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The olive-tree leaves as a source of high-added value molecules: Oleuropein
Chapter 5
The olive-tree leaves as a source of high-added value molecules: Oleuropein Lı´dia A.S. Cavacaa, Ignacio M. Lo´pez-Cocab, Guadalupe Silverob and Carlos A.M. Afonsoa,* a
Faculty of Pharmacy, Research Institute for Medicines (iMed.ULisboa), University of Lisbon, Lisbon, Portugal b Laboratory of Applied and Sustainable Organic Chemistry (Labasoc), Department of Organic and Inorganic Chemistry, University of Extremadura, Ca´ceres, Spain * Corresponding author. e-mail: [email protected]
Chapter Outline Introduction The olive tree The Olea europaea complex Origin of the olive tree Olive tree by-products Phenolic compounds Occurrence of phenolic compounds in olive tree Oleuropein Oleuropein chemistry Biosynthesis of oleuropein Biological activities of oleuropein and derivatives Factors that influence oleuropein and phenolic levels Cultivar and environment conditions Harvesting season and fruit size
132 132 132 134 135 137 137 139 139 140 141 143 147
Influence of olive leaf treatment conditions 149 Extraction, purification and analytical methods 151 Natural sources of oleuropein and other phenolic compounds 151 Polyphenols extraction, purification and analytical methods 152 Valorisation of oleuropein 158 General reactivity 158 Synthetic transformations of oleuropein 158 Conclusions 171 Acknowledgments 171 Abbreviations 172 References 173
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Studies in Natural Products Chemistry, Vol. 64. https://doi.org/10.1016/B978-0-12-817903-1.00005-X Copyright © 2020 Elsevier B.V. All rights reserved.
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Introduction Natural products include a large and diverse group of substances obtained from biological sources, such as marine organisms, bacteria, fungi and plants [1]. These compounds have become high-added value ingredients used in foods, dietary supplements, cosmetics and pharmaceuticals [2–5]. Regarding nutrition, some health benefits of the Mediterranean diet, such as lower prevalence of cancer and cardiovascular disease, are credited to the intake of phenolic compounds present in olive oil [6–11]. These phenolic compounds occur not only in oil, but also in other parts of the olive tree, such as leaves and twigs [12]. Olive leaves contain, indeed, a wide variety of phenolic compounds, such as simple phenols, flavonoids, and secoiridoids. Taking into account that these leaves constitute a copious by-product from the olive tree cultivation, their valorisation, as a source of value-added products, should be encouraged [13–15]. The major secoiridoid compound found in olive leaves is oleuropein. This substance, and some of its derivatives, possesses potent biological and pharmacological activities, such as anticancer, cardioprotective, neuroprotective, gastroprotective, hepato-protective, anti-diabetes, anti-obesity and radioprotective ones. These properties are in large part attributed to its presumed antioxidant and anti-inflammatory effects [16]. The fact that natural products, such as oleuropein, are able to interact with a broad range of biological macromolecules, makes them highly attractive in pharmaceutical research. Because of this, isolation, structural elucidation and formulation of key active compounds from natural sources have gained an increasing importance [17]. Furthermore, natural products can serve as powerful starting materials to generate drug substances with novel therapeutic utility, by means of synthetic modifications [18].
The olive tree The Olea europaea complex The family Oleaceae is composed of 25 genera. One of them is the genus Olea L., which comprises >40 species. The species Olea europaea L. is native to temperate regions in Europe, Africa, and Asia, although it has been cultivated in various places, with the appropriate climate, all around the world [19,20]. The details of this species were originally published in the Carl Linnaeus’ book entitled Species Plantarum [21]. There are six natural subspecies: subsp. europaea, in the Mediterranean basin; subsp. laperrinei, in the Sahara region; subsp. cuspidata, in South Africa, Southeast Asia, China, and Arabia; subsp. Maroccana, in Morocco; subsp. cerasiformis, in the Madeira archipelago; and subsp. guanchica, in the Canary Islands [20,22]. However, according to other sources, only two subspecies are accepted. These are the subsp. cuspidata, or African olive, and the subsp. europaea, or European olive [23].
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Two varieties are recognized within the O. europaea subsp. europaea: the cultivated olive tree (var. europaea), and the wild olive tree (var. sylvestris), known as oleaster. The term oleaster is used for both true wild forms and feral olive trees, as well. Overall, the true wild olive is not morphologically distinguishable from the feral form, and genetic markers has to be used for this purpose. Some botanists consider the wild variety as a valid species, named Olea oleaster, rather than a subspecies [20,24,25]. According to molecular results, the wild olive tree is to be considered the ancestor of the cultivated olive tree [26,27]. Olea europaea has 46 chromosomes and is a diploid species [28]. However, karyological studies are difficult since the chromosomes are small, similar in morphology and numerous. The presence of tetraploids and hexaploids was detected in subsp. cerasiformis and maroccana, respectively [29–31]. In addition, the coexistence of diploid and triploid individuals within the same population was also detected in a South Algerian population of the Laperrine’s olive. The triploid population does not represent >3% [32] Table 5.1. Olive constitutes an essential tree crop; production of olive oil and olive fruit is paramount in the Mediterranean basin, and the health benefits associated with its culinary use have prompted its cultivation in areas outside its original cradle [24]. The harvested area of olive trees is about 10.6 million
TABLE 5.1 The different taxa of the Olea europaea complex and their ploidy. Native geographic distribution
Ploidy
Oleaster, wild olive
Mediterranean basin
2
Olive, cultivated olive
Mediterranean basin
2
cuspidata
African olive
From South Africa to Southwest China
2
laperrinei
Saharan olive, Laperrine’s olive
Saharan mountains from south Algeria to northeast Sudan
2 (3)
guanchica
Canarian olive
Canary islands
2
cerasiformis
Madeiran olive
Madeira archipelago
4
maroccana
Moroccan olive
High Atlas, Morocco
6
Subspecies
Variety
Common name
europaea
sylvestris
europaea
europaea
Adapted from P.S. Green, Kew Bull. 57 (2002) 91–140; Integrated Taxonomic Information System on-line database, http://www.itis.gov. (Retrieved on July 2018).
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hectares, according to data from 2016; >96% of this area is concentrated in the Mediterranean region. Spain has 2.5 million hectares of olive groves, and it is the main producer of olive oil [34].
Origin of the olive tree Oleaster has thriven all along the Mediterranean shores. A study based on evaluation of archeological remains and examination of the wild relatives of the cultivated crops concluded that olive was most likely domesticated in the Near East in protohistoric time (fourth and third millennia B.C.) [35]. However, the controversy of the origin of the olive tree has come a long way since then; there are reports favoring the monofocal origin hypothesis followed by studies questioning it. In fact, evidences based upon wood charcoal and pollen sequences support the existence of oleaster both in the Levant and in Iberia during the late Pleistocene, proving that the wild olive is also genuine in the western Mediterranean [36–38]. Morphometric analysis of olive pits from both wild and cultivated olive trees from different regions shows the emergence of cultivation practices from the Neolithic and the Bronze Age in Spain [39,40]. Furthermore, study of archeological charcoal in Estremadura (Portugal) indicates that this region was a refuge area for Olea europaea during the colder periods of the Pleistocene [41]. Studies based on molecular analysis have challenged the single origin theory, too [42]. Nuclear DNA polymorphism in oleaster displays a gradient between the east and west of the Mediterranean basin, whereas in cultivars, the gradient is less visible owing to their diffusion and selection. According to this study, no unidirectional flux of domesticated olives would have taken place [27,43,44]. Diversity from the East thus enriched the biodiversity existing in the West by hybridization with local forms [45]. The structure of genetic polymorphism between the eastern-western parts of Mediterranean basin is in favor of a large-scale geographic differentiation of olive tree, whereas the crosses between the different taxa suggest that gene flows between them could occur during more or less recent stages of their differentiation [26]. The findings of the study of maturase K (matK) gene sequence of 46 populations of the Olea europaea complex support the grouping of subsp. europaea, cerasiformis, guanchica, maroccana, and laperrinei in one group separate from subsp. cuspidata. According to the same report, Inter-Simple Sequence Repeats (ISSR) data provide evidence for either gene flow between var. europaea and var. sylvestris, or for the repeated domestication of the cultivated olive [46]. Allozyme markers provide evidence of the survival of indigenous oleaster populations, particularly in the western part of the basin. Genetic diversity values over the domesticated olives, feral trees, and wild olives are consistent
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with the interpretation that domesticated olive trees represent a sample of the genetic variation in genuinely wild olive populations that persist today [24]. In Andalusia (Spain) where large areas of oleaster forests are present [47,48], genetic diversity and relationships between local cultivars and wild olive trees indicated a clear differentiation of Andalusian wild olives from cultivars of this area [38]. Moreover, the results obtained show that wild olive genetic resources of Andalusia represent a clearly differentiated gene pool either from cultivars of the same area and wild olive populations of other regions studied. Andalusian wild olives represent an interesting gene pool for olive breeders, taking into account that traits, such as resistance to pathogens and parasites, low plant vigor and adaptation to adverse environments, are rarely found in cultivated germplasm [49]. When it comes to gene pool sources for olive breeding, the Laperrine’s olive emerges as a subspecies of great interest; it thrives in the extremely arid and harsh conditions found in center Sahara desert, at an altitude between 1400 and 2800 m. It reproduces through vegetative or clonal growth. In fact, plastid DNA analyses indicate that Laperrine’s olive has been used during the secondary diversification of the cultivated olive tree in the region [50,51]. Nuclear microsatellite and plastid DNA data show a high genetic differentiation between europaea and laperrinei. Although no first-generation hybrid between them is detected, recent reciprocal admixture between Mediterranean and Saharan subspecies is found in a few accessions, thus confirming that laperrinei has been involved in the diversification of cultivated olives [52]. The complex process of propagation of olive clones along the Mediterranean makes it difficult to unambiguously identify the pedigree of different cultivars. Moreover, it is very likely that different cultivars have interbred with wild olive trees, therefore, contributing to the genetic diversity of the species [48]. The questions on where and how often has the crop been domesticated remain open to debate and more studies are needed. There are studies that supported up to nine different primary domestication events across the Mediterranean [43,53,54]. However, more recent reports favor the theory of the unique domestication site [47,50,52], or the two-focal domestication scenario [48]. The olive genomic era is advancing at a high rate. Undoubtedly, the recent report on the genome sequencing of the oleaster, and the expected resequencing of several olive varieties will provide a valuable resource for the study of the evolution and domestication processes of the olive tree [55]. Moreover, it will enhance breeding programs and the formation of new varieties [56,57].
Olive tree by-products Industries generate a large quantity of by-products as a waste containing highadded value chemical compounds.
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SCHEME 5.1 By-products from olive tree production and olive processing industry. Adapted from J. Meirinhos, B.M. Silva, P. Valenta˜o, R.M. Seabra, J.A. Pereira, A. Dias, P.B. Andrade, F. Ferreres, Nat. Prod. Res. 2005, 19, 189-195.
The main by-products arising from both olive tree cultivation and olive processing industry are divided into oil extraction by-products (i.e., crude, exhausted “solvent-extracted” and partly destoned olive cakes, olive pulp, vegetation waters, and leaves collected at olive mills), as well as pruning and harvest residues (i.e., olive leaves, large branches and twigs), Scheme 5.1. The world crude olive cake production is estimated at about 2.9 106 tons. Usually, all types of olive cake are used in animal feeding. Exhausted olive cake has extended application as fuel or furfural source, Scheme 5.1. Substantial quantities of olive leaves are generated every year (10–30 kg tree1, 6 108 trees worldwide) found in large amounts during olive oil production, when leaves are separated from olives (5% of the weight of olives), and during olive tree pruning (25% of the weight of olives). The global amount accumulated annually may exceed 18 million tons (2006–2013 average) [58]. Olive leaves are mostly used as animal feed or simply directly thrown away, burned, or grinded and scattered on the field, potentially causing environmental damage, and with increasing cost for producers due to their removal, storage and elimination [59]. Olive by-products represent a considerable but insufficiently exploited resource and their potential should not be neglected. The generation of large amounts of these by-products make the search for new applications a demand. In addition, their content in high-added value compounds must encourage the valorisation of these bio-renewable resources.
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Phenolic compounds Occurrence of phenolic compounds in olive tree Plants are one of the most important sources of natural bioactive products. They have developed an effective defense system against predators, involving the production of a large number of different phytochemical compounds [1]. Phytochemicals are high-added value natural substances used as ingredients in foods, dietary supplements, cosmetics and pharmaceuticals. The consumers’ preference for products containing natural additives, generally perceived as safer and healthier, has increased the demand for phytochemical ingredients [3–5]. Polyphenols constitute one of the most common groups of phytochemicals in plants [60]. Olive biophenols include a major group of secondary metabolites distinguished by their water solubility and high molecular weights. They are of considerable physiological and morphological importance in plants, displaying a wealth of both structural variety and diversity of key activities. Actually, their excellent properties are a consequence of their function in the tree, namely reactivity against pathogens attack and response to insect injury [13,61]. Polyphenols are present in almost all parts of the olive tree but their nature and concentration vary greatly among the tissues. In olive fruit/olive oil several phenolic compounds are identified— flavonols (e.g., quercetin 3-rutinoside, luteolin 7-O-glucoside, luteolin 5-Oglucoside, apigenin 7-O-glucoside), phenolic acids (e.g., chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, syringic acid, p-coumaric acid, o-coumaric acid, ferulic acid, sinapic acid, benzoic acid, cinnamic acid, gallic acid), phenolic alcohols (e.g., 3,4-DHPEA, p-HPEA) and secoiridoids (e.g., oleuropein, demethyloteuropein, ligstroside, nuzhenide), Fig. 5.1. The main hydroxycinnamic acid derivative is verbascoside. Hydroxytyrosol and tyrosol are present at highest contents in comparison to other phenolic compounds. Oleuropein is the major secoiridoid in unripe olive fruit [62]. Five groups of phenolic compounds are mainly identified in olive leaves: oleuropeosides (oleuropein and verbascoside), flavones (luteolin 7-O-glucoside, apigenin 7-O-glucoside, diosmetin 7-O-glucoside, luteolin and diosmetin), flavonols (rutin), flavan-3-ols (catechin) and substituted phenols (tyrosol, hydroxytyrosol, vanillin, vanillic acid and caffeic acid). Additionally, the dialdehydic deacetoxy forms of the secoiridoid compounds oleuropein and ligstroside, oleocanthal and oleacein can be found, Fig. 5.1. Phenolic compounds such as luteolin 7-O-rutinoside, luteolin 4’-O-glucoside, apigenin 7-O-glucoside and apigenin 7-O-rutinoside are also reported, Fig. 5.1. The most abundant compound in olive leaves is oleuropein, followed by hydroxytyrosol, the flavone-7-glucosides of luteolin, apigenin and verbascoside. A typical composition of the lyophilized extract of olive leaf is shown in Table 5.2 [63].
FIG. 5.1 Examples of polyphenols in olive tree. Glc—Glucose; Rut—Rutinose. Adapted from N. Talhaoui, A. Taamalli, A.M. Go´mez-Caravaca, A. Ferna´ndezGuti errez, A. Segura-Carretero. Food Res. Int. 77 (2015) 92–108.
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TABLE 5.2 Phenolic compounds, in mg/kg, in olive leaf lyophilized extract. Compound
mg/kga
Oleuropein
26,471.4 1760.2
Luteolin 7-O-glucoside
4208.9 97.8
Apigenin 7-O-glucoside
2333.1 74.7
Luteolin 40 -O-glucoside
1355.9 75.9
Verbascoside
966.1 18.1
Rutin
495.9 12.2
Caffeic acid
220.5 23.3
Sum of the determined compounds
36,051.8
a Results are expressed as mean standard deviation of three determinations. Adapted from A.P. Pereira, I.C.F.R. Ferreira, F. Marcelino, P. Valenta˜o, P.B. Andrade, R. Seabra, L. Estevinho, A. Bento, J.A. Pereira, Molecules 12 (2007) 1153–1162.
Oleuropein Oleuropein chemistry Oleuropein is included in a specific group of coumarin-like compounds, the secoiridoids, and is the most abundant biophenol in olive leaves. Bourquelot and Vintilesco detected oleuropein in olives for the first time in 1908 [64], but its chemical structure was assigned by Panizzi et al. in 1960 [65]. It was specified as the heterosidic ester of β-glucosylated elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol) and is responsible for bitterness taste in olive oil. It has a stereocenter at C-5 with (S) configuration and an exocyclic 8,9-double bond with (E) configuration, characteristic of secoiridoids. The molecular structure of oleuropein can be divided into three subunits: hydroxytyrosol, monoterpene and glucose moieties, Fig. 5.2. [10,66–69]. The low-energy conformation of oleuropein was investigated through semiempirical and ab initio calculations and confirmed by molecular dynamics simulations. The minimum energy conformation of oleuropein in vacuo has a closed geometry in which the glucose moiety is in close proximity to the hydroxytyrosol unit. The structure is stabilized by hydrogen bonds between the catechol hydrogen atoms and the hydroxy groups of the sugar ˚ short-axis radius and 8.22 A ˚ moiety, forming a hydrophobic cavity of 3.56 A
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FIG. 5.2 Molecular structure of oleuropein. Representation of hydroxytyrosol (red), monoterpene (green) and glucoside (black) moieties.
