A low-melting-point solvent-polymer aqueous two-phase system for high-yield co-extraction and highly selective separation of citrus flavonoids

Abstract

This invention belongs to the field of plant component extraction technology, specifically relating to a low-eutectic solvent-polymer aqueous two-phase system for high-yield co-extraction and high-selectivity separation of citrus flavonoids. It provides a high-yield DES for extracting citrus flavonoids, composed of benzyltrimethylammonium chloride and xylitol; and also provides a low-eutectic solvent-polymer aqueous two-phase system for highly selective separation of citrus flavonoids, composed of a polymer, water, and the aforementioned DES. The DES exhibits high extraction yield of citrus peel flavonoids, with a maximum total flavonoid extraction rate of up to 16.784 mg / g. The low-eutectic solvent-polymer aqueous two-phase system achieves a partition coefficient of 14.34 and an extraction rate of 93.48% for hesperidin, and a partition coefficient of 2.23 and an extraction rate of 99.05% for nonotrimonin, with a selectivity coefficient of 732.76 for both.

Description

Technical Field

[0001] This invention belongs to the field of plant component extraction technology, specifically relating to a low eutectic solvent-polymer aqueous two-phase system for high-yield co-extraction and high-selectivity separation of citrus flavonoids. Background Technology

[0002] The extraction of citrus flavonoids often uses organic solvents (such as methanol), which have drawbacks such as high toxicity, high volatility, and low extraction rates. Demodified eutectic solvents (DES), as a novel green solvent, often exhibit good extraction effects of bioactive substances and are suitable for extracting flavonoids from citrus peels. Currently, choline chloride-type DES are the main extraction reagents, and there are no literature reports on the use of benzyltrimethylammonium chloride-type DES for the extraction of citrus flavonoids. Because DES are composed of hydrogen bond donors and acceptors, there are many types, and no research has yet been conducted on this type of DES. Furthermore, some reported DES have lower extraction efficiency than benzyltrimethylammonium chloride-type DES, presumably because benzyltrimethylammonium chloride has the ability to dissolve plant cell walls and often has lower viscosity, making it easier to release citrus flavonoids.

[0003] However, it should be noted that DES extracts are often mixed systems containing multiple components. For example, extracts from citrus peel pomace typically contain two types of components: glycosylated flavonoids and polymethoxylated flavonoids, but their biological activities and applications are quite different. Therefore, further separation of the target components in the DES mixture system is essential. Liquid-liquid extraction technology based on aqueous two-phase systems (ATPS) has advantages such as low cost, simple operation, and mild conditions, and has been widely used in food, pharmaceutical, and chemical industries. Due to the salting-out effect of inorganic salts, DES and inorganic salt ATPS are very easy to form and are currently the most reported aqueous two-phase systems. However, in the DES-inorganic salt system, the strong salting-out effect of inorganic salts can easily precipitate all solutes simultaneously, and the eutectic solvent's ability to generate specific affinity for different solutes is insufficient, making selective extraction of the target analyte difficult to achieve. Summary of the Invention

[0004] [Technical Issues]

[0005] Current methods for extracting flavonoids from citrus peel suffer from drawbacks such as high toxicity, high volatility, or insufficient extraction rates. Citrus peel flavonoids are mixtures containing multiple components, necessitating further separation and purification of the target components. A low-cost, simple-to-operate, and mild-condition method for extracting and separating citrus peel flavonoids is needed to achieve high flavonoid extraction rates and highly selective flavonoid separation.

[0006] [Technical Solution]

[0007] The first objective of this invention is to provide a low eutectic solvent system (DES) for high extraction efficiency of citrus flavonoids, consisting of a hydrogen bond donor and a hydrogen bond acceptor, wherein the hydrogen bond acceptor is benzyltrimethylammonium chloride and the hydrogen bond donor is xylitol.

[0008] In one embodiment of the present invention, the molar ratio of hydrogen bond acceptor to hydrogen bond donor of DES is 3:1 to 1:3.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned DES, comprising the steps of: mixing a hydrogen bond donor and a hydrogen bond acceptor, and stirring at 90-120°C until the system is a clear and transparent viscous liquid.

