A method for extracting a low eutectic solvent of a montana cake polyphenol

Abstract

This invention discloses a low-eutectic solvent extraction method for polyphenols from *Vernicia fordii* seed cake, belonging to the field of agricultural and forestry by-product processing technology. Using *Vernicia fordii* seed cake, an oilseed processing by-product, as raw material, the method involves pre-treatment with defatting followed by polyphenol extraction using a low-eutectic solvent, and purification using AB-8 macroporous resin. This invention employs a multi-component low-eutectic system, resulting in polyphenol extraction efficiency far exceeding that of traditional water and ethanol processes. The purified polyphenols exhibit high purity and contain various phenolic substances such as chlorogenic acid and rutin. The preparation process is simple, the solvent used is green and biodegradable, and the entire process can be easily mass-produced under normal pressure. The obtained highly stable polyphenols can be widely used as natural antioxidants and functional food ingredients, demonstrating promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of agricultural and forestry by-product processing technology, and in particular to a method for preparing polyphenols from *Vernicia fordii* seed cake using a eutectic solvent. Background Technology

[0002] As a woody oilseed tree species, *Vernicia fordii* produces a large amount of cake as a byproduct after its fruit is pressed for oil. Currently, the conventional methods for processing this cake are mainly to make livestock feed or to compost it directly on-site, with some processing stages resulting in the direct disposal of the cake material. This type of processing not only leads to a significant waste of natural active resources such as polyphenols, high-quality protein, and various trace elements within the material, but also causes the cake to produce a pungent odor during long-term fermentation, and the leachate can easily pollute the surrounding water and soil. Existing component analysis studies have confirmed that *Vernicia fordii* cake is rich in phenolic acids and flavonoid polyphenols. These polyphenols possess physiological activities such as antioxidant activity, inhibition of sugar metabolism enzymes, and inhibition of xanthine oxidase, and can be used as natural food antioxidants and functional dietary ingredients. However, due to the lack of mature, scalable integrated processing technology, the polyphenols in the cake are difficult to separate and purify efficiently.

[0003] Currently, the mainstream processes for extracting polyphenols from *Vernicia fordii* seed cake are water extraction and ethanol extraction. Both traditional solvent-based processes have several inherent drawbacks. Pure water, with its single polarity, can only dissolve a small amount of highly polar phenolic acid components and has poor solubility for moderately and weakly polar polyphenols such as flavonoids and flavonoid aglycones, resulting in low polyphenol extraction yields. The crude extract also contains a large amount of polysaccharides, proteins, and inorganic salt impurities, making subsequent purification and separation difficult. While 80% ethanol extraction is slightly better than water extraction, the overall dissolution efficiency remains limited. The production process consumes a large amount of solvent, and heating during the concentration stage easily leads to polyphenol oxidation and loss. Furthermore, ethanol is difficult to dissociate bound polyphenols within the raw material, resulting in a final polyphenol purity of only 60%–67%. Although auxiliary extraction methods such as ultrasound, microwave, and supercritical fluid extraction can slightly increase polyphenol dissolution, the equipment purchase and operating costs are high. The localized high temperatures generated by microwaves and ultrasound can easily damage the polyphenol molecular structure, and supercritical extraction requires high-pressure, sealed, specialized equipment, making it unsuitable for small- to medium-sized production scenarios. Furthermore, the crude polyphenols obtained by traditional solvents have a high impurity content, which can interfere with the physiological activity of the polyphenols themselves. As a result, the antioxidant, sugar control, and uric acid reduction properties of the products do not meet the standards for use as functional raw materials.

[0004] Eutectic solvents are a green extraction medium that has emerged in recent years. With advantages such as low toxicity, biodegradability, the ability to form multiple hydrogen bonds with polyphenols, and excellent extraction efficiency, they are gradually being applied to various plant polyphenol extraction experiments. However, current research on eutectic solvents for polyphenols from *Vernicia fordii* seed cake has technical shortcomings. Existing studies lack systematic process optimization, and most studies neglect the purification step of the crude extract. Directly freeze-dried crude extracts from eutectic solvents contain large amounts of sugars and pigment impurities, with polyphenol purity below 70%, making them unsuitable for high-end food ingredients. Existing literature only compares the polyphenol extraction yields of different solvents, completely neglecting to examine the stability differences of polyphenols prepared with different eutectic solvents under storage and processing conditions. It also lacks systematic research on polyphenol structure and solubility, failing to address practical application challenges such as polyphenol thermal degradation and rapid degradation.

[0005] Polyphenols possess inherently heat- and pH-sensitive physicochemical properties, which is a common pain point limiting their industrial application. Polyphenol molecules contain numerous phenolic hydroxyl groups, making them highly susceptible to dissociation in neutral or weakly alkaline environments, leading to oxidation and polymerization reactions that produce physiologically inactive, dark-colored polymers. Food processing commonly employs short-time sterilization and baking processes at 60-65°C, which significantly accelerate polyphenol oxidation and decomposition. Current industry methods for improving polyphenol stability primarily involve microencapsulation and chemical structure modification, which are cumbersome and involve high costs for excipients. Existing research on polyphenols from *Vernicia fordii* (tung oil) cake meal lacks a low-cost, excipient-free stabilization control scheme. There is a lack of a complete preparation process for polyphenols from *Vernicia fordii* cake meal that integrates high extraction rates, high purity, and high processing stability. A green, low-cost, end-to-end integrated technology is urgently needed to transform oil extraction byproducts into high-quality polyphenol functional raw materials. Summary of the Invention

[0006] The purpose of this invention is to provide a method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for eutectic solvent extraction of polyphenols from *Vernicia fordii* seed cake, characterized by comprising the following steps: (1) Raw material degreasing pretreatment: Take the tung seed cake, crush and sieve it, and degrease it to obtain degreased cake powder; (2) Preparation of eutectic solvent: Use any one of betaine, proline and choline chloride as hydrogen bond acceptor and any one of lactic acid and levulinic acid as hydrogen bond donor, mix and melt to prepare eutectic solvent; (3) Extraction: The defatted cake powder is mixed with the eutectic solvent for extraction, and then centrifuged to obtain a crude polyphenol extract; (4) Purification: The crude polyphenol extract was loaded onto the pretreated AB-8 macroporous resin for adsorption. After washing with water to remove impurities, it was eluted with ethanol. After desolvation of the eluent, it was freeze-dried to obtain purified polyphenols from the tung oil cake.

[0008] Furthermore, the eutectic solvent comprises any one of the following three systems: System 1: Proline to levulinic acid molar ratio 1:2; System 2: Betaine to lactic acid molar ratio 1:2.5; System 3: The molar ratio of choline chloride to levulinic acid is 1:1.5.

[0009] Pure polyphenols from *Vernicia fordii* seed cake were prepared using a eutectic solvent with a betaine-lactic acid molar ratio of 1:2.5. After purification, the total polyphenol purity was ≥82%. Under the conditions of pH 4.0 and heating at 65 ℃ for 30 min, the polyphenol retention rate was ≥86%, and the water solubility was ≥98%.

[0010] Furthermore, the hydrogen bond acceptor and hydrogen bond donor are mixed and stirred at 80°C for 1-2 h to form the eutectic solvent.

[0011] Furthermore, the temperature extracted in step (3) is 60°C.

[0012] Furthermore, the pretreatment method for the AB-8 macroporous resin includes the following steps: The AB-8 macroporous resin was soaked in 95% ethanol, and after removing the alcohol, it was successively soaked in 4 wt% hydrochloric acid and 4 wt% sodium hydroxide with shaking for 8 hours each, and finally washed with water until neutral.

[0013] Furthermore, the defatted cake powder and the diluted eutectic solvent were mixed at a ratio of 1:10 g / mL, extracted at 60°C for 1.5 h, and centrifuged at 8000 r / min for 10 min.

[0014] The present invention also provides polyphenols extracted from eutectic solvent extraction of the above-mentioned polyphenols from tung oil cake.

[0015] The present invention discloses the following technical effects: This invention constructs an integrated preparation process combining eutectic solvent extraction and macroporous resin purification, effectively solving many defects of traditional processes such as low polyphenol extraction rate, insufficient product purity, and easy degradation during processing. This invention uses a specific eutectic solvent to replace traditional extraction media such as water and ethanol, significantly improving polyphenol dissolution efficiency; then, AB-8 macroporous resin is used to deeply remove impurities such as polysaccharides and proteins, resulting in high-purity polyphenols after purification.

[0016] The entire process of this invention is carried out under normal pressure, using a low-eutectic solvent that is green and biodegradable. It uses oilseed cake as raw material, resulting in low production costs and facilitating large-scale production. The obtained highly stable polyphenols can be widely used as natural antioxidants and functional food ingredients, and have good prospects for industrial application. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The effect of different extraction solvents on the extraction yield of polyphenols from *Vernicia fordii* seed cake.

[0019] Figure 2 The effect of DES ratio on the extraction yield of polyphenols from *Vernicia fordii* cake meal.

