Ionic liquid-assisted subcritical water enhances the extraction of phenolics from brown seaweed and its antioxidant activity

Separation and Purification Technology xxx (2017) xxx–xxx

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Ionic liquid-assisted subcritical water enhances the extraction of phenolics from brown seaweed and its antioxidant activity Tri Vo Dinh a, Periaswamy Sivagnanam Saravana a, Hee Chul Woo b, Byung Soo Chun a,⇑ a b

Department of Food Science and Technology, Pukyong National University, 45 Yongso-ro, Namgu, Busan 608-737, Republic of Korea Department of Chemical Engineering, Pukyong National University, 365 Sinseon-ro, Namgu, Busan 608-737, Republic of Korea

a r t i c l e

i n f o

Article history: Received 11 May 2017 Received in revised form 31 May 2017 Accepted 4 June 2017 Available online xxxx Keywords: Subcritical water extraction Brown seaweed Ionic liquid Phenolic compounds Antioxidant activity

a b s t r a c t Natural phenolic substances have recently been recovered from brown seaweeds using various techniques. Particularly, subcritical water extraction (SWE) has been developed to replace conventional processes because of its sustainable properties. In addition, ionic liquids (IL) have recently been proved to have potential capabilities in extracting bioactive compounds from natural resources. This study explores the power of the IL-assisted SWE method (SWE + IL) in obtaining different phenolic compounds from the brown seaweed Saccharina japonica. The imidazolium-based IL 1-butyl-3-methylimidazolium tetrafluoroborate [C4C1im][BF4] was provided as a catalyst in the SWE system at different temperatures (100– 250 °C) and concentrations (0.25–1.00 M) to examine its thermal and quantitative effects. Solid–liquid extraction (SLE) and SWE were additionally provided as references for the considered method. Highperformance liquid chromatography analysis of the SWE and SWE + IL extracts showed high contents of gallic, chlorogenic, gentisic, protocatechuic, p-hydroxybenzoic, and caffeic acids along with minor amounts of vanillic and syringic acids. The highest total content of phenolics was determined at 175 °C in two SWE techniques; however, in conventional SLE, phenolic compounds were poorly detected. Various antioxidant-determining assays such as 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay, 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay, total antioxidant capacity assay, and ferric reducing antioxidant power assay indicated the high antioxidant activity of extracting samples from SWE and SWE + IL; however, limited capacity was observed from conventional SLE. Correlation testing and principal component analysis indicated a strong connection between phenolic content and antioxidant activity, thereby demonstrating the high quality of phenolic bioactive extracts from brown seaweeds. Ó 2017 Elsevier B.V. All rights reserved.

1. Introduction For over decades, brown seaweeds have been increasingly consumed worldwide for their richness in polysaccharides, proteins and amino acids, dietary fibers, minerals, polyunsaturated fatty acids, vitamins, fucoxanthin, and natural antioxidant compounds [1–3]. The functional bioactivities (such as antitumor, antioxidant, anti-inflammatory, antiobese, hepatoprotective, antiangiogenic, and antifungal effects) of numerous species in brown seaweeds have been identified [4–7]. With a large aquaculture size and rapid growing rate, brown seaweeds are ideally considered as an alternative resource for human nutrients, cosmetics, and medical supplements in an effort to prevent terrestrial plants from overexploitation [8,9]. ⇑ Corresponding author.

Natural phenolic compounds, mostly including one aromatic ring with two or more hydroxyl groups and functional derivatives, have been comprehensively classified as typical antioxidant compounds [10–12]. The phenolic unique structure can play the role of (i) a hydrogen atom donor that can bind to lipid radicals and decrease the rate of the autoxidation process [13] or (ii) an electron donor that, after scavenging the free oxidants, can turn into a radical cation with higher stability so that it cannot react with the substrates [14]. Recently, various phenolic substances in different types of seaweeds have been determined. For instance, 16 different species, including red, green, and brown seaweeds in the Danish coastal area, were defined as rich in gallic, protocatechuic, gentisic, p-hydroxybenzoic, chlorogenic, vanillic, syringic, caffeic, salicylic, coumaric, and ferulic acids (Fig. 1) [15]. Marine seaweeds, by means of their massive aquaculture scale, can contribute an enormous volume of natural phenolic compounds for futuristic applications.

E-mail address: [email protected] (B.S. Chun). http://dx.doi.org/10.1016/j.seppur.2017.06.009 1383-5866/Ó 2017 Elsevier B.V. All rights reserved.

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T. Vo Dinh et al. / Separation and Purification Technology xxx (2017) xxx–xxx

Fig. 1. Structures of common phenolic substances extracted from the Danish coastal seaweeds.

Several procedures such as solid–liquid extraction (SLE), enzyme-assisted extraction, microwave-assisted extraction, ultrasound-assisted extraction, and subcritical water extraction (SWE) have been carried out in an attempt to extract natural phenolic compounds [16–22]. Among these techniques, SWE has currently received much attention in extracting secondary metabolites from plants and algae [22–24]. Under regular conditions, water has a high polarity, which is not suitable for the removal of organic compounds from raw materials [23]. However, under subcritical environments where the temperature and pressure are significantly increased, an excessive reduction in the dielectric constant (e) of water occurs (e = 80 at 25 °C to e = 27 at 250 °C and 50 bar) [23,25]. This phenomenon results in a noteworthy increase in diffusivity, which allows water to act like an organic solvent in extracting bioactive compounds. Nevertheless, a clean and green process with non-flammable and non-toxic solvent,

quick reaction time, and excellent extraction capacity has designated SWE as a highly preferable technique for the extraction of valuable products [24,26]. With the urgent demand for sustainable technology, alternative green solvents are being highly exploited to replace conventional harmful reagents. Ionic liquids (IL), in particular, have been successfully applied in several food and medical extracting methods [27,28]. The physicochemical properties of IL, that is, negligible vapor pressure, high thermal and electrochemical stability, wide solvating range, and strong miscibility with aqueous substances, are sufficient to extract functional bioactive compounds [29,30]. Different techniques have already been applied together with IL in microwave and ultrasound complex systems [31], liquid–liquid microextraction [32], IL-based silicas and polymers [27], etc. In the IL-based ultrasound-assisted extraction of phenolic compounds from S. japonica [33], 1-butyl-3-methylimidazolium

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this limitation. Toward this aim, a combination of SWE assisted by IL (SWE + IL) might be a possible approach for IL application. In addition, high thermal stability up to 400 °C could be another appropriate property of IL when applying in SWE [38]. In this study, the SWE method was conducted to extract phenolic substances from S. japonica. A wide range of temperatures from 100 to 250 °C was used to examine the impact of high thermal conditions on final product quality. IL [C4C1im][BF4] at concentrations from 0.25 to 1.00 M were subsequently applied as a catalyst solvent in the SWE + IL process. Moreover, SLE with conventional solvents and IL was simultaneously provided. Extract samples were evaluated by their phenolic content, individual phenolic compositions, and antioxidant bioactivity. Fig. 2. Chemical structure of [C4C1im][BF4] IL.