FIG. 5.3 Representation of the hydrogen bond network between hydroxytyrosol and glucoside moieties of oleuropein. Dashed lines represent hydrogen bonds. Adapted from E. Gikas, F.N. Bazoti, A. Tsarbopoulos, J. Mol. Struc.-THEOCHEM 821 (2007) 125–132.
long-axis radius, and by hydrophilic regions on the exterior of the molecule, Fig. 5.3. Nevertheless, these simulations did not consider the effect of solvent on oleuropein’s conformation [70]. Recently, molecular dynamics simulations showed extensive changes in oleuropein’s conformation depending on the microenvironment [vacuum, water, or triolein (oil)/water systems], showing the amphiphilic character of this compound [71].
Biosynthesis of oleuropein Iridoids (A) are monoterpenes characterized by a bicyclic fused ring system comprising a six-membered heterocyclic ring fused to a cyclopentane ring. Secoiridoids (B) are derived from iridoids by the opening of the cyclopentane ring at the 7,8 bond, Fig. 5.4 [72]. Oleuropein and other secoiridoids characterized by an exocyclic 8,9olefinic functionality are termed oleosidic secoiridoids or oleosides and
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FIG. 5.4 General representation of iridoids (A) and secoiridoids (B) molecular structures.
are unique to oleaceous plants. Secoiridoid conjugates that contain an esterified phenolic moiety result from a branching in the mevalonic acid pathway in which oleoside moiety (monoterpene) synthesis and phenylpropanoid metabolism (phenolic unit) merge [72]. Mevalonic acid is formed from the initial condensation of three acetylSCoA molecules, originating the ester β-hydroxy-β-methylglutaryl-CoA (HMG-CoA), which produces mevalonic acid after hydrolysis and an enzymatic reduction [73]. The proposed biosynthetic pathway for oleuropein formation in Oleaceae is shown in Scheme 5.2, via 7-epi-loganic acid and ligstroside as direct precursor [72].
Biological activities of oleuropein and derivatives Secoiridoids are known to exhibit a wide range of biological and pharmacological activity [74]. Olive leaf extract gained popularity in the global nutraceutical market due to a range of claimed health attributes, traditionally consumed for the treatment of a wide spectrum of ailments [16]. Oleuropein is responsible for most of the extract attributes and has potent biological and pharmacological properties, including anticancer, cardioprotective, neuroprotective, gastroprotective, hepato-protective, anti-diabetes, antiobesity and radioprotective. These properties are in large part attributed to its putative antioxidant and anti-inflammatory effects [16,63]. Oleuropein derivatives were also found to have biological properties. The accumulation of free radicals on cellular membrane lipids play a major role in various pathological disorders, including atherosclerosis, cancer, aging, rheumatoid arthritis and inflammation [75]. Compounds sharing an ortho-diphenolic (catecholic) structure are known to possess antioxidant activity. In this way the high antioxidant capacity of oleuropein is conferred by the catecholic structure, having the ability to scavenge free radicals, and consequently acts in the treatment of some pathologies associated with oxidative stress, Fig. 5.5 [16]. Prevention of free radical formation by oleuropein may be due to its ability to chelate metal ions, such as Cu and Fe, which catalyze
SCHEME 5.2 Proposed biosynthetic pathway for the formation of oleuropein. Adapted from H.K. Obied, P.D. Prenzler, D. Ryan, M. Servili, A. Taticchi, S. Esposto, K. Robards, Nat. Prod. Rep. 25 (2008) 1167–1179.
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FIG. 5.5 Bioactive functional groups of oleuropein and olive leaf extract health attributes. Adapted from L. Gentile, N.A. Ucella, G. Sivakumar, Curr. Med. Chem. 24 (2017) 4315–4328.
free radical generation reactions, as well as its ability to inhibit several inflammatory enzymes, such as lipoxygenases, without affecting the cyclo-oxygenase pathway [76]. The health benefits associated to olive leaf extract, oleuropein and its derivatives are presented in Table 5.3, compiling several studies performed in vitro in cell lines and in vivo using animal models and human volunteers. All these valuable biological properties make oleuropein and its derivatives promising compounds for pharmaceutical, food and cosmetic application. Therefore, it is important to stimulate research from different areas to enhance the preparation of compounds with additional interest.
Factors that influence oleuropein and phenolic levels Oleuropein and total phenolic levels are affected by abiotic (non-living chemical and physical parts of the environment that affect the tree, such as soil, water, air, temperature, moisture, etc.) and biotic (other plants, fungi and bacteria, animals and human influences) factors [11]. In this way, oleuropein and other phenolic content depend, essentially of cultivar, harvesting season, maturity of leaves, proportion of branches on the tree, geographical origin and moisture content [97–101]. These changes are widely reported in literature, where different extraction and analytical methods are used for content determination [11,58,68,88, 100,102–117].
TABLE 5.3 Health benefits of olive leaf extract, oleuropein and its derivatives. Studies
Compounds
Subjects/cell models
Effects
In vivo
Olive leaf extract
Animal studies
Adult male Swiss mice; whole-body irradiated with a single dose of 48 cGy
Antioxidant and radioprotective effects; hydroxyl radical scavenging capacity [77]
Oleuropein
Adult male Spargue-Dawley rats; oleuropein plus ethanol (12 mg/kg body weight; 10 days)
Antioxidant effect by scavenging of ROS, produced by ethanol that initiate lipid peroxidation [78]
Oleuropein and Hydroxytyrosol
Adult male Wistar rats; diabetes induced with alloxan
Anti-diabetic and antioxidant effects [79]
Hydroxytyrosol and its triacetylated derivative
Wistar rats fed a standard laboratory diet or cholesterol-rich diet
Lipid-lowering and antioxidant effects [80]
Olive leaf extract
Wistar rats exposed to cold restraint stress; olive leaf extract (80 mg/kg daily) dissolved in distilled water
Modulation of cold restraint stress oxidative changes in rat liver by inhibition of lipid peroxidation [81]
Olive leaf extract
Sprague-Dawley rats receiving gentamycin (25 mg/kg, 50 mg/kg or 100 mg/kg daily, 12 days)
Amelioration of gentamycin nephrotoxicity by inhibition of lipid peroxidation [82]
Oleuropein
Female C57BL/6 mice; azoxymethane (AOM)/ dextran sulfate sodium (DSS)-induced colorectal cancer; oleuropein (50 mg/kg or 100 mg/kg) dissolved in distilled water
Protection from AOM/DSS-induced colorectal cancer associated with acute colitis; suppression of the growth and multiplicity of colonic tumors [83]
Hydroxytyrosol
Adult male Wistar rats; C6 glioma cell implantation; subcutaneous injections of 100 μg oleuropein, 100 μg hydroxytyrosol or both daily, for 5 days
Only hydroxytyrosol inhibited tumor growth [84]
Oleuropein
Visceral leishmaniosis model L. donovaniinfected BALB/c mice; intraperitoneal injection of oleuropein 14 times
Oleuropein and hydroxytyrosol selectivity for L. donovani; Oleuropein gave parasite depletion of >95% in liver and spleen after 6 weeks [85]
In vivo
Oleuropein
10 healthy female 20–30 years having skin Fitzpatrick types II and III
Soothing effect in the treatment of UVBinduced erythema [86]
In vitro
Oleuropein
Animal/ microbial cell lines
Normal mouse hepatocyte FL83B cells; HepG2 and FL83B cells
Decrease of the number size of lipid droplets in free fatty acid-treated cells and reduced intracellular triglyceride accumulation [87]
Olive leaf extract
Bacillus cereus CECT 148; B. subtilis CECT 498; Staphylococcus aureus ESA 7; Escherichia coli CECT 101; Pseudomonas aeruginosa CECT 108; Klebsiella pneumoniae ESA 8.
Antimicrobial effects observed with contribution of oleuropein and hydroxytyrosol [88]
Human studies
Candida albicans CECT 1394; Cryptococcus neoformans ESA 3. In vitro
Oleuropein
Whole blood of 11 healthy male volunteers
Inhibition of platelet activation by scavenging of H2O2 produced in arachidonic acid metabolism cascade that leads to platelet aggregation [89]
Oleuropein
Human breast cancer cell line MCF-7
Apoptotic cell death of human breast cancer MCF-7 cells [90]
Oleuropein and its semisynthetic peracetylated derivatives
Human breast cancer cell lines (MCF-7 and T-47D)
Anti-proliferative and antioxidant effects; the peracetylated compounds exerted higher antiproliferative effects than oleuropein [91]
Hydroxytyrosol rich extract
MCF-7 human breast cancer cells
A dose-dependent growth inhibition of MCF-7 cells [92]
Human cell lines
and Hydroxytyrosol
Continued
TABLE 5.3 Health benefits of olive leaf extract, oleuropein and its derivatives.—Cont’d Studies
Compounds
Subjects/cell models
Effects
Olive leaf extract
Peripheral blood leukocytes from six healthy volunteers
Protective effect on the peripheral blood leukocytes against adrenaline induced DNA damage [93]
Olive leaf extract
Human endothelial cells from bovine brain, MCF-7 cells and T-24 cells (human urinary bladder carcinoma)
Antiproliferative effect against cancer and endothelial cells [94]
Olive leaf extract (oleuropein and apigenin-7glucoside)
Human HL-60 cells
Apigenin-7-glucoside of the olive leaf extract was mainly responsible for the HL-60 differentiation and oleuropein showed to exert an influence over this differentiation [95]
Oleacein
Human recombinant 5-lipooxygenase (5-LO)
Inhibition of 5-LO (IC50 ¼ 2 μM), acting as anti-inflammatory agent [96]
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Cultivar and environment conditions The composition of olives varies with cultivar and environment conditions. Olive cultivar affects the absolute concentration of specific polyphenols, while the total phenolic profile remains practically unchanged. Oleuropein is always present in the fruit of all cultivars, but verbascoside is an example of a cultivar-dependent compound. Environment conditions such as temperature, moisture, altitude and leaves age also alter the amount of oleuropein and phenolic profile. Cold stress and freezing cause severe dehydration and induce cell membrane damage in olive leaves. In lightly cold stressed leaves, oleuropein level is lower than in non-stressed samples. On the other hand, in moderately and heavily cold stressed leaves, oleuropein concentration was higher after exposure to cold stress. The accumulation of oleuropein and its antioxidant capacity may protect against oxidative damage induced by freezing. The activity of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) is another element in the response of the tree to such stress. PAL is involved in the mechanism of plant recovery if the cold stress is low or medium. If the cold stress is high, PPO activity avoid serious oxidative damage [118]. The effect of water deficit on olive leaf phenolic composition was studied. Water deficit stress induces accumulation of phenolic compounds, especially oleuropein, suggesting their role as antioxidants [109]. Oleuropein is also involved in the olive tree protection against salinity stress in leaves, serving as a glucose-reservoir for osmoregulation and high energy-consuming processes required for plant adaptation to salinity, increasing its content [119]. Total phenolic levels in leaves decline as the geographical altitude decreases, a factor related with changes in climatic conditions with geographical altitude. Leaves from trees cultivated in windy and humid air have lower levels of phenolic compounds [120]. An important biotic factor affecting the phenolic content is the age of the leaves. Oleuropein amount is higher in younger leaves than in mature ones, which suggest gradual oleuropein degradation with the leaves progressive age [103,104].
Harvesting season and fruit size Oleuropein levels are affected by harvesting season. During the growth period of olives, oleuropein levels decrease reaching a minimum in July, when the olive fruit is fully developed. Unexpectedly, the total phenolic content increase in July, suggesting a degradation of oleuropein and appearance of other phenolic substances [121]. During ripening, while elenolic acid glucoside (jaspolyside) (A) and demethyloleuropein (B) accumulate in olive fruits, oleuropein levels decrease. Oleuropein biodegradation into these glycosylated derivatives involves cleavage
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SCHEME 5.3 Molecular structures of elenolic acid glucoside (A) and demethyloleuropein (B), formed by esterase action, and aglycone forms (C and D) and oleuropein aglycone (E) by β-glucosidase action.
by specific endogenous esterases, through hydrolysis of hydroxytyrosol ester or methyl ester, respectively. Activation of endogenous β-glucosidase during crushing or malaxation might produce the aglycone forms (C, D and E) by cleavage of glucose, Scheme 5.3 [122–125]. Ryan et al. have paid attention to other tissues, suggesting that different tissues have different phenolic composition, and as such, the metabolism of each plant tissue is characteristic [104]. On the contrary, Briante et al. claim that the biodegradation of oleuropein is similar in olive leaves. Most of the molecules isolated from olive leaves are thought to have originated from oleuropein where the above mentioned endogenous enzymes played an important role [67]. Recently, De Leonardis et al. found that the enzyme polyphenol oxidase (PPO) is also involved in oleuropein degradation in olive leaves. Fresh leaves and dried leaves showed different enzymatic activity on oleuropein. Extract from fresh leaves provided both β-glucosidase and PPO activity. However, the decrease of oleuropein concentration in dried leaves was mainly attributable to the PPO activity, leading exclusively to the formation of oxidation products [126].
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Oleuropein concentration can also be affected by the dimensions of the fruits, which is intrinsically related with fruit maturity, since larger fruits are associated with maturation. Small-fruit varieties usually have high oleuropein and low verbascoside contents; the opposite is characteristic of largefruit varieties [11,15,88,127,128].
Influence of olive leaf treatment conditions The conditions in which olive leaves are treated influence the phenolic content. Currently, there are no widely accepted guidelines for the drying of olive leaves and data from literature are insufficient or contradictory. However, in order to stabilize the by-product, avoid quality losses and undesirable degradation during storage, the immediate dehydration of olive leaves is an important operation. Traditional methods of drying as shade or sun drying are still practiced, but this operation is not well controlled, influencing the final quality of the product. Thus, for industrial purposes, hot air drying is the most used method, allowing an accurate control of the process variables [129]. Several studies have been interested in the investigation and modeling of the drying behavior of olive leaves. The effect of blanching and infrared drying on the leaves color, total phenol content and moisture removal rate of four olive tree varieties was studied, using an infrared dryer at 40 °C, 50 °C, 60 °C and 70 °C. Infrared drying seemed promising, inducing a considerable moisture removal from the fresh leaves (>85%) and short drying time (15 min at 70 °C). Total phenol content of dried olive leaves increased compared to fresh leaves. In the specific case of Chemlali variety, total phenols increased from 13.8 0.2 to 21.3 2.9 mg GAE/g d.w. (+ 54.35%) after IR drying at 40 °C. Infrared drying is then suggested for preserving olive leaves during storage [130]. Erbay et al. studied the use of hot air to dry olive leaves in a pilot-scale heat pump conveyor dryer and evaluated the effect of drying on product quality. Optimum operating conditions were found to be at 53.43 °C, air velocity of 0.64 m/s, and process time of 288.32 min. At these conditions, was observed a total phenolic content decrease (9.77%), moisture loss of 94% and antioxidant activity loss of 44.25% [131]. The effect of freeze- (liquid N2 or conventional freezing 28 °C) and hotair (70 °C or 120 °C) drying on the concentration of olive leaves polyphenols was studied. Both drying methods significantly influenced the concentration of polyphenols. Freeze drying was not considered an adequate technique to promote extraction since the leach of phenolic compounds from olive leaves was not promoted. The antioxidant potential was reduced compared with that of fresh leaves, due to activation of oxidative enzymes. N2-freeze treatment was better than conventional, increasing oleuropein content by 448% compared with fresh leaves, and providing total phenolic contents of 37 2 and
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36.3 1.4 mg GAE/g d.w., respectively. Hot-air drying at 120 °C provided a higher phenolic content (45 2 mg GAE/g d.w.) than freeze-drying, especially in oleuropein. The reduction of the drying temperature to 70 °C results in a significant decrease of total phenols (45 2 mg GAE/g d.w.). Hot-air drying was evaluated on leaves previously affected by N2-freezing. Freezing prior to hot-air drying significantly reduced total phenolic content (37 2 mg GAE/g d.w., 70 °C and 49 2 mg GAE/g d.w., 120 °C) [132]. The effect of solar drying conditions on the drying time and some quality parameters of olive leaves, such as color, total phenol content and radical scavenging activity, was investigated using a laboratory convective solar dryer. Temperatures of 40 °C, 50 °C and 60 °C and air velocities of 1.6 and 3.3 m3/min were employed in the study. The drying time required to reduce the moisture content to 0.10 kg kg1 d.w. depended on the temperature. Total phenol content was affected by drying conditions and olive leaves variety. In general, temperature of 60 °C and air velocity of 3.3 m3/min resulted in higher content of polyphenols among the four varieties (16.86–24.08 mg caffeic acid/ 1 g d.w.), although was observed a decrease in all values compared with that of fresh leaves (22.03–26.06 mg caffeic acid/1 g d.w.) [133]. Microwave radiation is also used in leaves drying. Aouidi et al. used a microwave oven and dried leaves twice for 2 min at a maximum power of 800 W. Lyophilization was adopted for oleuropein determination in olive leaves [134]. Recently, microwave was seen as a reasonable selection for drying olive leaves preserving phenolic compounds during storage. Optimum conditions for microwave drying were found to be 2.085 g of sample at 459.257 W for 6 min to achieve maximum yields of total phenolic content (38.712 mg GAE/g d.w.) and oleuropein (203.561 mg/g d.w.). Microwave drying was considered a promising technique compared to other methods, namely freeze-, vacuum-, oven-, and ambient air-drying [135]. The influence of an ultrasound energy system was also investigated on the drying process of olive leaves. Air drying experiments (40 °C, 1 m s1) were performed without or with ultrasound application (8, 16, 25 and 33 kW/m3). The use of ultrasound on drying olive leaves could represent a way to increase the drying rate [136]. Kamran et al. studied the recovery of phenolic compounds from fresh, air-dried, freeze-dried and oven-dried (60 °C and 105 °C) olive leaves. Extracts of oven-dried leaves at 105 °C showed the highest phenol recoveries ( 140 mg/g d.w.). Olive leaves oven-dried at 105 °C for 3 h increased oleuropein recovery as compared with fresh olive leaves [14]. Erbay et al. determined and tested the most appropriate thin-layer drying model with air temperatures of 50 °C, 60 °C and 70 °C, and air velocities of 0.5, 1.0 and 1.5 m s–1, to understand the drying behavior of olive leaves. The drying depends on the velocity and temperature of the air. The temperature has great influence, in a way that the drying rate increases with increased temperature [137].
