Oriented Deep Eutectic Solvents as Efficient Approach for Selective Extraction of Bioactive Saponins from Husks of Xanthoceras sorbifolia Bunge

The husks of Xanthoceras sorbifolia Bunge (X. sorbifolia), as by-products of industrial production, have brought a severe burden to the environment and caused an enormous waste of resources. Bioactive triterpenoid saponins are rich in the husks. To reuse the husks and gain high-quality saponin products, saponin-oriented deep eutectic solvents (DESs), as an efficient and selective extraction strategy from X. sorbifolia husks, were designed for the first time. The enhancement of the extraction rate was investigated by screening solvents from acidic DESs and response surface methodology (RSM) optimization. As a result, the tetrapropylammonium bromide-lactic acid (TPMBr-La) was the most efficient DESs, with an extraction efficiency of up to 135% higher than 70% ethanol. A maximum extraction rate of 72.11 ± 0.61 mg Re/g dw was obtained under the optimized parameters. Scanning electron microscope graphs revealed that damage to the microstructure caused by DESs enhanced the extraction efficiency. Moreover, the recovery of total saponins with D101 macroporous resin was consistent with the pseudo-second-order kinetic model. Seven saponins were also identified by HPLC-MS analysis. Finally, TPMBr-La extracts exhibited 92.30 ± 1.10% DPPH radical scavenging rate at 100 μg/mL, and 92.20 ± 0.30% ABTS radical scavenging rate at 1200 μg/mL. Our current research proposes a selective and high-efficiency substitute for the extraction of saponins and might contribute to further DESs application in the recycling of by-products.


Introduction
Xanthoceras sorbifolia Bunge. (X. sorbifolia), belonging to the Sapindaceae family, is a native woody oil crop and has a planting history of more than 1000 years in China [1]. Due to its strong survival ability and adaptability [2], it is very suitable for planting in different climatic conditions and is also an ecological tree species and windproof against the sand. In recent decades, X. sorbifolia has attracted significant attention due to its promising potential for the production of high-quality biodiesel [3], pharmaceutical [4][5][6] and for ecological purposes [7] worldwide. As of 1998, the planting area of X. sorbifolia has been expanded to 800,000 hm 2 [8]. At present, the development of biodiesel made from the seeds of the X. sorbifolia faces two major problems: the high number of low value-added wastes, such as fruit husks, produced in the production process, and the high cost of production. The mass of X. sorbifolia husks accounts for about 50% of the overall fruit mass [9]. The annual Chcl-Ga Choline chloride Glycolic acid 1:2 4 Bet-Ga Betaine Glycolic acid 1:2 5 Bet-La Betaine Lactic acid 1:2 6 Eacl-La Ethylamine hydrochloride Lactic acid 1:2 7 Eacl-Ga Ethylamine hydrochloride Glycolic acid 1:2 8 TPMBr-La Tetrapropylammonium bromide Lactic acid 1:2 9 TPMBr-Ga Tetrapropylammonium bromide Glycolic acid 1:2 10 TPMBr-Mal Tetrapropylammonium bromide Malonic acid 1:2 11 Eg-La Ethylene glycol Lactic acid 1:2 12 Eg-Ma Ethylene glycol Malic acid 1:2 13 Eg-Mal Ethylene glycol Malonic acid 1:2 14 Eg-Ga Ethylene glycol Glycolic acid 1:2

Extraction of Total Saponins and Total Flavonoids
A total of 100 mg of X. sorbifolia powder and 2 mL of solvents with 30% water content (DESs, ethanol, and water) were added to a 10-mL centrifugal tube. The mixtures were shaken in a water-bath shaker at 60 • C for 30 min. Then, they were centrifuged at 12,000 rpm for 20 min to take the supernatant for determination.

Microstructure of Plant Material
The microstructure graphs of the X. sorbifolia powder treated by different solvents (water, ethanol, and TPMBr-La) were detected under thermal field emission scanning electron microscope JSM-7600F (Japan). The extracts were centrifuged to extract the precipitate and dried at low temperature in an oven. After being covered with gold, the microstructure was observed under a 3000-times magnification.