[0010] A third objective of this invention is to provide the application of the DES in the extraction of citrus flavonoids.

[0011] The fourth objective of this invention is to provide a method for ultrasound-assisted extraction of flavonoids from citrus peel using DES, comprising the following steps: mixing citrus peel with an aqueous DES solution containing 10-85% vt, wherein the ratio of citrus peel powder to DES solution is 1 g / 10 mL to 1 g / 30 mL, ultrasonicating at 45-75°C and 320-640 W for 15-60 min, centrifuging or allowing to stand for separation, and taking the supernatant to obtain the citrus peel flavonoid extract.

[0012] The fifth objective of this invention is to provide a low-eutectic solvent-polymer aqueous two-phase system for highly selectively separating citrus flavonoids, comprising a polymer, water, and the aforementioned DES, wherein the DES concentration of the aqueous two-phase system is 22-32 wt%, the polymer concentration is 38-48 wt%, and the balance is water.

[0013] In one embodiment of the present invention, the polymer is polypropylene glycol (PPG) or polyoxyethylene polyoxypropylene ether (EOPO).

[0014] In one embodiment of the present invention, the molecular weight of PPG is 400-2000, and the molecular weight of EOPO is 1100-2900.

[0015] In one embodiment of the present invention, when the polymer is PPG, the DES concentration of the eutectic solvent-polymer aqueous two-phase system is preferably 28-30 wt%, the polymer concentration is preferably 42-44 wt%, and the balance is water.

[0016] In one embodiment of the present invention, when the polymer is EOPO, the DES concentration of the eutectic solvent-polymer aqueous two-phase system is preferably 26-30 wt%, the polymer concentration is preferably 40-44 wt%, and the balance is water.

[0017] The sixth object of the present invention is to provide a method for preparing the above-mentioned eutectic solvent-polymer aqueous two-phase system, comprising the steps of:

[0018] Prepare an aqueous solution of DES by thoroughly mixing the aqueous solution of DES, the polymer, and water to obtain a eutectic solvent-polymer aqueous two-phase system.

[0019] In one embodiment of the present invention, the DES concentration in the DES aqueous solution is 70-90 wt%.

[0020] A seventh object of the present invention is to provide an application of the above-mentioned eutectic solvent-polymer aqueous two-phase system; said application includes the selective separation of different flavonoids; further, said application includes the selective separation of hesperidin and nobiletin.

[0021] The eighth object of the present invention is a method for separating flavonoids using the above-described eutectic solvent-polymer aqueous two-phase system, comprising the steps of:

[0022] Mix DES, polymer, water and the flavonoid to be separated thoroughly, and let stand at 15~35℃ until the distribution equilibrium is reached; after mixing, the concentration of DES is 22~32 wt%, the concentration of polymer is 38~48 wt%, and the remainder is water.

[0023] In one embodiment of the present invention, the flavonoids to be separated are hesperidin and nobiletin; the flavonoids to be separated are derived from citrus peel.

[0024] Beneficial effects:

[0025] The benzyltrimethylammonium chloride-xylitol eutectic solvent prepared in this invention has a high extraction yield of flavonoids from citrus peel, with the highest total flavonoid extraction rate reaching 16.784 mg / g, and the extraction effect is significantly better than other eutectic solvents.

[0026] Traditional DES / salt-ATPS, while achieving high extraction rates by driving most compounds into a single phase due to the strong salting-out effect, suffers from poor selectivity due to significant differences in phase composition. This invention utilizes DES / polymer-ATPS, which forms two phases with significantly different hydrophobicities but milder compositions (a DES-rich phase is hydrophilic, and a polymer-rich phase is hydrophobic), resulting in significantly higher selectivity. Nonocitretin tends to partition into the polymer-rich phase, while hesperidin tends to partition into the DES-rich phase. This achieves integrated extraction and dispersive processing in a low-cost, simple, and mild manner. For hesperidin, the partition coefficient reaches 14.34, with an extraction rate of 93.48%; for nonocitretin, the partition coefficient reaches 2.23, with an extraction rate of 99.05%, and the selectivity coefficient for both reaches 732.76. Attached Figure Description

[0027] Figure 1Effect of the molar ratio of hydrogen bond donors and acceptors in DES on the total extraction rate of citrus flavonoids.