[0020] Figure 3 The effect of moisture content on the extraction yield of polyphenols from *Vernicia fordii* seed cake.

[0021] Figure 4 To investigate the effect of extraction temperature on the polyphenol extraction yield from *Vernicia fordii* seed cake.

[0022] Figure 5 The effect of extraction time on the polyphenol extraction yield from *Vernicia fordii* seed cake.

[0023] Figure 6 The effect of different solvent extraction on the leakage point of polyphenols in tung oil cake.

[0024] Figure 7 UV absorption spectra of polyphenols extracted from *Vernicia fordii* seed cake using different solvents.

[0025] Figure 8 Infrared spectra of polyphenols extracted from *Vernicia fordii* seed cake using different solvents.

[0026] Figure 9 Scanning electron microscopy (SEM) images of polyphenols extracted from *Vernicia fordii* seed cake using different solvents were observed at magnifications of 5.00 K and 10.0 K. A represents the water-extracted polyphenol sample, B represents the 80% ethanol-extracted polyphenol sample, and C, D, and E correspond to the three eutectic solvent extracts DES-4, DES-9, and DES-10, respectively.

[0027] Figure 10 Total ion chromatogram (TIC) of polyphenols from five types of *Vernicia fordii* seed cake.

[0028] Figure 11The UV-Vis absorption spectra of polyphenols from three types of *Vernicia fordii* seed cake under pH and heat treatment are shown.

[0029] Figure 12 Infrared spectra of polyphenols from three types of *Vernicia fordii* seed cake under pH and heat treatment.

[0030] Figure 13 The color changes of three polyphenol solutions from *Vernicia fordii* cake under pH and heat treatment are shown. From left to right in the figure are polyphenol solutions under the conditions of pH = 4, pH = 4 heating, pH = 7, and pH = 7 heating treatment.

[0031] Figure 14 The values ​​of L*, a*, b* and total color difference ΔE are given for three polyphenol solutions of *Vernicia fordii* cake under pH and heat treatment.

[0032] Figure 15 The transmittance of polyphenols from three types of *Vernicia fordii* seed cake under pH and heat treatment is given.

[0033] Figure 16 The retention rates of polyphenols in three types of *Vernicia fordii* cake meal under different pH and heat treatment conditions. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0040] The tung oil cake used in this invention comes from the Key Laboratory of Tung Oil Cake of the State Forestry and Grassland Administration.

[0041] Example 1 (1) Pretreatment of tung oil cake meal: First, the *Vernicia fordii* seed cake was pulverized using a pulverizer and then passed through a 60-mesh sieve to obtain *Vernicia fordii* seed cake powder. Next, the *Vernicia fordii* seed cake powder was mixed with n-hexane at a material-to-liquid ratio of 1:5 g / mL, and the mixture was placed on a magnetic stirrer for degreasing. This degreasing process was repeated twice, with each degreasing process lasting 2 hours. After degreasing, the mixture was filtered, and the filter residue was collected and placed in a fume hood for 48 hours of ventilation to finally obtain defatted *Vernicia fordii* seed cake powder.

[0042] (2) Preparation of eutectic solvent: Using choline chloride, betaine, and proline as hydrogen bond acceptors, and six hydrogen bond donors (glucose, lactic acid, levulinic acid, ethylene glycol, glycerol, and citric acid), twelve eutectic solvents were prepared in specific molar ratios (Table 1). The prepared mixtures were placed in an 80 °C constant-temperature water bath and stirred magnetically for 1–2 h until a clear, transparent solution was formed. After the solution cooled naturally to room temperature, a stable, transparent eutectic solvent system without stratification was obtained.

[0043] Table 1 (3) Extraction of polyphenols from tung oil seed cake meal: Accurately weigh 5 g of defatted *Vernicia fordii* seed cake powder, and add different types of extraction solvents (water, 80% ethanol, and DES) at a material-to-liquid ratio of 1:10 g / mL. Place the mixture in a 60 ℃ water bath for 1.5 h. After extraction, centrifuge at 8000 r / min for 10 min. Collect the supernatant after centrifugation; this supernatant is the crude polyphenol extract from *Vernicia fordii* seed cake.

[0044] 1. Screening of eutectic solvents: To screen suitable eutectic solvent systems for polyphenol extraction from *Vernicia fordii* seed cake, this invention uses polyphenol extraction yield as an evaluation index to screen 12 prepared eutectic solvents. The solvents were grouped according to hydrogen bond acceptor type, and the system with the highest extraction efficiency was selected as the target solvent for subsequent studies.

[0045] The determination of polyphenol extract content from *Vernicia fordii* seed cake is as follows: The preparation steps of the standard curve are as follows: Gallic acid reference standard was prepared into a 1 mg / mL standard stock solution, and then serially diluted to obtain reference standard working solutions with concentrations of 10, 20, 30, 40, and 50 µg / mL. 1 mL of each of the above reference standard solutions of different concentrations was transferred to a 25 mL colorimetric tube, and 1 mL of 0.1 mol / L Folin-Ciocalteu solution and 4 mL of 7.5% sodium carbonate solution were added sequentially. After thorough mixing, the solution was diluted to the mark with pure water and allowed to stand in a dark environment for 1 h to complete color development. The absorbance of each solution was then measured at a wavelength of 765 nm. A standard curve was plotted with gallic acid concentration (x) as the abscissa and the corresponding absorbance (y) as the ordinate.

[0046] The method for determining the polyphenol extraction yield from *Vernicia fordii* seed cake is as follows: Take 0.05 mL of crude polyphenol extract from *Vernicia fordii* seed cake and dilute it to 5 mL with pure water. Transfer 0.2 mL of the diluted extract to a colorimetric tube, add 0.2 mL of 0.1 mol / L Folin-Ciocalteu solution, mix thoroughly, and let stand for 5 min. Then add 0.8 mL of 7.5% sodium carbonate solution, shake well, and let stand in the dark for 1 h. Measure the absorbance of the solution at 765 nm. Calculate the polyphenol concentration using the standard curve described above, and then calculate the polyphenol extraction yield using the following formula: (1) In the formula: W-polyphenol extraction yield, mg / g; C - The mass concentration of polyphenols in the tung oil seed cake meal calculated according to the standard curve, mg / mL; V - Volume of the extract after dilution, in mL; N - Dilution factor; Mass of M-mountain tung seed cake defatted powder, g.

[0047] This invention uses the Folin-Ciocalteu colorimetric method to determine the polyphenol content of *Vernicia fordii* seed cake, establishing a standard curve using gallic acid as a reference standard. A linear regression was performed with gallic acid concentration (x) on the x-axis and corresponding absorbance (y) on the y-axis, yielding the regression equation y = 9.8036x + 0.0088 (R²). 2= 0.9992), indicating a good linear relationship between total polyphenol concentration (mg / mL) and absorbance, which can be used for accurate quantification of polyphenol content in samples.

[0048] Figure 1 The effect of different extraction solvents on the extraction yield of polyphenols from *Vernicia fordii* seed cake was investigated. Different lowercase letters in the bar chart indicate significant differences (P < 0.05), while identical lowercase letters indicate no significant differences (P > 0.05) (the same applies to the other figures in this invention). Figure 1 It can be seen that different extraction solvents have significant differences in the extraction yield of polyphenols from *Vernicia fordii* seed cake. P <0.05), when water and ethanol were used as extraction solvents, the polyphenol extraction content was at a low level, while various eutectic solvents showed better extraction effects. Among them, DES-9 had the highest polyphenol extraction yield, reaching 71.39±1.52 mg / g, which was significantly better than other solvent systems. DES-8, DES-10, and DES-12 were next, with extraction yields of 63.39±0.70 mg / g, 60.01±0.47 mg / g, and 56.88±1.05 mg / g, respectively, all of which were significantly higher than those of the ethanol extract (50.94±0.58 mg / g) and the water extract (40.22±1.52 mg / g). P <0.05). This may be because DES, through the synergistic effect of hydrogen bond donors and acceptors, can effectively disrupt the binding of polyphenols with cellulose and lignin in plant cell walls, thereby enhancing the dissolution efficiency of polyphenols. The differences in hydrogen bond strength, polarity, and viscosity among different DES components lead to significant differences in their ability to dissolve and extract polyphenols. Among them, DES-9 exhibits the best extraction effect due to its solvent effect being more compatible with the molecular structure of polyphenols in *Vernicia fordii* cake meal. Traditional water extraction and ethanol extraction, due to their single solvent polarity and limited cell wall disruption ability, yielded significantly lower extraction amounts than the DES system, further confirming the advantages of DES as a novel green solvent in the extraction of polyphenols from *Vernicia fordii* cake meal.

[0049] 2. Single-factor experiment on the extraction of polyphenols from *Vernicia fordii* seed cake using eutectic solvents: To optimize the process conditions for DES extraction of polyphenols from *Vernicia fordii* cake meal, the effects of four factors on the extraction yield of polyphenols from *Vernicia fordii* cake meal were investigated (Table 2).