2. Materials and methods tetrafluoroborate [C4C1im][BF4] (with the structure shown in Fig. 2) was identified as the most adequate catalyst for phenolic extraction. A similar process according to Lu et al. [34] demonstrated that tetrafluoroborate [BF 4 ] was the most suitable anion among chloride [Cl], bromide [Br], and hexafluorophosphate [PF in 6] alkylimidazolium-based IL for the extraction of phenolic alkaloids from Nelumbo nucifera. Vidal et al. [35] also mentioned the high ability of imidazolium-based tetrafluoroborate IL in extracting p-hydroxybenzoic acid, phenol, and tyrosol at temperatures from 15 to 40 °C. However, most referential studies were carried out at ambient temperature and pressure. Up to the present time, the combination of IL in SWE has been used for the recovery of lipids from the microalgae species Scenedesmus [36,37]. Therefore, the effects of IL under pressurized hot situations are not well investigated. On the other hand, high viscosity was noted as a great disadvantage of IL [30]. Pure IL with long alkyl chain and large size of non-polar part could lead to an increase in the van der Waal’s interactions, which is not a compatible property of an ideal extracting medium. Hence, mixing with a low-viscosity solvent such as water could minimize

2.1. Sample preparation S. japonica was freshly collected in June 2016 in Guemil-eup, Wando-gun, Jeollanam-do, South Korea. The sample was carefully washed with tap water to remove contamination before being freeze-dried at 110 °C for 72 h using an Eyela FDU-2100 model (Tokyo Rikakikai Co., Ltd., Japan). Dried seaweed was then ground using an SMKA-4000 model blender (PN Co., Ltd., Korea) and sieved to a mesh size of 710 lm. Fine powder was stored below 20 °C prior to extraction. 2.2. Chemicals Phenolic standards such as gallic acid monohydrate (98.0%), chlorogenic acid (95.0%), gentisic acid (98.0%), protocatechuic acid (97.0%), p-hydroxybenzoic acid (99.0%), caffeic acid (95.0%), and syringic acid (98.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1-Butyl-3-methylimidazolium

Fig. 3. Schematic diagram of the laboratory-scale subcritical water apparatus.

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T. Vo Dinh et al. / Separation and Purification Technology xxx (2017) xxx–xxx Table 1 TPC of S. japonica extracts using SLE with various solvents. TPC (mg PGE/g DW) Solvent

Conventional extraction

Water Ethanol Acetone Dichloromethane Diethyl ether [C4C1im][BF4]

2.42 ± 0.01a 0.33 ± 0.01d 0.32 ± 0.01d 0.21 ± 0.01e 0.56 ± 0.01c 2.09 ± 0.02b

Data are expressed as the mean of triplicate ± SD. Subscript letters within a column indicate significant differences between samples at the level of p < 0.05. Fig. 4. TPC of the S. japonica extracts at various concentrations of [C4C1im][BF4] in SWE + IL. Subscript letters indicate significant differences between samples at the level of p < 0.05.

Table 2 TPC of S. japonica extracts using SWE and SWE + IL. TPC (mg PGE/g DW) Temperature (°C)

SWE

SWE + IL

100 125 150 175 200 225 250

10.71 ± 0.49 g 22.58 ± 0.38f 25.55 ± 0.53e 39.27 ± 0.38b 39.52 ± 0.27a 38.10 ± 0.35c 28.42 ± 0.23d

8.02 ± 0.57g 23.12 ± 0.31f 39.39 ± 0.30b 39.55 ± 0.46a 37.72 ± 0.38c 34.72 ± 0.49d 32.69 ± 0.42e

Data are expressed as the mean of triplicate ± SD. Subscript letters within a column indicate significant differences between samples at the level of p < 0.05.

tetrafluoroborate [C4C1im][BF4] (99.0%) was purchased from Chem-Tech Research Incorporation (Namyang, South Korea). Nitrogen gas (99.99%) was obtained from KOSEM (Yangsan, South Korea). All the reagents used were of either analytical or highperformance liquid chromatography (HPLC) grade. 2.3. Solid–liquid extraction 2.3.1. Conventional solvent extraction Conventional solvents used for the extraction of phenolic compounds included distilled water, ethanol, acetone, dichloromethane, and diethyl ether, varying from high polarity to very low polarity. First, a solid–liquid mixture at a ratio of 1:32 (w/v) was prepared. Then, the mixture was poured into a 100 mL glass beaker prior to magnetic stirring (24 h, 500 rpm, room conditions). Finally, aqueous extracts were obtained by filtering the supernatant through membrane filters (0.45 lm pore size, Fisher Scientific) and storing at 4 °C prior to analysis. 2.3.2. IL extraction An aqueous solution of 0.5 M [C4C1im][BF4] in distilled water was prepared as an alternate solvent in SLE. Extracting conditions were constantly maintained as follows: 1:32 w/v mixing ratio, 24 h stirring process, and 500 rpm stirring speed under room conditions. Finally, aqueous extracts were thoroughly filtered and stored at 4 °C prior to analysis. 2.4. Subcritical water extraction SWE was carried out using a laboratory-scale subcritical water apparatus (Phosentech, Daejeon, South Korea), as described in Fig. 3. A mixture of 5 g seaweed powder and 160 mL distilled water was poured into a cylindrical reactor (200 cm3) made of C276 alloy. The reactor was then tightly closed before increasing the

temperature using an electric heater. A four-blade agitator was stirred at 200 rpm rotation speed to homogenize the inner solution. Working pressure (50 bar) was maintained by filling compressed nitrogen gas (99.99% purity) into the reactor. Seven temperature conditions (from 100 to 250 °C at 25 °C intervals) were separately applied to investigate the thermal impact on phenolic extraction capacity of SWE. The preheating period to reach the desired temperature fluctuated from 12 to 35 min, proportionally with temperature. The reaction time for each run was 5 min, and hot reacting solutions were immediately collected by passing through a circulated cooler system. Finally, aqueous extracts were vacuum filtered and preserved below 4 °C prior to analysis. 2.5. Ionic liquid-assisted subcritical water extraction An IL solution of 0.5 M [C4C1im][BF4] in distilled water was alternatively used as a catalyst in SWE + IL experiments. Pressure, mixing ratio, and stirring speed were correspondingly maintained as in SWE along with a temperature range of 100–250 °C at 25 °C intervals. The preheating period to reach the desired temperature fluctuated from 14 to 40 min, proportionally with temperature. Collected extracts were vacuum filtered and then freeze-stored at 4 °C prior to analysis. The most suitable temperature was then selected to apply the molarity variation impact of [C4C1im][BF4] solvent (0.25–1.00 M) on phenolic extraction in SWE + IL. 2.6. Total phenolic content determination The total phenolic content (TPC) of extracts was determined using the Folin–Ciocalteu reagent (FCR) based on the method of Matanjun et al. [39], with a few modifications. Briefly, an aliquot of 1 mL extract was mixed with 1 mL of 0.2 N FCR prepared solution. The mixture was then incubated in the dark for 4 min before adding 0.8 mL of 7.5% sodium carbonate. The solution mixture was then kept in the dark for 2 h before centrifuging at 5500 rpm and 25 °C for 10 min to separate all the residues. The supernatant was then collected, and the absorbance at 765 nm wavelength was measured using multi-mode microplate readers (Synergy 2, BioTeK, Vermont, USA). Distilled water was used as the blank, and phloroglucinol was used as the reagent standard. The final phenolic content was expressed as mg phloroglucinol equivalent/ g dry weight (mg PGE/g DW). 2.7. High-performance liquid chromatography analysis The HPLC system used was the Hitachi HPLC system (Hitachi America Ltd., New York, USA) with a Hitachi L-2130 pump, an L-

