Preparation method of mulberry root-bark flavone, mulberry root-bark flavone and application

Abstract

The application discloses a preparation method of mulberry root bark flavones, the mulberry root bark flavones and application. The preparation method comprises the following steps: mixing choline chloride and lactic acid to obtain a deep eutectic solvent in a molar ratio of 1:2 to 1:3; mixing mulberry root bark powder and the deep eutectic solvent to obtain a mulberry root bark flavone DES crude extract in a mass ratio of 1:30 to 1:50; and purifying the mulberry root bark flavone DES crude extract by using a magnetic molecular imprinting resin to obtain the mulberry root bark flavones. The magnetic molecular imprinting resin comprises a magnetic core, an intermediate protective layer and a molecular imprinting polymer layer, imprinting template molecules are composed of sanggenon C and sang flavone G, the average thickness of the molecular imprinting polymer layer is 20 nm to 30 nm, and the average thickness of the intermediate protective layer is 20 nm to 30 nm. The preparation method is synergized by deep eutectic solvent extraction and the magnetic molecular imprinting resin, and the prepared mulberry root bark flavones have high purity and good cell autophagy promotion effect.

Description

Technical Field

[0001] This application belongs to the field of plant active ingredient extraction technology, specifically relating to a method for preparing mulberry root bark flavonoids, mulberry root bark flavonoids and their applications. Background Technology

[0002] Traditional extraction processes for flavonoids from mulberry root bark mainly involve ethanol reflux extraction and ultrasound-assisted ethanol extraction. These processes have significant drawbacks: they easily leave organic solvent residues, resulting in poor extraction efficiency and selectivity, and they can also damage the molecular structure of heat-sensitive flavonoids.

[0003] Furthermore, crude flavonoids are often purified using conventional macroporous adsorption resins such as D101 and AB-8. These resins rely on van der Waals forces for non-specific adsorption, which, while enriching flavonoids, also adsorbs large amounts of impurities such as polysaccharides, lignin, and pigments. The resulting product is dark in color and has low purity, increasing subsequent refining steps and driving up overall production costs. Traditional processes and single-process methods are insufficient to simultaneously meet the requirements of high extraction rates, high product purity, and environmentally friendly industrial production. Summary of the Invention

[0004] To address the aforementioned issues, this application provides a method for preparing mulberry root bark flavonoids, the mulberry root bark flavonoids themselves, and their applications. The preparation method of this application, through the synergistic effect of deep eutectic solvent extraction and magnetic molecular imprinting resin, not only effectively improves the extraction rate and time of mulberry root bark flavonoids but also achieves selective adsorption of four target flavonoids, exhibiting superior adsorption capacity and extraction rate. The resulting mulberry root bark flavonoids possess high purity and demonstrate excellent autophagy-promoting effects.

[0005] In a first aspect, embodiments of this application provide a method for preparing flavonoids from mulberry root bark, comprising: Choline chloride and lactic acid are mixed in a molar ratio of 1:2 to 1:3 and stirred at 60°C to 70°C until transparent to obtain a deep eutectic solvent.

[0006] Mulberry root bark powder was mixed with a deep eutectic solvent at a mass ratio of 1:30 to 1:50. The mixture was extracted for 30 to 60 minutes at 50 to 65°C and with an ultrasonic power of 200 W to 300 W. The supernatant was collected by centrifugation to obtain crude extract of mulberry root bark flavonoids (DES).

[0007] The crude extract of mulberry root bark flavonoids (DES) was purified using magnetic molecular imprinting resin to obtain mulberry root bark flavonoids.

[0008] The magnetic molecularly imprinted resin includes a magnetic core, an intermediate protective layer covering the surface of the magnetic core, and a molecularly imprinted polymer layer grafted onto the surface of the intermediate protective layer. The molecularly imprinted polymer layer is a polymer network with specific recognition sites formed by free radical polymerization of imprinted template molecules, functional monomers, and crosslinking agents under the action of a porogen. The imprinted template molecules are composed of mulberry root ketone C and mulberry ginseng ketone G, and the molar ratio of mulberry root ketone C to mulberry ginseng ketone G is 1:1 to 2:1. The average thickness of the molecularly imprinted polymer layer is 20 nm to 30 nm.

[0009] The magnetic core includes at least one of Fe3O4, γ-Fe2O3, CoFe2O4 and NiFe2O4, and the average thickness of the intermediate protective layer is 20 nm to 30 nm. The intermediate protective layer includes at least one of SiO2, Al2O3 and ZrO2.

[0010] In some optional embodiments, the step of purifying the crude extract of mulberry root bark flavonoids (DES) using magnetic molecular imprinting resin to obtain mulberry root bark flavonoids includes: A magnetic molecularly imprinted resin was prepared into a resin homogenate and packed into a glass chromatography column. A ring magnet was fitted onto the outer wall of the glass chromatography column.

[0011] The crude DES extract of mulberry root bark flavonoids was fed upward from the bottom of the glass chromatography column at a flow rate of 5 BV / h to 7 BV / h. At the same time, a ring magnet was moved up and down to form a fluidized bed. The absorbance of the effluent at 280 nm was monitored online. When the effluent reached 10% of the loading solution, the loading was stopped.

[0012] An axial gradient magnetic field is applied to the glass chromatography column using a ring magnet. The column is then eluted with 3 column volumes of 10% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. The eluent is discarded until the absorbance value is <0.05. The magnetic field strength at the top of the column is 0.5 T to 0.6 T, and the magnetic field strength at the bottom of the column is 0.2 T to 0.3 T.

[0013] A pulsed magnetic field of 1 Hz and 50% duty cycle is applied to a glass chromatography column using a ring magnet. Elution is carried out with 4 column volumes of 70% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. The elution peaks with absorbance values ​​>0.1 are collected to obtain the eluent.

[0014] The eluent was concentrated under reduced pressure at 45°C and dried under vacuum to obtain mulberry root bark flavonoids.

[0015] In some alternative embodiments, the magnetic core comprises Fe3O4 and the intermediate protective layer comprises SiO2.

[0016] In some optional embodiments, the functional monomer is selected from one or more of methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, 4-vinylpyridine, 2-vinylpyridine, vinylimidazole, acrylamide, N-isopropylacrylamide, methacrylamide, styrene, and p-vinylbenzoic acid. The crosslinking agent is selected from one or more of ethylene glycol dimethacrylate, divinylbenzene, N,N'-methylenebisacrylamide, 1,4-butanediol dimethacrylate, trimethylolpropane trimethacrylate, triallyl isocyanurate, and pentaerythritol tetraacrylate. The porogen is selected from a choline chloride-ethylene glycol eutectic solvent, a choline chloride-glycerol eutectic solvent, or a choline chloride-ethylene glycol-ethanol eutectic solvent.

[0017] In some alternative embodiments, the pore-forming agent includes a choline chloride-ethylene glycol-ethanol eutectic solvent, wherein the molar ratio of choline chloride, ethylene glycol, and ethanol is from 1:2:1.5 to 1:3:2.

[0018] In some alternative embodiments, the ratio of the total molar amount of template molecules, the molar amount of functional monomers, and the molar amount of crosslinking agent is 1:8:20 to 1:8:40.

[0019] In some alternative embodiments, the magnetic molecularly imprinted resin is prepared by the following steps: The magnetic core was prepared by coprecipitation.

[0020] A mesophase protective layer is coated onto the surface of the magnetic core to obtain mesophase protective layer coated particles.

[0021] The surface double bonds of the mesophase protective layer-coated particles were modified using a silane coupling agent to obtain the particles to be grafted.

[0022] Morus root ketone C, morrhus ketone G and functional monomers were dissolved in a porogen and stirred at room temperature in the dark to allow the template molecules to form hydrogen-bonded complexes with the functional monomers, thus obtaining a pre-assembled solution.

[0023] The particles to be grafted, a crosslinking agent, and an initiator are added to the pre-assembled solution, and the reaction is carried out under a protective atmosphere to obtain a crude product of magnetic molecularly imprinted resin.

[0024] The crude product of the magnetic molecularly imprinted resin was collected by magnetic separation and then Soxhlet extracted with a methanol-acetic acid mixture until the eluent showed no absorption at 280 nm.

[0025] Wash with methanol until neutral, then vacuum dry to obtain magnetic molecularly imprinted resin.

