3.1. DES Synthesis and the Effect of HBD:HBA Molar Ratio ()
The selection of an appropriate
is important in the synthesis of DES because the molar proportion between HBD and HBA may critically affect DES extraction performance [
13,
14]. Earlier investigations outlined that DES composed of L-lactic acid (LA) and glycine (Gly) were not stable at
≤ 3 and tended to form plastic solid at room temperature [
21]. Following examinations pointed out that stability (no crystallization) of DES composed of LA and Gly could be assured at
≥ 5 [
22]. In a recent study, it was clearly showed that switching
from 5 up to 13, extraction efficiency may be significantly impacted [
13]. Thus, in this study, screening of DES with
ranging from 5 to 13, was the first step towards the development of an effective solvent. All DES were tested as 70% (
w/
v) aqueous mixtures and the results obtained are presented in
Figure 1. The DES with
= 5 was proven to be the highest-performing system, giving significantly increased Y
TP (
p < 0.05).
To obtain a more integrated picture, the efficiency of LA-Gly (5:1) was further appraised by comparing its performance with that of two other green solvents, namely 60% (
v/
v) aqueous ethanol and water, but also with a commonly used solvent, 60% (
v/
v) aqueous methanol. Apart from Y
TP, the Y
TFn, A
AR, and P
R were also considered, and the outcome is depicted in
Figure 2. LA-Gly (5:1) gave higher Y
TP and Y
TFn, which were statistically significant (
p < 0.05) (
Figure 2A,B). Furthermore, the EBF extracts obtained with LA-Gly (5:1) had higher, but statistically non-significant (
p > 0.05) A
AR and P
R, (
Figure 2C,D). Considering all these results together, it was concluded that LA-Gly (5:1) was the highest-performic system.
3.2. Optimization of Extraction Performance
The experimental design was set up to evaluate the influence of three key extraction variables (
CDES, R
L/S, S
S) on the DES performance for polyphenol recovery. The scope was the generation of a polynomial equation (model) based on the experimental data, to deliver a concrete statistical prevision. Validity of the fitted model was assessed by both ANOVA and lack-of-fit tests (
Table 2). All non-significant terms were omitted from the equation derived, and thus its final form was the following:
The square correlations coefficient (R
2) and the
p-value provide an indication of the total variability around the mean calculated by the model. Since R
2 was 0.94 and the
p value (considering a confidence interval of 95%) was highly significant, it could be argued that the model displayed a sound fitting to the experimental data. Measured and predicted Y
TP values for each design point are analytically given in
Table 3.
The three-dimensional plots crafted using the model, show at-a-glance variations of the response (Y
TP) as a function of changes in the three model variables (
Figure 3). The use of the desirability function permitted the optimization of the levels of all three variables simultaneously, to achieve maximum system performance and enabled the calculation of the set of conditions that would allow for attaining the highest theoretical yield (121.24 ± 8.77 mg GAE g
−1 dm). These conditions were
CDES = 85% (
w/
v), R
L/S = 60 mL g
−1 and S
S = 200 rpm. Confirmation of the validity of the model was done by carrying out three extractions under the optimal conditions, which gave Y
TP of 114.96 ± 5.02.
ANOVA revealed that for
CDES (X
1), only the quadratic effect was significant; increasing R
L/S (X
2) had a positive effect on Y
TP, whereas the effect of S
S (X
3) was negative. No cross effects between process variables were found to be significant, evidence that every variable exerted distinguishable influence on the extraction yield. The optimized predicted
CDES levels were in line with previous results on polyphenol extraction with DES, suggesting 80% (
w/
v) to be the most suitable
CDES for effective polyphenol recovery [
23,
24].
Appropriate mixing of DES with water is a key step in regulating critical DES properties, such as viscosity and polarity [
25]. Yet, water cannot exceed a certain level because this would provoke DES disintegration and abolishment of its intrinsic characteristics [
26].
