Method for accurately quantifying content of isoflavone components in astragalus membranaceus and application

Abstract

The invention discloses a method for accurately quantifying the content of isoflavone components in astragalus membranaceus and application, and belongs to the technical field of isoflavone content detection. The method comprises the following steps: (1) preparing a reference substance solution by taking CG as a reference substance, and obtaining a standard linear equation of the reference substance solution; (2) crushing an astragalus membranaceus sample, and performing ultrasonic extraction and centrifugation to obtain a solution to be detected; (3) performing chromatographic analysis on the to-be-detected solution and the reference substance solution to obtain peak areas; and (4) on the basis of the CG standard linear equation, obtaining a standard linear equation of each isoflavone component to be detected, and then obtaining the content of isoflavone including unstable components CGM and FGM in the sample to be detected according to the peak area of the isoflavone to be detected. According to the method, the contents of six main isoflavone components including unstable components CGM and FGM in the astragalus membranaceus can be detected by using a single reference substance, the method can be used for evaluating the quality of the astragalus membranaceus, and the storage life of the astragalus membranaceus can be detected on the basis of the contents of the CGM and the FGM.

Description

Technical Field

[0001] The present invention belongs to the technical field of isoflavone content detection, and particularly relates to a method and use for accurately quantifying the content of isoflavone components in Astragalus membranaceus Background Art

[0002] Astragalus membranaceus is a commonly used bulk medicinal material, known as "nine out of ten medicines contain Astragalus membranaceus". There are about 150 compound preparations containing Astragalus membranaceus included in the Chinese Pharmacopoeia. The flavonoid components in Astragalus membranaceus are mainly isoflavone components, mainly calycosin-7-O-β-D-glucoside (CG) and its structurally similar isoflavone components such as calycosin (C), ononin (FG), formononetin (F), and unstable components calycosin-7-O-β-D-glucoside-6”-O-malonate (CGM) and 6"-O-malonylononin (FGM). These isoflavone components are widely present in Astragalus membranaceus and have various pharmacological activities, such as anti-inflammatory, antioxidant, neuroprotective, and anti-tumor effects, and are commonly used to treat various diseases, such as cardiovascular diseases, nervous system diseases, inflammatory diseases, and tumors.

[0003] In the quality standard of Astragalus membranaceus medicinal materials / slices in the Chinese Pharmacopoeia, the sample preparation method is "heating under reflux for 4 hours", and calycosin-7-O-β-D-glucoside is the quality control index component. Existing studies have shown that the unstable components CGM and FGM will gradually degrade into CG and FG during the storage and extraction preparation of the slices, resulting in the absence of CGM and FGM in Astragalus membranaceus preparations, but the contents of CG and FG are much higher than those in the slices, thus resulting in a transfer rate of CG and FG far higher than 100%. At the same time, CG and FG will be respectively converted into C and F under the action of intestinal bacteria in the body, and at the same time, C can also be converted into F. Therefore, only measuring the content of calycosin-7-O-β-D-glucoside in Astragalus membranaceus cannot reflect the content of isoflavone components that play a role in the body after Astragalus membranaceus enters the body. It is necessary to establish a multi-index simultaneous quantification analysis method and simplify the sample preparation method, so as to provide a more scientific and applicable quality control method for the quality evaluation of Astragalus membranaceus.

[0004] Existing studies have established a method for simultaneously determining the contents of multiple flavonoid components in Astragalus membranaceus slices / herbs, and simplified the extraction method of Astragalus membranaceus slices / herbs to ultrasonic extraction. Although there are obvious chromatographic peaks of CGM and FGM in the liquid chromatography of the samples obtained by ultrasonic extraction, due to the inability to obtain the reference substances of CGM and FGM, their contents cannot be determined, and thus the accurate quantification of the isoflavone components in Astragalus membranaceus cannot be achieved. At present, some scholars have established a "one measure for multiple evaluations" method to determine the contents of multiple isoflavone components in Astragalus membranaceus. However, due to the lack of reference substances, the relative correction factors of CGM and FGM cannot be calculated, and thus the accurate quantification of CGM and FGM cannot be carried out. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a method and use for accurately quantifying the contents of isoflavone components in Astragalus membranaceus, which can use a single reference substance to detect the contents of multiple isoflavone components including the unstable components CGM and FGM in Astragalus membranaceus, and can detect the storage years of Astragalus membranaceus based on the contents of the unstable components CGM and FGM.

