Screening method of eutectic solvent and application of eutectic solvent in selective demethylation of polymethoxylated flavonoids

Abstract

The invention belongs to the technical field of bioactive components, and particularly relates to a screening method of a deep-eutectic solvent and application of the deep-eutectic solvent in selective demethylation of polymethoxylated flavonoids. According to the method, the five eutectic solvents are screened on the basis of physicochemical properties and thermodynamic properties, the eutectic solvent capable of being efficiently demethylated is obtained, and the requirement for obtaining hydroxylated PMFs is met; meanwhile, the concentration, the reaction temperature and the reaction time of the demethylation reagent are optimized, and the optimal reaction condition is obtained, so that the technical problem that a high-performance demethylation special reagent is lacked in the prior art is solved.

Description

Technical Field

[0001] This application belongs to the field of bioactive ingredient technology, and in particular relates to a method for screening eutectic solvents and its application in the selective demethylation of polymethoxyflavones. Background Technology

[0002] Polymethoxyflavones (PMFs) are a class of special flavonoids naturally found in the peels of citrus plants in the Rutaceae family. Their basic skeleton contains multiple methoxy groups, which endows them with low polarity and a near-planar molecular structure, thus giving them excellent biomembrane permeability and stronger membrane transport capacity. Based on these properties, PMFs exhibit a wider range of biological activities. However, their molecular structure with multiple methoxy substitutions also leads to strong hydrophobicity, which significantly reduces their water solubility and oral bioavailability.

[0003] Demethylation of polymethoxyflavones (PMFs), by replacing one or two methoxy groups with hydroxyl groups to form hydroxylated PMFs, can combine the advantages of both methoxy and hydroxyl groups. While maintaining comparable permeability and membrane transport capacity to PMFs, it also enhances the polarity and water solubility of PMFs, thus effectively improving their bioavailability. Furthermore, many studies have shown that hydroxylated PMFs possess stronger biological activities than their counterparts, including anticancer and anti-inflammatory activities. Demethylation of PMFs includes natural and artificial demethylation methods. During the natural drying and storage of dried tangerine peel, PMFs... Natural demethylation of tangerine peel occurs slowly, which enhances its bioactivity with prolonged storage. However, natural demethylation is affected by various factors such as storage temperature, humidity, and microbial distribution, resulting in low demethylation efficiency and making it difficult to achieve large-scale production. In addition, although artificial chemical acid hydrolysis demethylation has the advantage of large-scale production, it suffers from problems such as numerous byproducts and organic reagent residues, limiting its application in the food and pharmaceutical fields. Furthermore, the large number of byproducts also affects the yield and efficiency of hydroxylated PMFs. While enzyme-catalyzed demethylation can achieve selective demethylation at specific sites, it suffers from slow reaction, low yield, and high cost.

[0004] Therefore, current methods of natural demethylation, chemical acid hydrolysis demethylation, and enzyme-catalyzed demethylation are insufficient to meet the requirements for high-efficiency, large-scale acquisition of hydroxylated PMFs. It is necessary to develop efficient, low-cost, and environmentally friendly selective demethylation reagents and methods, which are of great significance for the large-scale acquisition of hydroxylated PMFs. However, high-performance demethylation reagents and methods are currently lacking. Summary of the Invention

[0005] In view of this, this application provides a method for screening eutectic solvents and their application in the selective demethylation of polymethoxyflavones, in order to solve the technical problem of the lack of high-performance demethylation reagents in the prior art.

[0006] The first aspect of this application provides a method for screening eutectic solvents, comprising the following steps:

[0007] Offers a range of eutectic solvent combinations;

[0008] Based on the physicochemical and / or thermodynamic properties of the eutectic solvents, an optimal combination of eutectic solvents is selected from a series of provided combinations as the demethylating agent.

[0009] Preferably, the series of eutectic solvent combinations includes five eutectic solvents, with choline chloride as the hydrogen bond acceptor and one of malic acid, DL-tartaric acid, glycolic acid, phosphoric acid, and oxalic acid as the hydrogen bond donor.

[0010] Preferably, the molar ratio of hydrogen bond acceptor to hydrogen bond donor in the eutectic solvent is 1 to 2:1.

[0011] Preferably, the screening process includes: selecting an optimal eutectic solvent as a demethylating agent from a series of provided eutectic solvent combinations based on one of the physicochemical properties of the eutectic solvent, namely pH, conductivity, viscosity, density, and polarity, as a screening parameter.

