Optimization through Response Surface Methodology of Dynamic Maceration of Olive ( Olea europaea L.) Leaves †

: Bioactive compounds derived from plants are secondary metabolites that can act through various bioactivities, namely as antioxidant, antimicrobial, anti-inﬂammatory, anti-hypertensive, and hypoglycemic agents. Combined with the pressure generated by consumers for more natural products with beneﬁcial effects on health, these compounds may be suitable candidates to act as preservatives in food products. For this purpose, the extraction process becomes essential for the acquisition of a quality extract with efﬁciency and with the desired ﬁnal properties. Therefore, the main objective of this work was to perform the optimization of the extraction yield of olive leaves ( Olea europaea L.), by applying response surface methodology (RSM) and employing dynamic maceration as extraction technique. Three factors were analyzed: time (F1), temperature (F2), and solvent (F3), ranging from 5 to 120 min, 25 to 100 ◦ C, and from 0 to 100% ethanol, respectively. The study used the Box Behnken design, relying on 17 individual randomized runs. The response was the dry weight of the extract (Y1), which ranged from 21.1 to 90.5 mg. The optimization studies pointed to the increase of yield with the increase of time and temperature, but inversely by applying higher time and lower temperature values and higher temperature and lower time values. The highest yield of the dry extract was achieved at 120 min (F1), 25 ◦ C (F2), and 87% (F3) of ethanol:water. Future studies will be carried out to analyze the preservative effects of incorporating olive extract in foods, as well as analysis of other response for optimizing the best food preserving extract.


Introduction
Olea europaea L., a species of Oleaceae and known as olive tree, is one of the most popular plants in the Mediterranean region. Besides its use in the food industry, its leaves, essential oil, and extracts are important sources for the pharmaceutical and cosmetic industry [1]. Olive leaves represent a large part of agricultural waste generated. Thus, with the recovery of discarded leaves, extracts with high bioactive capacity can be obtained [2]. These bioactive compounds may be potential substitutes for synthetic preservatives, and this shift is largely driven by consumer demand for more natural and healthful products [3]. The antioxidant, antimicrobial, anti-inflammatory, anti-hypertensive, and hypoglycemic properties of olive leaf extracts have been reported by several authors, and these characteristics are attributed to the phenolic compounds present in the leaves of this species [2,[4][5][6][7][8]. Previous studies carried out on olive leaf extracts revealed the significant presence of active phenolics, namely oleuropeosides (oleuropein and verbascoside); flavonoids (apigenin-7-O-glucoside, apigenin-7-O-rutinoside, luteolin-7-O-glucoside, luteolin-7-O-rutinoside, rutin, luteolin, apigenin, diosmetin), hydroxycinnamic acid derivatives, with a predominance of verbascoside and substituted phenols (hydroxytyrosol, tyrosol, caffeic and vanillic acids) [5,6,9]. In this scenario, the adequate extraction becomes important to acquire a quality extract with a good functionalization in the final product [10]. Several methods have been applied for the extraction of these compounds from O. europaea L. leaves, such as maceration [5][6][7]11,12], ultrasound-assisted extraction [7,13], and microwave-assisted extraction [7,14]. Although conventional extraction by maceration has some important disadvantages, such as long extraction times and high levels of energy consumption, it is still a widely used technique considering its simplicity and the possible application of green solvents to the process. Therefore, the main objective of this work was to perform the optimization of the extraction yield of olive leaves (Olea europaea L.), through dynamic maceration as extraction technique, by applying response surface methodology (RSM).

Reagents and Plant Material
Reagents with analytical grade were used and purchased from scientific suppliers. The olive leaves (Olea europaea L.) after being purchased were dried and ground to a powder of approximately 20 mesh using a grinder (Moulinex A320, Mayenne, France).

Response Surface Methodology (RSM)
Response surface methodology (RSM) was the technique applied for optimization with dynamic maceration as the extractive method and Box Behnken design as the model used. Three analysis factors were defined: time (F1) ranging from 5 to 120 min, temperature (F2) from 25 to 100 • C, and solvent (F3) from 0 to 100% ethanol. The response variable (Y1) was the dry weight, expressed in mg. Seventeen independent runs with distinct conditions were performed (Table 1), with each factor having minimum, medium, and maximum values based on the literature, that is, upper and lower limits and center point.

Dynamic Maceration (DM)
The maceration was carried out in a thermostatic bath in which the temperature was controlled. Stirring was carried out with the aid of submersible magnetic stirrers (Micro Stirrers, Thermo Scientific Cimarec, Thermo Fisher Scientific, Waltham, MA, USA). The solid/liquid ratio was 30 g/L and constant for all runs applied. After the extraction process, the solution was filtered through Whatman paper filter #4 and then stored for the dry weight procedure. For dry weight, 5 mL of the extraction solution was added to adequately tared crucibles. The crucibles were placed in an oven and remained for four days for complete drying. At the end, the dry crucibles were weighed a second time and the dry weight of each sample was calculated.

Results and Discussion Extraction Optimization Studies
The purpose of the extraction was to obtain the highest yield as a function of the dry weight. Table 2 presents the obtained values of Y1, with the response ranging from 21.1 to 90.5 mg/mL. The coded values were analysed using the Design Expert software, with no transformation, using a quadratic model. The model showed and adequate p-value and an insignificant lack of fit. Figure 1 shows the optimal points with the highest extraction yield, ploting two factors in each graph, maintaining the third at the optimal point. Thus, it is clear that in Figure 1a, low temperature, and 120 min promote extraction yield, which is in acordance with Figure 1b Using the maximize function, the optimum point was set at 25 °C (F1), 120 min (F2), and 87% ethanol (F3). The maceration extraction technique is a conventional and simple technique in which 120 min becomes time consuming, and thus other non-conventional Using the maximize function, the optimum point was set at 25 • C (F1), 120 min (F2), and 87% ethanol (F3). The maceration extraction technique is a conventional and simple technique in which 120 min becomes time consuming, and thus other non-conventional techniques (e.g., ultrasound-assisted extraction) could be applied and may provide the same or higher yield in shorter times. The main advantage presented in the maceration optimization was in relation to the temperature factor, since at 25 • C the need for heating becomes minimal. In industrial applications, the absence of heating avoids additional costs for the extraction process, which is an attractive aspect.
Although a higher dry weight value might not indicate a higher amount of phenolic compounds and higher bioactivity of the extract due to the eventual presence of other substances, it may be an indication of a trend towards a higher presence of bioactive compounds as demonstrated in previous studies in the literature [15][16][17]. Therefore, the optimization performed serves as a basic study for further comparative analyses. Nevertheless, there is also a need for further studies involving the optimization and identification based on the phenolic content present in the sample, as well as antioxidant and antimicrobial analyses to associate with the bioactivity and obtain more concrete results for application.

Conclusions
The optimization performed by RSM indicated the optimal extractive point as a function of dry weight at 120 min (F1), 25 • C (F2) and 87% (F3) of ethanol:water. According to this study, the yield increases inversely with temperature. In addition, for industrial applications, the temperature value found for maceration becomes an attractive factor, considering that it avoids additional costs. Future studies will be conducted to optimize for specific phenolic compounds and by applying other extractive methods to analyze the potential of the extract as a natural preservative. Funding: This research was funded by the Foundation for Science and Technology (FCT, Portugal) and the European Fund for Regional Development (Fundo Europeu de Desenvolvimento Regional (FEDER)) through the Programa Operacional Regional Norte 2020, within the "PlantCovid" project (NORTE-01-02B7-FEDER-054870).
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data available on request.