[0028] Figure 2 The effect of different solvents on the extraction rate of citrus flavonoids.

[0029] Figure 3 The effect of DES aqueous solution concentration on the extraction rate of citrus flavonoids.

[0030] Figure 4 This is the phase diagram of PPG and DES.

[0031] Figure 5 This is the phase diagram of EOPO and DES.

[0032] Figure 6 is the partition coefficient of the aqueous two-phase system.

[0033] Figure 7 The extraction rate is the value of the aqueous two-phase system.

[0034] Figure 8 is the selectivity coefficient for the aqueous two-phase system. Detailed Implementation

[0035] Test methods

[0036] Flavonoid extraction rate: The flavonoid extract from Wogan mandarin fruit peel was diluted 20 times with chromatographic grade methanol solution. The sample solution was filtered through a 0.22 μm filter membrane and then placed into a brown sample vial for RSLC analysis to calculate the flavonoid extraction rate.

[0037] Allocation coefficient:

[0038] Extraction rate:

[0039] Selectivity coefficient:

[0040] in, C t and C b These represent the concentrations (mg / mL) of the compounds in the upper and lower phases, respectively. K e 、K f They represent compounds e, f The allocation coefficient.

[0041] Example 1

[0042] A method for preparing different DES and extracting citrus flavonoids using a 70% vt DES aqueous solution includes the following steps:

[0043] Preparation of S1 and DES: Accurately weigh HBD (hydrogen bond donor) and HBA (hydrogen bond acceptor) into a beaker according to the molar ratio and mix them. Then place the mixture in an oil bath at 90-120°C and heat and stir for 2-3 hours until the mixture solution becomes clear and transparent and stable. The hydrogen bond donor, hydrogen bond acceptor, and the molar ratio of hydrogen bond acceptor to hydrogen bond donor of DES are shown in Table 1. After the DES cools naturally, add water to prepare a DES aqueous solution with a DES content of 70% VT.

[0044] S2. Extraction of flavonoids from Wogan mandarin peel using ultrasound-assisted eutectic solvent technology: Accurately weigh 0.1 g of Wogan mandarin peel powder into a 5 mL centrifuge tube, add 2 mL of DES aqueous solution, vortex for 5-10 s, then place the mixed sample in an ultrasonic instrument under the following conditions: 60℃, 320 W, 30 min. Centrifuge the ultrasonically extracted solution at 9000 rpm for 10 min, collect the supernatant after centrifugation, and obtain the flavonoid extract from Wogan mandarin peel.

[0045] Table 1 Composition of different DES

[0046]

[0047] Example 2

[0048] The only difference from Example 1 is that the DES aqueous solution in step S2 is replaced with water, 70% ethanol, 70% methanol, DES-11 aqueous solution with a DES content of 55 vt%, and DES-16~DES-24 aqueous solution, and the ultrasonic conditions are 75°C, 320 W, 15 min.

[0049] The total flavonoid extraction rate of DES-1~DES-10 and DES-12 is as follows: Figure 1 As shown in Table A, the total extraction rates of citrus flavonoids from water, ethanol, methanol, DES-11, and DES-16 to DES-24 are shown in Table 2. When the hydrogen bond acceptor is benzyltrimethylammonium chloride, DES with xylitol as the hydrogen bond donor has a higher total extraction rate of citrus flavonoids.