[0012] Preferably, the screening process includes: selecting an optimal eutectic solvent as a demethylating agent from a series of provided eutectic solvent combinations based on the thermal stability of the eutectic solvent's thermodynamic properties.

[0013] The second aspect of this application provides the application of the eutectic solvent obtained by the screening method described in the first aspect in the selective demethylation of polymethoxyflavones.

[0014] Preferably, the application in the selective demethylation of polymethoxyflavones specifically includes: mixing the eutectic solvent obtained by the screening method described in the first aspect with the polymethoxyflavones to carry out a selective demethylation reaction, thereby obtaining hydroxylated polymethoxyflavones with demethylated C-5 positions.

[0015] Preferably, the demethylation reaction takes 15-60 minutes and is carried out at a temperature of 70-110°C.

[0016] Preferably, in step S2, the polymethoxyflavonoid is selected from at least one of hesperidin, sweet orange flavonoid, 3,5,6,7,3',4'-hexamethoxyflavonoid, and 3,5,6,7,8,3',4'-heptamethoxyflavonoid.

[0017] Preferably, in step S2, the volume ratio of the demethylating agent to the polymethoxyflavone is 1~10:1.

[0018] The third aspect of this application provides the use of the hydroxylated polymethoxyflavonoids obtained from the application described in the second aspect in the preparation of antioxidant products.

[0019] Compared with the prior art, the eutectic solvents screened by the screening method provided in this application, used as demethylating agents for the selective demethylation of polymethoxyflavones, have at least the following beneficial effects: a screening method for eutectic solvents and its application in the selective demethylation of polymethoxyflavones.

[0020] 1. The five eutectic solvents provided in this application all have selective demethylation effects on polymethoxyflavones and are high-performance demethylation reagents.

[0021] 2. The method for selective demethylation of polymethoxyflavones using a eutectic solvent provided in this application is based on screening of physicochemical and thermodynamic properties. Choline chloride / oxalic acid (Ch / OA) was selected from five eutectic solvents as the demethylating agent because it has a higher proton release, faster ion mobility, and better thermal stability.

[0022] 3. In the method for selective demethylation of polymethoxyflavones using a eutectic solvent provided in this application, the reaction temperature, time and concentration have been optimized to obtain reaction conditions with better demethylation effect and meet the requirements for achieving selective demethylation of polymethoxyflavones C-5. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 In the screening step provided in Example 2 of this application, the schematic diagram of the thermodynamic property analysis results of choline chloride / oxalic acid (Ch / OA) is shown. Figure A shows the analysis results of differential scanning calorimetry, and Figure BC shows the analysis results of thermogravimetric analysis.

[0025] Figure 2 The diagram shows the conversion rate and conversion ratio of nobiletin using five eutectic solvents provided in Example 2 of this application; Figure A shows the characteristic peaks of the demethylation reaction products of the five eutectic solvents, and Figure B shows the statistical results of the conversion rate and conversion ratio.

[0026] Figure 3 Pearson correlation coefficient heatmap of the physicochemical properties of five eutectic solvents provided in Example 2 of this application with the conversion rate and conversion ratio of nobiletin;

[0027] Figure 4 The infrared spectrum of choline chloride / oxalic acid (Ch / OA) provided in Example 3 of this application;

[0028] Figure 5 The effect of reaction temperature on the demethylation of nobiletin during the choline chloride / oxalic acid (Ch / OA) demethylation reaction provided in Example 3 of this application; Figure A shows the characteristic peaks of the demethylation reaction product, and Figure B shows the statistical results of conversion rate and conversion ratio;

[0029] Figure 6 The effect of reaction time on the demethylation of nobiletin during the choline chloride / oxalic acid (Ch / OA) demethylation reaction provided in Example 3 of this application; Figure A shows the characteristic peaks of the demethylation reaction product, and Figure B shows the statistical results of conversion rate and conversion ratio;

[0030] Figure 7 The effect of the concentration of choline chloride / oxalic acid (Ch / OA) used in the demethylation reaction of choline chloride / oxalic acid (Ch / OA) provided in Example 3 of this application on the demethylation of nobiletin; Figure A shows the characteristic peaks of the demethylation reaction product, and Figure B shows the statistical results of conversion rate and conversion ratio;

[0031] Figure 8 The liquid chromatograms of four polymethoxyflavones before and after the demethylation reaction of choline chloride / oxalic acid (Ch / OA) provided in Example 4 of this application are shown.