[0050] Table 2 Among choline chloride solvents, DES-4 showed significantly higher extraction yields than DES-3, DES-5, and other solvents in the same system, and was therefore selected as the representative of this class. For proline solvents, DES-9 was selected as the representative due to its optimal extraction efficiency. For betaine solvents, DES-10 was chosen as the representative due to its highest extraction yield, ensuring the scientific validity and representativeness of subsequent process optimization experiments. Table 3 shows that the three eutectic solvents exhibit significant differences in physicochemical properties such as viscosity, pH, density, and polarity, which collectively determine their extraction capabilities for polyphenols from *Vernicia fordii* seed cake. All three DES solvents are strongly acidic, with DES-10 having the lowest pH (2.40 ± 0.37) and DES-4 the highest (3.10 ± 0.05). This strongly acidic environment effectively inhibits polyphenol oxidative degradation and promotes cell wall swelling and rupture, providing favorable conditions for polyphenol dissolution. In terms of viscosity, DES-9 had the highest viscosity (486.23 ± 0.49 mPa·s), which stems from the dense hydrogen bond network formed by the proline multifunctional group and levulinic acid. DES-4 had the lowest viscosity (314.16 ± 0.57 mPa·s), which is related to the relatively small steric hindrance of its choline chloride quaternary ammonium salt structure. DES-10 had a moderate viscosity, consistent with the typical characteristics of betaine-lactic acid DES. Regarding density, all three DES types had higher densities than water, and the density of molecular packing was positively correlated with the strength of hydrogen bonds, with DES-10 having the highest density (1.24 ± 0.06 g / cm³). 3 The lowest concentration of DES-4 was (1.20 ± 0.14 g / cm³). 3 Regarding polarity, DES-10 exhibits the strongest polarity (54.37 ± 0.55 kJ / mol), primarily contributed by the strong polar hydroxyl and carboxyl groups of lactic acid. DES-9 shows the weakest polarity (48.45 ± 0.71 kJ / mol), with the hydrophobic fatty ring of proline weakening the system's polarity. Polarity is one of the core driving forces affecting polyphenol dissolution. While DES-10 has the strongest polarity, its hydrogen bond matching with polyphenols from *Vernicia fordii* seed cake is not optimal. In contrast, the hydrogen bond donor-acceptor structure of proline and levulinic acid in DES-9 can more precisely form multiple hydrogen bonds with the phenolic hydroxyl and carboxyl groups of polyphenols, thereby more efficiently disrupting the binding of polyphenols to cell wall polysaccharides and proteins. This is the key to its highest extraction efficiency. Viscosity has a dual impact on extraction efficiency. Although DES-9 has the highest viscosity, its strong hydrogen bond binding ability offsets the negative impact of diffusion resistance. DES-4 has the lowest viscosity, but its insufficient polarity and hydrogen bond donor-acceptor structure result in the lowest extraction efficiency. DES-10 strikes a balance between polarity and viscosity, resulting in moderate extraction efficiency. Furthermore, the strongly acidic environment of all three DES types ensures stable dissolution of polyphenols, a key reason why their extraction efficiency is significantly superior to traditional solvents such as water and ethanol.

[0051] Table 3 Composition and physicochemical properties of DES Note: Different lowercase letters in the same column indicate significant differences (P<0.05), and the same lowercase letters in the same column indicate no significant differences (P>0.05).

[0052] Figure 2 This study demonstrates the differences in polyphenol extraction yields of three DES formulations (DES-4, DES-9, and DES-10) at different DES ratios. The composition ratio of the eutectic solvent significantly affects the polyphenol extraction efficiency. Different uppercase letters in the bar chart indicate significant differences among the three polyphenols (P<0.05), while different lowercase letters indicate significant differences among different DES ratios for the same polyphenol (P<0.05). The study shows that the interaction between hydrogen bond donors and acceptors in DES changes with the ratio, thus affecting solvent polarity, viscosity, and other properties, ultimately impacting the polyphenol solubility. As shown in the figure, for DES-9, the polyphenol extraction yield reaches its peak (73.54±1.51 mg / g) at a ratio of 1:2. DES-10 exhibits a relatively high extraction yield (66.22±0.70 mg / g) at a ratio of 1:2.5. DES-4 showed a relatively high extraction yield (58.89 ± 0.79 mg / g) at a ratio of 1:1.5. DES-9 may benefit from the optimal synergistic effect of hydrogen bond donors and acceptors at a 1:2 ratio, which promotes polyphenol solubility. DES-10, on the other hand, exhibits better overall solvent penetration and solubility at a 1:2.5 ratio. Therefore, the DES ratio is a key factor affecting polyphenol extraction yield, and different DES formulations have their own suitable extraction ratios due to their unique composition.

[0053] Figure 3The changes in polyphenol extraction yield of three DES groups (DES-4, DES-9, and DES-10) under different water contents are shown. Water content in DES is a crucial factor affecting polyphenol extraction. For DES-9, the polyphenol extraction yield showed a significant upward trend as the water content increased from 0% to 30%, reaching a peak at 30% water content (69.43±2.09 mg / g). Subsequently, the extraction yield decreased with further increases in water content. This may be because appropriate amounts of water can improve the polarity and diffusion properties of the eutectic solvent, thereby enhancing its ability to dissolve and extract polyphenols. However, excessive water dilutes the effective components of the eutectic solvent, reducing extraction efficiency. For DES-10, the polyphenol extraction yield reached a relatively high level (59.91±1.28 mg / g) at a water content of 40%. At a 40% water content, the solvent microstructure of DES-10 improved, promoting the interaction between the solute and solvent, thus improving extraction efficiency. The polyphenol extraction yield of DES-4 fluctuated relatively little across the entire range of moisture content. The highest polyphenol extraction yield (55.14 ± 1.04 mg / g) was achieved at a moisture content of 30%, but it was still lower than that of DES-9 and DES-10.

[0054] Figure 4 The effect of different extraction temperatures on the polyphenol extraction yield of five groups of samples was investigated. As shown in the figure, with increasing extraction temperature, the overall polyphenol extraction yield of various solvents initially increased and then decreased, but differences existed between different solvents. The control group yield was generally lower than that of DES-type solvents. Water, as a polar solvent, has a certain solubility for polar components like polyphenols. Increased temperature accelerates molecular motion, slightly improving solubility; however, due to the lack of hydrogen bonding interactions between water and polyphenols, as seen in eutectic solvents, the improvement in extraction efficiency is limited. Ethanol is a commonly used polyphenol extraction solvent, with polarity between water and pure ethanol. It has good solubility for polyphenols; increased temperature promotes the penetration of ethanol molecules into the material and the dissolution of polyphenols. However, compared to DES-type solvents, the interaction between ethanol and polyphenols is relatively simple, limiting further increases in extraction yield. The polyphenol extraction yield of all five groups of samples reached its peak at 60 ℃. Increasing temperature can enhance molecular thermal motion, reduce solvent viscosity, promote solvent penetration into the material and solute dissolution, thereby increasing extraction yield. However, excessively high temperatures may lead to polyphenol oxidative degradation. Studies have shown that eutectic solvents, due to their unique hydrogen bond network structure, can significantly improve the solubility and mass transfer of polyphenols when heated appropriately. Compared to water and ethanol, the three groups of eutectic solvent samples interacted with polyphenol molecules more efficiently during the heating process, resulting in higher extraction yields.

[0055] Figure 5The effect of different extraction times on the polyphenol extraction yield of five groups of samples (water, ethanol, DES-4, DES-9, and DES-10) was investigated. As shown in the figure, the polyphenol extraction yield of all solvents initially increased and then decreased with prolonged extraction time, with significant differences among the different solvents. The extraction yield of all five groups reached its peak at 1.5 h, possibly because longer extraction time allows for more thorough contact between the solvent and the material, promoting solute dissolution. However, once the solute reaches dissolution equilibrium in the solvent, further extending the extraction time has limited effect on increasing the extraction yield, and may even lead to a decrease in extraction yield due to reactions such as polyphenol oxidation. For the samples extracted with water and ethanol, the extraction yield increased somewhat with time, but was still significantly lower than that extracted with DES-type solvents. Water, as a polar solvent, allows polyphenol dissolution primarily through molecular diffusion; the dissolution rate is slow in the initial stages of extraction, gradually increasing over time. However, due to the lack of hydrogen bonds and other interactions between water and polyphenols, the increase in extraction yield is limited. While ethanol, due to its moderate polarity, has good solubility for polyphenols and its molecules can quickly penetrate into the material to dissolve polyphenols, its extraction yield is limited by the singularity and intensity of the interaction between ethanol and polyphenols, making it difficult to significantly increase the extraction yield after reaching a certain level. DES-4, DES-9, and DES-10, compared to water and ethanol, can achieve higher extraction yields more quickly during the extraction process. Therefore, eutectic solvents are generally superior to traditional solvents, and different DESs have their own optimal extraction times to achieve higher extraction yields.