R
L/S is also a parameter that could profoundly affect solid–liquid extraction, since R
L/S defines the concentration gradient of the solute (polyphenols) between the solid particles and the liquid phase. This gradient is considered to be the driving force for diffusion, which governs polyphenol entrainment from the inner of the solid to the liquid. Diffusivity may be increased by raising R
L/S [
27]; however, the optimum R
L/S found for polyphenol extractions with DES may vary from 29.5 [
28] to as high as 100 mL g
−1 dm [
29,
30]. The optimal R
L/S determined for EBF (60 mL g
−1) is in accordance with recent studies on polyphenol extraction with DES from saffron processing wastes (60 mL g
−1) [
15] and hop (59 mL g
−1) [
13].
S
S is a variable with crucial role in solid–liquid extraction, and it has been proven that careful S
S setting could provide higher extraction yields [
27,
31]. In a recent study where S
S was considered as one of the variables for constructing experimental design, it was found to exert a statistically significant effect on the polyphenol extraction yield [
15]. It has been proposed that appropriately set S
S may create sufficient turbulence in the extraction tank to increase mass transfer rate. Such an effect has been demonstrated to increase polyphenol diffusivity [
27]. On the other hand, optimization of polyphenol extraction has shown that, in some cases, low S
S (300 rpm) may favor increased extraction yield, as opposed to higher S
S (900 rpm), which apparently was hindering in this regard [
30]. In other recent examinations, the findings indicated quite the opposite [
15,
29]. Since the phenomena associated with the effect of S
S may be related with factors such as the nature of the solid material, the solid particle diameter, the solute (polyphenols species), the viscosity of the liquid phase (solvent), etc., the actual effect of S
S on extraction yield would be a subject of case experimentation.
3.3. Temperature Effects
Extraction temperature is a variable that should be carefully used, because polyphenols are generally considered to be thermosensitive substances. Although, in general, increased temperature may contribute in achieving higher extraction yields, it is not a universal rule that temperature rising generates proportional effect on the extraction yield and antioxidant activity. This argument may be exemplified by results drawn from the extraction of various plant materials, including
Moringa oleifera leaves [
23], onion solid wastes [
32,
33], chickpea sprouts [
34] and red grape pomace [
35]. This being the case, the investigation of the effect of temperature on the extraction yield and the antioxidant activity of the extracts merits particular attention.
Thus, EBF was extracted under optimal conditions, at temperatures ranging from 40 to 80 °C, and the extracts produced were examined by determining Y
TP, Y
TFn, A
AR, and P
R. Switching temperature from 40 to 80 °C did afford higher Y
TP, and the value obtained at 80 °C was statistically different (
Figure 4A), which pointed emphatically to a strong temperature effect. Likewise, the extracts produced at 80 °C displayed significantly higher A
AR (
Figure 4C), but for the P
R, no statistical difference was seen between the levels acquired at 70 and 80 °C (
Figure 4D). Contrary to those findings, significantly higher Y
TFn was recorded at 50 °C (
Figure 4B). The overall picture dictated that extraction temperature up to 80 °C could be used to enrich EBF extracts in polyphenols and enhance their antioxidant activity.
3.4. Effect of Ultrasound-Assisted Pretreatment
The pretreatment consisted of ultrasonicating the samples prior to performing batch-stirred tank extraction under optimized conditions, at 80 °C. Ultrasonication was carried out for a period varying from 5 to 40 min at ambient temperature (23 ± 1 °C), and the results are portrayed in
Figure 5. After the ultrasonication step, the Y
TP was, at best, almost 50% lower than that achieved with the stirred-tank extraction. This finding strongly emphasized that ultrasonication is ineffective as a standalone extraction methodology, which is in accordance with previous observations [
12,
14], although contradictory results have also been reported [
36]. However, when ultrasonication was accompanied by stirred-tank extraction, Y
TP determined was always significantly higher than that attained without ultrasonication pretreatment. It was also notable that Y
TP displayed statistically non-significant variations as a response to ultrasonication time. Thus, even the shortest ultrasonication period tested (5 min), resulted in a very important enhancement of the yield after 150 min of stirred-tank extraction. This is in line with recent kinetic data on the extraction of polyphenols from hop (
Humulus lupulus) using a glycerol/L-alanine DES and ultrasonication as a pretreatment step, which evidenced significant enhancement of subsequent stirred-tank extraction, at 80 °C [
13].
