Method for simultaneously determining eight flavonoid compounds in peach blossom

Abstract

The invention discloses a method for simultaneously determining eight flavonoid compounds in peach blossoms. According to the method, the content of the flavone compounds in the peach blossoms is measured by utilizing a high performance liquid chromatography, and a C18 chromatographic column with small filler particle diameter and long column length is selected, so that the separation degree is improved, and the detection time is shortened; according to the method, the contents of eight flavone compounds such as rutin, hyperoside, isoquercitrin, kaempferol-7-O-glucoside, quercitrin, trilobatin, violet cloud glycoside and afzelin in the peach blossom can be efficiently and simultaneously measured.

Description

Technical Field

[0001] The present invention belongs to the technical field of determination of active substance content in peach by-products, and particularly relates to a method for simultaneously determining 8 flavonoid compounds in peach blossoms. Background Art

[0002] Peach blossom is the flower of the deciduous tree peach in the Rosaceae family, which has the effects of beauty care and health care and is widely used in the food and medicine fields. Research shows that peach blossoms contain various chemical components such as flavonoids, polyphenols, polysaccharides, carotenoids, various vitamins, amino acids and trace elements, and have high nutritional and medicinal values. Flavonoid compounds have various pharmacological effects such as anti-tumor, anti-inflammatory and analgesic, immune regulation, blood sugar lowering, treatment of osteoporosis, anti-radiation, etc., and have broad application prospects.

[0003] According to literature reports, peach blossoms contain various flavonoid compounds such as afzelin, quercitrin, trifolin, Hyperoside, isoquercitrin, rutin, astragalin and Kaempferol-7-O-β-D-glucopyranoside. For the method of determining the flavonoid content in peach blossoms, only scholar Cui Lulu used acetonitrile as the mobile phase and adopted HPLC method to simultaneously detect the contents of 3 flavonoids, namely rutin, Hyperoside and isoquercitrin in peach blossoms. In addition to the above 3 flavonoids, peach blossoms are also rich in pharmacologically active flavonoid compounds such as quercitrin, trifolin, astragalin and afzelin, but there is no literature report.

[0004] In the actual sample detection, the polarities of quercitrin and astragalin in peach blossoms are similar, and their retention times in the C 18 chromatographic column are similar. Especially when using acetonitrile with stronger elution ability as the mobile phase, it is difficult to achieve baseline separation and cannot be accurately detected. In addition, the elution orders of flavonoid compounds with different polarities in the C 18 chromatographic column are inconsistent due to different mobile phases. Summary of the Invention

[0005] Aiming at the problem in the prior art that the polarities of quercitrin and astragalin in peach blossoms are similar, and their retention times in the C 18 chromatographic column are similar. Especially when using acetonitrile with stronger elution ability as the mobile phase, it is difficult to achieve baseline separation and cannot be accurately detected; the elution orders of flavonoid compounds with different polarities in the C 18There are problems such as inconsistent elution order due to different mobile phases in the chromatographic column and limitations in the detection method of peach blossom flavonoids. The present invention provides a method for simultaneously determining 8 flavonoid compounds in peach blossoms. This method uses high-performance liquid chromatography to determine the content of flavonoid compounds in peach blossoms. By selecting a C chromatographic column with a small filler particle diameter and a short column length 18 chromatographic column, screening a suitable mobile phase, thereby improving the separation efficiency and shortening the detection time, achieving the efficient simultaneous determination of the contents of 8 flavonoid compounds such as rutin, hyperoside, isoquercitrin, kaempferol-7-O-glucoside, quercitrin, trifolin, astragalin, and afzelin in peach blossoms, providing a reference basis for the determination of peach blossom flavonoid content, functional development, and rational utilization.

