molecules Article Extraction of High Value Triterpenic Acids from Eucalyptus globulus Biomass Using Hydrophobic Deep Eutectic Solvents Nuno H. C. S. Silva , Eduarda S. Morais, Carmen S. R. Freire, Mara G. Freire and Armando J. D. Silvestre * CICECO-Aveiro Institute of Materials, Chemistry Department, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal; [email protected] (N.H.C.S.S.); [email protected] (E.S.M.); [email protected] (C.S.R.F.); [email protected] (M.G.F.) * Correspondence: [email protected] Academic Editor: Mert Atilhan Received: 9 December 2019; Accepted: 31 December 2019; Published: 4 January 2020 Abstract: Triterpenic acids (TTAs), known for their promising biological properties, can be found in different biomass sources and related by-products, such as Eucalyptus globulus bark, and have been extracted using organic volatile solvents such as dichloromethane. Recently, deep eutectic solvents (DES) have been identified as promising alternatives for the extraction of value-added compounds from biomass. In the present work, several hydrophobic DES were tested for the extraction of TTAs from E. globulus bark. Initial solubility studies revealed that DES based on menthol and thymol as the most promising solvents for these compounds given the highest solubilities obtained for ursolic acid (UA) at temperatures ranging from room temperature up to 90 ◦C. Accordingly, an eutectic mixture of menthol:thymol (1:2) was confirmed as the best candidate for the TTAs extraction from E. globulus outer bark, leading to extraction yields (weight of TTA per weight of biomass) at room temperature of 1.8 wt% for ursolic acid, 0.84 wt% for oleanolic acid and 0.30 wt% for betulinic acid. These values are significantly higher than those obtained with conventional organic solvents under similar conditions. The results obtained using these DES are promising for the recovery of TTAs for nutraceutical and pharmacological applications, while reinforcing the potential of DES as promising solvents to be applied in biorefinery processes. Keywords: triterpenic acids; ursolic acid; Eucalyptus globulus outer bark; hydrophobic deep eutectic solvents; menthol; thymol 1. Introduction In the last decades, renewable resources have gathered a raising interest due to increasing concerns with the inevitable dwindling of fossil resources in the decades to come, as well as to the environmental impact resulting from their massive use, associated with global warming and pollution [1]. The development of value chains based on biomass, in the so-called biorefineries, requires the development of sustainable fractionation processes, allowing an integrated exploitation of all biomass fractions [2–4]. Furthermore, the development of biomass refining processes is also relevant to foster the valorization of side-streams and by-products of presently implemented industrial processes, while contributing to the development of the circular economy concept [5,6], fulfilling the UN Sustainable Goals [7]. The integrated exploitation of agroforest biomass and related by-products is nowadays a fundamental issue, in which the development of environmentally friendly strategies for the recovery of high-value compounds plays a significant role [2,5]. As far as forest biomass is concerned, pulp and Molecules 2020, 25, 210; doi:10.3390/molecules25010210 www.mdpi.com/journal/molecules Molecules 2020, 25, 210 2 of 11 paperMolecules industry 2019, 24, is x a key player in the development of these concepts given its importance worldwide,2 of 11 mainly due to the large amounts of woody biomass processed and by-products generated, and low benignworldwide, technologies mainly currentlydue to the used. large Furthermore, amounts of woody this sector biomass needs processed to implement and by-products breakthrough technologiesgenerated, and to low improve benign e ffitechnologiesciency, increase currently the us productsed. Furthermore, quality this and sector portfolio, needs whileto implement incrasing sustainabilitybreakthrough and technologies reducing to environmental improve efficiency, impact increase [8]. In the particular, products quality the pulp and and portfolio, paper industrywhile generatesincrasing considerable sustainability amounts and reducing of forest environmental biomass by-products, impact [8]. In such particular, as leaves, the branchespulp and andpaper bark. Theseindustry by-products generates are,considerable in part, leftamounts in the of forest forest for biomass soil fertilization by-products, or such are as burned leaves, in branches pulp mills and for powerbark. generation.These by-products are, in part, left in the forest for soil fertilization or are burned in pulp mills for Eucalyptuspower generation.species are amongst the most commonly