[0056] Physicochemical property testing of eutectic solvents: Viscosity testing: The viscosity of the eutectic solvent was measured using a rheometer at a temperature controlled at 25 ℃, within the range of 0.01-100 s. -1 The measurement was conducted continuously for 3 minutes at a variable shear rate, and finally stopped at 0.1 s. -1 The average of three measurements at the shear rate was taken as the final viscosity value of the DES.

[0057] pH value determination: To investigate the effect of the acidity or alkalinity of DES on the polyphenol extraction rate, a pH meter was used to characterize its pH value. Under constant temperature conditions of 25 ℃, the specific pH value of each DES was accurately measured by a pH electrode.

[0058] Density measurement: The binding of hydrogen bond donors and hydrogen bond acceptors will cause changes in solvent density. This invention uses a densitometer to accurately detect the density of each DES.

[0059] Solvent Polarity Test: The maximum absorption wavelength of Nile Red in the UV-Vis region is significantly correlated with solvent polarity. This invention quantifies and evaluates the polarity characteristics of DES by measuring the Molar Transition Energy (ENR) of this reagent in DES. 1 mL of DES is thoroughly mixed with 3 mL of a 10 μg / mL Nile Red-ethanol solution. The solution is then scanned using a UV spectrophotometer in the wavelength range of 400-800 nm, and the maximum absorption wavelength (λ) of the solution is recorded. max Substitute it into formula (2) to calculate the ENR value.

[0060] (2) In the formula: ENR is the molar transition energy of the solvent (kJ / mol); h is Planck's constant; c is the speed of light; NA is Avogadro's constant; λ max The maximum absorption wavelength is 10 nm.

[0061] 3. Static test of polyphenol purification from *Vernicia fordii* seed cake using macroporous resin Macroporous resin pretreatment: Appropriate amounts of AB-8, D101, and NKA-9 macroporous resins were weighed and soaked in 95% ethanol solution for 24 hours. After soaking, the resins were filtered and repeatedly rinsed with deionized water until no alcohol odor remained. Subsequently, the resins underwent acid-base pretreatment. First, the resins were placed in a 4% hydrochloric acid (HCl) solution and soaked in a constant-temperature shaker for 8 hours. After soaking, the resins were washed with deionized water until neutral. Then, the resins were transferred to a 4% sodium hydroxide (NaOH) solution and soaked in a constant-temperature shaker for 8 hours, followed by washing with deionized water until neutral. After filtration, the pretreated macroporous resins were stored in deionized water at room temperature using a wet method.

[0062] Table 4 Physical parameters of macroporous resins Macroporous resin purification of polyphenols from *Vernicia fordii* seed cake: Weigh 5 g of each of the three pretreated macroporous resins (AB-8, D101, and NKA-9) and place them in conical flasks along with 25 mL of crude polyphenol extract. Shake in the dark at 25 °C and 120 r / min for 24 h. After adsorption reaches equilibrium, filter the solution, collect the supernatant and measure its absorbance. Calculate the adsorption rate of each type of resin according to the corresponding formula.

[0063] After filtration, the resin was rinsed with deionized water, and the surface moisture was blotted dry with filter paper. The resin was then placed in an Erlenmeyer flask, and 25 mL of 70% ethanol solution was added. The solution was shaken in the dark at 25 °C and 120 r / min for 24 h. After shaking, the resin was filtered, and the absorbance of the desorbed solution was measured and the desorption rate of each resin was calculated.

[0064] (3) (4) In the formula: C0 is the initial polyphenol mass concentration, mg / mL; C1 is the polyphenol mass concentration at adsorption equilibrium, mg / mL; C2 is the polyphenol mass concentration in the desorption solution, mg / mL.

[0065] As shown in Table 5, there were significant differences in the adsorption rates of the three resins. Among them, AB-8 resin showed the best overall performance for DES extract, while the adsorption rates of DES-9 (89.93 ± 0.56%) and DES-4 (88.49 ± 0.34%) were significantly higher than those of the other resins. P The adsorption rate (<0.05) is attributed to the moderate π-π interaction between its weakly polar framework and the aromatic structure of polyphenols, which ensures both high adsorption capacity and easy desorption by ethanol. Zhao Dan et al. optimized the desorption process of AB-8 resin for jujube pulp polyphenols, achieving a desorption rate of 83.40%, consistent with the high desorption rate results of the DES system in this study. Although D101 resin had a relatively high desorption rate, its adsorption rate was generally low (55.11%-66.89%), consistent with the characteristics of weak adsorption and easy desorption. NKA-9 resin had the lowest adsorption rate (<45%) due to its strong polarity, and its desorption efficiency was unstable, indicating poor affinity for polyphenols. Therefore, the combination of AB-8 resin and a low eutectic solvent shows the best potential in polyphenol purification, especially the differentiated performance of DES-9 (high adsorption) and DES-4 (high desorption), providing a basis for targeted process optimization.

[0066] Table 5. Adsorption and desorption effects of different resin types on polyphenols in tung oil cake. Note: Different lowercase letters in the same column indicate significant differences (P<0.05), and the same lowercase letters in the same column indicate no significant differences (P>0.05).

[0067] 4. Dynamic test of polyphenol purification of *Vernicia fordii* seed cake meal using macroporous resin The macroporous resin pretreatment was carried out using the same method as in the "Static Test of Purification of Polyphenols from Cinnamomum camphora Seed Cake with Macroporous Resin".

[0068] AB-8 macroporous resin was used to uniformly purify the crude polyphenol extracts from *Vernicia fordii* seed cake obtained by DES extraction and traditional solvent extraction. 20.0 g of pretreated AB-8 macroporous resin was uniformly packed into a 300×40 mm chromatography column. During packing, the lower outlet of the column was simultaneously opened to allow distilled water to flow out at a uniform rate, ensuring a flat, bubble-free column bed and maintaining a stable chromatographic environment. After loading, the column was rinsed with three column volumes of distilled water to effectively remove residual proteins, polysaccharides, and inorganic salts that were not adsorbed by the resin, thus avoiding interference with subsequent purification. Crude polyphenol extracts of different concentrations obtained by different extraction methods were loaded onto the chromatography column at a constant flow rate of 1.0 mL / min for adsorption. The eluent was collected in 10 mL fractions, and the polyphenol concentration of each fraction was measured. A leakage curve was plotted with the sample volume on the x-axis and the eluent polyphenol concentration on the y-axis. The leakage point is identified when the polyphenol concentration in the effluent reaches 1 / 10 of the sample concentration, at which point sample loading is stopped. After adsorption, the resin surface is thoroughly rinsed with deionized water to remove residual impurities. Then, a 60% (v / v) ethanol solution is used for dynamic desorption at a flow rate of 0.5 mL / min. The desorbed eluent is placed at 45 °C, and the ethanol is removed by low-pressure rotary evaporation to obtain a concentrated polyphenol solution. The concentrate is then freeze-dried under vacuum to obtain a purified polyphenol powder, which is stored at -20 °C in the dark for later use.

[0069] Macroporous resins have limited adsorption capacity for target components. When the resin reaches saturation with polyphenols from *Vernicia fordii* seed cake, its adsorption capacity for target components decreases significantly, causing polyphenols in the injection solution to leak from the macroporous resin column. This experiment uses the polyphenol concentration in the effluent (C0.05) as the basis for this designation. t The ratio of the polyphenol concentration (C0) in the sample solution to the total polyphenol concentration in the loading solution is used as the criterion. When this ratio reaches 1 / 10, it is determined that a leak has been reached, at which point the resin can no longer effectively adsorb more polyphenol components. Figure 6 It was found that the earliest leakage point appeared in the polyphenols extracted from *Vernicia fordii* seed cake, followed by those extracted from ethanol. The three eutectic solvents appeared later, with DES-10 appearing last. Lower flow rates prolonged the residence time of the solution in the resin layer, promoting the interaction between polyphenols and the resin, thereby enhancing adsorption capacity. Higher flow rates shortened the contact time between polyphenols and the resin, reducing adsorption opportunities and decreasing adsorption efficiency. When an injection flow rate of 1.0 mL / min was used, the leakage point appeared at approximately 65 mL, and the polyphenol concentration in the effluent remained at a low level at this point. This indicates that AB-8 resin has a strong adsorption capacity and high adsorption efficiency for polyphenols extracted from *Vernicia fordii* seed cake using eutectic solvents.

[0070] 5. Calculation of polyphenol purity in tung oil cake meal The purity of polyphenols from *Vernicia montana* seed cake was calculated based on the static and dynamic tests of "purification of polyphenols from *Vernicia montana* seed cake using macroporous resin". Ethanol precipitation was used to remove and purify some impurities such as proteins and polysaccharides from the crude polyphenol extract. First, the crude polyphenol extract was concentrated to a suitable volume under reduced pressure. Then, 80% ethanol solution was added, and the mixture was thoroughly mixed and allowed to stand for 12 hours. After the impurities had fully precipitated, the supernatant was separated by centrifugation. To ensure complete removal of impurities, this operation was repeated three times. The supernatant after three precipitation treatments was concentrated again under reduced pressure to remove excess ethanol. Then, it was purified using AB-8 macroporous resin. After purification, it was freeze-dried to obtain purified polyphenol powder. An appropriate amount of the freeze-dried powder was taken and prepared into a solution of the appropriate concentration. The purity of the polyphenols was calculated using relevant tests.