Optimal DESs Extraction Conditions for Total Saponins
Extraction variables and ranges were determined based on previous single-factor experiments (Supplement Materials Figure S1). Box-Behnken design experiment was conducted to optimize the extraction conditions. Extraction time (min), extraction temperature ( • C), liquid-solid ratio (mL/g), and water content (%) were selected as independent variables, and the extraction rate of total saponins (mg/g) was used as the response value. Data were analyzed by software Design Expert 10.0.7. The obtained experimental data were consistent with the following second-order polynomial model: where Y is the response value, X i and X j are the independent variables, β 0 is a constant, β i, β ii , β ij are coefficients for linear, quadratic, and interaction parameters. The quantitative determination of total saponins was performed by the method previously reported in the literature [27]. Mixed 0.5 mL saponin extract with 0.2 mL 5% vanillin and 0.8 mL perchloric acid solution in a 10-mL test tube. The tubes were kept at 60 • C for 15 min and cooled for 2 min; the sample absorbances were measured at 552 nm. According to the relationship between absorbance value and different concentration of ginsenoside Re, the standard curve equation was acquired as follows: y = 14.043x + 0.0833 (0.2 < x < 1 mg/mL), R 2 = 0.9997. The extraction rate was represented by mg ginsenoside Re equivalent (mg Re/g dw).

Determination of Total Flavonoids Content
The quantitative determination of total flavonoids was performed by the method previously reported in the literature [28]. A total of 1 mL extracted flavonoid samples were well-mixed with 0.3 mL of 5% NaNO 2 solution; then, the sample was left for 6 min. Then, 0.3 mL of 10% Al(NO) 3 solution was added, shook well, and placed in the sample for 6 min. Next, 4 mL of 4% NaOH solution was mixed, and the volume was fixed to 10 mL with 80% ethanol solution. Finally, samples were left at room temperature for 15 min and absorbances were measured at 510 nm. According to the relationship between absorbance value and different rutin concentrations, the standard curve equation was acquired as follows: y = 6.1043x + 0.0492 (0.04 < x < 1.5), R 2 = 0.9994, indicating that the linearity of rutin was good in the concentration range from 0.04 to 1.5 mg/mL. This method can be used for the quantitative analysis of flavonoids; the extraction rate was represented by mg rutin equivalent (mg rutin /g dw), and calculated as follows: Extraction rate (mg/g) = mass of the crude saponins or flavonoids mass of the shell powder (2)

Qualitative Analysis of Recovered Saponins Composition by HPLC-ESI-MS
Qualitative analysis of recovered saponins was performed on an HPLC chromatographic system (Agilent, Palo Alto, CA, USA) coupled with an LTQ-Qrbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The chromatographic with a ZORBAX SB-Aq C 18 column (250 × 4.6 mm, 5 µm, Agilent, Palo Alto, CA, USA). The mobile phase were methyl alcohol (C) and 0.01% formic acid water (D); the flow rate was 0.4 mL/min. Gradient elution was conducted as follows: 0-30 min, 50-70% C; 30-60 min, 70-80% C; 60-70 min, 80-50% C. Injection volume was 10 µL and injection temperature was 30 • C. MS parameters were as follows: capillary temperature, 250 • C; auxiliary gas heater temperature, 200 • C; spray voltage, 1.5 kV; scanning range, 300-2000 m/z. The recovery of saponins from DESs extract was carried out by adsorption and desorption with five kinds of macroporous resins, including DM301, NKA-9, D101, HPD100, and HP20. Saponins extracted by TPMBr-La were isolated and purified in accordance with the modified method [29]. A total of 2 g of the treated resin was weighed separately in a 50-mL conical bottle, and 10 mL of saponins solution extracted by TPMBr-La was added, before shaking at 25 • C with a speed of 80 rpm. The supernatant was sampled at regular intervals to detect the saponin content until the adsorption equilibrium was reached, before calculating the adsorption capacity (q e ) and adsorption rate (Q ar ). The saturated macroporous resin was washed, and 10 mL of 70% ethanol solution was used as desorption solution, and shaken a shaking table at 25 • C with a speed of 80 rpm to desorption equilibrium, before calculating the desorption rate (Q dr ).
where C 0 and C e are the concentration (mg/mL) of total saponins at the beginning and equilibrium of adsorption process, V a is the loading volume (mL) and W is the weight of macroporous resins (g). C d (mg/mL) represents the concentration of saponins in the desorption eluent and V d is the eluent volume (mL) in desorption experiments.

