The Effect of Different Extraction Protocols on Brassica oleracea var. acephala Antioxidant Activity, Bioactive Compounds, and Sugar Profile

The extraction of glucosinolates in boiling aqueous methanol from freeze dried leaf tissues is the most common method for myrosinase inactivation but can be hazardous because of methanol toxicity. Although freeze drying is the best dehydration method in terms of nutritional quality preservation, the main drawbacks are a limited sample quantity that can be processed simultaneously, a long processing time, and high energy consumption. Therefore, the aim of this study is to evaluate the effects of applying high temperature for myrosinase inactivation via hot air drying prior to the extraction step, as well as the effects of cold aqueous methanol extraction on total antioxidant activity, total glucosinolates, total phenolic content, and sugar profile in 36 landraces of kale. The results from our study indicate that cold aqueous methanol can be used instead of boiling aqueous methanol with no adverse effects on total glucosinolate content. Our results also show that hot air drying, compared to freeze drying, followed by cold extraction has an adverse effect on antioxidant activity measured by DPPH radical scavenging, total glucosinolate content, as well as on the content of all investigated sugars.


Introduction
Vegetables from the Brassicaceae family are known for their excellent nutritional value and are abundant in carbohydrates, vitamins (ascorbic acid, folic acid, β-carotene, α-tocopherol), macro and micro elements (iron, calcium, selenium, copper, manganese, zinc), as well as secondary metabolites, including glucosinolates, phenolics (tannins, phenolic acids, anthocyanidins, flavonols, coumarins, flavones), and other bioactive molecules, such as phytosterols and terpenoids [1,2]. They are also known for their antimicrobial and anticancerogenic activity [3,4]. One of the well-known members of the Brassicaceae family is kale (Brassica oleracea var. acephala). It originates and is traditionally used in Mediterranean countries, but has gained special attention and popularity in the U.S., and later worldwide over the last decade [5]. It is characterized by leaves, which do not form a head, unlike other leafy vegetables from the Brassicaceae family as white cabbage, savoy cabbage, Brussels sprouts, and Chinese cabbage [6].
The most abundant sulfur containing compounds in plants from the Brassicaceae family are glucosinolates and S-methylcysteine sulfoxide [7]. Cruciferous vegetables are extensively researched regarding their beneficial compounds especially ones active in cancer prevention. Isothiocyanates are degradation products from glucosinolates are known for their antioxidant, immunostimulatory, Both sucrose and fructose content was significantly lower in the freeze dried extracts obtained by the hot methanol extraction method (7.8 ± 0.3 g/100g DW and 4.4 ± 0.2 g/100g DW for sucrose and Figure 1. The effect of different extraction protocols on total glucosinolates content, total phenolic content, DPPH radical scavenging activity and ferric ion reducing antioxidant power in B. oleracea var. acephala leaf extracts. Values are expressed as mean ± SE (N = 324). The different letters above bars denote significant difference by Tukey's Unequal N Honestly Significant Difference (HSD) test, p < 0.05. TGls-total glucosinolates; TPC-total phenolic content; DPPH-DPPH radical scavenging activity; FRAP-Ferric ion Reducing Antioxidant Power.
The same effect was observed for total phenolic content, where significantly lower amounts were extracted with the hot methanol method (10.1 ± 0.2 mg gallic acid equivalents (GAEQ)/g DW) compared to the cold methanol extraction (10.6 ± 0.2 mg GAEQ/g DW) in freeze dried leaves ( Figure 1). On the other hand, total phenolic content was found to be higher in oven dried leaves (15.7 ± 0.2 mg GAEQ/g DW) compared to freeze dried B. oleracea var. acephala leaves followed by cold extraction (10.6 ± 0.2 mg GAEQ/g DW) ( Figure 1).

Multivariate Analysis of the Data Obtained by Different Extraction Protocols
The obtained data was processed with Partial Least Squares-Discriminant Analysis (PLS-DA) to further clarify the relations between different extraction protocols. The PLS-DA model was employed due to the supervised nature of the learning algorithm. Overall, the extracts which included oven drying leaf tissue following cold methanol extraction were separated on the primary axis from the extracts obtained from freeze dried leaf tissue followed by cold or hot methanol extraction ( Figure 3). The highest contribution to the sample placement on the primary axis had total phenolic content, DPPH radical scavenging activity, glucose, and sucrose content ( Figure 3). On the second axis, the extracts, which were obtained from freeze dried leaf tissues following hot or cold methanol extraction, were separated according to fructose, total glucosinolates, and FRAP values ( Figure 3).