[0071] (5) In the formula: C is the concentration of polyphenol solution, mg / mL; V is the volume of solution, mL; M is the mass of the purified lyophilized powder, g.

[0072] Table 6 shows the purity of polyphenols from *Vernicia fordii* seed cake after purification. After enrichment with AB-8 macroporous resin, the highest purity of polyphenols from the purified *Vernicia fordii* seed cake reached 82.88 ± 1.02%, which is 1.37 times that of the water extract (60.71 ± 0.67%).

[0073] Table 6. Purity of polyphenols extracted from *Vernicia fordii* seed cake and meal using different solvents after purification. Note: Different lowercase letters in the same row indicate significant differences (P<0.05), and the same lowercase letters in the same row indicate no significant differences (P>0.05).

[0074] 6. Analysis of polyphenol composition and bioactivity of *Vernicia fordii* seed cake meal (1) Ultraviolet-visible spectral analysis: Take the extracted polyphenol sample from the tung oil cake, dilute it with methanol to a concentration of 0.1 mg / mL, and use an ultraviolet-visible spectrophotometer to scan and detect it in the wavelength range of 200-400 nm. Set the scanning interval to 1 nm, and use methanol as a blank control reference to complete the ultraviolet spectral analysis of the sample.

[0075] Figure 7The UV absorption spectra of polyphenols from *Vernicia montana* seed cake extracted with five different solvents and gallic acid standards are presented. As shown in the figures, the polyphenol samples from *Vernicia montana* seed cake extracted with different solvents all exhibit typical UV absorption characteristics of polyphenols in the wavelength range of 200-400 nm, with a strong absorption peak appearing around 270 nm. This absorption peak mainly originates from the π-π electron transition of the benzene ring structure of the polyphenol molecule, which is a characteristic absorption peak of polyphenols, confirming that all samples are polyphenolic substances. Comparison of the spectral characteristics of extracts from different solvents shows that the absorbance of the DES extract is significantly higher than that of the water and ethanol extracts, with a sharper and stronger absorption peak. This indicates that the DES extraction system can more efficiently enrich the polyphenolic components in *Vernicia montana* seed cake, and has better solubility and structural retention of polyphenol molecules. The absorbance of the water and ethanol extracts is relatively low, reflecting the relatively weak extraction efficiency of traditional solvents for polyphenols. Combined with the comparative spectral analysis of gallic acid standard, the material structural properties of polyphenols from *Vernicia veitchii* seed meal were further verified, and it was also shown that there were significant differences in the enrichment degree of polyphenols by different extraction solvents. P <0.05).

[0076] (2) Fourier transform infrared spectroscopy analysis: Accurately weigh 1 mg of freeze-dried polyphenol powder from *Vernicia fordii* seed cake and 100 mg of potassium bromide. Mix them thoroughly and dry them in an oven until constant weight. Then grind them into a fine powder in a mortar and press them into thin sheets. Spectroscopy was performed using a Fourier transform infrared spectroscopy system at 4000-400 cm⁻¹. -1 The infrared spectrum of polyphenols from *Vernicia fordii* seed cake was obtained by scanning within the specified wavelength range. First, a background scan was performed on pure potassium bromide tablets, and then a scan was performed on polyphenol sample tablets.

[0077] Infrared spectroscopy can be used to identify the functional group structures in polyphenols. Figure 8The images show the infrared spectra of polyphenols extracted from *Vernicia fordii* seed cake using five different solvents. Several characteristic absorption peaks can be observed in the spectra. The broad absorption band at 3413.2–3420 cm⁻¹ is attributed to the stretching vibration of hydroxyl groups in the polyphenol molecule, consistent with the characteristic of polyphenols containing a large number of hydroxyl groups. A stretching vibration absorption peak at 2829.9–2833.3 cm⁻¹ indicates the presence of methylene groups. The absorption peak at 1595.2–1612.2 cm⁻¹ corresponds to the C=O stretching vibration of the benzene ring skeleton. The absorption peak near 1353.7–1362.2 cm⁻¹ corresponds to the stretching vibration of CO. The low wavenumber peaks at 773.8 cm⁻¹ and 619 cm⁻¹ correspond to the out-of-plane bending vibration of CH on the aromatic ring. These characteristic absorption peaks indicate that the polyphenols from *Vernicia fordii* seed cake contain a large number of hydroxyl groups, a benzene ring structure, and C=O bonds, consistent with the structural characteristics of polyphenols. Infrared spectroscopy analysis further confirmed that the meal of *Vernicia fordii* contains abundant polyphenols, which corroborated the results of ultraviolet-visible spectroscopy analysis.

[0078] (3) Scanning electron microscopy analysis: The freeze-dried *Vernicia fordii* seed cake polyphenol samples were sputter-coated with gold. After a uniform metal film formed on the sample surface, the samples were observed and photographed under a scanning electron microscope to determine their microstructure. This experiment referenced and slightly modified the research method of Belwal et al., and further analyzed the physicochemical properties of the samples by observing their microstructural characteristics.

[0079] The microstructure of polyphenols extracted from *Vernicia fordii* seed cake using different solvents was characterized by scanning electron microscopy at magnifications of 5.00 K and 10.0 K, respectively. The scanning electron micrographs are shown below. Figure 9 As shown, the polyphenols extracted from *Vernicia fordii* seed cake by water extraction, alcohol extraction, and extraction with eutectic solvents (DES-4, DES-9, DES-10) exhibit significant differences in microstructure. SEM images of the water-extracted polyphenols are shown below. Figure 9 A) shows that it mainly exhibits a plate-layer aggregate structure, with particles primarily in the form of irregular plates, edges with certain angularity, and a relatively smooth surface but with slight fragmentation. The size of individual plate-like particles ranges from 5 to 10 μm, with no obvious small particle aggregation. SEM images of alcohol-extracted polyphenols ( Figure 9 B) It exhibits a composite structure of sheet-like substrate and small particles. The substrate still retains a sheet-like morphology similar to that of water extraction, but the sheet-like surface is covered with a large number of dispersed fine particles. The particles are irregularly ellipsoidal and have good dispersibility.

[0080] DES extraction of SEM images of polyphenols ( Figure 9CE (concentrated phenolic extract) exhibits a typical spherical particle aggregate structure. The product uses near-spherical particles with a diameter of 0.5-2 μm as the basic unit. The particles have smooth surfaces, uniform size, and further aggregate to form dense clusters. This morphology originates from the unique solvent effect of DES. DES forms a stable solvated complex with polyphenol molecules through a hydrogen bond network. During the extraction process, polyphenol molecules easily self-assemble into regular spherical particles after solvent evaporation. At the same time, the low volatility of DES promotes particle aggregation, ultimately forming a highly aggregated spherical cluster structure, which also reflects that the enrichment efficiency of DES for polyphenols is significantly higher than that of water extraction and alcohol extraction.

[0081] (3) High performance liquid chromatography-tandem mass spectrometry analysis test Preparation of the test sample: Take the purified polyphenol powder from the tung oil cake and prepare a sample solution with a concentration of 1.0 mg / mL using chromatographic grade methanol. After serial dilution, draw the sample solution with a disposable sterile syringe and filter it through a 0.22 μm organic microporous membrane. The resulting filtrate is the test sample solution, which is ready for use.

[0082] Chromatographic conditions: Qualitative determination was performed using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS / MS). An Agilent UPLC1290 liquid chromatography system was used, paired with a Poroshell 120 EC-C18 column (2.1 mm × 100 mm × 2.7 μm). The column oven temperature was controlled at 35 ℃, the autosampler temperature was set to 4 ℃, and the injection volume was 5 μL. The mobile phase was set to phase A (1% formic acid aqueous solution) and phase B (0.1% formic acid-acetonitrile solution), and the elution flow rate was maintained at 300 μL / min. The gradient elution program is set as follows: 5%B (0-5 min), 5-7%B (5-10 min), 7-10%B (10-20 min), 10-30%B (20-40 min), 30-95%B (40-45 min), 95%B (45-50 min), 95-5%B (50-51 min), 5%B (51-56 min).

[0083] Mass spectrometry conditions: A high-resolution Q-TOF-MS / MS mass spectrometer was used for detection in full-scan mode with electrospray ionization (ESI) negative ion generation. The ion source drying gas temperature was controlled at 350 °C, the drying gas flow rate at 10 L / min, the nebulizer gas pressure at 40 psi, the sheath gas temperature at 400 °C, and the sheath gas flow rate at 12 L / min. The capillary lysis voltage was set to -3.5 kV, the capillary outlet voltage to 135 V, the collision energy to 30 ± 5 eV, and the mass spectrometry scan range to 100-1000 u, thus completing the qualitative analysis of polyphenol components.

