One-test multi-evaluation method for the determination of flavonoid components in fresh fruits of Abelmoschus esculentus

Abstract

The present invention discloses a method for multi-component determination of flavonoid components in young fruits of Abelmoschus esculentus, belonging to the technical field of traditional Chinese medicine detection. Using isoquercitrin as an internal reference substance, by calculating the relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside, the contents of 4 flavonoid components are detected quickly and accurately, solving the problems that reference substances of the other three components except isoquercitrin are difficult to obtain and expensive, resulting in high detection costs; solving the problem that the solution state of quercetin-3-O-(6”-O-malonyl)-glucoside is unstable and easily converted into isoquercitrin, resulting in inaccurate content determination; solving the problem of changes in the content of flavonoid components caused by traditional drying methods, and being able to accurately reveal the true situation of flavonoid components in young fruits of Abelmoschus esculentus.

Description

Technical Field

[0001] The invention relates to a one-test-multiple-evaluation method for determining the content of flavonoid components in young okra fruits, and belongs to the technical field of traditional Chinese medicine detection. Background Art

[0002] Okra, also known as okra, coffee okra, lady's finger, and coffee okra, is an annual herbaceous plant in the Malvaceae family. Okra is a edible and medicinal plant rich in flavonoids, which exhibit various biological activities, including antioxidant, anti-inflammatory, and anti-tumor properties. Detection of flavonoids in okra can provide a deeper understanding of the types, contents, and potential biological activities of these compounds, providing a scientific basis for research on okra's pharmacological and health-promoting properties.

[0003] Flavonoids, in particular, have attracted significant attention in the fields of health foods, pharmaceuticals, and cosmetics due to their unique biological activities. Therefore, determining the types and contents of flavonoids in okra will contribute to the industrial development of okra, promote improvements in its cultivation and processing techniques, facilitate the selection of okra varieties with higher flavonoid content and superior quality, and provide cultivation recommendations for farmers. Furthermore, this will help optimize okra processing, better preserve flavonoids, and increase the product's added value. Therefore, determining flavonoids in okra is crucial for gaining a deeper understanding of its pharmacological effects, evaluating its quality, promoting its industrial development, and improving cultivation and processing techniques.

[0004] Currently, methods for detecting flavonoids in okra require the preparation of multiple reference substances, resulting in high testing costs. Furthermore, the inventors discovered during their research that quercetin-3-O-(6-"-O-malonyl)-glucoside is unstable in solution and easily converted into isoquercetin, leading to inaccurate test results. Furthermore, when preparing the test solution, dried okra must first be obtained. The currently used drying method can cause changes in the flavonoid content and cannot accurately reveal the true content of the flavonoid composition.

[0005] It should be noted that the above content falls within the technical knowledge of the inventor and does not necessarily constitute prior art. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a one-test-multiple-evaluation method for determining the content of flavonoid components in young okra fruits, which reduces the detection cost, improves the detection efficiency, and can accurately reveal the true situation of the flavonoid components in young okra fruits.

[0007] The present invention achieves the above-mentioned purpose by adopting the following technical solutions:

[0008] A method for simultaneous determination of multiple flavonoid components in young fruits of Abelmoschus esculentus (L.) Moench, comprising the following steps:

[0009] S1. Determine the chromatographic conditions for high performance liquid chromatography analysis:

[0010] The chromatographic column is packed with octadecylsilyl silica gel; acetonitrile is used as mobile phase A, and 0.2% phosphoric acid solution is used as mobile phase B for gradient elution; the detection wavelength is 258 nm, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL;

[0011] S2. Prepare a series of reference substance solutions, including a series of mixed reference substance solutions of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin, and a series of reference substance solutions of quercetin-3-O-(6”-O-malonyl)-glucoside:

[0012] S3. Determine the relative correction factors:

[0013] S3.1 Respectively absorb the series of mixed reference substance solutions and the series of reference substance solutions of quercetin-3-O-(6”-O-malonyl)-glucoside, inject and analyze them successively according to the chromatographic conditions in step S1, record the chromatographic peak areas, take the peak areas as the ordinate and the concentrations of the reference substance solutions as the abscissa, plot the standard curves, and obtain the linear regression equations of each component to be measured;