[0006] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is as follows:

[0007] The object of the present invention is to provide a method for accurately quantifying the contents of isoflavone components in Astragalus membranaceus, which comprises the following steps:

[0008] (1) Prepare a reference substance solution using CG as the reference substance to obtain its standard linear equation;

[0009] (2) Crush the Astragalus membranaceus sample to be tested into powder, then carry out ultrasonic extraction, filter and centrifuge to obtain a solution to be tested;

[0010] (3) Carry out chromatographic analysis on the solution to be tested and the reference substance solution to obtain the peak areas of the isoflavones to be tested;

[0011] (4) Based on the CG standard linear equation, obtain the standard linear equations of each isoflavone component to be tested according to the molar mass coefficient method, and then obtain the contents of several isoflavones including the unstable components CGM and FGM in the sample to be tested according to the peak areas of the isoflavones to be tested.

[0012] Further, the specific process of step (2) is as follows:

[0013] S1. Crush the Astragalus membranaceus sample to be tested and pass it through a sieve, add methanol, and then carry out ultrasonic extraction at 40 - 50 kHz for 30 - 50 min at room temperature, centrifuge, and collect the filtrate and residue respectively;

[0014] S2. After washing the residue with methanol, ultrasonic extraction is carried out again under the conditions of S1, filtered, and the filtrate is combined with the filtrate obtained in S1, and then filtered through a microporous filter membrane to obtain the solution to be tested.

[0015] Furthermore, the astragalus samples to be tested are astragalus cut pieces, preparations, raw astragalus or processed astragalus.

[0016] Furthermore, when performing chromatographic analysis in step (3), the chromatographic conditions are as follows: T3 chromatographic column;

[0017] The mobile phase is 0.1% formic acid acetonitrile (A) and 0.1% formic acid water (B);

[0018] Gradient elution program: 0 - 5 min, 5% - 17% A; 5 - 7.5 min, 17% - 17% A; 7.5 - 11.5 min, 17% - 22% A; 10.5 - 14 min, 22% - 24% A; 14 - 21.5 min, 24% - 45% A; 21.5 - 24 min, 45% - 95% A; 24 - 26 min; 95% - 5% A, flow rate 0.3 mL·min -1 , injection volume 2 μL, column temperature 40 °C, detection wavelength 260 nm.

[0019] Furthermore, the standard linear equation of CG in step (1) is: y = 18911x + 3850.9;

[0020] The conversion equation for obtaining the standard linear equation of each isoflavone component to be tested is:

[0021]

[0022] Among them, M r is the molar mass of CG, M 测 is the molar mass of the isoflavone to be tested, y 测 is the peak area of the isoflavone to be tested, x 测 is the mass concentration of the isoflavone to be tested.

[0023] Furthermore, after obtaining the mass concentration of the isoflavone to be tested, its content in the astragalus sample to be tested is calculated according to the following equation:

[0024] Among them, x 测 is the mass concentration of the isoflavone to be tested, L 测 is the volume of the solution to be tested, m 测 is the mass of the astragalus sample to be tested.

[0025] Furthermore, the types of isoflavones detected in step (4) include CG, C, FG, F, CGM, and FGM.

[0026] Another object of the present invention is to provide the use of the above method in evaluating the quality of Astragalus membranaceus and / or determining the storage life of Astragalus membranaceus.

[0027] Furthermore, the quality of Astragalus membranaceus is evaluated according to the contents of six isoflavone components, namely CG, C, FG, F, CGM and FGM, in Astragalus membranaceus.