[0084] To identify the types of phenolic monomers in the polyphenols extracted from *Vernicia fordii* cake meal using five different solvents, UPLC-ESI-QTOF-MS was used to perform qualitative analysis on the polyphenol components of the purified *Vernicia fordii* cake meal. Figure 10 Total ion chromatograms (TICs) of five polyphenols from *Vernicia montana* seed cake were analyzed. By comparing the retention times of each chromatographic peak, the information of quasi-molecular ion peaks from primary mass spectrometry (PMS), and the information of fragment ions from secondary mass spectrometry (MSS) with a self-built database and relevant literature reports, a total of 21 phenolic compounds were identified. The TICs and analysis results show that the polyphenolic compounds in *Vernicia montana* seed cake can be divided into five major categories: phenolic acids, flavonoids, flavanols, flavonols, and anthocyanins. Flavonoids showed significantly higher detection numbers and chromatographic response intensities than other categories. Phenolic acids showed the second highest number of detected compounds and the next highest signal intensity. Based on the distribution of compound detection numbers, chromatographic peak response intensities, and structural categories, flavonoids were identified as the main components, with rutin, isoquercitrin, and hesperidin as core components. Phenolic acids were the minor components, mainly including chlorogenic acid, 2-hydroxycinnamic acid, neochlorogenic acid, protocatechuic acid, and caffeic acid.

[0085] Significant differences in polyphenol components were observed in the extracts of *Vernicia fordii* seed cake obtained using different solvents. Only five components were detected in the water extract (gallic acid, protocatechuic acid, chlorogenic acid, neochlorogenic acid, and rutin). Nine components were detected in the ethanol extract, with four new components added compared to the water extract: ferulic acid, isoquercetin, caffeic acid, and p-coumaric acid. The extracts obtained using eutectic solvents showed a significantly higher variety of components than those obtained using traditional solvents. DES-4 detected 13 components, with four new components added compared to the ethanol extract: 2-hydroxycinnamic acid, hesperidin, catechin, and epicatechin. DES-9 detected 12 components, with 2-hydroxycinnamic acid being less detected than in DES-4. DES-10 had the most components, with 17 detected, except for four free flavonoid aglycones: quercetin, kaempferol, isorhamnetin, and gallocatechin gallate. This difference is likely due to the combined effects of extraction solvent, extraction efficiency, and detection methods. The water extract showed strong selectivity for highly polar phenolic acids, but weak extraction ability for flavonoid glycosides and aglycones. The ethanol extract had some enrichment effect on moderately polar polyphenols, but the overall variety was limited. While DES-4 and DES-9, two eutectic solvents, were superior to traditional solvents in improving the extraction of phenolic acids and flavonoid glycosides, their dissolution of weakly polar flavonoid aglycones remained limited. DES-10, with its quaternary ammonium salt structure and synergistic hydrogen bonding, could simultaneously disrupt the cell walls of plant matrix and form stable bonds with polyphenols of different polarities, achieving comprehensive and efficient extraction of phenolic acids, flavanols, flavonoid glycosides, flavonoid aglycones, and anthocyanins. It is more suitable for the comprehensive extraction and subsequent activity evaluation of polyphenols from *Vernicia fordii* seed cake. Preliminary assignment was completed based on the retention time (RT) of the total ion chromatogram and the quasi-molecular ion peak information from primary mass spectrometry. In negative ion scanning mode, [MH] was extracted from each chromatographic peak.- The quasi-molecular ion peaks were precisely determined by high-resolution mass spectrometry (M / Z), and the compounds were preliminarily identified by comparing their relative molecular mass, molecular formula, and data from databases (Metlin, HMDB, MassBank). Simultaneously, the chromatographic retention patterns of different polyphenol classes were used to preliminarily classify the compounds. Structural confirmation and precise identification were achieved through characteristic fragment ions obtained by secondary mass spectrometry (MS²). For each quasi-molecular ion peak, the MS² fragment spectra generated by collision-induced dissociation (CID) were acquired, and the structure was analyzed by combining these fragmentation patterns with those of polyphenolic compounds.

[0086] For phenolic acids, chlorogenic acid and neochlorogenic acid are isomers, and their quasi-molecular ion peaks are both [MH]. - (m / z 353.0873) The parent ion undergoes ester bond cleavage, losing quinic acid residues (C7H). 12 O6, 192 Da), producing characteristic fragment ions (m / z 191.0561), and further fragmentation to produce caffeic acid anions (m / z 179.0350). Ultimately, the difference in retention time is used to distinguish chlorogenic acid from neochlorogenic acid. Quasi-molecular ion peak [MH] - (m / z 163.0401), in MS², the parent ion loses a CO2 molecule (44 Da), producing a characteristic fragment ion (m / z 119.0503), accompanied by further fragmentation after decarboxylation (m / z 91.0552), consistent with the typical fragmentation pathway of cinnamic acid compounds, thus identifying it as 2-hydroxycinnamic acid. Quasi-molecular ion peak [MH] - (m / z 169.0140), in MS², the parent ion loses a CO2 molecule (44 Da) to produce a characteristic fragment ion (m / z 125.0244), a hallmark cleavage product of gallic acid. Quasi-molecular ion peak [MH] - (m / z 153.0193), in MS², the parent ion loses a CO2 molecule (44 Da), producing a characteristic fragment ion (m / z 109.0295), corresponding to the characteristic cleavage of dihydroxybenzoic acid, thus identified as protocatechuic acid. Quasi-molecular ion peak [MH] - (m / z 193.0506), in MS², the parent ion loses a CO2 molecule (44 Da), producing a characteristic fragment ion (m / z 149.0373), accompanied by demethylation (15 Da) producing a fragment (m / z 178.0273), consistent with the fragmentation pattern of ferulic acid. Quasi-molecular ion peak [MH] -(m / z 179.0350), in MS², the parent ion loses a CO2 molecule (44 Da) to produce a characteristic fragment ion (m / z 135.0451), a hallmark cleavage product of caffeic acid. Quasi-molecular ion peak [MH] - (m / z 163.0401), the parent ion in MS² loses a CO2 molecule (44 Da) to produce a characteristic fragment ion (m / z 119.0502), which is identified as p-coumaric acid by distinguishing it from 2-hydroxycinnamic acid by retention time.

[0087] For flavonoids, the quasi-molecular ion peak [MH] - (m / z 609.1400), in MS², the parent ion successively loses rutinyl groups (C). 12 H 22 O 10 The peak value is 308 Da, which produces a characteristic fragment ion of quercetin aglycone (m / z 300.0276). Further fragmentation produces fragments (m / z 271.0249), representing the classic fragmentation pathway of rutin. A quasi-molecular ion peak [MH] is observed. - (m / z 463.0776), the precursor ion in MS² loses a glucose moiety (C6H). 10 O5, 162 Da), producing quercetin aglycone (m / z 300.0277), which further cleaved to produce fragments (m / z 271.0255, 179.0348). These fragments, distinguished from rutin by retention time, were identified as isoquercetin. Quasi-molecular ion peak [MH] - (m / z 609.1905), in MS², the parent ion loses a rutinyl group (308 Da), producing hesperidin aglycone (m / z 301.0455), which further cleaves to produce fragments (m / z 271.0255, 125.024), thus confirming the structure of hesperidin. Quasi-molecular ion peak [MH] - (m / z 463.0834), the precursor ion in MS² loses a glucose unit (162 Da), producing quercetin aglycone (m / z 300.0276), which further cleaves to produce fragments (m / z 271.0249, 151.0037). These fragments, distinguished from isoquercetin by retention time, were identified as quercetin-3-O-glucoside. Quasi-molecular ion peak [MH] - (m / z 593.1469), the precursor ion in MS² loses a rutinyl group (308 Da), producing kaempferol aglycone (m / z 285.0405), which further cleaves to produce fragments (m / z 255.0299, 227.0349), suggesting the presence of kaempferol aglycone. According to Abdulla et al.

[120] Based on the analytical method established by the researchers, combined with the characteristic that the peak intensity at m / z 285 is higher than that at m / z 284 and the difference in mass number, it is preliminarily inferred that it is kaempferol-3-O-rutin.

[0088] For flavanols, the quasi-molecular ion peak is [MH]. - (m / z 289.0718) In MS², the parent ion undergoes characteristic fragmentation of the flavanol nucleus, losing CO₂ (44 Da) to produce (m / z 245.0820), and further losing C₂H₂O (42 Da) to produce (m / z 203.0714). Based on the typical fragmentation pathway of flavanols and comparison results reported in the literature, these were identified as catechin and epicatechin, respectively. Epicatechin was confirmed by direct comparison with the retention time and mass spectrometry data of the standard. Quasi-molecular ion peak [MH] - (m / z 457.0776) In MS², the parent ion loses gallic acid residues (C7H6O5, 170 Da), producing characteristic fragment ions of gallocatechin (m / z 305.0350), which further cleave to produce fragments (m / z 179.0348, 125.0247), consistent with the typical cleavage pattern of gallocatechin gallate (EGCG), thus confirming the structure as EGCG.

