A method for detecting the antioxidant capacity of Radix Astragali broken pieces based on HPLC-ABTS + · Online detection method of antioxidant capacity of Radix Astragali broken pieces

Abstract

The present application belongs to the technical field of traditional Chinese medicine quality detection, and particularly relates to a method for detecting the antioxidant capacity of Radix Astragali broken wall decoction pieces based on HPLC-ABTS + The method is based on HPLC-ABTS + The analysis system is established, the regression equation of the Trolox concentration and the inverted peak area is established according to the inverted peak formed by the change of the ABTS response value after the reaction, the antioxidant capacity of the Radix Astragali broken wall decoction pieces is obtained through conversion, and the inverted peak area of each active index component is substituted into the regression equation to obtain the antioxidant capacity of each index component; the method can simply and intuitively reflect the strength of the antioxidant capacity of each active component and the total antioxidant capacity of the Radix Astragali broken wall decoction pieces, and then reflect the product quality from the antioxidant activity, and the automatic detection can be realized through the instrument setting, the operation is simple, safe and economical, and the method is suitable for mass quality detection of the Radix Astragali broken wall decoction pieces.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine quality detection. More specifically, it relates to a method for online detection of the antioxidant capacity of Radix Astragali broken wall decoction pieces based on HPLC-ABTS + ·a method for online detection of the antioxidant capacity of Radix Astragali broken wall decoction pieces. BACKGROUND

[0002] Radix Astragali has the functions of tonifying qi and raising yang, consolidating the exterior and stopping sweating, promoting water and reducing swelling, producing saliva and nourishing blood, removing stagnation and relieving obstruction, removing pus and discharging pus, and astringing and generating muscle, and is known as a qi-tonifying saint medicine. Since ancient times, there has been a saying that nine out of ten medicines are Radix Astragali. As a medicine-food homologous variety, it is widely used as a medicine, a health food, a soup base, etc. and has a large annual export and domestic sales volume, and is a bulk medicinal material. Modern pharmacological studies have shown that Radix Astragali has multiple pharmacological effects such as regulating immunity, inhibiting inflammatory factors, antiviral, reducing blood sugar, and anti-tumor. Radix Astragali broken wall decoction pieces are ultrafine powders with a particle size D 90 <45μm processed from Radix Astragali medicinal materials as raw materials by modern broken wall crushing technology, and the Radix Astragali ultrafine powders are made into 30-100 mesh uniform dry granular decoction pieces without adding any solidifying substances. Radix Astragali broken wall decoction pieces significantly improve the dissolution rate of effective components while retaining the original traditional decoction piece substances, and have the advantages of safety, high efficiency, uniformity, and convenient use, and are a new type of traditional Chinese medicine decoction piece and a safe health product popular in modern life, and have a huge market prospect.