[0028] Furthermore, the storage life of Astragalus membranaceus is determined according to the content changes of the unstable components CGM and FGM in Astragalus membranaceus.

[0029] Advantages of the present invention:

[0030] 1. The detection method constructed by the present invention can accurately detect the contents of six chemical components (CG, FG, C, F, CGM and FGM) with the same chromophore in Astragalus membranaceus by using a single CG reference substance without the premise of other reference substances. In the prior art, multiple standard curve methods and multi-component determination by single marker method require reference substances for each component to be measured. Due to the lack of reference substances for CGM and FGM, only four components (CG, C, F, GF) can be quantified. It can be seen that the method established by the present invention can quantify components without reference substances, especially for the unstable components CGM and FGM for which reference substances cannot be obtained, and without measuring the relative correction factor or multiple standard curves, saving workload and improving efficiency.

[0031] 2. On the basis of ultrasonic extraction for sample preparation, the present invention has established a method for simultaneous determination of the contents of calycosin glycosides (CG, C, CGM, FG, F and FGM) in Astragalus membranaceus. This method realizes the determination of the contents of unstable components CGM and FGM without reference substances, thus providing a scientific and economical method for the accurate determination of isoflavone components in Astragalus membranaceus and its preparations, especially providing a method for quantitatively monitoring the dynamic changes of unstable components during the process of decoction pieces and preparations.

[0032] 3. The present invention modifies the sample preparation method from "heating under reflux for 4 hours" to "ultrasonic extraction for 30 minutes", and at the same time, the quantitative component changes from CG to the sum of CG + CGM quantified by CG, and the contents of other isoflavonols with similar structures are increased. The pharmacopoeia method fails to calculate the content of potential CG, that is, the content of the untransformed precursor component CGM, and partial loss of isoflavone components will occur during the sample preparation process, resulting in a low determination result of CG in Astragalus membranaceus. Based on this, the present invention modifies the sample preparation method from "heating under reflux for 4 hours" to "ultrasonic extraction for 30 minutes", greatly shortening the sample preparation time, and ultrasonic extraction is convenient for simultaneous preparation of multiple batches of samples. Therefore, the sample preparation method and quantitative method proposed by the present invention are superior to the current pharmacopoeia method and are convenient for rapid detection of a large number of samples in production practice. Description of the Drawings

[0033] Figure 1 The detection method established for the present invention is used to evaluate the quality of Astragalus membranaceus.

[0034] Figure 2 Comparison of the determination results between the detection method established for the present invention and the multi-component quantification by single marker method.

[0035] Figure 3 UPLC chromatogram of isoflavone components in Astragalus membranaceus at 260 nm; wherein, A are four reference substances; B are samples of Astragalus membranaceus extract before and after heat treatment; in the figure, 1 is CG, 2 is CGM, 3 is FG, 4 is C, 5 is FGM, 6 is F.

[0036] Figure 4 UPLC chromatogram of Astragalus membranaceus samples treated by two different extraction methods at 260 nm; wherein, A is the method of the present invention; B is the method in the pharmacopoeia; in the figure, 1 is CG, 2 is CGM, 3 is FG, 4 is C, 5 is FGM, 6 is F.

[0037] Figure 5 UPLC chromatogram of Astragalus membranaceus samples with different storage years at 260 nm, wherein, A is the determination in July 2023; B is the determination in January 2025; in the figure, 1 is CG, 2 is CGM, 3 is FG, 4 is C, 5 is FGM, 6 is F. Detailed implementation manners

[0038] The following describes the detailed implementation manners of the present invention to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the detailed implementation manners. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions created using the concept of the present invention are within the scope of protection.

