Method for identifying active flavone compound group in chenopodium album and rapidly detecting active flavone compound group in chenopodium album

Abstract

The invention relates to the field of plant component detection, in particular to a rapid detection method for identifying active flavone component groups in chenopodium album. According to the invention, chemical system separation and active site tracking separation are adopted; according to the present invention, the five active flavone components such as ilicin A, quercetin-3-O-beta-D-glucuronic acid, quercetin-3-O-beta-D-glucoside, kaempferol-3-O-beta-D-glucuronic acid, and isorhamnetin-3-O-beta-D-glucuronic acid in the chenopodium album are firstly determined as the evaluation indexes of the functional components such as oxidation resistance and gout treatment; a UPLC synchronous detection method for five active flavone components is established, the defects of an existing evaluation system and an existing quality control method are overcome, and a scientific basis is provided for further processing and utilization and quality control of the chenopodium album.

Description

Technical Field

[0001] This invention relates to the field of plant component detection, specifically to the establishment of quality evaluation indicators for five active flavonoid compound groups in quinoa and a rapid detection method for them using ultra-high performance liquid chromatography (UPLC) with a five-in-one mixed standard. Background Technology

[0002] Quinoa ( Chenopodium quinoa Quinoa, a traditional crop from the Andes Mountains with a history spanning millennia, has recently garnered global attention for its comprehensive nutritional qualities. Its nutritional matrix boasts three core advantages: high protein content, a balanced ratio of essential amino acids, and gluten-free properties, making it an ideal choice for special dietary needs. In particular, quinoa is rich in functional components such as flavonoids, saponins, and polyphenols, demonstrating significant potential in chronic disease prevention and functional food development. Among the bioactive components of quinoa, flavonoids serve as the core functional unit, with a significantly higher total content than traditional grains like rice and wheat, exhibiting remarkable antioxidant and anti-inflammatory multi-target bioactivity. These natural products hold significant application prospects in areas such as functional food formulation optimization, drug precursor development, and innovative raw materials for anti-aging cosmetics. However, current research on flavonoids in quinoa primarily focuses on quinoa grains, while the regulatory mechanisms of flavonoid synthesis pathways and the characteristics of monomeric components in quinoa sprouts (the most metabolically active phenological stage) remain unclear. Of particular interest is the lack of reports on the functional evaluation and rapid detection methods of monomeric flavonoid components in quinoa sprouts.

[0003] Existing reports on the detection of flavonoids, primarily using ultraviolet spectrophotometry or liquid chromatography, suffer from the following technical bottlenecks: weak specificity for flavonoid components, low accuracy of results, and the inadequacy of single-component content determination to comprehensively reflect the group characteristics of flavonoid components in *Chenopodium album*. Chinese patent document CN112666302A discloses a method for identifying and rapidly detecting active flavonoid components in barley seedlings. However, due to the diversity of chemical structures of flavonoid components in *Chenopodium album* and barley seedlings, differences in extraction methods, and varying levels of research on detection standardization, the separation conditions for barley seedlings, based on long-term basic research and standardization, are highly adapted to known flavonoid structures. In contrast, *Chenopodium album* requires further phytochemical analysis and condition optimization to establish a detection system specifically targeting its flavonoid characteristics. Currently, no reports have been published on the identification and rapid detection methods for active flavonoid compounds in *Chenopodium album*. Summary of the Invention

[0004] The purpose of this invention is to address the current lack of evaluation indicators and rapid quantitative detection methods for active flavonoids in quinoa, especially the lack of evaluation indicators for their antioxidant and gout-treating functions, and to provide a method for identifying and rapidly detecting active flavonoid compounds in quinoa.

[0005] Previously, we isolated 16 flavonoids from the n-butanol fraction, five of which were isolated and identified from *Chenopodium album* for the first time and are among the most abundant components in the plant. This invention employs a research approach of active compound tracking and separation, along with structural identification. It comprehensively utilizes chromatographic and spectroscopic methods to systematically separate and identify flavonoids from *Chenopodium album*. Using established antioxidant and gout-treating activity testing models, the bioactivity of the flavonoids is evaluated. Five representative flavonoid compounds are selected as evaluation indicators to establish a method for evaluating the quality of *Chenopodium album*, thus providing a solid foundation and scientific basis for the in-depth development of *Chenopodium album*.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A. The chemical components in quinoa were systematically isolated, and the antioxidant activity and the n-butanol fraction for treating gout were evaluated and tracked using DDPH in vitro experiments and xanthine oxidase inhibition rate.

