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Enhanced aqueous two-phase extraction of proanthocyanidins from grape seeds by using ionic liquids as adjuvants
Separation and Purification Technology 226 (2019) 154–161
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Enhanced aqueous two-phase extraction of proanthocyanidins from grape seeds by using ionic liquids as adjuvants
T
Lu Rana, Chong Yanga, Meilin Xua, Zhibiao Yib, Dabing Rena, , Lunzhao Yia, ⁎
a b
⁎
Yunnan Food Safety Research Institute, Kunming University of Science and Technology, Kunming 650500, PR China Dongguan Mathematical and Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan 523808, PR China
ARTICLE INFO
ABSTRACT
Keywords: Grape seed Proanthocyanidins Aqueous two-phase extraction Ionic liquids
Grape seeds are rich in proanthocyanidins, which are well-known natural antioxidants. In this work, an aqueous two-phase system (ATPS) based on ethanol/(NH4)2SO4 was proposed to extract and recover proanthocyanidins from grape seeds. Ionic liquids (IL) were used as adjuvants of ATPS to improve the extraction performance. The extraction efficiencies (EE) of proanthocyanidins were dramatically raised from 47.89 to 52.42% to 97.23 – 97.79% by adding 5 wt% [C6mim]BF4 into ATPS. Extraction conditions were optimized as follows: 29 wt% ethanol, 19 wt% (NH4)2SO4, 4 wt% [C6mim]BF4, solid–liquid ratio = 1:20 g/mL, temperature (T) = 40 °C, pH = 5.0, and extraction time (t) = 20 min. Under optimum conditions, the yields of catechin, epicatechin, and procyanidin B2 reached 1.21, 1.22, and 0.14 mg/g dry weight, respectively, with EE of over 99.01%. Compared with conventional organic solvents such as methanol, ethanol, and isopropanol, the ATPS using ILs as adjuvants provided higher extraction yields. Results suggested that ethanol-based ATPS can extract value-added compounds from waste residues in the fruit-processing industry. In addition, low amount of IL addition can significantly enhance the extraction performance of ATPS.
1. Introduction Grape, one of the highest-yielding fruits worldwide, is mainly used to produce raisins and wines. Large amounts of byproducts, including skins, seeds, and rachis, are inevitably produced during processing [1]. These byproducts contain several bioactive compounds, such as phenolic acids, anthocyanins, and proanthocyanidins. Proanthocyanidins are oligomeric phenolics (e.g., dimers and trimers of monomeric catechins) and are identified as the most abundant chemical constituents in grape seeds [2]. Previous studies confirmed that proanthocyanidins possess health properties, such as antioxidant [3], antibacterial [4], and anticancer activities [5]. Therefore, extracting and recovering proanthocyanidins from waste grape seeds bears importance as both processes can increase the value of grape-derived products and reduce the food processing-induced environmental burden. Over the past decades, aqueous two-phase systems (ATPSs) have been intensively used as a novel alternative to traditional organic solvent in extraction, separation, and purification [6]. Compared with several organic solvents, ATPSs present higher biocompatibility and a more environment-friendly nature [7]. ATPSs are typically formed by mixing aqueous solutions of two immiscible solutes: polymer + polymer, polymer + salt, salt + salt, short-chain aliphatic ⁎
alcohols + salt, and ionic liquid + salt [7–9]. Among these systems, polymer-based ATPSs have been largely exploited since the 1980s. However, shortcomings related to high viscosities of coexisting phases, easy emulsification and limited polarity ranges, have been a bottleneck in the application of polymer-based ATPS [6]. In recent years, ATPSs based on small-molecule organic solvents (e.g., ethanol, acetone, and npropanol) have drawn considerable attention [10]. Small-molecule organic solvent-based ATPSs generally feature low viscosity, low cost, and rapid phase separation. To date, ethanol-based ATPSs have been successfully applied to extraction of natural products, such as phenolic acids, flavonoids, and alkaloids [11–13]. However, the limited polarity ranges of ethanol-based ATPSs may result in poor selectivity and low extraction efficiency. Ionic liquids (ILs) are a novel type of green solvents that can replace traditional organic solvents in extraction, separation, and purification processes [9,14]. Compared with traditional organic solvents, ILs feature negligible vapor pressure, non-flammability, high chemical stability, and strong dissolving capacity [9]. Rogers and coworkers firstly reported ATPS formation by mixing aqueous solutions of ILs and inorganic salts [15]. From this report, substantial efforts have been exerted to develop IL-based ATPSs. Given the excellent properties of ILs, IL-based ATPSs possess more advantages, namely, low viscosity, fast
Corresponding authors. E-mail addresses: [email protected] (D. Ren), [email protected] (L. Yi).
https://doi.org/10.1016/j.seppur.2019.05.089 Received 24 July 2018; Received in revised form 27 March 2019; Accepted 29 May 2019 Available online 30 May 2019 1383-5866/ © 2019 Elsevier B.V. All rights reserved.
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phase separation, high extraction efficiency, and tunable selectivity, than polymer-based systems [9]. In the past decade, IL-based ATPSs have shown outstanding performance in extracting natural-derived compounds (e.g., flavonoids, polyphenols, and alkaloids) and biological molecules (e.g., proteins, amino acids, and nucleic acids) [9,16]. However, IL synthesis is expensive and tedious. IL purification also presents difficulty, thus hindering the development and application of IL-based ATPSs in large-scale extraction and separation [17]. Apart from being used as phase-forming components, a small amount of IL is also added to polymer-based ATPSs as additive to enhance the extraction efficiency (EE) of amino acids, proteins, and pharmaceutical ingredients [18,19]. A recent work demonstrated that adding ILs as adjuvants in ethanol-based ATPSs can enhance the EE of sinomenine from Sinomenium acutum [20]. When used as additives or adjuvants, ILs can improve the physicochemical properties of ATPS and thus influence the partition behavior of target molecules. Simultaneously, adding a small amount of IL can reduce the cost of IL-based ATPSs. In this work, ethanol-based ATPSs were developed and applied to extract proanthocyanidins from grape seeds. To improve the extraction performance, 10 ILs with different anions and cations were evaluated as adjuvants. Extraction conditions, including solid–liquid ratio, extraction time, temperature, and pH, were also optimized.
2.4. Aqueous two-phase extraction The aqueous solutions of 40 wt% (NH4)2SO4 and 70 wt% ethanol were used to prepare ATPSs. The aqueous solutions of (NH4)2SO4, ethanol, IL, and water were added into a 15 mL centrifugal tube on the basis of predetermined mass fractions. For thorough equilibration, the centrifuge tube was shaken vigorously and settled for 2 h. For extraction of proanthocyanidins from grape seeds, a certain amount of grape seed powder was added to the prepared ATPS, and ultrasonic-assisted extraction (UAE) method was used. After performing UAE, the ATPS was centrifuged (8000 rpm/min for 10 min), and the top and bottom phases were separated. The proanthocyanidin concentrations in each phase were measured using the ultra-performance liquid chromatography (UPLC)-MS. The volume of each phase was determined for the calculation of phase ratio (R). The R, partition coefficient (K), EE, and extraction yield (Y) of proanthocyanidins were calculated using Eqs (1)–(4), respectively.