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Malik and Bradford compared freeze-, ambient (25 °C) air-, and hot (60 °C) air-drying of olive leaves and their effect on oleuropein and other phenolic stability and recovery. Air-drying at ambient temperature revealed a fully preservation of oleuropein and verbascoside levels, while freeze- and hot air-drying caused a reduction in polyphenolic content, including oleuropein [freezing—53.5–95.4% loss (time dependent); hot air—19–32% loss] [102]. Another factor studied is the effect of blanching process, which involves the treatment by means of form of heat, usually either steam or boiling water [138]. Recently was reported that the water blanching of olive leaves (1:4 (w/v), 90–95 °C, 20 s) increased phenolic content up to 0.329– 0.532 mg/g GAE (61.7%) when compared with fresh, solar-dried and ovendried leaves [139].
Extraction, purification and analytical methods Natural sources of oleuropein and other phenolic compounds Plants from Oleaceae family are a natural source of oleuropein and other phenolic compounds. As described above, the olive tree (O. europaea L.) is considered the main source of oleuropein, which can be found in several parts of the plant (i.e., leaves, olives and branches), Fig. 5.6. Argania spinosa L. was identified as another natural source of oleuropein. However, only the argan oil is reported to have this natural product. Pressed cake, leaves or fruit pulp from this plant have no oleuropein [140], Fig. 5.6. The use of olive leaves as natural source of oleuropein is advantageous comparing to other natural sources, since they are highly available as a by-product (see Section Olive tree by-products) throughout the year and have a higher content in oleuropein [0.5–2% (w/w) on dry basis]. Therefore, olive leaves are considered a cheap and easily available natural source of oleuropein, and their valorisation must be encouraged [2,11,13,97,141–144].
FIG. 5.6 Natural sources of oleuropein.
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Polyphenols extraction, purification and analytical methods The recovery of phenols depends on important parameters (e.g., extraction method, temperature, solvent and time). Sample handling, processing, clean-up and storage conditions, extract stability, analytical technique sensitivity and the purity of standards used in the preparation of calibration curves are commonly factors that can account for the wide variation of values in literature. It is also difficult to develop a single method for optimum extraction of all phenolic compounds since their polarities vary significantly.
Extraction of polyphenols Extraction is one of the most important steps in sample pre-treatment for polyphenols analysis. The extraction begins with olive leaves drying operations (see Section Harvesting season and fruit size), size reduction and homogenization of the samples [145]. Solid/liquid extraction (e.g., Soxhlet extraction) has been the most common procedure, where olive leaves are macerated in a solvent (e.g., methanol, ethanol, ethyl acetate, diethyl ether or aqueous alcohol mixtures). These methods require large amounts of toxic solvents and long extraction times, involving high costs, time waste and environmental pollution. Being time consuming and having poor efficiency, some degree of heating is required, which could easily lead to thermo-sensitive ingredients, with loss of biological activity or degradation into other substances [146]. Subsequently, in order to minimize these disadvantages, other extraction techniques were developed. These include microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), superheated liquid extraction (SHLE), liquid/liquid extraction, pressurized liquid extraction, derivatized polar extraction, fractionation by solid-phase extraction and dynamic ultrasound-assisted extraction (UAE). Microwaves are nonionizing electromagnetic radiation with a frequency in the range of 300–300,000 MHz, which corresponds to wavelength (λ) of 1 mm–1 m [147]. In the microwave, the heating occurs in a targeted and selective manner with practical no heat being lost due to the closed system. When the moisture is heated up inside the plant cells, water evaporates and generates tremendous pressure on the cells wall, leading to rupture and facile leaching out of the phytoconstituents [148]. In microwave-assisted extraction (MAE) the heat is dissipated volumetrically and all molecules exposed to the radiation are affected directly [149]. A correct choice of the solvent is fundamental for obtaining an optimal extraction process. Solvent choice for MAE is dictated by the solubility of the target analyte, interaction between solvent and plant matrix and by the microwave absorbing properties of the solvent. Consequently, the solvent need to have good selectivity toward the analyte of interest and exclude unwanted matrix components; be compatible with further chromatographic
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analytical steps; and to have appropriated dielectric properties. In general, polar substances (e.g., water, methanol and ethanol) absorb microwave radiation, whereas the less polar substances are not recommended for extraction in the microwave [147]. The volume of the solvent is also a critical factor. It must be sufficient to ensure that the plant matrix is always entirely immersed. The heating efficiency of the solvent under microwave should also be considered and depends on solvent evaporation [148]. The unique heating mechanism of MAE can significantly reduce the extraction time and therefore minimize the degradation of target components [11]. It also provides higher extraction efficiency and selectivity, involving low consumption of organic solvents [13]. MAE is then a promising technique compared with conventional extraction methods and presents different physical and chemical phenomena [150]. Supercritical fluid extraction (SFE) is the process of separating one component from a matrix using supercritical fluids as the extracting solvent. A supercritical fluid possesses the properties of a gas as well as that of a liquid. Therefore, the gas-like mass transfer and the liquid-like solvating power give supercritical fluids an edge over other extracting solvents [151]. Among several possible fluids [e.g., ethane and propane (low-critical temperature solvents); alkanes, methanol and water (high-critical temperature solvents)], carbon dioxide (CO2) has been the most studied solvent for supercritical processes because it is safe, non-toxic, non-flammable, highly selective and has moderate critical conditions (304 K, 7.4 MPa) that allows low temperature extraction conditions, avoiding thermal degradation of the product [152]. Recently, oleuropein was extracted from olive leaves using ethanol as extractant solvent. Oleuropein extraction yield was 5.4% (w/w dry leaves) and 20% (w/w extract). Supercritical antisolvent extraction (SAE) was further performed on ethanol extract, operating at different pressures (100–200 bar) and temperatures (35 °C and 60 °C). SAE produced a powder rich in oleuropein up to 36% (w/w) at 35 °C and 150 bar [153]. SFE has received increasing interest, because supercritical fluids provide high solubility, improved mass-transfer rates and the operation can be manipulated by changing the temperature or pressure. However, this technique is limited to compounds of low or medium polarity [128]. Recently was also observed that the use of CO2-SFE alone, during oleuropein extraction from olive leaves, was not satisfactory, requiring a polar modifier to improve yield and selectivity of the process [154]. Superheated liquid extraction (SHLE) is the extraction using aqueous or organic solvents at high pressure and temperature without reaching the critical point. A static mode (i.e., the volume of extractant is fixed), a dynamic mode (i.e., the extractant flows continuously through the sample) and a static-dynamic mode (i.e., combination of the two modes) can be considered [155]. The combination of static and dynamic modes was found to reduce the extraction time and provide better extraction efficiency [156,157].
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Superheated water (i.e., water under pressure and above 100 °C but below its critical temperature of 374 °C; and pressure high enough to maintain the liquid state) has been used for this extraction process. Under these conditions, water is less polar and so the organic compounds are more soluble than at room temperature [158]. The advantages of using superheated water is the shorter extraction time, higher extraction ability for polar compounds, higher quality of the extract and lower costs. However, it is not suitable for thermally labile compounds [151,159]. SHLE has higher extraction efficiency compared with conventional extraction methods due to the increased capacity to disrupt matrix-analyte interaction at high temperatures and pressure. At high temperatures, the surface tension and the viscosity of the extractant are reduced, increasing mass transfer and enhancing extraction. Other advantages include the reduction of solvents and the high selectivity of this method, which has been used to extract phenols from olive leaves [156]. Liquid-liquid extraction using a microfluidic system (water, ambient temperature, flow rate ratio 1, residence time 0.1293 min) was employed to extract oleuropein from ethyl acetate into aqueous phase. This method has simple operation, is cost effective and environmentally friendly, compared with conventional extraction methods [160]. Ultrahigh pressure extraction (UPE) is a novel technique that utilizes high-pressure conditions (100–500 MPa) and temperature (20–50 °C) to extract active ingredients from plants [161]. It increases the mass transfer rate by changing the concentration gradient and diffusivity, causing damage to the plant cell membrane and increasing its permeability. This results in a shorter processing safety, and achievement of higher compound yields [146]. Reduced-pressure boiling extraction (60 °C, 20 min, material/liquid 1:30, 85% ethanol) was used to extract oleuropein in high yield from olive leaves [162]. Ultrasound-assisted extraction (UAE) uses sound waves that propagate in frequencies from 20 to 100 kHz, creating expansion and compression cycles (i.e., mechanical). In a liquid, they produce negative pressure cycles (bubbles or cavitation) causing permanent physical and chemical changes, by disrupting the stability of solid at the interface liquid-liquid and liquid-gas systems [163]. The mass transfer of the solute from the matrix into the solvent occurs by diffusion and osmotic processes [150]. High-frequency ultrasound standing waves were applicated in olive oil extraction from olive fruits. Megasonic standing waves have shown to have a positive effect in enhancing olive oil extractability before or after malaxation. The combination of low-frequency sonication to promote cell wall disruption pre-malaxation followed by megasonic standing wave (400 and 600 kHz, 57–67 min, 18–21 kJ/kg) application post-malaxation, resulted more advantageous to enhance oil extractability [164].
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UAE of oleuropein and related biophenols from olive leaves was considered faster and more efficient than traditional methods, because cavitation favors penetration and transport at the interface between aqueous or organic liquid phase subjected to ultrasound energy and a solid matrix [165]. The increase in the temperature caused by ultrasonic irradiation did not degrade oleuropein [61]. Recently, an integrated approach based on UAE and spray drying was suggested. The extraction of olive leaves was performed using an ultrasound sonicator and the concentrated extract was further spray dried (149 and 156 °C for inlet air temperature, 21.35 and 23.10 m3/h for drying air flow rate, and 683 and 638 L/h for atomizing agent flow rate) to be included in food formulations [166]. UAE allows shorts extraction times, low extraction temperature and high extraction yields. This technique is efficient, simple, cheap, and a good alternative to conventional extraction processes. Moreover, the equipment is easy to handle and less expensive compared to other methods, such as microwave or supercritical fluid [167]. The disadvantages include the lack of uniformity in the distribution of ultrasonic energy and the loss of power during the process [163]. The recovery of sugars and natural antioxidants from olive leaves using steam-explosion has been addressed in a recent work [168]. Under the studied conditions, grinding of olive leaves is not required so a stage in the process is removed, which leads to a saving of time, energy and equipment and improves the economic viability. Under working conditions (180 °C, 8.3 min), make 70% of the initial sugars to be recovered with a very low formation of inhibitory compounds due to little degradation. Furthermore, several compounds with many applications in food and pharmaceutical industries were recovered, including oleuropein, hydroxytyrosol and flavonoids (luteolin, apigenin, together with their corresponding 7-O-glucosides) with antioxidant capacity. Luque-Garcı´a and De Castro designed and constructed an ultrasoundassisted Soxhlet extractor combined with supercritical CO2 extraction with improved rates and yields [169]. Dang et al. investigated the optimal operating conditions for oleuropein yield from olive leaves by testing supercritical CO2 fluid extraction, microwave-assisted extraction and Soxhlet extraction techniques, showing short extraction time for microwave-assisted technique [170]. Comparison of all of the different extraction techniques has shown MAE and conventional solid/liquid extraction to be the best choices for extraction of more polar compounds such as oleuropein and its derivatives from olive leaves. Although MAE has been seen as a promising alternative technique, there are still some general limitations, such as capital cost due to the need for specialized equipment, scale-up problems, difficulties in continuous operation, and degradation of sensitive compounds when longer times or higher
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power are used. In addition, the use of domestic devices may lead to poor reproducibility of the extraction method because of the heterogeneity of the microwave field [129,171–173]. Laboratory-scale methods use preferably polar solvents in the extraction of oleuropein and derivatives from olive leaves. Methanol-water mixtures are usually employed, although the use of less polar solvents such as hexane was also reported. Nevertheless, the increased human use of these compounds makes mandatory the development of green extraction methods based on nontoxic extractants (e.g., water) [61]. Recently, Apostolakis et al. studied the efficiency of heated water/glycerol mixtures in extracting polyphenols from dried olive leaves. Glycerol is a bio-solvent, with no toxicity and it is a natural constituent of foods. Authors concluded that the use of a heated aqueous glycerol solution was more efficient as compared with a hydro alcoholic solution in extracting olive leaf polyphenols, providing the same differentiated selectivity [2]. Green approaches have also been tried. Recently, biopolymers and polymeric absorbents have been used to separate and purify target compounds from natural plant sources, with low cost and harmless to operator’s health. Altiok et al. and Bayc¸ın et al. used the polymer silk fibroin, which have been used since the 1940s by its hydrophobic and bonding characteristics, in order to increase the purity of oleuropein from olive leaves [174,175]. Beside biopolymers, macroporous resins are important as polymeric absorbent, usually applied in separation and purification of bioactive compounds from natural sources [144]. One example was the use of a continuous adsorption/nanofiltration hybrid process and the use of imprinted polymer with in situ recovery of solvent [176]. Molecular imprinted polymers were also used to selectively recover several biophenols, including oleuropein [177]. An oleuropein rich extract was obtained from olive leaves by combining microfiltration, ultrafiltration and nanofiltration processes. The water extract of olive leaves was subjected to microfiltration process (0.2 μm) to remove larger particles, followed by ultrafiltration (removal of molecules larger than 5 kDa), and finally a nanofiltration process (300 Da), allowed the concentration of polyphenols, mainly oleuropein (1685 mg/100 g extract) [178]. The nanofiltration process may be an appealing one to explore for largescale operation due to its low energy consumption, easy scale-up, and use of mild conditions. Also, this process has been described as being economic and to have easy and simple operation [176,179]. However, some of the identified limitations such as large molecular-weight gap, long screening, high solvent consumption, and unpredictability of solvent effects created more difficulties in the specific case of extraction of olive leaves [180]. The consumption of solvents can be minimized by the potential for their recovery, and improvements in membrane composition allowed a broad range of molecular-weight cut-off that can be determined more efficiently [181].
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Two computational methods (RSM and ANN) were used to predict the total phenolic content and oleuropein in olive leaf extracts obtained by solvent-free MAE. The optimal conditions to obtain a maximal yield of oleuropein (0.060 0.012 ppm) were 250 W, 2 min of extraction time and 5 g of olive leaves [182]. RSM was used for prediction of the optimal values of parameters affecting the extraction of oleuropein through microchannel. Optimum conditions of F ¼ 80 kHz, 25 °C and P ¼ 100 w resulted in 96.29% of oleuropein extraction, more than other applied methods. Microchannel continuous system has several advantages, including low solvent consumption, short time for extraction and high efficiency [183].
Analysis and purification of polyphenols The colourimetric Folin-Ciocalteau assay is the most used and rapid quantitative technique for the determination of total polar phenolics in olive leaves. High performance liquid chromatography (HPLC), usually in reversed-phase mode and coupled with several detectors (UV–Vis, MS, NMR, DAD, etc.), is used for the determination of individual compounds [129]. Nuclear magnetic resonance spectroscopy (NMR) is used in the analysis of complex mixtures without previous separation of the individual components. Gas chromatography (GC) has lower detection limits and better separation, but needs sample pre-treatments, using derivatization reagents. Nevertheless, high/ultra-performance liquid chromatography (HPLC/UPLC) coupled to diode-array detection (DAD) and/or coupled to mass spectrometry (MS) is the most used to quantify and characterize phenolic compounds [11]. The quantification of oleuropein is mainly performed through analytical HPLC, usually by reversed-phase chromatography with use of a gradient elution method. Gradient elution mode is commonly utilized, because the complexity of the phenolic profile makes it not able to be well separated by the isocratic elution mode. UV–Vis detection system continued to be one of the most used detection systems for phenolic compounds, generally using as wavelength 280 nm. However, it should be considered that no universal absorbance maximum exists for olive leaf phenolics [129]. The isolation of oleuropein from olive leaves usually involves purification through column chromatography mainly on silica [184,185]. Published values for oleuropein recovery from olive leaves varied massively, from 5.6 to 108.6 mg/g dry weight, depending on some factors as discussed before in Section Factors that influence oleuropein and phenolic levels. The structural characterization of oleuropein has been performed by 1H, 13 C, and two-dimensional H-C heteronuclear NMR spectroscopy ([D6]DMSO [176,184,186], [D4]methanol [187,188], D2O [189] and CDCl3 [190]), by mass spectrometry (ESI, TOF, IT, CI, CID, EI, FAB) [176,191–193], and by FTIR [176,194,195] and UV/Vis [191,194,195] spectroscopy.