[0008] B. Separate and purify the main flavonoid components of the active site, and identify their structures.

[0009] C. Evaluation of the antioxidant and gout treatment activities of isolated flavonoid monomer components.

[0010] D. Establishment of a UPLC method for simultaneous detection of five active flavonoid components.

[0011] Dry samples of *Chenopodium album* were extracted with 80% ethanol using a room-temperature percolation method. The total extract was then fractionally extracted using extraction solvents of different polarities (petroleum ether, dichloromethane, ethyl acetate, and n-butanol). Combined with DPPH antioxidant and xanthine oxidase activity evaluation experiments, the n-butanol extract fraction was identified as the enriched fraction of flavonoids with antioxidant and xanthine oxidase inhibitory activities. The n-butanol fraction of *Chenopodium album* was separated and purified using a chromatography column packed with silica gel, MCI, Sephades LH-20 dextran gel, and ODS. Based on physicochemical properties and spectroscopic data, the structures of the compounds were identified, and five active flavonoid components in *Chenopodium album* were determined: holmarin A, quercetin-3- O-β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β -D-glucuronic acid. A UPLC method for the simultaneous detection of five isolated active flavonoid components was established, using these five components as reference standards.

[0012] The application adopts chemical system separation combined with active site tracking, firstly determines 5 active flavone components in the leaf mustard as the functional component evaluation indexes for anti-oxidation and gout treatment, establishes an UPLC synchronous detection method of the 5 active flavone components, makes up the deficiency of the existing evaluation system and quality control method, and provides a scientific basis for further processing and utilization and quality control of the leaf mustard.

[0013] In a first aspect of the application, a method for identifying and rapidly detecting active flavone compound groups in leaf mustard is provided, comprising the following steps:

[0014] A, 5 kg of leaf mustard is added into 80% ethanol, and is extracted by percolation method, and the extract is concentrated to obtain total extract, which is suspended in deionized water and then extracted with petroleum ether, dichloromethane, ethyl acetate and n-butanol in sequence to obtain n-butanol extract;

[0015] B, the n-butanol extract is subjected to chromatography on a CHP20 macroporous resin column, and gradient elution is performed with ethanol-water to obtain flavone compounds;

[0016] C, the n-butanol extract of the leaf mustard is separated and purified by chromatography on a chromatographic column filled with silica gel, MCI, Sephadex LH-20 dextran gel and ODS, the structure of the compounds is identified according to the physical and chemical properties and spectral data, and the main flavone monomer components with antioxidant activity and for gout treatment are obtained by combining the evaluation of antioxidant activity and xanthine oxidase inhibitory activity, and it is determined that quercetin-3-O-rutinoside A, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glucuronide and isorhamnetin-3-O-glucuronide are the functional component evaluation indexes of flavone antioxidant and gout treatment in the leaf mustard; O-β O-β O-β O-β

[0017] D, quercetin-3-O-rutinoside A, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, kaempferol-3-O-glucuronide and isorhamnetin-3-O-glucuronide are used as the control samples, and the content of the flavone components is detected by the method of synchronous detection of active flavone components in the leaf mustard by using the ultra-high performance liquid chromatography. O-β O-β O-β O-β

[0018] ​​​​​​​​Further, the step C adopts the method of DPPH antioxidant and xanthine oxidase inhibition activity tracking to determine the active site; and then the chromatographic column with silica gel, MCI, Sephadex LH-20 dextran gel and ODS as the filler is used for separation and purification to obtain the antioxidant monomer compound; the monomer compound for treating gout is obtained by combining the evaluation of xanthine oxidase inhibition activity; and finally the monomer flavone compound with high content and good activity is selected as the quality evaluation index of the functional flavone ingredient group in the S. viridis.

[0019] Further, the step D adopts the ultra-high performance liquid chromatography method for synchronous detection of the active flavone ingredient mixed standard in the S. viridis, which comprises the following steps.