R=
Vt Vb
(1)
K=
Ct Cb
(2)
EE =
2. Materials and methods
Ct Vt × 100% Ct Vt + Cb Vb
Ct Vt Mt
(3)
2.1. Materials and reagents
Y=
Catechin, epicatechin, and procyanidin B2 were purchased from Chengdu Mansite Bio-technology Co., Ltd (Chengdu, China). ILs, including [C4mim]Br, [C6mim]Br, [C8mim]Br, [C4mim]NO3, [C4mim]TOS, [C4mim][CH3SO3], [C4mim][HSO4], [C2mim]BF4, [C4mim]BF4, [C6mim]BF4, [C8mim]BF4, and [C10mim]BF4, were bought from Chengjie Chemical Co., Ltd (Shanghai, China). Table S1 presents the chemical structures of the studied ILs. Mass spectrometrygrade acetonitrile and formic acid were purchased from Merck KGaA (Germany) and Sigma-Aldrich (USA), respectively. HPLC-grade methanol, ethanol, isopropanol, and ethyl acetate were bought from TCI (Shanghai, China). Inorganic salts, namely, ammonium sulfate ((NH4)2SO4), sodium sulfate (Na2SO4), sodium carbonate (Na2CO3), sodium dihydrogen phosphate (NaH2PO4), and potassium phosphate dibasic (K2HPO4), were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd (Tianjin, China).
where Vt and Vb represent the volumes (mL) of top and bottom phases, respectively. Ct and Cb represent the concentration (mg/mL) of proanthocyanidins in top and bottom phases, respectively. And Mt is the total mass of grape seed powder (g) added into ATPS.
(4)
2.5. Screening of ILs Ten ILs, including [C4mim]Br, [C4mim]NO3, [C4mim]TOS, [C4mim] [CH3SO3], [C4mim][HSO4], [C2mim]BF4, [C4mim]BF4, [C6mim]BF4, [C8mim]BF4, and [C10mim]BF4, were investigated as additives. A 5 wt% sample of each IL was added to the ATPS containing 21 wt% ethanol and 19 wt% (NH4)2SO4, and K and EE values were measured. The IL resulting in the highest K and EE values was then studied in the range of 1–5 wt% additions to determine the optimum IL dose. The ATPS without IL was used as control, and all measurements were performed in triplicate. The R, K, EE, and Y values of representative proanthocyanidins were determined and used to investigate the effects of IL addition.
2.2. Preparation of crude sample
2.6. Extraction with different solvents
Grape seed samples were obtained from Xinjiang province, China. The grape seeds were dried in a drying oven at 60 °C for 24 h. The dried seed samples were then powdered by a disintegrator. Subsequently, the powders were sieved to 100 mesh particle size and stored in a dry place.
The proposed ATPS with IL addition was compared with seven other traditional solvents: pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate. A 0.5 g sample powder was extracted with 10 mL of each solvent in a 15 mL centrifuge tube under ultrasonic condition (40 °C for 20 min). The resulting extracts were centrifuged (8000 rpm/min for 10 min), and the supernatant was collected, diluted with 50% acetonitrile, and filtered (0.22 µm) for subsequent UPLC-MS analysis. Each extraction was performed in triplicate.
2.3. Phase diagrams The binodal curves were determined by cloud-point titration at 25 ± 1 °C [21]. Initially, 0.5 mL of salt solution with known concentration was added into a 10 mL centrifugal tube. Then, 70% ethanol solution was added dropwise until the mixture became cloudy. After forming a turbid solution, a known mass of water was added to clarify the mixture again. This procedure was repeated to obtain sufficient data to construct binodal curves. For ATPSs with ILs as additives, salt or aqueous ethanol solutions were prepared by adding a certain amount of ILs (e.g., 25 wt% salt + 5 wt% IL). The solutions containing ILs were used to determine binodal curves, following the procedure described here.
2.7. Ultra-HPLC (UHPLC)-MS analysis The grape seed samples were qualitatively and quantitatively analyzed using UHPLC-MS. In this study, catechin, epicatechin, and procyanidin B2 were selected as representatives to evaluate the extraction performance of ATPS. For qualitative analysis, a high-resolution MS (HRMS) technique (Q Exactive Focus, Thermo Fisher, USA) was 155
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employed to identify proanthocyanidins in grape seeds. For quantitative analysis, a triple quadrupole MS system (LCMS-8040, Shimadzu, Japan) was applied to determine the concentrations of three representative proanthocyanidins in extracts. Detailed instrument parameters and experimental conditions are summarized in Supplemental Materials.
ATPS in the current work. Five inorganic salts, such as (NH4)2SO4, Na2SO4, Na2CO3, NaH2PO4, and K2HPO4, were examined as phaseforming components. The binodal curves of ATPSs composed of ethanol, and each salt were determined and illustrated in Fig. 2(a). The binodal curves aid in the comparison of the phase-forming capabilities of different salts. A closer binodal curve position to the origin of coordinates indicates a stronger salt capability to induce ATPS formation. Fig. 2(a) reveals that the phase-forming capability of the investigated salts follows the order: Na2CO3 > Na2SO4 > K2HPO4 > (NH4)2SO4 > NaH2PO4. With the exception of K2HPO4 (ΔGhyd = –2379 kJ/mol), the phase-forming capabilities of these salts agree well with their Gibbs free energies of hydration: Na2CO3 (ΔGhyd = –2045 kJ/mol) > Na2SO4 (ΔGhyd = –1810 kJ/mol) > (NH4)2SO4 (ΔGhyd = –1650 kJ/mol) > NaH2PO4 (ΔGhyd = –830 kJ/ mol) [22,23]. Here, the anion and cation contributed to the salts’ Gibbs free energies of hydration. The correlation between Gibbs free energies of hydration and phase-forming capabilities of salts indicate that salts with high salting-out capability consistently present a strong phaseforming property. To evaluate the extraction performance of different ATPSs, this work measured the K and EE values at a constant ATPS composition, namely, 19 wt% ethanol + 25 wt% salts. Only (NH4)2SO4, K2HPO4, and Na2CO3 formed ATPS when 25 wt% salts were mixed with 19 wt% ethanol. Therefore, 25 wt% Na2SO4 was easily precipitated, whereas 25 wt% K2HPO4 was completely dissolved. Thus, the K and EE values were measured in ATPSs composed of (NH4)2SO4, K2HPO4, and Na2CO3, and the results are illustrated in Fig. 2(b, c). The K and EE values of proanthocyanidins varied considerably with different types of salts. The ethanol/(NH4)2SO4 system yielded higher K (8.94 – 19.13) and EE (> 91.67%) values than ATPSs containing K2HPO4 and Na2CO3. The differences in K and EE values were associated with the stabilities of proanthocyanidins under different solution environments. Proanthocyanidins are acidic polyphenols which are stable in acidic systems, such as ethanol/(NH4)2SO4 system (pH ≈ 5.63) [24]. By contrast, ethanol/K2HPO4 (pH ≈ 9.98) and ethanol/Na2CO3 (pH ≈ 12.22) are alkaline systems that possibly react with acidic constituents, thus destroying the chemical structures of proanthocyanidins. Therefore, (NH4)2SO4 is the best option to extract proanthocyanidins in terms of EE and solute stability.