[0039] Example 1

[0040] A method for accurately quantifying the content of isoflavone components in Astragalus membranaceus, which comprises the following steps:

[0041] (1) Preparation of the reference substance solution: Accurately weigh 1.0 mg of the CG reference substance, dissolve it in 1.0 mL of methanol, shake well, and prepare a reference substance solution with a concentration of 1.0 mg·mL -1 . Store it at -20 °C for later use, and dilute it to a suitable concentration with methanol before injection; meanwhile, obtain its standard linear equation: y = 18911x + 3850.9;

[0042] (2) Preparation of test solution: Powder the Astragalus membranaceus pieces shown in Table 1. Weigh accurately 1.0 g of the powder (passing through the 4th pharmacopoeia sieve) and place it in a 50 mL centrifuge tube. Add 10 mL of methanol and extract ultrasonically for 30 min (room temperature, 40 kHz), then centrifuge for 5 min (3600 r·min -1 ), filter, wash the residue with methanol twice, adding 2.5 mL of methanol each time, extract ultrasonically for 5 min, and centrifuge for 5 min (3600 r·min -1 ), filter, combine the filtrates and mix well, then centrifuge for 10 min (10000 r·min -1 ). The supernatant is filtered through a 0.22 μm microporous membrane to obtain the test solution;

[0043] Table 1 Specific information of Astragalus membranaceus pieces

[0044]

[0045]

[0046] (3) Perform chromatographic analysis on the test solution and the reference solution to obtain the peak areas of the isoflavones to be measured; the chromatographic conditions are as follows:

[0047] T3 chromatographic column (2.1 mm × 100 mm, 1.6 μm); the mobile phase is 0.1% formic acid acetonitrile (A) and 0.1% formic acid water (B); gradient elution program: 0 - 5 min, 5% - 17% A; 5 - 7.5 min, 17% - 17% A; 7.5 - 11.5 min, 17% - 22% A; 10.5 - 14 min, 22% - 24% A; 14 - 21.5 min, 24% - 45% A; 21.5 - 24 min, 45% - 95% A; 24 - 26 min; 95% - 5% A, flow rate 0.3 mL·min -1 , injection volume 2 μL, column temperature 40 °C, detection wavelength 260 nm;

[0048] (4) Based on the CG standard linear equation, according to the molar mass coefficient method, obtain the standard linear equations of each isoflavone (C, FG, F, CGM, and FGM) component to be measured through the conversion equation. The standard linear equations of each isoflavone component to be measured are shown in Table 2, and the conversion equation is as follows:

[0049]

[0050] Among them, M r is the molar mass of CG, M 测 is the molar mass of the isoflavone to be measured, y 测 is the peak area of the isoflavone to be measured, and x 测 is the mass concentration of the isoflavone to be measured;

[0051] Table 2 Standard linear equations of each isoflavone to be measured

[0052]

[0053]

[0054] (5) Substitute the peak areas of each isoflavone to be measured obtained in step (3) into the corresponding standard linear equation. After obtaining the mass concentration of the isoflavone to be measured, calculate its content in the astragalus sample to be measured according to the following equation: The results are shown in Table 3. The quality evaluation of astragalus can be achieved through the contents of the above 6 components. For example Figure 1 , it can be seen that the quality of astragalus No. 11 is significantly different from that of the other astragalus.

[0055] where x 测 is the mass concentration of the isoflavone to be measured, L 测 is the volume of the solution to be measured, m 测 is the mass of the astragalus sample to be measured.

[0056] Table 3 Isoflavone content

[0057]

[0058] Comparative Example 1 (Traditional standard curve quantitative method, ES)

[0059] Preparation of standard solution: Weigh 1.0 mg of each of the reference substances CG, C, FG, and F accurately, dissolve them in 1.0 mL of methanol, shake well, and prepare reference substance solutions with concentrations of 1.0 mg·mL -1 respectively. Store them at -20 °C for later use and dilute them to an appropriate concentration with methanol before injection.

[0060] Preparation of test solution: The same as the preparation method of the test solution in Example 1.

[0061] Chromatographic conditions: The same as the chromatographic conditions in Example 1.

[0062] Precisely pipette 2 μL of each of the test solution and the series of mixed standard solutions (CG, FG, F, C) and inject them into the high-performance liquid chromatograph for chromatographic analysis. Establish the standard curve linear equations of different components and determine the contents of four isoflavones in astragalus medicinal materials by the multi-standard curve method.