[0014] Among them, the reference substance solution of quercetin-3-O-(6”-O-malonyl)-glucoside is used within 12 h after preparation;

[0015] S3.2 Using isoquercitrin as the internal reference substance, calculate the relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside according to the linear regression equations;

[0016] S4. Prepare the test solution:

[0017] S4.1 Take the young fruits of Abelmoschus esculentus (L.) Moench, and make them into dried medicinal materials of Abelmoschus esculentus (L.) Moench by vacuum freeze-drying;

[0018] S4.2 Pulverize the dried medicinal materials of Abelmoschus esculentus (L.) Moench to obtain powder, sieve it, take 1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol, weigh it, ultrasonically treat it for 30 min, take it out, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake well, filter, and take the subsequent filtrate to obtain the test solution;

[0019] S5. Determine the contents of the components to be measured in the test solution:

[0020] Absorb the test sample solution and the mixed reference substance solution, perform high-performance liquid chromatography analysis according to the chromatographic conditions in step S1. Locate each chromatographic peak based on the relative retention time of each component to be measured relative to isoquercitrin. Using the concentration and peak area of the isoquercitrin reference substance solution as a reference, calculate the contents of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside in the test sample solution respectively according to the relative correction factor determined in step S3;

[0021] Among them, the test sample solution is used within 24 hours after preparation.

[0022] Optionally, in step S1, the specific program of gradient elution is: 0 - 15 min: 5% A, 95% B; 15 - 35 min: 5% - 25% A, 95% - 75% B; 35 - 45 min: 25% - 50% A, 75% - 50% B.

[0023] Optionally, in step S2, the preparation method of the series of mixed reference substance solutions of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin includes the following steps:

[0024] Take appropriate amounts of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin reference substances, and add 70% methanol to make a mixed reference substance stock solution containing 208.544 μg of quercetin-3-O-gentiobioside, 91.532 μg of quercetin-3-O-xylose-(1-2)-glucose, and 152.829 μg of isoquercitrin per 1 mL;

[0025] Using the stepwise dilution method, accurately pipette the mixed reference substance stock solution of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin, and dilute it with 70% methanol solution to prepare a series of mixed reference substance solutions with dilutions of 1, 2.5, 5, 10, 20, and 40 times respectively.

[0026] Optionally, in step S2, the preparation method of the series of quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solutions includes the following steps:

[0027] Accurately weigh the quercetin-3-O-(6”-O-malonyl)-glucoside reference substance, and add 70% methanol to make a reference substance stock solution containing 100.352 μg of quercetin-3-O-(6”-O-malonyl)-glucoside per 1 mL;

[0028] Precisely pipette the reference substance stock solution of quercetin-3-O-(6”-O-malonyl)-glucoside, and dilute it with 70% methanol solution to prepare a series of reference substance solutions of quercetin-3-O-(6”-O-malonyl)-glucoside diluted 1, 2, 4, 8, 16, and 36 times respectively.

[0029] Optionally, in step S3.2, the relative correction factor f i / s is calculated by the formula f i / s = A s C i / (A i C s) .

[0030] Wherein, s is the internal reference substance isoquercitrin, i is the component to be measured, A s is the peak area of the internal reference substance reference, C s is the concentration of the internal reference substance reference, A i is the peak area of the reference substance of the component to be measured, C i is the concentration of the reference substance of the component to be measured.

[0031] Optionally, in step S3.2, the relative correction factor of quercetin-3-O-gentiobioside is 1.60, the relative correction factor of quercetin-3-O-xylose-(1-2)-glucose is 1.42, and the relative correction factor of quercetin-3-O-(6”-O-malonyl)-glucoside is 1.32.

[0032] Optionally, in step S4.1, the temperature of vacuum freeze-drying is -20°C, the vacuum degree is -0.1 Mpa, and the drying time is 72 h.

