within the detected concentration range.

[0159] The regression equations, linear ranges, correlation coefficients (r), and limits of detection (LOD) for the six carbohydrate reference standards are shown in Table 2.

[0160] Table 2

[0161]

[0162] 2.7 Recovery rate analysis

[0163] Take 0.3g of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang sample (S12) with clearly defined content of the components to be tested, and add 2mL of a mixed solution of six sugar reference standards consisting of 0.110 5mg rhamnose, 0.726 3mg fructose, 0.014 2mg mannitol, 0.090 2mg D-anhydrous glucose, 0.763 3mg sucrose, and 0.0736mg raffinose. Prepare the test solution according to the preparation method of the test solution of Qingzhi Huayu Fang in section 2.6. Perform six parallel tests and inject the samples under the chromatographic conditions specified in section 2.1. Calculate the recovery rate and RSD of each sugar component based on the test results. The results show that the recovery rates of the six sugars are all between 92.01% and 107.54%, indicating that the accuracy of the detection method provided in this embodiment meets the detection requirements.

[0164] The detailed amounts of the six sugars added, the measured amounts, the recovery rates, the average values, and the RSD values ​​are listed in Table 3.

[0165] Table 3

[0166]

[0167]

[0168] 2.8 Establishment and Similarity Analysis of Standard Liquid Chromatograms for Lipid-Lowering Traditional Chinese Medicine Formula for Clearing Lipids and Removing Blood Stasis

[0169] Fifteen batches of lipid-lowering traditional Chinese medicine decoctions containing the formula qingzhihuayufang, prepared by Shanghai Huanghai Pharmaceutical Co., Ltd., with batch numbers 210820~210834 / S1~S15, were first prepared into dried water extracts of the formula according to the preparation method in section "2.2". Then, test solutions of the formula were prepared according to the preparation method in section "2.6". Finally, high-performance liquid chromatography-electrospray ionization was performed under the chromatographic conditions specified in section "2.1" to obtain their respective chromatograms.

[0170] Then, the chromatographic data of the 15 batches of lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (version 2012.130723) in "*.cdf" format. Using the S1 characteristic chromatogram as a control, the mean method was employed, with a time window width of 0.1 min. After multi-point correction, 14 common characteristic chromatographic peaks in each chromatogram were selected as Mark peaks for matching, generating a standard liquid chromatogram (R) of the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction. This yielded the following results: Figure 3 The image shown is a comparison of the high performance liquid chromatography-electrospray ionization (HPLC-ESI) chromatogram and its standard characteristic chromatogram of a multi-batch lipid-lowering traditional Chinese medicine decoction for clearing lipids and removing blood stasis, provided in an embodiment of the present invention.

[0171] It should be noted that: although Figure 3 The comparison chart was obtained using a lipid-lowering traditional Chinese medicine decoction for clearing lipids and removing blood stasis as the test sample. However, according to the experiment, a lipid-lowering traditional Chinese medicine decoction for clearing lipids and removing blood stasis, prepared as provided in this embodiment, was obtained by decocting Chinese medicinal herbs including Scutellaria baicalensis (fried), Typha pollen, Polygonatum sibiricum, Capsella bursa-pastoris fruit, Polygonum cuspidatum, and Curcuma longa, and then adding pharmaceutically acceptable excipients. The test solution was prepared according to the methods in sections "2.2" and "2.6", and then tested according to the requirements in section "2.1". The results were also obtained as described in section "2." Figure 3 The same chromatograms and standard characteristic chromatograms were used for each test sample.

[0172] Table 4 lists the similarity values ​​of the chromatograms of 15 batches of lipid-lowering traditional Chinese medicine decoctions for clearing lipids and removing blood stasis.

[0173] Table 5 lists the relative areas of characteristic peaks in the chromatograms of 15 batches of lipid-lowering traditional Chinese medicine decoctions for clearing lipids and removing blood stasis.

[0174] Table 6 lists the relative retention times of each characteristic peak in the chromatograms of 15 batches of lipid-lowering traditional Chinese medicine decoctions containing Qingzhi Huayu.

[0175]

[0176]

[0177]

[0178] from Figure 3 As can be seen from the data listed in Tables 4 to 6:

[0179] The chromatograms of 15 batches of lipid-lowering traditional Chinese medicine Qingzhi Huayu decoctions all had 14 common sugar characteristic chromatographic peaks, with a similarity greater than 0.98. The RSD of the peak area was between 7.8% and 14.6%, and the RSD of the relative retention time was less than 1.5%.