[0020] d1, preparation of the control sample stock solution: accurately take a proper amount of five control samples, and add 1 ml of 70% methanol solution to prepare the mixed control sample solution with the concentration of 0.52 mg / ml of wintergreen glycoside A, 0.21 mg / ml of quercetin-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide, 0.03 mg / ml of quercetin-3-O-rhamnopyranosyl-3'-O-β-D-glucoside, 0.07 mg / ml of kaempferol-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide and 0.025 mg / ml of isorhamnetin-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide. O-β O-β d1, preparation of the control sample stock solution: accurately take a proper amount of five control samples, and add 1 ml of 70% methanol solution to prepare the mixed control sample solution with the concentration of 0.52 mg / ml of wintergreen glycoside A, 0.21 mg / ml of quercetin-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide, 0.03 mg / ml of quercetin-3-O-rhamnopyranosyl-3'-O-β-D-glucoside, 0.07 mg / ml of kaempferol-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide and 0.025 mg / ml of isorhamnetin-3-O-rhamnopyranosyl-3'-O-β-D-glucuronide. O-β O- β

[0021] d2, preparation of the sample solution: accurately take 1.0 g of the sample in a round-bottom flask, accurately transfer 10 ml of 70% methanol solution, weigh, reflux in a 70 ℃ water bath for 0.5 h, cool to room temperature, supplement the weight, take the filtrate through a 0.22 μm microporous filter to obtain the sample solution.

[0022] d3, determination of the content of the five active flavone ingredients in the S. viridis: the control sample solution and the sample solution are analyzed by the ultra-high performance liquid chromatograph under the same chromatographic conditions, and the content of the five flavone ingredients is calculated by the peak area external standard method.

[0023] Further, in the step d2, the optimal solid-liquid ratio of the S. viridis powder and the added 70% methanol solution is 1:10 (g / mL). In a preferred embodiment of the present application, the sample solution preparation method in the step d2 is as follows: take 1.0 g of the sieved S. viridis powder, accurately add 10 mL of 70% methanol solution, weigh, reflux in a 70 ℃ water bath for 1 h, take out and cool, supplement the weight after cooling to room temperature, shake, filter, take the filtrate through a 0.22 μm microporous filter as the test sample solution.

[0024] ​​​Furthermore, in step d3, the chromatographic column used is: Waters ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 × 100 mm); detection wavelength: 360 nm; mobile phase A: methanol, mobile phase B: 0.4% glacial acetic acid water; column temperature: 20 ℃; injection volume: 1 μL.

[0025] Furthermore, in step d3, the mobile phase in the liquid chromatography analysis is A: methanol, mobile phase B: 0.4% glacial acetic acid water, the gradient elution program is shown in Table 1, and the flow rate is 0.2 mL / min.

[0026] Table 1 Gradient elution program

[0027]

[0028] In a second aspect, the present invention provides holmarin A and quercetin-3- O-β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β Application of -D-glucuronic acid as a quality evaluation index for flavonoids with antioxidant and gout-treating properties in the quality evaluation of active flavonoid components in quinoa.

[0029] In a third aspect, this invention provides a method for evaluating the quality of active flavonoid components in quinoa. This method uses the following five active flavonoid monomers as quality evaluation indicators for flavonoids in quinoa that possess antioxidant and gout-treating properties, and identifies and detects them using the method described above: holmarin A, quercetin-3- O-β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β -D-glucuronic acid.

[0030] In a fourth aspect, this invention provides a method for screening quinoa varieties with antioxidant and gout-treating properties. This method employs the aforementioned identification and rapid detection method for active flavonoid compounds in quinoa to detect holmarin A and quercetin-3- O-β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β The content of -D-glucuronic acid was determined by selecting five varieties with high content of active flavonoid components.

[0031] The advantages of this invention are:

[0032] 1. This invention targets the chemical components in quinoa, using solvents of different polarities for extraction and systematic separation, employing DPPH in vitro experiments to track antioxidant activity sites, evaluating xanthine oxidase inhibition rate to determine that the n-butanol extraction site is the enriched site of antioxidant and gout-treating flavonoids, and providing a technical route for the extraction and separation of active flavonoids in quinoa and reference indicators for quality evaluation.

[0033] 2. This invention identifies five main antioxidant flavonoid components in quinoa: holmyl glycoside A, quercetin-3- O- β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β -D-glucuronic acid was identified as a quality evaluation index for flavonoids in quinoa that have antioxidant and gout-treating properties.

[0034] 3. This invention establishes a UPLC method for simultaneous detection of five active flavonoid components by precisely defining the separation and preparation of five flavonoid reference standards and the conditions for ultra-high performance liquid chromatography. The chromatographic separation effect is good, with a resolution of >1.5 and a theoretical plate number of not less than 7000.