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T. Vo Dinh et al. / Separation and Purification Technology xxx (2017) xxx–xxx Table 3 Linear regression equations of the eight phenolic calibration curves and retention time of each standard. Standard

Retention time

Equation

R2 value

Gallic acid Chlorogenic acid Gentisic acid Protocatechuic acid p-Hydroxybenzoic acid Vanillic acid Caffeic acid Syringic acid

4.90 ± 0.21 6.29 ± 0.33 8.59 ± 0.35 9.22 ± 0.41 12.45 ± 0.12

y = 250703x  865011 y = 109550x + 106 y = 10840x + 13871 y = 152691x  661137 y = 11575x  65179

R2 = 0.9969 R2 = 0.9981 R2 = 0.9999 R2 = 0.9997 R2 = 0.9993

13.25 ± 0.23 15.46 ± 0.67 16.18 ± 0.85

y = 163514x  106 y = 255918x  106 y = 276614x + 35437

R2 = 0.9959 R2 = 0.9954 R2 = 1.0000

Data are expressed as the mean of retention time ± SD.

2420 UV–VIS detector, and Hitachi EZ-Chrom Elite software. A Macherey-Nagel Nucleosil 100-5 C8 reversed-phase column (250 mm  4.6 mm, 5 lm) was selected to determine phenolic compounds. The analysis method was programmed based on the method of Saravana et al. [40], with some modifications. The detection wavelength was selected as 280 nm, which is sufficient for phenolic detection. Eluent solvents included water with 0.1% glacial acetic acid (solvent A) and acetonitrile with 0.1% glacial acetic acid (solvent B). The gradient of the two eluents was programmed as follows: 8% solvent B, 92% solvent A (0–2 min); 10% solvent B, 90% solvent A (2–25 min); 30% solvent B, 70% solvent A (25–27 min) and then returning to the initial percentage of 8% solvent B and 92% solvent A (27–30 min). The total running time for each trigger was 30 min with a mobile-phase flowrate of 0.8 mL/min. Phenolic compounds were detected by matching their spectrum and retention time with reference phenolic standards. Quantification of each individual compound was calculated using corresponding standard calibration curves and expressed as milligram/gram dry weight (mg/g DW). 2.8. Antioxidant activity analysis 2.8.1. 2,2-Diphenyl-1-picrylhydrazyl radical scavenging activity The antioxidant activities of all extracts were determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay as described by Pereira et al. [41], with slight modifications. Briefly, 3.9 mL of 60 lM DPPH solution (ethanol as solvent) was thoroughly mixed with 0.1 mL aqueous extracting sample. The mixture was then kept in the dark for 30 min before the absorbance was measured at 517 nm wavelength. Trolox solution was prepared in ethanol and used as an equivalent calibration standard. The final results were expressed as milligram Trolox equivalent/gram dry weight (mg TE/g DW). 2.8.2. 2,20 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical scavenging assay The 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS.+) radical scavenging assay was carried out based on the method of Foo et al. [42], with a few changes. Briefly, 7 mM ABTS.+ solution (0.0384 g ABTS/10 mL distilled water) and 2.45 mM potassium persulfate solution (0.00662 g K2S2O8/10 mL distilled water) were prepared and mixed in a 1:1 equal volume. The solution mixture was then kept in the dark for 16 h at room temperature to generate ABTS.+ radical cations. The desired solution was then stored at 4 °C. On the day of the assay, the absorbance of ABTS.+ solution was measured at 734 nm wavelength. The OD value of the testing solution was maintained at 0.7 ± 0.02 by diluting in ethanol. To determine antioxidant activity, 0.1 mL sample was mixed with 3.9 mL ABTS+ solution. The mixture was then kept in

the dark for 6 min. The absorbance measured at 734 nm wavelength, expressed as milligram gallic acid (mg GAE/g DW). Distilled water in the blank.

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of the reacting mixture was and the final results were equivalent/gram dry weight ABTS.+ solution was used as

2.8.3. Total antioxidant capacity The total antioxidant capacity (TAC) assay was carried out according to the method of Hifney et al. [43]. Briefly, 0.1 mL extract was added to 3 mL prepared solution containing 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The mixture was incubated at 95 °C for 90 min in a water bath. The absorbance of the reacting mixture was measured at 695 nm wavelength. Ascorbic acid was used as the reference standard, and the final results were expressed as milligram ascorbic acid equivalent/gram dry weight (mg AAE/g DW). 2.8.4. Ferric reducing antioxidant power The ferric reducing antioxidant power (FRAP) assay was conducted according to the method of Thaipong et al. [44]. Briefly, 300 mM acetate at pH 3.6, 10 mM 2,4,6-tris[2-pyridyl]-s-triazine (TPTZ) in 40 mM hydrochloric acid, and 20 mM ferric chloride solution were prepared on the day of the assay. FRAP reagent was generated by mixing 200 mL acetate buffer with 20 mL prepared TPTZ solution and ferric chloride solution, together with 24 mL distilled water. The mixing process was maintained at 37 °C, and fresh mixture should be straw color. To detect antioxidant activity, 0.15 mL extracting sample was mixed with 2.85 mL FRAP reagent, and then the mixture was incubated at 37 °C in the dark for 30 min before measuring the absorbance at 593 nm wavelength. Trolox reagent was used as the reference standard. The final results were calculated and expressed as milligram Trolox equivalent/gram dry weight (mg TE/g DW). 2.9. Statistical analysis One-way analysis of variance (ANOVA) was performed to analyze statistical mean values. Triplicate data (n = 3) were collected for all analysis procedures and expressed as mean ± standard deviation using SPSS software (version 20 for Windows, IBM, Chicago, IL, USA). Statistical analysis was performed using the Tukey test in which p < 0.05 was considered as sufficient. Pearson correlation analysis and principal component analysis (PCA) were performed using XLSTAT (version 2015.4, Redmond, Washington, USA). 3. Results and discussion In this study, we focused on the extraction of natural phenolic compounds using various extraction methods. The objectives were to evaluate (i) the ability of SWE and SWE + IL to extract phenolic compounds and (ii) the potential antioxidant activity of collected extracts in comparison with conventional SLE. 3.1. SLE of phenolic compounds 3.1.1. Conventional solvent extraction Five solvents (i.e., water and four organic solvents of dissimilar polarities) were included in the experimentations. The TPCs are shown in Table 1. Water gave the highest TPC value (2.42 ± 0.01 mg PGE/g DW) among ethanol (0.33 ± 0.01 mg PGE/g DW), acetone (0.32 ± 0.01 mg PGE/g DW), dichloromethane (0.21 ± 0.01 mg PGE/g DW), and diethyl ether (0.56 ± 0.01 mg PGE/g DW). A similar observation was reported by Nihal et al. [45], in which the polyphenol content in mate tea was obtained with highly polar solvents such as water or mixtures of water with