used species by the pulp industry in South WesternEucalyptus Europe (Portugalspecies are and amongst Spain), the South most Americacommonly (Brazil used andspecies Chile), by the South pulp Africa, industry Japan, in South among Western Europe (Portugal and Spain), South America (Brazil and Chile), South Africa, Japan, among others [9]. E. globulus is the dominant species used in Portugal due to its high productivity and high others [9]. E. globulus is the dominant species used in Portugal due to its high productivity and high quality of the pulp fibers produced [10]. However, Eucalyptus by-products could be further valorized if quality of the pulp fibers produced [10]. However, Eucalyptus by-products could be further valorized high value compounds could be extracted prior to their energy conversion. It has been reported that if high value compounds could be extracted prior to their energy conversion. It has been reported several Eucalyptus species outer barks contain large amounts of triterpenic acids (TTAs), such as ursolic that several Eucalyptus species outer barks contain large amounts of triterpenic acids (TTAs), such as (UA),ursolic betulinic (UA), betulinic (BA) and (BA) oleanolic and oleanolic (OA) acids (OA) [11 acids–14] (Figure[11–14]1 (Figure), which 1), display which relevantdisplay relevant biological activities,biological such activities, as antimicrobial, such as antimicrobial, antitumor, antitu hepatoprotective,mor, hepatoprotective, anti-inflammatory, anti-inflammatory, cytotoxic, cytotoxic, anti-allergic andanti-allergic anti-HIV activitiesand anti-HIV [15–21 activities]. Accordingly, [15–21]. theseAcco highrdingly, value these compounds high value have compounds been studied have in been a wide rangestudied of applications in a wide range in the of applications food, biomedical in the andfood, pharmaceutical biomedical and fieldspharmaceutical [22–24]. fields [22–24]. Ursolic acid Oleanolic acid Betulinic acid FigureFigure 1. 1.Chemical Chemical structure of of the the three three ma mainin triterpenic triterpenic acids acids present present in Eucalyptus in Eucalyptus speciesspecies outer outerbark. bark. TTAsTTAs areare usuallyusually extractedextracted from biomass using using volatile volatile organic organic solvents, solvents, such such as as dichloromethanedichloromethane [12 [12],], n -hexanen-hexane [ 25[25],], ethanol ethanol [ 26[26]] oror chloroformchloroform [[18],18], andand using extraction processes processes as simpleas simple solid-liquid solid-liquid extraction extraction and and Soxhlet Soxhlet extraction. extraction. For instance, extraction extraction yields yields of of1.4% 1.4% of the of the threethree main main TTAs TTAs (UA,(UA, BA,BA, OA), from from which which 0.82% 0.82% partakes partakes to UA to UA [12], [12 have], have been been obtained obtained from E. from E.globulus globulus outerouter bark bark by bySoxhlet Soxhlet extraction extraction using using dichlo dichloromethane.romethane. However, However, these solvents these solventsare often are oftentoxic, toxic, and andsome some of these of these processes processes are areenergy energy dema demanding.nding. Therefore, Therefore, it is it important is important to consider to consider greenergreener extraction extraction technologies, technologies, wherewhere novel solvents solvents like like supercritical supercritical CO CO2 may2 may lead lead to an to anefficient efficient andand sustainable sustainable extraction extraction ofof thesethese compounds [11, [1127,28].,27,28]. For For example, example, DominguesDomingues et al. et al.[11][11 have] have optimizedoptimized the the extraction extraction of of TTAs TTAs from fromE. globulusE. globulusouter outer bark bark using using supercritical supercritical CO CO2, under2, under 200 200 bar, bar, 40 ◦C and40 5%°C ethanol,and 5% but ethanol, with extraction but with yields extraction 20% lower yields than 20% those lower obtained than with those dichloromethane obtained with [12 ]. Ionicdichloromethane liquids have also[12]. beenIonic recently liquids studiedhave also for been the extractionrecently studied of TTAs, for achieving the extraction higher of extraction TTAs, yieldsachieving when higher compared extraction to conventional yields when solvents compared [29]. Thisto conventional last study opened solvents new [29]. perspectives This last study to study alternativeopened new and perspectives tunable solvents to study for alternative the extraction and of tu TTAsnable fromsolventsE. globulus for the extractionouter bark, of as TTAs ionic from liquids butE. alsoglobulus deep outer eutectic bark, solvents as ionic (DES)liquids [ 30but]. also deep eutectic solvents (DES) [30]. AA deep deep eutectic eutectic solvent solvent isis composedcomposed of, at