A method for determining flavonoid glycosides and flavonoid aglycones in plant-derived products.

Abstract

This invention discloses a method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products, belonging to the field of chemical detection technology. This method enables simultaneous and efficient quantitative detection of 10 common flavonoid glycosides or flavonoid aglycones in plant-derived products. After sample extraction, a high-performance liquid chromatography (HPLC) method under specific conditions is used for testing. This method is applicable to plant-derived product samples of various properties, provides highly accurate results, and is time-efficient, allowing for real-time or periodic monitoring of flavonoid compounds in products.

Description

Technical Field

[0001] This invention relates to the field of chemical detection technology, specifically to a method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products. Background Technology

[0002] Flavonoids are a class of compounds formed by two benzene rings linked by three carbon atoms. They are abundant in plants and often exist in the form of flavonoid glycosides and flavonoid aglycones. Flavonoid aglycones have higher antioxidant activity, anti-inflammatory activity, and bioavailability than flavonoid glycosides. Therefore, many current research and development technologies, after extracting flavonoids from plants and preparing them into products, employ microbial or chemical transformation processes to convert some flavonoid glycosides into flavonoid aglycones. To ensure product process stability or optimize process conditions (e.g., by feeding back the real-time conversion status of flavonoid glycosides to the process conditions for optimization) and product efficacy, the flavonoid aglycones and flavonoid glycosides in these plant-derived products need to be quantitatively detected in real-time or periodically during production and storage.

[0003] However, there are many types of flavonoids. Some existing detection methods for flavonoids, such as T / ISAS 001-2019 "Quality Standard for Seabuckthorn Flavonoids" and DB34 / T2743-2016 "Determination of Total Flavonoid Content in Sophora japonica and its Products - Spectrophotometric Method", are only applicable to the determination of total flavonoid content in samples and cannot quantify specific flavonoid glycosides and flavonoid aglycones. Although NY / T3950-2021 "Determination of 10 Flavonoids in Plant-Derived Foods - High Performance Liquid Chromatography-Tandem Mass Spectrometry" discloses a test method for determining various flavonoids such as epicatechin, hesperidin, daidzein, quercetin, genistein, luteolin, apigenin, myricetin, isorhamnetin, and naringenin in plant-derived foods, this method yields low concentrations and has poor applicability to some plant extracts or fermented products. Furthermore, this method uses high performance liquid chromatography-tandem mass spectrometry equipment, which is costly.

[0004] On the other hand, current testing methods for flavonoids mainly focus on several common flavonoid aglycones such as quercetin, kaempferol, and isorhamnetin. Common flavonoid glycosides include isorhamnetin-3-O-glucoside, isorhamnetin-3-O-gluco-7-O-rhamnoside, isoquercitrin, narcissin, rutin, and myricetin. However, there is virtually no method for detecting coexisting flavonoid glycosides. For example, patent CN 107389814 A discloses a method for testing flavonoid aglycones, but this method uses hydrochloric acid to hydrolyze flavonoid glycosides in the pretreatment, making it impossible to determine the flavonoid glycosides in the sample. Furthermore, existing conventional detection methods are time-consuming to process samples, making it impossible to achieve real-time or periodic monitoring of component content. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products. This method enables simultaneous and efficient quantitative detection of 10 common flavonoid glycosides or flavonoid aglycones in plant-derived products. After sample extraction, a high-performance liquid chromatography (HPLC) method under specific conditions is employed for testing. This method is applicable to plant-derived product samples of various properties, provides highly accurate test results, and is time-efficient, making it suitable for real-time or periodic monitoring of flavonoid compounds in products.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products includes the following steps:

[0008] (1) Sample pretreatment: The plant-derived product was mixed with methanol and ultrasonically treated. The resulting mixture was then diluted to a final volume to obtain the sample solution to be tested.

[0009] (2) High-performance liquid chromatography (HPLC) detection: Prepare a reference solution, and perform HPLC detection on the reference solution and the sample solution to be tested; the reference solution includes isorhamnetin-3-O-glucose-7-O-rhamnoside solution, dihydromyricetin solution, rutin solution, isoquercetin solution, narcissin solution, isorhamnetin-3-O-glucose solution, myricetin solution, quercetin solution, kaempferol solution, and isorhamnetin solution;

[0010] (3) Compare the chromatograms of the sample test solution and the reference solution to perform quantitative analysis of plant-derived products;

[0011] The mobile phase conditions for the high-performance liquid chromatography detection are as follows:

[0012] Acetonitrile was used as mobile phase A, and a 0.4–0.8 wt% phosphoric acid aqueous solution was used as mobile phase B for gradient elution. The gradient elution conditions were as follows: 0–5 min, the volume ratio of mobile phase A to mobile phase B was 20:80; 5–15 min, the volume ratio of mobile phase A to mobile phase B gradually changed to (45:55)–(50:50); 15–22 min, the volume ratio of mobile phase A to mobile phase B remained constant; 22–22.5 min, the volume ratio of mobile phase A to mobile phase B gradually changed to 95:5; 22.5–26 min, the volume ratio of mobile phase A to mobile phase B remained constant; 26–26.1 min, the volume ratio of mobile phase A to mobile phase B gradually changed to 20:80; 26.1–30 min, the volume ratio of mobile phase A to mobile phase B remained constant.

