Method for extracting isoorientin from corn stigma through collaborative optimization of natural eutectic solvent and ultrasonic

Abstract

The invention provides a method for extracting isoorientin from corn stigma, and relates to the technical field of extraction of active ingredients. The method comprises the following steps: carrying out drying and crushing pretreatment on corn stigma, fully extracting corn stigma powder by using a natural eutectic solvent, designing three single factors to be combined with ultrasonic treatment, and carrying out vacuum filtration on the treated sample to further enrich the extract. Compared with a traditional chemical synthesis method for preparing the isoorientin, the isoorientin prepared by the method has the advantages that the purity is higher, chemical substances in the product are fewer, the residual quantity of harmful substances is low, the extraction efficiency of the isoorientin is further improved, effective components in the corn stigma can be quickly and fully dissolved out in a short time, and auxiliary products of corn can be recycled.

Description

Technical Field

[0001] The invention relates to the technical field of natural product extraction, and in particular to a method for extracting corn silk flavonoid compounds by natural eutectic and ultrasound synergistically and the optimization of process conditions. Background Art

[0002] Corn silk is the style and stigma of Zea mays L., a grass plant. It tastes sweet and mild, is neutral in nature, and enters the kidney, liver, and gallbladder meridians. It is rich in saponins, flavonoids, chelate elements, and other active ingredients. It can regulate blood sugar levels by promoting insulin secretion and enhancing the biological activity of insulin. It also has antioxidant activity that reduces the production of free radicals and protects the cardiovascular system. Previously, isoorientin extracted from bamboo leaves and hawthorn leaves has anti-inflammatory and antioxidant activities. Studies have shown that isoorientin is also present in corn silk, which may also be the main active ingredient of corn silk that can reduce swelling, but it is usually discarded as waste in the corn processing process.

[0003] Isorientin is a natural flavonoid glycoside compound with important biological activity. Its chemical name is luteolin-6-C-glucoside and its molecular formula is C 21 H 20 O 11 , molecular weight is 448.38, usually yellow or off-white crystalline powder, density 1.755g / cm 3 , boiling point 857.7℃, slightly soluble in methanol, needs to be stored in the dark and refrigerated to maintain stability. It is widely found in plants such as oriental grass, selfheal, chrysanthemum, pepper, peppermint, cassia seed, bamboo leaf, passion flower, etc. It has biological activities such as antioxidant, anti-inflammatory and immunomodulatory, cardiovascular protection, anti-tumor activity, neuroprotection and metabolic regulation, and as a natural compound, it has higher safety and fewer adverse reactions than synthetic compounds.

[0004] Corn silk has attracted much attention due to its low cost, rich biological activity and no toxic side effects. However, the traditional organic solvent reflux extraction method has great limitations, such as long time and poor extraction efficiency. Therefore, a method that can save time and improve extraction efficiency is crucial for the extraction of compounds.

[0005] Natural deep eutectic solvents (DES) represent a mixture of hydrogen bond acceptors and donors with a defined molar ratio, their preparation is simple, only stirring and heating are needed to produce additional value. Composed of compounds of natural origin, they are called natural deep eutectic solvents (NADES), as a result of their application, there are no toxic solvents in the atmosphere, the economic cost of the extraction process is reduced, and they are safe and biodegradable. Summary of the invention

[0006] The object of the present invention is to provide a method for extracting isoorientin from corn silk and the optimization of its process conditions. By adopting the process flow and condition setting described in the present invention for the extraction of isoorientin from corn silk, the problems of poor extraction effect, time-consuming, and difficult to control in the traditional process can be effectively solved, and the extraction amount can be significantly increased.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] A method for synergistically optimizing the extraction of isoorientin from corn silk by a natural deep eutectic solvent and ultrasound, the specific steps are as follows: (1) drying the fresh corn silk in an oven at 60 °C, pulverizing and sieving to obtain powder; (2) preparing a natural deep eutectic solvent by mixing a hydrogen bond acceptor, a hydrogen bond donor compound and water in a certain molar ratio; (3) adding the powder obtained in step (1) to the natural deep eutectic solvent to obtain a mixed solution; (4) placing the mixed solution obtained in step (3) in a shaker and shaking and soaking for 2 hours; (5) subjecting the mixed solution treated in step (4) to ultrasonic treatment, centrifuging and taking the supernatant; (6) filtering the supernatant with a vacuum pump to obtain a preliminary extract; (7) analyzing with an enzyme-linked immunosorbent assay (ELISA) reader and high performance liquid chromatography (HPLC) to detect the total flavonoid content and the content of isoorientin.