[0003] According to the free radical theory proposed by British scholar Denham Harman in 1956, more and more studies have found that free radicals are involved in many disease processes. Free radicals generated during normal cell metabolism attack life macromolecules, causing tissue cell damage and body degenerative changes, accelerating aging, and leading to the occurrence of malignant diseases such as tumors. Studies have also shown that many traditional Chinese medicines have antioxidant capacity, and the efficacy of traditional Chinese medicines is closely related to their antioxidant effects. The active ingredients such as flavonoids, glycosides and polysaccharides contained in Huangqi have certain antioxidant activity. Therefore, there is a close correlation between the antioxidant capacity of Huangqi and its efficacy, and the antioxidant capacity of Huangqi can largely reflect the quality of Huangqi. The existing technology related to the study of the antioxidant capacity of active ingredients of Huangqi mainly focuses on the antioxidant capacity of Huangqi polysaccharides. For example, scholars such as Liang Wannian (Liang Wannian, Li Haichi, Zhong Chao, et al. Optimization of high-pressure crushing extraction process of Astragalus polysaccharides and in vitro antioxidant activity [J]. China Pharmaceutical Industry, 2022, 31 (11): 28-32.) disclosed the optimization of the high-pressure crushing extraction process of Huangqi polysaccharides, and the in vitro antioxidant activity of Huangqi polysaccharides was detected by taking the clearance capacity of 1,1-diphenyl-2-trinitrobenzene hydrazine (DPPH) and 2,2-azobis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt (ABTS) free radicals as indicators. However, this method cannot quickly determine the antioxidant capacity of multiple active ingredients in Huangqi, and it cannot simultaneously compare the antioxidant capacity differences of various active ingredients in Huangqi. In addition, since the broken wall decoction pieces of traditional Chinese medicines do not have the morphological characteristics of traditional Chinese medicine decoction pieces, the dissolution of the active ingredients or index components and other chemical components will change after being broken, so the identification and quality detection methods of traditional decoction pieces cannot be completely applied. Therefore, the method in the above prior art cannot be used to determine the antioxidant capacity of multiple active ingredients in Huangqi broken wall decoction pieces, and it cannot simultaneously compare the antioxidant capacity differences of various active ingredients in Huangqi broken wall decoction pieces. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies in the prior art that the detection method cannot quickly determine the antioxidant capacity of multiple active ingredients in Huangqi broken wall decoction pieces, and cannot simultaneously compare the antioxidant capacity differences of various active ingredients in Huangqi broken wall decoction pieces. The present application provides a method for on-line detection of the antioxidant capacity of Huangqi broken wall decoction pieces based on HPLC-ABTS + · The method for on-line detection of the antioxidant capacity of Huangqi broken wall decoction pieces based on HPLC-ABTS + · The on-line activity detection method quickly analyzes the antioxidant capacity of Huangqi broken wall decoction pieces, simultaneously reflects the spectrum-effect relationship of the internal material basis of the new type of traditional Chinese medicine decoction pieces, and screens the substances with antioxidant activity as index components, so as to quickly and effectively evaluate the quality.

[0005] The purpose of the present application is to provide the application of the method in the quality detection of Huangqi broken wall decoction pieces.

[0006] The above object of the present application is achieved by the following technical solutions.

[0007] The present application protects a kind of HPLC-ABTS + The method for on-line detection of the antioxidant capacity of Radix Astragali broken decoction includes the following steps:

[0008] S1, preparation of test solution: accurately weigh Radix Astragali broken decoction, add methanol, heat to reflux at 70-85℃ to obtain crude extract, concentrate, redissolve and filter the crude extract, and the filtrate is the test solution;

[0009] S2, HPLC-ABTS + Analysis system: confirm the HPLC chromatographic detection conditions, and ABTS + Solution and sample solution are fully reacted, the sample solution before and after reaction is determined by the HPLC chromatographic detection conditions, the sample solution after reaction forms a chromatographic inverted peak on the chromatogram, and the chromatographic inverted peak area is recorded;

[0010] S3, establishment of regression equation: Trolox solution is passed through the HPLC-ABTS + Analysis system in step S2, and a regression equation of the relationship between Trolox concentration and chromatographic inverted peak area is established;

[0011] S4, determination of antioxidant capacity of Radix Astragali broken decoction: the test solution obtained in step S1 is passed through the HPLC-ABTS + Analysis system in step S2, the chromatographic inverted peak area is obtained, substituted into the regression equation obtained in step S3, and the antioxidant capacity of Radix Astragali broken decoction is obtained by conversion;

[0012] In step S2, the chromatographic detection conditions are as follows: a reversed-phase chromatographic column is used, 0.1-0.3vol% formic acid aqueous solution is used as mobile phase A, and acetonitrile is used as mobile phase B, and detection is carried out under the following gradient elution conditions:

[0013]

[0014] In step S2, the ABTS + Solution is determined alone at any wavelength of 730-760nm, and the absorbance is 1.0. Since the absorbance of ABTS + Solution determined alone at the set detection wavelength is 1.0, the absorbance of ABTS + Solution after reaction decreases, and an inverted peak is shown in the chromatogram.

[0015] Further, in step S2, the ABTS +• The solution is measured alone at a detection wavelength of 750 nm to make the absorbance 1.0.

[0016] Preferably, the mobile phase A is 0.15 vol% aqueous formic acid and the mobile phase B is acetonitrile.

