A broccoli leaf flavone and its deep eutectic solvent-based extraction method and application

Abstract

The present application relates to the broccoli leaf flavonoids technical field, especially in kind of broccoli leaf flavonoids and its based on deep eutectic solvent extraction method and application, including the following steps: after mixing choline chloride and glucose, add water, intermittent stirring to get uniform transparent liquid, after cooling, the liquid is not solidified to get deep eutectic solvent;The broccoli leaf is ground and sieved, add deep eutectic solvent mixture, put into microwave oven and carry out intermittent heating, ultrasonic extraction after centrifugation to obtain supernatant;After macroporous resin activation, column packing, supernatant, distilled water washing after ethanol elution and collection of eluate;Rotary evaporation to remove ethanol, pre-freezing and freeze-drying to obtain broccoli leaf flavonoids powder.The present application adopts the above steps, constructs choline chloride-glucose based deep eutectic solvent, combines microwave-ultrasonic synergistic auxiliary extraction, makes the extraction rate of broccoli leaf flavonoids increase by 2.54 times compared with traditional ethanol method, and the detected flavonoid type increases by 2.38 times.

Description

Technical Field

[0001] This invention relates to the field of broccoli leaf flavonoids technology, and in particular to a method for extracting broccoli leaf flavonoids based on deep eutectic solvents and its application. Background Technology

[0002] Traditional natural flavonoid extraction techniques mainly employ water-alcohol mixed solvents (such as 70% ethanol) for extraction, reflux extraction, or ultrasound-assisted extraction. These methods are simple to operate and easily scaled up industrially, but they suffer from the following drawbacks: Insufficient extraction efficiency and selectivity: The ethanol system has weak solubility for certain highly polar or bound flavonoids, resulting in low extraction yields and poor selectivity of target components. Unsatisfactory preservation of purity and functional activity: Traditional systems struggle to effectively separate impurities, leading to high levels of non-target components in the extract, which in turn affects downstream purification and activity utilization. Energy and safety issues: Volatile organic solvents such as ethanol have high vapor pressures, requiring energy recovery after extraction, and posing safety and environmental emission risks.

[0003] The aforementioned defects mainly stem from the limitations of the physicochemical properties of traditional solvents, namely, the limited range of solvent polarity and insufficient interaction with flavonoid molecules, making it difficult to achieve efficient dissolution and selective extraction of the complex structure of flavonoids. Summary of the Invention

[0004] The purpose of this invention is to provide a method and application for the extraction of flavonoids from broccoli leaves based on deep eutectic solvents. By constructing a choline chloride-glucosyl deep eutectic solvent system and combining it with microwave-ultrasound synergistic extraction, the extraction rate of flavonoids from broccoli leaves is increased by 2.54 times compared with the traditional ethanol method, and the number of detected flavonoid species increases by 2.38 times.

[0005] To achieve the above objectives, the present invention provides a method for extracting flavonoids from broccoli leaves and their extraction based on a deep eutectic solvent, comprising the following steps: S1. Mix choline chloride and glucose, add water, and incubate at 60-120℃ with intermittent stirring until a uniform and transparent liquid is obtained. After cooling, the liquid does not solidify, thus obtaining a deep eutectic solvent. S2. Grind the broccoli leaves and pass them through a 50-70 mesh sieve. Add them to the deep eutectic solvent obtained in S1 and mix. Place the mixture in a microwave oven for intermittent heating, then transfer it to an ultrasonic cleaner for ultrasonic extraction. Centrifuge at room temperature to obtain the supernatant. S3. After activating the macroporous resin with ethanol, hydrochloric acid and sodium hydroxide in sequence, pack the column, rinse with distilled water and then load the supernatant from S2 onto the column. After loading, let it stand, rinse with distilled water and then elute with ethanol, and collect the eluent. S4. The eluent from S3 is removed by rotary evaporation to remove ethanol, pre-freeze and then freeze-dry to obtain broccoli leaf flavonoid powder.

[0006] Preferably, in S1, the molar ratio of choline chloride to glucose is 1:1-3, and the amount of water added is 20-50% of the total mass of choline chloride and glucose.