[0009] Further, in step (1), the mesh number of the sieve selected for sieving the corn silk powder is 60 mesh.

[0010] Further, in step (2), the natural deep eutectic solvent is prepared according to the molar ratio of choline chloride: ethylene glycol: water of 1:1:3.

[0011] Further, in step (3), the material-liquid ratio of the corn silk powder to the natural deep eutectic solvent is 1:10 - 1:30 g / mL.

[0012] Further, in step (5), the ultrasonic time is 5 - 25 min, and the ultrasonic power is 150 - 650 W. Ice is used to ensure that the ultrasonic temperature does not change with the power.

[0013] Further, in step (7), the method for detecting the total flavonoid content is as follows: First, dilute the extract 5 times, take 80 μL of the diluted solution and mix it with 10 μL of 5% NaNO2 in a 96-well cell culture plate, shake well and let it stand at room temperature for 6 min; then add 10 μL of 10% Al(NO3)3, mix well and continue to stand for 10 min; finally, add 100 μL of 5% NaOH, shake well and let it stand for 15 min. Measure the absorbance at 510 nm with an enzyme-linked immunosorbent assay (ELISA) reader. Using rutin as a standard product to draw a standard curve (A = 0.0028C + 0.0515, R 2= 0.996), where A is the absorbance, C is the concentration of rutin equivalent (RE) in mg / mL, and the calculation result is expressed as mg rutin equivalent / g dry weight (mg RE / g DW).

[0014] Further, in step (7), the method for determining the content of isoorientin is as follows: Mobile phase A: acetonitrile solution and B: 0.1% phosphoric acid aqueous solution, using binary high-pressure gradient (A:B = 14:86) elution, injecting 10 μL of the extract into a C-18 column, setting the flow rate at 1.0 mL / min, and the column temperature at 35 ± 1 °C.

[0015] Compared with the traditional technology, the beneficial effects of the present invention are as follows:

[0016] 1. The present invention provides a method for extracting isoorientin from corn silk. Compared with the method of preparing isoorientin by traditional chemical synthesis, it has higher purity, fewer chemical substances in the product, and low residue of harmful substances. In addition, the natural deep eutectic solvent is non-toxic and can be well degraded. Compared with alcohol and water, it has a higher and more stable extraction rate. Under the treatment of the combined ultrasonic cell disruption technology, the efficiency of extracting isoorientin from corn silk is higher, and the active ingredients in corn silk can be quickly and fully dissolved within a short time.

[0017] 2. The present invention not only enables the waste in the corn processing process to be recycled, but also provides a strong impetus for the development and high-value utilization of corn waste resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the process flow chart of the present invention;

[0019] Figure 2 is the graph of the total flavonoid extraction amount at different solid-liquid ratios;

[0020] Figure 3 is the graph of the total flavonoid extraction amount at different ultrasonic powers;

[0021] Figure 4 is the graph of the total flavonoid extraction amount at different ultrasonic times;

[0022] Figure 5 is the 3D graph of the response surface for single-factor process optimization;

[0023] Figure 6 HPLC graph of extracting isoorientin. DETAILED DESCRIPTION OF THE INVENTION

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0025] Example 1

[0026] Experiment on the influence of different solid-liquid ratios on the extraction amount of isoorientin in corn silk

[0027] (1) The fresh corn silk is dried in an oven at 60 °C to remove moisture. The dried corn silk is ground with an automatic grinder to obtain corn silk powder, which is passed through a 60-mesh sieve.

[0028] (2) Weigh 5 portions of 5 g of corn silk powder each, and add 50, 75, 100, 125, and 150 mL of natural eutectic solvent to each portion. Shake in a shaker for 2 hours to allow the natural eutectic solvent to fully extract the corn silk powder, obtaining a mixed solution.

[0029] (3) The mixed solution obtained after sufficient treatment in (2) is ultrasonically treated with an ultrasonic cell disruptor at a power of 350 W for 10 minutes.