[0026] Secondly, this application provides a mulberry root bark flavonoid, which is prepared by the method described in the first aspect.

[0027] Thirdly, embodiments of this application provide the application of mulberry root bark flavonoids prepared by the method of the first aspect of this application or mulberry root bark flavonoids of the second aspect of this application in the preparation of cosmetics.

[0028] In some alternative implementations, the cosmetic has the function of promoting autophagy in skin cells.

[0029] The preparation method of this application has at least the following advantages over the prior art: The method uses choline chloride as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor to prepare a deep eutectic solvent. The chloride ions and hydroxyl groups of choline chloride form multiple intermolecular hydrogen bonds with the carboxyl and hydroxyl groups of lactic acid, disrupting the original crystal lattice structure and forming a homogeneous deep eutectic solvent in a room-temperature liquid state. This system has a stable hydrogen bond network, and its polarity can be precisely controlled by the molar ratio of the raw materials. It is highly compatible with the polarity of flavonoids from mulberry root bark, enabling efficient flavonoid extraction.

[0030] Simultaneously, morula root ketone C and morula flavonoid G were selected as template molecules to prepare magnetic surface-imprinted resins. After template elution, imprinted cavities matching the size and spatial structure of the target flavonoid molecules were formed on the resin surface. On the one hand, impurities such as polysaccharides, pigments, and lignin could not enter the imprinted cavities due to their mismatched spatial structures, achieving efficient sieving. On the other hand, after flavonoid molecules entered the cavities, they could form multiple binding forces through hydrogen bonding, electrostatic interactions, and π-π stacking interactions. Combined with an imprinted polymer layer of a specific thickness and an intermediate protective layer, this further enhanced the adsorption strength, adsorption capacity, and selectivity specificity.

[0031] Furthermore, the target substances bound to the magnetic surface molecular imprints are readily accessible to the eluent, making them easy to elute. Weakly bound, non-specific impurities are largely removed during the rinsing step before analysis. Magnetic surface molecular imprinted resins are more robust due to their "rigid core-thin shell" structure, smooth spherical surfaces, and good stability, resulting in significantly improved reusability. Attached Figure Description

[0032] Figure 1 This is the HPLC-DAD spectrum of flavonoids from mulberry root bark in Example 1. Detailed Implementation

[0033] To make the purpose, technical solution, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the embodiments described in this specification are merely for explaining this application and are not intended to limit it.

[0034] For simplicity, this application only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form a range not explicitly stated; and any lower limit can be combined with other lower limits to form a range not explicitly stated, just as any upper limit can be combined with any other upper limit to form a range not explicitly stated. Furthermore, although not explicitly stated, every point or individual value between the endpoints of the range is included within that range. Therefore, each point or individual value can be used as its own lower or upper limit and combined with any other point or individual value or with other lower or upper limits to form a range not explicitly stated.

[0035] In the description of this application, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0036] The foregoing description of this application is not intended to describe every disclosed implementation or method. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are representative only and should not be construed as exhaustive.

[0037] Traditional extraction processes for flavonoids from mulberry root bark mainly involve ethanol reflux extraction and ultrasound-assisted ethanol extraction. These processes have significant drawbacks: they easily leave organic solvent residues, resulting in poor extraction efficiency and selectivity, and they can also damage the molecular structure of heat-sensitive flavonoids.

[0038] Furthermore, crude flavonoids are often purified using conventional macroporous adsorption resins such as D101 and AB-8. These resins rely on van der Waals forces for non-specific adsorption, which, while enriching flavonoids, also adsorbs large amounts of impurities such as polysaccharides, lignin, and pigments. The resulting product is dark in color and has low purity, increasing subsequent refining steps and driving up overall production costs. Traditional processes and single-process methods are insufficient to simultaneously meet the requirements of high extraction rates, high product purity, and environmentally friendly industrial production.

[0039] While there are reports of using deep eutectic solvents to extract flavonoids or molecularly imprinted resins to separate flavonoids in existing technologies, traditional molecular imprinting employs bulk polymerization, where templates, functional monomers, and crosslinking agents are mixed, polymerized, and then ground and sieved to obtain particles. In traditional bulk molecular imprinting, most of the binding sites are embedded within the polymer. This results in the binding sites of the molecular imprints being located inside the polymer, leading to a slow diffusion rate of the target analyte into the polymer. Furthermore, it is difficult to elute completely quickly; some target analytes are encapsulated in "closed pores" within the polymer, preventing contact with the elution solvent and forming permanent adsorption. Additionally, the large number of lipophilic impurities adsorbed in the non-imprinted hydrophobic regions within the polymer cause the elution solvent to elute both the target analyte and impurities during the elution process.

[0040] To address the aforementioned issues, this application provides a method for preparing mulberry root bark flavonoids, the mulberry root bark flavonoids themselves, and their applications. The preparation method of this application, through the synergistic effect of deep eutectic solvent extraction and magnetic molecular imprinting resin, not only effectively improves the extraction rate and time of mulberry root bark flavonoids but also achieves selective adsorption of four target flavonoids, exhibiting superior adsorption capacity and extraction rate. The resulting mulberry root bark flavonoids possess high purity and demonstrate excellent autophagy-promoting effects.

[0041] In a first aspect, embodiments of this application provide a method for preparing flavonoids from mulberry root bark, comprising: Step 100: Mix choline chloride and lactic acid in a molar ratio of 1:2 to 1:3, and stir at 60°C to 70°C until transparent to obtain a deep eutectic solvent.

[0042] Step 200: Mix mulberry root bark powder with a deep eutectic solvent at a mass ratio of 1:30 to 1:50, extract for 30 to 60 minutes at 50°C to 65°C and ultrasonic power of 200W to 300W, centrifuge and collect the supernatant to obtain crude mulberry root bark flavonoid DES extract.

[0043] Step 300: After purifying the crude extract of mulberry root bark flavonoids (DES) using magnetic molecular imprinting resin, mulberry root bark flavonoids are obtained.

[0044] The magnetic molecularly imprinted resin includes a magnetic core, an intermediate protective layer covering the surface of the magnetic core, and a molecularly imprinted polymer layer grafted onto the surface of the intermediate protective layer. The molecularly imprinted polymer layer is a polymer network with specific recognition sites formed by free radical polymerization of imprinted template molecules, functional monomers, and crosslinking agents under the action of a porogen. The imprinted template molecules are composed of mulberry root ketone C and mulberry ginseng ketone G, and the molar ratio of mulberry root ketone C to mulberry ginseng ketone G is 1:1 to 2:1. The average thickness of the molecularly imprinted polymer layer is 20 nm to 30 nm.

[0045] The magnetic core includes at least one of Fe3O4, γ-Fe2O3, CoFe2O4 and NiFe2O4, and the average thickness of the intermediate protective layer is 20 nm to 30 nm. The intermediate protective layer includes at least one of SiO2, Al2O3 and ZrO2.

[0046] As an example, in step 100, the molar ratio of choline chloride to lactic acid in the deep eutectic solvent can be 1:2, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.8, 1:2.9, 1:3, or any range of the above values. The mixing temperature of choline chloride and lactic acid can be 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, or any range of the above values.

[0047] As an example, in step 200, the mass ratio of mulberry root bark powder to the deep eutectic solvent can be 1:30, 1:32, 1:35, 1:38, 1:40, 1:42, 1:45, 1:48, 1:50, or any range of the above values. The extraction temperature can be 50℃, 52℃, 55℃, 58℃, 60℃, 62℃, 65℃, or any range of the above values. The ultrasonic power can be 200W, 220W, 240W, 260W, 280W, 300W, or any range of the above values. The extraction time can be 30min, 35min, 40min, 45min, 50min, 55min, 60min, or any range of the above values.

[0048] As an example, the molar ratio of mulberry root ketone C to mulberry senna ketone G in the molecularly imprinted polymer layer can be 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, or any range of the above values.

[0049] As an example, the average thickness of the molecularly imprinted polymer layer can be 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, or any range of the above values. The average thickness of the intermediate protective layer can be 20 nm, 21 nm, 22 nm, 23 nm, 24 nm, 25 nm, 26 nm, 27 nm, 28 nm, 29 nm, 30 nm, or any range of the above values.