[0032] Figure 9 The diagram shows a comparison of the effects of choline chloride / oxalic acid (Ch / OA) on the demethylation of four polymethoxyflavones provided in Example 4 of this application; Figure A shows the degradation amount of polymethoxyflavones and the amount of hydroxylated polymethoxyflavones generated, and Figure B shows the statistical results of conversion rate and conversion ratio.

[0033] Figure 10 The graph shows the test results of DPPH free radical scavenging ability, ABTS free radical scavenging ability, and ferric ion reducing ability (FRAP) of choline chloride / oxalic acid (Ch / OA) provided in Example 5 of this application. Detailed Implementation

[0034] This application provides a method for screening eutectic solvents and its application in the selective demethylation of polymethoxyflavones, in order to solve the technical problem of the lack of high-performance demethylation reagents in the prior art.

[0035] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] Existing demethylation methods, such as natural transformation, chemical acid hydrolysis, and enzyme catalysis, suffer from problems including numerous byproducts, low yields, and high costs. Currently, there is a lack of highly efficient reagents, making the preparation of hydroxylated PMFs difficult. Therefore, this application provides a method for screening eutectic solvents and its application in the selective demethylation of polymethoxyflavones. The screened series of eutectic solvent compositions consist of choline chloride as a hydrogen bond acceptor and at least one of malic acid, DL-tartaric acid, glycolic acid, phosphoric acid, and oxalic acid as a hydrogen bond donor.

[0037] Eutectic solvents are a class of solvents with unique properties formed by intermolecular hydrogen bonding between hydrogen bond donors and acceptors. The eutectic solvents provided in this application use choline chloride as a hydrogen bond acceptor and one of malic acid, DL-tartaric acid, glycolic acid, phosphoric acid, or oxalic acid as a hydrogen bond donor. Combining these two types yields a series of eutectic solvents, such as choline chloride / malic acid (Ch / MA), choline chloride / tartaric acid (Ch / TA), choline chloride / glycolic acid (Ch / GA), choline chloride / phosphoric acid (Ch / PA), and choline chloride / oxalic acid (Ch / OA). These eutectic solvents possess both strong proton release capabilities and excellent ion mobility. This property enables them to generate high concentrations of protons (H+). + This process effectively promotes proton attack on the C-5 methoxy group of polymethoxyflavones (PMFs), thereby achieving selective demethylation at the C-5 position and generating the corresponding hydroxylated PMFs. The eutectic solvents provided in this application all exhibit high conversion rates and excellent selectivity, demonstrating their potential as high-performance demethylating agents in the efficient preparation of hydroxylated PMFs. Furthermore, this application also provides a method for the selective demethylation of polymethoxyflavones using eutectic solvents, including a screening step and a demethylation step.

[0038] For the screening step in the method, this application uses the physicochemical and thermodynamic properties of eutectic solvents as the basis for screening. pH and conductivity are the two most significant physicochemical properties affecting the demethylation effect of eutectic solvents. The active protons in the eutectic solvent (DES) are an important factor affecting the hydrolysis reaction activity. The lower the pH of the eutectic solvent (DES), the stronger its ability to release protons, thus making the demethylation reaction activity of polymethoxyflavones stronger. Conductivity directly reflects the migration ability of ions; the higher the conductivity of the eutectic solvent (DES), the higher its ion mobility. These two properties work together to promote the demethylation process of polymethoxyflavones. Simultaneously, the viscosity of the eutectic solvent (DES) affects conductivity; low-viscosity eutectic solvents (DES) promote ion migration within the eutectic solvent (DES) and increase conductivity. Furthermore, the density of the eutectic solvent (DES) affects the conversion ratio. High-density DES increases the degradation of polymethoxyflavones, leading to a higher conversion ratio, moving it further away from a conversion ratio of 1. Conversely, low-density DES brings the conversion ratio closer to 1. It is worth noting that polarity also affects pH and conductivity; eutectic solvents with low polarity values ​​have low pH and high conductivity. Therefore, in the method for selective demethylation of polymethoxyflavones using eutectic solvents provided in this application, the screening step should select eutectic solvents based on low pH, high conductivity, low polarity, low viscosity, and low density. Furthermore, since demethylation requires heating, after screening based on the above physicochemical properties, eutectic solvents with good thermal stability should also be selected. Good thermal stability ensures that the molecular structure is not destroyed during subsequent heating and demethylation, forming a dense hydrogen bond network. This network, through hydrogen bond interactions, forms a eutectic solvent with a certain degree of thermal stability. After screening based on the physicochemical and thermodynamic properties of the eutectic solvents, the optimal eutectic solvent combination (choline chloride / oxalic acid) achieves a hydroxylation conversion rate of 87.39% for various polymethoxyflavones, including nobiletin, with a conversion ratio closer to 1.