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ethanol, acetone, dimethylformamide, and methanol. Therefore, solvent polarities might have significant impacts on the final phenolic content of the extracts. On the other hand, different phenolics might have different solubilities with the solvents used. As reported by Mota et al. [46], hydroxybenzoic phenolics such as gal-

lic acid and salicylic acid were highly soluble in water whereas phenylpropenoic acids such as ferulic acid, caffeic acid, and trans-cinnamic acid were poorly dissolved. Diethyl ether in this study performed higher TPC, as compared with less non-polar solvent such as acetone, dichloromethane and ethanol, might be

Fig. 5. HPLC chromatographs of (A) phenolic standards including gallic(1), chlorogenic(2), gentisic(3), protocatechuic(4), p-hydroxybenzoic(5), vanillic(6), caffeic(7), and syringic(8) acids; (B) SLE extract with water as the solvent; (C) SLE extract with 0.5 M [C4C1im][BF4] in water as the solvent; (D) SWE extract at 175 °C; E) SWE + IL at 175 °C; (F) SWE extract at 200 °C; and (G) SWE + IL extract at 200 °C.

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because of its high solvation with hydrophobic phenolic compounds. Hence, the phenolic composition of S. japonica was also believed to have a certain influence on the final extraction efficiency of SLE. Details of phenolic composition are discussed in the HPLC analysis section. 3.1.2. IL extraction As shown in Table 1, IL in SLE resulted in lesser phenolic content (2.09 ± 0.02 mg PGE/g DW) when compared with water. This is in contrast with the previous conclusion by Fan et al. [47], who believed that hydrogen-bonding interactions between hydroxyl groups in phenols and [BF 4 ] anions as well as hydrophobic interactions between imidazolium cations and phenols would contribute to increasing the extraction capacity of the IL–water aqueous solution. Vidal et al. [35] also indicated a high efficiency of the imidazolium-based water aqueous system to obtain hydroxybenzoic acid. However, the solid–liquid mixing ratio as well as the particle size of samples might be the reason for the low recovery of phenolic compounds in extracts [48]. In this study, IL at room temperature were not a sufficient catalyst for water in extracting phenolic compounds. 3.2. SWE of phenolic compounds Temperature, pressure, modifiers/additives, and reaction time can have remarkable effects on the final properties of extracting products using SWE [25]. Altogether, temperature is considered a critical parameter that can create significant differentiation in the extraction efficiency of water [25]. In this study, a wide array of temperatures from 100 to 250 °C was used in the experiments. The phenolic contents are reported in Table 2. From 100 to 175 °C, the TPC values steadily increased from 10.71 ± 0.49 mg PGE/g DW to 39.27 ± 0.38 mg PGE/g DW and remained constant at approximately 39 mg PGE/g DW from 175 to 225 °C before being rapidly reduced to 28.42 ± 0.23 mg PGE/g DW at 250 °C. The disparity in TPC between SWE at 200 °C and SLE could reach approximately 16-fold with water as the solvent or 19-fold with 0.5 M [C4C1im][BF4] in water as the solvent. The decrease in the dielectric constant of water in SWE could be the reason for the high shift of TPC when compared with SLE. The obtained results are somehow opposite to those of the previous study on extraction of phenolics in Irish seaweeds, where the phenolic content in Fucus spiralis using SWE (90.79 ± 1.16 mg PGE/g DW) was lower than in SLE with water as the solvent (130.58 ± 2.78 mg PGE/g DW) [49]. Besides, the reduction of phenolic content at high temperature (approximately 250 °C) might be linked to the degradation of phenolic compounds. A similar observation was reported by HassasRoudsari et al. [50] when trying to extract bioactive compounds from canola meal. The TPC from canola extracts was observed to be higher at 110 °C but lower at 160 °C, which was believed to be due to the degradation of phenolic compounds at 160 °C. Moreover, semi-polar phenolic compounds in canola extracts were likely to be more soluble at the lower thermal condition of 110 °C. The review of Teo et al. [25] on SWE also mentioned that thermal impact would lead to the degradation of certain classes of compound at certain temperatures. Details of the thermal impact on phenolic composition were precisely determined in HPLC analysis. 3.3. SWE+IL of phenolic compounds Different studies on the decomposition of [C4C1im][BF4] have similarly concluded that the thermal stability of the imidazolium IL used was generally high. Fredlake et al. [51] measured the decomposition temperature of imidazolium-based IL using thermal gravimetric analysis (TGA). The results for the onset and start