[0007] Preferably, in S1, the intermittent stirring time is 20-40 min, the interval between intermittent stirring is 1-2 min, and the total stirring time is 8-20 min.

[0008] Preferably, in S2, the power of intermittent heating is 300-400W, the intermittent heating time is 1-5min, the interval between intermittent heating is 20-40s, and the heating time of intermittent heating is 0.5-3min; The ultrasonic extraction frequency is 20-50kHz, the power is 100-200W, the temperature is 50-80℃, and the time is 30-60min; The centrifugation speed is 8000-10000 rpm, and the time is 10-60 min.

[0009] Preferably, in S3, the flavonoid concentration in the supernatant of the sample is 0.5-2 mg / mL, and the sample loading flow rate is 2-3 mL / min.

[0010] Preferably, in S4, the pre-freezing temperature is -50°C to -30°C.

[0011] The above-mentioned broccoli leaf flavonoids were prepared by an extraction method based on a deep eutectic solvent.

[0012] The above-mentioned flavonoids from broccoli leaves can be used to prepare flavonoid microcapsules or sunscreens.

[0013] Preferably, in preparing flavonoid microcapsules, flavonoid powder from broccoli leaves is added to a sodium alginate solution, followed by the addition of ethanol to obtain a mixture. The mixture is then placed into a vibrating nozzle of an encapsulation device, which pumps the mixture into a calcium chloride solution to obtain microcapsules. The microcapsules are then placed in a chitosan solution and stirred. After washing with sterile distilled water, the mixture is freeze-dried to obtain flavonoid microcapsules.

[0014] Preferably, when used in the preparation of sunscreen, the aqueous phase is slowly added to the oil phase containing a mixture of titanium dioxide and zinc oxide powders while stirring. The mixture is homogenized at 3000-5000 rpm for 2-3 minutes to obtain an emulsion. After cooling to below 40°C, flavonoid microcapsules are added and stirred at low speed until the mixture is homogeneous to obtain the sunscreen.

[0015] Therefore, the present invention employs the above-mentioned extraction method and application of flavonoids from broccoli leaves and based on deep eutectic solvent, and its beneficial effects are as follows: 1. The extraction method provided by this invention, by constructing a choline chloride-glucosyl deep eutectic solvent system and combining it with microwave-ultrasound synergistic extraction, increases the flavonoid extraction rate of broccoli leaves by 2.54 times compared with the traditional ethanol method, and increases the number of detected flavonoid species by 2.38 times; after dynamic adsorption-elution purification with AB-8 macroporous resin, the product purity reaches 72.33±1.11%. 2. In application, this invention employs a two-step encapsulation method using sodium alginate and chitosan, which increases the flavonoid encapsulation rate by 12% and significantly enhances thermal stability and antioxidant activity (ABTS / DPPH scavenging rates increase by 9.7% and 12.2%, respectively). When applied to sunscreen, its combination with TiO2 / ZnO increases the SPF value by 17% to 28%.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This is a distribution diagram of flavonoid species in the flavonoid powder from broccoli leaves in Example 1 of the present invention; Figure 2 This is a distribution diagram of flavonoid species in the flavonoid powder of broccoli leaves in Comparative Example 1 of this invention; Figure 3 This is a columnar comparison chart of the flavonoid types in the flavonoid powder of broccoli leaves in Example 1 and Comparative Example 1 of the present invention; Figure 4 This is a graph showing the ABTS free radical scavenging rate of Example 1, Comparative Example 1, Comparative Example 4, and Vitamin C in this invention; Figure 5 This is a graph showing the DPPH free radical scavenging rate of Example 1, Comparative Example 1, Comparative Example 4, and Vitamin C in this invention; Figure 6 This is a schematic diagram showing the types and extraction amounts of some flavonoids in broccoli leaves from Example 1 and Comparative Example 1 of the present invention. Detailed Implementation

[0018] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0019] This invention provides a method for extracting flavonoids from broccoli leaves and their extraction based on a deep eutectic solvent, comprising the following steps: S1. Mix choline chloride and glucose, add water, and incubate at 60-120℃. Stir intermittently until a uniform and transparent liquid is obtained. After cooling, the liquid does not solidify, and a deep eutectic solvent DES is obtained. Choline chloride (quaternary ammonium salt) in DES forms a hydrogen bond network with glucose (polyhydroxy), which can effectively destroy plant cell walls and release intracellular flavonoids.