[0017] In step S2, the sample solution is ABTS + • The solution is measured alone at a detection wavelength of 750 nm to make the absorbance 1.0.

[0018] Preferably, in step S2, the detection wavelength used before the reaction is 250-260 nm.

[0019] More preferably, in step S2, the detection wavelength used before the reaction is 254 nm.

[0020] Preferably, in step S2, the detection wavelength used after the reaction is 730-760 nm. The detection wavelength used after the reaction is the same as the wavelength used for measuring the ABTS + • The wavelength used for measuring the solution alone to make the absorbance 1.0 is the same as the wavelength used for measuring the solution.

[0021] More preferably, in step S2, the detection wavelength used after the reaction is 750 nm.

[0022] Preferably, in step S1, the mass-volume ratio of the Astragalus membranaceus broken cell pieces to methanol is 1:3-3.5 (g / mL).

[0023] More preferably, in step S1, the mass-volume ratio of the Astragalus membranaceus broken cell pieces to methanol is 1:3 (g / mL).

[0024] Preferably, in step S2, the column temperature for detection is 25-30°C.

[0025] Preferably, in step S1, the heating reflux time is 4 h. The sample peak shape is better after 4 h of reflux extraction. The effective component content is lower than 4 h, and the effective component content changes little more than 4 h, so the heating reflux time is 4 h.

[0026] Preferably, the filtration is performed using an organic filter membrane with a pore size of 0.2 μm.

[0027] Preferably, in step S2, the reverse-phase chromatographic column is Capcell Pak MG-C 18 .

[0028] Preferably, in step S2, the injection volume for detection is 10-20 μl.

[0029] Preferably, in step S2, the flow rate of the mobile phase for detection is 0.5-0.7 ml / min.

[0030] More preferably, in step S2, the flow rate of the detected mobile phase is 0.6 ml / min.

[0031] Preferably, in step S2, the ABTS + solution enters the HPLC-ABTS + system at a flow rate of 0.2-0.4 ml / min.

[0032] Preferably, in step S2, the ABTS + solution enters the HPLC-ABTS + system at a flow rate of 0.3 ml / min.

[0033] Further, the ABTS + solution is prepared as follows: 2,2-azino-di(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt 7 mM is dissolved in pure water and made up to 50 ml in a brown volumetric flask, and potassium persulfate 5 mM is dissolved in pure water and made up to 50 ml in a brown volumetric flask, and the two are mixed in equal volumes (1:1, v / v) and reacted at 4°C in the dark for 12 h. The solution is diluted with anhydrous ethanol to an absorbance of 1.0 at any one of the wavelengths of 730-760 nm determined separately before use.

[0034] Specifically, in step S2, the HPLC-ABTS + system is as shown in Figure 1 , and the specific analysis method is as shown in Figure 1 : the mobile phase is pumped into the pipeline by pump 1 at a flow rate of 0.5-0.7 ml / min, and the ABTS + solution is pumped into the pipeline by pump 2 at a flow rate of 0.2-0.4 ml / min, while the separated substances from the sample solution after entering the sample injector and passing through the high-performance liquid chromatography column are discharged from the UV detector 1 (i.e., UV1, with a detection wavelength of 250-260 nm) and enter another pipeline, and the ABTS + solution is mixed with the separated substances by a three-way joint and enters the reaction coil, and the sample solution and the ABTS + solution fully react at the position of the reaction coil, and the reaction solution is discharged into the UV detector 2 (i.e., UV2, with a detection wavelength of 730-760 nm). Since the ABTS + solution has an absorbance of 1.0 at the set detection wavelength, the absorbance of the ABTS + solution after the reaction decreases, and an inverted peak is shown in the chromatogram.

[0035] Specifically, in step S4, the establishment of the regression equation specifically comprises the following steps: 20 mg of Trolox powder is placed in a 100 ml volumetric flask, and methanol solution is added to the calibration mark and shaken uniformly; 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 7.5 ml and 10 ml of standard solution are accurately measured and placed in 20 ml volumetric flasks, and methanol solution is added to the calibration mark and shaken uniformly for standby. ABTS + · As the Trolox solution, a series of concentration gradients of Trolox standard solution are passed through HPLC-ABTS + · Analysis system, get Trolox solution and ABTS + · The inverted peak area after the solution reaction, taking the inverted peak area as the vertical coordinate and the Trolox solution concentration as the horizontal coordinate, draws a standard curve to obtain the regression equation of the relationship between the Trolox concentration and the inverted peak area.