[0033] Optionally, in step S4.2, the power of ultrasonic treatment is 300 W, and the frequency is 40 KHz.

[0034] Optionally, in step S5, the relative retention times of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside are 0.914, 0.931, and 1.042 respectively.

[0035] The beneficial effects of this application include but are not limited to:

[0036] The method for simultaneous determination of multiple components of flavonoids in young fruits of Abelmoschus esculentus provided by the present invention realizes the content determination of 4 flavonoid components including quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin, and quercetin-3-O-(6”-O-malonyl)-glucoside, which is convenient for evaluating the quality and medicinal efficacy of Abelmoschus esculentus.

[0037] Specifically, in the present invention, isoquercitrin is used as an internal reference substance, and the relative retention times and relative correction factors of the other three components to be measured and isoquercitrin are calculated to quickly and accurately detect the contents of four flavonoid components, solving the problems that reference substances of the other three components to be measured except isoquercitrin are difficult to obtain and expensive, resulting in high detection costs, reducing the detection costs and improving the detection efficiency. Secondly, it solves the problem that the solution state of quercetin-3-O-(6”-O-malonyl)-glucoside is unstable and easily converted into isoquercitrin, resulting in inaccurate content determination. At the same time, the present invention improves the preparation method of the test solution, solves the problem of the change in the content of flavonoid components caused by the traditional drying method, and can accurately reveal the true situation of flavonoid components in the young fruits of okra. Description of the Drawings

[0038] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0039] Figure 1 are the chromatographic peak attribution of flavonoid components in okra, where 1 is quercetin-3-O-gentiobioside; 2 is quercetin-3-O-xylose-(1-2)-glucose; 3 is isoquercitrin; 4 is quercetin-3-O-(6”-O-malonyl)-glucoside;

[0040] Figure 2 is the chromatogram for investigating the solution stability of quercetin-3-O-(6”-O-malonyl)-glucoside. Detailed Embodiments

[0041] The present invention will be further described in detail below. However, it should be noted that the following specific embodiments only give specific operation examples of the present invention in an exemplary manner, but the protection scope of the present invention is not limited thereto. The protection scope of the present invention is only defined by the claims. Those skilled in the art can obviously think that various other improvements and substitutions can be made to the embodiments described in the present invention within the protection scope defined by the claims of the present invention, and still be able to achieve the same technical effects and reach the ultimate technical purpose of the present invention.

[0042] Unless otherwise specified, the raw materials in this specification are purchased through commercial channels, and the raw materials and instruments used are as follows:

[0043] I. Instruments and Reagents

[0044] 1.1 Instruments

[0045] Agilent 1260 high performance liquid chromatography, Agilent Technologies Co., Ltd.; BSA224S-CW electronic balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; TS8606 freeze dryer, FVICO Co., Ltd.; XS105 1 / 100,000 electronic balance, Mettler Toledo Technology (China) Co., Ltd.; KQ-800VSW dual-frequency silent ultrasonic cleaner, Kunshan Ultrasonic Instruments Co., Ltd.

[0046] 1.2 Reagents and test drugs

[0047] Quercetin-3-O-gentiobioside reference substance (batch number 080100-202309, content 98%, Shanghai Hongyong Biotechnology Co., Ltd.); Quercetin-3-O-xylose-(1-2)-glucose (batch number DSTDH038201, content 98%, Lemeitian Medicine | Dest Bio); Isoquercitrin (batch number 111809-202205, content 96.3%, National Institutes for Food and Drug Control); Quercetin-3-O-(6”-O-malonyl)-glucoside (batch number DSTDH043701, content 98%, Lemeitian Medicine | Dest Bio).

[0048] Methanol, acetonitrile, and phosphoric acid are chromatographically pure, and water is ultrapure water.

[0049] 15 batches of okra, which are the tender fruits of Abelmoschus esculentus Moench, a plant of the genus Abelmoschus in the family Malvaceae.