[0013] The chromatographic column used in the high-performance liquid chromatography detection was C18. 18-AQ or T3 chromatographic column.

[0014] Currently, flavonoids are the main active ingredients in most plant-derived products, contributing to their bio-health benefits. However, existing methods for testing, especially quantifying, flavonoids in products mainly fall into two categories: one tests the total flavonoid content, i.e., the total content of flavonoid glycosides and flavonoid aglycones; the other only tests for flavonoid aglycones with significant biological activity in the sample. Neither method can simultaneously detect flavonoid glycosides and flavonoid aglycones in the product, making it impossible for those skilled in the art to simultaneously monitor the content of these two substances in such products. This is especially problematic when some plant-derived products further convert flavonoid glycosides into flavonoid aglycones. Without this information, it is impossible to effectively feed back into process parameter improvement procedures to quickly increase the effective conversion rate of flavonoid glycosides in continuous production lines. Although some existing technologies combine multiple detection methods, testing the total flavonoid content and flavonoid aglycone content of the sample and then performing secondary calculations to confirm the flavonoid glycoside content, this approach involves many error factors and has low accuracy in real-time testing.

[0015] Therefore, in the technical solution of this invention, the inventors use methanol as the extraction solution to extract plant-derived products, and then select 10 common flavonoid glycosides or flavonoid aglycones as reference target species. A specific high-performance liquid chromatography column is used to quantitatively detect them under special conditions. This detection method has high adaptability to plant-derived products and will not have significant differences in detection accuracy due to different samples. Simultaneously, the characteristic peaks of each active ingredient in the detection results have high separation degree, and the detection results have high accuracy and stability. The separation time is short (only 30 minutes as described in the technical solution), resulting in high overall cost-effectiveness. It is very suitable for real-time or periodic monitoring of flavonoid glycosides and flavonoid aglycones during the production, processing, and preservation of plant-derived products.

[0016] Preferably, the plant-derived product includes at least one of sea buckthorn-derived products, sophora japonica-derived products, and jujube-derived products.

[0017] Sea buckthorn is currently a popular raw material for pharmaceuticals and health products. Its flavonoids are effective active ingredients for treating or controlling hypertension and hyperlipidemia, and improving vascular elasticity. Therefore, it is essential to conduct component testing and monitoring on finished or semi-finished products (such as raw liquid) made from sea buckthorn. Sophora japonica buds and Japanese raisin tree fruit are also plant species with high levels of flavonoid active ingredients and can be combined with other types of plant raw materials. Therefore, the testing and monitoring of the components, especially flavonoids, in these plant products is equally crucial.

[0018] More preferably, the plant-derived product is a fermented product.

[0019] As mentioned above, although existing plant-derived products contain flavonoid glycosides and flavonoid aglycones, the latter has higher functional activity. Therefore, existing technologies further employ processes such as fermentation after obtaining the plant extract. During fermentation, some flavonoid glycosides are converted into flavonoid aglycones. The testing method described in this invention, based on the quantitative sensitivity and accuracy of both substances, is very suitable for testing samples where the types of flavonoid compounds change and transform. For example, simultaneous testing of plant extract and fermentation broth can confirm the transformation of flavonoid compounds during fermentation, thereby providing feedback to the fermentation conditions for optimization and assisting in the efficient conversion of flavonoid glycosides at the fermentation stage.

[0020] Preferably, the plant-derived product is a liquid;

[0021] More preferably, in step (1), the volume ratio of the plant-derived product to methanol is (1:15) to (1:5), and the ultrasonic treatment time is 25 to 35 minutes.

[0022] Preferably, in step (2), the UV detection wavelength during high performance liquid chromatography is 0-8.5 min, 290 nm-300 nm (preferably 292 nm); 8.5-30 min, 350-370 nm (preferably 360 nm).

[0023] Preferably, the flow rate of the mobile phase during high-performance liquid chromatography detection in step (2) is 0.8 to 1 mL / min.

[0024] The inventors' experiments revealed that the separation degree of the 10 flavonoid glycosides or flavonoid aglycones varied with different elution flow rates. However, within the range of 0.8 to 1 mL / min, the separation degree of each detection substance reference standard was greater than 1.5, indicating good separation effect. Those skilled in the art can adjust the separation degree within this range according to the different key detection substances to achieve a relatively higher separation degree.