[0050] It should be noted that the average thickness of the molecularly imprinted polymer layer and the intermediate protective layer refers to the average value calculated by statistically analyzing the thickness of 10 magnetic molecularly imprinted resins at three corresponding locations for each resin in a transmission electron microscope (TEM) image.

[0051] The method for preparing flavonoids from mulberry root bark provided in this application uses choline chloride as a hydrogen bond acceptor and lactic acid as a hydrogen bond donor, mixed in a specific molar ratio to prepare a deep eutectic solvent (DES). The chloride ions and hydroxyl groups of choline chloride form multiple intermolecular hydrogen bonds with the carboxyl and hydroxyl groups of lactic acid, disrupting the original crystal lattice structure and forming a homogeneous deep eutectic solvent in a room-temperature liquid state. This system has a stable hydrogen bond network, and its polarity can be precisely controlled by the molar ratio of the raw materials, highly compatible with the polarity of mulberry root bark flavonoids, enabling efficient flavonoid extraction. Furthermore, the polarity of impurities such as polysaccharides, proteins, and lignin is incompatible with DES, preventing the formation of stable complexes, and these impurities are difficult to dissolve after cell wall rupture. Under ultrasound-assisted conditions, the cavitation effect generated by ultrasound can further enhance cell wall rupture, accelerate the penetration of DES into the cell interior, shorten the mass transfer distance, and increase the dissolution rate of the DES-flavonoid complex, achieving short-time and efficient extraction. The entire process is conducted at low temperatures and is gentle, without damaging the chemical structure of the flavonoids.

[0052] Simultaneously, magnetic surface-imprinted resin was prepared using mulberry root ketone C and mulberry flavonoid G as template molecules. After template elution, imprinted cavities matching the size and spatial structure of the target flavonoid molecules were formed on the resin surface. On the one hand, impurities such as polysaccharides, pigments, and lignin could not enter the imprinted cavities due to their mismatched spatial structures, achieving efficient sieving. On the other hand, after flavonoid molecules entered the cavities, they could form multiple binding forces through hydrogen bonding, electrostatic interactions, and π-π stacking interactions. Combined with an imprinted polymer layer of a specific thickness and an intermediate protective layer, the adsorption strength, adsorption capacity, and selectivity were further enhanced. The inventors of this application discovered that using mulberry root ketone C and mulberry flavonoid G in a molar ratio of 1:1 to 2:1 as imprinted template molecules resulted in the best cross-recognition ability and a wider coverage range, enabling better recognition of all structurally similar Diels-Alder adducts in mulberry bark, including mulberry flavonoid G, L, O, and H. Compared to using a single imprinted template molecule or a combination of multiple imprinted template molecules, the adsorption capacity and adsorption selectivity for the four target flavonoids were optimal.

[0053] Furthermore, the target substances bound to the surface by the magnetic surface molecular imprinting site are easily accessible to the eluent and easily eluted. Most of the weakly bound non-specific impurities are removed in the rinsing step before analysis. The magnetic surface molecular imprinting resin is more robust due to its "rigid core-thin shell" structure, smooth spherical surface, good stability, and greatly improved reusability.

[0054] In some embodiments, step 300 includes: Step 330: Take the magnetic molecularly imprinted resin and prepare a resin homogenate, then fill it into a glass chromatography column. Several ring magnets are fitted on the outer wall of the glass chromatography column.

[0055] Step 340: The crude DES extract of mulberry root bark flavonoids is allowed to flow upward from the bottom of the glass chromatography column at a flow rate of 5 BV / h to 7 BV / h. At the same time, the ring magnet moves up and down to form a fluidized bed. The absorbance value of the effluent at 280 nm is monitored online. When the sample reaches 10% of the loading solution, the loading is stopped.

[0056] Step 350: Apply an axial gradient magnetic field to the glass chromatography column using a ring magnet, and elute with 3 column volumes of 10% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. Discard the eluent until the absorbance value is <0.05. The magnetic field strength at the top of the column is 0.5T to 0.6T, and the magnetic field strength at the bottom of the column is 0.2T to 0.3T.

[0057] Step 360: Apply a pulsed magnetic field of 1 Hz and 50% duty cycle to the glass chromatography column using a ring magnet, and elute with 4 column volumes of 70% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. Collect the elution peaks with absorbance values ​​> 0.1 to obtain the eluent.

[0058] Step 370: The eluent is concentrated under reduced pressure at 45°C and dried under vacuum to obtain mulberry root bark flavonoids.

[0059] In some embodiments, before step 330, the method may include step 310, which involves activating the magnetic molecularly imprinted resin, and step 320, which involves magnetically pre-clarifying the crude DES extract.

[0060] Step 310: Use 3 to 5 times the volume of ethanol to sonicate and soak the magnetic molecular imprinted resin; under the action of an external magnetic field, separate the magnetic molecular imprinted resin, discard the ethanol solution, and wash the magnetic molecular imprinted resin 2 to 3 times with 3 to 5 times the volume of deionized water or diluted deep eutectic solvent to obtain the activated magnetic molecular imprinted resin.

[0061] Step 320: Add the activated magnetic molecular imprinting resin to the crude extract of mulberry root bark flavonoids DES, stir for 5 to 10 minutes and apply an external magnetic field for 10 to 15 seconds, separate the supernatant, and dilute it with deionized water at a volume ratio of 1:1 for later use.

[0062] As an example, in step 310, the volume of ethanol can be 3 times, 3.5 times, 4 times, 4.5 times, 5 times, or any combination of the above values ​​of the magnetic molecular imprinting resin volume.

[0063] As an example, in step 310, the volume of deionized water or diluted deep eutectic solvent can be 3 times, 3.5 times, 4 times, 4.5 times, 5 times, or any combination of the above values ​​of the magnetic molecular imprinted resin.

[0064] As an example, in step 320, the stirring time can be 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or any range of the above times. The time for applying the external magnetic field can be 10 s, 11 s, 12 s, 13 s, 14 s, 15 s, or any range of the above values.

[0065] As an example, in step 340, the flow rate of the crude extract of mulberry root bark flavonoids DES can be 5 BV / h, 5.2 BV / h, 5.5 BV / h, 5.8 BV / h, 6 BV / h, 6.2 BV / h, 6.5 BV / h, 6.8 BV / h, 7 BV / h, or any range of the above values.

[0066] As an example, in step 350, the rinsing flow rate of the 10% ethanol aqueous solution can be 2 BV / h, 2.2 BV / h, 2.5 BV / h, 2.8 BV / h, 3 BV / h, 3.2 BV / h, 3.5 BV / h, 3.8 BV / h, 4 BV / h, or any range of the above values. The magnetic field strength at the top of the column can be 0.5 T, 0.52 T, 0.54 T, 0.56 T, 0.58 T, 0.6 T, or any range of the above values. The magnetic field strength at the bottom of the column can be 0.2 T, 0.22 T, 0.24 T, 0.26 T, 0.28 T, 0.3 T, or any range of the above values.

[0067] As an example, in step 360, the elution flow rate of the 70% ethanol aqueous solution can be 2 BV / h, 2.2 BV / h, 2.5 BV / h, 2.8 BV / h, 3 BV / h, 3.2 BV / h, 3.5 BV / h, 3.8 BV / h, 4 BV / h, or any range of the above values.

[0068] In some embodiments, the magnetic core comprises Fe3O4 and the intermediate protective layer comprises SiO2.

[0069] In some embodiments, the functional monomer is selected from one or more of methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, 4-vinylpyridine, 2-vinylpyridine, vinylimidazole, acrylamide, N-isopropylacrylamide, methacrylamide, styrene, and p-vinylbenzoic acid.

[0070] In some embodiments, the crosslinking agent is selected from one or more of ethylene glycol dimethacrylate, divinylbenzene, N,N'-methylenebisacrylamide, 1,4-butanediol dimethacrylate, trimethylolpropane trimethacrylate, triallyl isocyanurate, and pentaerythritol tetraacrylate.

[0071] In some embodiments, the pore-forming agent is selected from choline chloride-ethylene glycol eutectic solvent, choline chloride-glycerol eutectic solvent, or choline chloride-ethylene glycol-ethanol eutectic solvent.