[0039] For the demethylation step in the method, conditions such as excessively short reaction time or excessively high heating temperature should be avoided, otherwise it will be difficult to effectively generate hydroxylated polymethoxyflavones. The optimized process conditions used in this application are: reaction time 15~60 min, reaction temperature 70~110℃. Accordingly, the selective demethylation method of polymethoxyflavones using the eutectic solvent provided in this application yields C-5 hydroxylated polymethoxyflavones with stronger DPPH radical scavenging ability, ABTS radical scavenging ability, and iron ion reducing ability, thus making them applicable in the field of antioxidants.

[0040] The method for selective demethylation of polymethoxyflavones using a eutectic solvent provided in this application will be described in detail below with reference to embodiments.

[0041] Example 1

[0042] This embodiment provides a set of methods for preparing eutectic solvents. The preparation method is based on the formula shown in Table 1. After mixing hydrogen bond acceptor (choline chloride) and hydrogen bond donor (one of malic acid, DL-tartaric acid, glycolic acid, phosphoric acid or oxalic acid), the mixture is first stirred in a constant temperature water bath at 60°C until the solid becomes liquid. Then, it is placed at 100°C and magnetically stirred for 8 hours until it becomes clear and transparent, thus obtaining five kinds of eutectic solvents.

[0043] Table 1: Eutectic Solvent Formulation

[0044]

[0045] Example 2

[0046] This embodiment provides a method for selective demethylation of polymethoxyflavones using a eutectic solvent, the method including a screening step and a demethylation reaction step.

[0047] The screening step includes: selecting eutectic solvents with superior conversion rates and conversion ratios of hydroxylated polymethoxyflavones from a group of eutectic solvents provided in Example 1;

[0048] The demethylation reaction step includes: mixing a set of eutectic solvents provided in Example 1 with nobiletin to carry out a demethylation reaction.

[0049] The results of the density, viscosity, polarity, conductivity, and pH tests of the eutectic solvent in the screening step are shown in Table 2, and include the following processes:

[0050] Density testing includes:

[0051] The density of 1 mL of eutectic solvent (DES) was determined at room temperature using the isovolute method, as shown in the following formula:

[0052]

[0053] In the formula, ρ DESs m3 is the density of DES, g / mL; m1 is the mass of the centrifuge tube and DES, g; m2 is the mass of the centrifuge tube and deionized water, g; ρ w ρ is the density of water, in g / mL.

[0054] Viscosity testing includes:

[0055] The DES was placed at room temperature, and its viscosity was measured using a digital rotational viscometer. Different types of rotors were used to measure the sample at different speeds to ensure that the measured values ​​did not exceed the full scale.

[0056] Polarity testing includes:

[0057] Prepare a 1 mg / mL Nile red ethanol solution by dissolving 10.0 mg of Nile red in 10.0 mL of anhydrous ethanol. Store at 4°C protected from light. Dilute 10-fold before use. Add 100 μL of the diluted Nile red solution to 2 mL of eutectic solution and mix thoroughly. Then, use a multi-mode microplate reader to scan the sample at wavelengths of 400-800 nm and record the maximum absorption wavelength. Calculate the Nile red polarity parameter E using the following formula. NR :

[0058]

[0059] E in the formula NR λ is the polarity parameter of Nile red, kcal / mol; h is Planck's constant; c is the speed of light in vacuum, m / s; NA is Avogadro's constant; λ max This is the wavelength of maximum ultraviolet absorption.

[0060] Conductivity testing includes:

[0061] The conductivity of the eutectic solvent (DES) was determined at room temperature using a conductivity meter.

[0062] pH testing includes:

[0063] The pH of the eutectic solvent (DES) was determined using a pH meter at room temperature.