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The development of existing methods of separation and introduction of new techniques of high resolution are needed to give rise to the discovery of new effective compounds from phytopharmaceutical sources and improve its separation [148]. A novel ionic liquid modified absorbent with a structure of an interpenetrating polymer network (PS/PVIm) was designed and used to enrich oleuropein. The high affinity of PS/PVIm for oleuropein and the introduction of an ionic liquid imidazolium group and interpenetration in polymer network improved the adsorption efficiency, however the adsorption rate decreased. The design and preparation of appropriate resins with ionic liquids and high absorption efficiency to enrich oleuropein from olive leaves extract have significant importance [196]. A compilation of values of oleuropein content regarding different extraction, purification and analytic methods, according to leaves conditions and olive tree variety are presented in Table 5.4.
Valorisation of oleuropein General reactivity As reported in Section Oleuropein chemistry, oleuropein molecular structure can be divided in three subunits: hydroxytyrosol, monoterpene, and glucose moieties. Secoiridoids react readily with solvents and are transformed into different compounds during extraction procedures. This is due to the easy opening of the secoiridoid ring after hydrolysis of the glucose moiety. Scheme 5.4 presents an overview of proposed transformation pathways of the oleuropein aglycon in olive leaves during extraction and observed in solution (i.e., methanol or chloroform), and complete transformation of 3,4-DHPEA-DETA (10) into 3,4-DHPEA-EDA (8) in acidic medium [203,204]. Transformations include oleuropein aglycone (2) equilibrium forms from opening of the secoiridoid ring (3a/b and 6a/b); intramolecular reaction of (3a) to oleuropein-aglycone mono-aldehyde (3,4-DHPEA-EA) (4), and transformation into (5) in methanol; methyl ester hydrolysis of (6a/b) into (7a/b) and decarbomethoxylation into oleuropein-aglycone di-aldehyde (3,4DHPEA-EDA) (8) and transformation into (11) in methanol; and conversion of equilibrium form (9) into (12) under acidic conditions, Scheme 5.4.
Synthetic transformations of oleuropein The monoterpene unit in oleuropein is a highly functionalized moiety that includes two esters (i.e., including the bond between the hydroxytyrosol and the monoterpene subunits), one alkene, one enol ether, one acetal (i.e., bond between the glucoside and the monoterpene subunits) and a stable chiral center at C-5, Fig. 5.7.
TABLE 5.4 Published values of oleuropein concentration according to extraction method. Leaves conditions
Leccino, Moraiolo and Frantoio
Fresh leaves
Moraiolo
Dried leaves (freeze-dried with liquid N2)
Variety
N3 (Don Carlo)
Extraction Method
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)a
Solid-liquid extraction
Purification
CH3OH, 1 week, rt. Then, (CH3)2CO/H2O (1:1) Extraction with n-pentane, CH3Cl and ethyl acetate
Column chromatography with silica gel CH3Cl/CH3OH (9:1) then CH3Cl/CH3OH (4:1)
Solid-liquid extraction Different volume ratios of H2O/Ethanol
Observations
References
2.95
Oleuropein isolated from ethyl acetate extract
Gariboldi et al. [184]
Analytical
14.4
HPLC
8.39
Best extraction solvent mixture: H2O/Ethanol (1:1) Moraiolo variety possesses the highest oleuropein content
Briante et al. [67]
N2
7.06
Coratina
6.10
Nociara
3.70
Frantoio
3.19
I-77
3.03
Leccino
1.05
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety
Leaves conditions
Unknown
Dried leaves
Extraction Method Microwave-assisted extraction (MAE) 100–200 W, 5–15 min Ethanol 80%–100%
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Analytical
23.2
HPLC-DAD (280, 330, 340, 350 nm)
Observations
References
MAE—faster extraction method
Japo´n-Luja´n [197]
Optimal extractant— Ethanol/H2O (8:2)
Lichrospher 100 RP18 column (250 4 mm, 5 μm); Kromasil 5 C18 (15 4,6 mm, 5 μm) 6% acetic acid, 2 mM sodium acetate in H2O, and ACN Flow: 0.8 mL/min Gradient
Unknown
Dried leaves (40 °C, 8 h)
Superheated liquid extraction (SHLE) Variables: temperature, static and dynamic extraction time, extractant flow-rate and extractant composition
Analytical
23.0
Pressure: 6 bar
HPLC-DAD (280, 330, 340 and 350 nm)
Solvent—EtOH: H2O (7:3), 140 °C, 6 min
Lichrospher 100 RP18 (250 4 mm, 5 μm)
Dynamic mode, extractant: 7 min at 1 mL/min
Kromasil 5 C18 column (15 4.6 mm, 5 μm) 6% acetic acid, 2 mM sodium acetate in H2O, and ACN Gradient Flow: 0,8 mL/min
Extraction time: 13 min
Japo´n-Luja´n et al. [198]
Unknown
Dried leaves
Ultrasound-assisted extraction 20KHz, 450 W Variables: irradiation time, extraction flow-rate, Ethanol%, probe position, temperature, radiation amplitude and duty cycle
Analytical
22.6
HPLC-DAD (280, 330, 340 and 350 nm) Lichrospher 100 RP18 (250 4 mm, 5 μm) Kromasil 5 C18 column (15 4,6 mm, 5 μm)
Optimal conditions: irradiation time— 25 min; extraction flowrate—5 mL/min; 59% EtOH; Probe position— 4 cm; temperature—40 ° C;
Japo´n-Luja´n et al. [61]
Faster and more efficient than traditional methods
6% acetic acid, 2 mM sodium acetate in H2O, and ACN Gradient Flow: 0.8 mL/min Unknown (December 2004)
Dried leaves (37 °C, 3 days)
Solid-liquid extraction
Analytical
Ethanol, CH3OH, acetone and their aqueous forms (10%/90%, v/v)
HPLC-UV (280 nm) C18 Lichrospher 100 analytical column (250 4 mm, 5 μm), 30 °C
74.5
70% EtOH: best extractant solvent Silk fibroin as promising adsorbent for oleuropein purification.
Baic¸ın et al. [175]
Flow: 1 mL/min Acetic acid/H2O (2.5:97.5) and ACN Gradient, 60 min
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety
Leaves conditions
Chemlali
Dried leaves
Extraction Method
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Observations
References
Solid-liquid extraction
Analytical
43.2
–
CH3OH/H2O (4:1)
HPLC
Jemai et al. [199]
24 h
Shim-pack VP-ODS (250 4.6 mm), 40 °C
24.4
–
Jemai et al. [79]
8.80–21.7
Optimal extraction time: 10 min giving high oleuropein yield (21,7 103 mg/g)
Procopio et al. [185]
0.1% phosphoric acid in H2O, and 70% ACN in H2O Flow: 0.5 mL/min Gradient, 40 min Dried leaves
Solid-liquid extraction
Analytical
CH3OH/H2O (4:1) 24 h
HPLC Shim-pack VP-ODS (250 4.6 mm), 40 °C 0.1% phosphoric acid in H2O, and 70% acetonitrile in H2O Flow: 0.5 mL/min Gradient, 40 min
Coralina
Dried leaves
Microwave-assisted extraction H2O, 800 W, 10 min
Purification LC Supelco Versa Flash CH2Cl2/MeOH (8:2)
Koroneiki
Air dried leaves
Supercritical fluid extraction (SFE) 30 MPa Extraction: 50 °C Separation: 55 °C Solvent-to-feed ratio: 120 or 290 Co-solvent: 5% or 20%
Analytical
51.0
HPLC-DAD (248 nm) SupelcoAnalytical Discovery HS C18 (250 4.6 mm; 5 μm) 25 °C
Best solvent-to-feed ratio: 290 Co-solvent: 20% EtOH
Xynos et al. [200]
Extraction: 50 °C Separation: 55 °C
H2O + 1% acetic acid, and CH3OH
Pressure: 30 MPa
Gradient Flow: 1 mL/min
Unknown (November 2010)
Fresh leaves Steam and hot water blanching; air-dried at 60 ° C
Solid-liquid extraction
Analytical
Hot water blanched
Ethanol/H2O (7:3)
HPLC-DAD (280 nm)
4.63–5.06
Pinnacle II RP C18 (150 4.6 mm, 3 μm), 40 °C
Non-blanched
Oleuropein content was higher when steam or hot water blanching were used
Stamatopoulous et al. [201]
Mid-infrared spectroscopy as rapid tool to predict oleuropein content
Aouidi et al. [134]
0.210–2.14
Kromasil 100–5 C18 (3.0/4.6 mm) 0.02 M sodium acetate pH 3.2 + acetic acid, and ACN Flow: 1 mL/min Chemlali, Chetoui, Meski, Sayali and Zarrazi
Dried leaves (MW-assisted, 800 W, 2 2 min)
Solid-liquid extraction
Analytical
109.2 to
CH3OH/H2O (4:1)
Mid-infrared spectroscopy (4000 cm1 and 700 cm1)
172
24 h
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety Serrana
Extraction Method
Oleuropein Quantification/ Purification
Fresh leaves
Solid-liquid extraction
Analytical
Fresh HAD 70 °C
Hot air (HAD) or freeze (FD) drying
Ethanol/H2O (4:1)
HPLC-DAD/MS-MS (240, 280 and 330 nm)
69.0
24 h
Lichrospher 100 RP18 (250 4 mm, 5 μm)
Air dried leaves
Pressurized liquid extraction (PLE)
Linear gradient
3.00
Flow: 1 mL/min
FD N2
ESI—negative mode
16.4
Desolvation temperature: 365 °C
HAD 70 °C + FD N2
Vaporiser temperature: 400 °C
30.1
Variables: temperature, static time, extraction cycles and Ethanol % Pressure: 1500 psi
Analytical HPLC-DAD (248 nm) Supelco Analytical Discovery HS C18 (250 4,6 mm; 5 μm) 25 °C H2O + 1% acetic acid, and CH3OH Gradient Flow: 1 mL/min
References
Hot air drying of leaves gives high oleuropein recovery
Ahmad-Qasem et al. [132]
Extraction yield affected by temperature, static time and extraction cycles
Xynos et al. [202]
109 FD conventional
Nebulizer: 4.83 bar
Observations
Fresh HAD 120 °C
2.5% acetic acid in H2O, and acetonitrile
Dry gas: N2 12 L/min
Koroneiki
Oleuropein Concentration (mg. g21)
Leaves conditions
HAD 120 °C + FD N2 48.0 261
Optimal conditions for oleuropein extraction: 190 °C, EtOH: H2O (56:44)
Sevillana
Air dried leaves
Solid-liquid extraction
Analytical
EtOAc, r.t., 24 h
HPLC/UV–Vis (254 nm)
Liquid-liquid extraction/MiS
C18 analytical column (Shim-Pack VP-ODS, 250 mm, 4.6 mm i.d., Shimadzu, Japan)
H2O, flow rate ratio 1 Residence time 0.1293 min
687
Microfluidic system (MiS)
Naleini et al. [160]
Extraction yield depends on residence time, pH, temperature and EtOAc/H2O ratio
Phosphate buffer (0.05 mol/L and pH 3 adjusted with orthophosphoric acid) and acetonitrile (70:30, v/v), gradient Injection volume: 10 μL 10 min, r.t., flow: 1.0 mL/min
Chemlali (July)
MW dried leaves 1200 W, 70 °C, 10 min
Ultrasound-assisted extraction (UAE) vs. SolidLiquid Extraction (SLE) H2O, 10 min 10–70 °C
Analytical
57.5 (UAE)
HPLC-DAD/MS-MS (240, 280, 230 nm, ESI)
51.5 (SLE)
UAE intensified the initial extraction rate
Khemakhem et al. [165]
Merck Lichrospher 100RP18 (5 μm, 250 4 mm) column 2.5% acetic acid and acetonitrile, gradient Flow: 1 mL/min, 50 min
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety Chemlali
Leaves conditions MW dried leaves 1200 W, 70 °C, 10 min
Extraction Method Microfiltration (0.2 μm), Ultrafiltration (5 kDa) and Nanofiltration (300 Da) H2O, 30 °C, 1 h
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Analytical
Observations
References
16.9
Production of an oleuropein concentrate
Khemakhem et al. [178]
10.7 (Alcoholic Extraction) 19.2 (SAE)
Hybrid process Oleuropein degraded at 60 °C
Baldino et al. [153]
HPLC-UV (254 nm) C18 column (4.6 mm 250 mm) 70% acetonitrile/H2O and 0.1% phosphoric acid/H2O 40 °C Flow: 0.6 mL/min
Unknown
Water content
Solid-Liquid Extraction
Analytical
8% (w/w)
EtOH, 20 °C
HPLC-UV/Vis (280 nm)
Supercritical Antisolvent Extraction (SAE)
C18 LiChrospher
SC-CO2, 150 bar, 35 °C Rate: 2.5%
column (150 mm 3.0 mm i. d., 5 μm, Phenomenex) 2.5% acetic acid in water and 10% acetonitrile Gradient Injection volume: 20 μL Flow: 1 mL/min
a
Mass of oleuropein in mg per gram of olive leaves dry weight.
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SCHEME 5.4 Proposed transformations of the oleuropein aglycone in olive leaves during olive extraction and observed in solution (methanol, chloroform and acidic solutions). Solvents are identified in dashed boxes. Adapted from L.A.S. Cavaca, C.A.M. Afonso, Eur. J. Org. Chem. (2018) 581-589.
FIG. 5.7 Monoterpene moiety chemical features. Reactive positions of the monoterpene moiety are represented by arrows; stereocenters represented by (*).
168 Studies in Natural Products Chemistry
This multifunctional structure makes it difficult to be obtained by other means than extraction from natural sources. The seven potential reactive positions available for synthetic chemical modifications facilitate the valorisation of oleuropein towards the synthesis of diverse and synthetically rich building blocks with potential applications (e.g., pharmaceutical, cosmetic, food additives, etc.), Fig. 5.7. The glucose unit confers to oleuropein solubility in water. However, the presence of such polar moiety difficult the synthetic procedures for oleuropein valorisation, mostly accomplished in organic solvent medium. In addition, it might complicate further purification processes. Therefore, to fully take advantage of all oleuropein chemical features it is essential to remove this problematic unit and produce more handleable molecules. Several authors, Scheme 5.5, have reported a variety of synthetic transformations of oleuropein [205]. The cleavage of the glucose moiety in oleuropein is usually performed by using enzymes or by chemical hydrolysis. The endogenous olive tree β-glucosidase and its commercially available counterpart from almonds have been employed for this purpose, leading to the formation of mixtures of aglycone compounds with different structures [143,206,207]. An enzymatic tailoring of oleuropein was performed using commercial available lipase and thermophilic β-glucosidase. Degradation products of oleuropein using β-glucosidase (i.e., oleuropein aglycone and glucose), lipase (i.e., elenolic acid glucoside and hydroxytyrosol) or both enzymes (i.e., glucose, elenolic acid and hydroxytyrosol) were identified by HRMS/MS spectrometry [208]. The acid treatment of oleuropein has also been reported using sulfuric acid, anhydrous hydrochloric acid, and erbium(III) trifluoromethanesulfonate (F-H, Scheme 5.5) [184,185,209]. In general, complex mixtures of oleuropein aglycone derivatives are obtained, including elenolic acid (7) and compound (8). The use of lanthanides (H, Scheme 5.5) leads to the assignment of several products’ molecular structures, revealing the presence of two isomers (i.e., hemiacetal (8) and dialdehyde), as well as subsequent products resulting from the rearrangement of the oleuropein aglycon, possibly through 1,4-addition of the enol form of the aldehyde closer to methyl ester to the double bond (8a and 8b, Scheme 5.5) [186]. The chemical hydrolysis of β-glycosidic bond with sulfuric acid in dioxane produced elenolic acid (7) in 7% yield (F, Scheme 5.5), which is of particular interest because of its potent and broad-range antiviral activity. These conditions allowed the removal of the glucose moiety together with the hydrolysis of the hydroxytyrosol ester to a carboxylic acid [185]. Erbium(III) was also applied as a catalyst for the peracetylation of oleuropein under mild conditions, affording a yield of 65% [186]. Peracetylated oleuropein is of additional interest due to its antiproliferative effects [210]. The hydrolysis of the hydroxytyrosol ester to afford the corresponding carboxylic acid (A, Scheme 5.5) was performed under basic conditions with
SCHEME 5.5 Synthetic transformations of oleuropein. Adapted from L.A.S. Cavaca, C.A.B. Rodrigues, S.P. Simeonov, R.F.A. Gomes, J.A.S. Coelho, G.P. Romanelli, A.G. Sathicq, J.J. Martı´nez, C.A.M. Afonso, ChemSusChem 11 (2018) 2300–2305. (The reference numbers correspond to those of article [219]).