[0072] In some embodiments, the pore-forming agent comprises a choline chloride-ethylene glycol-ethanol eutectic solvent, wherein the molar ratio of choline chloride, ethylene glycol, and ethanol is from 1:2:1.5 to 1:3:2. As an example, the molar ratio of choline chloride, ethylene glycol, and ethanol may be 1:2:1.5, 1:2:2, 1:3:1.5, 1:3:2, or any range of the above values.

[0073] In some embodiments, the ratio of the total molar amount of template molecules, the molar amount of functional monomers, and the molar amount of crosslinking agent is from 1:8:20 to 1:8:40. As an example, it can be 1:8:20, 1:8:22, 1:8:25, 1:8:28, 1:8:30, 1:8:32, 1:8:35, 1:8:38, 1:8:40, or any range of the above values.

[0074] In some embodiments, the magnetic molecularly imprinted resin is prepared by the following steps: Step S100: The magnetic core is prepared by co-precipitation method.

[0075] Step S200: A mesophase protective layer is coated on the surface of the magnetic core to obtain mesophase protective layer coated particles.

[0076] In step S300, the surface double bonds of the mesophase protective layer coated particles are modified using a silane coupling agent to obtain the particles to be grafted.

[0077] In step S400, mulberry root ketone C, mulberry senna ketone G and functional monomers are dissolved in a porogen and stirred at room temperature in the dark to allow the template molecules to form hydrogen-bonded complexes with the functional monomers, thus obtaining a pre-assembled solution.

[0078] In step S500, the particles to be grafted, the crosslinking agent, and the initiator are added to the pre-assembled solution, and the reaction is carried out under a protective atmosphere to obtain a crude product of magnetic molecular imprinted resin. The initiator can be azobisisobutyronitrile.

[0079] Step S600: Collect the crude product of magnetic molecularly imprinted resin by magnetic separation, and extract it with a methanol-acetic acid mixture using Soxhlet extraction until the eluent shows no absorption at 280 nm.

[0080] Step S700: Wash with methanol until neutral, then vacuum dry to obtain magnetic molecular imprinted resin.

[0081] In some embodiments, step 200 further includes pulverizing the dried mulberry root bark and passing it through a 40-60 mesh sieve to obtain mulberry root bark powder.

[0082] Secondly, this application provides a mulberry root bark flavonoid, which is prepared by the method described in the first aspect.

[0083] Thirdly, the embodiments of this application provide the application of mulberry root bark flavonoids prepared by the method of the first aspect or the mulberry root bark flavonoids of the second aspect in the preparation of cosmetics.

[0084] In some embodiments, cosmetics have the effect of promoting skin cell autophagy.

[0085] Example The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on mass, and all reagents used in the examples are commercially available or synthesized by conventional methods and can be used directly without further processing. The instruments used in the examples are also commercially available.

[0086] Example 1 In this embodiment, the magnetic molecularly imprinted resin is prepared through the following steps: Step S100, Preparation of Fe3O4 magnetic nanoparticles: 2.7 g FeCl3·6H2O and 1 g FeCl2·4H2O were dissolved in 100 mL of deoxygenated water. The mixture was heated to 80 °C under nitrogen protection, and 10 mL of concentrated ammonia was rapidly added dropwise. The mixture was stirred for 30 minutes. Magnetic separation was performed, and the nanoparticles were washed three times each with deionized water and ethanol. The nanoparticles were then vacuum dried at 60 °C for 12 hours to obtain black Fe3O4 powder.

[0087] Step S200, SiO2 coating: 1.0 g of the above Fe3O4 was dispersed in a mixture of 160 mL ethanol and 40 mL deionized water, sonicated for 30 minutes, and then 50 mL concentrated ammonia was added and stirred until homogeneous. 4 mL of tetraethyl orthosilicate (TEOS) was slowly added dropwise, and the mixture was stirred at room temperature for 6 hours. After magnetic separation, the mixture was washed three times with ethanol and dried under vacuum at 60 °C to obtain Fe3O4-SiO2. TEM characterization showed that the average thickness of the SiO2 coating layer was approximately 25 nm.

[0088] Step S300, surface double bond modification: 1.0 g of the above Fe3O4-SiO2 was dispersed in 1 L of toluene, and 3 mL of KH-570 (γ-methacryloyloxypropyltrimethoxysilane) was added. The mixture was refluxed under nitrogen protection for 12 hours. After magnetic separation, the mixture was washed three times each with toluene and ethanol, and dried under vacuum at 60 °C to obtain the double bond modified Fe3O4-SiO2-MPS.

[0089] Step S400, dual-template surface imprinting polymerization: Dissolve 0.1 mmol mulberry root ketone C + 0.1 mmol mulberry root ketone G (total template 0.2 mmol) and 1.6 mmol 4-vinylpyridine in 40 mL of a DES eutectic solvent choline chloride-ethylene glycol-ethanol (molar ratio 1:2:2) mixture and stir at room temperature in the dark for 2 hours to allow the template and functional monomer to fully form hydrogen bond complexes, thus obtaining a pre-assembled solution.

[0090] In step S500, 1.0 g of the double-bond modified Fe3O4-SiO2-MPS was added to the pre-assembled solution and ultrasonically dispersed for 30 minutes. Then, 4 mmol of ethylene glycol dimethacrylate (EGDMA) and 40 mg of azobisisobutyronitrile (AIBN) were added, and the mixture was reacted in a water bath at 60°C for 24 hours under nitrogen protection.

[0091] Step S600, template elution: magnetic separation is used to collect the crude product of magnetic molecular imprinted resin, and Soxhlet extraction with methanol-acetic acid (9:1) is performed for 48 hours until the eluent shows no absorption at 280 nm.

[0092] Step S700, Drying: Wash the polymer with methanol until neutral, and dry it under vacuum at 60°C for 12 hours to obtain the final dual-template magnetic surface molecularly imprinted resin (MMIPs).

[0093] A type of mulberry root bark flavonoid is prepared by the following steps: Step 100: Choline chloride and lactic acid are mixed in a molar ratio of 1:2.5 and stirred in a water bath at 65°C until clear to obtain DES.

[0094] Step 200: Dry mulberry root bark is pulverized and passed through a 50-mesh sieve to obtain mulberry root bark powder. Take 10g of crude mulberry root bark powder and extract it using a choline chloride-lactic acid eutectic solvent (molar ratio 1:2.5, 20% water content) at a mass ratio of 40:1 (DES:mulberry root bark powder) at 50℃ for 30 minutes. Filter to obtain crude DES extract of mulberry root bark flavonoids.

[0095] Step 310: Use 4 times the resin volume of ethanol to sonicate and soak the magnetic molecular imprinted resin. Step 320: Under the action of an external magnetic field, separate the magnetic molecular imprinted resin, discard the ethanol solution, and wash the magnetic molecular imprinted resin three times with four times the volume of deionized water or the diluted deep eutectic solvent to obtain the activated magnetic molecular imprinted resin.

[0096] Step 330: Take the magnetic molecularly imprinted resin and prepare a resin homogenate, then fill it into a glass chromatography column. Several ring magnets are fitted on the outer wall of the glass chromatography column.

[0097] Step 340, Column packing: The crude DES extract of mulberry root bark is fed upward from the bottom of the glass chromatography column at a flow rate of 5 BV / h. At the same time, the ring magnet is slowly moved up and down to form a fluidized bed. The absorbance of the effluent at 280 nm is monitored online. When the sample reaches 10% of the loading solution, the loading is stopped.

[0098] Step 350: Apply an axial gradient magnetic field (0.5T at the top of the column and 0.2T at the bottom of the column) to the glass chromatography column using a ring magnet, and elute with 3 column volumes of 10% ethanol aqueous solution at a flow rate of 3 BV / h. Discard the eluent until the absorbance value is <0.05.

[0099] Step 360: A pulsed magnetic field (1 Hz, 50% duty cycle) is applied to the glass chromatography column using a ring magnet. Elution is performed with 4 column volumes of 70% ethanol aqueous solution at a flow rate of 2 BV / h. The elution peaks with absorbance values ​​>0.1 are collected. The eluent is concentrated under reduced pressure at 45°C and dried under vacuum to obtain mulberry root bark flavonoids.