2.8. Statistical analysis SPSS statistics software version 22.0 (SPSS Inc., Chicago, IL, USA) was used for analysis of variance. Calculated p values were used to analyze whether K and EE were significantly affected by experimental conditions. 3. Results and discussion 3.1. UPLC-MS analysis of proanthocyanidins in grape seed 3.1.1. Chemical composition of grape seeds HRMS can provide rich information (accurate m/z value and MS/MS spectra) for structural elucidation of compounds in complex samples. Hence, this work utilized HRMS to identify proanthocyanidins occurring in grape seed. Fig. 1 illustrates the total ion chromatogram of grape seed extract. By comparing elemental compositions, retention times, and MS/MS spectra with chemical standards, five proanthocyanidins, such as procyanidin B1 (1), catechin (2), procyanidin B2 (3), epicatechin (4), and epicatechin gallate (5), were identified. Fig. 1 depicts the chemical structures of the identified proanthocyanidins. 3.1.2. Quantitative determination of proanthocyanidins in extracts Catechin, epicatechin, and procyanidin B2 were selected as representative proanthocyanidins in quantitative determination experiments. Quantitative analyses of catechin, epicatechin, and procyanidin B2 were performed by multiple-reaction monitoring mode (as displayed in Fig. S1) on a Shimadzu triple quadrupole MS system. The standard curve of each compound was determined in the range of 0.0001–0.01 mg/mL (Figs. S2–S4). The established standard curves are as follows: catechin, y = 70000000x − 1890.7 (R2 = 0.9977); epicatechin, y = 100000000x − 25495 (R2 = 0.9924); procyanidin B2, y = 90000000x − 21223 (R2 = 0.9937).
3.3. Optimization of ethanol and salt concentrations
3.2. Screening of salts
Proanthocyanidins were mainly extracted in the upper ethanol-rich phase (as displayed in Fig. S1). ATPS composition is a major factor
As a low-toxicity organic solvent, ethanol was used to construct
Fig. 1. Total ion chromatogram (TIC) of grape seeds sample and structure of procyanidin B1 (1), catechin (2), procyanidin B2 (3), epicatechin (4), and epicatechin gallate (5). 156
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Fig. 2. Phase diagrams for the different salt-based ATPS at 25 °C (a), and effects of salt type on the partition coefficient (b) and extraction efficiency (c) of proanthocyanidins in ATPS containing 19 wt% salt and 25 wt% ethanol.
Fig. 3. Effects of ethanol concentration (a, b) and (NH4)2SO4 concentration (c, d) on the partition coefficient and extraction efficiency of proanthocyanidins.
influencing extraction performances, namely, K, EE, and Y. Thus, this work evaluated the effects of ethanol and (NH4)2SO4 concentrations by determining the K and EE values of representative proanthocyanidins. The results are presented in Fig. 3(a–d). As observed in Fig. 3(a, b), K and EE values evidently increased as ethanol concentration increased in the range of 21–29 wt% and reached the maximum at 29 wt% ethanol. In practice, a large volume of ethanolrich phase was observed when ethanol concentration increased, promoting the dissolution of several proanthocyanidins and thus enhancing EE. Fig. 3(c, d) indicate that the variation in (NH4)2SO4 concentration in the range of 16–20 wt% also led to the increase in K and EE values. Both
K and EE values of proanthocyanidins reached the maximum at 19 wt% of (NH4)2SO4. Salting-out effect is considered a major driving force enriching solutes in the ethanol-rich phase [13,25]. The increase in salt concentration can increase the salting-out capability, which benefits the distribution of proanthocyanidins in the ethanol-rich phase. Thus, K and EE values increased in the range of 16–19 wt% of (NH4)2SO4 as salting-out capability was enhanced. However, excessive amounts of salt (> 19 wt%) led to the decrease in water in the ethanol-rich top phase, and this condition can weaken the affinity of ethanol-rich phase to proanthocyanidins and thus cause the decrease in K and EE values [11]. Therefore, 29 wt% ethanol and 19 wt% (NH4)2SO4 were employed to construct ATPSs in subsequent experiments. 157
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Fig. 4. Effects of IL anions and cations on K (a) and EE (b) of proanthocyanidins (21 wt% ethanol + 19 wt% (NH4)2SO4 + 5 wt% IL).
3.4. Effect of ILs
formation of IL aggregates or clusters weakens the interaction between solutes and ILs, explaining the decrease in K and EE values of ATPS composed of [C8mim][BF4] and [C10mim][BF4]. Therefore, [C6mim] BF4 was selected as the most suitable adjuvant as this IL provided the highest K (Kcatechin = 32.87, Kepicatechin = 20.86, Kprocyanidin B2 = 27.50) and EE (> 96.35%) values.
This work used ILs as adjuvants to manipulate the partition behavior of proanthocyanidins in ethanol-based ATPSs. To improve the extraction performance of ATPSs, this study thoroughly investigated the effects of 10 ILs with different anions and cations. Table S1 summarizes the full names and chemical structures of the used ILs. Using an ATPS containing 21 wt% ethanol and 19 wt% (NH4)2SO4 as the control system, the K and EE values of proanthocyanidins were determined with 5 wt% IL additions, and the results are presented in Fig. 4(a–b). ILs were mainly present in the ethanol-rich phase. The anion effects of ILs were evaluated by changing the anions [BF4]–, [NO3]–, Br–, TOS–, [SO3]–, and [HSO4]– while keeping the [C4mim]+ cation. Fig. 4(a, b) illustrate that the K and EE values of proanthocyanidins varied considerably with different anions. Except for [TOS]– and [HSO4]–, the addition of ILs containing other anions promoted the enrichment proanthocyanidins in the ethanol-rich phase, as indicated by high K and EE values. For the six tested ILs with different anions, the K and EE values increased in the following order: [C4mim] BF4 > [C4mim]NO3 > [C4mim]Br > [C4mim][TOS] > [C4mim] [CH3SO3] > [C4mim][HSO4]. This result suggests that anion-type ILs play important roles in manipulating the partition behavior of solutes when using ILs as adjuvants. In comparison with the ATPS without IL addition (K < 3.09, EE < 64.49%), [C4mim]BF4 provided the highest K (Kcatechin = 11.83, Kepicatechin = 9.85, and Kprocyanidin B2 = 10.50) and EE (> 92.08%) values. Thus, [BF4]– was selected as the suitable anion for subsequent evaluation. In the current work, the IL cation effects were analyzed by changing the cations [Cnmim]+ (n = 2, 4, 6, 8, 10) while maintaining [BF4]– anions. Fig. 4(a, b) demonstrate the variation in K and EE values of proanthocyanidins with five ILs containing different cations. Proanthocyanidins can migrate to the ethanol-rich phase in the following order: no IL < [C2mim]BF4 < [C4mim]BF4 < [C10mim] BF4 < [C8mim]BF4 < [C6mim]BF4. Increasing the alkyl chain length of IL cation can increase the hydrophobicity of ILs [26]. Increasing hydrophobicity (n = 2–6) initially enhanced the molecular interactions between solute and IL. Such interaction promoted the partition of proanthocyanidins in the top phase containing IL and ethanol [27]. However, a strong hydrophobicity (n = 8, 10) can cause the self-aggregation of ILs, thus forming IL aggregates or clusters [28]. The
3.5. Optimization of extraction conditions Extraction parameters, namely, the IL dose, solid–liquid ratio, extraction time, temperature, and pH, exert significant effects on the extractability of ATPS. This work used a single-factor experiment to optimize these important factors, and catechins, epicatechin, and procyanidin B2 were used as representatives to evaluate the extraction performance. 3.5.1. Effect of IL dose Apart from the chemical structures of IL, IL dose also substantially influenced the improvement of EE. Here, we further investigated the dose effect of [C6mim]BF4 on EE within the range of 1–5 wt%, and the results are presented in Fig. 5(a, b). Both figures indicate that the K and EE values of proanthocyanidins increased as IL content increased up to 4 wt%. Such an increase may also be attributed to the enhancement of affinity of ethanol-rich phase to the solute caused by addition of IL [18]. Beyond 4 wt%, a higher amount of [C6mim]BF4 caused no further enhancement in EE. Therefore, 4 wt% [C6mim]BF4 was selected and added to 29 wt% ethanol/19 wt% (NH4)2SO4 ATPS for further experiments. 3.5.2. Effect of solid–liquid ratio. Solid–liquid ratio (g/mL), which is a major factor affecting EE, was optimized in the ratio of 1:10–1:50, and the results are presented in Fig. 6. As indicated in the figure, solid–liquid ratio at 1:20 presented the highest K (43.53–99.60) and EE (> 98.50) values. When the solid–liquid ratio exceeded 1:20, the K and EE values of catechin and procyanidin decreased significantly (p < 0.05), whereas those of epicatechin showed no evident change (p > 0.05). Solid–liquid ratio is an important parameter during extraction. For a certain amount of sample (1 g of grape seed in this work), a low volume of solvent (< 20 mL) cannot completely extract the target solute from the solid sample 158
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Fig. 5. Effects of IL dose on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; solid-liquid ratio: 1:20 mg/mL; T = 30 °C; pH: no adjustment; time: 20 min).