Multivariate Analysis of the Data Obtained by Different Extraction Protocols
The obtained data was processed with Partial Least Squares-Discriminant Analysis (PLS-DA) to further clarify the relations between different extraction protocols. The PLS-DA model was employed due to the supervised nature of the learning algorithm.
Overall, the extracts which included oven drying leaf tissue following cold methanol extraction were separated on the primary axis from the extracts obtained from freeze dried leaf tissue followed by cold or hot methanol extraction ( Figure 3). The highest contribution to the sample placement on the primary axis had total phenolic content, DPPH radical scavenging activity, glucose, and sucrose content ( Figure 3). On the second axis, the extracts, which were obtained from freeze dried leaf tissues following hot or cold methanol extraction, were separated according to fructose, total glucosinolates, and FRAP values ( Figure 3). methanol. Sucrose was the fifth most important variable where the oven dried leaf tissues had lower yields of the compound compared to extracts from freeze dried tissues. The sixth variable, according to the discriminating power, was DPPH radical scavenging, where again the extracts obtained from oven dried tissues exhibited lower antioxidant activity compared to extracts obtained from freeze dried leaf tissues. The variable with the lowest discriminating power according to the obtained model was total glucosinolates where cold methanol extraction from freeze dried Brassica tissue exhibited the highest yield compared to the other two extraction protocols.

Correlations between Bioactive Compounds, Antioxidant Activity, and Sugar Content in Brassica Extracts
Statistically significant correlations (p ≥ 0.05) were observed between bioactive compounds content, antioxidant activity, as well as between sugar content (Table 1). Positive correlations were observed between total phenolic content, total glucosinolates, and FRAP values (Table 1). Negative correlations were observed between total phenolic content and sucrose, glucose, and fructose content, The model showed that the largest difference between extraction protocols was in fructose content where the freeze drying and cold methanol combination yielded the highest amount of the compound. Total phenolic content was the second most important variable in the differentiation between the observed extraction protocols where the oven dried and cold methanol combination exhibited the highest yield of phenolic compounds. The third most important variable in differentiating among extraction protocols was glucose where similar amounts were extracted with either hot or cold methanol from freeze dried leaf tissue, whereas several-fold lower amounts were extracted from oven dried tissue with cold methanol. The next most important variable was FRAP where the highest values were observed in extracts obtained from oven dried tissues by cold methanol. Sucrose was the fifth most important variable where the oven dried leaf tissues had lower yields of the compound compared to extracts from freeze dried tissues. The sixth variable, according to the discriminating power, was DPPH radical scavenging, where again the extracts obtained from oven dried tissues exhibited lower antioxidant activity compared to extracts obtained from freeze dried leaf tissues. The variable with the lowest discriminating power according to the obtained model was total glucosinolates where cold methanol extraction from freeze dried Brassica tissue exhibited the highest yield compared to the other two extraction protocols.

Correlations between Bioactive Compounds, Antioxidant Activity, and Sugar Content in Brassica Extracts
Statistically significant correlations (p ≥ 0.05) were observed between bioactive compounds content, antioxidant activity, as well as between sugar content (Table 1). Positive correlations were observed between total phenolic content, total glucosinolates, and FRAP values (Table 1). Negative correlations were observed between total phenolic content and sucrose, glucose, and fructose content, as well as total glucosinolates and glucose content (Table 1). DPPH scavenging activity correlated positively with sugar content but FRAP correlated negatively with sucrose and glucose content (Table 1). Positive correlations were observed between sucrose, glucose, and fructose content (Table 1).