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temperatures of [C4C1im][BF4] were 380 and 285 °C, respectively. Ammam and Fransaer [52] also recorded that the [C4C1im] cation starts to decompose at temperatures above 300 °C, and for [C4C1im][BF4] the major loss in weight was detected from 400 to 600 °C by TGA. Thus, at temperatures below 250 °C, [C4C1im] [BF4] can be utilized as a safe controlled catalyst from SWE + IL without any conversion into undesired components. In Table 2, TPC values appeared similarly between SWE with and without the assistance of [C4C1im][BF4]. The highest content was identified at 175 °C with an equivalent amount of 39.55 ± 0.46 mg PGE/g DW. However, TPC nearly reached the maximum value (39.39 ± 0.30 mg PGE/g DW) at 150 °C in SWE + IL; in SWE, the value was 25.55 ± 0.53 mg PGE/g DW at 150 °C and kept increasing to the maximum value of 39.52 ± 0.27 mg PGE/g DW equivalent at 200 °C. Furthermore, the rate of decrease in TPC in samples with the presence of IL was slower at 250 °C (32.69 ± 0.42 mg PGE/g DW, while in SWE the value was only 28.42 ± 0.23 mg PGE/g DW). Hence, IL added into water appeared to increase the extraction ability of the entire subcritical solvent. This phenomenon might be due to the high solvating property of IL in interactions with complex matrixes [27]. The effect of IL concentration on the SWE method was inspected at 175 °C. The TPC values were 58.91 ± 0.20 mg PGE/g DW at 0.25 M [C4C1im][BF4], 39.61 ± 0.10 mg/g DW at 0.50 M, 32.87 ± 0.10 mg PGE/g DW at 0.75 M, and 20.48 ± 0.12 mg PGE/g DW at 1.00 M. The obtained results showed a proportional reduction in TPC with an increase in [C4C1im][BF4] molarity (as demonstrated in Fig. 4). Similar results were reported by Han et al. [33], in which the high extraction efficiency of phenolic compounds was determined at 0.5 M [C4C1im][BF4] solution at a concentration range from 0.5 to 1.00 M in an ultrasound-based extraction system. A similar study by Jiao et al. [53] also reported a rapid decrease in total phenol extraction content with an increase in the mole ratio of imidazole (0.3–1.2) in a complex model oil formed by dissolving various phenolic compounds (such as phenols, o-cresol, p-cresol, and m-cresol) in hexane. 3.4. HPLC analysis of individual phenolic compounds from various methods Eight phenolic standards were selected based on earlier research by Farvin et al. [15], who mentioned that gallic, gentisic, chlorogenic, protocatechuic, p-hydroxybenzoic, vanillic, caffeic, and syringic acids were frequently detected in different species of brown seaweed. The phenolic standards were separately injected into the HPLC system to define retention time and calculate the regression equation with R2 values as demonstrated in Table 3. As can be seen in Fig. 5, SLE showed poor peak quality in both water and IL extractions. As mentioned before in TPC, water was among the best of the conventional solvents used. However, only gallic acid could be clearly observed from the chromatograph; the other peak signals were essentially minor. When using IL as the extracting solvent, different peaks could be observed of gallic, chlorogenic, gentisic, and protocatechuic acids. Quantification of all the major phenolic compounds detected is reported in detail in Table 4. In the case of SWE, subcritical water seemed to give higher extracting capability than conventional SLE solvents. At 100 °C, the peak chromatograph was obtained as in the SLE method, where the peak quality was poorly determined as compared with at higher temperature. The major phenolic compounds were identified from 125 °C and rapidly increased in content above 150 °C. This could be due to the effect of hot water on the destruction of cell wall structure. Lou et al. [54] observed that a heating temperature of 150 °C could increase the contents of both soluble pheno-

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lics such as gallic acid and insoluble phenolics such as ferulic and p-coumaric acids. Gentisic acid (35.94 ± 0.59 mg/g DW, 225 °C), p-hydroxybenzoic acid (26.92 ± 1.76 mg/g DW, 250 °C), and protocatechuic acid (6.44 ± 0.48 mg/g DW, 225 °C) were the major substances obtained from SWE. Syringic acid (0.67 ± 0.06 mg/g DW, 225 °C) and vanillic acid (2.34 ± 0.06 mg/g DW, 225 °C) were found to be the lowest in content. Obviously, almost all phenolic compounds showed the highest content at temperatures around 200– 225 °C, before being reduced at 250 °C. However, certain phenolics were reduced in content earlier. The contents of gallic acid and chlorogenic acid were significantly decreased beyond 175 °C. At higher temperatures, the preheating period extended, and this is believed to cause unnecessary reactions or decompositions of certain target phenolic compounds. In a similar study, Liazid et al. [55] observed the reduction in stability of various phenolic compounds (such as gentisic, gallic, p-hydroxybenzoic, vanillic, and caffeic acids) that were recovered from grape skin and seeds by using a

high thermal microwave-assisted extraction system. The decreases were recognized by a reduction in TPC from 125 °C (95% phenolic recovery) to 175 °C (34.7% phenolic recovery). In the SWE + IL process, a deficit alteration with SWE was observed. The number of detected phenolics increased when the temperature was beyond 100 °C. The clearest and highest chromatograph of extract was defined at 200 °C (totally 160.62 ± 3.04 mg/g DW). As can be seen from Table 4, chlorogenic acid (64.16 ± 0.99 mg PGE/g DW, 200 °C), gentisic acid (19.53 ± 0.79 mg PGE/g DW, 200 °C), protocatechuic acid (34.37 ± 0.50 mg PGE/g DW, 175 °C), caffeic acid (14.00 ± 0.62 mg PGE/g DW, 225 °C), and p-hydroxybenzoic acid (19.68 ± 0.53 mg/ g DW, 200 °C) were the major defined phenolics along with minor compounds such as syringic acid (3.83 ± 0.07 mg PGE/g DW, 200 °C) and vanillic acid (1.29 ± 0.07 mg PGE/g DW, 125 °C). At 100 °C, the performance of SWE + IL was lesser as compared with SWE, whereas similar observation was not detected at higher tem-

Table 4 Quantification of individual phenolic compounds (mg/g DW) from different extracts using HPLC. Extraction method SLE Water Ethanol Acetone Dichloromethane Diethyl ether [C4C1im][BF4] SWE 100 °C 125 °C 150 °C 175 °C 200 °C 225 °C 250 °C SWE + IL 100 °C 125 °C 150 °C 175 °C 200 °C 225 °C 250 °C

Gallic

Chlorogenic

Gentisic

Protocatechuic

p-Hydroxybenzoic

Vanillic

Caffeic

Syringic

Total

0.85 ± 0.03l 0.03 ± 0.01p 0.14 ± 0.03 n,o 0.04 ± 0.01p 0.06 ± 0.01o,p 0.18 ± 0.02n

0.34 ± 0.02j ND ND 0.15 ± 0.01j 0.28 ± 0.02j 0.56 ± 0.02i,j

ND ND ND ND ND ND

0.06 ± 0.01i ND ND ND 0.10 ± 0.02i 0.27 ± 0.02i

0.15 ± 0.03h ND ND ND ND ND

0.07 ± 0.01f ND ND 0.05 ± 0.01f 0.06 ± 0.01f 0.06 ± 0.01f

0.12 ± 0.03h ND ND ND 0.12 ± 0.03h 0.13 ± 0.05h

ND ND ND ND 0.02 ± 0.01i ND

1.59 ± 0.07m,n 0.03 ± 0.01 n 0.14 ± 0.03n 0.23 ± 0.01n 0.63 ± 0.04m,n 1.20 ± 0.07m,n

1.18 ± 0.02k 3.57 ± 0.04g 5.99 ± 0.04d 5.28 ± 0.04f 2.72 ± 0.03h 1.72 ± 0.02i 0.89 ± 0.02 L

3.43 ± 0.15g 7.14 ± 0.10f 15.64 ± 0.51d 11.26 ± 0.85e 2.74 ± 0.07g,h 1.99 ± 0.13g,h,i 1.44 ± 0.20 h,i,j