Fig. 6. Effects of solid-liquid ratio on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; T = 30 °C; pH: no adjustment; time: 20 min).
Fig. 7. Effects of temperature on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; pH: no adjustment; time: 20 min).
matrix. However, an excess volume (> 20 mL) can cause a dilution effect on solutes although they can be completely extracted. Thus, dilution effect was the major reason for the reduced K and EE values when the solid–liquid ratio exceeded 1:20 [29]. Thus, 1:20 was selected as the best solid–liquid ratio and used for subsequent studies.
extraction of proanthocyanidins. As high temperature generally benefits the mass transfer of solute, raising EE to the highest value possible is suggested under mild temperature (e.g., 40 °C in this work). However, high temperature also possibly induces the degradation of unstable compounds and evaporation of solvents (e.g., ethanol and water), both of which can lead to the decrease in extraction performance. In addition, high temperature must consume additional energy. Therefore, 40 °C was optimized as the suitable extraction temperature.
3.5.3. Effect of temperature This research analyzed the effect of temperature on the extraction performance in the range of 20–60 °C, and the results are presented in Fig. 7. As depicted in the figure, increasing temperature from 20 °C to 40 °C enhanced the K and EE values of proanthocyanidins, whereas both values decreased at a temperature beyond 40 °C. These results indicate that high temperature is unsuitable for the aqueous two-phase
3.5.4. Effect of pH This study investigated the effects of pH on the K and EE values of proanthocyanidins in ATPS, and the results are illustrated in Fig. 8. HCl and NaOH aqueous solutions were used to adjust the system pH in the 159
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Fig. 8. Effects of pH on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; T: 40 °C; time: 20 min).
Fig. 9. Effects of extraction time on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; T: 40 °C; pH: 5).
range between 3.0 and 10.0. Fig. 8 reveals that acidic conditions (pH < 7) provided higher K and EE values than alkaline conditions. At pH 5, the K and EE values reached their maximum. By changing the electrical charge form of solutes, pH can affect the electrostatic interactions between solutes and solvents and thus cause a significant effect on the partition behavior of solutes in ATPS [30]. Essentially, proanthocyanidins are oligomeric phenolics with weak acidity and are stable in weak acidic environment. In alkaline solutions, the phenolic hydroxyl group of proanthocyanidins can be dissociated, resulting in an adverse effect on the partition of proanthocyanidins in the ethanol-rich phase. As a result, pH 5.0 was selected for the following experiments.
the highest Y of catechin (1.21 mg/g), epicatechin (1.22 mg/g), and procyanidin B2 (0.14 mg/g). Among the conventional solvents, 70% methanol exhibited the best performance, but its Y value remained remarkably lower than that of ATPS (p < 0.05). The amounts of reagent consumptions were also compared, as indicated in Fig. 10(b). The ATPS method consumed the lowest amount of volatile organic solvent (2.9 g of ethanol) over other methods. Considering the total amount of consumed reagent, the amounts of consumed in ATPS were similar with those of 70% ethanol and 70% methanol (p > 0.05) and lower than those of other organic solvents (p < 0.05). Although the proposed ATPS failed to achieve the lowest cost, it still featured a high EE and consumed minimal amount of organic solvent. Thus, ATPS remains a good choice for extracting proanthocyanidins from grape seeds.
3.5.5. Optimization of extraction time The effect of extraction time on the EE of proanthocyanidins was also studied in the range of 5–60 min, and the results are indicated in Fig. 9. The maximum K (Kcatechin = 97.45, Kepicatechin = 65.78, and Kprocyanidin B2 = 148.55) and EE (> 99.01%) were obtained at 20 min. When the extraction time exceeded 20 min, the K and EE values decreased. Such decreases may have resulted from solute degradation and ethanol evaporation under prolonged ultrasound condition [31]. Hence, 20 min was selected as the proper extraction time.
4. Conclusions In this work, an ATPS based on ethanol/(NH4)2SO4 was proposed to extract proanthocyanidins from grape seeds. By adding 5 wt% [C6mim] BF4 as an adjuvant to the initial ATPS composed of 21 wt% ethanol and 19 wt% (NH4)2SO4, remarkable increases in K (average from 2.55 to 27.08) and EE (average from 59.65% to 97.12%) were observed. Optimal extraction performance was obtained with 29 wt% ethanol, 19 wt% (NH4)2SO4, 4 wt% [C6mim] BF4, solid–liquid ratio = 1:20 g/ mL, T = 40 °C, pH = 5.0, and t = 20 min. As a result, the Y of three representative proanthocyanidins, namely, catechin, epicatechin, and procyanidin B2, reached 1.21, 1.22, and 0.14 mg/g, respectively. On the contrary, the EE of three compounds amounted to values up to 99%. Compared with conventional solvents (pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate), the proposed ATPS using ILs as adjuvants achieved a higher Y but consumed less amount of organic solvents. In conclusion, ethanol-based
3.6. Comparison of different solvents The optimized ATPS (29% ethanol + 19 wt% (NH4)2SO4 + 4% [C6mim]BF4, w/w) was compared with other traditional solvents (pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate) regarding their Y of proanthocyanidins, and the results are presented in Fig. 10(a). All extractions were performed under identical conditions: solid–liquid ratio = 1:20, T = 40 °C, pH = 5.0, and t = 20 min. Fig. 10(a) illustrates that the optimized ATPS provided 160
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[5] [6] [7]
[8] [9] [10] [11] [12]
[13] [14] [15]
[16] [17]
Fig. 10. (a) The effect of different solvent systems on the extraction yield of proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4 + 4 wt% IL, w/w); (b) the consumption of different solvent (pure water was excluded).
[18] [19]
ATPSs using ILs as adjuvants provide a promising strategy to extract and recover value-added compounds from waste residues of food processing. In future studies, a method must be developed to recover and reuse solvents (e.g., IL, salt, and ethanol). The proposed method benefits the minimization of extraction cost.
[20] [21] [22]
Acknowledgements
[23]
The present work was financially supported by the Applied Basic Research Project of Yunnan Province (No. 2018FD033), National Natural Science Foundation of China (No. 21775058) and Guangdong Social Development Program (2016108101043).
[24]
Appendix A. Supplementary material
[25] [26]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.seppur.2019.05.089.
