2.2. Quantitative Composition of the Obtained Extracts
Graphical illustration of the efficiency of extraction methods and each solvent system applied in the present study towards particular catechin and gallic acid was presented in
Figure 1. While
Table 2 presents concentration of each compound in the obtained extracts and the total amount of all investigated substances (sum of all catechins and gallic acid).
GA–gallic acid; EGCG–epigallocatechin gallate; EGC–epigallocatechin; ECG–epicatechin gallate; EC–epicatechin; C–catechin; MW–maceration with water; MEW–maceration with ethanol:water (1:1 v/v); ME–maceration with ethanol; MM–maceration with methanol; MO–maceration with ethyl acetate; MA–maceration with acetone:water (5:1 v/v); UW–ultrasound extraction with water; UEW–ultrasound extraction with ethanol:water (1:1 v/v); UE–ultrasound extraction with ethanol; UM–ultrasound extraction with methanol; UO–ultrasound extraction with ethyl acetate; UA–ultrasound extraction with acetone:water (5:1 v/v); AW–Accelerated Solvent Extraction (ASE) with water; AEW–ASE with ethanol:water (1:1 v/v); AE–ASE with ethanol; AM–ASE with methanol; AO–ASE with ethyl acetate; AA–ASE with acetone:water (5:1 v/v)
Chromatographic analysis of green tea extracts obtained by various methods and using different solvent systems revealed the presence of six main polyphenols: C, EC, ECG, EGC, EGCG and GA. EGCG was the dominant catechin, whose content significantly exceeded the concentration of other compounds. Moreover, EGC and ECG were determined in high amounts. This is in agreement with other studies suggesting these two catechins being major catechins in green and black tea [
8,
15,
16,
17]. The EGCG concentration in the obtained extracts was in a fairly wide range of 5.44–406 mg/200 mL. The lowest efficiency was obtained using ultrasounds and ethyl acetate, while the highest applying ASE and a mixture of ethanol and water. EGCG was proved to be a major catechin in all extracts, except UEW in which the dominant catechin was EGC and MO and AO in which ECG was determined in the highest concentration. The third major catechin in all extract was ECG (with the exceptions mentioned above), while the concentration of C, EC and GA was much lower in comparison to the three major compounds. Qualitative composition of the extracts was similar to catechin profile of the green tea infusions obtained in other studies, however quantitative profile was significantly different, which emphasizes the important influence of the extraction method and applied solvents [
9,
18,
19]. In addition, the parameter regarding the total content of catechins and gallic acid in all extracts was very different (10.2–748 mg/200 mL), which shows how important the method of extraction is, as well as the application of an appropriate solvent. The highest was observed when using ASE and ethanol:water mixture and the lowest in the case of using ultrasound extraction and ethyl acetate.
Based on the obtained results, it can be concluded that all the extraction techniques studied showed similar efficiency in the extraction of the tested catechins. However, larger differences occur when using different solvents. The results herein obtained are in agreement with the previous findings reported by Perva-Uzunalic et al. analyzing the effect of different temperatures and solvents on the efficiency of extraction of catechins from green tea who saw that EGCG was the dominant compound extracted from green tea in different solvent and temperature systems. They also showed that pure methanol and ethanol better extract catechins from the tested raw material, while in the case of acetone the addition of 20% of water significantly improved the extraction efficiency. They also observed that the use of organic solvents and their aqueous mixtures increases the efficiency of extraction of catechins from green tea compared to pure water [
15]. Results of the present study are in agreement with these findings. The presented results show that of the solvent systems used, ethyl acetate turned out to be the solvent that extracted the least amount of polyphenol compounds, while the extract most rich in polyphenols was obtained using ethanol/water mixture. The present study shed also a new light on the extraction efficiency of particular catechins, when using different extraction models. From the data presented in the
Table 2 it appears that the most effective solvent system for the extraction of EGCG and EGC was by far the mixture of ethanol and water. Application of maceration or ASE gave similar results, which were not statistically significant. However, in the case of ultrasound extraction, the mixture of acetone and water was proved to be the best for the extraction of EGCG. This solvent system was also proved to be the most efficient for ECG for all extraction techniques. Regarding the concentration of C, EC and GA the most effective was hot water, which was proved by PCA analysis. In the case of GA, combination of ultrasound maceration or ASE with water resulted in statistically significant higher extraction efficiency in comparison to simple maceration. Lan-Sook and co-workers applied Response Surface Methodology (RSM) for the optimization of extraction efficiency of phenolics from green tea. The predicted optimal conditions for the highest antioxidant activity and minimum caffeine level were found at 19.7% ethanol, 26.4 min extraction time and 24.0 °C extraction temperature [
20]. They also revealed that the ratio of (EGCG + ECG)/EGC was identified a major factor contributing to the antioxidant activity of green tea extracts, which is in agreement with results of the present study.