170 Studies in Natural Products Chemistry
sodium hydroxide, followed by acetylation to afford the oleoside monomethyl ester peracetate (2, Scheme 5.5). This compound is involved in the stereocontrolled synthesis of the secoiridoid glucosides nudiflosides D and A, natural compounds extracted from the plant Jasminum nudiflorum (Oleaceae) [210]. Transformations of the monoterpene core for the synthesis of different scaffolds with additional importance have been published. The enzymatic acetal cleavage by β-glucosidase formed either pyridine alkaloid jasminine in 20% yield (4; C, Scheme 5.5) [211] or compound 5 in 5% yield (D, Scheme 5.5) [143,207,208], depending on the ammonium salt used. The phytopharmacological profile of Jasminum grandiflorum Linn. (Oleaceae), the natural source of jasminine (4), has been known to be antimicrobial, an angiotensin converting enzyme inhibitor, anti-ulcer and antioxidant [212–214]. Treatment of oleuropein with sodium borohydride in water produces oleuropeinol in 85% yield (3, B, Scheme 5.5), with reduction of both the hydroxytyrosol ester and the methyl ester to their primary alcohols. The acid rearrangement of oleuropeinol in the presence of aqueous hydrochloric acid removes the glucose moiety, and an equilibrium between the closed acetal (3a, 20% yield, Scheme 5.5) and the open dialdehyde (3b, 80% yield, Scheme 5.5) forms is observed. The mixture was further acetylated affording the monoacetyl derivative of the open form (3c, 98% yield, Scheme 5.5). This means that the equilibrium shifts towards the open form, which is further acetylated. The same mixture was also reduced with sodium borohydride in methanol, transforming the aldehyde into alcohol 3d (98% yield, Scheme 5.5) [215]. The only reported modification of oleuropein in methanol has been the transesterification of the hydroxytyrosol ester to a methyl ester. In contrast the acid rearrangement of oleuropein gave two major products. One results from the formation of a cyclic hemiacetal moiety between the primary alcohol function and the aldehyde function, which affords the cyclic acetal. This reaction produces a mixture of compounds 3e and 3f, with compound 3e being formed in larger amounts (40% yield). This suggests the existence of an equilibrium between these two compounds, with compound 3f converting slowly into the more stable compound 3e [216]. An efficient procedure for the semisynthesis of oleacein (6, Ea, Scheme 5.5) from oleuropein through Krapcho demethoxycarbonylation was developed. The possible conversion of oleuropein into oleacein involves three main transformations: cleavage of the glucose moiety, secoiridoid ring-opening followed by formation of two aldehyde groups and demethoxycarbonylation. This reaction was applied in order to perform these three steps in one-pot fashion in 22% yield [95]. Recently, a simple and environment friendly microwave-assisted method (180 °C, 20 min) to produce oleacein (6, Eb, Scheme 5.5) in a good yield (48%) starting from the easily available oleuropein. Oleacein has several reported biological activities, including antioxidant and antimicrobial [216].
The olive-tree leaves as a source of high-added value molecules Chapter
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A set of oleuropein aglycone derivatives were synthesized by transacetylation under mild conditions in 60–90% yield (12a-i, J, Scheme 5.5). Oleuropein aglycone in closed ring form is obtained by hydrolysis of oleuropein using Lewis acid [Er(OTf)3] catalysis [217]. Recently, the acid-promoted methanolysis of oleuropein was studied using a variety of homogeneous and heterogeneous acid catalysts. Exclusive cleavage of the acetal bond between the glucoside and the monoterpene subunits [(S,S)-9 and (S,R)-9, I, Scheme 5.5] or further hydrolysis of the hydroxytyrosol ester and subsequent intramolecular rearrangement (10, I, Scheme 5.5) were observed upon identification of the most efficient catalyst and experimental conditions. Formation of ()-methyl elenolate (11, Scheme 5.5) was also observed, which is a reported precursor for the synthesis of the antihypertensive drug ()-ajmalicine [218].
Conclusions This chapter gives an overview about the olive-tree leaves exploitation as a cheap and easily available source of polyphenols, mainly the high-added value natural compound oleuropein. An insight is made about the olive tree and related by-products as natural sources of bioactive compounds, in particular about oleuropein. The occurrence, biosynthesis, biodegradation, quantification, extraction and purification, as well as biological activities and synthetic transformations of oleuropein are reviewed in this chapter. Oleuropein is a natural product that can be extracted in quite large amounts from olive tree (O. europaea L.) leaves, which are a cheap and easily available source of this bioactive compound. The chemical features of the monoterpene ring make oleuropein a valuable bio-renewable synthetic building block for the generation of structurally more diverse derivatives, which might allow the identification of new molecules with valuable biological activities or with application in cosmetic and food research areas. The potential of oleuropein should be explored, and studies at the level of synthetic modifications of this valuable compound deserve further development taking advantage of the rich and close functionalities. Additionally, the isolation of pure oleuropein from olive leaf extracts with potential scaleup application without the requirement for chromatographic methods or complex separation processes is still an open issue.
Acknowledgments I.M.L.-C. and G.S. are grateful for the financial support of the Regional Government of Extremadura (Junta de Extremadura) and the European Regional Development Fund through grants GR15094 and IB16167.
172 Studies in Natural Products Chemistry
Abbreviations AFPL AOM CI CID DAD DHPEA-EA DHPEA-EDA DMSO DNA DSS EI ESI FAB FD GAE GC HAD HPEA-EA HPEA-EDA HPLC HRMS IGS ISSR IT LO MAE matK MS NMR PAL PPO RAPD RFLP RNA ROS SFE SHLE SSR TOF UAE UPE
amplified fragment length polymorphism azoxymethane chemical ionization collision-induced dissociation diode array detector oleuropein-aglycone mono-aldehyde oleuropein-aglycone dialdehyde dimethylsulfoxide deoxyribonucleic acid dextran sulfate sodium electron ionization electron spray ionization fast atom bombardment freeze drying gallic acid equivalents gas chromatography hot air drying ligstroside-aglycone mono-aldehyde ligstroside-aglycone dialdehyde high performance liquid chromatography high resolution mass spectrometry inter gene spacer inter simple sequence repeat ion trap lipooxygenase microwave-assisted extraction Maturase K mass spectrometry nuclear magnetic resonance phenylalanine ammonia-lyase polyphenol oxidase randomly-amplified polymorphic DNA restriction fragment length polymorphism ribonucleic acid reactive oxygen species supercritical fluid extraction superheated liquid extraction simple sequence repeat time-of-flight ultrasound-assisted extraction ultrahigh pressure extraction
The olive-tree leaves as a source of high-added value molecules Chapter
UPLC UV/Vis UVB
5 173
ultra performance liquid chromatography ultraviolet/visible ultraviolet B
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The olive-tree leaves as a source of high-added value molecules: Oleuropein Lı´dia A.S. Cavacaa, Ignacio M. Lo´pez-Cocab, Guadalupe Silverob and Carlos A.M. Afonsoa,* a
Faculty of Pharmacy, Research Institute for Medicines (iMed.ULisboa), University of Lisbon, Lisbon, Portugal b Laboratory of Applied and Sustainable Organic Chemistry (Labasoc), Department of Organic and Inorganic Chemistry, University of Extremadura, Ca´ceres, Spain * Corresponding author. e-mail: [email protected]
Chapter Outline Introduction The olive tree The Olea europaea complex Origin of the olive tree Olive tree by-products Phenolic compounds Occurrence of phenolic compounds in olive tree Oleuropein Oleuropein chemistry Biosynthesis of oleuropein Biological activities of oleuropein and derivatives Factors that influence oleuropein and phenolic levels Cultivar and environment conditions Harvesting season and fruit size
132 132 132 134 135 137 137 139 139 140 141 143 147
Influence of olive leaf treatment conditions 149 Extraction, purification and analytical methods 151 Natural sources of oleuropein and other phenolic compounds 151 Polyphenols extraction, purification and analytical methods 152 Valorisation of oleuropein 158 General reactivity 158 Synthetic transformations of oleuropein 158 Conclusions 171 Acknowledgments 171 Abbreviations 172 References 173
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Introduction Natural products include a large and diverse group of substances obtained from biological sources, such as marine organisms, bacteria, fungi and plants [1]. These compounds have become high-added value ingredients used in foods, dietary supplements, cosmetics and pharmaceuticals [2–5]. Regarding nutrition, some health benefits of the Mediterranean diet, such as lower prevalence of cancer and cardiovascular disease, are credited to the intake of phenolic compounds present in olive oil [6–11]. These phenolic compounds occur not only in oil, but also in other parts of the olive tree, such as leaves and twigs [12]. Olive leaves contain, indeed, a wide variety of phenolic compounds, such as simple phenols, flavonoids, and secoiridoids. Taking into account that these leaves constitute a copious by-product from the olive tree cultivation, their valorisation, as a source of value-added products, should be encouraged [13–15]. The major secoiridoid compound found in olive leaves is oleuropein. This substance, and some of its derivatives, possesses potent biological and pharmacological activities, such as anticancer, cardioprotective, neuroprotective, gastroprotective, hepato-protective, anti-diabetes, anti-obesity and radioprotective ones. These properties are in large part attributed to its presumed antioxidant and anti-inflammatory effects [16]. The fact that natural products, such as oleuropein, are able to interact with a broad range of biological macromolecules, makes them highly attractive in pharmaceutical research. Because of this, isolation, structural elucidation and formulation of key active compounds from natural sources have gained an increasing importance [17]. Furthermore, natural products can serve as powerful starting materials to generate drug substances with novel therapeutic utility, by means of synthetic modifications [18].
The olive tree The Olea europaea complex The family Oleaceae is composed of 25 genera. One of them is the genus Olea L., which comprises >40 species. The species Olea europaea L. is native to temperate regions in Europe, Africa, and Asia, although it has been cultivated in various places, with the appropriate climate, all around the world [19,20]. The details of this species were originally published in the Carl Linnaeus’ book entitled Species Plantarum [21]. There are six natural subspecies: subsp. europaea, in the Mediterranean basin; subsp. laperrinei, in the Sahara region; subsp. cuspidata, in South Africa, Southeast Asia, China, and Arabia; subsp. Maroccana, in Morocco; subsp. cerasiformis, in the Madeira archipelago; and subsp. guanchica, in the Canary Islands [20,22]. However, according to other sources, only two subspecies are accepted. These are the subsp. cuspidata, or African olive, and the subsp. europaea, or European olive [23].
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Two varieties are recognized within the O. europaea subsp. europaea: the cultivated olive tree (var. europaea), and the wild olive tree (var. sylvestris), known as oleaster. The term oleaster is used for both true wild forms and feral olive trees, as well. Overall, the true wild olive is not morphologically distinguishable from the feral form, and genetic markers has to be used for this purpose. Some botanists consider the wild variety as a valid species, named Olea oleaster, rather than a subspecies [20,24,25]. According to molecular results, the wild olive tree is to be considered the ancestor of the cultivated olive tree [26,27]. Olea europaea has 46 chromosomes and is a diploid species [28]. However, karyological studies are difficult since the chromosomes are small, similar in morphology and numerous. The presence of tetraploids and hexaploids was detected in subsp. cerasiformis and maroccana, respectively [29–31]. In addition, the coexistence of diploid and triploid individuals within the same population was also detected in a South Algerian population of the Laperrine’s olive. The triploid population does not represent >3% [32] Table 5.1. Olive constitutes an essential tree crop; production of olive oil and olive fruit is paramount in the Mediterranean basin, and the health benefits associated with its culinary use have prompted its cultivation in areas outside its original cradle [24]. The harvested area of olive trees is about 10.6 million
TABLE 5.1 The different taxa of the Olea europaea complex and their ploidy. Native geographic distribution
Ploidy
Oleaster, wild olive
Mediterranean basin
2
Olive, cultivated olive
Mediterranean basin
2
cuspidata
African olive
From South Africa to Southwest China
2
laperrinei
Saharan olive, Laperrine’s olive
Saharan mountains from south Algeria to northeast Sudan
2 (3)
guanchica
Canarian olive
Canary islands
2
cerasiformis
Madeiran olive
Madeira archipelago
4
maroccana
Moroccan olive
High Atlas, Morocco
6
Subspecies
Variety
Common name
europaea
sylvestris
europaea
europaea
Adapted from P.S. Green, Kew Bull. 57 (2002) 91–140; Integrated Taxonomic Information System on-line database, http://www.itis.gov. (Retrieved on July 2018).
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hectares, according to data from 2016; >96% of this area is concentrated in the Mediterranean region. Spain has 2.5 million hectares of olive groves, and it is the main producer of olive oil [34].
Origin of the olive tree Oleaster has thriven all along the Mediterranean shores. A study based on evaluation of archeological remains and examination of the wild relatives of the cultivated crops concluded that olive was most likely domesticated in the Near East in protohistoric time (fourth and third millennia B.C.) [35]. However, the controversy of the origin of the olive tree has come a long way since then; there are reports favoring the monofocal origin hypothesis followed by studies questioning it. In fact, evidences based upon wood charcoal and pollen sequences support the existence of oleaster both in the Levant and in Iberia during the late Pleistocene, proving that the wild olive is also genuine in the western Mediterranean [36–38]. Morphometric analysis of olive pits from both wild and cultivated olive trees from different regions shows the emergence of cultivation practices from the Neolithic and the Bronze Age in Spain [39,40]. Furthermore, study of archeological charcoal in Estremadura (Portugal) indicates that this region was a refuge area for Olea europaea during the colder periods of the Pleistocene [41]. Studies based on molecular analysis have challenged the single origin theory, too [42]. Nuclear DNA polymorphism in oleaster displays a gradient between the east and west of the Mediterranean basin, whereas in cultivars, the gradient is less visible owing to their diffusion and selection. According to this study, no unidirectional flux of domesticated olives would have taken place [27,43,44]. Diversity from the East thus enriched the biodiversity existing in the West by hybridization with local forms [45]. The structure of genetic polymorphism between the eastern-western parts of Mediterranean basin is in favor of a large-scale geographic differentiation of olive tree, whereas the crosses between the different taxa suggest that gene flows between them could occur during more or less recent stages of their differentiation [26]. The findings of the study of maturase K (matK) gene sequence of 46 populations of the Olea europaea complex support the grouping of subsp. europaea, cerasiformis, guanchica, maroccana, and laperrinei in one group separate from subsp. cuspidata. According to the same report, Inter-Simple Sequence Repeats (ISSR) data provide evidence for either gene flow between var. europaea and var. sylvestris, or for the repeated domestication of the cultivated olive [46]. Allozyme markers provide evidence of the survival of indigenous oleaster populations, particularly in the western part of the basin. Genetic diversity values over the domesticated olives, feral trees, and wild olives are consistent
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with the interpretation that domesticated olive trees represent a sample of the genetic variation in genuinely wild olive populations that persist today [24]. In Andalusia (Spain) where large areas of oleaster forests are present [47,48], genetic diversity and relationships between local cultivars and wild olive trees indicated a clear differentiation of Andalusian wild olives from cultivars of this area [38]. Moreover, the results obtained show that wild olive genetic resources of Andalusia represent a clearly differentiated gene pool either from cultivars of the same area and wild olive populations of other regions studied. Andalusian wild olives represent an interesting gene pool for olive breeders, taking into account that traits, such as resistance to pathogens and parasites, low plant vigor and adaptation to adverse environments, are rarely found in cultivated germplasm [49]. When it comes to gene pool sources for olive breeding, the Laperrine’s olive emerges as a subspecies of great interest; it thrives in the extremely arid and harsh conditions found in center Sahara desert, at an altitude between 1400 and 2800 m. It reproduces through vegetative or clonal growth. In fact, plastid DNA analyses indicate that Laperrine’s olive has been used during the secondary diversification of the cultivated olive tree in the region [50,51]. Nuclear microsatellite and plastid DNA data show a high genetic differentiation between europaea and laperrinei. Although no first-generation hybrid between them is detected, recent reciprocal admixture between Mediterranean and Saharan subspecies is found in a few accessions, thus confirming that laperrinei has been involved in the diversification of cultivated olives [52]. The complex process of propagation of olive clones along the Mediterranean makes it difficult to unambiguously identify the pedigree of different cultivars. Moreover, it is very likely that different cultivars have interbred with wild olive trees, therefore, contributing to the genetic diversity of the species [48]. The questions on where and how often has the crop been domesticated remain open to debate and more studies are needed. There are studies that supported up to nine different primary domestication events across the Mediterranean [43,53,54]. However, more recent reports favor the theory of the unique domestication site [47,50,52], or the two-focal domestication scenario [48]. The olive genomic era is advancing at a high rate. Undoubtedly, the recent report on the genome sequencing of the oleaster, and the expected resequencing of several olive varieties will provide a valuable resource for the study of the evolution and domestication processes of the olive tree [55]. Moreover, it will enhance breeding programs and the formation of new varieties [56,57].
Olive tree by-products Industries generate a large quantity of by-products as a waste containing highadded value chemical compounds.
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SCHEME 5.1 By-products from olive tree production and olive processing industry. Adapted from J. Meirinhos, B.M. Silva, P. Valenta˜o, R.M. Seabra, J.A. Pereira, A. Dias, P.B. Andrade, F. Ferreres, Nat. Prod. Res. 2005, 19, 189-195.
The main by-products arising from both olive tree cultivation and olive processing industry are divided into oil extraction by-products (i.e., crude, exhausted “solvent-extracted” and partly destoned olive cakes, olive pulp, vegetation waters, and leaves collected at olive mills), as well as pruning and harvest residues (i.e., olive leaves, large branches and twigs), Scheme 5.1. The world crude olive cake production is estimated at about 2.9 106 tons. Usually, all types of olive cake are used in animal feeding. Exhausted olive cake has extended application as fuel or furfural source, Scheme 5.1. Substantial quantities of olive leaves are generated every year (10–30 kg tree1, 6 108 trees worldwide) found in large amounts during olive oil production, when leaves are separated from olives (5% of the weight of olives), and during olive tree pruning (25% of the weight of olives). The global amount accumulated annually may exceed 18 million tons (2006–2013 average) [58]. Olive leaves are mostly used as animal feed or simply directly thrown away, burned, or grinded and scattered on the field, potentially causing environmental damage, and with increasing cost for producers due to their removal, storage and elimination [59]. Olive by-products represent a considerable but insufficiently exploited resource and their potential should not be neglected. The generation of large amounts of these by-products make the search for new applications a demand. In addition, their content in high-added value compounds must encourage the valorisation of these bio-renewable resources.