[0036] Further, in step S4, the determination of the antioxidant capacity of the Astragalus membranaceus broken wall decoction piece specifically comprises the following steps: 1×10 -3 mg / ml Trolox solution corresponding to the inverted peak area as a unit of activity (u), and the test product solution is injected into the above HPLC-ABTS + · Analysis system, the total inverted peak area obtained is substituted into the regression equation of the relationship between the Trolox concentration and the inverted peak area to obtain the number of units of activity corresponding to the total inverted peak area of each active ingredient as the antioxidant capacity of the Astragalus membranaceus broken wall decoction piece. At the same time, the inverted peak area of each active index component is substituted into the above regression equation to obtain the antioxidant capacity of each index component.

[0037] The application also protects the application of the method in the quality detection of the Astragalus membranaceus broken wall decoction piece.

[0038] The application has the following beneficial effects:

[0039] The application discloses a method for online detection of the antioxidant capacity of an Astragalus membranaceus broken wall decoction piece, which is based on high performance liquid chromatography combined with 2,2-diazene-di(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt technology (namely HPLC-ABTS +· Analysis system is established), according to the change of ABTS response value after reaction, the inverse peak is formed, the regression equation of Trolox concentration and inverse peak area relationship is established, and the number of activity units corresponding to the total area of each active ingredient inverse peak is calculated as the antioxidant capacity of Radix Astragali broken pieces, and each active index component inverse peak area is substituted into the above regression equation, and the antioxidant capacity of each index component is calculated; this method can simply and intuitively reflect the antioxidant capacity of each active ingredient and the total antioxidant capacity of Radix Astragali broken pieces, and then reflect the product quality from the aspect of antioxidant activity, and automatic detection can be realized through instrument setting, which is simple, safe and economical, and is suitable for large-scale quality detection of Radix Astragali broken pieces. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 HPLC-ABTS + · Analysis system schematic diagram.

[0041] Figure 2 Data statistical graph of the regression equation of Trolox concentration and inverse peak area in Example 1.

[0042] Figure 3 HPLC-ABTS + · Online analysis chromatogram.

[0043] Figure 4 HPLC-ABTS + · Online analysis chromatogram.

[0044] Figure 5 HPLC-ABTS + · Online analysis chromatogram.

[0045] Figure 6 HPLC-ABTS + · Online analysis chromatogram.

[0046] Figure 7 HPLC-ABTS + · Online analysis chromatogram.

[0047] Figure 8 HPLC-ABTS + · Online analysis chromatogram. DETAILED DESCRIPTION

[0048] The present application is further described in conjunction with the accompanying drawings and specific examples, which do not in any way limit the scope of the present application. Unless otherwise specified, the reagents, methods and apparatus used in the following examples are those conventional in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Trolox powder: Aladdin, Lot#k2225823;

[0051] Radix Astragali broken wall decoction pieces: Zhongzhi Chinese Herbal Pieces Co., Ltd. of Zhongshan City, 2211022A, 2211042A 2211062A 2211082A, 2211102A, 2211122A;

[0052] Calycosin: China Institute for Food and Drug Control, 111703-201504.

[0053] Calycosin: China Institute for Food and Drug Control, 111703-201504.

[0054] Calycosin: China Institute for Food and Drug Control, 111703-201504.

[0055] Calycosin: China Institute for Food and Drug Control, 111703-201504.

[0056] Example 1 HPLC-ABTS + · Online detection method of antioxidant capacity of Radix Astragali broken wall decoction pieces

[0057] (1) Preparation of the sample to be tested: 50 g of Radix Astragali broken wall decoction pieces was accurately weighed, 150 ml of methanol was added, and it was refluxed in a 70°C water bath for 4 hours. The crude extract was filtered and placed in an evaporating dish to evaporate to dryness. 10 ml of methanol was used to dissolve the residue, and the concentrated solution was filtered through an organic filter membrane to obtain the sample to be tested.