[0180] In addition, through with Figure 1 or Figure 2 The comparison of the high performance liquid chromatography-electrospray ionization (HPLC-ESI) chromatograms of the six sugar reference standards shown in the figure, with peak 9 (sucrose peak) as the S peak and a relative retention time of 1.00, shows that the relative retention times of peaks 1, 3, 5, 6, and 12 are 0.37, 0.54, 0.65, 0.73, and 1.27, respectively. This confirms that they are rhamnose peak, fructose peak, mannitol peak, D-anhydrous glucose peak, and raffinose peak, respectively.

[0181] Therefore, it can be concluded that the accuracy and repeatability of the detection method provided in this embodiment meet the detection requirements. Figure 3The standard characteristic chromatogram (R) in it can be used as the standard characteristic chromatogram for the qualitative detection of the decoction / medicine of Qingzhi Huayu Prescription, a traditional Chinese medicine for reducing lipid, and the saccharide components contained therein, and is used for the qualitative detection of the decoction / medicine of Qingzhi Huayu Prescription, a traditional Chinese medicine for reducing lipid, and the saccharide components contained therein.

[0182] Example 2

[0183] This example provides a method for the qualitative detection of saccharide components contained in the decoction / medicine of Qingzhi Huayu Prescription, a traditional Chinese medicine for reducing lipid.

[0184] A method for the qualitative detection of saccharide components contained in the decoction / medicine of Qingzhi Huayu Prescription, a traditional Chinese medicine for reducing lipid, includes the following steps:

[0185] Obtain the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription according to the detection method of the decoction / medicine of Qingzhi Huayu Prescription provided in Example 1 above, that is Figure 3 the liquid chromatogram R in it, and calibrate the chromatographic peak with a relative retention time of 0.37 min as the rhamnose peak, the chromatographic peak with a relative retention time of 0.54 min as the fructose peak, the chromatographic peak with a relative retention time of 0.65 min as the mannitol peak, the chromatographic peak with a relative retention time of 0.73 min as the D-glucose anhydrous peak, the chromatographic peak with a relative retention time of 1.00 min as the sucrose peak, and the chromatographic peak with a relative retention time of 1.27 min as the raffinose peak;

[0186] Obtain the dry product of the water extract of the decoction / medicine from the tested decoction / medicine according to the method provided in Example 1 above and prepare the corresponding tested solution of the decoction / medicine, and then detect the tested solution of the decoction / medicine according to the method for obtaining the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription in Example 1 to obtain the liquid chromatogram of the tested decoction / medicine;

[0187] Compare the liquid chromatogram of the tested decoction / medicine with the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription;

[0188] For the chromatographic peaks that appear at the positions corresponding to the rhamnose peak, fructose peak, mannitol peak, D-glucose anhydrous peak, sucrose peak, and raffinose peak marked in the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription on the liquid chromatogram of the tested decoction / medicine, and there is only an error allowed by the experiment between the relative retention time of this chromatographic peak and the relative retention time of the corresponding chromatographic peak in the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription, then this chromatographic peak is the sugar species represented by the chromatographic peak with this relative retention time in the standard liquid chromatogram of the decoction / medicine of Qingzhi Huayu Prescription.

[0189] From the above description, it can be seen that:

[0190] The qualitative detection method for sugar components in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine provided in this embodiment is the first time that a high-performance liquid chromatography-electrospray detector has been used to qualitatively detect sugar components in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine. The method is reliable, highly sensitive, and has good repeatability, and can provide a basis for the quality control and quality standard formulation of the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine.

[0191] Example 3

[0192] This embodiment provides a method for quantitative detection of carbohydrate components in lipid-lowering traditional Chinese medicine decoctions / prepared medicines.

[0193] A method for quantitatively detecting carbohydrate components in a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, used for the quantitative detection of carbohydrate components in the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, comprising the following steps:

[0194] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis was obtained according to the detection method provided in Example 1 above. The chromatographic peaks with relative retention times of 0.37 min were identified as rhamnose peak, 0.54 min as fructose peak, 0.65 min as mannitol peak, 0.73 min as D-anhydrous glucose peak, 1.00 min as sucrose peak, and 1.27 min as raffinose peak.