ND 0.73 ± 0.06h 2.04 ± 0.24g 2.30 ± 0.27g 15.02 ± 0.27c 35.94 ± 0.59a 6.20 ± 0.34f

0.20 ± 0.02i 1.08 ± 0.05h,i 1.07 ± 0.02h,i 4.48 ± 0.34f,g 5.73 ± 0.25e,f 6.44 ± 0.48e 3.62 ± 0.25 g

0.35 ± 0.03j,h 2.30 ± 0.21f 0.36 ± 0.03j,h 9.68 ± 0.44d,e 19.42 ± 0.57c 24.72 ± 0.56b 26.92 ± 1.76a

ND 0.25 ± 0.04e 0.25 ± 0.02e 0.32 ± 0.06e 1.74 ± 0.13b 2.34 ± 0.06a 1.74 ± 0.10b

0.39 ± 0.07h 0.54 ± 0.02g,h 0.52 ± 0.03g,h 2.15 ± 0.12e,f 2.61 ± 0.29e 2.90 ± 0.24e 1.40 ± 0.22f,g

0.01 ± 0.01i 0.10 ± 0.03i 0.08 ± 0.01i 0.40 ± 0.05g,h 0.60 ± 0.06f,g 0.67 ± 0.06f 0.34 ± 0.03 h

5.56 ± 0.22l 15.70 ± 0.45k 25.94 ± 0.77i 35.87 ± 0.93h 50.58 ± 0.40f 76.73 ± 0.63c 42.55 ± 1.77 g

0.35 ± 0.02m 1.38 ± 0.02j 10.72 ± 0.03a 6.23 ± 0.02c 6.48 ± 0.03b 5.97 ± 0.03d 5.62 ± 0.07e

0.72 ± 0.10i,j 6.25 ± 0.27f 11.44 ± 0.38e 52.68 ± 0.72b 64.16 ± 0.99a 51.13 ± 1.17c 14.53 ± 0.54d

ND ND 6.75 ± 0.48f 10.71 ± 1.03d 19.53 ± 0.79b 8.46 ± 0.34e 6.62 ± 0.22f

2.15 ± 0.07h 10.97 ± 0.40d 26.18 ± 0.14c 34.37 ± 0.50a 33.52 ± 0.86a 31.43 ± 1.41b 11.23 ± 0.67d

ND 0.51 ± 0.04 j,h 1.84 ± 0.11f,g 8.22 ± 0.67e 19.68 ± 0.53c 9.96 ± 0.36d 8.21 ± 0.40e

ND 1.29 ± 0.07c 0.85 ± 0.10d ND ND ND ND

0.62 ± 0.02g,h 0.55 ± 0.02g,h 6.89 ± 0.19d 11.01 ± 0. 75b 13.43 ± 0.55a 14.00 ± 0.62a 7.88 ± 0.50c

0.02 ± 0.01i 1.33 ± 0.12e 1.73 ± 0.16d 2.03 ± 0.17c 3.83 ± 0.07a 3.23 ± 0.10b 1.26 ± 0.09e

3.85 ± 0.08l,m 22.28 ± 0.58 j 66.42 ± 1.13d 125.25 ± 1.53b 160.62 ± 3.04a 124.18 ± 2.64b 55.33 ± 1.04e

Data are expressed as the mean of triplicate ± SD. Subscript letters within a column indicate significant differences between samples at the level of p < 0.05.

Fig. 6. Individual phenolic contents of SWE + IL extracts at various concentrations of [C4C1im][BF4]. Subscript letters indicate significant differences between samples at the level of p < 0.05.

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T. Vo Dinh et al. / Separation and Purification Technology xxx (2017) xxx–xxx Table 5 Antioxidant activities of obtained extracts by DPPH, ABTS.+, TAC, and FRAP assays. Extraction method SLE Water Ethanol Acetone Dichloromethane Diethyl ether [C4C1im][BF4] SWE 100 °C 125 °C 150 °C 175 °C 200 °C 225 °C 250 °C SWE + IL 100 °C 125 °C 150 °C 175 °C 200 °C 225 °C 250 °C

DPPH (mg TE/g DW)

ABTS

2.00 ± 0.18e 0.70 ± 0.12 h,i 0.99 ± 0.16 g,h 1.45 ± 0.11f 0.86 ± 0.21 h 0.29 ± 0.14i

.+

(mg GAE/g DW)

TAC (mg AAE/g DW)

FRAP (mg TE/g DW)

6.83 ± 2.44b ND ND ND ND ND

27.37 ± 0.17 j 3.52 ± 0.13 m 2.59 ± 0.08 m,n 1.62 ± 0.04 n 1.67 ± 0.05 n 2.15 ± 0.01m,n

2.04 ± 0.20i ND ND ND ND 0.45 ± 0.06 j,k

ND 3.05 ± 0.12d 4.79 ± 0.21c 5.46 ± 0.13b 5.46 ± 0.12b 5.30 ± 0.14b 4.84 ± 0.23c

ND 21.76 ± 4.80a 18.56 ± 4.80a 23.36 ± 3.20a 22.83 ± 7.21a 21.76 ± 3.20a 24.96 ± 1.60a

13.32 ± 0.11l 24.57 ± 0.14 k 42.75 ± 0.18g,h 48.41 ± 0.17c,d 43.44 ± 0.17g 43.85 ± 0.17f,g 41.04 ± 0.25 h

3.07 ± 0.11h 8.15 ± 0.28 g 10.24 ± 0.26f 13.21 ± 0.27e 15.53 ± 0.32d 14.98 ± 0.18d 7.57 ± 0.19 g

1.40 ± 0.18f,g 4.59 ± 0.14c 6.17 ± 0.05a 6.39 ± 0.04a 6.39 ± 0.05a 6.51 ± 0.05a 6.39 ± 0.03a

ND 23.36 ± 3.20a 22.29 ± 3.33a 26.56 ± 3.20a 24.96 ± 3.20a 23.36 ± 1.60a 20.69 ± 4.03a

31.09 ± 1.25i 45.46 ± 0.57e,f 47.14 ± 1.32d,e 52.38 ± 0.67a 51.20 ± 1.07a,b 50.03 ± 0.54b,c 43.40 ± 1.05g

0.81 ± 0.05 j 10.74 ± 0.31f 17.96 ± 0.47b,c 21.97 ± 0.29a 18.37 ± 0.40b 17.30 ± 0.30c 15.13 ± 0.34d

Data are expressed as the mean of triplicate ± SD, ND: Not determined. Subscript letters within a column indicate significant differences between samples at the level of p < 0.05.