[27]
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Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur
Enhanced aqueous two-phase extraction of proanthocyanidins from grape seeds by using ionic liquids as adjuvants
T
Lu Rana, Chong Yanga, Meilin Xua, Zhibiao Yib, Dabing Rena, , Lunzhao Yia, ⁎
a b
⁎
Yunnan Food Safety Research Institute, Kunming University of Science and Technology, Kunming 650500, PR China Dongguan Mathematical and Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan 523808, PR China
ARTICLE INFO
ABSTRACT
Keywords: Grape seed Proanthocyanidins Aqueous two-phase extraction Ionic liquids
Grape seeds are rich in proanthocyanidins, which are well-known natural antioxidants. In this work, an aqueous two-phase system (ATPS) based on ethanol/(NH4)2SO4 was proposed to extract and recover proanthocyanidins from grape seeds. Ionic liquids (IL) were used as adjuvants of ATPS to improve the extraction performance. The extraction efficiencies (EE) of proanthocyanidins were dramatically raised from 47.89 to 52.42% to 97.23 – 97.79% by adding 5 wt% [C6mim]BF4 into ATPS. Extraction conditions were optimized as follows: 29 wt% ethanol, 19 wt% (NH4)2SO4, 4 wt% [C6mim]BF4, solid–liquid ratio = 1:20 g/mL, temperature (T) = 40 °C, pH = 5.0, and extraction time (t) = 20 min. Under optimum conditions, the yields of catechin, epicatechin, and procyanidin B2 reached 1.21, 1.22, and 0.14 mg/g dry weight, respectively, with EE of over 99.01%. Compared with conventional organic solvents such as methanol, ethanol, and isopropanol, the ATPS using ILs as adjuvants provided higher extraction yields. Results suggested that ethanol-based ATPS can extract value-added compounds from waste residues in the fruit-processing industry. In addition, low amount of IL addition can significantly enhance the extraction performance of ATPS.
1. Introduction Grape, one of the highest-yielding fruits worldwide, is mainly used to produce raisins and wines. Large amounts of byproducts, including skins, seeds, and rachis, are inevitably produced during processing [1]. These byproducts contain several bioactive compounds, such as phenolic acids, anthocyanins, and proanthocyanidins. Proanthocyanidins are oligomeric phenolics (e.g., dimers and trimers of monomeric catechins) and are identified as the most abundant chemical constituents in grape seeds [2]. Previous studies confirmed that proanthocyanidins possess health properties, such as antioxidant [3], antibacterial [4], and anticancer activities [5]. Therefore, extracting and recovering proanthocyanidins from waste grape seeds bears importance as both processes can increase the value of grape-derived products and reduce the food processing-induced environmental burden. Over the past decades, aqueous two-phase systems (ATPSs) have been intensively used as a novel alternative to traditional organic solvent in extraction, separation, and purification [6]. Compared with several organic solvents, ATPSs present higher biocompatibility and a more environment-friendly nature [7]. ATPSs are typically formed by mixing aqueous solutions of two immiscible solutes: polymer + polymer, polymer + salt, salt + salt, short-chain aliphatic ⁎
alcohols + salt, and ionic liquid + salt [7–9]. Among these systems, polymer-based ATPSs have been largely exploited since the 1980s. However, shortcomings related to high viscosities of coexisting phases, easy emulsification and limited polarity ranges, have been a bottleneck in the application of polymer-based ATPS [6]. In recent years, ATPSs based on small-molecule organic solvents (e.g., ethanol, acetone, and npropanol) have drawn considerable attention [10]. Small-molecule organic solvent-based ATPSs generally feature low viscosity, low cost, and rapid phase separation. To date, ethanol-based ATPSs have been successfully applied to extraction of natural products, such as phenolic acids, flavonoids, and alkaloids [11–13]. However, the limited polarity ranges of ethanol-based ATPSs may result in poor selectivity and low extraction efficiency. Ionic liquids (ILs) are a novel type of green solvents that can replace traditional organic solvents in extraction, separation, and purification processes [9,14]. Compared with traditional organic solvents, ILs feature negligible vapor pressure, non-flammability, high chemical stability, and strong dissolving capacity [9]. Rogers and coworkers firstly reported ATPS formation by mixing aqueous solutions of ILs and inorganic salts [15]. From this report, substantial efforts have been exerted to develop IL-based ATPSs. Given the excellent properties of ILs, IL-based ATPSs possess more advantages, namely, low viscosity, fast
Corresponding authors. E-mail addresses: [email protected] (D. Ren), [email protected] (L. Yi).
https://doi.org/10.1016/j.seppur.2019.05.089 Received 24 July 2018; Received in revised form 27 March 2019; Accepted 29 May 2019 Available online 30 May 2019 1383-5866/ © 2019 Elsevier B.V. All rights reserved.
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phase separation, high extraction efficiency, and tunable selectivity, than polymer-based systems [9]. In the past decade, IL-based ATPSs have shown outstanding performance in extracting natural-derived compounds (e.g., flavonoids, polyphenols, and alkaloids) and biological molecules (e.g., proteins, amino acids, and nucleic acids) [9,16]. However, IL synthesis is expensive and tedious. IL purification also presents difficulty, thus hindering the development and application of IL-based ATPSs in large-scale extraction and separation [17]. Apart from being used as phase-forming components, a small amount of IL is also added to polymer-based ATPSs as additive to enhance the extraction efficiency (EE) of amino acids, proteins, and pharmaceutical ingredients [18,19]. A recent work demonstrated that adding ILs as adjuvants in ethanol-based ATPSs can enhance the EE of sinomenine from Sinomenium acutum [20]. When used as additives or adjuvants, ILs can improve the physicochemical properties of ATPS and thus influence the partition behavior of target molecules. Simultaneously, adding a small amount of IL can reduce the cost of IL-based ATPSs. In this work, ethanol-based ATPSs were developed and applied to extract proanthocyanidins from grape seeds. To improve the extraction performance, 10 ILs with different anions and cations were evaluated as adjuvants. Extraction conditions, including solid–liquid ratio, extraction time, temperature, and pH, were also optimized.
2.4. Aqueous two-phase extraction The aqueous solutions of 40 wt% (NH4)2SO4 and 70 wt% ethanol were used to prepare ATPSs. The aqueous solutions of (NH4)2SO4, ethanol, IL, and water were added into a 15 mL centrifugal tube on the basis of predetermined mass fractions. For thorough equilibration, the centrifuge tube was shaken vigorously and settled for 2 h. For extraction of proanthocyanidins from grape seeds, a certain amount of grape seed powder was added to the prepared ATPS, and ultrasonic-assisted extraction (UAE) method was used. After performing UAE, the ATPS was centrifuged (8000 rpm/min for 10 min), and the top and bottom phases were separated. The proanthocyanidin concentrations in each phase were measured using the ultra-performance liquid chromatography (UPLC)-MS. The volume of each phase was determined for the calculation of phase ratio (R). The R, partition coefficient (K), EE, and extraction yield (Y) of proanthocyanidins were calculated using Eqs (1)–(4), respectively.