[0064] The thermodynamic properties of eutectic solvents in the screening process included: analyzing the thermodynamic properties of eutectic solvents (DES) using thermogravimetric analysis and differential scanning calorimetry, with results as follows: Figure 1 As shown, it includes the following steps:

[0065] The thermogravimetric analysis process includes: spreading 5-10 mg of eutectic solvent evenly in an alumina crucible, setting the heating rate to 10 °C / min, and heating from 25 °C to 500 °C under a flow of 50 mL / min N2.

[0066] The differential scanning calorimeter analysis process includes: weighing approximately 5 mg of eutectic solvent into an aluminum crucible, cooling the sample temperature from 25 °C to -70 °C at a rate of 10 °C / min under N2 protection, holding at this temperature for 10 min, and then heating it to 80 °C at a rate of 10 °C / min.

[0067] Table 2: Physicochemical properties of different DES

[0068]

[0069] As shown in Table 2, the physicochemical properties test results, including density, viscosity, polarity, conductivity, and pH, indicate that choline chloride / oxalic acid (Ch / OA) is a low-molecular-weight eutectic solvent (DES) with low pH, high conductivity, low polarity, low viscosity, and low density. Meanwhile, the thermodynamic analysis results of choline chloride / oxalic acid (Ch / OA) are as follows... Figure 1 As shown, from Figure 1 The differential scanning calorimetry (DSC) analysis results shown in Figure C indicate that choline chloride / oxalic acid (Ch / OA) exhibits a melting peak around 30℃, a melting point distinct from choline chloride or oxalic acid, indicating the successful synthesis of a eutectic solvent. Further analysis reveals... Figure 1 The thermogravimetric analysis results shown in the AB diagram indicate that a small amount of mass loss occurs within the range of 50~150℃, which is due to its strong hygroscopicity leading to dehydration during the heating process. The three thermal decomposition step temperatures of 214.5℃, 252.8℃, and 315.7℃ are higher than the demethylation reaction temperature, indicating that choline chloride / oxalic acid (Ch / OA) has good thermal stability and its molecular structure is not easily destroyed during the demethylation reaction.

[0070] As can be seen from the above screening steps, in this embodiment, choline chloride / oxalic acid (Ch / OA) was selected as the most promising demethylating agent from a group of eutectic solvents provided in Example 1 for screening of physicochemical and thermodynamic properties. At the same time, in order to verify whether the screening conditions for physicochemical and thermodynamic properties are correct, the demethylation reaction step was carried out on a group of eutectic solvents provided in Example 1.

[0071] In this embodiment, the demethylation reaction step includes:

[0072] The polymethoxyflavonoid used was noriheptacorlina. To reasonably evaluate the demethylation effect of the eutectic solvents provided in Example 1, this application used both conversion rate and conversion ratio as indicators for comprehensive characterization; the formulas for conversion rate and conversion ratio are as follows:

[0073]

[0074]

[0075] In the formula, C0 is the initial concentration of hydroxylated PMFs before the reaction, in μg / mL; C1 is the final concentration of hydroxylated PMFs after the reaction, in μg / mL; C2 is the initial concentration of PMFs before the reaction, in μg / mL; C3 is the final concentration of PMFs after the reaction, in μg / mL; C p M represents the concentration of hydroxylated PMFs generated after complete conversion of PMFs. p M is the molar mass of PMFs; d denoted as the molar mass of the hydroxylated PMFs.

[0076] The demethylation reaction steps include:

[0077] 5 mg of norepinephrine was accurately weighed and placed in 5 mL of different eutectic solvents. The sample was dispersed evenly using magnetic stirring and reacted in an oil bath at 100 °C for 1 h. After the reaction, the reaction solution was quickly diluted 10-fold with chromatographic grade methanol. The contents of norepinephrine and hydroxylated norepinephrine in the reaction solution were then determined by HPLC (Venusil ASB C18 reversed-phase column, column temperature 25 °C) to calculate the conversion rate and conversion ratio. The results are as follows: Figure 2 As shown.

[0078] from Figure 2 It can be seen that choline chloride / oxalic acid (Ch / OA) is the best demethylating agent for nonocitretin in terms of both conversion rate and conversion ratio, with a conversion rate of 80.48% and a conversion ratio around 1; at the same time Figure 3 The Pearson correlation also shows the relationship between the pH, conductivity, polarity, viscosity, and density of the eutectic solvent and the conversion rate and conversion ratio of nobiletin. This indicates that this application should screen eutectic solvents with low pH, high conductivity, low polarity, low viscosity, and low density as demethylating agents through physicochemical and thermodynamic property screening.