[0025] More preferably, the column temperature for high-performance liquid chromatography detection is 30–40°C.

[0026] The separation of the flavonoids to be detected varied under different column temperatures. The separation degree of each substance was relatively high in the range of 30 to 40°C. As the temperature increased from 30°C, the separation degree of each substance further improved and reached its maximum value at 35°C. However, the separation degree decreased as the temperature increased further. Overall, the effect was best in this range.

[0027] Preferably, the specific steps for preparing the reference solution in step (2) are as follows: dissolve isorhamnetin-3-O-gluco-7-O-rhamnoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucoside, myricetin, quercetin, kaempferol, and isorhamnetin in methanol, and make up to volume to obtain reference solutions of isorhamnetin-3-O-gluco-7-O-rhamnoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucoside, myricetin, quercetin, kaempferol, and isorhamnetin.

[0028] More preferably, the step of preparing the reference solution in step (2) is as follows: weigh isorhamnetin-3-O-glucoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucoside, myricetin, quercetin, kaempferol, and isorhamnetin, respectively, and dissolve them in methanol to prepare a stock solution. Then, transfer the stock solution to a 20mL volumetric flask, shake well, and dilute to volume to obtain isorhamnetin-3-O-glucoside with a concentration of 21.9μg / mL. A mixed reference solution of -O-glucose-7-O-rhamnoside, 87.0 μg / mL dihydromyricetin, 54.0 μg / mL rutin, 45.8 μg / mL isoquercetin, 46.6 μg / mL narcissin, 45.2 μg / mL isorhamnetin-3-O-glucose, 45.8 μg / mL myricetin, 57.6 μg / mL quercetin, 45.2 μg / mL kaempferol, and 47.8 μg / mL isorhamnetin.

[0029] For the preparation of the reference solution, the above-mentioned preferred technical solutions can be used in the embodiments, but it is not limited to this. It can also be prepared by a method that can obtain a reference solution with the same properties. In actual preparation, for the detection substance, plant-derived products containing flavonoids, raw materials corresponding to different active substances can be used to prepare the reference solution, or commercial standard reference standards can be used directly as the reference solution.

[0030] Preferably, the mobile phase B used in step (2) for high performance liquid chromatography detection is a phosphoric acid aqueous solution with a mass content of 0.8 wt%.

[0031] Through experiments, the inventors discovered that the tailing factors of each component differ when the mass content of phosphoric acid in mobile phase B is different. The recommended range for the tailing factor is generally 0.95 to 1.05. When using pure water as mobile phase B, although the tailing factors of some flavonoids such as isorhamnetin-3-O-glucose-7-O-rhamnoside are within the range, the tailing factors of substances such as myricetin and narcissin are too large and do not meet the requirements. With the introduction of phosphoric acid and the increase of its concentration, the separation of each component increases, the tailing factor basically meets the requirements, and it is also more conducive to the cleaning and recovery of the chromatographic column.

[0032] Preferably, the chromatographic column used in step (2) for high performance liquid chromatography detection has a size of 5μm×4.6mm×250mm.

[0033] Another object of the present invention is to provide a method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products for real-time or periodic monitoring of the content of flavonoid glycosides and flavonoid aglycones during the production and preservation of plant-derived products.

[0034] As mentioned above, the main functional active component of flavonoids in plant-derived products is the flavonoid aglycone. Therefore, if the proportion of flavonoid aglycones in some high-flavonoid plant-derived products is low, they may not achieve high functional activity. On the other hand, some plant-derived products undergo further processing after extraction from plants, converting some flavonoid glycosides into flavonoid aglycones. The testing method described in this invention, applied to component detection or real-time monitoring of the preparation process in these products, can fully reveal the real-time content of each flavonoid compound. For example, for products, it can better assess the functional effects of the product, or determine the stability of flavonoid compounds under different storage conditions. For the process, the results can be directly fed back to the process end in real time, thereby positively controlling process parameters to ensure the conversion efficiency and stability of flavonoid glycosides.

[0035] The beneficial effects of this invention are that it provides a method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products. This method enables simultaneous and efficient quantitative detection of 10 common flavonoid glycosides or flavonoid aglycones in plant-derived products. After sample extraction, a high-performance liquid chromatography (HPLC) method under specific conditions is used for testing. This method is applicable to plant-derived product samples of various properties, and the detection results are highly accurate. At the same time, the testing method is time-saving and can be used for real-time or periodic monitoring of flavonoid compounds in products. Attached Figure Description

[0036] Figure 1 The scan spectrum of dihydromyricetin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0037] Figure 2 The scan spectrum of isorhamnetin-3-O-glucose-7-O-rhamnoside, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0038] Figure 3 The scan spectrum of rutin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0039] Figure 4The scan spectrum of isoquercetin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0040] Figure 5 This is a scan spectrum of narcissin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, in the wavelength range of 190 nm to 400 nm.