[0035] 4. This invention is the first to achieve simultaneous detection of five flavonoid components in quinoa. The method is simple and quick to operate, with high accuracy, high precision, and good repeatability. It can be used to simultaneously determine the content of five flavonoids in different quinoa samples using mixed standards. This provides a basis for the screening of quinoa varieties rich in active flavonoids, the development and quality control of quinoa products, and provides a scientific basis for the breeding of quinoa varieties with high active flavonoid content and the further development and quality control of quinoa-related products. It has high application value. Attached Figure Description

[0036] Figure 1 : DPPH scavenging rate of different concentrations of quinoa; A: total extract; B: dichloromethane; C: ethyl acetate; D: n-butanol.

[0037] Figure 2 Chromatogram for specificity assessment of quinoa; 1: Ilex glycoside A; 2: Quercetin-3- O-β -D-glucuronic acid; 3: Quercetin-3- O-β -D-glucoside; 4: Kaempferol-3- O-β -D-glucuronic acid; 5:Isorhamnetin-3- O-β -D-glucuronic acid; a: Chenopodium album sample solution; b: control solution; c: 70% methanol solution. Detailed Implementation

[0038] The specific embodiments provided by the present application are described in detail below with reference to the examples.

[0039] Example 1: Identification and rapid detection of active flavonoid components in goosefoot

[0040] 1. Test material

[0041] The test material was goosefoot, provided by the Plant Cell Engineering Laboratory of the Biotechnology Institute of Shanghai Agricultural Academy.

[0042] 2. Methods

[0043] 2.1 Extraction of goosefoot and determination of active extraction fractions

[0044] Preparation of goosefoot extract: Goosefoot was placed in a stainless steel filter of BXG-400, and 80% ethanol 470L was used for percolation extraction. The combined extract was concentrated under reduced pressure, and TLC was used to detect whether the extraction was complete. A total of 1.07 kg of goosefoot extract was obtained, with an extraction yield of 21%.

[0045] Preparation of goosefoot extract: Goosefoot was placed in a stainless steel filter of BXG-400, and 80% ethanol 470L was used for percolation extraction. The combined extract was concentrated under reduced pressure, and TLC was used to detect whether the extraction was complete. A total of 1.07 kg of goosefoot extract was obtained, with an extraction yield of 21%.

[0046] Preparation of goosefoot extract: Goosefoot was placed in a stainless steel filter of BXG-400, and 80% ethanol 470L was used for percolation extraction. The combined extract was concentrated under reduced pressure, and TLC was used to detect whether the extraction was complete. A total of 1.07 kg of goosefoot extract was obtained, with an extraction yield of 21%.

[0047] The DPPH free radical scavenging experiment was performed as follows:

[0048] DPPH solution was prepared and used immediately, with 95% ethanol to prepare a standard reagent with a concentration of 0.2 mM, and stored in the dark. The reaction system was divided into sample group, control group and solvent group, 250 μl of 80% ethanol solution was added to the solvent group, 50 μl of sample solution was taken from the sample group, 50 μl of 95% ethanol solution was taken from the control group, and finally 200 μl of DPPH solution was added to the sample group and the control group at the same time, and the absorbance A was measured at 530 nm wavelength after incubation in the dark for 30 min. Three groups of experiments were determined in parallel, and the average value was calculated to calculate the clearance rate. The formula is as follows:

[0049] Clearance rate = [(A control group - (A sample group - A solvent group)) / A control group] x 100%

[0050] Evaluation of xanthine oxidase inhibitory activity

[0051] A 96-well cell culture plate was used, and the reaction system was divided into blank control group, enzyme group and sample group. 160 μL of PBS was added to 40 μL of xanthine solution in the blank control group, 120 μL of PBS was added to 40 μL of xanthine solution in the enzyme group, 80 μL of PBS was added to 40 μL of xanthine solution and 40 μL of sample solution in the sample group, and finally 40 μL of xanthine oxidase solution was added to the enzyme group and the sample group at the same time. The time was counted from the addition of xanthine oxidase solution, and each group was measured every 20 s, the reaction time was 5 min, and the absorbance A was measured at 295 nm wavelength. Three groups of experiments were determined in parallel, and the average value was calculated to calculate the inhibition rate. The formula is as follows:

[0052] Inhibition rate = (K 酶 -K 样品 ) / (K 酶 -K 空白 ) x 100%

[0053] Note: K: the slope of the linear fitting of the ultraviolet display data with time as the abscissa and absorbance as the ordinate.