[0020] S2. Grind the broccoli leaves and pass them through a 50-70 mesh sieve. Add them to the deep eutectic solvent obtained in S1 and mix. Place the mixture in a microwave oven for intermittent heating, then transfer it to an ultrasonic cleaner for ultrasonic extraction. Centrifuge at room temperature to obtain the supernatant. S3. After activating the macroporous resin with ethanol, hydrochloric acid and sodium hydroxide in sequence, pack the column, rinse with distilled water and then load the supernatant from S2 onto the column. After loading, let it stand, rinse with distilled water and then elute with ethanol, and collect the eluent. S4. The eluent from S3 is rotary evaporated to remove ethanol, pre-frozen, and then freeze-dried to obtain broccoli leaf flavonoid powder. Water is removed by low-temperature vacuum sublimation to maintain the integrity of the flavonoid structure.

[0021] In some embodiments of the present invention, in S1, the molar ratio of choline chloride to glucose is 1:1-3, and the amount of water added is 20-50% of the total mass of choline chloride and glucose. Adding 20-50% water can adjust the polarity of DES and enhance its solubility for flavonoids.

[0022] In some embodiments of the present invention, in S1, the intermittent stirring time is 20-40 min, the interval between intermittent stirring is 1-2 min, and the total stirring time is 8-20 min. This prevents local overheating and promotes uniform DES formation.

[0023] In some embodiments of the present invention, in S2, the power of intermittent heating is 300-400W, the intermittent heating time is 1-5min, the interval between intermittent heating is 20-40s, and the heating time of intermittent heating is 0.5-3min; to avoid continuous high temperature damaging the flavonoid structure, microwave heating utilizes the rapid rotation of polar molecules in the microwave field to generate heat, destroy cell structure, and promote DES penetration.

[0024] The ultrasonic extraction frequency is 20-50kHz, the power is 100-200W, the temperature is 50-80℃, and the time is 30-60min; the cavitation effect generates local high temperature and high pressure, which breaks the cells and accelerates mass transfer.

[0025] Centrifuge at 8000-10000 rpm for 10-60 minutes. Remove cell debris and impurities, and obtain a clear supernatant.

[0026] In S3, the macroporous resin is one of AB-8 resin, D101 resin, and NIKA-2 resin. AB-8 resin is a non-polar adsorption resin, suitable for adsorbing moderately polar flavonoids. AB-8 resin has moderate polarity and specific surface area, forming hydrogen bonds and hydrophobic interactions with flavonoids, resulting in high adsorption capacity.

[0027] In some embodiments of the present invention, in step S3, the flavonoid concentration in the supernatant is 0.5-2 mg / mL, and the loading flow rate is 2-3 mL / min. After the flavonoids are adsorbed on the resin, they are eluted with 70% ethanol, and separation and purification are achieved by utilizing polarity changes.

[0028] In some embodiments of the present invention, in step S4, the pre-freezing temperature is -50°C to -30°C. This ensures that the sample remains in a glassy state throughout the freeze-drying process, preventing structural collapse.

[0029] In some embodiments of the present invention, the broccoli leaf flavonoids are prepared by the above-mentioned extraction method based on deep eutectic solvent.

[0030] In some embodiments of the present invention, the above-mentioned broccoli leaf flavonoids are used to prepare flavonoid microcapsules or sunscreens.

[0031] In some embodiments of the present invention, when preparing flavonoid microcapsules, flavonoid powder from broccoli leaves is added to a sodium alginate solution, and then ethanol is added to obtain a mixture. The mixture is placed in a vibrating nozzle of an encapsulation device, and the vibrating nozzle pumps the mixture into a calcium chloride solution to obtain microcapsules. The microcapsules are then placed in a chitosan solution and stirred. After being washed with sterile distilled water, they are freeze-dried to obtain flavonoid microcapsules.