Study on Adsorption Kinetics of D101 Macroporous Resin
A total of 2 g of treated D101 macroporous resin was weighed into a 50 mL conical flask. Then, a total of 10 mL DESs extract was added, and shaken at 25 • C and 80 rpm for 7 h in a constant-temperature shaker. The supernatant was sampled at every 30 min to detect the saponin content until the adsorption equilibrium was reached. The adsorption rate and mechanism were studied by fitting the intra-particle diffusion, pseudo-first-order, and pseudo-second-order kinetic models. The linear equations of the three models are as follows: Intra-particle diffusion : q = k Pseudo-first-order : ln q e − q = −k 1 t + lnq e Pseudo-second-order : where q e and q are adsorption capacity (mg/g) at the equilibrium and t min; k i , k 1 , and k 2 represent the adsorption rate constants of intra particle diffusion, pseudo-first-order, and pseudosecond-order kinetic models; C is the kinetic constant of intra particle diffusion, mg/g.

2.
9. Determination of Antioxidant Activity 2.9.1. DPPH Radical Scavenging Assay DPPH radical scavenging assay was determined by this method with slight modification [30]. The sample was prepared into a series of concentrations with methanol. In brief, 100-µL sample solutions were mixed with the same volume of DPPH solution. The mixture was placed away from light for 30 min; then, the absorbances were read at 517 nm. The DPPH radical scavenging rate of samples was assessed in accordance with the following equation:

ABTS Radical Scavenging Assay
The ABTS radical scavenging capacity was determined by referring to the instructions of Total Antioxidant Capacity Assay Kit. ABTS could generate ABTS radical cations (ABTS + ) in the presence of an oxidant. A total of 200 µL ABTS working solution and 10-µL diluted samples were mixed in a clear 96-well microplate. The microplate was allowed to stand for 6 min at room temperature and the absorbance was measured at 734 nm. The ABTS radical scavenging rate of the sample was calculated in accordance with Equation (9).

Statistical Analysis
Except for HPLC-ESI-MS analyses, all experiments were performed in triplicate. Data were expressed as mean ± standard deviation. The statistically significant differences (p < 0.05) were conducted by analysis of variance (ANOVA).