Discussion
Biological activity of kale containing phytochemicals are associated with antioxidant, anti-cancerogenic activity and protection of gastrointestinal and cardiovascular system [4,[31][32][33]. Sikora et al. found that kale had much higher antioxidant activity than broccoli, Brussels sprouts, and cauliflower [34]. Šamec et al. on the other hand found that white cabbage and kale sprouts had significantly higher antioxidant capacity, polyphenol and glucosinolate content compared to arugula, broccoli, and Chinese cabbage sprouts [20]. Kale is also abundant in organic acids, including citric, malic, pyruvic, shikimic fumaric, and aconitic acids [35].
The freeze drying process is regarded as the best method for sample dehydration because it can prolong shelf life without compromising nutritional quality [36]. The major drawback of this method is limited sample quantity that can be processed simultaneously, long processing time (up to several days) and high energy consumption [37]. On the other hand, oven drying is readily accessible to most laboratories, sample quantity is seldom an issue and the drying time can be as low as one hour but rarely exceeds 24 h.
In our study, freeze dried leaf tissues followed by cold extraction yielded significantly higher total glucosinolate content compared to either oven dried leaf tissue followed by cold extraction or freeze dried leaf tissue followed by hot extraction. The results are in accordance with Rutnakornpituk et al., who investigated the effect of freeze and oven drying on phenolic and glucosinolate content, as well as, antioxidant activity in five cruciferous vegetables where the authors concluded that freeze dried leaf tissues yielded higher glucosinolate content in all investigated species [38]. On the other hand, Tetteh et al. studied the effect of different various drying methods on post-harvest glucosinolate content in Moringa oleifera leaf tissues where no differences were observed between oven dried and freeze dried leaf tissue samples [39]. Doheny-Adams et al. investigated the effect of hot and cold methanol extraction on glucosinolate content in five Brassicaceae species [12]. The results from their study showed that the hot extraction step can be replaced with cold extraction with no losses in glucosinolate content, which is in line with the findings presented in our study [12].
Oven drying, compared to freeze drying followed by cold extraction yielded significantly higher total phenolic content and FRAP values and lower total glucosinolate content and DPPH scavenging activity in B. oleracea var. acephala leaf extracts. Similarly, Managa et al. showed that the majority of investigated phenolic compounds in Chinese cabbage significantly increased in content in oven dried compared to freeze dried samples [40]. Although the phenolic compound content in Chinese cabbage was higher, the antioxidant activity measured by FRAP, DPPH, and ABTS, was significantly lower in the oven dried compared to freeze dried samples [40]. On the other hand, Korus found that freeze dried kale had significantly higher total phenolic content and antioxidant activity compared to air dried kale, albeit at a lower temperature (55 • C) compared to this study (105 • C) [41]. Papoutsis et al. found higher total phenolic content and DPPH scavenging activity in hot air dried compared to freeze dried lemon [28]. Que et al. also found higher total phenolic content and antioxidant activity in hot air dried compared to freeze dried pumpkin flour [27]. Das et al. studied the antioxidant properties of freeze dried and oven dried wheatgrass where higher phenolic content but lower FRAP and DPPH radical scavenging activity was found in hot air dried compared to freeze dried samples [42]. Hot air drying also exhibited elevated total phenolic content and antioxidant activity compared to freeze drying in olive leaves as shown by Ahmad-Qasem et al. [43]. Hot air dried leaf tissue extracts tend to exhibit higher phenolic compounds content, as found in this study, but the link with antioxidant capacity is not clear. In our study we found FRAP having significant correlation with total glucosinolate content as well as total phenolic content, but no significant correlation with DPPH scavenging activity. Interestingly, we found at the same time the highest DPPH radical scavenging activity and lowest FRAP values in freeze dried tissues followed by hot extraction, while exactly the opposite was determined in hot air dried samples followed by cold extraction. The obtained results are contrary to previously published [42,44,45] data where FRAP and DPPH radical scavenging assays have high correlations, especially since it is known that both depend mainly on the electron transfer mechanism which measures the antioxidant's reducing ability [46,47]. FRAP values correlated highly with total phenolic content and negatively with sucrose and fructose content while the data obtained by the DPPH radical scavenging assay correlated positively with the samples' sugar content possibly indicating interferences with the meta-products of Maillard reactions occurring during the hot air drying process [48].
Hot air drying B. oleracea var. acephala leaf tissues followed by cold extraction induced significantly lower content of all investigated sugars compared to freeze drying followed by cold extraction. If freeze dried tissues were subjected to hot extraction sucrose and fructose levels were significantly lower compared to the cold extraction process but higher than oven dried leaf tissues except for fructose. Fante and Zapata Norena studied the quality of hot air dried and freeze dried garlic and found that inulin content decreased with hot air drying while glucose and fructose content increased compared to freeze dried garlic [49]. Iombor et al. studied changes in soursop flour composition as affected by oven and freeze drying and found 40% decrease in carbohydrate content in hot air dried samples [50]. Zhang et al. studied the changes in chestnut starch properties during different drying methods and found lower starch but higher reducing sugar content in oven dried compared to freeze dried samples [51]. Karaman et al. found lower fructose and glucose content in oven dried compared to freeze dried persimmon powders [52]. On the other hand, Gao et al. showed that there was no difference in sucrose, glucose and fructose content in oven and freeze dried jujube samples [53]. The observed decrease in sugar content in our study could be attributed to Maillard reactions occurring at elevated temperatures during hot air drying [54] or possibly the caramelization of sugars [55]. Michalska et al. determined that increased drying temperature of different plum cultivars resulted in decreased total sugar content while early and intermediate Maillard reaction products increased in content [48]. Similarly, Li et al. observed a decrease in reducing sugar content during hot air drying opposed to freeze drying of instant Tremella fuciformis, which the authors attributed to an intense Maillard reaction occurring between the carbonyl group of the reducing sugars and amino acids at elevated temperatures [56]. Previously published data on oligosaccharide and simple sugars extraction in various solvent mixtures and extraction temperatures showed that, with increased temperature, especially at the solvent boiling point, sugar content yields were higher [57]. We, on the other hand, observed lower yields of sucrose and fructose in freeze dried leaves hot extracts, compared to the cold extraction step while glucose levels remained unaffected.