[0006] The object of the present invention is achieved through the following technical solutions:

[0007] A method for simultaneously determining 8 flavonoid compounds in peach blossoms, comprising the following steps:

[0008] S1. Preparation of the test solution: Crush and sieve the dried peach blossoms, weigh 200.0 mg of peach blossom powder and place it in a 5.0 mL brown hard centrifuge tube, add 3.5 mL of 80% (v / v) methanol aqueous solution, extract ultrasonically for 30.0 min, extract 2 times, combine the extracts and make up the volume to 10.0 mL, and filter through a 0.45 μm microporous filter membrane to obtain the test solution of the peach blossom sample;

[0009] S2. Content determination:

[0010] S2-1. Liquid phase conditions:

[0011] Liquid chromatograph: Thermo Scientific Vanquish Core;

[0012] Chromatographic column: YMC-Pack ODS-AM 4.6×150 mm C 18 , 3 μm;

[0013] Mobile phase: methanol (A) and 0.1% formic acid aqueous solution (B);

[0014] Time gradient:

[0015] 0 - 35 min, 29% A;

[0016] 35 - 40 min, 29% A - 34% A;

[0017] 40 - 55 min, 34% A;

[0018] 55 - 60 min, 34% - 45%;

[0019] 60 - 65 min, 45% A;

[0020] 65 - 72 min, 100% A;

[0021] 72 - 80 min, 29%;

[0022] Flow rate: 1.0 mL / min;

[0023] Detection wavelength: 254 nm;

[0024] Column temperature 30°C;

[0025] Injection volume 3.0 μL;

[0026] S2 - 2, Standard working curve and content determination:

[0027] Prepare 8 kinds of flavonoid standard samples with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, and detect according to the steps in S2. Draw a standard curve with the standard concentration as the ordinate (Y) and the peak area as the abscissa (X), and calculate the content of each flavonoid compound in the peach blossom sample according to the standard working curve;

[0028] The 8 kinds of flavonoid compounds are hyperin, isoquercitrin, rutin, kaempferol - 7 - O - glucoside, trifolin, quercitrin, astragalin, and afzelin.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The method for simultaneously determining 8 kinds of flavonoid compounds in peach blossom according to the present invention has more flavonoid compounds determined, higher accuracy, shorter detection time compared with the methods reported in the literature, and corrects the elution order of flavonoid compounds caused by different solvents, providing a reference basis for the determination of flavonoid content in peach blossom, functional development, and pharmacological utilization. Brief description of the drawings

[0031] Figure 1 is the chromatogram of 8 kinds of flavonoid compounds in column A when acetonitrile is used as the mobile phase in the method for simultaneously determining 8 kinds of flavonoid compounds in peach blossom according to the embodiment of the present invention;

[0032] Figure 2 is the chromatogram of 8 kinds of flavonoid compounds in column B when acetonitrile is used as the mobile phase in the method for simultaneously determining 8 kinds of flavonoid compounds in peach blossom according to the embodiment of the present invention;

[0033] Figure 3 is the chromatogram of 8 kinds of flavonoid compounds in column A when the mobile phase is methanol in the method for simultaneously determining 8 kinds of flavonoid compounds in peach blossom according to the embodiment of the present invention;

[0034] Figure 4This is the chromatogram of 8 flavonoid compounds in column B when the mobile phase is methanol in the method for simultaneously determining 8 flavonoid compounds in peach blossoms according to the embodiments of the present invention. Detailed implementation manners

[0035] The following further describes the detailed implementation manners of the present invention in conjunction with embodiments.

[0036] Embodiment:

[0037] A method for simultaneously determining 8 flavonoid compounds in peach blossoms includes the following steps:

[0038] 1. Materials:

[0039] 1.1 Test materials:

[0040] Peach blossoms (Shuibaitao), dried, pulverized, and sieved.