The results obtained in a Folin–Ciocalteu test, which was used to determine the total content of polyphenolic compounds in the samples tested and may also characterize the antioxidant activity of the extracts, were confirmed by the chromatographic determinations described above. Ethyl acetate is by far the least effective extractant, since only small amounts of catechins were detected in the samples tested, regardless of the extraction technique used. Unlike ethyl acetate, among all the solvents tested, a mixture of ethanol/water was the most effective in delivering extracts richest in catechins. Application of simple maceration and ASE gave comparable effects using individual solvents. A slightly lower degree of extraction was noted using ultrasounds, except for extraction using mixture of acetone and water, in which this method proved to be the most effective, as these extracts contained the largest amount of polyphenolic compounds regarding F–C determinations. Results of antioxidant activity of extracts obtained using various extraction models including F–C method were presented in
Table 3. Obtained results of F–C analysis were in agreement with our previous data for green tea infusions [
9], showing that this product is very rich in polyphenols and therefore presents high antiradical activity, similarly to
Vaccinium meridionale—a berry known for its high polyphenols content and antioxidant activity (up to 724.24 mg gallic acid/L) [
21]. A very high concentration of phenolics, determined using F–C reagent, was also revealed in the cryoconcentrates obtained from fruits of another berry-maqui-berries (
Aristotelia chilensis), which contained up to 4311.8 mg gallic acid/100 g [
22]. Bilberry and blackberry pomace extracts were also proved to contain high amounts of protocatechuic acid (3.36–35.18 mg g
−1) and gallic acid (9.57–31.98 mg g
−1) and therefore presented high antioxidant activity, confirmed with electron spin resonance (ESR) spectroscopy [
23]. In addition to tea or different species of berries, coffee also may be characterized by high content of phenolics and significant antioxidant properties. Recently, Beder-Belkhiri and co-workers revealed that filtered Algerian coffee contained up to 690 mg of gallic acid equivalents/100 g and Turkish coffee exhibited the highest antiradical activity, with 73.34% towards DPPH radical [
24].
However, not only is the high content of compounds with antioxidant properties such as polyphenols a crucial factor to take into consideration for polyphenols to exert their health beneficial properties, but equally important is also the actual bioavailability of these compounds (which may be influenced by other nutrients, temperature, pH or gastric digestion) [
25,
26].
The antioxidant capacity of the obtained green tea extracts was additionally evaluated using DPPH and expressed both in percentage of the radicals scavenged by the extracts and Trolox equivalents. Overall, the extracts produced by ultrasounds were characterized by lower antioxidant activity in comparison to other techniques. It can be assumed that ultrasounds negatively affect the stability of polyphenolic compounds, therefore the antioxidant potential of these extracts is lower, which was also shown in previous studies [
27,
28]. In addition, Sun and co-investigators—who were studying the influence of sonification on various parameters of fresh apple juice—noticed a significant decrease of phenolic content and antioxidant activity measured by oxygen radical absorption capacity assay (ORAC). They concluded that, indeed, application of ultrasound increased the extraction of polyphenols, but later free radicals which were produced by sonification increased the degradation of polyphenols [
29]. On the other hand, several studies revealed that application of ultrasounds significantly increased concentration of polyphenols and antioxidant activity of the extracts [
30,
31,
32,
33,
34]. However, according to Setyaningsih et al. 2016, an increase in antioxidant potential and phenolics concentration is observed to a temperature of 60–70 °C; above this level, a significant deterioration of polyphenols may be observed, which also decreases antioxidant activity of the samples [
27]. Zapata and co-investigators—who studied the effect of pH, temperature and time of extraction on the antioxidant properties of
Vaccinium meridionale—observed that an increase of temperature resulted in more efficient extraction of polyphenols. However, anthocyanins were degraded above 80 °C and within 20 min of processing [
21]. In addition, in this method, ethyl acetate was characterized by the weakest extraction capacity, and the extracts obtained using this solvent present activity below 10% (Trolox equiv. <0.5 mM/L), and these samples were rated as inactive. Therefore, their activity was not marked in
Figure S1. Only in the case of ethyl acetate extracts obtained using ASE, some minor antiradical activity was observed, and these values were presented in
Figure S1. In general, the results of antioxidant activity of the obtained extracts were in agreement with chromatographic determinations. The highest ability to deactivate the DPPH radical was demonstrated by extracts obtained using a mixture of ethanol and water (1:1
v/v), as well as a mixture of acetone and water (5:1
v/v). The high antioxidant capacity of these extracts can be associated with the high content of EGCG and EGC as revealed by chromatographic results.
2.3. PCA Analysis
From the obtained results, a matrix made of columns (content of compounds, F–C method, DPPH, Trolox equivalent) and rows (type of extract) was created and subjected to PCA analysis. The PCA carried out explains 85.27% of the variability in the first two principal components (61.39% and 23.88%, respectively) (
Figure 2 and
Figure 3).
The first component (PC1) is related to the overall extraction efficiency and shows the differences between MO, UO, AO and other methods MW, MEW, ME, MM, MA, UW, UEW, UE, UM, UA, AW, AEW, AE, AM, AA. In addition, it can be concluded that the best methods were AEW and MEW. The PC1 component shows one group containing compounds found in large quantities in green tea (EGC, EGCG, ECG and TOT-sum of all compounds) and the activity of the tested extracts (DPPH, F–C method, Trolox equivalent) which are strongly and positively correlated (
Table S1).
The second component (PC2) reveals another group (GA, C, EC) which contains compounds found in a small amount in green tea compared to the other ingredients tested. In addition, the PC2 component separates the MW, UW, AW methods (all using water as solvent) as the best methods for C, EC and GA extraction from among the solvents used (
Figure S1).