[0050] II. Determination method

[0051] The multi-component determination method for flavonoid components in the tender fruits of okra provided by the present invention includes the following steps:

[0052] S1. Determine the chromatographic conditions of high performance liquid chromatography analysis:

[0053] The chromatographic column is packed with octadecylsilane-bonded silica gel, specifically using an Agilent ZORBAX SB-C18 (4.6×250 mm, 5 μm) chromatographic column; acetonitrile is used as mobile phase A, and 0.2% phosphoric acid solution is used as mobile phase B for gradient elution; the detection wavelength is 258 nm, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL;

[0054] The specific conditions for gradient elution are as follows:

[0055] 0 - 15 min: 5% A, 95% B; 15 - 35 min: 5% - 25% A, 95% - 75% B; 35 - 45 min: 25% - 50% A, 75% - 50% B.

[0056] S2. Prepare the reference substance solution:

[0057] S2.1 Preparation of the mixed reference stock solution of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose and isoquercitrin:

[0058] Accurately weigh appropriate amounts of the reference substances of quercetin-3-O-gentiobioside (HT-1), quercetin-3-O-xylose-(1-2)-glucose (HT-2), and isoquercitrin (HT3). Add 70% methanol to prepare a mixed reference stock solution containing 208.544 μg of quercetin-3-O-gentiobioside, 91.532 μg of quercetin-3-O-xylose-(1-2)-glucose, and 152.829 μg of isoquercitrin per 1 mL;

[0059] Using the stepwise dilution method, accurately pipette the mixed reference stock solution of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin, and add 70% methanol solution to dilute it into a series of mixed reference solutions with dilution factors of 1, 2.5, 5, 10, 20, and 40 times respectively;

[0060] S2.2 Preparation of the reference solution of quercetin-3-O-(6”-O-malonyl)-glucoside:

[0061] Accurately weigh the reference substance of quercetin-3-O-(6”-O-malonyl)-glucoside (HT-4). Add 70% methanol to prepare a reference stock solution containing 100.352 μg of quercetin-3-O-(6”-O-malonyl)-glucoside per 1 mL;

[0062] Accurately pipette the reference stock solution of quercetin-3-O-(6”-O-malonyl)-glucoside, and add 70% methanol solution to dilute it into a series of reference solutions of quercetin-3-O-(6”-O-malonyl)-glucoside with dilution factors of 1, 2, 4, 8, 16, and 36 times respectively.

[0063] S3. Determine the relative correction factor:

[0064] Respectively pipette the series of mixed reference solutions and the series of reference solutions of quercetin-3-O-(6”-O-malonyl)-glucoside, and inject them for analysis in sequence according to the chromatographic conditions in step S1. Record the chromatographic peak areas. Taking the peak areas as the ordinate (Y) and the concentrations of the reference solutions as the abscissa (X), plot the standard curves to obtain the linear regression equations and correlation coefficients of each component to be measured. The results are shown in Table 1.

[0065] Among them, the reference solution of quercetin-3-O-(6”-O-malonyl)-glucoside should be used within 12 hours after preparation.

[0066] Table 1 Regression equations, correlation coefficients and linear ranges

[0067]

[0068] The results in Table 1 show that the linear relationships between the peak areas and concentrations of the components to be measured are good within the corresponding concentration ranges.

[0069] S3.2 Using isoquercitrin as the internal reference substance, calculate the relative correction factors f of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside according to the linear regression equation i / s , and the results are shown in Table 2.

[0070] The calculation formula for the relative correction factor is f i / s = A s C i / (A i C s) .

[0071] Among them, s is the internal reference substance isoquercitrin, i is the component to be measured, A s is the peak area of the internal reference substance reference, C s is the concentration of the internal reference substance reference, A i is the peak area of the component to be measured reference, and C i is the concentration of the component to be measured reference.

[0072] Specifically, the average value of the relative correction factor of quercetin-3-O-gentiobioside is 1.60, the average value of the relative correction factor of quercetin-3-O-xylose-(1-2)-glucose is 1.42, and the average value of the relative correction factor of quercetin-3-O-(6”-O-malonyl)-glucoside is 1.32.