[0041] 1.2 Drugs and reagents:

[0042] Hyperoside (CAS#482-36-0), isoquercitrin (CAS#21637-25-2), rutin (CAS#153-18-4), kaempferol-7-O-glucoside (CAS#16290-07-6), trifolin (CAS#23627-87-4), quercitrin (CAS#522-12-3), astragalin (CAS#480-10-4), and afzelin (CAS#482-39-3) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0043] 2. Experimental method:

[0044] 2.1 Preparation of test solution:

[0045] Weigh 200.0 mg of peach blossom powder and place it in a 5.0 mL brown hard centrifuge tube. Add 3.5 mL of an 80% methanol aqueous solution, extract ultrasonically for 30.0 min, extract twice, combine the extracts and make up the volume to 10.0 mL, and filter through a 0.45 μm microporous filter membrane to obtain the test solution of the peach blossom sample.

[0046] 2.2 Optimization of liquid phase conditions:

[0047] Use a liquid chromatograph (Thermo Scientific Vanquish Core) to optimize and investigate the liquid phase conditions. The chromatographic column investigated was column A (YMC-Pack ODS-AM 4.6×150 mm C 18 , 3 μm) and column B (Thermo Acclaim120 4.6×250 mm C 18,5μm); the aqueous phase in the mobile phase is 0.1% formic acid aqueous solution, and the organic phase is methanol and acetonitrile; the separation between the chromatographic peaks of flavonoid compounds in the sample is optimized by adjusting the proportion of the organic phase in the mobile phase. The other conditions are the same, the column temperature is 30℃, the injection volume is 3μL, the detection wavelength is 254nm, and the flow rate is 1.0mL / min. The optimal chromatographic detection conditions are determined based on the separation and elution order of the eight flavonoid compounds in the peach blossom sample.

[0048] 2.3 Linear relationship investigation:

[0049] Prepare standard concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, perform detection according to the optimal chromatographic detection conditions, plot a standard curve with the standard concentration as the ordinate (y) and the peak area as the ordinate (x), and examine the linear relationship of the detection method.

[0050] 2.4 Precision test:

[0051] The peach blossom sample solution was prepared according to the method in 2.1, and the sample was injected 6 times under the optimal chromatographic detection conditions. The relative standard deviation (RSD) of the peak areas of the eight flavonoid compounds in the peach blossom sample was calculated to evaluate the precision of the instrument.

[0052] 2.5 Repeatability test:

[0053] Accurately weigh 100 mg, 200 mg, and 300 mg of peach blossom powder of the same variety, prepare peach blossom sample test solution according to the method in 2.1, analyze using the optimal chromatographic detection conditions, calculate the relative standard deviation of the peak area, and evaluate the repeatability of the detection method.

[0054] 2.6 Stability test:

[0055] Accurately weigh 200 mg of peach blossom powder, prepare peach blossom sample test solution according to the method under "2.1", accurately pipette 3.0 μL, determine the peak areas of 8 flavonoids under the optimal chromatographic detection conditions at 0, 2, 4, 6, 12, and 24 h, calculate the relative standard deviation of the peak area, and evaluate the stability of the method.

[0056] 2.7 Sample recovery test:

[0057] Accurately weigh 3 portions of peach blossoms of the same variety with known content, each portion is 200 mg, and peach blossom sample solution is prepared according to the method under "2.1.2". High, medium and low quality flavonoid compound standards are added at a content of about 20% to 50% respectively. The total content after adding samples is detected using the optimal chromatographic detection conditions, and the recovery rate is calculated.