[0041] Figure 6 The scan spectrum of isorhamnetin-3-O-glucoside, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0042] Figure 7 The image shows the scanning spectrum of myricetin, the reference substance in the reference solution of the test method described in Example 1 of this invention, in the wavelength range of 190 nm to 400 nm.

[0043] Figure 8 The scan spectrum of quercetin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0044] Figure 9 The scan spectrum of kaempferol, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0045] Figure 10 The scan spectrum of isorhamnetin, the reference substance in the reference solution of the test method described in Example 1 of the present invention, is in the wavelength range of 190 nm to 400 nm.

[0046] Figure 11 The chromatogram shows the detection results of the test sample plant extract 1 (marked as sea buckthorn sample in the figure) and the blank control standard by the test method described in Example 1 of the present invention.

[0047] Figure 12 This is a chromatographic comparison of the detection results of the test sample plant extract 1 (marked as sea buckthorn sample in the figure) before and after spiking, as described in Example 1 of the present invention.

[0048] Figure 13 The chromatogram shows the detection results of the test sample plant extract 2 (marked as Sophora japonica flower sample) and the blank control standard by the test method described in Example 2 of the present invention.

[0049] Figure 14 This is a chromatographic comparison of the detection results of the plant extract 2 (marked as Sophora japonica flower sample) before and after spiking according to the test method described in Example 2 of the present invention.

[0050] Figure 15The chromatogram shows the detection results of the test sample plant extract 3 (marked as Hovenia dulcis sample in the figure) and the blank control at a wavelength of 360 nm using the test method described in Example 3 of the present invention.

[0051] Figure 16 The chromatogram shows the detection results of the test sample plant extract 3 (marked as Hovenia dulcis sample in the figure) and the blank control at a wavelength of 292 nm using the test method described in Example 3 of the present invention.

[0052] Figure 17 This is a chromatographic comparison of the detection results of the test sample plant extract 3 (marked as Hovenia dulcis sample) before and after spiked at a wavelength of 360 nm using the test method described in Example 3 of the present invention.

[0053] Figure 18 This is a chromatographic comparison of the detection results of the test sample plant extract 3 (marked as Hovenia dulcis sample) before and after spiked at a wavelength of 292 nm using the test method described in Example 3 of the present invention.

[0054] Figure 19 This is a chromatographic comparison of the detection results at different mobile phase flow rates during high-performance liquid chromatography (HPLC) testing in Example 2 of the present invention.

[0055] Figure 20 This is a chromatographic comparison of the detection results under different mobile phase selections during high-performance liquid chromatography testing in Example 3 of the present invention.

[0056] Figure 21 This is a chromatographic comparison of the detection results under different gradient elution programs in the high-performance liquid chromatography test method of Example 4 of the present invention.

[0057] Figure 22 This is a chromatographic comparison of the detection results at different temperatures during high-performance liquid chromatography testing in Example 5 of the present invention.

[0058] Figure 23 This is a chromatographic comparison of the detection results under different column types selected during high-performance liquid chromatography testing in Example 6 of the present invention. Detailed Implementation

[0059] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments and comparative examples. The purpose of this description is to provide a detailed understanding of the invention, not to limit it. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Unless otherwise specified, the experimental reagents and instruments designed in the embodiments and comparative examples of this invention are commonly used reagents and instruments.

[0060] The plant-derived products used in the specific embodiments of the present invention include plant extracts 1 to 3;

[0061] The method for preparing the plant extract is as follows: Take the washed and dried plant extracts (sea buckthorn, sophora japonica, and jujube fruit) and add water at a ratio of 10 times the mass of the plant extracts to prepare a solution. Add calcium carbonate to adjust the pH value to 4.0-5.0, add white sugar at a mass ratio of 6% of the obtained plant extract, and stir until dissolved to obtain the plant extract.

[0062] Plant extract 1 is sea buckthorn extract, plant extract 2 is sophora japonica extract, and plant extract 3 is Japanese raisin tree extract.

[0063] Example 1

[0064] An embodiment of the method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products of the present invention includes the following steps:

[0065] (1) Sample pretreatment: Add 2 mL of plant extract 1 to a 25 mL volumetric flask, mix with 15 mL of methanol and sonicate for 30 min. Make up the volume of the mixture to obtain sample solution 1. Take the supernatant and filter it through a 0.22 μm filter membrane before testing.