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Phenolic compounds Occurrence of phenolic compounds in olive tree Plants are one of the most important sources of natural bioactive products. They have developed an effective defense system against predators, involving the production of a large number of different phytochemical compounds [1]. Phytochemicals are high-added value natural substances used as ingredients in foods, dietary supplements, cosmetics and pharmaceuticals. The consumers’ preference for products containing natural additives, generally perceived as safer and healthier, has increased the demand for phytochemical ingredients [3–5]. Polyphenols constitute one of the most common groups of phytochemicals in plants [60]. Olive biophenols include a major group of secondary metabolites distinguished by their water solubility and high molecular weights. They are of considerable physiological and morphological importance in plants, displaying a wealth of both structural variety and diversity of key activities. Actually, their excellent properties are a consequence of their function in the tree, namely reactivity against pathogens attack and response to insect injury [13,61]. Polyphenols are present in almost all parts of the olive tree but their nature and concentration vary greatly among the tissues. In olive fruit/olive oil several phenolic compounds are identified— flavonols (e.g., quercetin 3-rutinoside, luteolin 7-O-glucoside, luteolin 5-Oglucoside, apigenin 7-O-glucoside), phenolic acids (e.g., chlorogenic acid, caffeic acid, p-hydroxybenzoic acid, protocatechuic acid, vanillic acid, syringic acid, p-coumaric acid, o-coumaric acid, ferulic acid, sinapic acid, benzoic acid, cinnamic acid, gallic acid), phenolic alcohols (e.g., 3,4-DHPEA, p-HPEA) and secoiridoids (e.g., oleuropein, demethyloteuropein, ligstroside, nuzhenide), Fig. 5.1. The main hydroxycinnamic acid derivative is verbascoside. Hydroxytyrosol and tyrosol are present at highest contents in comparison to other phenolic compounds. Oleuropein is the major secoiridoid in unripe olive fruit [62]. Five groups of phenolic compounds are mainly identified in olive leaves: oleuropeosides (oleuropein and verbascoside), flavones (luteolin 7-O-glucoside, apigenin 7-O-glucoside, diosmetin 7-O-glucoside, luteolin and diosmetin), flavonols (rutin), flavan-3-ols (catechin) and substituted phenols (tyrosol, hydroxytyrosol, vanillin, vanillic acid and caffeic acid). Additionally, the dialdehydic deacetoxy forms of the secoiridoid compounds oleuropein and ligstroside, oleocanthal and oleacein can be found, Fig. 5.1. Phenolic compounds such as luteolin 7-O-rutinoside, luteolin 4’-O-glucoside, apigenin 7-O-glucoside and apigenin 7-O-rutinoside are also reported, Fig. 5.1. The most abundant compound in olive leaves is oleuropein, followed by hydroxytyrosol, the flavone-7-glucosides of luteolin, apigenin and verbascoside. A typical composition of the lyophilized extract of olive leaf is shown in Table 5.2 [63].
FIG. 5.1 Examples of polyphenols in olive tree. Glc—Glucose; Rut—Rutinose. Adapted from N. Talhaoui, A. Taamalli, A.M. Go´mez-Caravaca, A. Ferna´ndezGuti errez, A. Segura-Carretero. Food Res. Int. 77 (2015) 92–108.
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TABLE 5.2 Phenolic compounds, in mg/kg, in olive leaf lyophilized extract. Compound
mg/kga
Oleuropein
26,471.4 1760.2
Luteolin 7-O-glucoside
4208.9 97.8
Apigenin 7-O-glucoside
2333.1 74.7
Luteolin 40 -O-glucoside
1355.9 75.9
Verbascoside
966.1 18.1
Rutin
495.9 12.2
Caffeic acid
220.5 23.3
Sum of the determined compounds
36,051.8
a Results are expressed as mean standard deviation of three determinations. Adapted from A.P. Pereira, I.C.F.R. Ferreira, F. Marcelino, P. Valenta˜o, P.B. Andrade, R. Seabra, L. Estevinho, A. Bento, J.A. Pereira, Molecules 12 (2007) 1153–1162.
Oleuropein Oleuropein chemistry Oleuropein is included in a specific group of coumarin-like compounds, the secoiridoids, and is the most abundant biophenol in olive leaves. Bourquelot and Vintilesco detected oleuropein in olives for the first time in 1908 [64], but its chemical structure was assigned by Panizzi et al. in 1960 [65]. It was specified as the heterosidic ester of β-glucosylated elenolic acid and 3,4-dihydroxy-phenylethanol (hydroxytyrosol) and is responsible for bitterness taste in olive oil. It has a stereocenter at C-5 with (S) configuration and an exocyclic 8,9-double bond with (E) configuration, characteristic of secoiridoids. The molecular structure of oleuropein can be divided into three subunits: hydroxytyrosol, monoterpene and glucose moieties, Fig. 5.2. [10,66–69]. The low-energy conformation of oleuropein was investigated through semiempirical and ab initio calculations and confirmed by molecular dynamics simulations. The minimum energy conformation of oleuropein in vacuo has a closed geometry in which the glucose moiety is in close proximity to the hydroxytyrosol unit. The structure is stabilized by hydrogen bonds between the catechol hydrogen atoms and the hydroxy groups of the sugar ˚ short-axis radius and 8.22 A ˚ moiety, forming a hydrophobic cavity of 3.56 A
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FIG. 5.2 Molecular structure of oleuropein. Representation of hydroxytyrosol (red), monoterpene (green) and glucoside (black) moieties.
FIG. 5.3 Representation of the hydrogen bond network between hydroxytyrosol and glucoside moieties of oleuropein. Dashed lines represent hydrogen bonds. Adapted from E. Gikas, F.N. Bazoti, A. Tsarbopoulos, J. Mol. Struc.-THEOCHEM 821 (2007) 125–132.
long-axis radius, and by hydrophilic regions on the exterior of the molecule, Fig. 5.3. Nevertheless, these simulations did not consider the effect of solvent on oleuropein’s conformation [70]. Recently, molecular dynamics simulations showed extensive changes in oleuropein’s conformation depending on the microenvironment [vacuum, water, or triolein (oil)/water systems], showing the amphiphilic character of this compound [71].
Biosynthesis of oleuropein Iridoids (A) are monoterpenes characterized by a bicyclic fused ring system comprising a six-membered heterocyclic ring fused to a cyclopentane ring. Secoiridoids (B) are derived from iridoids by the opening of the cyclopentane ring at the 7,8 bond, Fig. 5.4 [72]. Oleuropein and other secoiridoids characterized by an exocyclic 8,9olefinic functionality are termed oleosidic secoiridoids or oleosides and
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FIG. 5.4 General representation of iridoids (A) and secoiridoids (B) molecular structures.
are unique to oleaceous plants. Secoiridoid conjugates that contain an esterified phenolic moiety result from a branching in the mevalonic acid pathway in which oleoside moiety (monoterpene) synthesis and phenylpropanoid metabolism (phenolic unit) merge [72]. Mevalonic acid is formed from the initial condensation of three acetylSCoA molecules, originating the ester β-hydroxy-β-methylglutaryl-CoA (HMG-CoA), which produces mevalonic acid after hydrolysis and an enzymatic reduction [73]. The proposed biosynthetic pathway for oleuropein formation in Oleaceae is shown in Scheme 5.2, via 7-epi-loganic acid and ligstroside as direct precursor [72].
Biological activities of oleuropein and derivatives Secoiridoids are known to exhibit a wide range of biological and pharmacological activity [74]. Olive leaf extract gained popularity in the global nutraceutical market due to a range of claimed health attributes, traditionally consumed for the treatment of a wide spectrum of ailments [16]. Oleuropein is responsible for most of the extract attributes and has potent biological and pharmacological properties, including anticancer, cardioprotective, neuroprotective, gastroprotective, hepato-protective, anti-diabetes, antiobesity and radioprotective. These properties are in large part attributed to its putative antioxidant and anti-inflammatory effects [16,63]. Oleuropein derivatives were also found to have biological properties. The accumulation of free radicals on cellular membrane lipids play a major role in various pathological disorders, including atherosclerosis, cancer, aging, rheumatoid arthritis and inflammation [75]. Compounds sharing an ortho-diphenolic (catecholic) structure are known to possess antioxidant activity. In this way the high antioxidant capacity of oleuropein is conferred by the catecholic structure, having the ability to scavenge free radicals, and consequently acts in the treatment of some pathologies associated with oxidative stress, Fig. 5.5 [16]. Prevention of free radical formation by oleuropein may be due to its ability to chelate metal ions, such as Cu and Fe, which catalyze
SCHEME 5.2 Proposed biosynthetic pathway for the formation of oleuropein. Adapted from H.K. Obied, P.D. Prenzler, D. Ryan, M. Servili, A. Taticchi, S. Esposto, K. Robards, Nat. Prod. Rep. 25 (2008) 1167–1179.
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FIG. 5.5 Bioactive functional groups of oleuropein and olive leaf extract health attributes. Adapted from L. Gentile, N.A. Ucella, G. Sivakumar, Curr. Med. Chem. 24 (2017) 4315–4328.
free radical generation reactions, as well as its ability to inhibit several inflammatory enzymes, such as lipoxygenases, without affecting the cyclo-oxygenase pathway [76]. The health benefits associated to olive leaf extract, oleuropein and its derivatives are presented in Table 5.3, compiling several studies performed in vitro in cell lines and in vivo using animal models and human volunteers. All these valuable biological properties make oleuropein and its derivatives promising compounds for pharmaceutical, food and cosmetic application. Therefore, it is important to stimulate research from different areas to enhance the preparation of compounds with additional interest.
Factors that influence oleuropein and phenolic levels Oleuropein and total phenolic levels are affected by abiotic (non-living chemical and physical parts of the environment that affect the tree, such as soil, water, air, temperature, moisture, etc.) and biotic (other plants, fungi and bacteria, animals and human influences) factors [11]. In this way, oleuropein and other phenolic content depend, essentially of cultivar, harvesting season, maturity of leaves, proportion of branches on the tree, geographical origin and moisture content [97–101]. These changes are widely reported in literature, where different extraction and analytical methods are used for content determination [11,58,68,88, 100,102–117].
TABLE 5.3 Health benefits of olive leaf extract, oleuropein and its derivatives. Studies
Compounds
Subjects/cell models
Effects
In vivo
Olive leaf extract
Animal studies
Adult male Swiss mice; whole-body irradiated with a single dose of 48 cGy
Antioxidant and radioprotective effects; hydroxyl radical scavenging capacity [77]
Oleuropein
Adult male Spargue-Dawley rats; oleuropein plus ethanol (12 mg/kg body weight; 10 days)
Antioxidant effect by scavenging of ROS, produced by ethanol that initiate lipid peroxidation [78]
Oleuropein and Hydroxytyrosol
Adult male Wistar rats; diabetes induced with alloxan
Anti-diabetic and antioxidant effects [79]
Hydroxytyrosol and its triacetylated derivative
Wistar rats fed a standard laboratory diet or cholesterol-rich diet
Lipid-lowering and antioxidant effects [80]
Olive leaf extract
Wistar rats exposed to cold restraint stress; olive leaf extract (80 mg/kg daily) dissolved in distilled water
Modulation of cold restraint stress oxidative changes in rat liver by inhibition of lipid peroxidation [81]
Olive leaf extract
Sprague-Dawley rats receiving gentamycin (25 mg/kg, 50 mg/kg or 100 mg/kg daily, 12 days)
Amelioration of gentamycin nephrotoxicity by inhibition of lipid peroxidation [82]
Oleuropein
Female C57BL/6 mice; azoxymethane (AOM)/ dextran sulfate sodium (DSS)-induced colorectal cancer; oleuropein (50 mg/kg or 100 mg/kg) dissolved in distilled water
Protection from AOM/DSS-induced colorectal cancer associated with acute colitis; suppression of the growth and multiplicity of colonic tumors [83]
Hydroxytyrosol
Adult male Wistar rats; C6 glioma cell implantation; subcutaneous injections of 100 μg oleuropein, 100 μg hydroxytyrosol or both daily, for 5 days
Only hydroxytyrosol inhibited tumor growth [84]
Oleuropein
Visceral leishmaniosis model L. donovaniinfected BALB/c mice; intraperitoneal injection of oleuropein 14 times
Oleuropein and hydroxytyrosol selectivity for L. donovani; Oleuropein gave parasite depletion of >95% in liver and spleen after 6 weeks [85]
In vivo
Oleuropein
10 healthy female 20–30 years having skin Fitzpatrick types II and III
Soothing effect in the treatment of UVBinduced erythema [86]
In vitro
Oleuropein
Animal/ microbial cell lines
Normal mouse hepatocyte FL83B cells; HepG2 and FL83B cells
Decrease of the number size of lipid droplets in free fatty acid-treated cells and reduced intracellular triglyceride accumulation [87]
Olive leaf extract
Bacillus cereus CECT 148; B. subtilis CECT 498; Staphylococcus aureus ESA 7; Escherichia coli CECT 101; Pseudomonas aeruginosa CECT 108; Klebsiella pneumoniae ESA 8.
Antimicrobial effects observed with contribution of oleuropein and hydroxytyrosol [88]
Human studies
Candida albicans CECT 1394; Cryptococcus neoformans ESA 3. In vitro
Oleuropein
Whole blood of 11 healthy male volunteers
Inhibition of platelet activation by scavenging of H2O2 produced in arachidonic acid metabolism cascade that leads to platelet aggregation [89]
Oleuropein
Human breast cancer cell line MCF-7
Apoptotic cell death of human breast cancer MCF-7 cells [90]
Oleuropein and its semisynthetic peracetylated derivatives
Human breast cancer cell lines (MCF-7 and T-47D)
Anti-proliferative and antioxidant effects; the peracetylated compounds exerted higher antiproliferative effects than oleuropein [91]
Hydroxytyrosol rich extract
MCF-7 human breast cancer cells
A dose-dependent growth inhibition of MCF-7 cells [92]
Human cell lines
and Hydroxytyrosol
Continued
TABLE 5.3 Health benefits of olive leaf extract, oleuropein and its derivatives.—Cont’d Studies
Compounds
Subjects/cell models
Effects
Olive leaf extract
Peripheral blood leukocytes from six healthy volunteers
Protective effect on the peripheral blood leukocytes against adrenaline induced DNA damage [93]
Olive leaf extract
Human endothelial cells from bovine brain, MCF-7 cells and T-24 cells (human urinary bladder carcinoma)
Antiproliferative effect against cancer and endothelial cells [94]
Olive leaf extract (oleuropein and apigenin-7glucoside)
Human HL-60 cells
Apigenin-7-glucoside of the olive leaf extract was mainly responsible for the HL-60 differentiation and oleuropein showed to exert an influence over this differentiation [95]
Oleacein
Human recombinant 5-lipooxygenase (5-LO)
Inhibition of 5-LO (IC50 ¼ 2 μM), acting as anti-inflammatory agent [96]
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Cultivar and environment conditions The composition of olives varies with cultivar and environment conditions. Olive cultivar affects the absolute concentration of specific polyphenols, while the total phenolic profile remains practically unchanged. Oleuropein is always present in the fruit of all cultivars, but verbascoside is an example of a cultivar-dependent compound. Environment conditions such as temperature, moisture, altitude and leaves age also alter the amount of oleuropein and phenolic profile. Cold stress and freezing cause severe dehydration and induce cell membrane damage in olive leaves. In lightly cold stressed leaves, oleuropein level is lower than in non-stressed samples. On the other hand, in moderately and heavily cold stressed leaves, oleuropein concentration was higher after exposure to cold stress. The accumulation of oleuropein and its antioxidant capacity may protect against oxidative damage induced by freezing. The activity of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) is another element in the response of the tree to such stress. PAL is involved in the mechanism of plant recovery if the cold stress is low or medium. If the cold stress is high, PPO activity avoid serious oxidative damage [118]. The effect of water deficit on olive leaf phenolic composition was studied. Water deficit stress induces accumulation of phenolic compounds, especially oleuropein, suggesting their role as antioxidants [109]. Oleuropein is also involved in the olive tree protection against salinity stress in leaves, serving as a glucose-reservoir for osmoregulation and high energy-consuming processes required for plant adaptation to salinity, increasing its content [119]. Total phenolic levels in leaves decline as the geographical altitude decreases, a factor related with changes in climatic conditions with geographical altitude. Leaves from trees cultivated in windy and humid air have lower levels of phenolic compounds [120]. An important biotic factor affecting the phenolic content is the age of the leaves. Oleuropein amount is higher in younger leaves than in mature ones, which suggest gradual oleuropein degradation with the leaves progressive age [103,104].