Design and Screening of Saponin-Oriented DESs
With the advantages of acidic DESs in saponin extraction [24], we selected lactic acid, malonic acid, glycolic acid, and malic acid as HBDs. Choline chloride, betaine, ethylamine hydrochloride, tetrapropylammonium bromide, and ethylene glycol were chosen as HBAs. To facilitate analysis, fourteen designed DESs were classed into five groups based on HBAs. In contrast with traditional organic solvents, the high viscosity of the DESs affected the mass transfer rate, so 30% water was added to the DESs to ease the viscosity [31]. Meanwhile, considering the difference in the extraction efficiency of DESs for saponins and flavonoids, flavonoids, as an important component in X. sorbifolia husks, were also chosen to estimate the specific and efficient DESs for saponins. The extraction rates of the total saponins and total flavonoids with different DESs groups and conventional efficient solvents, i.e., 70% ethanol and water are exhibited in Figure 1. It is indicated that the component of DESs extensively impacted the extraction efficiency for the total saponins and total flavonoids, respectively. As shown in Figure 1a, the Chcl-based DESs group had the highest overall extraction efficiency for saponins, followed by the TPMBr-based, Eacl-based, and Bet-based groups. The Eg-based group showed the lowest extraction efficiency for saponins. The extraction efficiency of most of the acidic DESs for saponins was higher than when using 70% ethanol and water, and this finding is in accordance with the previous report [32]. Among the 14 kinds of DESs, TPMBr-La (67.01 ± 0.57 mg Re/g dw) showed the highest extraction rate for saponins, up to 135% higher than the use of 70% ethanol (28.50 ± 0.02 mg Re/g dw) and 296% higher than the use of water (16.90 ± 0.60 mg Re/g dw). Moreover, Chcl-Ga, Bet-La, Eg-La, and Eacl-La also displayed an efficient extraction rate (60.72 ± 2.53 mg Re/g dw, 54.24 ± 1.99 mg Re/g dw, 51.49 ± 2.53 mg Re/g dw, and 51.14 ± 2.90 mg Re/g dw).
Bromine ion can connect the hydroxyl H atom on saponins and the solvent, to reduce the spatial effect around saponins and form more hydrogen bonds [33]. The comprehensive score for TPMBr-La for the total saponins was optimal, which could be attributed to the growth in the number of hydrogen bonds and more stable average noncovalent interaction.
The overall extraction efficiency of the acidic DESs for total flavonoids was obviously lower than that for saponins. In Figure 1b, the Chcl-Ga, Bet-La, and Eg-La (41.99 ± 0.63 mg Rutin/g dw, 33.82 ± 1.22 mg Rutin/g dw, 36.36 ± 0.03 mg Rutin/g dw, respectively) showed a significant extraction efficiency rate for flavonoids. However, TPMBr-La (23.57 ± 3.40 mg Re/g dw) displayed a lower extraction efficiency for flavonoids, suggesting that TPMBr-La is selective for the extraction of saponins. Taken together, TPMBr-La was the best DESs to specificly extract bioactive saponins from X. sorbifolia and would be applied in the follow-up experiments.

Preliminary Extraction Mechanism for the Efficiency and Specificity of Saponins Extraction
To better understand why TPMBr-La was the most effective method for the extraction of saponins, the different extraction solvents, including water, 70% ethanol, and TPMBr-La, were investigated. The microstructure graphs of the X. sorbifolia powder before and after treatment using the above extraction solvents were recorded using thermal field emission scanning electron microscope. The X. sorbifolia powder without extraction displayed a flat and undamaged surface (Figure 2a), whereas the powders extracted by other solvents displayed different degrees of damage. The X. sorbifolia powder treated by water displayed many strip folds without crack (Figure 2b). The powder treated by ethanol and TPMBr-La showed visible pores and cracks, as shown in Figure 2c,d, respectively. Compared to the ethanol treatment, the TPMBr-La treatment showed more severe and pronounced damage. The damage was caused by the dissolution of cellulose and broken lignin after extraction with DES, which may be more conducive to the infiltration of DESs and the overflow of bioactive substances. This finding may account for the higher extraction efficiency of the DESs as compared to the organic solvent, which is consistent with the finding of polydatin extraction by DESs [34].
Antioxidants 2022, 11, x FOR PEER REVIEW Figure 1. The extraction rate of total saponins and total flavonoids from X. sorbifolia husk different solvents: (a) the extraction rate of total saponins; (b) the extraction rate of total flav There is a significant difference when * p < 0.05, ** p < 0.01 and *** p < 0.001 versus 70% etha