[0066] (2) High-performance liquid chromatography (HPLC) detection: Prepare a reference solution, and perform HPLC detection on the reference solution and the sample test solution 1; the reference solution includes isorhamnetin-3-O-glucose-7-O-rhamnoside solution, dihydromyricetin solution, rutin solution, isoquercitrin solution, narcissin solution, isorhamnetin-3-O-glucose solution, myricetin solution, quercetin solution, kaempferol solution, and isorhamnetin solution;

[0067] The reference solution was prepared as follows: weigh isorhamnetin-3-O-glucoside-7-O-rhamnoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucoside, myricetin, quercetin, kaempferol, and isorhamnetin, and dissolve them in methanol to prepare the stock solution. Then, transfer the stock solution to a 20 mL volumetric flask, mix, shake well, and dilute to volume.

[0068] The preparation parameters are shown in Table 1:

[0069] Table 1

[0070]

[0071]

[0072] Pipette 0.2 mL, 0.3 mL, 0.5 mL, 1.0 mL, 2.0 mL, and 5 mL of the mixed reference solution into 5 mL volumetric flasks, respectively, and dilute to 5 mL with methanol to obtain a series of control solutions for the preparation of the standard curve;

[0073] The standard curves were prepared by diluting methanol to six concentration levels and then plotting the standard curves to determine the linear range of each component. The correlation coefficients of the standard curves for the 10 components met the condition R > 0.999, indicating good linearity for all 10 references within their respective ranges. The linear ranges of each component are shown in Table 2.

[0074] Table 2

[0075]

[0076] The mobile phase conditions for the high-performance liquid chromatography detection are as follows:

[0077] Gradient elution was performed using acetonitrile as mobile phase A and 0.4 wt% phosphoric acid aqueous solution as mobile phase B. The gradient elution conditions are shown in Table 3.

[0078] Table 3

[0079] Time (min) Flowability A Acetonitrile (volume %) Flowability B 0.4% Phosphoric Acid (volume %) 0 20 80 5 20 80 15 50 50 22 50 50 22.5 95 5 26 95 5 26.1 20 80 30 20 80

[0080] The chromatographic column used for the high-performance liquid chromatography detection was Hungpu C. 18 AQ(2) column chromatography column, size 5μm×4.6mm×250mm, flow rate 1mL / min, injection volume 10μL, column temperature 35℃;

[0081] The UV detection wavelength settings for high performance liquid chromatography are as follows: 0–8.5 min, wavelength set to 292 nm; 8.5–30 min, wavelength set to 360 nm.

[0082] The detection wavelength of the reference solution was determined based on a full-wavelength scan of the diode array detector, with a scanning range of 190 nm to 400 nm. The scan spectrum is shown below. Figures 1-10 The maximum absorption wavelengths correspond to the following positions: isorhamnetin-3-O-glucoside at 372 nm and 254 nm, dihydromyricetin at 292 nm, rutin at 354 nm and 256 nm, isoquercetin at 354 nm and 256 nm, narcissin at 356 nm and 254 nm, isorhamnetin-3-O-glucoside at 354 nm and 254 nm, myricetin at 374 nm and 254 nm, quercetin at 372 nm and 256 nm, kaempferol at 366 nm and 266 nm, and isorhamnetin at 372 nm and 254 nm. Except for dihydromyricetin, all other components have two maximum absorption wavelengths and strong absorption in the 350-370nm range. Therefore, 360nm was selected as the detection wavelength for the nine reference standards except dihydromyricetin, and 292nm was selected as the detection wavelength for dihydromyricetin.

[0083] (3) Compare the chromatograms of the sample test solution and the reference solution to perform quantitative analysis of plant-derived products;

[0084] Meanwhile, a spiked recovery test of a blank control, distilled water, was set up during the chromatographic test to examine the accuracy of the test results. Distilled water was used instead of the sample, and an appropriate amount of mixed reference solution was added and mixed thoroughly. Seven samples were prepared repeatedly according to the above preparation method, and then tested under the above chromatographic conditions. The spiked recovery rate of each reference standard was calculated, and the results are shown in Table 4.

[0085] Table 4

[0086]

[0087] Subsequently, samples 2–6 were prepared in parallel and subjected to the same tests as described above. The test results for each sample are as follows: Figure 11 As shown in Table 5, no chromatographic peaks with the same retention times as those of the reference solutions were observed in the chromatograms of the blank control solution, proving that there was no interference.

[0088] It can be seen that eight flavonoids were detected in plant extract 1, and the RSD values ​​of the seven parallel samples were all less than 10%, which demonstrates that the method has good repeatability and high precision for the detection of plant extract 1.