[0073] Table 2 Relative correction factor f i / s Calculation results

[0074]

[0075] S4. Preparation of the test solution:

[0076] S4.1 Take the young fruits of okra and use vacuum freeze-drying to prepare dried okra medicinal materials; specifically, the temperature of vacuum freeze-drying is -20 °C, the vacuum degree is -0.1 Mpa, and the drying time is 72 h;

[0077] S4.2 Pulverize the dried okra medicinal materials to obtain powder. Take about 1 g of the powder (passing through No. 2 sieve), accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol, weigh it, ultrasonically treat it (power 300 W, frequency 40 KHz) for 30 min, take it out, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0078] S5. Determination of the content of the component to be measured in the test solution:

[0079] Absorb the test solution and the mixed reference substance solution, and perform high-performance liquid chromatography analysis according to the chromatographic conditions in step S1. Locate each chromatographic peak according to the relative retention time of each component to be measured relative to isoquercitrin. Using the concentration and peak area of the isoquercitrin reference substance solution as a reference, and according to the relative correction factor determined in step S3, calculate the contents of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside in the test solution respectively.

[0080] Among them, the test solution is used within 24 hours after preparation.

[0081] The relative retention time is the average value of the relative retention times of each component to be measured relative to isoquercitrin under different flow rates, different column temperatures, different instruments, and chromatographic column conditions. Specifically, the relative retention times of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside are 0.914, 0.931, and 1.042 respectively.

[0082] III. Results of content determination:

[0083] Using the external standard method (ESM) and the quantitative analysis of multi-components by single marker (QAMS) respectively, according to the chromatographic conditions in step S1, determine the contents of 4 flavonoid components, namely quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin, and quercetin-3-O-(6”-O-malonyl)-glucoside in 15 batches of okra. The results are shown in Table 3.

[0084] Table 3 Results of content determination of 15 batches of okra

[0085]

[0086] It can be seen that the relative deviations of the contents of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside measured by the two methods are all less than 2%, indicating that there is no significant difference in the detection results of the two methods, and the QAMS method has good accuracy.

[0087] IV. Investigation on the drying method of fresh okra fruits

[0088] Take an appropriate amount of fresh okra fruits, cut them into 1-mm-thick slices, mix well, take 10 g (with a water content of 90%, and the dried weight is about 1 g), and prepare test solution 1 according to the test solution preparation method in step S4.

[0089] The fresh fruits of okra were dried by three methods: vacuum freeze-drying (lyophilization), vacuum drying at 60 °C under reduced pressure, and drying at 60 °C in an oven. The test solution 2-4 was prepared according to the preparation method of the test sample in S4.2.

[0090] The samples were analyzed by injection according to the chromatographic conditions in step S1, and the differences in the flavonoid component contents between okra dried by different methods and fresh okra were compared, as shown in Table 4.

[0091] Table 4 Flavonoid component contents in fresh fruits of okra and okra dried by different methods

[0092]

[0093] It can be seen that after drying the fresh fruits of okra by different methods, the flavonoid component contents all decreased to varying degrees. Among them, the content decreased the most by the oven-drying method, which may be related to the longer drying time. The flavonoid content in the lyophilized sample was close to that in the fresh okra sample, indicating that the freeze-drying method had the least impact on the flavonoid components in okra. Therefore, using the freeze-drying method in this application to process the samples can retain the flavonoid components in okra to the greatest extent, and the test results are more representative.

[0094] V. Methodology investigation

[0095] 5.1 Specificity investigation

[0096] The test solution of okra was prepared according to the preparation method of the test solution in step S4, and 70% methanol solution was used as the blank solvent. Another appropriate amount of the reference stock solution in step S2 was diluted 5 times as the reference solution. The samples were analyzed by injection according to the chromatographic conditions in step S1. By comparing with the reference chromatographic peaks, the chromatographic peaks in the okra test sample were assigned. As shown in, there was no interference at the corresponding position in the blank solvent, indicating that the method had good specificity. Figure 1 The blank solvent showed no interference at the corresponding position, indicating that the method had good specificity.