[0054] 2.2 Separation and purification of spinach extract

[0055] The n-butanol part was dissolved with water, and MCI column chromatography elution was carried out with different concentrations of ethanol solution. After elution, TLC thin layer chromatography detection was carried out, and similar fractions were collected and combined. The method of DPPH antioxidant activity tracking was used to determine the active part. Various separation methods such as MCI reverse phase column chromatography, ODS column chromatography, silica gel column chromatography and ultra-high performance liquid chromatography were used for active tracking, separation and purification to obtain monomer compounds.

[0056] 2.3 Determination of flavonoid reference substance

[0057] The monomer compounds were subjected to DPPH antioxidant and xanthine oxidase inhibition activity evaluation, and the monomer flavonoid compounds with high content and good activity were selected as the control.

[0058] 2.4 Preparation of control stock solution

[0059] An appropriate amount of control was accurately weighed and added to 70% methanol to prepare a mixed control stock solution with a certain mass concentration.

[0060] 2.5 Preparation of test sample solution

[0061] The extraction method was investigated by taking the control, and on this basis, the extraction time, solvent volume fraction, and solid-liquid ratio were investigated by single factor. By comparing the peak area of each component, the optimal preparation method of the test sample solution was determined.

[0062] 2.6 Determination of active flavonoid content

[0063] The control solution and test sample solution were analyzed by ultra-high performance liquid chromatograph under the same chromatographic conditions. The liquid chromatography conditions were as follows,

[0064] Chromatographic column: Waters ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 x 100 mm);

[0065] Detection wavelength: 360 nm;

[0066] Mobile phase A: methanol, mobile phase B: 0.4%-acetic acid water;

[0067] Column temperature 20 ℃; injection volume 1 μL.

[0068] The gradient elution program was: 0-18 min, 75-81% A, flow rate 0.2 mL / min; 18-36 min, 64-74% A, flow rate 1.2-1.5 mL / min; 36-40 min, 55-64% A, flow rate 0.2 mL / min.

[0069] 3 Results

[0070] 3.1 Extraction of goosefoot and determination of active extraction fraction

[0071] The different solvent extraction fractions of goosefoot alcohol extract were evaluated for antioxidant activity and gout treatment activity by DPPH free radical scavenging experiment and xanthine oxidase inhibition activity. From the results (Table 1) Figure 1 and Table 2), the n-butanol fraction was the active extraction fraction.

[0072] Table 2 Inhibition activity evaluation of goosefoot series extracts on xanthine oxidase

[0073]

[0074] 3.2 Isolation and purification of Chenopodium album extract

[0075] The active site was determined using the DPPH antioxidant and xanthine oxidase inhibitory activity evaluation method. Figure 2 The active components were then separated and purified using various chromatographic methods to obtain monomeric flavonoid compounds.

[0076] 3.3 Determination of Flavonoid Reference Standards

[0077] Among monomeric flavonoids, holmarin A and quercetin-3- O-β -D-glucuronic acid, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronic acid, isorhamnetin-3- O-β D-glucuronic acid has high activity and content. In order to comprehensively consider the quality of quinoa, quality control studies were conducted on it from the aspects of activity and composition. Five active flavonoid components were identified as reference standards (Table 3), and a UPLC mixed standard simultaneous detection method for the five active flavonoid components was established.

[0078] Table 3. DPPH scavenging rate and inhibition rate of xanthine oxidase activity of five monomeric compounds.

[0079]

[0080] 3.4 Preparation of reference stock solution

[0081] Accurately weigh appropriate amounts of five reference standards and add them to 1 ml of 70% methanol solution to prepare a solution with a concentration of 0.52 mg / ml for holmarin A and 0.21 mg / ml for quercetin-3-. O-β -D-glucuronic acid, 0.03 mg / ml quercetin-3- O-β -D-glucoside, 0.07 mg / ml kaempferol-3- O-β -D-glucuronic acid, 0.025 mg / ml isorhamnetin-3- O-β A mixed reference solution of -D-glucuronic acid.

[0082] 3.5 Preparation of the test sample solution

[0083] (1) Investigation of extraction method: Accurately weigh 1.0 g of sample and place it in a round bottom flask. Accurately transfer 10 ml of 70% methanol solution into the flask, weigh it, sonicate for 0.5 h, reflux in a 70 ℃ water bath for 0.5 h, cool to room temperature and weigh the remaining sample. Take the filtrate and filter it through a 0.22 μm microporous membrane for chromatographic analysis.