[0089] Table 5

[0090]

[0091]

[0092] Furthermore, the accuracy of the method was examined through a spiked recovery test of the test solutions: 2 mL of the plant extract was placed in a 25 mL volumetric flask, an appropriate amount of mixed reference solution was added, followed by 15 mL of methanol and sonication for 30 min. The mixture was then brought to volume with methanol. Seven test solutions (1a to 7a) were prepared in parallel according to the above sample preparation method, and the same tests were performed under the same chromatographic conditions. The spiked recoveries of each reference were calculated, and the results are shown in Table 6. Sample 1 was used for chromatogram fitting comparison before and after spiked analysis, and the results are as follows... Figure 12 As shown in Table 6, the spiked recoveries of all references reached 90–110%, meeting the requirements of GB / T27417—2017 "Guideline for Conformity Assessment and Validation of Chemical Analysis Methods".

[0093] Table 6

[0094]

[0095] Example 2

[0096] The only difference between this embodiment and Embodiment 1 is that plant extract 1 is replaced with plant extract 2. The test sample solutions 1-7 are prepared in the same manner, and the test results are as follows: Figures 13-14 The results are shown in Table 7 (Precision Test of Parallel Samples 1-7) and Table 8 (Spiked Test of Parallel Samples, with Parallel Spiked Samples 1b-7b). The results indicate that the precision and accuracy of the detection results for the plant extract prepared from Sophora japonica flowers are also high.

[0097] Table 7

[0098]

[0099] Table 8

[0100]

[0101] Example 3

[0102] The only difference between this embodiment and Embodiment 1 is that plant extract 1 is replaced with plant extract 3. The test sample solutions 1-7 are prepared in the same manner, and the test results are as follows: Figures 15-18 The results are shown in Table 9 (Precision Test of Parallel Samples 1-7) and Table 10 (Spiked Test of Parallel Samples, Parallel Spiked Samples 1c-7c). Since only three types of flavonoids were detected in plant extract 3, and it also contained dihydromyricetin, the detection wavelengths were set at both 360nm and 292nm. The results indicate that the precision and accuracy of the detection results for plant extracts prepared from Hovenia dulcis seeds are also high.

[0103] Table 9

[0104]

[0105]

[0106] Table 10

[0107]

[0108] Example 1

[0109] To verify the sensitivity of the test method described in this invention for detecting the dynamic changes of flavonoids in plant extracts during processing, plant extracts 1-3 were fermented. The processing steps were as follows: the plant extract samples were heated to 90-100°C before fermentation and kept at that temperature for 1 hour. After cooling, fermentation bacteria (lactic acid bacteria, viable count of 10%) were inoculated. 7 cfu / mL to 10 9 After obtaining the cfu / mL concentration, fermentation began for 12 days at 35℃ and a rotation speed of 50–100 rpm. During fermentation, the viable cell count in the fermentation broth was maintained at 10%. 7cfu / mL to 10 9 The concentration of cfu / mL was increased, and the product was sterilized after fermentation to obtain the fermented plant extract sample.

[0110] For each plant extract, samples 1 to 3 in parallel groups were fermented to prepare three groups of samples. Then, the flavonoids in the samples were qualitatively and quantitatively analyzed according to the methods described in Examples 1 to 3. The results are shown in Tables 11 to 13.

[0111] Table 11

[0112]

[0113] Table 12

[0114]

[0115]

[0116] Table 13

[0117]

[0118] The test results show that after fermentation, the content of four flavonoid glycosides—isorhamnoside-3-O-glucose-7-O-rhamnoside, rutin, isoquercitrin, and narcissin—in Plant Extract 1 decreased, while the content of two flavonoid aglycones—quercetin and isorhamnoside—increased. This indicates that for the sea buckthorn extract of Plant Extract 1, the fermentation process can, to some extent, convert some flavonoid glycosides into flavonoid aglycones with higher functional activity. Similarly, after fermentation, the content of two flavonoid aglycones—quercetin and isorhamnoside—in Plant Extract 2 also increased, indicating some conversion of flavonoid glycosides. After fermentation, the content of dihydromyricetin, originally contained in Hovenia dulcis, in Plant Extract 3 decreased significantly, while the content of myricetin and quercetin increased significantly. This also demonstrates that the detection method described in this invention has real-time sensitivity and accuracy for detecting the conversion process of plant-derived products containing these flavonoid compounds during processing.

[0119] Example 2

[0120] To verify the optimal mobile phase flow rate in the high-performance liquid chromatography (HPLC) method described in this invention, a parallel experimental example 1 was set up. The detection was performed using the same method as in Example 1, but the mobile phase flow rate was adjusted to 0.8 mL / min for gradient elution. The resolution of each analyte was then compared, and the results are as follows: Figure 19 As shown in Table 14.