[0030] (4) The solution treated in (3) is centrifuged, and the supernatant is taken. The extraction solution is obtained by vacuum filtration with a vacuum pump, and it is appropriately diluted by corresponding multiples for the determination of the total flavonoid content and the determination of the isoorientin content.

[0031] The detection results are as Figure 2 shown. When the solid-liquid ratio is 1:15, the total flavonoid content is 4.7667 ± 0.117 mg RE / gDW, and the extraction amount of isoorientin is 0.8691 mg / g. Therefore, the extraction amount is higher under the condition of a solid-liquid ratio of 1:15.

[0032] Example 2

[0033] Experiment on the influence of different ultrasonic powers on the extraction amount of isoorientin in corn silk

[0034] (1) The fresh corn silk is dried in an oven at 60 °C to remove moisture. The dried corn silk is ground with an automatic grinder to obtain corn silk powder, which is passed through a 60-mesh sieve.

[0035] (2) Weigh 5 portions of 5 g of corn silk powder each, and add 75 mL of natural eutectic solvent to each portion. Shake in a shaker for 2 hours to allow the natural eutectic solvent to fully extract the corn silk powder, obtaining a mixed solution.

[0036] (3) The mixed solution after being fully processed in (2) is subjected to ultrasonic treatment for 10 minutes at powers of 250W, 350W, 450W, 550W, and 640W using an ultrasonic cell disruptor.

[0037] (4) The solution after being processed in (3) is centrifuged, the supernatant is taken, and the extraction solution is obtained by suction filtration through a vacuum pump. It is appropriately diluted by a corresponding multiple for the determination of the total flavonoid content and the determination of the isoorientin content.

[0038] The test results are as Figure 3 shown. As the ultrasonic power increases, the total flavonoid content gradually increases. When the ultrasonic power is 450W, the total flavonoid content is 7.9818 + 0.307 mg RE / g DW. As the power increases, the extraction amount shows a significant decrease. Under these conditions, the extraction amount of isoorientin is 1.1070 mg / g. Therefore, the extraction amount is higher under the condition of an ultrasonic power of 450W.

[0039] Example 3

[0040] Experiment on the effect of different ultrasonic times on the extraction amount of isoorientin in corn silk

[0041] (1) The fresh corn silk is dried in an oven at 60°C to remove moisture. The dried corn silk is ground using an automatic grinder to obtain corn silk powder, which is passed through a 60-mesh sieve.

[0042] (2) Weigh 5 portions of 5g of corn silk powder each, add 75 mL of natural deep eutectic solvent to each portion, and shake in a shaker for 2 hours to allow the natural deep eutectic solvent to fully extract the corn silk powder, obtaining a mixed solution.

[0043] (3) The mixed solution after being fully processed in (2) is subjected to ultrasonic treatment for 5, 10, 15, 20, and 25 minutes at a power of 450W using an ultrasonic cell disruptor.

[0044] (4) The solution after being processed in (3) is centrifuged, the supernatant is taken, and the extraction solution is obtained by suction filtration through a vacuum pump. It is appropriately diluted by a corresponding multiple for the determination of the total flavonoid content and the determination of the isoorientin content.

[0045] The test results are as Figure 4 shown. As the ultrasonic time increases, the total flavonoid content gradually increases. When the ultrasonic extraction time is 15 minutes, the total flavonoid content is 7.1384 ± 0.325 mg RE / g DW. As the ultrasonic time increases, the extraction amount shows a significant decrease. Under these conditions, the extraction amount of isoorientin is 1.2853 mg / g. Therefore, the extraction amount is higher under the condition of an ultrasonic time of 15 minutes.

[0046] Example 4

[0047] Experiment on the Influence of the Extraction Yield of Isorientin from Corn Silk under Optimal Conditions

[0048] (1) The fresh corn silk was dried in an oven at 60 °C to remove moisture. The dried corn silk was ground with an automatic grinder to obtain corn silk powder, which was passed through a 60-mesh sieve.

[0049] (2) 5 g of corn silk powder was weighed, and 75 mL of natural deep eutectic solvent was added to each. The mixture was shaken in a shaker for 2 hours to allow the natural deep eutectic solvent to fully extract the corn silk powder, obtaining a mixed solution.

