Method for extracting flavonoid compounds from wild rice by using deep eutectic solvent

[0041] In the present invention, the preparation method of the deep eutectic solvent preferably includes the following steps: first mixing choline chloride and hydrogen bond donor, and then adding water for second mixing to obtain the deep eutectic solvent. In the present invention, the method of the first mixing and the second mixing is preferably stirring and mixing; the speed of the stirring and mixing is preferably 200 to 800 rpm, more preferably 350 to 550 rpm; the temperature of the first mixing is preferably 45 ~80℃, more preferably 60~80℃, most preferably 80℃; the time of the first mixing is preferably 30~360min, more preferably 60~240min; the temperature of the second mixing is preferably 25~35 ℃, the time is preferably 10-60 min. In the present invention, the deep eutectic solvent is a stable, uniform and transparent liquid. The deep eutectic solvent provided by the present invention is an extractant with strong polarity, which can efficiently break the cell wall of wild rice and promote the dissolution of flavonoids of wild rice, and the deep eutectic solvent is non-toxic, biodegradable, and does not cause secondary pollution. It is green and environmentally friendly, and can be recycled and reused, which greatly reduces the extraction cost. The extractant has high extraction efficiency and low detection limit for flavonoids in wild rice.

[0042] In the present invention, the stirring is preferably carried out using an IKA magnetic stirrer (IKA company, Germany).

[0043] In the present invention, the material-to-liquid ratio of the wild rice powder and the deep eutectic solvent is preferably 10-100 mg/mL, more preferably 20-100 mg/mL, most preferably 30 mg/mL, 40 mg/mL, 50 mg/mL , 60mg/mL, 70mg/mL or 80mg/mL.

[0044] In the present invention, the balance used for weighing is preferably a BSA124S-CW electronic balance (sensitivity 0.0001g, Sartorius Scientific Instruments Beijing Co., Ltd., China).

[0045] In the present invention, the extraction method is preferably ultrasonic extraction. In the present invention, the temperature of the extraction is preferably 30-60°C, more preferably 40-60°C, and most preferably 50°C; the extraction time is preferably 1-20 min, more preferably 5-15 min, most preferably Preferably it is 10 minutes. In the present invention, the power of the ultrasonic extraction is preferably 120-600W, more preferably 150-500W, and most preferably 200W, 300W or 400W; the frequency of the ultrasonic extraction is preferably 20-25kHz, more preferably 21- 25kHz, most preferably 22kHz, 23kHz or 24kHz. In the present invention, the ultrasonic extraction is preferably performed in a KQ-500GVDV type Shuangpin constant temperature numerically controlled ultrasonic generator (Kunshan Ultrasonic Instrument Co., Ltd., China).

[0046] In the present invention, the solid-liquid separation method is preferably centrifugal separation, and the rotation speed of the centrifugal separation is preferably 2500-3000 rpm, more preferably 2600-3000 rpm, most preferably 3000 rpm; the time of the centrifugal separation is preferably 5 ~10min, more preferably 10min.

[0047] After obtaining the flavonoid extract, the present invention performs liquid chromatography to separate the flavonoid extract to obtain flavonoids.

[0048] In the present invention, the flavonoids preferably include catechins, procyanidins and quercetin.

[0049] In the present invention, preferably, the flavone extract is diluted with methanol, filtered, and then separated by liquid chromatography.

[0050] In the present invention, the volume ratio of the flavone extract to methanol is preferably 1:(1 to 4), more preferably 1:(2 to 4), and most preferably 1:(3 to 4). In the present invention, the pore size of the filtration membrane is preferably 0.22 μm.

[0051] In the present invention, the detection conditions of the liquid chromatography include: the mobile phase A is preferably an acetonitrile-acetic acid mixed solution, the volume fraction of acetic acid in the acetonitrile-acetic acid mixed solution is preferably 0.1%; the mobile phase B is preferably acetic acid Aqueous solution, the volume fraction of acetic acid in the aqueous solution of acetic acid is preferably 0.1%; the gradient elution procedure is preferably: Gradient elution procedure: the volume percentage of mobile phase A is increased from 5% to 10% in 0-5min, 5-7min The volume percentage of internal mobile phase A increases from 10% to 20%, the volume percentage of mobile phase A increases from 20% to 60% within 7-8 minutes, and the volume percentage of mobile phase A increases from 60% to 100% within 8-9 minutes. The volume percentage of mobile phase A decreases from 100% to 5% within 9-10 minutes, and the volume percentage of mobile phase A remains at 95% within 10-12 minutes; the flow rate is 0.3mL/min; the injection volume is 1μL; the column temperature is 25°C; the flow rate is preferably 0.3mL/min; the injection volume is preferably 1μL; the chromatographic column is preferably Acquity UPLC BEH C18column (50mm×2.1mm, 1.7μm), and the column temperature is preferably 25°C.

[0052] In the present invention, in order to facilitate the confirmation of the structures of the above five flavonoids obtained by separation, mass spectrometry is preferably used for detection; further preferably, liquid chromatography-mass spectrometry (HPLC-MS) is used for separation and detection, and the instrument used is preferably It is a Waters liquid phase mass spectrometer (Waters company, USA).