Preliminary Extraction Mechanism for the Efficiency and Specificity of Saponins Extr
To better understand why TPMBr−La was the most effective method for the tion of saponins, the different extraction solvents, including water, 70% ethan TPMBr−La, were investigated. The microstructure graphs of the X. sorbifolia pow fore and after treatment using the above extraction solvents were recorded using t field emission scanning electron microscope. The X. sorbifolia powder without ext displayed a flat and undamaged surface (Figure 2a), whereas the powders extrac other solvents displayed different degrees of damage. The X. sorbifolia powder trea water displayed many strip folds without crack (Figure 2b). The powder treated b nol and TPMBr−La showed visible pores and cracks, as shown in Figure 2c,d, respe Compared to the ethanol treatment, the TPMBr−La treatment showed more seve pronounced damage. The damage was caused by the dissolution of cellulose and lignin after extraction with DES, which may be more conducive to the infiltration o and the overflow of bioactive substances. This finding may account for the higher tion efficiency of the DESs as compared to the organic solvent, which is consiste the finding of polydatin extraction by DESs [34]. The extraction rate of total saponins and total flavonoids from X. sorbifolia husks using different solvents: (a) the extraction rate of total saponins; (b) the extraction rate of total flavonoids. There is a significant difference when * p < 0.05, ** p < 0.01 and *** p < 0.001 versus 70% ethanol.

Box-Behnken Design (BBD) Optimization
To assess the influence of four variables (extraction time, extraction temperature, liquid-solid ratio, and water content in the DESs) on the extraction efficiency of TPMBr-La from X. sorbifolia husks, a Box-Behnken design was conducted, and the levels of the factors are presented in Table 2. The experimental orders, levels of variables, and response values are shown in the Supplement Materials, Table S1. The quadratic regression equation for the extraction rate of total saponins (Y) and variables (A, B, C, and D) is as follows: The analysis of variance (ANOVA) for the regression model is displayed in Table 3. The p-value of the model was 0.0003 (p < 0.001), demonstrating that the model level was extremely significant. The coefficient of determination (R 2 ) was 0.9616, and the adjusted Rsquare (Adj R 2 ) was 0.8849, indicating that the experimental and predicted values correlate well. In addition, the p-value of the lack-of-fit value was not significant (0.9541), which showed that unknown factors had little interference with the test results. It can also be seen from the analysis of variance that the primary term, secondary term, and the interaction term of each factor had different effects on the extraction process. The impact degree of the independent variables was in the order of B > A > C > D. It was clear that the extraction temperature exhibited an extremely significant effect on the extraction rate (p < 0.0001). The p-values of the quadratic term coefficients A 2, B 2 , C 2 , and D 2 were less than 0.05, indicating that these factors had a significant impact on the extraction rate. However, the cross-product coefficients (AB, AC, BC, BD, and CD) were not significant (p > 0.05).

Box-Behnken Design (BBD) Optimization
To assess the influence of four variables (extraction time, extraction temperature, liquid-solid ratio, and water content in the DESs) on the extraction efficiency of TPMBr-La from X. sorbifolia husks, a Box-Behnken design was conducted, and the levels of the factors are presented in Table 2. The experimental orders, levels of variables, and response values are shown in the Supplement Materials, Table S1. The quadratic regression equation for the extraction rate of total saponins (Y) and variables (A, B, C, and D) is as follows: The analysis of variance (ANOVA) for the regression model is displayed in Table 3.  The 3D response plots illustrated the interaction effects between the variables (Figure 3). The curve in the extraction temperature was the steepest, indicating the greatest effect on the extraction rate, and the curve of the water content was the flattest, indicating that the influence was the lowest. This conclusion is also consistent with the results of the analysis of variance. Figure 3a is used as an example of the analysis of the mutual effect of the extraction time and temperature on the extraction rate of total saponins. The increase in temperature could improve the extraction rate of total saponins, but a further increase in temperature led to a decrease in the extraction rate. This may be due to the increase in temperature, which decreased the viscosity of the deep eutectic solvents and improved the mass transfer efficiency. When the temperature is too high, the extracted saponins may be degraded, resulting in a reduction in the extraction efficiency. The highest point can be seen in the graph, suggesting that the extraction rate had the highest value. The optimum TPMBr-La conditions for the extraction of total saponins from X. sorbifolia were as follows: extraction time of 28 min, extraction temperature of 78 • C, liquid-solid ratios of 26 mL/g, and water content of 35%. The actual extraction rate under optimal conditions was 72.11 ± 0.61 mg Re/g dw, closely matching the predicted value of 73.06 mg Re/g dw. It is suggested that this model can simulate and predict the actual extraction of bioactive saponins from X. sorbifolia.