[0050] Table 2 Total extraction rate of flavonoids from Wogan mandarin orange peel using different solvents

[0051]

[0052] Flavonoid extraction rates from DES-11 to DES-15 are as follows: Figure 1 As shown in B. For DES prepared from benzyltrimethylammonium chloride and xylitol, the highest extraction rates of hesperidin and nobiletin were achieved when the molar ratio of hydrogen bond acceptor to hydrogen bond donor was 3:1. The extraction rates in Example 2 are as follows. Figure 2As shown, the extraction rates of nonotrimonin and hesperidin from some DES samples were better than those from 70% ethanol and 70% methanol, with TMBAC-Xyl (DES-11) exhibiting the highest total extraction rate.

[0053] Example 3

[0054] The method for preparing DES aqueous solutions of different concentrations using TMBAC-Xyl to extract citrus flavonoids differs from Example 1 only in that the hydrogen bond donor and acceptor in step S1 are benzyltrimethylammonium chloride and xylitol, the molar ratio of hydrogen bond acceptor to hydrogen bond donor is 3:1, and water is added to prepare DES aqueous solutions with a water content of 15~90% (i.e., DES 10~85% VT).

[0055] The flavonoid extraction rate in Example 3 is as follows: Figure 3 As shown, when the moisture content is 30-45% (i.e., DES 70-55% VT), the extraction rate of hesperidin from the flavonoid extract of Wogan mandarin orange peel is the highest, and the extraction rate of norepinephrine is also relatively good. When the moisture content is 60%, the extraction rate of norepinephrine from the flavonoid extract of Wogan mandarin orange peel is the highest.

[0056] Example 4

[0057] Accurately weigh 0.1 g of Wogan orange peel powder and place it in a 5 mL centrifuge tube. Add a certain amount of DES aqueous solution (molar ratio of 3:1, water content of 45%), mix evenly, and then place the mixture in an ultrasonic instrument for extraction. An orthogonal experiment was designed to investigate the effects of different ultrasonic time, ultrasonic power, ultrasonic temperature, and solid-liquid ratio on the extraction rate. The specific parameters are shown in Table 3.

[0058] Table 3. Factors and levels in the orthogonal experiment

[0059]

[0060] The results are shown in Table 4. The highest total extraction rate of flavonoids was achieved when the ultrasonic power was 640 W, the ultrasonic time was 15 min, the temperature was 75℃, and the material-to-liquid ratio was 1 g / 20 mL, which was 16.001 mg / g.

[0061] Table 4. Results of the orthogonal experiment

[0062]

[0063] Table 5 shows the results of the orthogonal experimental variance analysis. It indicates that within each parameter range designed in this study, the degree of influence on the extraction rates of hesperidin and nonotrimonin in Wogan mandarin peel is C>B>D, i.e., ultrasonic temperature>ultrasonic time>solid-liquid ratio. A (i.e., ultrasonic power) has no significant effect on the extraction rate at a significance level of 0.05. Based on the experimental results, the optimal process is A3B1C3D2. However, considering economic costs and the fact that ultrasonic power has no significant effect on the extraction rate, and referring to the overall experimental results, the optimal process conditions were finally determined to be A1B1C3D2, i.e., ultrasonic power 320W, time 15 min, temperature 75℃, and solid-liquid ratio 1:20 g / mL.

[0064] Table 5. Results of orthogonal experiment variance analysis

[0065]

[0066] Example 5

[0067] Phase diagram construction of eutectic solvent-polymer aqueous two-phase system:

[0068] At 25±1℃, add 2 g of 85 wt% DES-12 aqueous solution to a 15 mL centrifuge tube. Add 85 wt% PPG or EOPO solution dropwise while shaking until the mixture is fully homogenized, and record the weight. Add the aqueous solution dropwise to the mixture while shaking until the solution becomes completely clear, and record the mass of water added. Repeat the above process until sufficient data is obtained. Plot a phase diagram with DES mass fraction and polymer mass fraction as the x and y axes, respectively, as shown below. Figure 4 , Figure 5 As shown. The selected PPG molecular weights were 400, 600, 800, 1000, and 2000, and the selected EOPO molecular weights were 1100, 2000, and 2900, respectively.