[0097] 5.2 Precision test

[0098] Precisely pipette the mixed reference solution of quercetin-3-O-gentiobioside (41.709 μg / mL), quercetin-3-O-xylose-(1-2)-glucose (18.306 μg / mL), isoquercitrin (30.566 μg / mL), and the reference solution of quercetin-3-O-(6”-O-malonyl)-glucoside (25.088 μg / mL) under item 5.1, and repeat the injection 6 times respectively according to the chromatographic conditions in step S1. Record the peak areas of each reference substance and calculate that the RSD values are all less than 2.0%, indicating that the instrument precision is good.

[0099] 5.3 Repeatability test

[0100] Accurately weigh 6 portions of okra samples of the same batch number, prepare the test solution according to the method in step S4, and determine according to the chromatographic conditions in step S1. The RSD values of the mass fractions of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin, and quercetin-3-O-(6”-O-malonyl)-glucoside are 1.41%, 1.14%, 1.34%, and 1.40% respectively, indicating that the method has good repeatability.

[0101] 5.4 Stability test

[0102] 5.4.1 Stability of the test solution

[0103] Take the okra test solution, and inject and determine according to the chromatographic conditions in step S1 at 0, 2, 4, 8, 12, and 24 h after preparation. The RSD values of the peak areas of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin, and quercetin-3-O-(6”-O-malonyl)-glucoside are 0.37%, 0.54%, 0.44%, and 1.93% respectively. The results are all less than 2.0%, indicating that the test solution has good stability within 24 h. Among them, the RSD values of the peak areas of isoquercitrin and quercetin-3-O-(6”-O-malonyl)-glucoside are relatively large, indicating the relatively poor stability of this component. The test solution needs to be detected as soon as possible within 24 h after preparation and should be prepared and used immediately to ensure the accuracy of the test results.

[0104] 5.4.2 Stability of the reference solution

[0105] Precisely pipette the reference solution under item 5.1 (with the same concentration as the precision test under item 5.2), and inject and determine according to the chromatographic conditions in step S1 at 0, 12, 24, and 48 h. The results are as Figure 2 shown. The RSD of the peak areas of the reference substances of quercetin-3-O-gentiobioside (HT-1), quercetin-3-O-xylose-(1-2)-glucose (HT-2), and isoquercitrin (HT-3) is less than 2% within 48 h, indicating that the reference solution is relatively stable; while the RSD of the peak area of quercetin-3-O-(6”-O-malonyl)-glucoside (HT-4) is greater than 10%, indicating that the reference solution is unstable and easily converted into isoquercitrin. It needs to be prepared and used immediately and detected within 12 h.

[0106] 5.5 Sample addition recovery

[0107] Take about 2 g of okra powder with known content, weigh it precisely, add reference substances of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin and quercetin-3-O-(6”-O-malonyl)-glucoside according to the ratio of sample to reference substance content of 1:1, prepare the test solution according to the method in step S4, prepare 6 portions in parallel, inject and analyze according to the chromatographic conditions in step S1, calculate the spike recovery rates and RSD values of the 4 components to be measured, and the results are shown in Table 5.

[0108] Table 5 Spike Recovery Rates

[0109]

[0110]

[0111] The results show that the average spike recovery rates of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin and quercetin-3-O-(6”-O-malonyl)-glucoside are 100.64%, 99.14%, 102.49% and 98.47% respectively, and the RSD values are 0.96%, 0.87%, 0.47% and 2.05% respectively, all of which are less than 3%, indicating that the accuracy of this analysis method is good.

[0112] 5.6 Investigation on Method Durability

[0113] 5.6.1 Investigation at Different Flow Rates

[0114] Taking isoquercitrin as a reference, calculate the relative retention times and relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose and quercetin-3-O-(6”-O-malonyl)-glucoside at different flow rates (0.8 mL / min, 1.0 mL / min, 1.2 mL / min), and the results are shown in Table 6.