[0100] Example 2 The difference between Example 2 and Example 1 is that γ-Fe₂O₃ is used for the magnetic core. Step S100 includes: dissolving 2.7g FeCl₃·6H₂O in 100mL deionized water, adding 1.0g polyethylene glycol, and stirring until homogeneous. Ammonia water is added dropwise until pH=10, and the mixture is stirred for 30 minutes. The mixture is transferred to a reaction vessel and hydrothermally reacted at 180℃ for 6 hours. After cooling to room temperature, magnetic separation is performed, followed by washing and drying to obtain γ-Fe₂O₃ powder.

[0101] Example 3 The difference between Example 3 and Example 1 is that the magnetic core is CoFe2O4. Step S100 includes: dissolving 2.7g FeCl3·6H2O and 1.2g CoCl2·6H2O in 100mL deionized water, adding 1.0g polyethylene glycol, and stirring until homogeneous. Ammonia water is added dropwise until pH=10, and the mixture is stirred for 30 minutes. The mixture is transferred to a reaction vessel and hydrothermally reacted at 180℃ for 6 hours. After cooling to room temperature, magnetic separation is performed, followed by washing and drying to obtain CoFe2O4 powder.

[0102] Example 4 The difference between Example 4 and Example 1 is that Al2O3 is used as the intermediate protective layer. Step S200 includes: dispersing 1.0 g of Fe3O4 in 100 mL of ethanol and sonicating for 30 minutes. Adding 2 mL of aluminum isopropoxide and stirring until homogeneous. Adding a small amount of ammonia and stirring at room temperature for 6 hours. Magnetic separation, washing three times with ethanol, and vacuum drying at 60 °C to obtain Fe3O4-Al2O3.

[0103] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that no magnetic core was used; instead, imprinting polymerization was performed directly on the surface of SiO2 microspheres. This yielded non-magnetic surface molecularly imprinted resins (SMMIPs).

[0104] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that a conventional bulk molecularly imprinted resin was used, which was prepared by the following method: 0.1 mmol of mulberry root ketone C + 0.1 mmol of mulberry root ketone G, 1.6 mmol of 4-vinylpyridine, 4.0 mmol of EGDMA and 40 mg of AIBN were dissolved in 40 mL of acetonitrile-methanol (9:1) mixed solution, and reacted in a water bath at 60°C for 24 hours under nitrogen protection. A block polymer was obtained, ground, sieved, and extracted with methanol-acetic acid (9:1) using a Soxhlet extractor for 48 hours. After drying, the conventional bulk molecularly imprinted resin (BMMIPs) was obtained.

[0105] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that polyamide resin was used. Step 300 includes: soaking the polyamide resin in ethanol to remove impurities and activate it, discarding the ethanol, washing it with deionized water until there is no ethanol odor, and then packing it into a column (column volume BV = 20 mL); loading the crude extract of mulberry root bark at a flow rate of 1.5 BV / h, washing the column with 2 BV of deionized water; using 75% ethanol at a flow rate of 1 BV / h to elute, collecting 3.5 BV of eluent; concentration and drying: concentration under reduced pressure at 60°C, and vacuum drying at 60°C to obtain the mulberry root bark flavonoid product.

[0106] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that in step S400, only 0.2 mmol of morinone C was used as the template molecule, and morinone G was not added. A magnetic molecularly imprinted resin was obtained.

[0107] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that in step S400, only 0.2 mmol of linalool G was used as the template molecule, and linalool C was not added, resulting in a magnetic molecularly imprinted resin.

[0108] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that in step S400, 0.05 mmol mulberry root ketone C + 0.05 mmol mulberry root ketone D + 0.05 mmol mulberry root ketone G + 0.05 mmol mulberry root ketone L are used as template molecules, and the total template amount is still 0.2 mmol, thus obtaining magnetic molecular imprinted resin.

[0109] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that 2 mL of tetraethyl orthosilicate (TEOS) was added in step S200. TEM characterization showed that the average thickness of the SiO2 coating layer was approximately 10 nm, yielding a magnetic molecularly imprinted resin.

[0110] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that 6 mL of tetraethyl orthosilicate (TEOS) was added in step S200. TEM characterization showed that the average thickness of the SiO2 coating layer was approximately 40 nm, yielding a magnetic molecularly imprinted resin.

[0111] Comparative Example 9 The difference between Comparative Example 9 and Example 1 is that 0.05 mmol of mulberry root ketone C + 0.05 mmol of mulberry root ketone G, 0.8 mmol of 4-vinylpyridine, and 2.0 mmol of EGDMA were added in step S400. TEM characterization showed that the average thickness of the molecularly imprinted polymer layer was approximately 10 nm, yielding a magnetic molecularly imprinted resin.

[0112] Comparative Example 10 The difference between Comparative Example 10 and Example 1 is that 0.15 mmol of mulberry root ketone C + 0.15 mmol of mulberry root ketone G, 2.4 mmol of 4-vinylpyridine, and 6.0 mmol of EGDMA were added in step S400. TEM characterization showed that the average thickness of the molecularly imprinted polymer layer was approximately 40 nm, yielding a magnetic molecularly imprinted resin.

[0113] The performance of the resins in Examples 1 to 4 and Comparative Examples 1 to 10 was evaluated.

[0114] Adsorption capacity test: Weigh 20g of the resins prepared in each of the above examples and comparative examples, add 2000mL of a mixed standard solution of 100μg / mL mulberry root ketone C, mulberry root ketone D, mulberry flavonoid G, and mulberry flavonoid L (solvent being DES deep eutectic solvent choline chloride and lactic acid), and shake at room temperature for 20 minutes for adsorption. Centrifuge / magnetic separation (centrifugation with non-magnetic resin), take the supernatant, and determine the concentration of each flavonoid by HPLC. Calculate the adsorption capacity based on the change in flavonoid concentration in the solution before and after adsorption.

[0115] Detection of saturation magnetization: After magnetizing the sample in a uniform external magnetic field, it is made to vibrate slightly at a fixed frequency. The magnetic flux passing through the detection coil will change periodically, thereby generating an induced electromotive force proportional to the total magnetic moment of the sample. By calibrating the relationship between the induced electromotive force and the magnetic moment, the magnetization of the sample under different magnetic fields can be calculated, and finally, a complete hysteresis loop can be obtained.

[0116] The operation steps are as follows: 1. Thorough purification: Use a magnet to repeatedly separate and wash the resin sample (at least 3 times) to remove all uncoated free Fe3O4 nanoparticles until the supernatant is completely clear and free of black particles.

[0117] 2. Complete drying: Dry in a vacuum drying oven at 60℃ for more than 12 hours, or freeze dry for 24 hours to ensure no residual moisture (moisture will cause the mass to be too large and the Ms result to be too low).

[0118] 3. Grinding and sieving: Grind into fine powder using an agate mortar and pestle, then pass through a 100-mesh sieve to obtain uniform powder and avoid particle agglomeration.

[0119] 4. Accurate weighing: Weigh 5-20 mg of sample (10 mg recommended) using a 1 / 100,000 analytical balance and record the accurate mass (accurate to 0.01 mg).

[0120] 5. Sample installation: Place the sample into the non-magnetic quartz sample tube and gently press it down to ensure that the sample is centered at the bottom of the tube; fix the sample tube to the end of the VSM vibrating rod and adjust its position so that the sample is centered in the electromagnet's magnetic field.

[0121] 6. System calibration: Calibrate the instrument's magnetic moment and magnetic field scale using standard nickel plates or pure iron standards.

[0122] 7. Saddle point adjustment: Fine-tune the position of the sample in three-dimensional space to maximize and stabilize the output signal of the detection coil.

[0123] 9. Setting Parameters: Maximum Magnetic Field: ±1.5T (Fe3O4 saturates at around 1T, no higher magnetic field is needed). Test Temperature: 300K (room temperature). Scan Rate: 100~200 Oe / s (too fast will cause distortion of the hysteresis loop). Number of Data Points: 120~150 points, which can be appropriately increased near coercivity and remanence. Start Test: The system automatically scans the magnetic field, collects magnetization data under different magnetic fields, and generates hysteresis loops. Background Subtraction: The background signal of the empty sample tube under the same test conditions is subtracted from the sample data.

[0124] 10. Data processing and result calculation: Read the total saturation magnetic moment value (M_total, unit: emu) from the plateau region of the hysteresis loop.

[0125] Formula for calculating saturation magnetization: , where: M s Saturation magnetization, unit: emu / g (International unit: 1 emu / g = 1 A·m) 2 / kg), M total : Total saturated magnetic moment of the sample, unit: emu; m: Dry mass of the sample, unit: g.