[0032] In some embodiments of the present invention, when applied to the preparation of sunscreen, the aqueous phase is slowly added to the oil phase containing a mixture of titanium dioxide and zinc oxide powder while stirring. The mixture is homogenized at 3000-5000 rpm for 2-3 minutes to obtain an emulsion. After cooling to below 40°C, flavonoid microcapsules are added and stirred at low speed until the mixture is homogeneous to obtain the sunscreen.

[0033] Example 1 S1. Choline chloride and glucose were mixed in a molar ratio of 1:2, and then water was added, with the amount of water being 30% of the total mass of choline chloride and glucose. The mixture was incubated at 80°C with intermittent stirring for 20 minutes, twice within each 20-minute period, with a 1-minute interval between stirring. A homogeneous and transparent liquid was obtained. Upon cooling, the liquid did not solidify, yielding the deep eutectic solvent DES.

[0034] S2. Grind the broccoli leaves and pass them through a 60-mesh sieve. Add the deep eutectic solvent DES obtained in S1 and mix. The ratio of DES to broccoli leaf fragments is 30:1. Place the mixture in a microwave oven and heat intermittently for 2 minutes, twice within 2 minutes with a 30-second interval between heatings. The power of the intermittent heating is 350W. Afterward, transfer the mixture to an ultrasonic cleaner for ultrasonic extraction. The ultrasonic extraction frequency is 40kHz, the power is 150W, the temperature is 60℃, and the time is 40 minutes. Centrifuge at room temperature to obtain the supernatant. The centrifugation speed is 9000 rpm, and the time is 30 minutes.

[0035] S3. The AB-8 macroporous resin was activated sequentially with ethanol, hydrochloric acid, and sodium hydroxide before being packed into a column: The AB-8 macroporous resin was soaked in anhydrous ethanol for 24 hours, placed on a shaker, and washed with anhydrous ethanol until the eluent mixed with water at a 1:5 ratio was clear. Then, the AB-8 macroporous resin was washed with distilled water until no ethanol odor remained. Afterward, it was soaked in hydrochloric acid solution (5%, v:v) for 4 hours and washed with water until neutral. Then, it was soaked in sodium hydroxide solution (2%, v:v) for 4 hours and washed with water until neutral. It was then air-dried for later use. After pretreatment and activation, the crude flavonoid extract was packed into a column and enriched and purified using a dynamic adsorption-elution mode.

[0036] After rinsing the resin column with distilled water, the supernatant from column S2 was loaded with a flavonoid concentration of 1 mg / mL. The loading volume was 5 BV at a flow rate of 2.3 mL / min, and the column was allowed to stand for 4 h after loading. The column was then rinsed with distilled water until there was no ethanol odor and the liquid was clear. Elution was then performed with 3 BV of 70% ethanol at a flow rate of 1.5 mL / min, and the eluent was collected.

[0037] S4. The eluent from S3 is removed by rotary evaporation to remove ethanol, pre-frozen at -40℃ and then freeze-dried to obtain broccoli leaf flavonoid powder.

[0038] Example 2 The difference between this embodiment and Embodiment 1 is that the resin used in S3 is D101 resin, while the rest of the steps are the same as in Embodiment 1.

[0039] Example 3 The difference between this embodiment and Embodiment 1 is that the resin used in S3 is NIKA-2 resin, while the rest of the steps are the same as in Embodiment 1.

[0040] Example 4 To prepare flavonoid microcapsules, flavonoid powder from broccoli leaves was added to a 2 wt% sodium alginate solution, followed by the addition of 5% ethanol to obtain a mixture with a flavonoid concentration of 1 wt%. The mixture was then placed into a vibrating nozzle (150 μm diameter) of an encapsulation device (BÜCHIEncapsulator B-390), which pumped the mixture into a 4.5 wt% calcium chloride solution. The vibration frequency was set to 170 Hz, the voltage to 250 V, and the supply pressure to 130 mbar. After slowly stirring the calcium chloride solution for 30 min, microcapsules were obtained. These microcapsules were then placed in a 0.1% (w / v) chitosan solution and stirred for 30 min. After washing with sterile distilled water, the microcapsules were freeze-dried to obtain the flavonoid microcapsules.