[0121] Table 14

[0122]

[0123]

[0124] It can be seen that the separation degree of each detected component reached above 1.5 at both flow rates, indicating that the test results at this flow rate range were ideal. Simultaneously, other experimental groups were set up to compare with other flow rates, and the results showed that excessively fast or slow flow rates led to low separation degrees.

[0125] Example 3

[0126] To verify the optimal selection of mobile phase type in the high-performance liquid chromatography (HPLC) detection method of the present invention, parallel experiments 2 and 3 were set up. Detection was performed using the same method as in Example 1, but mobile phase B was adjusted to a gradient elution of 0.8 wt% phosphoric acid aqueous solution and pure water. The separation effects under the three mobile phases B were then compared with those in Example 1, and the tailing factors of each flavonoid compound were statistically analyzed. Figure 20 As shown in Table 15.

[0127] Table 15

[0128]

[0129] The results show that when 0.4wt%–0.8wt% phosphoric acid aqueous solution is used as mobile phase B, the tailing factor is within the range of 0.95–1.05, indicating that this mobile phase has excellent separation effect on various flavonoids. However, when pure water is used as mobile phase B, the tailing factor of some flavonoids, especially flavonoid aglycones, exceeds the range. Obviously, this mobile phase cannot accurately detect the content of flavonoids in each product in real time, and it cannot detect the conversion between flavonoid glycosides and flavonoid aglycones.

[0130] Example of effect 4

[0131] To verify the optimality of the elution program in the high-performance liquid chromatography (HPLC) detection method of the present invention, parallel experiments 4-7 were set up, and the detection was performed according to the same method as in Example 1, but the gradient elution conditions are shown in Tables 16-19 respectively:

[0132] Table 16

[0133] Time (min) Flowability A Acetonitrile (volume %) Flowability B 0.4% Phosphoric Acid (volume %) 0 20 80 5 20 80 15 40 60 22 40 60 22.5 95 5 26 95 5 26.1 20 80 30 20 80

[0134] Table 17

[0135] Time (min) Flowability A Acetonitrile (volume %) Flowability B 0.4% Phosphoric Acid (volume %) 0 20 80 5 20 80 15 45 55 22 45 55 22.5 95 5 26 95 5 26.1 20 80 30 20 80

[0136] Table 18

[0137] Time (min) Flowability A Acetonitrile (volume %) Flowability B 0.4% Phosphoric Acid (volume %) 0 20 80 5 20 80 15 55 45 22 55 45 22.5 95 5 26 95 5 26.1 20 80 30 20 80

[0138] Table 19

[0139] Time (min) Flowability A Acetonitrile (volume %) Flowability B 0.4% Phosphoric Acid (volume %) 0 20 80 5 20 80 15 60 40 22 60 40 22.5 95 5 26 95 5 26.1 20 80 30 20 80

[0140] The elution effect was compared with that described in Example 1. Figure 21 As shown in Table 20, the volume ratio of the mobile phase during elution has different effects on the elution effect of flavonoids during the middle stage of elution. When the acetonitrile concentration is 55% and 60%, the resolution of the active ingredient isorhamnetin is less than 1, and the qualified characteristic peak cannot be separated. When the acetonitrile concentration is 40%, kaempferol and isorhamnetin cannot be separated by elution during 15-22 min, and the peak can only be eluted when the acetonitrile concentration reaches 95%. However, the increase in acetonitrile concentration at this time is mainly to elute impurities in the chromatographic column. Therefore, impurities and the two target analytes may be eluted at the same time during this period. Therefore, this concentration is also not suitable. Therefore, the volume ratio of mobile phase A to mobile phase B in the range of (45:55) to (50:50) is most suitable during 15-22 min.

[0141] Table 20

[0142]

[0143] Example 5

[0144] To verify the appropriate temperature range for high-performance liquid chromatography (HPLC) in the detection method of this invention, parallel experiments 8-9 were set up. Detection was performed using the same method as in Example 1, but the column temperature was set to 30°C and 40°C respectively during gradient elution. The results were compared with those of the method described in Example 1. Figure 22 As shown, the separation effect of each target analyte is good at 30-40℃, indicating that this temperature is suitable for the test method described in this invention.

[0145] Example 6

[0146] To verify whether the type of chromatographic column used in the high-performance liquid chromatography (HPLC) detection method of the present invention is appropriate, parallel experimental examples 10-11 were set up. Detection was performed using the same method as in Example 1, but the chromatographic columns were replaced with Hungpu T3 and Aglient Extend C18 columns during elution. The results were compared with those described in Example 1. Figure 23 As shown in Table 21, it can be seen that the elution retention time and resolution of each target analyte when eluted using the Hungpu T3 column are basically the same as those used in Example 1. 18 The AQ(2) column was consistent, while the Aglient Extend C18 column had a faster elution time. However, the resolution of isorhamnetin-3-O-glucose-7-O-rhamnoside and isorhamnetin was less than 1, indicating that the column could not be effectively used in the elution of the test method described in this invention.