[0126] 11. Result Judgment: Superparamagnetism Judgment: Qualified magnetic molecular imprinted resin should exhibit superparamagnetism, characterized by a closed hysteresis loop, meaning that the resin will not aggregate after the external magnetic field is removed. M s Low: Sample not completely dried, coating layer too thick, magnetic particle content too low.s High values: Indicate the presence of free Fe3O4 nanoparticles or sample contamination by ferromagnetic impurities. Asymmetrical hysteresis: Indicates the sample is not centered in the magnetic field or the instrument is not calibrated. Generally, M... s Rapid magnetic separation (solid-liquid separation completed within 1-2 minutes) can be achieved with ≥5 emu / g, meeting the requirements of solid-phase extraction and adsorption experiments.

[0127] Reusability test: The saturated resin was eluted with methanol-acetic acid (9:1) until the eluent showed no absorption at 280 nm. The adsorption experiment was then repeated 10 times, and the adsorption capacity was calculated for each iteration to evaluate the reusability of the resin. The results are shown in Table 1.

[0128] Table 1. Resin performance evaluation of Examples 1-4 and Comparative Examples 1-10

[0129] Note: In Table 1, " / " indicates that the corresponding substance or parameter is not included.

[0130] The results in Table 1 show that the magnetic molecular imprinting resin of Example 1 has an adsorption capacity that is 1.7 times that of traditional bulk imprinting resin and 2.1 times that of commonly used resin. The adsorption equilibrium time is shortened from 180-200 minutes to 20 minutes, the separation time is shortened from 10 minutes to 10 seconds, and the number of reuses is increased from 3-5 times to more than 10 times, showing significant advantages.

[0131] The dual-template magnetic molecularly imprinted resin of Example 1 exhibited the highest total adsorption capacity and best selectivity for the four target flavonoids. The single-template resins in Comparative Examples 4 and 5 only showed good adsorption capacity for flavonoids with similar template structures, while exhibiting poor adsorption capacity for the other type of flavonoid. The tetratemplate resin of Comparative Example 6, due to the excessive number of templates, reduced the imprinting sites per template, resulting in decreased adsorption capacity and selectivity. Polyamide resin adsorbed all four target flavonoids, but its adsorption capacity was significantly weaker than that of the magnetic molecularly imprinted resin of Example 1. According to Examples 1, 7, and 8, the resin exhibited optimal adsorption performance when the SiO2 coating thickness was 25 nm. When the SiO2 layer was too thin (10 nm), the protection of the Fe3O4 core was insufficient, making it easily corroded under acidic conditions, leading to decreased magnetism and site destruction. When the SiO2 layer was too thick (40 nm), it reduced the saturation magnetization of the material, affecting the magnetic separation rate and increasing mass transfer resistance. According to Examples 1, 9, and 10, the optimal balance between adsorption capacity and mass transfer rate of the resin was achieved when the thickness of the molecularly imprinted polymer (MMIP) layer was 25 nm. When the MMIP layer was too thin (10 nm), the number of imprinted sites was insufficient, resulting in low adsorption capacity. When the MMIP layer was too thick (40 nm), the mass transfer resistance increased, the adsorption equilibrium time was prolonged, and the internal template was difficult to elute, leading to an increased template leakage rate.

[0132] As shown in Examples 1 to 3, Fe3O4 has advantages such as low cost, simple preparation, and good magnetic responsiveness, making it the best choice. γ-Fe2O3 has slightly better chemical stability than Fe3O4, but its cost is higher. CoFe2O4 has the highest saturation magnetization, but its cost is also the highest, and cobalt has a certain degree of toxicity. As shown in Examples 1 and 4, SiO2 is the most effective intermediate protective layer. The surface of SiO2 is rich in silanol groups, making it easy to modify, and it has good chemical stability, effectively protecting the magnetic core. Al2O3 is more difficult to modify on the surface, and its chemical stability is not as good as SiO2, resulting in slightly poorer adsorption performance.

[0133] Example 5 The difference between Example 5 and Example 1 is as follows: Step 100: Choline chloride and lactic acid are mixed in a molar ratio of 1:2 and stirred in a water bath at 65°C until clear to obtain DES.

[0134] Step 200: Dry mulberry root bark is pulverized and passed through a 50-mesh sieve to obtain mulberry root bark powder. Take 10g of crude mulberry root bark powder and extract it using a choline chloride-lactic acid eutectic solvent (molar ratio 1:2, 20% water content) at a mass ratio of 30:1 (DES:mulberry root bark powder) at 50℃ for 30 minutes. Filter to obtain crude DES extract of mulberry root bark flavonoids.

[0135] Example 6 The difference between Example 6 and Example 1 is: Step 100: Choline chloride and lactic acid are mixed in a molar ratio of 1:3 and stirred in a water bath at 65°C until clear to obtain DES.

[0136] Step 200: Dry mulberry root bark is pulverized and passed through a 50-mesh sieve to obtain mulberry root bark powder. Take 10g of crude mulberry root bark powder and extract it using a choline chloride-lactic acid eutectic solvent (molar ratio 1:3, 20% water content) at a mass ratio of 50:1 (DES:mulberry root bark powder) at 50℃ for 30 minutes. Filter to obtain crude DES extract of mulberry root bark flavonoids.

[0137] Comparative Example 11 The difference between Comparative Example 11 and Example 1 is that in step 100, choline chloride and lactic acid are mixed in a molar ratio of 1:1.5 and stirred in a water bath at 65°C until clear, thus obtaining DES.

[0138] Comparative Example 12 The difference between Comparative Example 12 and Example 1 is that in step 100, choline chloride and lactic acid are mixed in a molar ratio of 1:3.5 and stirred in a water bath at 65°C until clear, thus obtaining DES.

[0139] Comparative Example 13 The difference between Comparative Example 13 and Example 1 is as follows: In step 200, the dried mulberry root bark is pulverized and passed through a 50-mesh sieve to obtain mulberry root bark powder. 10g of crude mulberry root bark powder is taken and ultrasonically extracted at 50°C for 30 minutes using a choline chloride-lactic acid eutectic solvent (molar ratio 1:2.5, 20% water content) at a mass ratio of 25:1 (DES:mulberry root bark powder). After filtration, a crude DES extract of mulberry root bark flavonoids is obtained.

[0140] Comparative Example 14 The difference between Comparative Example 14 and Example 1 is as follows: In step 200, dried mulberry root bark is pulverized and passed through a 50-mesh sieve to obtain mulberry root bark powder. 10g of crude mulberry root bark powder is taken and ultrasonically extracted at 50°C for 30 minutes using a choline chloride-lactic acid eutectic solvent (molar ratio 1:2.5, 20% water content) at a mass ratio of 55:1 (DES:mulberry root bark powder). After filtration, a crude DES extract of mulberry root bark flavonoids is obtained.

[0141] Comparative Example 15 The difference between Comparative Example 15 and Example 1 is that urea (BUN) was used as the hydrogen bond donor in the DES.

[0142] Comparative Example 16 The difference between Comparative Example 16 and Example 1 is that ethylene glycol (EG) was used as the hydrogen bond donor in DES.

[0143] Comparative Example 17 The difference between Comparative Example 17 and Example 1 is that citric acid is used as the hydrogen bond donor in DES, and the molar ratio of choline chloride to citric acid is 1:1.

[0144] Comparative Example 18 The difference between Comparative Example 18 and Example 1 is that steps 100 to 200 are replaced with the following steps: Take 10g of mulberry root bark powder, add 70% ethanol, with a material-to-liquid ratio of 20:1 (solvent: mulberry root bark powder), reflux at 80℃ for 2h, extract twice, combine the extracts, centrifuge at 4000r / min, and take the supernatant to obtain crude ethanol extract.

[0145] Comparative Example 19 The difference between Comparative Example 19 and Example 1 is that the former uses traditional ethanol reflux + polyamide resin purification to extract mulberry root ketones. Specifically, 10g of mulberry root bark powder is added to 70% ethanol at a material-to-liquid ratio of 20:1 (solvent: mulberry root bark powder). The mixture is refluxed at 80℃ for 2 hours, extracted twice, and the extracts are combined. The mixture is centrifuged at 4000 rpm, and the supernatant is collected to obtain a crude ethanol extract. The crude ethanol extract is concentrated and loaded onto a polyamide resin column, washed with 2 BV of water to remove impurities, and then eluted with 75% ethanol. The eluent is collected, concentrated, and dried to obtain the mulberry root bark flavonoid product.