[0058] (2) ABTS + · Preparation of solvent: 7 mM of 2,2-azino-bis(3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt was dissolved and diluted with pure water to 50 ml in a brown volumetric flask. 5 mM of potassium persulfate was dissolved and diluted with pure water to 50 ml in a brown volumetric flask. They were mixed in equal volumes (1:1, v / v) and reacted at 4°C in the dark for 12 h. Before use, the solution was diluted with anhydrous ethanol to an absorbance of 1.0 at a wavelength of 750 nm.

[0059] (3) Preparation of Trolox (water-soluble vitamin E) standard solution: Take 20 mg of Trolox powder into a 100 ml volumetric flask, add methanol solution to the mark, shake well; accurately take 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 7.5 ml, 10 ml of the standard solution, respectively, into a 20 ml volumetric flask, add methanol solution to the mark, shake well and reserve for use.

[0060] (4) Preparation of control solution: accurately weigh appropriate amounts of calycosin-7-glucoside, ononin, calycosin and formononetin control substances, respectively, dissolve and quantitatively dilute with methanol to prepare four control solutions with concentrations of 0.1805 mg / ml, 0.9049 mg / ml, 0.8374 mg / ml and 0.9617 mg / ml, respectively.

[0061] (5) Chromatographic conditions: Capcell Pak MG-C 18 chromatographic column, and the column temperature was kept at 25℃ by a column oven. The mobile phase was 0.15% formic acid aqueous solution (A) and acetonitrile (B), and the gradient setting of the mobile phase is shown in Table 1:

[0062] Table 1 Gradient elution conditions

[0063]

[0064] The flow rate of the mobile phase was 0.6 ml / min, and ABTS + · The flow rate was 0.3 ml / min; the detection wavelength of UV1 was 254 nm, and the detection wavelength of UV2 was 750 nm.

[0065] (6) HPLC-ABTS + · Analysis system construction: as shown in Figure 1 , the mobile phase was pumped into the pipeline by pump 1 at a flow rate of 0.6 ml / min, 22.5 ml of ABTS + solution was pumped into the pipeline by pump 2 at a flow rate of 0.3 ml / min, at the same time, 10 μl of sample solution (which can be the above-mentioned sample to be tested, Trolox standard solution or control solution) was injected from the sample injector, and the separated substances after passing through the high-performance liquid chromatographic column were discharged from UV detector 1, entered another pipeline, and mixed with ABTS + and the separated substances through a three-way joint and entered the reaction coil, and the reaction solution after reaction was passed into UV detector 2. Since the ABTS + solution was set to have an absorbance of 1.0 at 750 nm, the absorbance of the ABTS + solution after reaction decreased, and an inverted peak was presented in the chromatogram.

[0066] (7) Regression equation of Trolox concentration and inverted peak area: Inject the standard solution prepared in step (3) into the HPLC-ABTS in step (6) as sample solution + · Analysis system, and the regression equation of Trolox concentration and inverted peak area was Y=0.0092x-3.9281, R + 2 =0.9999 (see Figure 2 ).

[0067] (8) Detection of total antioxidant capacity of Astragalus membranaceus broken pieces: Inject the sample prepared in step (1) into the HPLC-ABTS in step (6) as sample solution + · Analysis system, and the regression equation of Trolox concentration and inverted peak area was Y=0.0092x-3.9281, R -3 mg / ml Trolox solution as one unit of activity (u)), and the total antioxidant capacity of Astragalus membranaceus broken pieces was obtained. The results are shown in Tables 2 and 3.