[0041] Example 5 Oil phase: Add 0.70g of white petrolatum, 0.40g of cetearyl alcohol, 0.25g of glyceryl monostearate (GMS), and 0.15g of Tween 80 sequentially. Heat and stir at 75±2℃ until all components are completely melted and the system is clear. Then take about 0.25-0.35g of the melted oil phase and pre-grind it thoroughly in a mortar with a mixture of titanium dioxide and zinc oxide powder (TiO2 and ZnO, total amount 0.80g) to obtain a uniform thick paste slurry (no dry powder, no obvious oil separation). Then scrape the slurry back into the oil phase of the main pot, first wetting at low speed, then dispersing at medium speed, until the system is uniform and there are no visible agglomerates.

[0042] Aqueous phase: Heat 6.80g of deionized water to 75±2℃, take 0.30g of propylene glycol (PG) to fully wet xanthan gum and add it to the aqueous phase. Continue stirring under heating conditions until xanthan gum is completely hydrated and the system is homogeneous without fish-eye-like lumps.

[0043] When used in the preparation of sunscreen, the aqueous phase is slowly added to the oil phase at 75°C while stirring to obtain the initial emulsion. The emulsion is homogenized at 4000 rpm for 3 minutes to obtain a homogeneous emulsion. After cooling to below 40°C, 10% of the total mass of the sunscreen flavonoid microcapsules are added and stirred at low speed until the mixture is uniform to obtain the sunscreen.

[0044] Comparative Example 1 The difference between this comparative example and Example 1 is that 70% ethanol (EtOH) was used to extract flavonoids from broccoli leaves to obtain broccoli leaf flavonoid powder.

[0045] Comparative Example 2 The difference between this comparative example and Example 1 is that only microwave-assisted extraction of MAE is performed in S2, while the rest of the steps are the same as in Example 1.

[0046] Comparative Example 3 The difference between this comparative example and Example 1 is that only ultrasonic extraction of UAE is performed in S2, while the rest of the steps are the same as in Example 1.

[0047] Comparative Example 4 The difference between this comparative example and Example 1 is that ultrasonic extraction and microwave-assisted extraction were not performed in S2, while the rest of the steps are the same as in Example 1.

[0048] Performance testing a. such as Figure 1-3 As shown, the extraction rate of flavonoids from broccoli leaves using the microwave-ultrasound synergistic assisted glucose-based deep eutectic solvent extraction method in Example 1 was 5.51 ± 0.23 mg / g, which was 2.54 times higher than that of Comparative Example 1 (70% ethanol). The number of flavonoid species extracted in Example 1 was significantly increased compared to Comparative Example 1 (70% ethanol). Example 1 detected 62 types of extracted flavonoids, approximately 2.38 times that of the ethanol system in Comparative Example 1, and the storage stability was significantly improved.

[0049] Antioxidant tests were performed on Example 1, Comparative Example 1, Comparative Example 4, and Vitamin C (VC), such as... Figure 4 and Figure 5 As shown, the broccoli leaf flavonoid powder extracted in Example 1 exhibits superior antioxidant properties compared to the broccoli leaf flavonoid powders extracted in Comparative Examples 1 and 4. Furthermore, the broccoli leaf flavonoid powder extracted in Example 1 also demonstrates good antioxidant properties compared to vitamin C.

[0050] like Figure 6 As shown, the types and extraction amounts of flavonoids in the broccoli leaves extracted in Example 1 are significantly better than those extracted in Comparative Example 1.

[0051] Therefore, the present invention adopts the above-mentioned extraction method and application of flavonoids from broccoli leaves based on deep eutectic solvent. By constructing a choline chloride-glucosyl deep eutectic solvent system and combining it with microwave-ultrasound synergistic assisted extraction, the extraction rate of flavonoids from broccoli leaves is increased by 2.54 times compared with the traditional ethanol method, and the number of detected flavonoid types increases by 2.38 times.