Screening of Macroporous Resins
For the efficient recovery of total saponins from TPMBr-La extract solution, we investigated the adsorption and desorption rates of NKA-9, DM301, D101, HPD100, and HP20 macroporous resins. As described in Figure 4, D101, DM301, and NKA-9 macroporous resin displayed a superior adsorption rate (91.41%, 89.62%, 89.34%, respectively), indicating that the total saponin in the TPMBr-La extract can be adsorbed well. In the process of desorption, the D101 macroporous resin showed a better desorption rate (77.74%) for total saponins. The reason that the desorption rate is not particularly high may be attributed to the water-soluble and nonvolatile properties of DESs. Therefore, D101 resin was used to recover total saponins from the TPMBr-La extract solution.

Kinetic Analysis of the Adsorption Process
To better understand the mechanisms of saponin adsorption on D101 macroporous resin, a kinetic analysis was conducted. The static adsorption kinetics of D101 macroporous resin on total saponins at 25 • C is shown in Figure 5a. For the first 60 min, the adsorption capacity q t significantly increased as the time continued to increase. After 60 min, the adsorption rate of the resin decreased, and the adsorption capacity increased slowly as the adsorption time increased. The equilibrium adsorption qt was the highest at 10.89 mg/g when the duration was 360 min.

Screening of Macroporous Resins
For the efficient recovery of total saponins from TPMBr−La extract solution, we investigated the adsorption and desorption rates of NKA−9, DM301, D101, HPD100, and HP20 macroporous resins. As described in Figure 4, D101, DM301, and NKA−9 macroporous resin displayed a superior adsorption rate (91.41%, 89.62%, 89.34%, respectively), indicating that the total saponin in the TPMBr-La extract can be adsorbed well. In The adsorption process was analyzed by fitting the intra-particle diffusion, pseudofirst-order, and pseudo-second-order kinetic models. The three kinetic models fitted at 25 • C for total saponins on D101 resin are shown in Figure 5b-d. The kinetic equations of total saponin adsorption by the D101 resin and their related parameters are displayed in Table 4. C > 0 in the kinetic equation for intra-particle diffusion, suggesting that the adsorption rate of saponins partly depends on intra-particle diffusion and partly on the boundary layer diffusion [35]. The calculated values of the maximum adsorption capacity (q e calc) of the pseudo-first-order and pseudo-second-order models were 6.7201 and 11.5340 mg/g, respectively. The q e calc of the pseudo-second-order kinetic model was closer to the experimental q e value (10.90 mg/g), and the R 2 (0.9991) was the highest; it seemed that the adsorption process of the D101 macroporous resin was fitted to the pseudo-second-order kinetic model.

Kinetic Analysis of the Adsorption Process
To better understand the mechanisms of saponin adsorption on D101 macroporous resin, a kinetic analysis was conducted. The static adsorption kinetics of D101 macroporous resin on total saponins at 25 °C is shown in Figure 5a. For the first 60 min, the adsorption capacity qt significantly increased as the time continued to increase. After 60 min, the adsorption rate of the resin decreased, and the adsorption capacity increased slowly as the adsorption time increased. The equilibrium adsorption qt was the highest at 10.89 mg/g when the duration was 360 min.

Kinetic Analysis of the Adsorption Process
To better understand the mechanisms of saponin adsorption on D101 macro resin, a kinetic analysis was conducted. The static adsorption kinetics o macroporous resin on total saponins at 25 °C is shown in Figure 5a. For the first the adsorption capacity qt significantly increased as the time continued to increas 60 min, the adsorption rate of the resin decreased, and the adsorption capacity in slowly as the adsorption time increased. The equilibrium adsorption qt was the hi 10.89 mg/g when the duration was 360 min.