[0146] The total flavonoids in Examples 1, 5 to 6, and Comparative Examples 11 to 18 were tested using the UV method, and the flavonoid yield was calculated. The test method is as follows: Accurately weigh 5.00g of magnesium acetate reagent into a 1000mL beaker, add methanol solution to 500g, and sonicate to dissolve evenly to obtain a 1.0% magnesium acetate methanol solution.

[0147] Accurately weigh 0.0050 g of mulberry root ketone C standard (mulberry root ketone C standard, CAS: 80651-76-9, (w%) HPLC > 99%) into a 100 mL volumetric flask, add methanol and sonicate to dissolve, then dilute to the mark with methanol to obtain a 0.05 mg / mL mulberry root ketone C standard solution.

[0148] Establish the standard curve of mulberry root ketone C based on the contents of Table 2.

[0149] Table 2. Construction of the standard curve for mulberry root ketone C

[0150] The mulberry root bark flavonoid products from Examples 1, 5 to 6, and Comparative Examples 11 to 18 were prepared into a clear, transparent liquid at a concentration of 0.30 mg / mL using methanol, and then sonicated to dissolve it evenly for later use.

[0151] Pipette 0.40 mL of the above liquid into a 10 mL stoppered test tube, add 9.6 mL of 1.0% magnesium acetate methanol solution, shake well, let stand for 30 min, and use 0 mL of the reaction solution as a blank control. Perform zeroing and baseline scanning, and measure the absorbance value Ax at a wavelength of 316 nm.

[0152] Calculate total flavonoids using the following formula: Total flavonoid content (%) = 10(Ax-b)0.12a×100%.

[0153] In the formula: Ax—absorbance value of the sample to be tested; a—a in the standard curve; b—b in the standard curve.

[0154] The absolute difference between two independent measurements obtained under repeatability conditions shall not exceed 10% of the arithmetic mean.

[0155] For detailed results, please refer to Table 3.

[0156] Table 3 Effect of different extraction methods on flavonoid yield

[0157] Note: In Table 3, " / " indicates that the corresponding substance or parameter is not included.

[0158] Table 3 shows that steps 100 and 200 achieve the highest extraction rate of mulberry root ketone when meeting the requirements of this application. In Comparative Example 11, DES exhibits poor flowability, high viscosity, a denser intermolecular hydrogen bond network, increased mass transfer resistance, slower diffusion of flavonoids from plant tissue to the solvent, lower lactic acid content, and a weakened ability to form hydrogen bonds with the phenolic hydroxyl groups in flavonoid molecules. The overall polarity of the eutectic solvent decreases, reducing its solubility for polar flavonoids. In Comparative Example 12, excess lactic acid molecules preferentially form hydrogen bonds with choline chloride, reducing effective binding sites for flavonoid molecules. Numerous intermolecular hydrogen bonds also form between lactic acid molecules, further reducing the solubilization ability for flavonoids. The significantly increased acidity of the eutectic solvent system may lead to slight degradation of some flavonoid compounds. Although low viscosity is suitable as an extraction solvent, this advantage is completely offset by the three negative factors mentioned above. When the viscosity is very low, the mass transfer rate is no longer the rate-limiting step in the extraction process.

[0159] In Comparative Example 13, the amount of DES was too low, and the mulberry root bark powder could not be completely wetted. The high viscosity of the eutectic solvent hindered the diffusion of flavonoid molecules from the plant cells into the solvent bulk. The total solvent volume quickly reached the dissolution equilibrium of flavonoids, and it was impossible to dissolve more flavonoid components. In Comparative Example 14, the higher amount of DES did not bring higher extraction yield. Excessive solvent would extract some impurities, such as proteins, pigments, and tannins. These impurities would occupy the hydrogen bond binding sites of the eutectic solvent, reducing the chance of the eutectic solvent binding with flavonoids.

[0160] In Comparative Example 15, urea contains only amide groups with weak hydrogen bond donor capacity, lacking carboxyl groups and highly reactive hydroxyl groups. Its hydrogen bond donor strength is far weaker than that of lactic acid and ethylene glycol, making it unable to form stable competitive hydrogen bonds with the multiple phenolic hydroxyl groups of flavonoid molecules in mulberry root bark. This means it cannot break the bond between flavonoids and the cellulose and lignin of plant cell walls, nor can it effectively solubilize flavonoids, resulting in extremely poor intrinsic solubility of the flavonoid active components in mulberry. The system is nearly neutral (pH≈6.5~7.0), completely lacking acid hydrolysis capability, and unable to disrupt the dense pectin layer and cellulose structure of plant cell walls. Flavonoid components are difficult to release from the cells, resulting in extremely low mass transfer efficiency. At 25℃, the viscosity of the classic choline chloride-urea (1:2) ratio is as high as several hundred mPa·s, more than 10 times that of the choline chloride-lactic acid system. Even with the addition of water to reduce viscosity, its mass transfer resistance remains significantly higher than other systems, severely hindering the diffusion of flavonoids from plant cells to the solvent. In Comparative Example 16, ethylene glycol contains only two terminal hydroxyl groups and no carboxylic acid groups. Its hydrogen bond donor strength is far weaker than that of hydroxycarboxylic acids such as lactic acid and citric acid, making it unable to form stable competitive hydrogen bonds with the phenolic hydroxyl groups of flavonoids in mulberry root bark. Furthermore, ethylene glycol is a neutral compound, and this DES system is neutral overall, completely lacking the ability to acidify the pectin layer and cellulose of plant cell walls, making it difficult for flavonoid active ingredients to be released from the cells. In Comparative Example 17, the optimal molar ratio of choline chloride to citric acid to form a stable DES solution is 1:1, so this optimal ratio was used for extraction. Although citric acid contains three carboxyl groups and one hydroxyl group, providing abundant hydrogen bond donor sites, its large molecular weight and significant steric hindrance prevent it from forming efficient and stable competitive hydrogen bonds with the phenolic hydroxyl groups of flavonoids in mulberry root bark, unlike the smaller molecule lactic acid. This makes it difficult to break the bond between flavonoids and the cellulose and lignin of plant cell walls, limiting the flavonoid dissolution rate. In addition, this DES system has high viscosity and significant mass transfer resistance, severely hindering the diffusion of flavonoids from plant cells into the solvent. Citric acid is weaker than lactic acid and has a poorer ability to hydrolyze the pectin layer and cellulose of plant cell walls, thus failing to efficiently destroy cell structures and release flavonoids. Although Comparative Example 18 had a higher extraction rate, the extract contained a large amount of non-target product sangin flavonoids, as well as a large number of impurities, tannic acid, polysaccharides, proteins, volatile components, and other substances.

[0161] The mulberry root ketones obtained in Examples 1, 2, 3, 18 and 19 were subjected to an autophagy promotion test, and the test method is as follows.

[0162] 1) Seeding of keratinocytes / human melanocytes / human fibroblasts: After cell resuscitation, when the plating rate reaches approximately 80%, seed at a dose of 1×10⁻⁶ cells / cells. 5 Cells were seeded into 6-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0163] 2) Experimental grouping: The experiment included a blank control group and a sample group. Two concentration gradients were set for each sample, and two replicate wells were set for each concentration gradient.

[0164] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 4).

[0165] Table 4 Test Concentration Setting Table 4) Drug administration: Drug administration was performed after 24 hours of incubation. For the blank control group, 2 mL of complete culture medium (90% MEM solution, 10% premium fetal bovine serum, 1% penicillin-streptomycin) was added to each well; for the sample group, 2 mL of culture medium containing the corresponding concentration of the test sample was added to each well. After drug administration, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 hours.

[0166] 5) After the incubation period, discard the supernatant; wash twice with pre-cooled sterile PBS.

[0167] 6) RNA extraction (follow the RNA extraction kit procedure below).

[0168] ① Add 300 μL of lysis buffer to each well of a 6-well plate and gently blow it 5-10 times until the solid suspension dissolves and the solution becomes clear.

[0169] ② Add an equal volume of binding solution to the lysis buffer and gently invert to mix 3-5 times.