[0053] In the present invention, the detection conditions of the mass spectrometry include: preferably negative ionization ESI(-), the mass scanning range is preferably m/z 105-1500, the capillary voltage is preferably 3000V, the cone voltage is preferably 25V, and the ion source The temperature is preferably 100°C, the atomizing gas temperature is preferably 200°C, the cone gas flow rate is preferably 50 L/h, and the atomizing gas flow rate is preferably 400 L/h.

[0055] Example 1

[0056] (1) Preparation of deep eutectic solvent

[0057] Add choline chloride and 1,4-butanediol into a round-bottom flask at a molar ratio of 1:6, add a magnet to the round-bottom flask, and use an IKA magnetic stirrer to magnetically stir at 80°C for 60 minutes. A mixed solution of choline chloride-1,4-butanediol is obtained, and 30 wt% of water is added to the obtained mixed solution to obtain a deep eutectic solvent (abbreviated as DES-1).

[0058] (2) Wild rice pretreatment

[0059] The wild rice is ground with a pulverizer, dried at 40°C until constant weight, and the obtained wild rice powder is stored at -20°C and protected from light for later use.

[0060] (3) Analysis of wild rice powder

[0061] Use BSA124S-CW electronic balance to weigh 500mg of wild rice powder, add 10mL deep eutectic solvent to mix thoroughly, and place it in KQ-500GVDV Shuangpin constant temperature numerical control ultrasonic generator, at 50℃, ultrasonic power is 200W, ultrasonic frequency is Ultrasonic extraction at 25kHz for 15min to obtain a solid-liquid mixture. After cooling the solid-liquid mixture to room temperature, it was placed in a centrifuge and centrifuged at 3000rpm for 10min. The resulting supernatant was the flavonoid extract. The flavonoid extract was mixed with 4 mL of methanol, and the resulting mixture was filtered through a 0.22 μm filter membrane to obtain a brass extract.

[0062] (4) HPLC-MS separation and detection

[0063] The brass extract was subjected to HPLC-MS detection.

[0064] Among them, the liquid chromatography conditions: mobile phase A (0.1% acetic acid in acetonitrile, v/v) and mobile phase B (0.1% acetic acid in water, v/v); the gradient elution procedure is as follows: 0~5min, 5~10 % Mobile phase A; 5~7min, 10~20% mobile phase A; 7~8min, 20~60% mobile phase A; 9~10min, 100~5% mobile phase A; 10~12min, 95% mobile phase A The flow rate is preferably 0.3mL/min; the injection volume is preferably 1μL; the chromatographic column is Acquity UPLC BEH C18 column (50mm×2.1mm, 1.7μm), and the column temperature is 25℃; the HPLC-MS separation and detection adopts The instrument of is preferably a Waters liquid phase mass spectrometer (Waters company, USA);

[0065] Mass spectrometry conditions: negative ionization method ESI(-); mass scanning range m/z is 105~1500; capillary voltage is 3000V; cone voltage is 25V; ion source temperature is 100℃; atomization gas temperature is 200℃; cone The gas flow rate is 50L/h; the atomization gas flow rate is 400L/h.

[0066] (5) Preparation of standard solution

[0067] Mix catechins and methanol to prepare a standard solution with a concentration of 0.5-25μg/mL;

[0068] Mix proanthocyanidins and methanol to prepare a standard solution with a concentration of 0.5-20 μg/mL;

[0069] Mix quercetin and methanol to prepare a standard solution with a concentration of 0.5-20μg/mL;

[0070] Store the standard solution in a refrigerator at 4°C for later use.

[0071] The liquid phase mass spectra of catechins, proanthocyanidins and quercetin in the extract of flavonoids are as follows figure 1 As shown, the mass spectrometric parameter results of catechin, procyanidin and quercetin are shown in Table 1.

[0072] Table 1 Mass spectrometric parameters of flavonoids

[0073]

[0074] by figure 1 As shown in Table 1, the retention times of proanthocyanidins, catechins, and quercetin are 2.65 min, 3.2 min and 8.18 min, respectively, and the three substances have high separation degrees; and the three flavonoid precursor ions and product ions are consistent with the standard, which proves Procyanidins, catechins and quercetin were isolated.

[0075] Table 2 shows the test results of five flavonoids: catechin, procyanidin and quercetin.

[0076] Table 2 Test results of flavonoids

[0077]

[0078] It can be seen from Table 2 that the linear range of catechins is 0.5-25μg/g, the linear ranges of proanthocyanidins and quercetin are 0.5-20μg/g, and the detection limits of catechins, procyanidins and quercetin are 9.747ng respectively /mL, 4.360ng/mL and 0.479ng/mL, the three flavonoids have a wide linear range, low detection limit, good linearity, R 2 All are greater than 0.99, the method is credible, and the reproducibility is good.

[0079] Examples 2~3

[0080] The flavonoids were extracted and detected according to the method of Example 1. The difference from Example 1 is that the deep eutectic solvent is different. The composition of the deep eutectic solvent in Examples 1 to 3 is shown in Table 3. Catechin, proanthocyanidin and quercetin The extraction amounts of the three flavonoids of cortexin are shown in Table 4.

[0081] Table 3 The composition of deep eutectic solvents in Examples 1-7

[0082]