[0069] Depend on Figure 4 , 5 It is known that TMBAC-Xyl can form aqueous two-phase systems with PPG400~2000 and EOPO1100~2900 series, and the molecular weight of the polymer affects the phase-forming ability. Finally, the two systems with the best phase-forming ability were selected as DES-PPG400 and DES-EOPO1100.

[0070] Example 6

[0071] A method for highly selective separation of flavonoids using a eutectic solvent-polymer aqueous two-phase system includes the following steps:

[0072] Accurately weigh 2 mg each of hesperidin and terpene glycoside standards and dissolve them in 5 mL of 80 wt% DES-12 aqueous solution to obtain a DES stock solution with a concentration of 0.2 mg / mL. Thoroughly mix the DES stock solution, polymer, and water to prepare a eutectic solvent-polymer aqueous two-phase system. Allow the system to stand at 25°C for 2 h to reach partition equilibrium. Separate the upper and lower phases using a separatory funnel to obtain hesperidin and terpene glycoside partition solutions.

[0073] The raw material quality examples for the eutectic solvent-polymer aqueous two-phase system are shown in Table 6.

[0074] Table 6. Raw material concentrations for eutectic solvent-polymer aqueous two-phase systems

[0075]

[0076] Comparative Example 1

[0077] The method for separating citrus flavonoids using a Na2SO4-DES aqueous two-phase system includes the following steps:

[0078] Accurately weigh 2 mg each of hesperidin and terpene glycoside standards and dissolve them in 5 mL of 80 wt% DES solution to obtain a DES stock solution with a concentration of 0.2 mg / mL. Thoroughly mix the DES stock solution, Na2SO4, and water to prepare a Na2SO4-DES aqueous two-phase system. Allow the system to stand at 25°C for 2 h to reach partition equilibrium. Separate the upper and lower phases using a separatory funnel to obtain the hesperidin partition solution and the terpene glycoside partition solution.

[0079] In sample Na2SO4-1, the mass ratio of DES stock solution, salt, and water was 20:7:23; in sample Na2SO4-2, the mass ratio of DES stock solution, salt, and water was 22:7:21.

[0080] Comparative Example 2

[0081] The method for separating citrus flavonoids using a C6H5K3O7-DES aqueous two-phase system includes the following steps:

[0082] Accurately weigh 2 mg each of hesperidin and terpene glycoside standards and dissolve them in 5 mL of 80 wt% DES solution to obtain a DES stock solution with a concentration of 0.2 mg / mL. Thoroughly mix the DES stock solution, C6H5K3O7, and water to prepare a C6H5K3O7-DES aqueous two-phase system. Allow the system to stand at 25 °C for 2 h to reach partition equilibrium. Separate the upper and lower phases using a separatory funnel to obtain the hesperidin partition solution and the terpene glycoside partition solution.

[0083] In sample C6H5K3O7-1, the mass ratio of DES stock solution, salt, and water was 42:25:33; in sample C6H5K3O7-2, the mass ratio of DES stock solution, salt, and water was 40:25:35.

[0084] The concentrations of each substance in the upper and lower phases after partition equilibrium were determined in Example 6 and Comparative Examples 1 and 2, and the partition coefficient, extraction rate, and selectivity coefficient of the system were calculated. Compared with the traditional DES-inorganic salt system, the DES-polymer system has a wider hydrophilicity / hydrophobicity range and shows significant advantages in terms of partition coefficient, extraction rate, and selectivity coefficient of citrus flavonoids.

[0085] Figure 6 The partition coefficients are those of the aqueous two-phase systems in Example 6 and Comparative Examples 1 and 2. The TL4 and TL5 systems in the polymer exhibit higher partition coefficients, demonstrating better separation performance than the DES-salt system.

[0086] Figure 7 The extraction rates of hesperidin and nobiletin in the aqueous two-phase systems of Example 6 and Comparative Examples 1 and 2 are shown. The extraction rates of the polymers in the TL3, TL4, and TL5 systems were relatively high, all exhibiting good extraction effects.