[0063] The standard curve equations of each component are shown in Table 4. The comparison of the multi-standard curve determination results between Comparative Example 1 and Example 1 is shown in Table 5. RSD% reflects the difference between the multi-standard curve determination results of Example 1 and Comparative Example 1.

[0064] Table 4 Standard curve equations of four isoflavones determined in Comparative Example 1

[0065] Component Linear equation <![CDATA[R 2 > Linear range (μg / mL) CG y = 18911x + 3850.9 0.9998 1.99-119.50 C y = 29185x + 1032.1 0.9998 1.98-59.45 FG y = 20215x + 4092.5 0.9997 1.98-118.92 F y = 33611x - 1294.8 0.9996 0.25-58.80

[0066] Table 5 Comparison of the determination results of multi-standard curve of Comparative Example 1 and the determination results of Example 1

[0067]

[0068] From the above test results, since there are no reference substances for CGM and FGM, the conventional multi-standard curve quantitative method can only quantitatively analyze 4 of the components. However, the method established in Example 1 of the present invention can simultaneously detect the contents of six chemical components (CG, C, FG, F, CGM, and FGM) with the same chromophore in Astragalus membranaceus without CGM and FGM reference substances. Compared with the determination results of the multi-standard curve method, for the contents of the four chemical components (CG, C, FG, and F) provided in Example 1 of the present invention, the obtained RSD% values are all less than 3%, indicating that the method has accurate quantitative results for the isoflavone components with the same chromophore in Astragalus membranaceus and is close to the traditional multi-standard curve quantitative results.

[0069] Comparative Example 2 (Existing method for simultaneous determination of multiple components by single marker, SSDMC)

[0070] Preparation of standard solution: The same as the preparation of the standard solution in Comparative Example 1.

[0071] Preparation of test solution: The same as the preparation of the test solution in Example 1.

[0072] Chromatographic conditions: The same as the chromatographic conditions in Example 1.

[0073] Precisely pipette 2 μL of each of the test solution and the series of mixed standard solutions (CG, C, FG, F) and inject them into a high-performance liquid chromatograph for chromatographic analysis. The contents of four isoflavone components in Astragalus membranaceus were determined by the method for simultaneous determination of multiple components by single marker.

[0074] Determination of relative correction factor: Using CG as a single reference standard, calculate the respective relative correction factors (RCF) of C, F, and FG according to the following formula:

[0075]

[0076] where k r represents the calibration curve slope of CG, and k 测 represents the calibration curve slopes of C, F, and FG.

[0077] Substitute the peak area measured by the instrument, the relative correction factors in Table 6, and the calculated mass concentration x (μg / mL) of CG into the following formula:

[0078]

[0079] Wherein A r and X r represent the peak area and mass concentration of CG respectively, and A 测 and X 测 represent the peak area and mass concentration of C, F or FG respectively, and RCF represents the relative correction factor of C, F and FG respectively (Table 6)

[0080] Then, according to the above steps, the mass concentrations X of C, F and FG are obtained 测 , and then, according to the preparation method of the test solution, multiply by the final volume of the test solution, 15 mL, and divide by 1.0 g of the original medicinal material powder to obtain the parts per thousand content of C, F and FG in the medicinal material powder, that is, the data in Table 7

[0081] Table 6 Relative correction factors (RCF) of C, F and FG

[0082] Component RCF C 0.65 FG 0.94 F 0.56

[0083] Table 7 Comparison of determination results by the multi-component determination method with single internal standard in Example 1 and Comparative Example 2

[0084]

[0085]

[0086] It can be seen from the above detection results that, like Comparative Example 1, due to the lack of reference substances for CGM and FGM, the multi-component determination method with single internal standard can only quantitatively analyze 4 of these components, while the method of the present invention can simultaneously quantify 6 components without the corresponding reference substances. Comparing with the determination results by the multi-standard curve method, the determination results of C, FG and F by the existing multi-component determination method with single internal standard and the determination results of the detection method constructed in Example 1 of the present invention are all close to the traditional multi-standard curve quantitative results, with no significant difference ( Figure 2 ).