[0084] The results show that the peak area of each component under reflux extraction is significantly higher than that under ultrasonic extraction, so reflux extraction is selected as the final extraction method (Table 4).

[0085] Table 4 Results of extraction method investigation (n = 3)

[0086]

[0087] (2) Investigation of extraction solvent: 1.0 g of sample was precisely weighed into a round-bottom flask, and 10 ml of water, 50% methanol, 70% methanol solution, and 100% methanol was added, respectively. The weight was determined, and reflux was carried out at 70°C water bath for 1 h. After cooling to room temperature, the weight was made up, and the filtrate was filtered through a 0.22 μm microporous filter membrane to obtain the sample for chromatographic analysis.

[0088] The results show that when the extraction solvent is 70% methanol solution, the average peak area of each compound is the highest, so 70% methanol is selected as the best extraction solvent (Table 5).

[0089] Table 5 Results of extraction solvent investigation (n = 3)

[0090]

[0091] (3) Investigation of extraction time: 1.0 g of sample was precisely weighed into a round-bottom flask, and 10 ml of 70% methanol solution was precisely transferred. The weight was determined, and reflux was carried out at 70°C water bath for 0.5 h, 1 h, and 1.5 h, respectively. After cooling to room temperature, the weight was made up, and the filtrate was filtered through a 0.22 μm microporous filter membrane to obtain the sample for chromatographic analysis.

[0092] The results show that when the extraction time gradually increases, the peak area of the compounds increases slowly, and the average peak area of each compound is less than 5%, so 0.5 h is selected as the best extraction time in order to save time (Table 6).

[0093] Table 6 Results of extraction time investigation (n = 3)

[0094]

[0095] (4) Investigation of extraction temperature: 1.0 g of sample was precisely weighed into a round-bottom flask, and 10 ml of 70% methanol solution was precisely transferred. The weight was determined, and reflux was carried out at 60°C, 70°C, and 80°C water bath for 0.5 h, respectively. After cooling to room temperature, the weight was made up, and the filtrate was filtered through a 0.22 μm microporous filter membrane to obtain the sample.

[0096] The results showed that with the increase of extraction temperature, the content of compounds would rise, but the content of compounds at 70℃ and 80℃ extraction temperature tended to be the same, so 70℃ was selected as the final extraction temperature (Table 7).

[0097] Table 7 Investigation results of extraction temperature (n = 3)

[0098]

[0099] (5) Investigation of the ratio of solid to liquid: 1.0 g of sample was precisely weighed in a round-bottom flask, 10 ml, 15 ml, and 20 ml of 70% methanol solution was precisely transferred, the weight was determined, and it was refluxed in a 70℃ water bath for 0.5 h. After cooling to room temperature, the weight was made up, and the filtrate was filtered through a 0.22 μm microporous filter membrane to obtain the sample solution.

[0100] The results showed that when the ratio of solid to liquid was 1:10, 1:15, and 1:20, the content of each compound did not have an obvious upward trend, so 1:10 was selected as the best ratio of solid to liquid in order to save reagents (Table 8).

[0101] Table 8 Investigation results of the ratio of solid to liquid (n = 3)

[0102]

[0103] In summary, the final extraction method of the sample solution was determined as follows: 1.0 g of sample was precisely weighed in a round-bottom flask, 10 ml of 70% methanol solution was precisely transferred, the weight was determined, and it was refluxed in a 70℃ water bath for 0.5 h. After cooling to room temperature, the weight was made up, and the filtrate was filtered through a 0.22 μm microporous filter membrane to obtain the sample solution.

[0104] 3.6 Investigation of the methodology:

[0105] (1) Investigation of specificity:

[0106] The above sample solution and the control solution were injected into the ultra-high performance liquid chromatograph, and the sample was injected for chromatographic analysis. The specific results are shown in Table 9. Figure 2 The peak times of each component in the control and sample solutions were consistent, and there was no impurity peak interference.