R=
Vt Vb
(1)
K=
Ct Cb
(2)
EE =
2. Materials and methods
Ct Vt × 100% Ct Vt + Cb Vb
Ct Vt Mt
(3)
2.1. Materials and reagents
Y=
Catechin, epicatechin, and procyanidin B2 were purchased from Chengdu Mansite Bio-technology Co., Ltd (Chengdu, China). ILs, including [C4mim]Br, [C6mim]Br, [C8mim]Br, [C4mim]NO3, [C4mim]TOS, [C4mim][CH3SO3], [C4mim][HSO4], [C2mim]BF4, [C4mim]BF4, [C6mim]BF4, [C8mim]BF4, and [C10mim]BF4, were bought from Chengjie Chemical Co., Ltd (Shanghai, China). Table S1 presents the chemical structures of the studied ILs. Mass spectrometrygrade acetonitrile and formic acid were purchased from Merck KGaA (Germany) and Sigma-Aldrich (USA), respectively. HPLC-grade methanol, ethanol, isopropanol, and ethyl acetate were bought from TCI (Shanghai, China). Inorganic salts, namely, ammonium sulfate ((NH4)2SO4), sodium sulfate (Na2SO4), sodium carbonate (Na2CO3), sodium dihydrogen phosphate (NaH2PO4), and potassium phosphate dibasic (K2HPO4), were purchased from Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd (Tianjin, China).
where Vt and Vb represent the volumes (mL) of top and bottom phases, respectively. Ct and Cb represent the concentration (mg/mL) of proanthocyanidins in top and bottom phases, respectively. And Mt is the total mass of grape seed powder (g) added into ATPS.
(4)
2.5. Screening of ILs Ten ILs, including [C4mim]Br, [C4mim]NO3, [C4mim]TOS, [C4mim] [CH3SO3], [C4mim][HSO4], [C2mim]BF4, [C4mim]BF4, [C6mim]BF4, [C8mim]BF4, and [C10mim]BF4, were investigated as additives. A 5 wt% sample of each IL was added to the ATPS containing 21 wt% ethanol and 19 wt% (NH4)2SO4, and K and EE values were measured. The IL resulting in the highest K and EE values was then studied in the range of 1–5 wt% additions to determine the optimum IL dose. The ATPS without IL was used as control, and all measurements were performed in triplicate. The R, K, EE, and Y values of representative proanthocyanidins were determined and used to investigate the effects of IL addition.
2.2. Preparation of crude sample
2.6. Extraction with different solvents
Grape seed samples were obtained from Xinjiang province, China. The grape seeds were dried in a drying oven at 60 °C for 24 h. The dried seed samples were then powdered by a disintegrator. Subsequently, the powders were sieved to 100 mesh particle size and stored in a dry place.
The proposed ATPS with IL addition was compared with seven other traditional solvents: pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate. A 0.5 g sample powder was extracted with 10 mL of each solvent in a 15 mL centrifuge tube under ultrasonic condition (40 °C for 20 min). The resulting extracts were centrifuged (8000 rpm/min for 10 min), and the supernatant was collected, diluted with 50% acetonitrile, and filtered (0.22 µm) for subsequent UPLC-MS analysis. Each extraction was performed in triplicate.
2.3. Phase diagrams The binodal curves were determined by cloud-point titration at 25 ± 1 °C [21]. Initially, 0.5 mL of salt solution with known concentration was added into a 10 mL centrifugal tube. Then, 70% ethanol solution was added dropwise until the mixture became cloudy. After forming a turbid solution, a known mass of water was added to clarify the mixture again. This procedure was repeated to obtain sufficient data to construct binodal curves. For ATPSs with ILs as additives, salt or aqueous ethanol solutions were prepared by adding a certain amount of ILs (e.g., 25 wt% salt + 5 wt% IL). The solutions containing ILs were used to determine binodal curves, following the procedure described here.
2.7. Ultra-HPLC (UHPLC)-MS analysis The grape seed samples were qualitatively and quantitatively analyzed using UHPLC-MS. In this study, catechin, epicatechin, and procyanidin B2 were selected as representatives to evaluate the extraction performance of ATPS. For qualitative analysis, a high-resolution MS (HRMS) technique (Q Exactive Focus, Thermo Fisher, USA) was 155
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employed to identify proanthocyanidins in grape seeds. For quantitative analysis, a triple quadrupole MS system (LCMS-8040, Shimadzu, Japan) was applied to determine the concentrations of three representative proanthocyanidins in extracts. Detailed instrument parameters and experimental conditions are summarized in Supplemental Materials.
ATPS in the current work. Five inorganic salts, such as (NH4)2SO4, Na2SO4, Na2CO3, NaH2PO4, and K2HPO4, were examined as phaseforming components. The binodal curves of ATPSs composed of ethanol, and each salt were determined and illustrated in Fig. 2(a). The binodal curves aid in the comparison of the phase-forming capabilities of different salts. A closer binodal curve position to the origin of coordinates indicates a stronger salt capability to induce ATPS formation. Fig. 2(a) reveals that the phase-forming capability of the investigated salts follows the order: Na2CO3 > Na2SO4 > K2HPO4 > (NH4)2SO4 > NaH2PO4. With the exception of K2HPO4 (ΔGhyd = –2379 kJ/mol), the phase-forming capabilities of these salts agree well with their Gibbs free energies of hydration: Na2CO3 (ΔGhyd = –2045 kJ/mol) > Na2SO4 (ΔGhyd = –1810 kJ/mol) > (NH4)2SO4 (ΔGhyd = –1650 kJ/mol) > NaH2PO4 (ΔGhyd = –830 kJ/ mol) [22,23]. Here, the anion and cation contributed to the salts’ Gibbs free energies of hydration. The correlation between Gibbs free energies of hydration and phase-forming capabilities of salts indicate that salts with high salting-out capability consistently present a strong phaseforming property. To evaluate the extraction performance of different ATPSs, this work measured the K and EE values at a constant ATPS composition, namely, 19 wt% ethanol + 25 wt% salts. Only (NH4)2SO4, K2HPO4, and Na2CO3 formed ATPS when 25 wt% salts were mixed with 19 wt% ethanol. Therefore, 25 wt% Na2SO4 was easily precipitated, whereas 25 wt% K2HPO4 was completely dissolved. Thus, the K and EE values were measured in ATPSs composed of (NH4)2SO4, K2HPO4, and Na2CO3, and the results are illustrated in Fig. 2(b, c). The K and EE values of proanthocyanidins varied considerably with different types of salts. The ethanol/(NH4)2SO4 system yielded higher K (8.94 – 19.13) and EE (> 91.67%) values than ATPSs containing K2HPO4 and Na2CO3. The differences in K and EE values were associated with the stabilities of proanthocyanidins under different solution environments. Proanthocyanidins are acidic polyphenols which are stable in acidic systems, such as ethanol/(NH4)2SO4 system (pH ≈ 5.63) [24]. By contrast, ethanol/K2HPO4 (pH ≈ 9.98) and ethanol/Na2CO3 (pH ≈ 12.22) are alkaline systems that possibly react with acidic constituents, thus destroying the chemical structures of proanthocyanidins. Therefore, (NH4)2SO4 is the best option to extract proanthocyanidins in terms of EE and solute stability.