[0087] Example 2

[0088] Source of raw materials: 1 batch of Astragalus membranaceus prepared slices purchased from Tongrentang

[0089] Preparation of standard solution: The same as the preparation of the standard solution in Example 1

[0090] Preparation of test solution 1: The same as the preparation of the test solution in Example 1

[0091] Preparation of test solution 2 (4 h): The mixed filtrate was prepared from Astragalus membranaceus prepared slices from Tongrentang according to the method of test solution 1, weighed, heated in a water bath at 85 °C for 4 hours (the bottle mouth was covered but not tightened), cooled to room temperature, and the weight loss was made up with methanol, mixed well, and centrifuged for 10 min (10000 r·min -1) The supernatant was filtered through a 0.22 μm microporous filter membrane to obtain the test solution.

[0092] Chromatographic conditions: same as those in Example 1.

[0093] Precisely pipette 2 μL of each of the test solution and the series of single standard (CG) solutions and inject them into an ultra-high performance liquid chromatograph for chromatographic analysis.

[0094] Under the above chromatographic conditions, six peaks were detected in both the astragalus extract and the extract after heating for 4 h, namely CG, C, FG, F, CGM, and FGM. Before and after heating, the contents of CGM and FGM gradually decreased, while the contents of CG and FG increased significantly. See Figure 3 . The contents of CG and FG were calculated by the external standard method, and the contents of CGM and FGM were calculated using the molar mass coefficient (MMC) method established by the research group. The contents of the four components were converted into moles. It was found that the molar contents of CGM and FGM were the highest at 0 h, and the molar contents of CG and FG increased significantly after heating for 4 h, while CGM and FGM decreased significantly. Existing studies have confirmed that CGM is unstable and degrades to CG under heating conditions. Similarly, FGM also degrades to FG. Calculate the molar number contents of CGM + CG and FGM + FG before and after heating. It was found that the total molar numbers of the two components were the same before and after heating. This result indicates that CG can be used as an internal reference to accurately quantify the contents of CGM and FGM.

[0095] Previous studies found that the contents of CGM and FGM in astragalus slices were higher than those of CG and FG, but CGM and FGM could not be detected in astragalus formula granules and had all been converted to CG and FG. To ensure the accurate calculation of the transfer rate of CG in "astragalus herbs / slices - preparations", the content of CGM was converted into the equivalent content of CG, and then the total CG content was calculated, as shown in Table 8. The results showed that the thousandth content and molar content of total CG were consistent before and after heating, further verifying the feasibility of quantifying CGM with CG. Therefore, we selected CGM, CG, FGM, FG, G, and F in astragalus slices as indicators, and used CG and FG to quantify the total amounts of CG + CGM and FGM + FG respectively, so as to quantify the content of isoflavone components in astragalus.

[0096] Table 8 Determination results of the contents of each component before and after heating

[0097]

[0098] Example 3

[0099] Source of raw materials: 1 batch of astragalus slices purchased from Tongrentang.

[0100] Preparation of the standard solution: same as the standard preparation method in Example 1.

[0101] Preparation of the test solution: The same method as that for preparing the test sample in Example 1 was used.

[0102] Chromatographic conditions: The same chromatographic conditions as those in Example 1 were used.

[0103] Precisely pipette 2 μL of each of the test solution and the series of single standard (CG) solutions and inject them into an ultra-high performance liquid chromatograph for chromatographic analysis. Quantify the four components of CG (CG + CGM), FG (FG + FGM), C, and F based on the method proposed in Example 1.

[0104] Under the above chromatographic conditions, a total of six peaks were detected, namely CG, C, FG, F, CGM, and FGM, as shown in Figure 4 A. The quantification results of the total CG (CG + CGM), total FG (FG + FGM), C, and F of the four components are shown in Table 9.

[0105] Comparative Example 3 (Pharmacopoeia method)

[0106] Source of raw materials: The same as that in Example 3.