[0195] Accurately weigh appropriate amounts of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose reference standards, respectively, and add water to prepare reference standard solutions containing 2.7630 mg rhamnose, 3.6314 mg fructose, 1.4169 mg mannitol, 3.0080 mg D-anhydrous glucose, 3.8164 mg sucrose, and 2.4520 mg raffinose per ml, respectively. Then, according to the method and conditions for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula provided in Example 1, liquid chromatography detection was performed to obtain the reference standard liquid chromatogram. The chromatographic peaks of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose on the reference standard liquid chromatogram were determined by comparing the relative retention times, and the peak areas corresponding to their respective contents were recorded.

[0196] According to the method provided in Example 1, the decoction / prepared medicine water extract dried product is obtained from the tested decoction / prepared medicine and the corresponding decoction / prepared medicine test solution is prepared. Then, the decoction / prepared medicine test solution is tested according to the method for obtaining the standard liquid chromatogram of lipid-lowering traditional Chinese medicine Qingzhi Huayu formula to obtain the liquid chromatogram of the tested decoction / prepared medicine.

[0197] The liquid chromatogram of the tested decoction / prepared medicine was compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. By comparing the relative retention times, the sugars contained in the tested lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine and their peak areas were determined on the liquid chromatogram of the tested decoction / prepared medicine.

[0198] Then, the peak areas of each sugar in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine are calculated by correspondingly comparing the content and peak area of ​​the same sugar recorded in the liquid chromatogram of the reference standard, so as to obtain the content of each sugar in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine.

[0199] Specifically, 15 batches of lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction samples prepared by Shanghai Huanghai Pharmaceutical Co., Ltd. in Example 1 above can be accurately weighed, and their test solutions can be obtained according to the preparation methods in "2.2" and "2.6". The tests can then be performed as specified in "2.1", and the content can be calculated. The specific results are shown in Table 7.

[0200] Table 7

[0201] serial number Rhamnose fructose Mannitol D-Anhydrous Glucose sucrose Raffinose S1 0.93 5.74 0.05 0.88 5.35 0.18 S2 0.64 5.70 0.05 1.19 4.44 0.13 S3 0.68 4.54 0.05 0.66 3.88 0.15 S4 0.75 5.64 0.05 0.98 4.74 0.13 S5 0.55 4.08 0.04 0.97 3.83 0.11 S6 0.72 5.03 0.06 0.88 4.74 0.16 S7 0.77 4.68 0.05 0.94 4.59 0.15 S8 0.71 5.62 0.04 0.89 4.25 0.14 S9 0.70 5.83 0.05 1.09 4.48 0.12 S10 0.69 5.52 0.05 1.04 4.38 0.15 S11 0.67 5.53 0.05 0.85 3.92 0.16 S12 0.83 6.30 0.03 1.05 4.52 0.13 S13 0.65 5.16 0.05 0.89 3.82 0.12 S14 0.71 5.78 0.05 0.85 4.27 0.11 S15 0.68 5.36 0.04 0.87 4.06 0.12 average 0.71 5.37 0.05 0.94 4.35 0.14 RSD% 12.2 10.8 12.1 13.4 9.7 14.9

[0202] The results show:

[0203] The 15 batches of lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction samples prepared by Shanghai Huanghai Pharmaceutical Co., Ltd. showed that the rhamnose content ranged from 0.55% to 0.93%, the fructose content ranged from 4.08% to 6.30%, the mannitol content ranged from 0.03% to 0.06%, the D-anhydrous glucose content ranged from 0.66% to 1.19%, the sucrose content ranged from 3.82% to 5.35%, and the raffinose content ranged from 0.11% to 0.18%. Furthermore, the content of all six sugars was within 70% to 130% of the average, indicating that the established method was accurate and reliable, and the extraction process had good consistency. This also demonstrates that the quantitative detection method provided in this embodiment is reliable, highly sensitive, and has good repeatability, providing a basis for the quality control and quality standard formulation of the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction.

[0204] It should be noted that:

[0205] Although this embodiment only tested the sugar content of the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction, according to experiments, the lipid-lowering traditional Chinese medicine Qingzhi Huayu prepared medicine, prepared by decocting Chinese medicinal herbs including Scutellaria baicalensis (fried), Typha pollen, Polygonatum sibiricum, Capsella bursa-pastoris fruit, Polygonum cuspidatum, and Curcuma longa, as provided in this embodiment, and then adding pharmaceutically acceptable excipients, and obtaining its test solution according to the preparation methods in "2.2" and "2.6", and then conducting the test as specified in "2.1", can also obtain the sugar content test results of the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction. This shows that the quantitative detection method provided in this embodiment is reliable, highly sensitive, and has good repeatability, and can provide a basis for the quality control and quality standard formulation of the lipid-lowering traditional Chinese medicine Qingzhi Huayu prepared medicine.