2.8. Statistical analysis SPSS statistics software version 22.0 (SPSS Inc., Chicago, IL, USA) was used for analysis of variance. Calculated p values were used to analyze whether K and EE were significantly affected by experimental conditions. 3. Results and discussion 3.1. UPLC-MS analysis of proanthocyanidins in grape seed 3.1.1. Chemical composition of grape seeds HRMS can provide rich information (accurate m/z value and MS/MS spectra) for structural elucidation of compounds in complex samples. Hence, this work utilized HRMS to identify proanthocyanidins occurring in grape seed. Fig. 1 illustrates the total ion chromatogram of grape seed extract. By comparing elemental compositions, retention times, and MS/MS spectra with chemical standards, five proanthocyanidins, such as procyanidin B1 (1), catechin (2), procyanidin B2 (3), epicatechin (4), and epicatechin gallate (5), were identified. Fig. 1 depicts the chemical structures of the identified proanthocyanidins. 3.1.2. Quantitative determination of proanthocyanidins in extracts Catechin, epicatechin, and procyanidin B2 were selected as representative proanthocyanidins in quantitative determination experiments. Quantitative analyses of catechin, epicatechin, and procyanidin B2 were performed by multiple-reaction monitoring mode (as displayed in Fig. S1) on a Shimadzu triple quadrupole MS system. The standard curve of each compound was determined in the range of 0.0001–0.01 mg/mL (Figs. S2–S4). The established standard curves are as follows: catechin, y = 70000000x − 1890.7 (R2 = 0.9977); epicatechin, y = 100000000x − 25495 (R2 = 0.9924); procyanidin B2, y = 90000000x − 21223 (R2 = 0.9937).
3.3. Optimization of ethanol and salt concentrations
3.2. Screening of salts
Proanthocyanidins were mainly extracted in the upper ethanol-rich phase (as displayed in Fig. S1). ATPS composition is a major factor
As a low-toxicity organic solvent, ethanol was used to construct
Fig. 1. Total ion chromatogram (TIC) of grape seeds sample and structure of procyanidin B1 (1), catechin (2), procyanidin B2 (3), epicatechin (4), and epicatechin gallate (5). 156
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Fig. 2. Phase diagrams for the different salt-based ATPS at 25 °C (a), and effects of salt type on the partition coefficient (b) and extraction efficiency (c) of proanthocyanidins in ATPS containing 19 wt% salt and 25 wt% ethanol.
Fig. 3. Effects of ethanol concentration (a, b) and (NH4)2SO4 concentration (c, d) on the partition coefficient and extraction efficiency of proanthocyanidins.
influencing extraction performances, namely, K, EE, and Y. Thus, this work evaluated the effects of ethanol and (NH4)2SO4 concentrations by determining the K and EE values of representative proanthocyanidins. The results are presented in Fig. 3(a–d). As observed in Fig. 3(a, b), K and EE values evidently increased as ethanol concentration increased in the range of 21–29 wt% and reached the maximum at 29 wt% ethanol. In practice, a large volume of ethanolrich phase was observed when ethanol concentration increased, promoting the dissolution of several proanthocyanidins and thus enhancing EE. Fig. 3(c, d) indicate that the variation in (NH4)2SO4 concentration in the range of 16–20 wt% also led to the increase in K and EE values. Both
K and EE values of proanthocyanidins reached the maximum at 19 wt% of (NH4)2SO4. Salting-out effect is considered a major driving force enriching solutes in the ethanol-rich phase [13,25]. The increase in salt concentration can increase the salting-out capability, which benefits the distribution of proanthocyanidins in the ethanol-rich phase. Thus, K and EE values increased in the range of 16–19 wt% of (NH4)2SO4 as salting-out capability was enhanced. However, excessive amounts of salt (> 19 wt%) led to the decrease in water in the ethanol-rich top phase, and this condition can weaken the affinity of ethanol-rich phase to proanthocyanidins and thus cause the decrease in K and EE values [11]. Therefore, 29 wt% ethanol and 19 wt% (NH4)2SO4 were employed to construct ATPSs in subsequent experiments. 157
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Fig. 4. Effects of IL anions and cations on K (a) and EE (b) of proanthocyanidins (21 wt% ethanol + 19 wt% (NH4)2SO4 + 5 wt% IL).
3.4. Effect of ILs
formation of IL aggregates or clusters weakens the interaction between solutes and ILs, explaining the decrease in K and EE values of ATPS composed of [C8mim][BF4] and [C10mim][BF4]. Therefore, [C6mim] BF4 was selected as the most suitable adjuvant as this IL provided the highest K (Kcatechin = 32.87, Kepicatechin = 20.86, Kprocyanidin B2 = 27.50) and EE (> 96.35%) values.
This work used ILs as adjuvants to manipulate the partition behavior of proanthocyanidins in ethanol-based ATPSs. To improve the extraction performance of ATPSs, this study thoroughly investigated the effects of 10 ILs with different anions and cations. Table S1 summarizes the full names and chemical structures of the used ILs. Using an ATPS containing 21 wt% ethanol and 19 wt% (NH4)2SO4 as the control system, the K and EE values of proanthocyanidins were determined with 5 wt% IL additions, and the results are presented in Fig. 4(a–b). ILs were mainly present in the ethanol-rich phase. The anion effects of ILs were evaluated by changing the anions [BF4]–, [NO3]–, Br–, TOS–, [SO3]–, and [HSO4]– while keeping the [C4mim]+ cation. Fig. 4(a, b) illustrate that the K and EE values of proanthocyanidins varied considerably with different anions. Except for [TOS]– and [HSO4]–, the addition of ILs containing other anions promoted the enrichment proanthocyanidins in the ethanol-rich phase, as indicated by high K and EE values. For the six tested ILs with different anions, the K and EE values increased in the following order: [C4mim] BF4 > [C4mim]NO3 > [C4mim]Br > [C4mim][TOS] > [C4mim] [CH3SO3] > [C4mim][HSO4]. This result suggests that anion-type ILs play important roles in manipulating the partition behavior of solutes when using ILs as adjuvants. In comparison with the ATPS without IL addition (K < 3.09, EE < 64.49%), [C4mim]BF4 provided the highest K (Kcatechin = 11.83, Kepicatechin = 9.85, and Kprocyanidin B2 = 10.50) and EE (> 92.08%) values. Thus, [BF4]– was selected as the suitable anion for subsequent evaluation. In the current work, the IL cation effects were analyzed by changing the cations [Cnmim]+ (n = 2, 4, 6, 8, 10) while maintaining [BF4]– anions. Fig. 4(a, b) demonstrate the variation in K and EE values of proanthocyanidins with five ILs containing different cations. Proanthocyanidins can migrate to the ethanol-rich phase in the following order: no IL < [C2mim]BF4 < [C4mim]BF4 < [C10mim] BF4 < [C8mim]BF4 < [C6mim]BF4. Increasing the alkyl chain length of IL cation can increase the hydrophobicity of ILs [26]. Increasing hydrophobicity (n = 2–6) initially enhanced the molecular interactions between solute and IL. Such interaction promoted the partition of proanthocyanidins in the top phase containing IL and ethanol [27]. However, a strong hydrophobicity (n = 8, 10) can cause the self-aggregation of ILs, thus forming IL aggregates or clusters [28]. The
3.5. Optimization of extraction conditions Extraction parameters, namely, the IL dose, solid–liquid ratio, extraction time, temperature, and pH, exert significant effects on the extractability of ATPS. This work used a single-factor experiment to optimize these important factors, and catechins, epicatechin, and procyanidin B2 were used as representatives to evaluate the extraction performance. 3.5.1. Effect of IL dose Apart from the chemical structures of IL, IL dose also substantially influenced the improvement of EE. Here, we further investigated the dose effect of [C6mim]BF4 on EE within the range of 1–5 wt%, and the results are presented in Fig. 5(a, b). Both figures indicate that the K and EE values of proanthocyanidins increased as IL content increased up to 4 wt%. Such an increase may also be attributed to the enhancement of affinity of ethanol-rich phase to the solute caused by addition of IL [18]. Beyond 4 wt%, a higher amount of [C6mim]BF4 caused no further enhancement in EE. Therefore, 4 wt% [C6mim]BF4 was selected and added to 29 wt% ethanol/19 wt% (NH4)2SO4 ATPS for further experiments. 3.5.2. Effect of solid–liquid ratio. Solid–liquid ratio (g/mL), which is a major factor affecting EE, was optimized in the ratio of 1:10–1:50, and the results are presented in Fig. 6. As indicated in the figure, solid–liquid ratio at 1:20 presented the highest K (43.53–99.60) and EE (> 98.50) values. When the solid–liquid ratio exceeded 1:20, the K and EE values of catechin and procyanidin decreased significantly (p < 0.05), whereas those of epicatechin showed no evident change (p > 0.05). Solid–liquid ratio is an important parameter during extraction. For a certain amount of sample (1 g of grape seed in this work), a low volume of solvent (< 20 mL) cannot completely extract the target solute from the solid sample 158
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Fig. 5. Effects of IL dose on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; solid-liquid ratio: 1:20 mg/mL; T = 30 °C; pH: no adjustment; time: 20 min).