Table 6 Antioxidant activities of SWE + IL extracts at various concentrations of [C4C1im][BF4]. .+

Concentration (M)

DPPH (mg TE/g DW)

ABTS

0.25 0.50 0.75 1.00

6.53 ± 0.04a,b 6.39 ± 0.04b 6.63 ± 0.07a 6.62 ± 0.05a

22.83 ± 3.33a 26.56 ± 3.20a 24.43 ± 0.92a 24.96 ± 4.23a

(mg GAE/g DW)

TAC (mg AAE/g DW)

FRAP (mg TE/g DW)

140.18 ± 1.13a 59.38 ± 0.67b 57.69 ± 1.46b 58.36 ± 0.47b

20.49 ± 0.44b 21.97 ± 0.29a 20.87 ± 0.46b 19.92 ± 0.41b

Data are expressed as the mean of triplicate ± SD. Subscript letters within a column indicate significant differences between samples at the level of p < 0.05.

perature. This phenomenon could be due to the reduction of [C4C1im][BF4] viscosity with temperature [30]. It was also noted that vanillic acid was detected at 125 and 150 °C, but when the temperature increased, the phenolic appeared to be nondetectable. Phenolic compounds were believed to be extracted faster in SWE + IL than in SWE, having more retention time in subcritical water that lead to a quick decomposition after being extracted. Herein, vanillic acid appeared to be the most unstable phenolic compound. Gonzalez et al. [56], who investigated the decomposition of vanillic acid under subcritical conditions, pointed out that vanillic acid was readily converted to 2-methoxy-phenol when the temperature increased from 280 to 300 °C, with a very quick decarboxylation time of 60 s and only 15 s if the temperature reached 350 °C. Molarity screening of IL in SWE + IL gave similar results in the content of the individual phenolics. As can be seen in Fig. 6, there was a slight increase of chlorogenic, protocatechuic, and caffeic acids by IL molarities whereas the contents of gallic, gentisic, p-hydroxybenzoic, and syringic acids tended to slightly decrease under the same condition. Vanillic acid was not detected at low concentrations (less than 0.50 M [C4C1im][BF4]); however, from 0.75 M of [C4C1im][BF4], this phenolic was detected. By assumption, a low content of IL in subcritical water might result in higher extraction ability than in condensed IL. Similar results by Han et al. [33] on the extraction of 3,4-dihydroxyb enzaldehyde, p-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, p-hydroxybenzaldehyde, and salicylic acid showed a significant reduction in phenolic content when the molarity of [C4C1im][BF4]

increased from 0.5 to 1.0 M. The reason for this decrease in phenolic content might be due to the increase in viscosity at a high concentration of IL in water, thus reducing the penetration ability of extracting solvent with raw samples. 3.5. Antioxidant activity analysis Phenolic compounds are known for their high antioxidant activity because of their function against free radicals [57]. However, different phenolic compounds might have different antioxidant mechanisms or selective interactions with only certain free radicals; also, other components inside the extracts could contain extra antioxidant power. Herein, we performed various antioxidant testing assays to examine bioactive samples as well as the correlation between phenolic content and antioxidant activity. The final data are recorded and expressed in Table 5. In the DPPH free radical scavenging assay, the ability of antioxidants to transfer a hydrogen atom to DPPH radicals and convert to non-radical DPPH molecules can aid in estimating the potential activity of samples. In SLE, water gave the highest activity equivalent to a weight of 2.00 ± 0.18 mg TE/g DW. The opposite occurred for [C4C1im][BF4] with a content of 0.29 ± 0.14 mg TE/g DW, the lowest among all the screening solvents in SLE. In SWE, the extract sample at 100 °C was not detected in scavenging activity, whereas at higher temperature, the activity was varied from 3.05 ± 0.12 mg TE/g DW to 5.46 ± 0.13 mg TE/g DW (125–200 °C) and started to reduce when temperature increased to 250 °C. In the case of SWE + IL, the extract sample at 100 °C was detected in scavenging

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Table 7 Pearson’s correlation coefficients of TPC and DPPH, ABTS.+, TAC and FRAP assays. Trait TPC DPPH ABTS.+ TAC FRAP **

TPC 1 – – – –

DPPH **

0.943 1 – – –

ABTS.+ **

0.935 0.943** 1 – –

TAC

FRAP **

0.917 0.930** 0.908** 1 –

0.959** 0.950** 0.906** 0.896** 1

Correlation is significant at the 0.01 level.

activity (1.40 ± 0.18 mg TE/g DW). The activity at this time was rapidly increased from 1.40 ± 0.18 mg TE/g DW to 6.17 ± 0.04 mg TE/g DW at 150 °C and remained constant at around 6.39 mg TE/ g DW from 150 to 250 °C. For ABTS.+ scavenging activity, the more the extracts inhibited ABTS.+ free radical cations, the more the antioxidant activity. The extracts from SLE virtually indicated underestimated activity. The antioxidant activity from water extract was equivalent to 6.83 ± 2.44 mg GAE/g DW, but from other solvents, including [C4C1im][BF4], the activities were negatively detected. In SWE, a similar trend with the DPPH method occurred when it was not possible to detect antioxidant activity at 100 °C. From 125 to 250 °C, the antioxidant activity was observed to be close to each other at an equivalent weight around 22 mg GAE/g DW. At 250 °C, the activity was observed to be highest with an amount of 24.96 ± 1.60 mg GAE/g DW. SWE + IL also indicated nondetectable activity at 100 °C. From 125 to 250 °C, the activity now varied close to 23 mg GAE/g DW. The highest activity in SWE + IL was determined at 175 °C with an equivalent amount of 26.56 ± 3.20 mg GAE/g DW. The activity reduced gradually from 175 to 250 °C (20.69 ± 4.03 mg GAE/g DW). In the TAC assay, the activity can be determined according to the reduction reaction of phosphate molybdenum(VI) to phosphate molybdenum(V) by antioxidants. Generally, all the samples were observed to have antioxidant activity. In SLE, water again gave the highest activity (27.37 ± 0.17 mg AAE/g DW) as compared with organic solvents and IL, which indicated a very poor activity of approximately 2 mg AAE/g DW. The activity significantly increased in the case of SWE and SWE + IL. In SWE, the antioxidant activity increased significantly from 13.32 ± 0.11 mg AAE/g DW (100 °C) to 48.41 ± 0.17 mg AAE/g DW (175 °C) before being slowly reduced to 41.04 ± 0.25 mg AAE/g DW at 250 °C. In SWE + IL, the activity sharply increased from 31.09 ± 1.25 mg AAE/g DW at 100 °C to 52.38 ± 0.67 mg AAE/g DW at 175 °C. From 175 to 225 °C, the antioxidant activity fluctuated around an equivalent amount of 51 mg AAE/g DW before being slightly reduced to 43.40 ± 1.05 mg AAE/g DW at 250 °C. Hence, the activity from SWE + IL generally had certain advantages as compared with SWE. For the FRAP assay, a rapid reduction of ferric-tripyridyltriazine (FeIII-TPTZ) to ferrous-tripyridyltriazine (FeII-TPTZ) would help identify the existence of antioxidants in the concerned extracts. As shown in Table 5, except for water and [C4C1im][BF4], organic solvents with antioxidant activity were not detected. Water indicated an activity of 2.04 ± 0.20 mg TE/g DW when a lower amount of 0.45 ± 0.06 mg TE/g DW was defined from [C4C1im][BF4]. In SWE, the activity gradually increased from 3.07 ± 0.11 mg TE/g DW to 15.53 ± 0.32 mg TE/g DW at 100–200 °C before being rapidly reduced to 7.57 ± 0.19 mg TE/g DW at 250 °C. This was also the case in SWE + IL, where the activity proportionally increased from 0.81 ± 0.05 mg TE/g DW to 21.97 ± 0.29 mg TE/g DW at 100–175 °C before being reduced to 15.13 ± 0.34 mg TE/g DW at 250 °C. For the molarity variation of SWE + IL, the obtained data indicated a slight disparity (as expressed in Table 6). The low concentration of IL seemed to be more suitable for phenolic extraction. An increase in IL content might increase the viscosity of the entire solution, thus linking to negative effects on the final extraction effi-