[0107] Preparation of the standard solution: The same method as that for preparing the standard in Example 1 was used.

[0108] Preparation of the test solution (Pharmacopoeia method): Powder the Astragalus membranaceus pieces, precisely weigh 1.0 g of the powder (passed through the 4th Pharmacopoeia sieve), place it in a round-bottom flask, precisely add 50 mL of methanol, weigh, heat under reflux for 4 h, cool, weigh again, make up the lost weight with methanol, shake well, filter, precisely measure 25 mL of the subsequent filtrate, recover the solvent to dryness, dissolve the residue in methanol, transfer it to a 5 mL volumetric flask, add methanol to the scale, shake well, and obtain the solution.

[0109] Chromatographic conditions: The same chromatographic conditions as those in Example 1 were used.

[0110] Precisely pipette 2 μL of each of the test solution and the series of single standard (CG) solutions and inject them into an ultra-high performance liquid chromatograph for chromatographic analysis, and compare with the quantification results in Example 2.

[0111] Under the above chromatographic conditions, six peaks (CG, C, FG, F, CGM, and FGM) can be detected in the samples prepared by both methods, as shown in Figure 4 B. This result indicates that during the heating extraction process of the sample by the current Pharmacopoeia method, the unstable components CGM and FGM are gradually degraded into CG and FG, but not completely degraded. Therefore, the content of calycosin-7-O-β-D-glucoside determined by the method in the Chinese Pharmacopoeia cannot represent the total content of potential calycosin-7-O-β-D-glucoside in the pieces.

[0112] The contents of the four components, namely CG, FG, C, and F, determined in Comparative Example 3 were compared with the results of the total CG, total FG, G, and F in Example 3. It was found that the contents of each component in the test samples prepared by the pharmacopoeia method were lower than those in the test samples of Example 3, as shown in Table 9. This result indicates that the heating reflux extraction process not only causes the unstable components CGM and FGM to gradually degrade into CG and FG, but also causes partial degradation of G and F.

[0113] Table 9 Results of the Contents of Each Component in the Method of the Present Invention and the Pharmacopoeia Method

[0114]

[0115] Example 4

[0116] Source of raw materials: One batch of Astragalus membranaceus pieces purchased from Tongrentang was first measured in July 2023 and stored in a desiccator at room temperature in the dark in the laboratory until the second measurement in January 2025.

[0117] Preparation of standard solution: The same as the standard preparation method in Example 1.

[0118] Preparation of test solution: The same as the test solution preparation method in Example 1.

[0119] Chromatographic conditions: The same as the chromatographic conditions in Example 1.

[0120] Precisely pipette 2 μL of each of the test sample and the series of single standard (CG) solutions and inject them into an ultra-high performance liquid chromatograph for chromatographic analysis to investigate the contents of the main isoflavones in Astragalus membranaceus herbs under different storage years. The results are shown in Figure 5 and Table 10.

[0121] Table 10 Comparison of the Contents of Isoflavone Components in Tongrentang Samples under Different Storage Years

[0122]

[0123] Note: a Below the quantification limit and not determined.

[0124] According to the above test results, the detection method established by the present invention was used to quantify the main isoflavone components in the same batch of Astragalus membranaceus herbs from Tongrentang in July 2023 and January 2025 respectively. Under the above chromatographic conditions, six peaks were detected in the sample chromatogram in July 2023, namely CG, C, FG, F, CGM, and FGM, as shown in Figure 5 A; Four peaks were detected in the sample chromatogram in January 2025, namely CG, C, FG, and F, while the chromatographic peaks corresponding to CGM and FGM were already below the quantification limit, as shown in Figure 5 B.

[0125] The quantitative results in Table 10 show that the contents of CG and FG measured in January 2025 are higher than those in July 2023, and the unstable components CGM and FGM do not reach the quantification limit, indicating that the unstable components gradually degrade into CG and FG during the storage of Astragalus membranaceus, resulting in increased contents of CG and FG. The contents of C and F show no obvious change during this period, indicating that these two components are relatively stable. In summary, after one year and five months of storage, the unstable components of Astragalus membranaceus are almost completely degraded. Therefore, the storage years of Astragalus membranaceus can be roughly judged by detecting the contents of CGM and FGM in Astragalus membranaceus.