[0050] (3) The mixed solution obtained from (2) after sufficient treatment was ultrasonically treated with an ultrasonic cell disruptor at a power of 450 W for 15 minutes.

[0051] (4) The solution obtained from (3) was centrifuged, and the supernatant was taken. The extraction solution was obtained by vacuum filtration, and it was appropriately diluted by a corresponding multiple for the determination of the total flavonoid content and the determination of the isorientin content.

[0052] After optimization by response surface methodology, it was found that under the conditions of a solid-liquid ratio of 1:1525 (g / mL), an ultrasonic power of 430.8 W, and an ultrasonic time of 19.46 minutes, the total flavonoid content was 8.0341 ± 0.422 mg RE / g DW, and the extraction yield of isorientin was 1.3028 mg / g.

Claims

1. A method for synergistically optimizing the extraction of isoorientin from corn silk by a natural deep eutectic solvent and ultrasound, characterized in that, It includes the following steps: (1) drying fresh corn silk in an oven at 60 °C, pulverizing it, and sieving to obtain powder; (2) preparing a natural deep eutectic solvent by mixing a hydrogen bond acceptor, a hydrogen bond donor compound, and water in a certain molar ratio; (3) adding the powder obtained in step (1) to the natural deep eutectic solvent to obtain a mixed solution; (4) placing the mixed solution obtained in step (3) in a shaker and shaking and soaking for 2 hours; (5) subjecting the mixed solution processed in step (4) to ultrasonic treatment, centrifuging, and taking the supernatant; (6) filtering the supernatant with a vacuum pump to obtain a preliminary extract; (7) analyzing with an enzyme-linked immunosorbent assay (ELISA) reader and high-performance liquid chromatography (HPLC) to detect the total flavonoid content and the content of isoorientin.

2. A method for synergistically optimizing the extraction of isoorientin from corn silk by using a natural deep eutectic solvent and ultrasound, characterized in that, The mesh number of the sieve selected during sieving of the corn silk powder is 60 mesh.

3. A method for synergistically optimizing the extraction of isoorientin from corn silk by natural deep eutectic solvents and ultrasound according to claim 1, characterized in that, The natural deep eutectic solvent is prepared according to the molar ratio of choline chloride: ethylene glycol: water of 1:1:

3.

4. A method for synergistically optimizing the extraction of isoorientin from corn silk by using a natural deep eutectic solvent and ultrasound according to claim 1, characterized in that The material-liquid ratio of the corn silk powder to the natural deep eutectic solvent is 1:10 - 1:30 g / mL.

5. A method for synergistically optimizing the extraction of isoorientin from corn silk by using a natural deep eutectic solvent and ultrasound, characterized in that, The ultrasonic time is 5 - 25 min, and the ultrasonic power is 150 - 650 W. Ice is used to ensure that the temperature of the ultrasonic treatment does not change with the power.

6. A method for synergistically optimizing the extraction of isoorientin from corn silk by using a natural deep eutectic solvent and ultrasound, characterized in that, The total flavonoid content detection method is as follows: first, the extract is diluted 5 times, 80 μL of the dilution is mixed with 10 μL of 5% NaNO2 in a 96-well cell culture plate, and the mixture is allowed to stand at room temperature for 6 minutes after sufficient shaking; then 10 μL of 10% Al(NO3)3 is added, the mixture is allowed to stand for 10 minutes after mixing; finally, 100 μL of 5% NaOH is added, the mixture is allowed to stand for 15 minutes after sufficient shaking, and the absorbance is measured at 510 nm using an enzyme marker, and a standard curve (A=0.0028C+0.0515, R 2 =0.996), wherein A is absorbance, C is the concentration of rutin equivalent (RE), the unit is mg / mL, and the calculation result is expressed as mg rutin equivalent / g dry weight (mg RE / g DW).

7. A method for synergistically optimizing the extraction of isoorientin from corn silk by using a natural deep eutectic solvent and ultrasound according to claim 1, characterized in that The method for determining the content of isoorientin is as follows: mobile phase A: acetonitrile solution and B: 0.1% phosphoric acid aqueous solution, eluting with binary high-pressure gradient (A:B = 14:86), injecting 10 μL of the extract into a C-18 column, setting the flow rate at 1.0 mL / min, and the column temperature at 35 ± 1 °C.

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