[0107] (2) Investigation of linear relationship: 0.5 μg / ml, 1.0 μg / ml, 2.0 μg / ml, 4.0 μg / ml, 8.0 μg / ml, and 16.0 μg / ml of Ilex saponin A, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, kaempferol-3-O-glucuronide, kaempferol-3-O-glucoside, and isorhamnetin-3-O-glucuronide were precisely weighed and dissolved in methanol to prepare the standard solution. O-β O- β O-β O-β ​​​- D-glucuronie acid control substance was dissolved in 1 mL volumetric flask, added to the scale, shaken, and the control mother liquor was obtained. The control mother liquor was diluted according to the original concentration of 50%, 25%, 12.5%, 6.25% to prepare a series of gradient solutions, and the concentration (mg / mL) of the control solution was taken as the abscissa (X), and the peak area was taken as the ordinate (Y). The standard curves of quercetin-3-O-rhamnoside, quercetin-3-O-rhamnoside, kaempferol-3-O-rhamnoside, and isorhamnetin-3-O-rhamnoside were drawn, and the regression equation was calculated (Table 9). O-β - D-glucuronie acid, quercetin-3-O-rhamnoside, O-β - D-glucuronie acid, quercetin-3-O-rhamnoside, O-β - D-glucuronie acid, quercetin-3-O-rhamnoside, O-β - D-glucuronie acid, quercetin-3-O-rhamnoside,

[0108] Table 9 Linear range of different components (n=3)

[0109]

[0110] (3) Precision test: 6 parallel test solutions were prepared, 1 μL of the test solution was precisely taken, and chromatographic determination was performed. The results showed that the RSD values of the peak areas of each component were less than 3%, indicating that the instrument precision was good (Table 10).

[0111] Table 10 Precision test results (n=6)

[0112]

[0113] (4) Reproducibility test: the same batch of spinach sample was prepared according to the above method, and the sample was analyzed. The RSD values of the peak areas of the 5 components of each sample were calculated. The results showed that the RSD values of the peak areas of the six components were less than 2%, indicating that the reproducibility of the simultaneous determination of the 5 components by ultra-high performance liquid chromatography was good (Table 11).

[0114] Table 11 Reproducibility test results (n=6)

[0115]

[0116] (5) Stability test: the same batch of spinach sample was prepared according to the above method, and the sample was analyzed at 0, 2, 4, 8, 16, and 24 h, respectively. The RSD values of the peak areas of the 5 components of each sample were calculated. The results showed that the RSD values of the 5 components were less than 2%, indicating that the test solution was stable within 24 h (Table 12).

[0117] Table 12 Stability test results (n=6)

[0118]

[0119] (6) The sample recovery rate experiment: 0.5 g of amaranth sample was precisely weighed and placed in a round-bottom flask, and the corresponding control was added according to the 100% content in the sample, and the test solution was prepared according to the above conditions. Repeat the above operation to obtain 6 test solutions. Each was analyzed by injection according to the above chromatographic conditions. The unit peak area value was determined, and the content and average sample recovery rate were calculated (Table 13). The results are shown in the table below.

[0120] Table 13: Sample recovery rate test results (n = 6)

[0121]

[0122] 3.7 Determination results of different batches of samples

[0123] Table 14: Sample information table

[0124]

[0125] The different batches of samples (Table 14) were prepared in triplicate according to the above test solution preparation method, and were analyzed by injection according to the above chromatography. The peak areas of the 5 components to be tested were recorded in turn and the average value was calculated. The average value was substituted into the linear regression equation to obtain the content of each component in different batches (Table 15), and the specific data are shown in the table below:

[0126] Table 15: Determination results of 5 flavonoid components (mg / g, n = 3)

[0127]

[0128] The preferred embodiments of the present application have been described in detail above, but the present application is not limited to the described embodiments, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present application. These equivalent modifications or substitutions are all included in the scope defined by the claims of the present application.

Claims

1. A method for identifying and rapidly detecting an active flavonoid compound group in Chenopodium quinoa, characterized in that, It comprises the following steps: A, taking the leaf mustard and adding 80% ethanol, percolation extraction, the extract was concentrated to get the total extract, after suspended in deionized water, sequentially extracted with petroleum ether, dichloromethane, ethyl acetate, n-butanol, and the n-butanol extract was obtained; B, the n-butanol extract was chromatographed by CHP20 macroporous resin column, and eluted with ethanol-water gradient to obtain flavonoids; C, the silica gel, MCI, Sephadex LH-20 dextran gel, ODS as filler chromatographic column is used to the Chenopodium quinoa L. n-butanol fraction is separated and purified, according to the physicochemical properties and spectrum data identify compound structure, combined with antioxidant activity and xanthine oxidase inhibition activity evaluation, get the main antioxidant activity and for gout treatment flavonoid monomer composition, determine the Aescin, quercetin-3- O-β -D-glucuronide, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronide, isorhamnetin-3- O-β -D-glucuronide, isorhamnetin-3-, is the quality evaluation index of functional ingredient group of flavonoids antioxidant and gout treatment in Chenopodium quinoa L. D, quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide O-β D, quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide O-β D, quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide O- β D, quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide O-β The D-glucuronide monomer component is a control, and the content of the flavonoid component is detected by the method of synchronous detection of the active flavonoid component mixed standard in the goosefoot.