Irradiation with ultrasound is known to intensify solid–liquid extraction through generation of cavitation effects [
37]. The collapse of cavitation bubbles nearby or on the surface of the solid particles is considered to cause particle disruption and destruction of cell walls, as well as intense shaking at a macroscopic level (ultrasound streaming), which may contribute in fast washing of the superficial solute, solvent penetration into canals and pores of plant material, and eventually increased diffusivity, high entrainment of the solute into the liquid phase, and enhanced solubilization. All these phenomena may be responsible for increasing polyphenol extraction yield [
11].
3.5. Polyphenolic Composition
The richest EBF extract was produced with a 10 min ultrasonication pretreatment and then stirred-tank extraction under optimized conditions, at 80 °C, for 150 min (
Figure 5). This sample was chosen to profile its analytical polyphenolic composition, and the trace recorded at both 320 and 360 nm revealed the presence of several chlorogenate and flavonol derivatives (
Figure 6). By carrying out liquid chromatography–diode array–mass spectrometry analysis, it was made possible to tentatively identify eight polyphenolic compounds (
Table 4). A total ion chromatogram is also provided (
Figure S1). Concerning chlorogenates, peak #1 showed a pseudo-molecular ion at
m/
z = 355 and a diagnostic fragment at
m/
z = 163. Considering the retention time of the original standard, this compound was tentatively identified as neochlorogenic acid. In a similar fashion, peak #2 was identified as chlorogenic acid [
14]. Peak #5 displayed a pseudo-molecular ion at
m/
z = 517 and two fragment ions at
m/
z = 355 and 163. This structure was assigned to a di-caffeoylquinic acid [
38]. Peak #6 gave a pseudo-molecular ion at
m/
z = 485, and a diagnostic fragment at
m/
z = 147. This compound was identified as a di-
p-coumaroylquinic acid derivative [
39].
With regard to flavonols, peak #3 yielded a pseudo-molecular ion at m/z = 611 and fragment ion at m/z = 303. These data, along with the retention time of the original standard, enabled the identification of this substance as rutin (quercetin 3-O-rutinoside).
Likewise, peak #8 was identified as quercetin. Peak #4 gave a pseudo-molecular ion at
m/
z = 465 and fragment ion at
m/
z = 303, which pointed to the structure of quercetin 3-
O-glucoside (isoquercitrin). For peak #7, a pseudo-molecular ion was detected at
m/
z = 625, an adduct with Na
+ at
m/
z = 647 and a diagnostic fragment at
m/
z = 317. This structure was tentatively assigned to isorhamnetin 3-
O-rutinoside (narcissin) [
40].
On the basis of the quantitative analysis, the predominant constituents were rutin (17.36 mg g
−1 dm), di-
p-coumaroylquinic acid (13.06 mg g
-1 dm), and chlorogenic acid (10.76 mg g
−1 dm) (
Table 5).
According to a survey on flower composition of 16 different
S. nigra genotypes [
41], the average content of neochlorogenic acid, chlorogenic acid, rutin and isoquercitrin were 1.6, 15.2, 21.0, and 1.0 mg g
−1 dm, respectively. For neochlorogenic acid, chlorogenic acid, rutin, isoquercitrin and narcissin, corresponding content ranges were shown to be 1.06–1.60, 12.40–14.00, 15.70–23.90, 0.73–3.05, and 4.26–5.33 mg g
−1 dm [
42]. Data from another study on EBF extracts were in line, reporting contents for chlorogenic acid, rutin, isoquercitrin, and quercetin of 5.93, 15.28, 2.64, and 0.11 mg g
−1 dm [
43]. Rutin and isoquercitrin contents of 20.2 and 0.97 mg g
–1 have also been reported, in EBF extracts obtained with pressurized liquid extraction [
44]. The values reported herein are close to these levels. On the other hand, microwave- and ultrasound-assisted extraction of EBF with 50% ethanol has been reported to give contents for chlorogenic acid and rutin of 56.49 and 91.39 mg g
−1 dm, respectively [
45].