Identification of Barrigenol-Like Saponins in the TPMBr-La Extracts
Triterpenoids saponins are the major components in X. sorbifolia husks, and their backbones include the oleanane-type, lupine-type, glyseian-type, cycloatene-type, and lanolin-type [15]. The barrigenol-like triterpenoid saponins are multiple hydroxylated oleanane-type [36]. It is reported that barrigenol-like triterpenoid saponins are not only abundant in the husks of X. sorbifolia, but also the main responsible active components.
To find the presence of barrigenol-like triterpenoid saponins that were recovered from the TPMBr-La extracts, we performed an HPLC-ESI-MS analysis in the positive ion mode to identify the chemical compositions. The total ion chromatogram (TIC) of the saponins is shown in Figure 6. Seven types of triterpenoid saponins were identified, and their chemical structures are presented in Figure 7.
pacity (qe calc) of the pseudo−first−order and pseudo−second−order models were 6.7201 and 11.5340 mg/g, respectively. The qe calc of the pseudo−second−order kinetic model was closer to the experimental qe value (10.90 mg/g), and the R 2 (0.9991) was the highest; it seemed that the adsorption process of the D101 macroporous resin was fitted to the pseudo−second−order kinetic model.

Identification of Barrigenol-Like Saponins in the TPMBr−La Extracts
Triterpenoids saponins are the major components in X. sorbifolia husks, and their backbones include the oleanane-type, lupine-type, glyseian-type, cycloatene-type, and lanolin-type [15]. The barrigenol-like triterpenoid saponins are multiple hydroxylated oleanane-type [36]. It is reported that barrigenol-like triterpenoid saponins are not only abundant in the husks of X. sorbifolia, but also the main responsible active components.
To find the presence of barrigenol-like triterpenoid saponins that were recovered from the TPMBr−La extracts, we performed an HPLC-ESI-MS analysis in the positive ion mode to identify the chemical compositions. The total ion chromatogram (TIC) of the saponins is shown in Figure 6. Seven types of triterpenoid saponins were identified, and their chemical structures are presented in Figure 7.

Evaluation of the Antioxidant Activity of the TPMBr−La Extracts
The saponins contained in the recovered extract of the ethanol from the husks of X. sorbifolia have been reported to have antioxidant activity [41]. Nevertheless, the impact of As an example, xanthoceracide (6) was identified based on ionic fragments. The precursor ion m/z 1141.57 [M + H] + and 1163.57 [M + Na] + in the ESI(+) mode were given, and the molecular formula could be inferred to as C 57 H 88 O 23. The cleavage behavior of the triterpenoid saponins in the positive mode of the ESI source was highly similar. Basically, glycosides were gradually lost at first to obtain the aglycone excimer ion peaks, such as m/z 693.84 and 493.89. Moreover, the successive loss of H 2 O and angeloyl occurred in order to obtain a series of glycoside fragments, such as m/z 593.28 and 342.93. Therefore, based on similar cleavage patterns and reports in the literature [37][38][39][40] Table S2. The identification of and research into barrigenol-like triterpenoids saponins in X. sorbifolia husks are beneficial to the further development and utilization of the saponin.