[0052] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for extracting flavonoids from broccoli leaves using a deep eutectic solvent, characterized in that: Includes the following steps: S1. Mix choline chloride and glucose, add water, and incubate at 60-120℃ with intermittent stirring until a uniform and transparent liquid is obtained. After cooling, the liquid does not solidify, thus obtaining a deep eutectic solvent. S2. Grind the broccoli leaves and pass them through a 50-70 mesh sieve. Add them to the deep eutectic solvent obtained in S1 and mix. Place the mixture in a microwave oven for intermittent heating, then transfer it to an ultrasonic cleaner for ultrasonic extraction. Centrifuge at room temperature to obtain the supernatant. S3. After activating the macroporous resin with ethanol, hydrochloric acid and sodium hydroxide in sequence, pack the column, rinse with distilled water and then load the supernatant from S2 onto the column. After loading, let it stand, rinse with distilled water and then elute with ethanol, and collect the eluent. S4. The eluent from S3 is removed by rotary evaporation to remove ethanol, pre-freeze and then freeze-dry to obtain broccoli leaf flavonoid powder.

2. The method for extracting flavonoids from broccoli leaves based on a deep eutectic solvent according to claim 1, characterized in that: In S1, the molar ratio of choline chloride to glucose is 1:1-3, and the amount of water added is 20-50% of the total mass of choline chloride and glucose.

3. The method for extracting flavonoids from broccoli leaves based on a deep eutectic solvent according to claim 1, characterized in that: In S1, the intermittent stirring time is 20-40 min, the interval between intermittent stirring is 1-2 min, and the total stirring time for intermittent stirring is 8-20 min.

4. The method for extracting flavonoids from broccoli leaves based on a deep eutectic solvent according to claim 1, characterized in that: In S2, the power of intermittent heating is 300-400W, the intermittent heating time is 1-5min, the interval between intermittent heating is 20-40s, and the heating time of intermittent heating is 0.5-3min. The ultrasonic extraction frequency is 20-50kHz, the power is 100-200W, the temperature is 50-80℃, and the time is 30-60min; The centrifugation speed is 8000-10000 rpm, and the time is 10-60 min.

5. The method for extracting flavonoids from broccoli leaves based on a deep eutectic solvent according to claim 1, characterized in that: In S3, the flavonoid concentration in the supernatant of the sample is 0.5-2 mg / mL, and the loading flow rate is 2-3 mL / min.

6. The method for extracting flavonoids from broccoli leaves based on a deep eutectic solvent according to claim 1, characterized in that: In S4, the pre-freezing temperature is -50℃ to -30℃.

7. A flavonoid from broccoli leaves, characterized in that: The flavonoids from broccoli leaves were prepared using a deep eutectic solvent extraction method according to any one of claims 1-6.

8. An application of flavonoids from broccoli leaves, characterized in that: The flavonoids from broccoli leaves according to claim 7 can be used to prepare flavonoid microcapsules or sunscreens.

9. The application of flavonoids from broccoli leaves according to claim 8, characterized in that: To prepare flavonoid microcapsules, flavonoid powder from broccoli leaves was added to a sodium alginate solution, followed by the addition of ethanol to obtain a mixture. The mixture was then placed in a vibrating nozzle of an encapsulation device, which pumped the mixture into a calcium chloride solution to obtain microcapsules. The microcapsules were then placed in a chitosan solution and stirred. After washing with sterile distilled water, the mixture was freeze-dried to obtain flavonoid microcapsules.

10. The application of flavonoids from broccoli leaves according to claim 9, characterized in that: When used in the preparation of sunscreen, the aqueous phase is slowly added to the oil phase containing a mixture of titanium dioxide and zinc oxide powders while stirring. The mixture is homogenized at 3000-5000 rpm for 2-3 minutes to obtain an emulsion. After cooling to below 40°C, flavonoid microcapsules are added and stirred at low speed until the mixture is homogeneous to obtain the sunscreen.

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