ciency of phenolic compounds and other antioxidants in S. japonica. The antioxidant activity shown by all four assays (DPPH, ABTS.+, TAC, and FRAP) indicated an increase in the activity of SWE + IL as compared with SWE. Earlier studies had also reported the strong influence of IL on the extraction of antioxidant compounds. Li et al. [58] discovered that the IL-based microwave-assisted extraction (MAE) of bioflavonoids significantly reduced the IC50 value of DPPH from 81.09 ± 3.92 mg/g (for Soxhlet extraction) and 68.35 ± 1.77 mg/g (for MAE) to 56.17 ± 2.21 mg/g. Other research by Katsoura et al. [59] reported the increase in antioxidant activity of flavonoid derivatives of rutin after an enzymatically biocatalytic preparation using a binary medium of [C4C1im][BF4] with acetone. They also observed that using imidazolium-based IL with [BF 4 ] and [PF 6 ] as media for the synthesis of cinnamic acid derivatives can enhance the antioxidant activity of derivatives such as ferulic acid against low-density lipoprotein, high-density lipoprotein, and total serum oxidation in vitro [60]. These encouraging results toward antioxidant activity from SWE and SWE + IL extracts are also discussed in the following PCA section. 3.6. Correlation between phenolic content and antioxidant activity To verify the correlation between phenolic content and antioxidant activity of the obtained extracts, the Pearson correlation analysis among TPC and the different assays (i.e., DPPH, ABTS.+, TAC, and FRAP) was applied and is reported in Table 7. Generally,

Fig. 7. Correlation circle of TPC, DPPH, ATBS, TAC and FRAP on antioxidant activities.

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T. Vo Dinh et al. / Separation and Purification Technology xxx (2017) xxx–xxx

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Fig. 8. Correlation biplot of different extracts samples on antioxidant activities by PCA (Pearson (n)).

all four antioxidant determination assays showed high correlation coefficients with TPC, starting from FRAP (r2 = 0.959), DPPH (r2 = 0.943), ABTS (r2 = 0.935), and TAC (r2 = 0.917). Furthermore, the antioxidant assays were closely correlated, regardless of the different reaction mechanisms. Similar results were reported by Thaipong et al. [44] with significant correlations between phenolic-rich extracts from guava fruits with various antioxidant assays such as ABTS.+, FRAP, DPPH, and oxygen radical absorbance capacity. Another study by Chirinos et al. [61] on 27 different selected plants from the Peruvian Andean region also noted that TPC was mainly responsible for the antioxidant activity among other compounds such as flavanoids, flavonoids, and anthocyanins. 3.7. PCA of antioxidant activity and TPC PCA was introduced to analyze the correspondence between TPC and antioxidant activity. Analyzed data were illustrated in the circle of correlations (Fig. 7) and PCA loading biplot (Fig. 8). The first two principal components (expressing 96.58% of initial variances of TPC, DPPH, ABTS.+, FRAP, and TAC) were selected to evaluate the correlation. The first component (F1, 94.30%) expressed strong correlations with the five assays used: TPC, DPPH, ABTS.+, TAC, and FRAP. As shown in Fig. 8, DPPH, ABTS.+, FRAP, TAC, and TPC were highly loaded on component 1 (F1). The most significant parameter was DPPH with a loading of 0.981, followed by TPC, FRAP, ABTS.+, and TAC with loadings of 0.979, 0.971, 0.966, and 0.958, respectively. The second component (F2, 2.28%) was mostly contributed by TAC (loading of 0.243). In Fig. 8, high loadings were correlated with subcritical extracts, which were previously determined as rich in phenolics using HPLC. SLE and lowtemperature SWE (100 °C) got minor loadings, reasonably coinciding with previous HPLC measurements. A similar PCA was performed by Wong et al. [62] on the antioxidant abilities of 25 different tropical plants, in which the three parameters DPPH,

FRAP, and TPC were the main contributors to the antioxidant activities of aqueous extracts. 4. Conclusions The extraction efficiencies were obviously different between the SWE and SLE methods. TPC, HPLC analysis, and antioxidant activity screening proposed a higher quantity and quality of phenolic substances extracted in SWE + IL and SWE than in SLE. Therefore, water at the subcritical condition can be a prospective technique for the recovery of natural phenolic compounds. Compared with SWE, SWE + IL provided a progressive enhancement in extracting phenolics. At 175 °C, the contents of gallic, chlorogenic, gentisic, protocatechuic, caffeic, and syringic acids in extracting products were approximately 1.18-, 4.68-, 4.66-, 7.67-, 5.12-, and 5.08-fold higher than in SWE. However, phydroxybenzoic acid had a slight deficiency (0.85-fold at 175 °C). Furthermore, the decomposition of vanillic acid in SWE + IL required intensive investigation on the mechanism of IL in enhancing the decomposition rate of phenolic compounds so as to minimize this disadvantage. Antioxidant activity was also enhanced in the presence of IL in the subcritical system from DPPH, ABTS.+, TAC, and FRAP. The antioxidant activities were highly correlated with the phenolic content in the final extracts, thus proving the valuable quality of extracting components from brown seaweed. Therefore, IL might probably be a potential catalytic agent for SWE in the extraction of bioactive compounds from natural resources. Acknowledgements This work was financially supported by the Ministry of Oceans and Fisheries of Korea (Project No. 20140559).

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Please cite this article in press as: T. Vo Dinh et al., Ionic liquid-assisted subcritical water enhances the extraction of phenolics from brown seaweed and its antioxidant activity, Separ. Purif. Technol. (2017), http://dx.doi.org/10.1016/j.seppur.2017.06.009