[0126] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A method for accurately quantifying the content of isoflavone components in Astragalus membranaceus, characterized in that, It includes the following steps: (1) Prepare a reference solution with CG as the reference substance and obtain its standard linear equation; (2) Crush the sample of Astragalus membranaceus to be tested into powder, then perform ultrasonic extraction, filtration and centrifugation to obtain the solution to be tested; (3) Perform chromatographic analysis on the solution to be tested and the reference solution to obtain the peak areas of the isoflavones to be tested; (4) Based on the standard linear equation of CG, calculate the standard linear equations of the various isoflavone components to be tested according to the molar mass coefficient method, and then obtain the contents of several isoflavones including the unstable components CGM and FGM in the sample to be tested based on the peak areas of the isoflavones to be tested.

2. The method according to claim 1, wherein The specific process of step (2) is as follows: S1. After crushing the sample of Astragalus membranaceus to be tested and sieving it, add methanol, and then perform ultrasonic extraction at 40 - 50 kHz for 30 - 50 min at room temperature, followed by centrifugation, and collect the filtrate and residue separately; S2. After washing the residue with methanol, perform ultrasonic extraction again under the conditions of S1, filter, combine the filtrate with the filtrate obtained in S1, and then filter with a microporous filter membrane to obtain the solution to be tested.

3. The method according to claim 2, characterized in that The sample of Astragalus membranaceus to be tested is Astragalus membranaceus cut pieces, preparations, raw Astragalus membranaceus, stir-fried Astragalus membranaceus, Astragalus membranaceus extracts and preparations.

4. The method according to claim 1, wherein When performing chromatographic analysis in step (3), the chromatographic conditions are as follows: Chromatographic column; The mobile phase is 0.1% formic acid acetonitrile (A) and 0.1% formic acid water (B); Gradient elution program: 0 - 5 min, 5% - 17% A; 5 - 7.5 min, 17% - 17% A; 7.5 - 11.5 min, 17% - 22% A; 10.5 - 14 min, 22% - 24% A; 14 - 21.5 min, 24% - 45% A; 21.5 - 24 min, 45% - 95% A; 24 - 26 min; 95% - 5% A, flow rate 0.3 mL·min -1 , injection volume 2 μL, column temperature 40 °C, detection wavelength 260 nm.

5. The method according to claim 1, characterized in that, The standard linear equation of CG in step (1) is: y = 18911x + 3850.9; The conversion equation for obtaining the standard linear equations of the various isoflavone components to be tested is: Among them, M r is the molar mass of CG, M 测 is the molar mass of the isoflavone to be measured, y 测 is the peak area of the isoflavone to be measured, x 测 is the mass concentration of the isoflavone to be measured.

6. The method according to claim 5, characterized in that After obtaining the mass concentration of the isoflavone to be measured, its content in the astragalus sample to be measured is calculated according to the following equation: where x 测 is the mass concentration of the isoflavone to be measured, L 测 is the volume of the solution to be measured, m 测 is the mass of the Astragalus membranaceus sample to be measured.

7. The method according to any one of claims 1 to 6, characterized in that The types of isoflavones detected in step (4) include CG, C, FG, F, CGM and FGM.

8. Use of the method according to any one of claims 1 - 7 in evaluating the quality of Astragalus membranaceus and / or determining the storage years of Astragalus membranaceus.

9. The use according to claim 8, characterized in that, Evaluate the quality of Astragalus membranaceus based on the contents of the six isoflavone components CG, C, FG, F, CGM and FGM in Astragalus membranaceus.

10. The use according to claim 8, characterized in that, Determine the storage years of Astragalus membranaceus based on the content changes of the unstable components CGM and FGM in Astragalus membranaceus.

Images