[0115] Table 6 Determination Results of Relative Retention Times and Relative Correction Factors under Different Flow Rate Conditions

[0116]

[0117] The results show that the RSD of the relative retention times of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose and quercetin-3-O-(6”-O-malonyl)-glucoside is in the range of 0.008% - 0.025%, and the RSD of the relative correction factors is in the range of 0.003% - 1.096%, indicating that different flow rates have no significant effect on the relative retention times and relative correction factors of the above 3 components to be measured.

[0118] 5.6.2 Investigation at Different Column Temperatures

[0119] Using isoquercitrin as a reference, the relative retention times and relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside were calculated under different column temperature conditions (25 °C, 30 °C, 35 °C), and the results are shown in Table 7.

[0120] Table 7 Determination results of relative retention times and relative correction factors under different column temperature conditions

[0121]

[0122] The results showed that the RSDs of the relative retention times of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside were in the range of 0.008% - 0.400%, and the RSDs of the relative correction factors were in the range of 0.315% - 0.924%, indicating that different column temperatures had no significant effect on the relative retention times and relative correction factors of the above components to be measured.

[0123] 5.6.3 Investigation of different chromatographic columns

[0124] Using isoquercitrin as a reference, the relative retention times and relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside were calculated on 3 Agilent 1260 liquid chromatographs and 4 different models / batch numbers of chromatographic columns (Agilent ZORBAX SB-C18 USCL131503, USCL131494 and Agilent EclipsePlus C18 USUXA53374, USUXA58806), and the results are shown in Table 8.

[0125] Table 8 Determination results of relative retention times and relative correction factors for different instruments and chromatographic columns

[0126]

[0127]

[0128] The results showed that the RSDs of the relative retention times of the above three components to be measured were in the range of 0.019% - 0.066, and the RSDs of the relative correction factors were in the range of 0.386% - 1.371%, indicating that using different instruments and different chromatographic columns for detection had no significant effect on the relative retention times and relative correction factors of the components to be measured, and the method had good durability.

[0129] The above specific embodiments shall not be construed as limiting the scope of the present invention. For those skilled in the art of the present technology, any alternative improvements or transformations made to the embodiments of the present invention fall within the scope of protection of the present invention.

[0130] Matters not described in detail in the present invention are all well-known technologies to those skilled in the art of the present technology.

Claims

1. A method for simultaneous determination of multiple components for measuring the content of flavonoids in young fruits of Abelmoschus esculentus, characterized in that, It includes the following steps: S1. Determine the chromatographic conditions for high performance liquid chromatography analysis: The chromatographic column uses octadecylsilyl-bonded silica gel as the filler; acetonitrile is used as mobile phase A, and 0.2% phosphoric acid solution is used as mobile phase B for gradient elution; the detection wavelength is 258 nm, the flow rate is 1.0 mL / min, the column temperature is 30 °C, and the injection volume is 10 μL; the specific program for gradient elution is: 0 - 15 min: 5% A, 95% B; 15 - 35 min: 5% - 25% A, 95% - 75% B; 35 - 45 min: 25% - 50% A, 75% - 50% B; S2. Prepare a series of reference substance solutions, including a series of mixed reference substance solutions of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, isoquercitrin, and a series of quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solutions: S3. Determine the relative correction factor: S3.1 Respectively pipette the series of mixed reference substance solutions and the series of quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solutions, inject and analyze them successively according to the chromatographic conditions in step S1, record the chromatographic peak areas, use the peak areas as the ordinate and the concentrations of the reference substance solutions as the abscissa to plot the standard curves, and obtain the linear regression equations of each component to be measured; Among them, the quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solution is used within 12 h after preparation; S3.2 Use isoquercitrin as the internal reference substance, and calculate the relative correction factors of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside according to the linear regression equations; S4. Prepare the test sample solution: S4.1 Take the tender fruits of okra, perform vacuum freeze-drying to obtain dried okra medicinal materials; the temperature of vacuum freeze-drying is -20 °C, the vacuum degree is -0.1 Mpa, and the drying time is 72 h; S4.2 Crush the dried okra medicinal materials to obtain powder, sieve it, take 1 g, accurately weigh it, place it in a stoppered conical flask, accurately add 50 mL of 70% ethanol, weigh it, ultrasonically treat it for 30 min, take it out, let it cool, weigh it again, make up the lost weight with 70% ethanol, shake well, filter, and take the continuous filtrate to obtain the test sample solution; S5. Determine the content of the components to be measured in the test sample solution: Pipette the test sample solution and the mixed reference substance solution, perform high performance liquid chromatography analysis according to the chromatographic conditions in step S1, locate each chromatographic peak according to the relative retention time of each component to be measured relative to isoquercitrin, use the concentration and peak area of the isoquercitrin reference substance solution as the reference, and calculate the contents of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside in the test sample solution respectively according to the relative correction factor determined in step S3; Among them, the test sample solution is used within 24 h after preparation.