[0147] Table 21

[0148]

[0149]

[0150] Example 7

[0151] As can be seen from Example 1, the detection method of the present invention has high sensitivity and accuracy for the fermentation broth of plant extract. In Example 1 and Examples 1-3, both the plant extract and the fermentation broth were extracted with methanol. In order to study the special characteristics of this extract, the plant extract 2 after fermentation in Example 1 was pretreated and detected according to the method described in Example 1, but the methanol in the pretreatment was replaced with pure water and a 50% volume methanol-water mixture, respectively. Parallel experiments 12-13 were set up, and three sets of parallel samples were set up with reference to Example 1. The test results are shown in Table 22.

[0152] Table 22

[0153]

[0154] The results show that different pretreatment extraction solutions lead to different detection results for the final sample. It is evident that pure methanol extraction yields a higher degree of flavonoid extraction from the fermented plant extract. If water is used for extraction, it may not be possible to obtain accurate results of the true component content, and it can only be used for dynamic monitoring of component transformation.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products, characterized in that, Includes the following steps: (1) Sample pretreatment: The plant-derived product was mixed with methanol and ultrasonically treated. The resulting mixture was then diluted to a final volume to obtain the sample solution to be tested. (2) High-performance liquid chromatography (HPLC) detection: Prepare a reference solution, and perform HPLC detection on the reference solution and the sample solution to be tested; the reference solution includes isorhamnetin-3-O-glucose-7-O-rhamnoside solution, dihydromyricetin solution, rutin solution, isoquercetin solution, narcissin solution, isorhamnetin-3-O-glucose solution, myricetin solution, quercetin solution, kaempferol solution, and isorhamnetin solution; the flow rate of the mobile phase during HPLC detection is 0.8~1 mL / min; (3) Compare the chromatograms of the sample test solution and the reference solution to perform quantitative analysis of plant-derived products; The mobile phase conditions for the high-performance liquid chromatography detection are as follows: Acetonitrile was used as mobile phase A, and 0.8 wt% phosphoric acid aqueous solution was used as mobile phase B for gradient elution. The gradient elution conditions were as follows: 0-5 min, the volume ratio of mobile phase A to mobile phase B was 20:80; 5-15 min, the volume ratio of mobile phase A to mobile phase B gradually changed to (45:55) to (50:50); 15-22 min, the volume ratio of mobile phase A to mobile phase B remained constant; 22-22.5 min, the volume ratio of mobile phase A to mobile phase B gradually changed to 95:5; 22.5-26 min, the volume ratio of mobile phase A to mobile phase B remained constant; 26-26.1 min, the volume ratio of mobile phase A to mobile phase B gradually changed to 20:

80. 26.1~30 min, the volume ratio of mobile phase A to mobile phase B remains constant; The chromatographic column used in the high-performance liquid chromatography detection was C18. 18 -AQ or T3 chromatographic column; The plant-derived product is at least one of sea buckthorn-derived products, sophora japonica-derived products, and raisin tree fruit-derived products, or at least one of fermented products of sea buckthorn-derived products, sophora japonica-derived products, and raisin tree fruit-derived products. The UV detection wavelength settings for high performance liquid chromatography are as follows: 0~8.5 min, wavelength set to 292 nm; 8.5~30 min, wavelength set to 360 nm. The column temperature for high-performance liquid chromatography (HPLC) detection is 30-40℃.

2. The method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products as described in claim 1, characterized in that, The plant-derived product is a liquid.

3. The method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products as described in claim 2, characterized in that, In step (1), the volume ratio of plant-derived product to methanol is (1:15) to (1:5), and the ultrasonic treatment time is 25 to 35 minutes.

4. The method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products as described in claim 1, characterized in that, The specific steps for preparing the reference solutions in step (2) are as follows: dissolve isorhamnetin-3-O-glucose-7-O-rhamnoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucose, myricetin, quercetin, kaempferol, and isorhamnetin in methanol, and make up to volume to obtain reference solutions of isorhamnetin-3-O-glucose-7-O-rhamnoside, dihydromyricetin, rutin, isoquercetin, narcissin, isorhamnetin-3-O-glucose, myricetin, quercetin, kaempferol, and isorhamnetin.

5. The method for testing flavonoid glycosides and flavonoid aglycones in plant-derived products as described in any one of claims 1 to 4 is used for real-time or periodic monitoring of the content of flavonoid glycosides and flavonoid aglycones during the production and preservation of plant-derived products.

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