[0079] Example 3

[0080] This embodiment provides a method for selective demethylation of polymethoxyflavones using the eutectic solvent choline chloride / oxalic acid (Ch / OA), the method including hydrogen bond interaction characterization and demethylation reaction steps.

[0081] Interaction characterization was performed using infrared spectroscopy, specifically for choline chloride / oxalic acid (Ch / OA). The testing procedure involved uniformly coating choline chloride / oxalic acid (Ch / OA) onto a pre-prepared KBr pellet, and then measuring its FTIR spectra in transmission mode within the range of 400-4000 cm⁻¹. -1 The number of scans was set to 32, and the resolution was set to 4cm. -1 The test results are as follows Figure 3 As shown; from Figure 4 As can be seen from the data, the characteristic peak of choline chloride includes 1477.21 cm⁻¹. -1 (The symmetrical bending vibration peak of -CH3), 2800-3000cm -1 (Stretching vibration peak of -CH3) and 3405.67cm -1 (Stretching vibration peak of -OH), the characteristic peak of the oxalic acid component is at 3471.24 cm⁻¹. -1 (Stretching vibration peak of -OH) and 1685.48 cm⁻¹ -1(The stretching vibration peak of C=O); while the absorption peak of the eutectic solvent composed of choline chloride and oxalic acid, choline chloride / oxalic acid (Ch / OA), showed a significant shift, with the C=O absorption peak of oxalic acid in the eutectic solvent shifting from 1685.48 cm⁻¹. -1 The redshift reached 1631.48cm. -1 The absorption peak of -OH is from 3471.24 cm⁻¹. -1 The redshift reached 3436.53cm. -1 Furthermore, a broad absorption peak was formed, indicating that the -COOH of oxalic acid reacts with the N-hydroxyl group on choline chloride. + Cl- and -OH interact through hydrogen bonding to form a eutectic solvent.

[0082] The demethylation reaction steps include:

[0083] Demethylation under different temperature conditions: 5 mg of nobiletin was accurately weighed and placed in 5 mL of a eutectic solvent, choline chloride / oxalic acid (Ch / OA). The sample was dispersed evenly using magnetic stirring and reacted in an oil bath at different temperatures ranging from 60 to 110 °C for 1 h. After the reaction, the reaction solution was rapidly diluted 10-fold with chromatographic grade methanol and filtered through a 0.22 μm filter membrane. The product content in the reaction solution was then determined by HPLC (Venusil ASB C18 reversed-phase column, column temperature 25 °C). The results are as follows: Figure 5 As shown.

[0084] Demethylation under different time conditions: 5 mg of nobiletin was accurately weighed and placed in 5 mL of a eutectic solvent, choline chloride / oxalic acid (Ch / OA). The sample was dispersed evenly using magnetic stirring and reacted in an oil bath at 100 °C for 15–60 min. After the reaction, the reaction solution was rapidly diluted 10-fold with chromatographic grade methanol and filtered through a 0.22 μm filter membrane. The product content in the reaction solution was then determined by HPLC (Venusil ASB C18 reversed-phase column, column temperature 25 °C). The results are as follows: Figure 6 As shown.

[0085] Demethylation under different concentrations of choline chloride / oxalic acid (Ch / OA) conditions: 5 mg of nobiletin was accurately weighed and placed in 5 mL of eutectic solvent choline chloride / oxalic acid (Ch / OA) of different concentrations. After uniform dispersion using magnetic stirring, the mixture was reacted for 60 min in oil baths at different temperatures (100 °C). After the reaction, the reaction solution was rapidly diluted 10-fold with chromatographic grade methanol and filtered through a 0.22 μm filter membrane. Subsequently, the product content in the reaction solution was determined by HPLC (Venusil ASB C18 reversed-phase column, column temperature 25 °C). The results are as follows: Figure 7 As shown.