[0206] In conclusion, it can be seen that:

[0207] Traditional Chinese medicine preparations are the most important way of using traditional Chinese medicine. Polysaccharides are one of the important components that constitute the basis of their efficacy. However, due to the complexity and diversity of the structure of polysaccharide macromolecules, their separation, purification and structural analysis are extremely difficult.

[0208] The fingerprinting method for traditional Chinese medicine is an internationally recognized important means of characterizing known and unknown chemical component groups of natural drugs, Chinese medicinal materials and compound preparations of traditional Chinese medicine. It can effectively control product quality and evaluate the consistency of Chinese medicinal materials, intermediates and finished products.

[0209] Traditional methods for extracting carbohydrate components from Chinese medicine include water extraction, ultrasound, reflux extraction, and enzymatic extraction. Among these, water extraction inherits and follows the extraction methods of classic prescriptions, and can retain the effective components to the greatest extent.

[0210] Although there are many common methods for detecting traditional Chinese medicine (TCM), including the anthrone-sulfuric acid method, the phenol-sulfuric acid method, and the lichenol-sulfuric acid method, these methods often use anhydrous glucose as a control and react with sulfuric acid when detecting sugars in TCM. This poses safety risks in actual operation, and the measured data cannot accurately and effectively reflect the sugar content. In addition, existing detection methods such as ion chromatography, capillary electrophoresis chromatography, and high-performance anion exchange chromatography-pulse amperometry are relatively cumbersome in sample processing and have poor repeatability. However, the high-performance liquid chromatography-electrospray ionization detection method (HPLC-CAD method) of this invention can overcome the problem that sugars do not absorb ultraviolet light, while ensuring good sensitivity and reproducibility.

[0211] In the process of constructing characteristic chromatograms of carbohydrate components and determining their content, in order to establish the optimal chromatographic conditions and sample preparation methods and to characterize the relative content of the components to be measured to the greatest extent, this invention investigated various elution systems and finally determined that the best elution system was an aqueous solution system containing different volume percentages of acetonitrile.

[0212] Secondly, through experience and practice, the inventors ultimately determined that using a Dikma Polyamino HILIC (4.6mm × 250mm, 5μm film thickness) column, which offers a more stable baseline, detects more types of sugars, and provides better separation, is the optimal choice.

[0213] Furthermore, through experience and practice, the inventors discovered that using a 30% (v / v) methanol aqueous solution and ultrasonic extraction—specifically, selecting a liquid-to-solid ratio of 0.3:25 and a 30% (v / v) methanol aqueous solution as the extraction solvent and ultrasonic (500W, 53kHz) for 20 minutes as the preparation method for the test sample—resulted in a higher sugar content in the lipid-lowering traditional Chinese medicine formula Qingzhi Huayu Fang, and the method was simpler.

[0214] Furthermore, analysis of the differences in the content of indicative carbohydrate components in the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis revealed that the detection method provided in this invention yielded the lowest mannitol content, around 0.1%, while fructose and sucrose contents were the highest, around 5%, which is 50 times that of mannitol. Rhamnose, D-anhydrous glucose, and raffinose contents were around 0.7%, which is 7 times that of mannitol. This difference in content is mainly related to the source of the ingredients in the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis. Fructose and sucrose come from the six medicinal materials in the formula, while mannitol mainly comes from Scutellaria baicalensis, Polygonum cuspidatum, and Polygonatum sibiricum.

[0215] It can be seen that:

[0216] This invention establishes a characteristic chromatographic characterization method for the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula for the first time using HPLC-CAD. This method can qualitatively and / or quantitatively detect the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula and its sugar components. It overcomes the difficulties in detecting traditional Chinese medicine components, such as the lack of ultraviolet absorption of polysaccharides, the slightly poor sensitivity of ELSD detectors, and the cumbersome derivatization process. This invention can provide a method and standard for the detection of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine and its sugar components and content.

[0217] The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula established by the detection method provided by this invention is accurate and reliable. Moreover, the detection method of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine and its six sugar components has high sensitivity, good repeatability, and wide linear range. The accuracy meets the method validation guidelines of the Chinese Pharmacopoeia. It can provide a basis for the quality control and quality standard formulation of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula decoction / prepared medicine.

[0218] It should be noted that:

[0219] In the description of the above specification, the terms "this embodiment," "an embodiment of the present invention," "as shown," "further," etc., indicate that the specific features, structures, materials, or characteristics described in the embodiment are included in at least one embodiment of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment, and the specific features, structures, materials, or characteristics described can be combined or combined in a suitable manner in any one or more embodiments. Furthermore, without causing contradiction, those skilled in the art can combine or combine the different embodiments and features of the different embodiments described in this specification.