Evaluation of the Antioxidant Activity of the TPMBr-La Extracts
The saponins contained in the recovered extract of the ethanol from the husks of X. sorbifolia have been reported to have antioxidant activity [41]. Nevertheless, the impact of different extraction methods on the antioxidant activity of saponins is not clear. Thus, we evaluated the antioxidant properties of saponins extracted from X. sorbifolia using different methods (TPMBr-La, ethanol, and water) through a DPPH and ABTS free radical scavenging ability tests. Vitamin C (Vc) was considered as a positive control.
As shown in Figure 8, the DPPH and ABTS free radical scavenging activity of TPMBr-La extracts displayed dose-dependence. When the concentration of the TPMBr-La extract was in the range of 25~100 µg/mL, the DPPH scavenging ability enhanced with the increasing concentration. When the concentration of the extract was 100~800 µg/mL, its scavenging ability no longer improved and tended to remain stable. The DPPH freeradical scavenging activity of the TPMBr-La extracts at 100 µg/mL exhibited the highest DPPH inhibition (92.30 ± 1.10%), significantly higher than that of ethanol extraction (26.74 ± 1.00%). As can be seen from Table 5, the TPMBr-La extracts gave the lowest DPPH inhibition IC 50 value of 36.54 ± 0.46 µg/mL (p < 0.001) compared to the ethanol extracts, which had the IC 50 value of 215.35 ± 6.90 µg/mL (p < 0.05), while Vc demonstrated an intermediate DPPH inhibition with the IC 50 value of 59.43 ± 0.53 µg/mL (p < 0.001). The ABTS free-radical scavenging rate of the TPMBr-La extracts (92.20 ± 0.30%) at 1200 µg/mL was significantly higher than that of ethanol extracts (69.89 ± 0.07%). ABTS inhibition IC 50 value of the TPMBr-La extracts was 541.13 ± 0.03 µg/mL (p < 0.001), which was lower than the IC 50 value of the ethanol extracts 855.25 ± 1.66 µg/mL (p < 0.001). In sum, the TPMBr-La extracts exhibited superior antioxidant activity, which may be due to the high content of saponins in the DESs solvent-extraction and the improved bioavailability through enhanced antioxidant membrane transport [42].  There is a significant difference when * p < 0 < 0.01, and *** p < 0.001 versus ethanol.

Conclusions
The husks of X. sorbifolia, as bulky by-products of industrial production brought a severe burden to the environment and caused an enormous waste of res Additionally, X. sorbifolia husks are known for their abundant triterpenoid saponin ticularly barrigenol-like triterpenoid saponins resources, which are responsible for of pharmacological bioactivities. For the high-value utilization of X. sorbifolia hus attempted to design eco-friendly and saponins-oriented DESs to extract the bioacti onins from X. sorbifolia husks. TPMBr−La, as an efficient and selective solvent, w tained from the fourteen prepared acidic DESs. The saponins' extraction efficiency Figure 8. Determination of the DPPH and ABTS radical scavenging capacity of bioactive saponins recovered from water, ethanol, and TPMBr-La. There is a significant difference when * p < 0.05, ** p < 0.01, and *** p < 0.001 versus ethanol.

Conclusions
The husks of X. sorbifolia, as bulky by-products of industrial production, have brought a severe burden to the environment and caused an enormous waste of resources. Additionally, X. sorbifolia husks are known for their abundant triterpenoid saponins, particularly barrigenol-like triterpenoid saponins resources, which are responsible for a range of pharmacological bioactivities. For the high-value utilization of X. sorbifolia husks, we attempted to design eco-friendly and saponins-oriented DESs to extract the bioactive saponins from X. sorbifolia husks. TPMBr-La, as an efficient and selective solvent, was obtained from the fourteen prepared acidic DESs. The saponins' extraction efficiency was up to 135% higher than when using 70% ethanol. The extraction mechanism showed that severe damage to the treated plant powders may be more conducive to the infiltration of DESs and the overflow of bioactive substances, to give rise to efficient extraction. The extraction conditions were optimized by Box-Behnken design RSM, and a maximum extraction rate of 72.11 ± 0.61 mg Re/g dw was obtained under the optimized extraction varies. The resulting D101 exhibited good adsorption and desorption abilities and its adsorption kinetic was compatible with the pseudo-second-order kinetic equation. The recovered TPMBr-La extracts exhibited significantly superior efficacy in scavenging the DPPH and ABTS radicals compared with the use of ethanol, indicating the probable enrichment effect of DESs on saponins. In summary, this study has demonstrated that acidic DESs are efficient and selective solvents for the high-value utilization of X. sorbifolia husks. Our findings also contribute to a new approach to the further application of DESs in the recycling of by-products.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox11040736/s1, Figure S1: Five independent variables were determined for the preliminary range by single factor experiment. Table S1: The experimental orders, levels of variables, and response values in Box-Behnken design. Table S2

Data Availability Statement:
The data presented in this study are available in this manuscript.