2. The multi-component determination method for the content of flavonoids in fresh fruits of Abelmoschus esculentus according to claim 1, characterized in that, In step S2, the preparation method of the series of mixed reference substance solutions of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin includes the following steps: Take appropriate amounts of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin reference substances, and add 70% methanol to prepare a mixed reference substance stock solution containing 208.544 μg of quercetin-3-O-gentiobioside, 91.532 μg of quercetin-3-O-xylose-(1-2)-glucose, and 152.829 μg of isoquercitrin per 1 mL; Using the stepwise dilution method, accurately pipette the mixed reference substance stock solution of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and isoquercitrin, and add 70% methanol solution to dilute it into a series of mixed reference substance solutions with 1, 2.5, 5, 10, 20, and 40 times dilutions respectively.

3. The multi-component determination method for the content of flavonoids in fresh fruits of Abelmoschus esculentus according to claim 1, characterized in that, In step S3.2, the preparation method of the series of quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solutions includes the following steps: Accurately weigh the quercetin-3-O-(6”-O-malonyl)-glucoside reference substance, and add 70% methanol to prepare a reference substance stock solution containing 100.352 μg of quercetin-3-O-(6”-O-malonyl)-glucoside per 1 mL; Accurately pipette the quercetin-3-O-(6”-O-malonyl)-glucoside reference substance stock solution, and add 70% methanol solution to dilute it into a series of quercetin-3-O-(6”-O-malonyl)-glucoside reference substance solutions with 1, 2, 4, 8, 16, and 36 times dilutions respectively.

4. The multi-component determination method for the content of flavonoid components in fresh okra fruits according to claim 1, characterized in that, In step S3.2, the relative correction factor f i / s is calculated by the formula f i / s = A s C i / (A i C s)； Among them, s is isoquercitrin as the internal reference substance, i is the component to be measured, A s is the peak area of the internal reference substance reference standard, C s is the concentration of the internal reference substance reference standard, A i is the peak area of the component to be measured reference standard, C i is the concentration of the component to be measured reference standard.

5. The multi-component determination method for the content of flavonoids in young fruits of Abelmoschus esculentus according to claim 1, characterized in that, In step S3.2, the relative correction factor of quercetin-3-O-gentiobioside is 1.60, the relative correction factor of quercetin-3-O-xylose-(1-2)-glucose is 1.42, and the relative correction factor of quercetin-3-O-(6”-O-malonyl)-glucoside is 1.

32.

6. The multi-component determination method for the content of flavonoid components in young fruits of Abelmoschus esculentus according to claim 1, wherein In step S4.2, the power of the ultrasonic treatment is 300 W, and the frequency is 40 KHz.

7. The multi-component determination method for flavonoid components in young fruits of Abelmoschus esculentus according to claim 1, characterized in that In step S5, the relative retention times of quercetin-3-O-gentiobioside, quercetin-3-O-xylose-(1-2)-glucose, and quercetin-3-O-(6”-O-malonyl)-glucoside are 0.914, 0.931, and 1.042 respectively.

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