[0220] Finally, it should be noted that:

[0221] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions described in the embodiments of the present invention. Non-essential improvements, adjustments or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. A method for detecting a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, used for the qualitative detection of the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, wherein the decoction is prepared by decocting the following traditional Chinese medicinal materials in water: stir-fried Scutellaria baicalensis, Typha pollen, Polygonatum sibiricum, Caprifolium arvense fruit, Polygonum cuspidatum, and Curcuma longa; and the prepared medicine is prepared by decocting the following traditional Chinese medicinal materials in water to obtain an extract, and then adding pharmaceutically acceptable excipients. The method is characterized in that... Includes the following steps: The following traditional Chinese medicine decoctions were prepared by decocting and extracting a mixture of stir-fried Scutellaria baicalensis, Typha pollen, Polygonatum sibiricum, Citrus aurantium fruit, Polygonum cuspidatum, and Curcuma longa to obtain a lipid-lowering and blood-activating traditional Chinese medicine formula. A standard solution of this formula was then prepared and analyzed by high-performance liquid chromatography-electrospray ionization (HPLC-ESI) to obtain the standard chromatogram. The characteristic peaks were then identified. The preparation method of the dried product of the water extract of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is as follows: Take 120 g of stir-fried Scutellaria baicalensis, 120 g of Typha pollen, 100 g of Polygonatum sibiricum, 90 g of Citrus aurantium fruit, 50 g of Polygonum cuspidatum and 100 g of Curcuma longa, add 10 times the amount of water and soak for 1 hour, then decoct for 2 hours and take the decoction. Then add 9 times the amount of water and decoct for 2 hours and take the decoction. Combine the two decoctions, filter, concentrate under reduced pressure, and vacuum dry to obtain the dried product of the water extract of the lipid-lowering Chinese medicine Qingzhi Huayu Fang. The preparation method of the standard solution of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis is as follows: Accurately weigh approximately 0.3 g of the dried aqueous extract of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang, place it in a 25 mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20 min, let it cool to room temperature, add another 30% by volume methanol aqueous solution to the mark, shake well, then take the supernatant, centrifuge, and filter through a 0.45 µm microporous membrane. The filtrate is the standard solution of the lipid-lowering traditional Chinese medicine Qingzhi Huayu Fang. The high-performance liquid chromatography-electrospray detector uses a Dikma Polyamino HILIC column, and the theoretical plate number of the column, calculated based on the high-performance liquid chromatogram of sucrose, should be no less than 5000. The liquid chromatography-electrospray detection uses acetonitrile aqueous solution as the mobile phase for elution. The elution is a gradient elution, and its gradient is: For the first 10 min, use an acetonitrile aqueous solution containing 82-80% by volume; for the next 10-20 min, use an acetonitrile aqueous solution containing 80-75% by volume; for the next 20-30 min, use an acetonitrile aqueous solution containing 75-65% by volume; for the next 30-35 min, use an acetonitrile aqueous solution containing 65-40% by volume; for the next 35-36 min, use an acetonitrile aqueous solution containing 40-82% by volume; and for the next 36-45 min, use an acetonitrile aqueous solution containing 82% by volume. A water extract of the sample was obtained and dried and a test solution was prepared. The test solution was then tested using the same detection method and conditions as the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the sample. The method for preparing the dried aqueous extract of the sample is as follows: The decoction sample to be tested is filtered, then concentrated under reduced pressure, and then vacuum dried to obtain the dried water extract of the decoction sample; or The drug sample to be tested is crushed, then 10 times the amount of water is added and decocted for 15 minutes. The decoction is then filtered, concentrated under reduced pressure, and dried under vacuum to obtain the dried water extract of the drug sample. The method for preparing the tested solution is as follows: Accurately weigh approximately 0.3 g of the dried aqueous extract of the decoction sample or the dried aqueous extract of the prepared medicine sample, place it in a 25 mL volumetric flask, add an appropriate amount of methanol aqueous solution with a methanol content of 30% by volume, sonicate for 20 min, let it cool to room temperature, add more methanol aqueous solution with a content of 30% by volume to the mark, shake well, take the supernatant, centrifuge, and then filter through a 0.45 µm microporous membrane. The filtrate is the test solution. The liquid chromatogram of the tested sample is compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. If the liquid chromatogram of the tested sample contains the characteristic peaks of the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and the relative retention time of each characteristic peak in the liquid chromatogram of the tested sample is only experimentally permissible, and there is only an experimentally permissible error between the relative retention time of each corresponding characteristic peak in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, then the tested sample is the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. The characteristic peaks include: rhamnose peak, fructose peak, mannitol peak, D-anhydrous glucose peak, sucrose peak, and raffinose peak.