[0058] 3. Experimental results

[0059] 3.1 Test conditions

[0060] When methanol (mobile phase A) is used as the mobile phase, the optimal time gradient for column A is gradient I (0 - 35 min, 29% A; 35 - 40 min, 29% A - 34% A; 40 - 55 min, 34% A; 55 - 60 min, 34% - 45%; 60 - 65 min, 45% A; 65 - 72 min, 100% A; 72 - 80 min, 29% A), and the optimal time gradient for column B is gradient II (0 - 35 min, 32% A; 35 - 40 min, 32% A - 35% A; 40 - 60 min, 35% A; 60 - 65 min, 35% - 45%; 65 - 75 min, 45% A; 75 - 82 min, 100% A; 82 - 90 min, 32% A). When acetonitrile (mobile phase A) is used as the mobile phase, the optimal time gradient for column A is gradient III (0 - 30 min, 16% A; 30 - 32 min, 16% A - 22% A; 32 - 40 min, 22% A; 40 - 47 min, 100% A; 47 - 55 min, 16% A), and the optimal time gradient for column B is gradient IV (0 - 40 min, 16% A; 40 - 42 min, 16% A - 22% A; 42 - 55 min, 22% A; 55 - 62 min, 100% A; 62 - 70 min, 16% A). The retention times and resolutions of the peach blossom flavonoid compounds for column A and column B under different elution conditions are shown in Table 1:

[0061] Table 1 Retention times and resolutions of the peach blossom flavonoid compounds for column A and column B under different elution conditions

[0062]

[0063] a - h represent rutin, hyperoside, isoquercitrin, kaempferol - 7 - O - glucoside, trifolin, quercitrin, astragalin, and afzelin respectively; "-" indicates no resolution and the chromatographic peaks overlap.

[0064] According to Table 1, when the mobile phase is methanol, the elution order of the flavonoid compounds in peach blossom for both column A and column B is hyperoside, isoquercitrin, rutin, kaempferol - 7 - O - glucoside, trifolin, quercitrin, astragalin, afzelin. When the mobile phase is acetonitrile, the elution order is rutin, hyperoside, isoquercitrin, trifolin, quercitrin, astragalin, kaempferol - 7 - O - glucoside, afzelin. When acetonitrile is used as the mobile phase, the chromatographic peaks of quercitrin and astragalin for column A and column B overlap ( Figure 1 and Figure 2 ), and when the mobile phase is methanol, the chromatographic peaks of quercitrin and astragalin for column A and column B can be effectively separated ( Figure 3 and Figure 4 ). According to Figure 3 ​, when the mobile phase is methanol, the 8 chromatographic peaks of the peach blossom flavonoid compounds in column A can be effectively separated, while the retention times of isoquercitrin and hyperoside in column B are similar ( Figure 4 ), and the chromatographic peaks overlap and cannot be separated. In addition, when acetonitrile is used as the mobile phase, the chromatographic peak of kaempferol-7-O-glucoside in column A overlaps with the chromatographic peaks of other compounds, affecting the accuracy of detection. When methanol is used as the mobile phase, there are no overlapping chromatographic peaks of other compounds before and after the chromatographic peak of this compound, and the detection accuracy is high. Therefore, in the optimal liquid phase conditions, the chromatographic column: YMC (Pack ODS-AM 4.6×150mm C 18 , 3μm); mobile phase: methanol-0.1% formic acid aqueous solution; time gradient: 0-35 min, 29% A; 35-40 min, 29% A-34% A; 40-55 min, 34% A; 55-60 min, 34%-45%; 60-65 min, 45% A; 65-72 min, 100% A; 72-80 min, 29% A; column temperature: 30°C, injection volume: 3 μL, detection wavelength: 254 nm, flow rate: 1.0 mL / min;

[0065] Figure 1 is the chromatogram of 8 flavonoid compounds in column A when acetonitrile is used as the mobile phase in the method for simultaneously determining 8 flavonoid compounds in peach blossom described in the example;

[0066] Figure 2 is the chromatogram of 8 flavonoid compounds in column B when acetonitrile is used as the mobile phase in the method for simultaneously determining 8 flavonoid compounds in peach blossom described in the example;

[0067] Figure 3 is the chromatogram of 8 flavonoid compounds in column A when the mobile phase is methanol in the method for simultaneously determining 8 flavonoid compounds in peach blossom described in the example;

[0068] Figure 4 is the chromatogram of 8 flavonoid compounds in column B when the mobile phase is methanol in the method for simultaneously determining 8 flavonoid compounds in peach blossom described in the example.