[0087] Figure 8 The values ​​represent the selectivity coefficients for the aqueous two-phase systems of Example 6 and Comparative Examples 1 and 2. The TL5 system exhibits the highest selectivity coefficient among the polymers, indicating that it possesses the best selectivity, significantly higher than that of the DES-salt system.

[0088] Example 7

[0089] A method for highly selective separation of flavonoids from Wogan orange peel using a eutectic solvent-polymer aqueous two-phase system includes the following steps:

[0090] Accurately weigh 0.1 g of Wogan orange peel powder into a 5 mL centrifuge tube, and add 2 mL of a 45% vt DES aqueous solution. Vortex for 5–10 s, then place the mixture in an ultrasonic instrument at 75℃, 320 W, for 15 min. Centrifuge the extracted solution at 9000 rpm for 10 min. Collect the supernatant after centrifugation to obtain the Wogan orange peel flavonoid extract. Thoroughly mix the Wogan orange peel flavonoid extract, EOPO1100, and water to prepare a DES-EOPO1100 aqueous two-phase system. The mass ratio of DES, EOPO1100, and water in the aqueous two-phase system is 3:4:3. Let it stand at 25℃ for 2 h to reach partition equilibrium.

[0091] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A low-electrome extraction solvent (DES) for high-yield extraction of citrus flavonoids, characterized in that, It consists of a hydrogen bond donor and a hydrogen bond acceptor. The hydrogen bond acceptor is benzyltrimethylammonium chloride, and the hydrogen bond donor is xylitol. The molar ratio of hydrogen bond acceptor to hydrogen bond donor in DES is 3:1 to 1:

3.

2. The method for preparing DES according to claim 1, characterized in that, The steps include: mixing the hydrogen bond donor and the hydrogen bond acceptor, and stirring at 90~120℃ until the system is clear and transparent.

3. The application of the DES according to claim 1 in the extraction of citrus flavonoids.

4. The method for extracting flavonoids from citrus peel using ultrasound-assisted DES extraction according to claim 1, characterized in that, The steps include: mixing citrus peel with a DES aqueous solution containing 10-85% vt, with a material-to-liquid ratio of 1 g / 10 mL to 1 g / 30 mL, sonicating at 45-75℃ and 320-640 W for 15-60 min, centrifuging or allowing to stand for separation, and taking the supernatant to obtain the citrus peel flavonoid extract.

5. A low-eutectic solvent-polymer aqueous two-phase system for highly selective separation of citrus flavonoids, characterized in that, It is composed of a polymer, water and the DES as described in claim 1, wherein the DES concentration is 22-32 wt%, the polymer concentration is 38-48 wt%, and the balance is water.

6. The eutectic solvent-polymer aqueous two-phase system according to claim 5, characterized in that, The polymer is polypropylene glycol (PPG) or polyethylene oxide polyoxypropylene ether (EOPO); the molecular weight of polypropylene glycol is 400-2000, and the molecular weight of polyethylene oxide polyoxypropylene ether is 1100-2900; when the polymer is PPG, the DES concentration of the eutectic solvent-polymer aqueous two-phase system is 28-30 wt%, the polymer concentration is 42-44 wt%, and the balance is water; when the polymer is EOPO, the DES concentration of the eutectic solvent-polymer aqueous two-phase system is 26-30 wt%, the polymer concentration is 40-44 wt%, and the balance is water.

7. The method for preparing the eutectic solvent-polymer aqueous two-phase system according to claim 5, characterized in that, Including the following steps: Prepare an aqueous solution of DES by thoroughly mixing the aqueous solution of DES, the polymer, and water to obtain a eutectic solvent-polymer aqueous two-phase system.

8. The application of the eutectic solvent-polymer aqueous two-phase system of claim 5 in the selective separation of hesperidin and nobiletin.

9. A method for separating flavonoids using the eutectic solvent-polymer aqueous two-phase system as described in claim 5, characterized in that, Including the following steps: Mix DES, polymer, water and the flavonoid to be separated thoroughly, and let stand at 15~35℃ until the distribution is balanced.

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