[0086] from Figure 5-7It can be seen that the concentration of the eutectic solvent choline chloride / oxalic acid (Ch / OA), the reaction temperature, and the time all affect demethylation. Figure 7 As shown, when using a 100% concentration of choline chloride / oxalic acid (Ch / OA), i.e., without adding water, the demethylation effect of norepinephrine is better. This may be because the addition of water would disrupt the hydrogen bond network structure of choline chloride / oxalic acid (Ch / OA) and also cause the pH of choline chloride / oxalic acid (Ch / OA) to increase, thus hindering demethylation. Figure 5 As shown, demethylation is not significant at reaction temperatures of 60-70℃, but at temperatures above 90℃, nobiletin undergoes significant C-5 demethylation to yield hydroxylated nobiletin. This may be because higher temperatures enhance the proton ion migration ability of the eutectic solvent; while... Figure 6 As shown, significant C-5 demethylation can occur after a reaction time of 60 min or more, but the content of hydroxylated hesperidin cannot be significantly increased when the reaction time is 70 min. Therefore, considering all factors, a concentration of 100% choline chloride / oxalic acid (Ch / OA) should be selected as the demethylating agent, and demethylation should be carried out at 100℃ for 60 min.

[0087] Example 4

[0088] This embodiment provides a method for selective demethylation of polymethoxyflavones using a eutectic solvent (choline chloride / oxalic acid).

[0089] In this embodiment, the method for selective demethylation of polymethoxyflavonoids uses 100% choline chloride / oxalic acid (Ch / OA) as the demethylating agent, and the demethylation reaction temperature is 100℃ for 60 min.

[0090] The procedure included: accurately weighing 5 mg each of hesperidin, sweet orange flavonoids, and 3,5,6,7,3',4'-hexamethoxyflavonoids or 3,5,6,7,8,3',4'-heptamethoxyflavonoids, and demethylating them in 5 mL of choline chloride / oxalic acid (Ch / OA) demethylation reagent. The samples were then dispersed uniformly using magnetic stirring and reacted in oil baths at different temperatures (100℃) for 60 min. After the reaction, the reaction solution was rapidly diluted 10-fold with chromatographic grade methanol and filtered through a 0.22 μm filter membrane. The product content in the reaction solution was then determined by HPLC (Venusil ASB C18 reversed-phase column, column temperature 25℃). The results are as follows: Figure 8-9 As shown.

[0091] Following the demethylation catalysis of DES, all four PMF monomers underwent degradation and generated new peaks. Figure 8The liquid chromatograms before and after the demethylation reaction show that 5,6,7,8,3',4'-hexamethoxyflavonoids are converted to demethylated hesperidin (5-DNOB), 5,6,7,4'-tetramethoxyflavonoids are converted to 5-hydroxy-3',4',6,7-tetramethoxyflavonoids (5-DSIN), 3,5,6,7,3',4'-hexamethoxyflavonoids are converted to artemetin, and 3,5,6,7,8,3',4'-heptamethoxyflavonoids are converted to 5-OH-HxMF; from Figure 9 (A) It can be seen that the degradation of polymethoxyflavonoid PMFs monomers is greater than the generation of hydroxylated polymethoxyflavonoid PMFs. However, when choline chloride / oxalic acid (Ch / OA) is used as a demethylating agent, the conversion ratio of nobiletin NOB is close to 1, and it also has a high conversion ratio for the remaining three polymethoxyflavonoid PMFs; at the same time, combined with Figure 9 The statistics of conversion rates and conversion ratios shown in (B) indicate that when choline chloride / oxalic acid (Ch / OA) is used as a demethylating agent, the conversion rate of norepinephrine NOB is also relatively high, and the conversion rate of the remaining three polymethoxyflavones (PMFs) is also relatively high. This shows that the demethylating agent choline chloride / oxalic acid (Ch / OA) obtained by screening through physicochemical and thermodynamic properties such as density, viscosity, polarity, conductivity and pH in Example 2 of this application has a high conversion rate and conversion ratio for a variety of polymethoxyflavones, and can selectively convert the C-5 methoxy group of polymethoxyflavones to hydroxyl groups to obtain hydroxylated polymethoxyflavones.

[0092] Example 5

[0093] This embodiment tests the antioxidant properties of C-5 hydroxylated polymethoxyflavones obtained by selective demethylation of polymethoxyflavones using the eutectic solvent choline chloride / oxalic acid (Ch / OA).

[0094] The process of antioxidant performance testing includes:

[0095] Based on the characteristic that hydroxylated polymethoxyflavones are poorly soluble in water while eutectic solvents are readily soluble in water, an anti-solvent method was used to separate the eutectic solvent and hydroxylated polymethoxyflavones. The process involved mixing the four demethylation reaction product solutions provided in Example 4 with 10 times the volume of pure water and refrigerating them overnight at 4°C. After the solid precipitated, the mixture was separated by centrifugation and freeze-dried to finally obtain four hydroxylated polymethoxyflavone samples.