Fig. 6. Effects of solid-liquid ratio on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; T = 30 °C; pH: no adjustment; time: 20 min).
Fig. 7. Effects of temperature on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; pH: no adjustment; time: 20 min).
matrix. However, an excess volume (> 20 mL) can cause a dilution effect on solutes although they can be completely extracted. Thus, dilution effect was the major reason for the reduced K and EE values when the solid–liquid ratio exceeded 1:20 [29]. Thus, 1:20 was selected as the best solid–liquid ratio and used for subsequent studies.
extraction of proanthocyanidins. As high temperature generally benefits the mass transfer of solute, raising EE to the highest value possible is suggested under mild temperature (e.g., 40 °C in this work). However, high temperature also possibly induces the degradation of unstable compounds and evaporation of solvents (e.g., ethanol and water), both of which can lead to the decrease in extraction performance. In addition, high temperature must consume additional energy. Therefore, 40 °C was optimized as the suitable extraction temperature.
3.5.3. Effect of temperature This research analyzed the effect of temperature on the extraction performance in the range of 20–60 °C, and the results are presented in Fig. 7. As depicted in the figure, increasing temperature from 20 °C to 40 °C enhanced the K and EE values of proanthocyanidins, whereas both values decreased at a temperature beyond 40 °C. These results indicate that high temperature is unsuitable for the aqueous two-phase
3.5.4. Effect of pH This study investigated the effects of pH on the K and EE values of proanthocyanidins in ATPS, and the results are illustrated in Fig. 8. HCl and NaOH aqueous solutions were used to adjust the system pH in the 159
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Fig. 8. Effects of pH on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; T: 40 °C; time: 20 min).
Fig. 9. Effects of extraction time on K (a) and EE (b) proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4; IL dose: 4 wt%; solid-liquid ratio = 1:20 mg/mL; T: 40 °C; pH: 5).
range between 3.0 and 10.0. Fig. 8 reveals that acidic conditions (pH < 7) provided higher K and EE values than alkaline conditions. At pH 5, the K and EE values reached their maximum. By changing the electrical charge form of solutes, pH can affect the electrostatic interactions between solutes and solvents and thus cause a significant effect on the partition behavior of solutes in ATPS [30]. Essentially, proanthocyanidins are oligomeric phenolics with weak acidity and are stable in weak acidic environment. In alkaline solutions, the phenolic hydroxyl group of proanthocyanidins can be dissociated, resulting in an adverse effect on the partition of proanthocyanidins in the ethanol-rich phase. As a result, pH 5.0 was selected for the following experiments.
the highest Y of catechin (1.21 mg/g), epicatechin (1.22 mg/g), and procyanidin B2 (0.14 mg/g). Among the conventional solvents, 70% methanol exhibited the best performance, but its Y value remained remarkably lower than that of ATPS (p < 0.05). The amounts of reagent consumptions were also compared, as indicated in Fig. 10(b). The ATPS method consumed the lowest amount of volatile organic solvent (2.9 g of ethanol) over other methods. Considering the total amount of consumed reagent, the amounts of consumed in ATPS were similar with those of 70% ethanol and 70% methanol (p > 0.05) and lower than those of other organic solvents (p < 0.05). Although the proposed ATPS failed to achieve the lowest cost, it still featured a high EE and consumed minimal amount of organic solvent. Thus, ATPS remains a good choice for extracting proanthocyanidins from grape seeds.
3.5.5. Optimization of extraction time The effect of extraction time on the EE of proanthocyanidins was also studied in the range of 5–60 min, and the results are indicated in Fig. 9. The maximum K (Kcatechin = 97.45, Kepicatechin = 65.78, and Kprocyanidin B2 = 148.55) and EE (> 99.01%) were obtained at 20 min. When the extraction time exceeded 20 min, the K and EE values decreased. Such decreases may have resulted from solute degradation and ethanol evaporation under prolonged ultrasound condition [31]. Hence, 20 min was selected as the proper extraction time.
4. Conclusions In this work, an ATPS based on ethanol/(NH4)2SO4 was proposed to extract proanthocyanidins from grape seeds. By adding 5 wt% [C6mim] BF4 as an adjuvant to the initial ATPS composed of 21 wt% ethanol and 19 wt% (NH4)2SO4, remarkable increases in K (average from 2.55 to 27.08) and EE (average from 59.65% to 97.12%) were observed. Optimal extraction performance was obtained with 29 wt% ethanol, 19 wt% (NH4)2SO4, 4 wt% [C6mim] BF4, solid–liquid ratio = 1:20 g/ mL, T = 40 °C, pH = 5.0, and t = 20 min. As a result, the Y of three representative proanthocyanidins, namely, catechin, epicatechin, and procyanidin B2, reached 1.21, 1.22, and 0.14 mg/g, respectively. On the contrary, the EE of three compounds amounted to values up to 99%. Compared with conventional solvents (pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate), the proposed ATPS using ILs as adjuvants achieved a higher Y but consumed less amount of organic solvents. In conclusion, ethanol-based
3.6. Comparison of different solvents The optimized ATPS (29% ethanol + 19 wt% (NH4)2SO4 + 4% [C6mim]BF4, w/w) was compared with other traditional solvents (pure water, methanol, 70% methanol, ethanol, 70% ethanol, isopropanol, and ethyl acetate) regarding their Y of proanthocyanidins, and the results are presented in Fig. 10(a). All extractions were performed under identical conditions: solid–liquid ratio = 1:20, T = 40 °C, pH = 5.0, and t = 20 min. Fig. 10(a) illustrates that the optimized ATPS provided 160
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[5] [6] [7]
[8] [9] [10] [11] [12]
[13] [14] [15]
[16] [17]
Fig. 10. (a) The effect of different solvent systems on the extraction yield of proanthocyanidins (ATPS: 29% ethanol + 19% (NH4)2SO4 + 4 wt% IL, w/w); (b) the consumption of different solvent (pure water was excluded).
[18] [19]
ATPSs using ILs as adjuvants provide a promising strategy to extract and recover value-added compounds from waste residues of food processing. In future studies, a method must be developed to recover and reuse solvents (e.g., IL, salt, and ethanol). The proposed method benefits the minimization of extraction cost.
[20] [21] [22]
Acknowledgements
[23]
The present work was financially supported by the Applied Basic Research Project of Yunnan Province (No. 2018FD033), National Natural Science Foundation of China (No. 21775058) and Guangdong Social Development Program (2016108101043).
[24]
Appendix A. Supplementary material
[25] [26]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.seppur.2019.05.089.
[27]
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