[0068] Table 2 Determination results of inverted peak area of Trolox standard solution

[0069]

[0070] Table 3 Calculation results of antioxidant capacity of Astragalus membranaceus broken pieces of different batches

[0071]

[0072] (9) Detection of antioxidant capacity of four active ingredients: Inject the four control samples prepared in step (4) into the HPLC-ABTS in step (6) as sample solution + · Analysis system, and the regression equation of Trolox concentration and inverted peak area was Y=0.0092x-3.9281, R + · Online analysis of chromatogram to determine the peaks of calycosin-7-glucoside, ononin, calycosin and formononetin in the sample (see Figure 3 ). Taking one sample of Astragalus membranaceus broken pieces as an example, step (8) was repeated to convert the number of units of activity corresponding to the inverted peak area of the four active ingredients in the sample, and the antioxidant capacity of the four active ingredients in Astragalus membranaceus broken pieces was obtained. The results are shown in Table 4.

[0073] ​Table 4 calculation results of antioxidant capacity of four active ingredients in Radix Astragali broken wall decoction pieces

[0074]

[0075] It can be seen that in the detection process of the total antioxidant capacity of Radix Astragali broken wall decoction pieces, the chromatogram of each component is analyzed, not only the antioxidant capacity between each active ingredient can be compared, but also the antioxidant capacity difference between different batches of samples can be quickly compared, and the specific components causing the difference can be determined, which can provide a comprehensive and efficient quality evaluation for Radix Astragali broken wall decoction pieces.

[0076] Example 2 selection of sample weight

[0077] The difference between example 2 and example 1 is that in step (1), the weight of Radix Astragali broken wall decoction pieces is 40g or 60g.

[0078] Other steps and parameters refer to example 1.

[0079] ABTS is added to the sample + The chromatogram before and after the solution reaction is shown in Figure 4 and Figure 5 .

[0080] The results show that the broken wall decoction pieces of 50g are extracted by methanol, and the chromatogram obtained by HPLC-ABTS + · system analysis has good peak shape and separation degree Figure 3 , which can maximize the peak area within the limit of instrument overload, and improve the accuracy of the experiment; when the weight of Radix Astragali broken wall decoction pieces is 40g, the sample amount is small, the baseline of the inverted peak is not stable, the integration is difficult, and finally the determination of the chromatographic inverted peak area is affected, which is not conducive to the accurate quantification of the subsequent antioxidant capacity; when the weight of Radix Astragali broken wall decoction pieces is 60g, the sample amount is too large, and the highest part of the chromatogram before the reaction with ABTS + · solution shows multiple peak tops, which may be that the substances are not separated, resulting in that the inverted peak after oxidation reaction cannot be completely determined as the reaction of only one kind of substance, which is not conducive to the accurate calculation of the antioxidant capacity of single component.

[0081] Example 3 selection of gradient elution conditions

[0082] The difference between example 3 and example 1 is that in step (5), the gradient elution conditions are different, and the gradient elution conditions are shown in table 5 and table 6.

[0083] Table 5 gradient elution conditions-85min

[0084]

[0085] Table 6 Gradient elution conditions-60min

[0086]

[0087] Other steps and parameters refer to Example 1.

[0088] The results of the chromatogram of the sample determined are compared with the results of the chromatogram obtained by the method of Example 1, and the results are shown in Table 3. Figure 6

[0089] The results show that, in step (5), when the gradient elution conditions are as shown in Table 5 and Table 6, the sample activity peak of the obtained chromatogram is too concentrated, resulting in poor inverted peak shape, unstable baseline, or no obvious sample peak in the chromatogram, which all affect the calculation of the inverted peak area in the subsequent integration and are not conducive to the accurate quantification of the subsequent antioxidant capacity.

[0090] Example 4 ABTS + Selection of flow rate

[0091] Example 4 differs from Example 1 in that, in step (5), the ABTS + flow rate is 0.2, 0.4, 0.5 or 0.8 ml / min.

[0092] Other steps and parameters refer to Example 1.

[0093] The results of the chromatogram of the sample determined are compared with the results of the chromatogram obtained by the method of Example 1, and the results are shown in Table 3. Figure 7

[0094] The results show that, when the ABTS + flow rate is 0.2-0.4 ml / min, there are relatively obvious inverted peaks, the baseline fluctuation before and after the reaction is small, and the inverted peak shape is complete, which can all solve the technical problems of the present application, wherein the ABTS + flow rate is 0.3 ml / min, the peak shape is the best, and the baseline is the most stable, which is the best solution; when the ABTS + flow rate is 0.5 or 0.8 ml / min, the flow rate is too fast, resulting in that the sample is not reacted in time but is detected, which is not conducive to the accurate quantification of the subsequent antioxidant capacity.