[0096] Four hydroxylated polymethoxyflavonoid samples were tested for DPPH radical scavenging ability, ABTS radical scavenging ability, and ferric ion reducing ability (FRAP). The DPPH radical scavenging ability test was conducted by adding 50 μL of each of the four hydroxylated polymethoxyflavonoid samples and the polymethoxyflavonoid sample solution to 1.95 mL / 60 μM DPPH solution (dissolved in 95% ethanol). The ABTS radical scavenging ability test was conducted by adding 100 μL of each of the four hydroxylated polymethoxyflavonoid samples and the polymethoxyflavonoid sample solution to 2 mL of ABTS solution. + The test was conducted in the working solution; the ferric reducing power (FRAP) determination procedure was as follows: 200 μL of the four hydroxylated polymethoxyflavones and polymethoxyflavone sample solutions were added to 1.8 mL of FRAP working solution for testing; the antioxidant test results are as follows. Figure 10 As shown.

[0097] from Figure 10 It can be seen that, compared with the four polymethoxyflavones, the four hydroxylated polymethoxyflavones showed significantly improved DPPH radical scavenging ability, ABTS radical scavenging ability, and iron ion reducing ability. The number of free hydroxyl groups in flavonoid molecules and the presence of C2-C3 double bonds on the C ring are key conditions for their antioxidant and free radical scavenging activities. In this application, by using choline chloride / oxalic acid (Ch / OA) as a demethylating agent, the C-5 methoxy group of polymethoxyflavones was successfully selectively demethylated, introducing hydroxyl groups and improving antioxidant performance.

[0098] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for screening of a deep eutectic solvent, characterized by, The method comprises the following steps: providing a series of eutectic solvent combinations; selecting a eutectic solvent as a demethylation reagent from the provided series of eutectic solvent combinations according to the physicochemical properties and / or thermodynamic properties of the eutectic solvent.

2. The method for screening eutectic solvents according to claim 1, characterized in that, The series of eutectic solvent combinations comprises five eutectic solvents, the hydrogen bond acceptor of which is choline chloride, and the hydrogen bond donor of which is one of malic acid, DL-tartaric acid, glycolic acid, phosphoric acid, and oxalic acid.

3. The method for screening eutectic solvents according to claim 1, characterized in that, The molar ratio of the hydrogen bond acceptor to the hydrogen bond donor in the eutectic solvent is 1-2:

1.

4. The method for screening eutectic solvents according to claim 1, characterized in that, The selection process comprises selecting a eutectic solvent as a demethylation reagent from the provided series of eutectic solvent combinations according to one of pH, conductivity, viscosity, density, and polarity in the physicochemical properties of the eutectic solvent as a selection parameter.

5. The method of claim 1, wherein the method is characterized by, The selection process comprises selecting a eutectic solvent as a demethylation reagent from the provided series of eutectic solvent combinations according to thermal stability in the thermodynamic properties of the eutectic solvent.

6. Application of a eutectic solvent selected by the method of any one of claims 1-5 in selective demethylation of polymethoxyflavones.

7. Use of a deep eutectic solvent according to claim 6 for the selective demethylation of polymethoxylated flavones, characterized in that, The application specifically comprises mixing the eutectic solvent selected by the method of any one of claims 1-5 and polymethoxyflavones to perform a selective demethylation reaction, so as to obtain a hydroxylated polymethoxyflavone demethylated at the C-5 position.

8. Use of a deep eutectic solvent according to claim 7 for the selective demethylation of polymethoxylated flavones, characterized in that, The demethylation reaction is performed for 15-60 min at a temperature of 70-110℃.

9. Use of a deep eutectic solvent according to claim 7 for the selective demethylation of polymethoxylated flavones, characterized in that, The polymethoxyflavones are at least one selected from the group consisting of nobiletin, sweet orange flavone, 3,5,6,7,3',4'-hexamethoxyflavone, and 3,5,6,7,8,3',4'-heptamethoxyflavone.

10. Application of the demethylated hydroxylated polymethoxyflavone obtained in the application of any one of claims 6-9 in preparation of an antioxidant product.

Images