2. The detection method for the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis as described in claim 1, characterized in that: The number of characteristic peaks is at least 14, and the relative retention times of the 14 characteristic peaks are as follows: 0.37 min, 0.43 min, 0.54 min, 0.62 min, 0.65 min, 0.73 min, 0.78 min, 0.94 min, 1.00 min, 1.13 min, 1.23 min, 1.27 min, 1.38 min and 1.40 min.

3. The detection method for the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis as described in claim 1, characterized in that: The chromatographic column has an inner diameter of 4.6 mm, a length of 250 mm, a film thickness of 5 µm, and a column temperature of 35 °C.

4. The detection method for the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis as described in claim 1, characterized in that: The flow rate of the mobile phase is 0.8 mL / min.

5. A qualitative detection method for components contained in a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, used for the qualitative detection of components contained in the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, characterized in that, Includes the following steps: The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis was obtained according to the detection method of claim 1 above, and the chromatographic peaks with relative retention times of 0.37 min were identified as rhamnose peak, 0.54 min as fructose peak, 0.65 min as mannitol peak, 0.73 min as D-anhydrous glucose peak, 1.00 min as sucrose peak, and 1.27 min as raffinose peak. The decoction / prepared medicine was obtained as a dry water extract and the corresponding decoction / prepared medicine test solution was prepared. Then, the decoction / prepared medicine test solution was tested according to the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the decoction / prepared medicine. The liquid chromatogram of the tested decoction / prepared medicine was compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis; The chromatographic peaks appearing at the positions of the rhamnose peak, fructose peak, mannitol peak, D-anhydrous glucose peak, sucrose peak, and raffinose peak in the liquid chromatogram of the tested decoction / prepared medicine corresponding to the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, and the relative retention time of the chromatographic peak is within the experimentally permissible error compared to the relative retention time of the corresponding chromatographic peak in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula, are the components represented by the chromatographic peak with the corresponding relative retention time in the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula.

6. A method for quantitatively detecting the components contained in a lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, used for the quantitative detection of components contained in the decoction / prepared medicine of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis, characterized in that, Includes the following steps: The standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis was obtained according to the detection method of claim 1 above, and the chromatographic peaks with relative retention times of 0.37 min were identified as rhamnose peak, 0.54 min as fructose peak, 0.65 min as mannitol peak, 0.73 min as D-anhydrous glucose peak, 1.00 min as sucrose peak, and 1.27 min as raffinose peak. Accurately weigh appropriate amounts of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose reference standards, and add water to prepare reference standard solutions containing 2.7630 mg rhamnose, 3.6314 mg fructose, 1.4169 mg mannitol, 3.0080 mg D-anhydrous glucose, 3.8164 mg sucrose, and 2.4520 mg raffinose per ml, respectively. Then, perform liquid chromatography detection according to the method and conditions for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis as described in claim 1 above to obtain the liquid chromatogram of the reference standard. By comparing the relative retention times, determine the chromatographic peaks of rhamnose, fructose, mannitol, D-anhydrous glucose, sucrose, and raffinose on the liquid chromatogram of the reference standard, and record the peak areas corresponding to their respective contents. The decoction / prepared medicine was obtained as a dry water extract and the corresponding decoction / prepared medicine test solution was prepared. Then, the decoction / prepared medicine test solution was tested according to the method for obtaining the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine formula for clearing lipids and removing blood stasis to obtain the liquid chromatogram of the decoction / prepared medicine. The liquid chromatogram of the tested decoction / prepared medicine is compared with the standard liquid chromatogram of the lipid-lowering traditional Chinese medicine Qingzhi Huayu formula. The components and peak areas contained in the tested lipid-lowering traditional Chinese medicine Qingzhi Huayu formula are determined by comparing the relative retention times on the liquid chromatogram of the tested decoction / prepared medicine. Then, the peak areas of each component in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine are calculated in correspondence with the content and peak area of ​​the same component recorded in the liquid chromatogram of the reference standard, so as to obtain the content of each component in the lipid-lowering traditional Chinese medicine Qingzhi Huayu decoction / prepared medicine.

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