[0069] 3.2 Linear relationship

[0070]

[0071]

[0072] 3.3 Precision test

[0073] The sample was injected 6 times, and the relative standard deviations of the peak areas of 8 flavonoid compounds, namely hyperoside, isoquercitrin, rutin, kaempferol-7-O-glucoside, trifolin, quercitrin, astragalin, and afzelin, were 0.8%, 1.1%, 0.9%, 1.2%, 1.1%, 0.8%, 1.0%, and 1.2% respectively, indicating good instrument precision.

[0074] 3.4 Repeatability test

[0075] The relative standard deviations of the peak areas of 8 flavonoid compounds, namely hyperoside, isoquercitrin, rutin, kaempferol-7-O-glucoside, trifolin, quercitrin, astragalin, and afzelin, were measured to be 1.1%, 0.9%, 1.2%, 1.2%, 1.1%, 0.9%, 1.0%, and 1.0% respectively, indicating good repeatability of this method.

[0076] 3.5 Stability test

[0077] The relative standard deviations of the contents of 8 flavonoid compounds, namely hyperoside, isoquercitrin, rutin, kaempferol-7-O-glucoside, trifolin, quercitrin, astragalin, and afzelin, were measured to be 1.1%, 1.0%, 1.2%, 1.0%, 1.2%, 0.9%, 1.2%, and 1.3% respectively, indicating that the peach blossom sample solution was stable within 24 h at room temperature.

[0078] 3.6 Recovery test

[0079] The total content after spiking was detected under the optimal detection conditions, and the recoveries are shown in Table 3.

[0080] Table 3 Recoveries of different flavonoid compounds in peach blossom

[0081]

[0082]

[0083] The above results show that in the method for simultaneously determining 8 flavonoid compounds in peach blossom of the present invention, the linear relationship, repeatability, stability, precision, and recovery are good, and it has good usability.

[0084] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several improvements and changes are made, and these all belong to the protection scope of the present invention.

Claims

1. A method for simultaneously determining 8 flavonoid compounds in peach blossoms, characterized in that: It includes the following steps: S1. Preparation of the test solution: Crush and sieve the dried peach blossoms, weigh 200.0 mg of peach blossom powder and place it in a 5.0 mL brown hard centrifuge tube, add 3.5 mL of 80% v / v methanol aqueous solution, extract ultrasonically for 30.0 min, extract twice, combine the extracts and make up the volume to 10.0 mL, and filter through a 0.45 μm microporous membrane to obtain the test solution of the peach blossom sample; S2. Content determination: S2-1. Liquid phase conditions: Liquid chromatograph: Thermo Scientific Vanquish Core; Chromatographic column: YMC-Pack ODS-AM 4.6×150mm C 18 , 3μm; Mobile phase: methanol (A) and 0.1% formic acid aqueous solution (B); Time gradient: 0 - 35 min, 29% A; 35 - 40 min, 29% A - 34% A; 40 - 55 min, 34% A; 55 - 60 min, 34% - 45%; 60 - 65 min, 45% A; 65 - 72 min, 100% A; 72 - 80 min, 29%; Flow rate: 1.0 mL / min; Detection wavelength: 254 nm; Column temperature 30 °C; Injection volume 3.0 μL; S2-2. Standard working curve and content determination: Prepare 8 kinds of flavonoid standard samples with concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.5 mg / mL, detect according to step S2, draw a standard curve with the standard product concentration as the ordinate Y and the peak area as the abscissa X, and calculate the content of each flavonoid compound in the peach blossom sample according to the standard working curve; The 8 kinds of flavonoid compounds are hyperin, isoquercitrin, rutin, kaempferol-7-O-glucoside, trifolin, quercitrin, astragalin, and afzelin.

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