[0089] For flavonols, the quasi-molecular ion peak [MH] - (m / z 315.0506), in MS², the parent ion loses a methyl group (15 Da), producing quercetin aglycone (m / z 300.0277), which further fragments to produce fragments (m / z 271.0255, 179.0348), thus confirming the structure of isorhamnetin. Quasi-molecular ion peak [MH] - (m / z 301.0455), the parent ion in MS² underwent ring A and ring B fragmentation, producing characteristic fragment ions (m / z 271.0255, 179.0348, 151.0401), consistent with the fragmentation pattern of flavonol aglycones, and was identified as myrtol. Quasi-molecular ion peak [MH] - (m / z 285.0471) In MS², the parent ion underwent characteristic fragmentation, producing fragments (m / z 257.0456, 229.0507, 117.0346). These fragments were distinguished from kaempferol-3-O-rutin by retention time, and the structure was identified as kaempferol. Quasi-molecular ion peak [MH] - (m / z 301.0354) In MS², the parent ion undergoes ring A and ring B cleavage, producing characteristic fragment ions (m / z 273.0405, 178.9987, 151.0037), which are distinguished from quercetin-3-O-glucoside by retention time, and the structure is identified as quercetin.

[0090] For anthocyanin compounds, the quasi-molecular ion peak [MH] - (m / z 461.0978), the parent ion in MS² loses a glucose moiety (162 Da), producing a characteristic fragment ion of malvidin aglycone (m / z 301.0455), which further cleaves to produce fragments (m / z 271.0255, 179.0348), thus completing the structural identification of malvidin-3-glucoside.

[0091] Table 7 Identification of polyphenolic components in five types of tung oilseed cakes 7. Effects of pH and heat treatment on the stability of polyphenols in *Vernicia fordii* seed cake. (1) Standardized preparation of polyphenol stock solution: To eliminate differences in initial polyphenol concentration and ensure consistency in treatment starting point, a grouped direct standardization method was used to prepare the working solution. A 3 (extraction method) × 2 (pH level) × 2 (heat treatment) full factorial experimental design was adopted. Twelve portions of lyophilized polyphenols extracted by water, alcohol, and eutectic solvent (DES-10) were accurately weighed and placed in 50 mL centrifuge tubes. The solutions were dissolved in 0.5% (w / v) citric acid aqueous solution and brought to a final volume of 50 mL. The solutions were vortexed to obtain the standardized working solution (1.0 mg GAE / mL). All samples were stored at 4 ℃ protected from light and used within 24 h.

[0092] (2) pH adjustment and heat treatment: Accurately transfer 15 mL from each of the above working solutions into a new tube, and then adjust the pH. For each type of extract, adjust two of the four sample solutions to pH 4.0 with 0.1 M HCl / NaOH, and adjust the other two to pH 7.0. Then, bring each tube to a uniform initial volume with the corresponding pH citrate buffer.

[0093] Three samples (water, alcohol, and DES-10) at each pH level were placed in a 65 °C water bath and heated for 30 min. Three other samples at the same pH were incubated at room temperature (25 °C) in the dark for 30 min as a control. After heat treatment, the samples were immediately transferred to an ice-water bath for rapid cooling to room temperature, and the volume was replenished again with the corresponding pH buffer to ensure a consistent final treatment volume across all groups.

[0094] (3) Sample processing and preservation: After processing, the 12 groups of samples were immediately subjected to various index determinations, or freeze-dried into powder and stored at -4 ℃ in the dark, and the analysis was completed within one week.

[0095] (4) Ultraviolet-visible spectroscopy analysis: The polyphenol sample of the treated tung oil cake was diluted with methanol to 0.1 mg / mL and scanned in the wavelength range of 350-800 nm using an ultraviolet-visible spectrophotometer with a scanning interval of 1 nm. Methanol was used as a blank control.

[0096] Figure 11 The figures show the UV absorption spectra of polyphenols from three types of *Vernicia fordii* seed cakes after pH and heat treatment. As can be seen from the figures, the UV-Vis absorption spectra of the three polyphenol samples after pH and heat treatment all exhibit typical polyphenol characteristic absorption peaks, mainly concentrated in the 200-400 nm range. The strong absorption peak near 280 nm is attributed to the π-π electronic transitions of the benzene ring structure in polyphenols, while the shoulder peaks at 300-360 nm are related to the n-π transitions of phenolic hydroxyl groups and conjugated double bonds. Significant differences in the spectral characteristics of polyphenols exist under different pH conditions. P <0.05). Under acidic conditions (pH=4.0), the absorbance of each system was generally lower than that under neutral conditions (pH=7.0), and the intensity of the characteristic peak at 300-360 nm was weakened. This may be because acidic conditions inhibited the dissociation of phenolic hydroxyl groups, reduced the expansion of deprotonated polyphenols and conjugated systems, and thus reduced the UV absorption intensity.

[0097] Heat treatment further intensified the spectral changes. Under neutral pH, the absorbance of all extraction systems significantly increased at 280 nm and 300-360 nm after heating, with sharper peaks. This indicates that heat treatment promoted the structural expansion or partial polymerization of polyphenols, enhancing the conjugation effect. Under acidic pH, the effect of heat treatment on the spectrum was relatively mild, with a smaller increase in absorbance, suggesting that the acidic environment mitigated heat-induced structural changes in polyphenols to some extent. Comparing the three extraction systems, the polyphenols extracted with DES-10 showed the most significant increase in absorbance under heating and neutral pH conditions, followed by ethanol extraction, with water extraction showing the weakest increase. This reflects differences in the structural stability of polyphenols obtained from different extraction solvents, suggesting that DES-10 may retain more highly active polyphenolic components that are easily affected by heat and pH.

[0098] (5) Fourier transform infrared spectroscopy analysis: Accurately weigh 1 mg of freeze-dried polyphenol powder from *Vernicia fordii* seed cake and 100 mg of potassium bromide. Mix them thoroughly and dry them in an oven until constant weight. Then grind them into a fine powder in a mortar and press them into thin sheets. Spectroscopy was performed using a Fourier transform infrared spectroscopy system at 4000-400 cm⁻¹. -1 The infrared spectrum of polyphenols from *Vernicia fordii* seed cake was obtained by scanning within the specified wavelength range. First, a background scan was performed on pure potassium bromide tablets, and then a scan was performed on polyphenol sample tablets.

[0099] Figure 12Infrared spectra of polyphenols from three types of *Vernicia fordii* seed cakes after pH and heat treatment. 3400-3500 cm⁻¹ -1 The strong and broad absorption peak at 2830 cm⁻¹ is attributed to the stretching vibration of the phenolic hydroxyl group (-OH) in the polyphenol molecule. -1 The nearby absorption peak corresponds to the saturated CH stretching vibration, 1600-1650 cm⁻¹ -1 The characteristic peak at 1000 cm⁻¹ is due to the vibration of the benzene ring skeleton. -1 The nearby absorption peak belongs to the CO stretching vibration, 800 cm⁻¹ -1 The nearby peaks correspond to the out-of-plane bending vibration of the aromatic ring CH, and these features collectively confirm the typical structure of polyphenols from *Vernicia fordii* seed cake. Comparing different pH and heat treatment groups, it was found that at pH = 4.0 and after heat treatment, the 3400 cm⁻¹ peak... -1 The hydroxyl peak intensity decreased slightly and the peak shape narrowed, indicating that acidic conditions and heat treatment partially disrupted the hydrogen bonding between polyphenol molecules. However, the characteristic peak positions of the benzene ring skeleton and CO bonds remained relatively stable, suggesting that the core aromatic structure of polyphenols possesses a certain degree of stability. The infrared characteristic peak positions and intensities of polyphenols showed slight differences under different extraction solvents. Polyphenols extracted with DES-10 showed the following peak intensity at 3400 cm⁻¹. -1 The hydroxyl peak is broader and more intense, indicating that the DES system is more conducive to preserving the polymeric hydrogen bond structure of polyphenols. The benzene ring skeleton vibration peak (1615-1619 cm⁻¹) in the ethanol extraction group... -1 The intensity was slightly stronger, while the CO vibration peak of the water extraction group (1069-1089 cm⁻¹) was slightly stronger. -1 The effect of solvent polarity on the exposure of polyphenol functional groups is more significant, reflecting the influence of solvent polarity on the degree of polyphenol functional group exposure. Overall, after pH adjustment and heat treatment, no new characteristic peaks or disappearance of characteristic peaks were observed in polyphenols extracted by the three solvents. The changes were only in peak intensity and peak width, indicating that pH and heat treatment mainly affect the infrared characteristics by influencing intermolecular hydrogen bonds and changing the aggregation state of polyphenols, without destroying the basic skeletal structure of polyphenols.

[0100] (6) Color: Take an appropriate amount of purified *Vernicia fordii* seed cake polyphenol sample, place it under natural light, observe its appearance and take pictures. At the same time, use a colorimeter to measure the color of the polyphenol sample. Lay the sample flat on a standard white plate to eliminate background color interference, and measure the four parameters L*, a*, b*, and ΔE of the sample respectively.