[0170] ③ Transfer the mixture to a purification column, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0171] ④ Add 600 μL of washing solution I, centrifuge at 12000 rcf for 30 s, and discard the liquid in the collection tube.

[0172] ⑤ Add 600 μL of washing buffer II, centrifuge at 12000 rcf for 30 s, discard the liquid in the collection tube, and repeat this step.

[0173] ⑥ Centrifuge at the highest speed for 2 minutes to remove residual liquid.

[0174] ⑦ Place the RNA purification column in the RNA elution tube provided in the kit, add 30 μL of elution buffer, incubate at room temperature for 2-3 min, centrifuge at the highest speed for 30 s, and the resulting solution is the purified RNA.

[0175] ⑧ Add an appropriate amount of DEPC water to dissolve the mRNA, determine the concentration, and perform reverse transcription according to the instructions of the reverse transcription kit.

[0176] 7) Reverse transcription PCR System (20 μL): 4 µL of 5×PrimeScript buffer; 1 µL of PrimeScript RT Enzyme Mix 1; 2 µL of Random 6 mers; 13 µL of RNA and ddH2O, making the amount of RNA 1000 ng.

[0177] The reaction conditions were 37℃ for 15 min; 80℃ for 5 s; 4℃ cycle; Total: approximately 15 min.

[0178] 8) Quantitative Real-Time PCR (QPCR) Experiment: System (20 μL): 10 μL of 2×SYBR, 2-5 μL of template (cDNA obtained from reverse transcription diluted 10 times as template), 1-2 μL of primers, and ultrapure water to bring the system to 20 μL. Reaction conditions: Pre-denaturation 94℃ for 5 min; 40 cycles: denaturation 94℃ for 30 s, annealing 60℃ for 30 s, extension 72℃ for 30 s (real-time fluorescence photography); melting curve 94℃ for 30 s, 60℃ for 30 s, 72℃ for 1 s (real-time fluorescence photography during the heating process).

[0179] The results of promoting autophagy are shown in Table 5.

[0180] Table 5 Results of promoting autophagy

[0181] As shown in Table 5, the mulberry root ketone prepared by the method of this application has a significantly better effect on promoting autophagy than the mulberry root ketone in the comparative example. The DES extraction and magnetic molecular imprinting resin separation of this application have a good synergistic effect.

[0182] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing flavonoids from mulberry root bark, characterized in that, include: Choline chloride and lactic acid are mixed in a molar ratio of 1:2 to 1:3 and stirred at 60°C to 70°C until transparent to obtain a deep eutectic solvent. The mulberry root bark powder was mixed with the deep eutectic solvent at a mass ratio of 1:30 to 1:

50. The mixture was extracted for 30 to 60 minutes at 50°C to 65°C and ultrasonic power of 200W to 300W. The supernatant was collected by centrifugation to obtain crude extract of mulberry root bark flavonoids (DES). The crude extract of mulberry root bark flavonoids (DES) was purified using magnetic molecular imprinting resin to obtain the mulberry root bark flavonoids. The magnetic molecularly imprinted resin comprises a magnetic core, an intermediate protective layer covering the surface of the magnetic core, and a molecularly imprinted polymer layer grafted onto the surface of the intermediate protective layer. The molecularly imprinted polymer layer is a polymer network with specific recognition sites formed by free radical polymerization of imprinted template molecules, functional monomers, and crosslinking agents under the action of a porogen. The imprinted template molecules are composed of mulberry root ketone C and mulberry ginseng ketone G, and the molar ratio of mulberry root ketone C to mulberry ginseng ketone G is 1:1 to 2:

1. The average thickness of the molecularly imprinted polymer layer is 20 nm to 30 nm. The magnetic core comprises at least one of Fe3O4, γ-Fe2O3, CoFe2O4 and NiFe2O4, and the average thickness of the intermediate protective layer is 20 nm to 30 nm. The intermediate protective layer comprises at least one of SiO2, Al2O3 and ZrO2.

2. The preparation method according to claim 1, characterized in that, The step of purifying the crude DES extract of mulberry root bark flavonoids using magnetic molecular imprinting resin to obtain the mulberry root bark flavonoids includes: The magnetic molecularly imprinted resin was prepared into a resin homogenate and packed into a glass chromatography column. Several ring magnets were fitted on the outer wall of the glass chromatography column. The crude DES extract of mulberry root bark flavonoids is allowed to flow upward from the bottom of the glass chromatography column at a flow rate of 5 BV / h to 7 BV / h, while the ring magnet is moved up and down to form a fluidized bed. The absorbance value of the effluent at 280 nm is monitored online, and the loading is stopped when the sample reaches 10% of the loading solution. An axial gradient magnetic field is applied to the glass chromatography column using the ring magnet. The column is eluted with 3 column volumes of 10% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. The eluent is discarded until the absorbance value is <0.

05. The magnetic field strength at the top of the column is 0.5T to 0.6T, and the magnetic field strength at the bottom of the column is 0.2T to 0.3T. A pulsed magnetic field of 1 Hz and 50% duty cycle is applied to the glass chromatography column by the ring magnet, and elution is carried out with 4 column volumes of 70% ethanol aqueous solution at a flow rate of 2 BV / h to 4 BV / h. The elution peak with absorbance value > 0.1 is collected to obtain the eluent. The eluent was concentrated under reduced pressure at 45°C and then dried under vacuum to obtain the mulberry root bark flavonoids.

3. The preparation method according to claim 1, characterized in that, The magnetic core comprises Fe3O4, and the intermediate protective layer comprises SiO2.

4. The preparation method according to claim 1, characterized in that, The functional monomer is selected from one or more of methacrylic acid, acrylic acid, itaconic acid, hydroxyethyl methacrylate, 4-vinylpyridine, 2-vinylpyridine, vinylimidazole, acrylamide, N-isopropylacrylamide, methacrylamide, styrene, and p-vinylbenzoic acid; And / or, the crosslinking agent is selected from one or more of ethylene glycol dimethacrylate, divinylbenzene, N,N'-methylenebisacrylamide, 1,4-butanediol dimethacrylate, trimethylolpropane trimethacrylate, triallyl isocyanurate, and pentaerythritol tetraacrylate. And / or, the pore-forming agent is selected from choline chloride-ethylene glycol eutectic solvent, choline chloride-glycerol eutectic solvent, or choline chloride-ethylene glycol-ethanol eutectic solvent.

5. The preparation method according to claim 4, characterized in that, The pore-forming agent comprises a eutectic solvent of choline chloride, ethylene glycol, and ethanol, wherein the molar ratio of choline chloride, ethylene glycol, and ethanol is 1:2:1.5 to 1:3:

2.

6. The preparation method according to claim 1, characterized in that, The ratio of the total molar amount of the template molecule, the molar amount of the functional monomer, and the molar amount of the crosslinking agent is from 1:8:20 to 1:8:

40.

7. The preparation method according to claim 1, characterized in that, The magnetic molecularly imprinted resin is prepared by the following steps: The magnetic core was prepared by co-precipitation method; An intermediate phase protective layer is coated onto the surface of the magnetic core to obtain intermediate phase protective layer coated particles; The surface double bonds of the mesophase protective layer-coated particles were modified using a silane coupling agent to obtain the particles to be grafted. The mulberry root ketone C, the mulberry ginseng ketone G and the functional monomer are dissolved in the porogen, and stirred at room temperature in the dark to allow the template molecule to form a hydrogen-bonded complex with the functional monomer, thus obtaining a pre-assembled solution. The particles to be grafted, the crosslinking agent, and the initiator are added to the pre-assembled solution, and the reaction is carried out under a protective atmosphere to obtain a crude product of magnetic molecularly imprinted resin. The crude product of the magnetic molecularly imprinted resin was collected by magnetic separation and extracted with a methanol-acetic acid mixture using a Soxhlet extract until the eluent showed no absorption at 280 nm. The resin was washed with methanol until neutral and then dried under vacuum to obtain the magnetic molecular imprinted resin.

8. A flavonoid from mulberry root bark, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.

9. The use of mulberry root bark flavonoids prepared by the method according to any one of claims 1 to 7 or the mulberry root bark flavonoids according to claim 8 in the preparation of cosmetics.

10. The application according to claim 9, characterized in that, The cosmetic product promotes autophagy in skin cells.

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