[0095] Example 5 ABTS + Selection of mobile phase

[0096] Example 5 differs from Example 1 in that, in step (5), the mobile phase uses 0.15 vol% aqueous phosphoric acid (mobile phase A) and acetonitrile (mobile phase B); or the mobile phase uses water (A) and methanol (B); or the mobile phase uses water (mobile phase A) and acetonitrile (mobile phase B); ​​

[0097] Other steps and parameters refer to Example 1.

[0098] The results of the chromatogram of the sample determined are shown in Figure 8 .

[0099] The results show that when the mobile phase is water (mobile phase A) and methanol (mobile phase B); or the mobile phase is water (mobile phase A) and acetonitrile (mobile phase B), the baseline float before and after the reaction has a large drift, and there are many impurity peaks, which is not conducive to the accurate quantification of the antioxidant capacity of the subsequent active ingredients. Similarly, when the mobile phase is 0.15 vol% aqueous phosphoric acid (A) and acetonitrile (B), the baseline float before and after the reaction has a large drift, and there are many impurity peaks, which also cannot realize the accurate quantification of the antioxidant capacity of the subsequent active ingredients.

[0100] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for determining the antioxidant capacity of a sample based on HPLC-ABTS + • The method for detecting the antioxidant capacity of Radix Astragali broken cell pieces on line, characterized in that, It comprises the following steps: S1, preparation of test sample solution: precisely weigh the Astragalus membranaceus broken decoction pieces, add methanol, heat to reflux at 70-85°C to obtain a crude extract, concentrate, redissolve, filter, and take the filtrate to obtain the test sample solution; S2, HPLC-ABTS + • Construction of the analysis system: confirm the HPLC chromatographic detection conditions, determine the sample solution before and after reaction with ABTS + • The solution reacts with the sample solution, the sample solution before and after reaction is determined under the HPLC chromatographic detection conditions, the sample solution after reaction forms a chromatographic inverted peak on the chromatogram, and the chromatographic inverted peak area is recorded; S3, Establishment of regression equation: Trolox solution is passed through the HPLC-ABTS column as described in step S2 + • Analysis system, establish the regression equation of the relationship between Trolox concentration and chromatographic inverted peak area; S4, determination of the antioxidant capacity of the Astragalus membranaceus broken pieces: the test solution obtained in step S1 was subjected to HPLC-ABTS + • analysis system, obtain the chromatographic inverted peak area, substitute it into the regression equation obtained in step S3, and convert to obtain the antioxidant capacity of the Astragalus membranaceus broken pieces; In step S2, the conditions for chromatographic detection are as follows: a reversed-phase chromatographic column is used, 0.1-0.3 vol% formic acid aqueous solution is used as mobile phase A, acetonitrile is used as mobile phase B, and detection is performed under the following gradient elution conditions: In step S3, the ABTS + • The solution is measured alone at a detection wavelength of 730-760 nm, and the absorbance is 1.

0. In step S2, the detection wavelength before reaction is 250-260 nm; In step S2, the detection wavelength after reaction is 730-760 nm; In step S2, the reversed-phase chromatographic column is Capcell Pak MG-C 18 .

2. The method of claim 1, wherein, In step S1, the mass-volume ratio of Astragalus membranaceus broken decoction pieces to methanol is 1 g:(3-3.5) mL.

3. The method of claim 1, wherein, In step S3, the column temperature for detection is 25-30°C.

4. The method of claim 1, wherein, In step S2, the injection volume for detection is 10-20 μl.

5. The method of claim 1, wherein, In step S2, the flow rate of mobile phase for detection is 0.5-0.7 ml / min.

6. The method of claim 1, wherein, In step S2, the ABTS + • Solution enters HPLC-ABTS + • The flow rate of the analysis system is 0.2~0.4 ml / min.

7. Use of the method of any one of claims 1-6 in the quality detection of Astragalus membranaceus broken decoction pieces.

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