[0101] The polyphenols from three different types of *Vernicia fordii* seed cake showed significant differences in color under different pH and heat treatment conditions. P <0.05%, visually manifested as a gradual change in solution color from light yellow to brownish-red ( Figure 13 ). Figure 14The graph shows the changes in L*, a*, b* values ​​and total color difference ΔE of the three types of polyphenols from *Vernicia fordii* seed cake after pH and heat treatment (L* is the brightness value, ranging from 0 (black) to 100 (white). a* is the red-green hue, with positive values ​​for red and negative values ​​for green. b* is the yellow-blue hue, with positive values ​​for yellow and negative values ​​for blue). The L* values ​​of the three types of polyphenols from *Vernicia fordii* seed cake were significantly higher in the acidic environment (pH = 4.0) than in the neutral environment (pH = 7.0). P The L* value was <0.05), and the L* value further decreased after heat treatment. The water extract showed the lowest L* value under pH = 7.0 heating conditions, indicating that neutral pH and heat treatment jointly promoted the oxidative browning of polyphenols, leading to a decrease in solution brightness and a deepening of color. The a* and b* values ​​showed consistent trends with the treatment conditions. Neutral pH and heat treatment significantly increased both a* and b* values, with the most significant increase observed in the water extract, while the increase in the DES-10 extract was relatively gradual. This suggests that water-extracted polyphenols are more sensitive to pH and heat stress, easily undergoing oxidative polymerization to generate colored substances. The DES-10 extraction system provides better protection for polyphenols, effectively inhibiting excessive increases in redness and yellowness. The total color difference ΔE results further confirmed that pH = 7.0 heating treatment had the most severe impact on the color of each extraction system. The ΔE value of the water extract was significantly higher than that of the ethanol and DES-10 extracts, reflecting the differences in color stability of polyphenols obtained from different extraction solvents. DES-10 extract can maintain a relatively stable color under extreme treatment conditions, showing stronger resistance to browning, which is closely related to its preserved polyphenol structure and antioxidant properties.

[0102] (7) Solubility: Using distilled water as a blank control, the transmittance of a 0.5 mg / mL *Vernicia fordii* seed cake polyphenol solution was scanned in the wavelength range of 350-800 nm using a UV-Vis spectrophotometer to determine the optimal wavelength for characterizing solubility. Subsequently, at this optimal wavelength, the transmittance (Tλ, %) of polyphenol aqueous solutions of different concentrations was measured. The solubility of *Vernicia fordii* seed cake polyphenols was quantitatively characterized by the transmittance values.

[0103] Figure 15The transmittance of polyphenols from three types of *Vernicia fordii* seed cakes after pH and heat treatment is shown. The transmittance of the three polyphenols from *Vernicia fordii* seed cakes varied significantly with wavelength, pH, and heat treatment conditions (P<0.05). Overall, the transmittance in the visible light region (400-800 nm) increased with increasing wavelength, reflecting the influence of different treatments on the solubility and dispersion stability of polyphenols. Under unheated conditions, the transmittance of each extraction system was ranked as DES-10 > ethanol > water. Specifically, the DES-10 extract showed a transmittance of 98.88 ± 0.36% at 460 nm at pH = 4.0 and 97.09 ± 0.58% at 580 nm at pH = 7.0, both significantly higher than the ethanol and water extracts (P<0.05). This indicates that DES-10, as a eutectic solvent, can effectively improve the solubility and dispersion uniformity of polyphenols in aqueous solution, reduce molecular aggregation and sedimentation, and thus maintain higher transmittance.

[0104] The interaction between pH and heat treatment further affected the light transmittance of polyphenols. The overall transmittance of all systems was higher under acidic conditions than under neutral conditions. This is because acidic conditions inhibited the dissociation of polyphenols and intermolecular hydrogen bonding, reducing polyphenol aggregation and precipitation. After heat treatment, the transmittance of all extraction systems decreased to varying degrees, with the water extract showing the most significant decrease and the DES-10 extract showing the smallest decrease. This indicates that heat treatment promotes the structural expansion and aggregation of polyphenol molecules. The DES-10 extraction system stabilizes the polyphenol structure through intermolecular interactions, mitigating the heat-induced decrease in solubility. Even under heating conditions at pH = 7.0, it maintained a transmittance of 93.46 ± 0.23% (610 nm), exhibiting superior solubility and stability.

[0105] (8) Stability: Effects of pH and heat treatment on the stability of polyphenol extract from *Vernicia fordii* cake meal. A 1 mg / mL concentration of polyphenol extract from *Vernicia fordii* cake meal was taken, and the pH of distilled water was adjusted to 4.0 and 7.0 with hydrochloric acid and sodium hydroxide, respectively. The extract was then treated at room temperature and heated (65 ℃, 30 min), respectively, and stored at room temperature in the dark. The polyphenol retention rate was calculated at 0, 3, 7, and 14 days.

[0106] (6) In the formula: A1—the concentration of polyphenols after treatment; A0—the initial concentration of polyphenols.

[0107] Figure 16The retention rates of polyphenols from three types of *Vernicia fordii* seed cakes after pH and heat treatment were shown. The storage stability of the three polyphenols from *Vernicia fordii* seed cakes differed significantly under different pH and heat treatment conditions (P<0.05). Overall, the polyphenol retention rate gradually decreased with prolonged storage time (0-14 d), and the rate of decrease was influenced by the extraction solvent, pH, and heat treatment. Under unheated conditions, the polyphenol retention rates of each extraction system were ranked as follows: DES-10 > ethanol > water. The polyphenols extracted from *Vernicia fordii* seed cakes using DES-10 still maintained a retention rate of 86.62 ± 1.88% after 14 days of storage at pH = 4.0, which slightly decreased at pH = 7.0 (80.64 ± 0.97%), significantly higher than those extracted with ethanol and water (P<0.05). This indicates that DES-10, as a eutectic solvent, can stabilize the polyphenol structure through hydrogen bonding and hydrophobic interactions, effectively inhibiting its oxidative degradation and exhibiting the best basic storage stability.

[0108] pH and heat treatment further exacerbated the degradation of polyphenols. Under neutral conditions, the reduction in polyphenol retention was significantly greater than under acidic conditions. This is because neutral conditions promote the dissociation of phenolic hydroxyl groups, accelerating the auto-oxidation and polymerization of polyphenols. After heat treatment, the water extract showed the most drastic reduction after 14 days of storage at pH 7.0 with a retention rate of only 27.19 ± 2.98%, while the DES-10 extract maintained a retention rate of 65.25 ± 1.13%. This indicates that heat treatment pre-destroys the structural stability of polyphenols. However, the DES-10 extraction system can mitigate heat-induced structural damage through molecular inclusion and environmental buffering, effectively preserving polyphenol activity even under extreme treatment and long-term storage, demonstrating stronger stress resistance.

[0109] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent, characterized in that, Includes the following steps: (1) Raw material degreasing pretreatment: Take the tung seed cake, crush and sieve it, and degrease it to obtain degreased cake powder; (2) Preparation of eutectic solvent: Use any one of betaine, proline and choline chloride as hydrogen bond acceptor and any one of lactic acid and levulinic acid as hydrogen bond donor, mix and melt to prepare eutectic solvent; (3) Extraction: The defatted cake powder is mixed with the eutectic solvent for extraction, and then centrifuged to obtain a crude polyphenol extract; (4) Purification: The crude polyphenol extract was loaded onto the pretreated AB-8 macroporous resin for adsorption. After washing with water to remove impurities, it was eluted with ethanol. After desolvation of the eluent, it was freeze-dried to obtain purified polyphenols from the tung oil cake.

2. The method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent according to claim 1, characterized in that, The eutectic solvent comprises any one of the following three systems: System 1: Proline to levulinic acid molar ratio 1:2; System 2: Betaine to lactic acid molar ratio 1:2.5; System 3: The molar ratio of choline chloride to levulinic acid is 1:1.

5.

3. The method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent according to claim 1, characterized in that, The hydrogen bond acceptor and hydrogen bond donor are mixed and stirred at 80°C for 1-2 h to form the eutectic solvent.

4. The method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent according to claim 1, characterized in that, The temperature extracted in step (3) is 60℃.

5. The method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent according to claim 1, characterized in that, The pretreatment method for the AB-8 macroporous resin includes the following steps: The AB-8 macroporous resin was soaked in 95% ethanol, and after removing the alcohol, it was successively soaked in 4 wt% hydrochloric acid and 4 wt% sodium hydroxide with shaking for 8 hours each, and finally washed with water until neutral.

6. The method for extracting polyphenols from *Vernicia fordii* seed cake using a eutectic solvent according to claim 1, characterized in that, The defatted cake powder and diluted eutectic solvent were mixed at a ratio of 1:10 g / mL, extracted at 60℃ for 1.5 h, and centrifuged at 8000 r / min for 10 min.

7. The polyphenols extracted from the eutectic solvent extraction method of *Vernicia fordii* seed cake as described in any one of claims 1-6.

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