Harvesting season and fruit size Oleuropein levels are affected by harvesting season. During the growth period of olives, oleuropein levels decrease reaching a minimum in July, when the olive fruit is fully developed. Unexpectedly, the total phenolic content increase in July, suggesting a degradation of oleuropein and appearance of other phenolic substances [121]. During ripening, while elenolic acid glucoside (jaspolyside) (A) and demethyloleuropein (B) accumulate in olive fruits, oleuropein levels decrease. Oleuropein biodegradation into these glycosylated derivatives involves cleavage
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SCHEME 5.3 Molecular structures of elenolic acid glucoside (A) and demethyloleuropein (B), formed by esterase action, and aglycone forms (C and D) and oleuropein aglycone (E) by β-glucosidase action.
by specific endogenous esterases, through hydrolysis of hydroxytyrosol ester or methyl ester, respectively. Activation of endogenous β-glucosidase during crushing or malaxation might produce the aglycone forms (C, D and E) by cleavage of glucose, Scheme 5.3 [122–125]. Ryan et al. have paid attention to other tissues, suggesting that different tissues have different phenolic composition, and as such, the metabolism of each plant tissue is characteristic [104]. On the contrary, Briante et al. claim that the biodegradation of oleuropein is similar in olive leaves. Most of the molecules isolated from olive leaves are thought to have originated from oleuropein where the above mentioned endogenous enzymes played an important role [67]. Recently, De Leonardis et al. found that the enzyme polyphenol oxidase (PPO) is also involved in oleuropein degradation in olive leaves. Fresh leaves and dried leaves showed different enzymatic activity on oleuropein. Extract from fresh leaves provided both β-glucosidase and PPO activity. However, the decrease of oleuropein concentration in dried leaves was mainly attributable to the PPO activity, leading exclusively to the formation of oxidation products [126].
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Oleuropein concentration can also be affected by the dimensions of the fruits, which is intrinsically related with fruit maturity, since larger fruits are associated with maturation. Small-fruit varieties usually have high oleuropein and low verbascoside contents; the opposite is characteristic of largefruit varieties [11,15,88,127,128].
Influence of olive leaf treatment conditions The conditions in which olive leaves are treated influence the phenolic content. Currently, there are no widely accepted guidelines for the drying of olive leaves and data from literature are insufficient or contradictory. However, in order to stabilize the by-product, avoid quality losses and undesirable degradation during storage, the immediate dehydration of olive leaves is an important operation. Traditional methods of drying as shade or sun drying are still practiced, but this operation is not well controlled, influencing the final quality of the product. Thus, for industrial purposes, hot air drying is the most used method, allowing an accurate control of the process variables [129]. Several studies have been interested in the investigation and modeling of the drying behavior of olive leaves. The effect of blanching and infrared drying on the leaves color, total phenol content and moisture removal rate of four olive tree varieties was studied, using an infrared dryer at 40 °C, 50 °C, 60 °C and 70 °C. Infrared drying seemed promising, inducing a considerable moisture removal from the fresh leaves (>85%) and short drying time (15 min at 70 °C). Total phenol content of dried olive leaves increased compared to fresh leaves. In the specific case of Chemlali variety, total phenols increased from 13.8 0.2 to 21.3 2.9 mg GAE/g d.w. (+ 54.35%) after IR drying at 40 °C. Infrared drying is then suggested for preserving olive leaves during storage [130]. Erbay et al. studied the use of hot air to dry olive leaves in a pilot-scale heat pump conveyor dryer and evaluated the effect of drying on product quality. Optimum operating conditions were found to be at 53.43 °C, air velocity of 0.64 m/s, and process time of 288.32 min. At these conditions, was observed a total phenolic content decrease (9.77%), moisture loss of 94% and antioxidant activity loss of 44.25% [131]. The effect of freeze- (liquid N2 or conventional freezing 28 °C) and hotair (70 °C or 120 °C) drying on the concentration of olive leaves polyphenols was studied. Both drying methods significantly influenced the concentration of polyphenols. Freeze drying was not considered an adequate technique to promote extraction since the leach of phenolic compounds from olive leaves was not promoted. The antioxidant potential was reduced compared with that of fresh leaves, due to activation of oxidative enzymes. N2-freeze treatment was better than conventional, increasing oleuropein content by 448% compared with fresh leaves, and providing total phenolic contents of 37 2 and
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36.3 1.4 mg GAE/g d.w., respectively. Hot-air drying at 120 °C provided a higher phenolic content (45 2 mg GAE/g d.w.) than freeze-drying, especially in oleuropein. The reduction of the drying temperature to 70 °C results in a significant decrease of total phenols (45 2 mg GAE/g d.w.). Hot-air drying was evaluated on leaves previously affected by N2-freezing. Freezing prior to hot-air drying significantly reduced total phenolic content (37 2 mg GAE/g d.w., 70 °C and 49 2 mg GAE/g d.w., 120 °C) [132]. The effect of solar drying conditions on the drying time and some quality parameters of olive leaves, such as color, total phenol content and radical scavenging activity, was investigated using a laboratory convective solar dryer. Temperatures of 40 °C, 50 °C and 60 °C and air velocities of 1.6 and 3.3 m3/min were employed in the study. The drying time required to reduce the moisture content to 0.10 kg kg1 d.w. depended on the temperature. Total phenol content was affected by drying conditions and olive leaves variety. In general, temperature of 60 °C and air velocity of 3.3 m3/min resulted in higher content of polyphenols among the four varieties (16.86–24.08 mg caffeic acid/ 1 g d.w.), although was observed a decrease in all values compared with that of fresh leaves (22.03–26.06 mg caffeic acid/1 g d.w.) [133]. Microwave radiation is also used in leaves drying. Aouidi et al. used a microwave oven and dried leaves twice for 2 min at a maximum power of 800 W. Lyophilization was adopted for oleuropein determination in olive leaves [134]. Recently, microwave was seen as a reasonable selection for drying olive leaves preserving phenolic compounds during storage. Optimum conditions for microwave drying were found to be 2.085 g of sample at 459.257 W for 6 min to achieve maximum yields of total phenolic content (38.712 mg GAE/g d.w.) and oleuropein (203.561 mg/g d.w.). Microwave drying was considered a promising technique compared to other methods, namely freeze-, vacuum-, oven-, and ambient air-drying [135]. The influence of an ultrasound energy system was also investigated on the drying process of olive leaves. Air drying experiments (40 °C, 1 m s1) were performed without or with ultrasound application (8, 16, 25 and 33 kW/m3). The use of ultrasound on drying olive leaves could represent a way to increase the drying rate [136]. Kamran et al. studied the recovery of phenolic compounds from fresh, air-dried, freeze-dried and oven-dried (60 °C and 105 °C) olive leaves. Extracts of oven-dried leaves at 105 °C showed the highest phenol recoveries ( 140 mg/g d.w.). Olive leaves oven-dried at 105 °C for 3 h increased oleuropein recovery as compared with fresh olive leaves [14]. Erbay et al. determined and tested the most appropriate thin-layer drying model with air temperatures of 50 °C, 60 °C and 70 °C, and air velocities of 0.5, 1.0 and 1.5 m s–1, to understand the drying behavior of olive leaves. The drying depends on the velocity and temperature of the air. The temperature has great influence, in a way that the drying rate increases with increased temperature [137].
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Malik and Bradford compared freeze-, ambient (25 °C) air-, and hot (60 °C) air-drying of olive leaves and their effect on oleuropein and other phenolic stability and recovery. Air-drying at ambient temperature revealed a fully preservation of oleuropein and verbascoside levels, while freeze- and hot air-drying caused a reduction in polyphenolic content, including oleuropein [freezing—53.5–95.4% loss (time dependent); hot air—19–32% loss] [102]. Another factor studied is the effect of blanching process, which involves the treatment by means of form of heat, usually either steam or boiling water [138]. Recently was reported that the water blanching of olive leaves (1:4 (w/v), 90–95 °C, 20 s) increased phenolic content up to 0.329– 0.532 mg/g GAE (61.7%) when compared with fresh, solar-dried and ovendried leaves [139].
Extraction, purification and analytical methods Natural sources of oleuropein and other phenolic compounds Plants from Oleaceae family are a natural source of oleuropein and other phenolic compounds. As described above, the olive tree (O. europaea L.) is considered the main source of oleuropein, which can be found in several parts of the plant (i.e., leaves, olives and branches), Fig. 5.6. Argania spinosa L. was identified as another natural source of oleuropein. However, only the argan oil is reported to have this natural product. Pressed cake, leaves or fruit pulp from this plant have no oleuropein [140], Fig. 5.6. The use of olive leaves as natural source of oleuropein is advantageous comparing to other natural sources, since they are highly available as a by-product (see Section Olive tree by-products) throughout the year and have a higher content in oleuropein [0.5–2% (w/w) on dry basis]. Therefore, olive leaves are considered a cheap and easily available natural source of oleuropein, and their valorisation must be encouraged [2,11,13,97,141–144].
FIG. 5.6 Natural sources of oleuropein.
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Polyphenols extraction, purification and analytical methods The recovery of phenols depends on important parameters (e.g., extraction method, temperature, solvent and time). Sample handling, processing, clean-up and storage conditions, extract stability, analytical technique sensitivity and the purity of standards used in the preparation of calibration curves are commonly factors that can account for the wide variation of values in literature. It is also difficult to develop a single method for optimum extraction of all phenolic compounds since their polarities vary significantly.
Extraction of polyphenols Extraction is one of the most important steps in sample pre-treatment for polyphenols analysis. The extraction begins with olive leaves drying operations (see Section Harvesting season and fruit size), size reduction and homogenization of the samples [145]. Solid/liquid extraction (e.g., Soxhlet extraction) has been the most common procedure, where olive leaves are macerated in a solvent (e.g., methanol, ethanol, ethyl acetate, diethyl ether or aqueous alcohol mixtures). These methods require large amounts of toxic solvents and long extraction times, involving high costs, time waste and environmental pollution. Being time consuming and having poor efficiency, some degree of heating is required, which could easily lead to thermo-sensitive ingredients, with loss of biological activity or degradation into other substances [146]. Subsequently, in order to minimize these disadvantages, other extraction techniques were developed. These include microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), superheated liquid extraction (SHLE), liquid/liquid extraction, pressurized liquid extraction, derivatized polar extraction, fractionation by solid-phase extraction and dynamic ultrasound-assisted extraction (UAE). Microwaves are nonionizing electromagnetic radiation with a frequency in the range of 300–300,000 MHz, which corresponds to wavelength (λ) of 1 mm–1 m [147]. In the microwave, the heating occurs in a targeted and selective manner with practical no heat being lost due to the closed system. When the moisture is heated up inside the plant cells, water evaporates and generates tremendous pressure on the cells wall, leading to rupture and facile leaching out of the phytoconstituents [148]. In microwave-assisted extraction (MAE) the heat is dissipated volumetrically and all molecules exposed to the radiation are affected directly [149]. A correct choice of the solvent is fundamental for obtaining an optimal extraction process. Solvent choice for MAE is dictated by the solubility of the target analyte, interaction between solvent and plant matrix and by the microwave absorbing properties of the solvent. Consequently, the solvent need to have good selectivity toward the analyte of interest and exclude unwanted matrix components; be compatible with further chromatographic
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analytical steps; and to have appropriated dielectric properties. In general, polar substances (e.g., water, methanol and ethanol) absorb microwave radiation, whereas the less polar substances are not recommended for extraction in the microwave [147]. The volume of the solvent is also a critical factor. It must be sufficient to ensure that the plant matrix is always entirely immersed. The heating efficiency of the solvent under microwave should also be considered and depends on solvent evaporation [148]. The unique heating mechanism of MAE can significantly reduce the extraction time and therefore minimize the degradation of target components [11]. It also provides higher extraction efficiency and selectivity, involving low consumption of organic solvents [13]. MAE is then a promising technique compared with conventional extraction methods and presents different physical and chemical phenomena [150]. Supercritical fluid extraction (SFE) is the process of separating one component from a matrix using supercritical fluids as the extracting solvent. A supercritical fluid possesses the properties of a gas as well as that of a liquid. Therefore, the gas-like mass transfer and the liquid-like solvating power give supercritical fluids an edge over other extracting solvents [151]. Among several possible fluids [e.g., ethane and propane (low-critical temperature solvents); alkanes, methanol and water (high-critical temperature solvents)], carbon dioxide (CO2) has been the most studied solvent for supercritical processes because it is safe, non-toxic, non-flammable, highly selective and has moderate critical conditions (304 K, 7.4 MPa) that allows low temperature extraction conditions, avoiding thermal degradation of the product [152]. Recently, oleuropein was extracted from olive leaves using ethanol as extractant solvent. Oleuropein extraction yield was 5.4% (w/w dry leaves) and 20% (w/w extract). Supercritical antisolvent extraction (SAE) was further performed on ethanol extract, operating at different pressures (100–200 bar) and temperatures (35 °C and 60 °C). SAE produced a powder rich in oleuropein up to 36% (w/w) at 35 °C and 150 bar [153]. SFE has received increasing interest, because supercritical fluids provide high solubility, improved mass-transfer rates and the operation can be manipulated by changing the temperature or pressure. However, this technique is limited to compounds of low or medium polarity [128]. Recently was also observed that the use of CO2-SFE alone, during oleuropein extraction from olive leaves, was not satisfactory, requiring a polar modifier to improve yield and selectivity of the process [154]. Superheated liquid extraction (SHLE) is the extraction using aqueous or organic solvents at high pressure and temperature without reaching the critical point. A static mode (i.e., the volume of extractant is fixed), a dynamic mode (i.e., the extractant flows continuously through the sample) and a static-dynamic mode (i.e., combination of the two modes) can be considered [155]. The combination of static and dynamic modes was found to reduce the extraction time and provide better extraction efficiency [156,157].
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Superheated water (i.e., water under pressure and above 100 °C but below its critical temperature of 374 °C; and pressure high enough to maintain the liquid state) has been used for this extraction process. Under these conditions, water is less polar and so the organic compounds are more soluble than at room temperature [158]. The advantages of using superheated water is the shorter extraction time, higher extraction ability for polar compounds, higher quality of the extract and lower costs. However, it is not suitable for thermally labile compounds [151,159]. SHLE has higher extraction efficiency compared with conventional extraction methods due to the increased capacity to disrupt matrix-analyte interaction at high temperatures and pressure. At high temperatures, the surface tension and the viscosity of the extractant are reduced, increasing mass transfer and enhancing extraction. Other advantages include the reduction of solvents and the high selectivity of this method, which has been used to extract phenols from olive leaves [156]. Liquid-liquid extraction using a microfluidic system (water, ambient temperature, flow rate ratio 1, residence time 0.1293 min) was employed to extract oleuropein from ethyl acetate into aqueous phase. This method has simple operation, is cost effective and environmentally friendly, compared with conventional extraction methods [160]. Ultrahigh pressure extraction (UPE) is a novel technique that utilizes high-pressure conditions (100–500 MPa) and temperature (20–50 °C) to extract active ingredients from plants [161]. It increases the mass transfer rate by changing the concentration gradient and diffusivity, causing damage to the plant cell membrane and increasing its permeability. This results in a shorter processing safety, and achievement of higher compound yields [146]. Reduced-pressure boiling extraction (60 °C, 20 min, material/liquid 1:30, 85% ethanol) was used to extract oleuropein in high yield from olive leaves [162]. Ultrasound-assisted extraction (UAE) uses sound waves that propagate in frequencies from 20 to 100 kHz, creating expansion and compression cycles (i.e., mechanical). In a liquid, they produce negative pressure cycles (bubbles or cavitation) causing permanent physical and chemical changes, by disrupting the stability of solid at the interface liquid-liquid and liquid-gas systems [163]. The mass transfer of the solute from the matrix into the solvent occurs by diffusion and osmotic processes [150]. High-frequency ultrasound standing waves were applicated in olive oil extraction from olive fruits. Megasonic standing waves have shown to have a positive effect in enhancing olive oil extractability before or after malaxation. The combination of low-frequency sonication to promote cell wall disruption pre-malaxation followed by megasonic standing wave (400 and 600 kHz, 57–67 min, 18–21 kJ/kg) application post-malaxation, resulted more advantageous to enhance oil extractability [164].