Hot Methanol Extraction
Freeze dried plant material (30 mg) was preheated to 75 °C for 3 min in a heating/cooling dry block (Biosan CH100, Riga, Latvia) and 1.5 mL of preheated 70: 30 methanol:water (v/v) at 75 °C was added. The samples were incubated for 10 min at 75 °C and manually shaken every 2 min. Afterwards the samples were centrifuged at 15,000 G for 5 min (Domel Centric 350, Železniki, Slovenia) and the supernatant was filtered through a 0.22 µ m nylon filter and transferred to a clean tube. The samples were stored at −80 °C until further analysis.

Cold Methanol Extraction
Freeze dried or oven dried plant material (30 mg) was extracted with 1.5 mL of 80:20 methanol: water (v/v) at 20 °C over a period of 30 min in an ultrasonic bath (MRC 250H, Holon, Israel). The

Hot Methanol Extraction
Freeze dried plant material (30 mg) was preheated to 75 • C for 3 min in a heating/cooling dry block (Biosan CH100, Riga, Latvia) and 1.5 mL of preheated 70: 30 methanol:water (v/v) at 75 • C was added. The samples were incubated for 10 min at 75 • C and manually shaken every 2 min. Afterwards the samples were centrifuged at 15,000 G for 5 min (Domel Centric 350, Železniki, Slovenia) and the supernatant was filtered through a 0.22 µm nylon filter and transferred to a clean tube. The samples were stored at −80 • C until further analysis.

Cold Methanol Extraction
Freeze dried or oven dried plant material (30 mg) was extracted with 1.5 mL of 80:20 methanol: water (v/v) at 20 • C over a period of 30 min in an ultrasonic bath (MRC 250H, Holon, Israel). The samples were centrifuged at 15,000 G for 5 min (Domel Centric 350, Železniki, Slovenia) and the supernatant was transferred to a clean tube. The samples were stored at −80 • C until further analysis.

Determination of Total Glucosinolates and Total Phenolic Content
Total glucosinolates were determined according to Ishida et al. [60] with some modifications.

Statistical Analysis
To determine the effect of the investigated extraction protocols on total antioxidant activity, total glucosinolate, total phenolic and sugar content in B. oleracea var. acephala leaves the results were processed by analysis of variance (ANOVA) and reported as mean ± SE. Pearson's correlations were calculated to evaluate the connection between bioactive compounds content, antioxidant activity, and sugar content. Further investigation on the impact of different extraction protocols on the studied compounds was carried out by employing PLS-DA as a supervised multivariate method. All statistical analyses were performed using Statistica 13.4.0.14. (Tibco Inc., Palo Alto, CA, USA). Significant differences were determined at p ≤ 0.05 and homogenous group means were compared by Tukey-Kramer Unequal N HSD test.

Conclusions
The results from our study confirm the results published by Doheny-Adams et al. [12] that cold (ambient temperature) aqueous methanol can be used instead of boiling aqueous methanol with no adverse effects on total glucosinolate content. In addition to higher total glucosinolate content we observed an increase in antioxidant activity measured by FRAP, total phenolic content, sucrose content, and fructose content when the cold extraction step was applied on freeze dried B. oleracea var. acephala plant tissues while glucose levels remained unaffected. Our results also show that hot air drying, compared to freeze drying, followed by cold extraction has an adverse effect on antioxidant activity measured by DPPH radical scavenging, total glucosinolate content, as well as, on the content of all studied sugars in B. oleracea var. acephala leaves, indicating it would be inferior to freeze drying. On the other hand, if phenolics are the compounds of interest in B. oleracea var. acephala leaves, hot air drying may be a viable alternative to freeze drying.