2. The method for identifying and rapidly detecting active flavonoid compounds in Chenopodium quinoa according to claim 1, characterized in that, The step C adopts the method of DPPH antioxidant and xanthine oxidase inhibition activity tracking to determine the active fraction; then, the chromatography column with silica gel, MCI, Sephadex LH-20 dextran gel and ODS as the filler is used for separation and purification to obtain antioxidant monomer compounds; the monomer compounds for treating gout are obtained by combining the evaluation of xanthine oxidase inhibition activity, and finally the monomer flavone compounds with high content and good activity are selected as the quality evaluation index of the functional flavone component group in leaf mustard.

3. The method for identifying and rapidly detecting active flavonoid compounds in Chenopodium quinoa according to claim 1, characterized in that, The step D is an ultra-high performance liquid chromatography method for synchronous detection of active flavone components in leaf mustard, which comprises the following steps: d1, Preparation of the control stock solution: accurately weigh 5 kinds of control substances, add 1 ml of 70% methanol solution to prepare a mixture control solution with the concentration of 0.52 mg / ml of quercitrin A, 0.21 mg / ml of quercetin-3-O- O-β -D-glucuronide, 0.03 mg / ml of quercetin-3- O- β -D-glucoside, 0.07 mg / ml of kaempferol-3- O-β -D-glucuronide, 0.025 mg / ml of isorhamnetin-3- O-β -D-glucuronide d2, sample solution preparation: accurately weigh 1.0 g of sample in a round-bottom flask, accurately transfer 10 ml of 70% methanol solution, weigh, reflux in a 70 ℃ water bath for 0.5 h, cool to room temperature, supplement the weight, take the filtrate through a 0.22 μm microporous filter membrane, and obtain the sample solution; d3, determination of the content of five active flavone components in leaf mustard: the control sample solution and the sample solution are analyzed by ultra-high performance liquid chromatography under the same chromatographic conditions, and the content of five flavone components is calculated by peak area external standard method.

4. The method for identifying and rapidly detecting the active flavonoid compound group in Chenopodium quinoa according to claim 3, characterized in that, In the step d2, the ratio of leaf mustard powder to 70% methanol solution is 1:10 g / mL.

5. The method of identifying and rapidly detecting the active flavonoid compound group in Chenopodium quinoa according to claim 3, characterized in that, In the step d3, the chromatographic column type used is Waters ACQUITY UPLC BEH-C18; the detection wavelength is 360 nm; the mobile phase A is methanol, the mobile phase B is 0.4% glacial acetic acid water; the column temperature is 20 ℃; and the injection amount is 1 μL.

6. quercetin-3-O-β-D-glucuronide, quercetin-3-O-β-D-glucuronide O-β -D-glucuronide, quercetin-3-O-β-D-glucuronide O-β -D-glucuronide, quercetin-3-O-β-D-glucuronide O-β -D-glucuronide, quercetin-3-O-β-D-glucuronide O-β -D-glucuronide, quercetin-3-O-β-D-glucuronide 7. A method for evaluating the quality of an active flavonoid component group in Chenopodium quinoa, characterized by, The quality evaluation method is to use five active flavonoid monomer components of wintergreen A, quercetin-3- O-β -D-glucuronide, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronide, isorhamnetin-3- O-β -D-glucuronide as the quality evaluation index of the flavonoids in the Chenopodium that have antioxidant and gout treatment, and the method is identified and detected according to any one of claims 1-5.

8. A method for screening of Chenopodium quinoa varieties having antioxidant and anti- gout properties, characterized by, is to use the active flavonoid compound group in S. japonica L. as claimed in any one of claims 1-5 to detect wintergreen glycoside A, quercetin-3- O-β -D-glucuronide, quercetin-3- O-β -D-glucoside, kaempferol-3- O-β -D-glucuronide, isorhamnetin-3- O-β -D-glucuronide, and select 5 varieties with high content of active flavonoid components.

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