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UAE of oleuropein and related biophenols from olive leaves was considered faster and more efficient than traditional methods, because cavitation favors penetration and transport at the interface between aqueous or organic liquid phase subjected to ultrasound energy and a solid matrix [165]. The increase in the temperature caused by ultrasonic irradiation did not degrade oleuropein [61]. Recently, an integrated approach based on UAE and spray drying was suggested. The extraction of olive leaves was performed using an ultrasound sonicator and the concentrated extract was further spray dried (149 and 156 °C for inlet air temperature, 21.35 and 23.10 m3/h for drying air flow rate, and 683 and 638 L/h for atomizing agent flow rate) to be included in food formulations [166]. UAE allows shorts extraction times, low extraction temperature and high extraction yields. This technique is efficient, simple, cheap, and a good alternative to conventional extraction processes. Moreover, the equipment is easy to handle and less expensive compared to other methods, such as microwave or supercritical fluid [167]. The disadvantages include the lack of uniformity in the distribution of ultrasonic energy and the loss of power during the process [163]. The recovery of sugars and natural antioxidants from olive leaves using steam-explosion has been addressed in a recent work [168]. Under the studied conditions, grinding of olive leaves is not required so a stage in the process is removed, which leads to a saving of time, energy and equipment and improves the economic viability. Under working conditions (180 °C, 8.3 min), make 70% of the initial sugars to be recovered with a very low formation of inhibitory compounds due to little degradation. Furthermore, several compounds with many applications in food and pharmaceutical industries were recovered, including oleuropein, hydroxytyrosol and flavonoids (luteolin, apigenin, together with their corresponding 7-O-glucosides) with antioxidant capacity. Luque-Garcı´a and De Castro designed and constructed an ultrasoundassisted Soxhlet extractor combined with supercritical CO2 extraction with improved rates and yields [169]. Dang et al. investigated the optimal operating conditions for oleuropein yield from olive leaves by testing supercritical CO2 fluid extraction, microwave-assisted extraction and Soxhlet extraction techniques, showing short extraction time for microwave-assisted technique [170]. Comparison of all of the different extraction techniques has shown MAE and conventional solid/liquid extraction to be the best choices for extraction of more polar compounds such as oleuropein and its derivatives from olive leaves. Although MAE has been seen as a promising alternative technique, there are still some general limitations, such as capital cost due to the need for specialized equipment, scale-up problems, difficulties in continuous operation, and degradation of sensitive compounds when longer times or higher
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power are used. In addition, the use of domestic devices may lead to poor reproducibility of the extraction method because of the heterogeneity of the microwave field [129,171–173]. Laboratory-scale methods use preferably polar solvents in the extraction of oleuropein and derivatives from olive leaves. Methanol-water mixtures are usually employed, although the use of less polar solvents such as hexane was also reported. Nevertheless, the increased human use of these compounds makes mandatory the development of green extraction methods based on nontoxic extractants (e.g., water) [61]. Recently, Apostolakis et al. studied the efficiency of heated water/glycerol mixtures in extracting polyphenols from dried olive leaves. Glycerol is a bio-solvent, with no toxicity and it is a natural constituent of foods. Authors concluded that the use of a heated aqueous glycerol solution was more efficient as compared with a hydro alcoholic solution in extracting olive leaf polyphenols, providing the same differentiated selectivity [2]. Green approaches have also been tried. Recently, biopolymers and polymeric absorbents have been used to separate and purify target compounds from natural plant sources, with low cost and harmless to operator’s health. Altiok et al. and Bayc¸ın et al. used the polymer silk fibroin, which have been used since the 1940s by its hydrophobic and bonding characteristics, in order to increase the purity of oleuropein from olive leaves [174,175]. Beside biopolymers, macroporous resins are important as polymeric absorbent, usually applied in separation and purification of bioactive compounds from natural sources [144]. One example was the use of a continuous adsorption/nanofiltration hybrid process and the use of imprinted polymer with in situ recovery of solvent [176]. Molecular imprinted polymers were also used to selectively recover several biophenols, including oleuropein [177]. An oleuropein rich extract was obtained from olive leaves by combining microfiltration, ultrafiltration and nanofiltration processes. The water extract of olive leaves was subjected to microfiltration process (0.2 μm) to remove larger particles, followed by ultrafiltration (removal of molecules larger than 5 kDa), and finally a nanofiltration process (300 Da), allowed the concentration of polyphenols, mainly oleuropein (1685 mg/100 g extract) [178]. The nanofiltration process may be an appealing one to explore for largescale operation due to its low energy consumption, easy scale-up, and use of mild conditions. Also, this process has been described as being economic and to have easy and simple operation [176,179]. However, some of the identified limitations such as large molecular-weight gap, long screening, high solvent consumption, and unpredictability of solvent effects created more difficulties in the specific case of extraction of olive leaves [180]. The consumption of solvents can be minimized by the potential for their recovery, and improvements in membrane composition allowed a broad range of molecular-weight cut-off that can be determined more efficiently [181].
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Two computational methods (RSM and ANN) were used to predict the total phenolic content and oleuropein in olive leaf extracts obtained by solvent-free MAE. The optimal conditions to obtain a maximal yield of oleuropein (0.060 0.012 ppm) were 250 W, 2 min of extraction time and 5 g of olive leaves [182]. RSM was used for prediction of the optimal values of parameters affecting the extraction of oleuropein through microchannel. Optimum conditions of F ¼ 80 kHz, 25 °C and P ¼ 100 w resulted in 96.29% of oleuropein extraction, more than other applied methods. Microchannel continuous system has several advantages, including low solvent consumption, short time for extraction and high efficiency [183].
Analysis and purification of polyphenols The colourimetric Folin-Ciocalteau assay is the most used and rapid quantitative technique for the determination of total polar phenolics in olive leaves. High performance liquid chromatography (HPLC), usually in reversed-phase mode and coupled with several detectors (UV–Vis, MS, NMR, DAD, etc.), is used for the determination of individual compounds [129]. Nuclear magnetic resonance spectroscopy (NMR) is used in the analysis of complex mixtures without previous separation of the individual components. Gas chromatography (GC) has lower detection limits and better separation, but needs sample pre-treatments, using derivatization reagents. Nevertheless, high/ultra-performance liquid chromatography (HPLC/UPLC) coupled to diode-array detection (DAD) and/or coupled to mass spectrometry (MS) is the most used to quantify and characterize phenolic compounds [11]. The quantification of oleuropein is mainly performed through analytical HPLC, usually by reversed-phase chromatography with use of a gradient elution method. Gradient elution mode is commonly utilized, because the complexity of the phenolic profile makes it not able to be well separated by the isocratic elution mode. UV–Vis detection system continued to be one of the most used detection systems for phenolic compounds, generally using as wavelength 280 nm. However, it should be considered that no universal absorbance maximum exists for olive leaf phenolics [129]. The isolation of oleuropein from olive leaves usually involves purification through column chromatography mainly on silica [184,185]. Published values for oleuropein recovery from olive leaves varied massively, from 5.6 to 108.6 mg/g dry weight, depending on some factors as discussed before in Section Factors that influence oleuropein and phenolic levels. The structural characterization of oleuropein has been performed by 1H, 13 C, and two-dimensional H-C heteronuclear NMR spectroscopy ([D6]DMSO [176,184,186], [D4]methanol [187,188], D2O [189] and CDCl3 [190]), by mass spectrometry (ESI, TOF, IT, CI, CID, EI, FAB) [176,191–193], and by FTIR [176,194,195] and UV/Vis [191,194,195] spectroscopy.
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The development of existing methods of separation and introduction of new techniques of high resolution are needed to give rise to the discovery of new effective compounds from phytopharmaceutical sources and improve its separation [148]. A novel ionic liquid modified absorbent with a structure of an interpenetrating polymer network (PS/PVIm) was designed and used to enrich oleuropein. The high affinity of PS/PVIm for oleuropein and the introduction of an ionic liquid imidazolium group and interpenetration in polymer network improved the adsorption efficiency, however the adsorption rate decreased. The design and preparation of appropriate resins with ionic liquids and high absorption efficiency to enrich oleuropein from olive leaves extract have significant importance [196]. A compilation of values of oleuropein content regarding different extraction, purification and analytic methods, according to leaves conditions and olive tree variety are presented in Table 5.4.
Valorisation of oleuropein General reactivity As reported in Section Oleuropein chemistry, oleuropein molecular structure can be divided in three subunits: hydroxytyrosol, monoterpene, and glucose moieties. Secoiridoids react readily with solvents and are transformed into different compounds during extraction procedures. This is due to the easy opening of the secoiridoid ring after hydrolysis of the glucose moiety. Scheme 5.4 presents an overview of proposed transformation pathways of the oleuropein aglycon in olive leaves during extraction and observed in solution (i.e., methanol or chloroform), and complete transformation of 3,4-DHPEA-DETA (10) into 3,4-DHPEA-EDA (8) in acidic medium [203,204]. Transformations include oleuropein aglycone (2) equilibrium forms from opening of the secoiridoid ring (3a/b and 6a/b); intramolecular reaction of (3a) to oleuropein-aglycone mono-aldehyde (3,4-DHPEA-EA) (4), and transformation into (5) in methanol; methyl ester hydrolysis of (6a/b) into (7a/b) and decarbomethoxylation into oleuropein-aglycone di-aldehyde (3,4DHPEA-EDA) (8) and transformation into (11) in methanol; and conversion of equilibrium form (9) into (12) under acidic conditions, Scheme 5.4.
Synthetic transformations of oleuropein The monoterpene unit in oleuropein is a highly functionalized moiety that includes two esters (i.e., including the bond between the hydroxytyrosol and the monoterpene subunits), one alkene, one enol ether, one acetal (i.e., bond between the glucoside and the monoterpene subunits) and a stable chiral center at C-5, Fig. 5.7.
TABLE 5.4 Published values of oleuropein concentration according to extraction method. Leaves conditions
Leccino, Moraiolo and Frantoio
Fresh leaves
Moraiolo
Dried leaves (freeze-dried with liquid N2)
Variety
N3 (Don Carlo)
Extraction Method
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)a
Solid-liquid extraction
Purification
CH3OH, 1 week, rt. Then, (CH3)2CO/H2O (1:1) Extraction with n-pentane, CH3Cl and ethyl acetate
Column chromatography with silica gel CH3Cl/CH3OH (9:1) then CH3Cl/CH3OH (4:1)
Solid-liquid extraction Different volume ratios of H2O/Ethanol
Observations
References
2.95
Oleuropein isolated from ethyl acetate extract
Gariboldi et al. [184]
Analytical
14.4
HPLC
8.39
Best extraction solvent mixture: H2O/Ethanol (1:1) Moraiolo variety possesses the highest oleuropein content
Briante et al. [67]
N2
7.06
Coratina
6.10
Nociara
3.70
Frantoio
3.19
I-77
3.03
Leccino
1.05
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety
Leaves conditions
Unknown
Dried leaves
Extraction Method Microwave-assisted extraction (MAE) 100–200 W, 5–15 min Ethanol 80%–100%
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Analytical
23.2
HPLC-DAD (280, 330, 340, 350 nm)
Observations
References
MAE—faster extraction method
Japo´n-Luja´n [197]
Optimal extractant— Ethanol/H2O (8:2)
Lichrospher 100 RP18 column (250 4 mm, 5 μm); Kromasil 5 C18 (15 4,6 mm, 5 μm) 6% acetic acid, 2 mM sodium acetate in H2O, and ACN Flow: 0.8 mL/min Gradient
Unknown
Dried leaves (40 °C, 8 h)
Superheated liquid extraction (SHLE) Variables: temperature, static and dynamic extraction time, extractant flow-rate and extractant composition
Analytical
23.0
Pressure: 6 bar
HPLC-DAD (280, 330, 340 and 350 nm)
Solvent—EtOH: H2O (7:3), 140 °C, 6 min
Lichrospher 100 RP18 (250 4 mm, 5 μm)
Dynamic mode, extractant: 7 min at 1 mL/min
Kromasil 5 C18 column (15 4.6 mm, 5 μm) 6% acetic acid, 2 mM sodium acetate in H2O, and ACN Gradient Flow: 0,8 mL/min
Extraction time: 13 min
Japo´n-Luja´n et al. [198]
Unknown
Dried leaves
Ultrasound-assisted extraction 20KHz, 450 W Variables: irradiation time, extraction flow-rate, Ethanol%, probe position, temperature, radiation amplitude and duty cycle
Analytical
22.6
HPLC-DAD (280, 330, 340 and 350 nm) Lichrospher 100 RP18 (250 4 mm, 5 μm) Kromasil 5 C18 column (15 4,6 mm, 5 μm)
Optimal conditions: irradiation time— 25 min; extraction flowrate—5 mL/min; 59% EtOH; Probe position— 4 cm; temperature—40 ° C;
Japo´n-Luja´n et al. [61]
Faster and more efficient than traditional methods
6% acetic acid, 2 mM sodium acetate in H2O, and ACN Gradient Flow: 0.8 mL/min Unknown (December 2004)
Dried leaves (37 °C, 3 days)
Solid-liquid extraction
Analytical
Ethanol, CH3OH, acetone and their aqueous forms (10%/90%, v/v)
HPLC-UV (280 nm) C18 Lichrospher 100 analytical column (250 4 mm, 5 μm), 30 °C
74.5
70% EtOH: best extractant solvent Silk fibroin as promising adsorbent for oleuropein purification.
Baic¸ın et al. [175]
Flow: 1 mL/min Acetic acid/H2O (2.5:97.5) and ACN Gradient, 60 min
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety
Leaves conditions
Chemlali
Dried leaves
Extraction Method
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Observations
References
Solid-liquid extraction
Analytical
43.2
–
CH3OH/H2O (4:1)
HPLC
Jemai et al. [199]
24 h
Shim-pack VP-ODS (250 4.6 mm), 40 °C
24.4
–
Jemai et al. [79]
8.80–21.7
Optimal extraction time: 10 min giving high oleuropein yield (21,7 103 mg/g)
Procopio et al. [185]
0.1% phosphoric acid in H2O, and 70% ACN in H2O Flow: 0.5 mL/min Gradient, 40 min Dried leaves
Solid-liquid extraction
Analytical
CH3OH/H2O (4:1) 24 h
HPLC Shim-pack VP-ODS (250 4.6 mm), 40 °C 0.1% phosphoric acid in H2O, and 70% acetonitrile in H2O Flow: 0.5 mL/min Gradient, 40 min
Coralina
Dried leaves
Microwave-assisted extraction H2O, 800 W, 10 min
Purification LC Supelco Versa Flash CH2Cl2/MeOH (8:2)
Koroneiki
Air dried leaves
Supercritical fluid extraction (SFE) 30 MPa Extraction: 50 °C Separation: 55 °C Solvent-to-feed ratio: 120 or 290 Co-solvent: 5% or 20%
Analytical
51.0
HPLC-DAD (248 nm) SupelcoAnalytical Discovery HS C18 (250 4.6 mm; 5 μm) 25 °C
Best solvent-to-feed ratio: 290 Co-solvent: 20% EtOH
Xynos et al. [200]
Extraction: 50 °C Separation: 55 °C
H2O + 1% acetic acid, and CH3OH
Pressure: 30 MPa
Gradient Flow: 1 mL/min
Unknown (November 2010)
Fresh leaves Steam and hot water blanching; air-dried at 60 ° C
Solid-liquid extraction
Analytical
Hot water blanched
Ethanol/H2O (7:3)
HPLC-DAD (280 nm)
4.63–5.06
Pinnacle II RP C18 (150 4.6 mm, 3 μm), 40 °C
Non-blanched
Oleuropein content was higher when steam or hot water blanching were used
Stamatopoulous et al. [201]
Mid-infrared spectroscopy as rapid tool to predict oleuropein content
Aouidi et al. [134]
0.210–2.14
Kromasil 100–5 C18 (3.0/4.6 mm) 0.02 M sodium acetate pH 3.2 + acetic acid, and ACN Flow: 1 mL/min Chemlali, Chetoui, Meski, Sayali and Zarrazi
Dried leaves (MW-assisted, 800 W, 2 2 min)
Solid-liquid extraction
Analytical
109.2 to
CH3OH/H2O (4:1)
Mid-infrared spectroscopy (4000 cm1 and 700 cm1)
172
24 h
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety Serrana
Extraction Method
Oleuropein Quantification/ Purification
Fresh leaves
Solid-liquid extraction
Analytical
Fresh HAD 70 °C
Hot air (HAD) or freeze (FD) drying
Ethanol/H2O (4:1)
HPLC-DAD/MS-MS (240, 280 and 330 nm)
69.0
24 h
Lichrospher 100 RP18 (250 4 mm, 5 μm)
Air dried leaves
Pressurized liquid extraction (PLE)
Linear gradient
3.00
Flow: 1 mL/min
FD N2
ESI—negative mode
16.4
Desolvation temperature: 365 °C
HAD 70 °C + FD N2
Vaporiser temperature: 400 °C
30.1
Variables: temperature, static time, extraction cycles and Ethanol % Pressure: 1500 psi
Analytical HPLC-DAD (248 nm) Supelco Analytical Discovery HS C18 (250 4,6 mm; 5 μm) 25 °C H2O + 1% acetic acid, and CH3OH Gradient Flow: 1 mL/min
References
Hot air drying of leaves gives high oleuropein recovery
Ahmad-Qasem et al. [132]
Extraction yield affected by temperature, static time and extraction cycles
Xynos et al. [202]
109 FD conventional
Nebulizer: 4.83 bar
Observations
Fresh HAD 120 °C
2.5% acetic acid in H2O, and acetonitrile
Dry gas: N2 12 L/min
Koroneiki
Oleuropein Concentration (mg. g21)
Leaves conditions
HAD 120 °C + FD N2 48.0 261
Optimal conditions for oleuropein extraction: 190 °C, EtOH: H2O (56:44)
Sevillana
Air dried leaves
Solid-liquid extraction
Analytical
EtOAc, r.t., 24 h
HPLC/UV–Vis (254 nm)
Liquid-liquid extraction/MiS
C18 analytical column (Shim-Pack VP-ODS, 250 mm, 4.6 mm i.d., Shimadzu, Japan)
H2O, flow rate ratio 1 Residence time 0.1293 min
687
Microfluidic system (MiS)
Naleini et al. [160]
Extraction yield depends on residence time, pH, temperature and EtOAc/H2O ratio
Phosphate buffer (0.05 mol/L and pH 3 adjusted with orthophosphoric acid) and acetonitrile (70:30, v/v), gradient Injection volume: 10 μL 10 min, r.t., flow: 1.0 mL/min
Chemlali (July)
MW dried leaves 1200 W, 70 °C, 10 min
Ultrasound-assisted extraction (UAE) vs. SolidLiquid Extraction (SLE) H2O, 10 min 10–70 °C
Analytical
57.5 (UAE)
HPLC-DAD/MS-MS (240, 280, 230 nm, ESI)
51.5 (SLE)
UAE intensified the initial extraction rate
Khemakhem et al. [165]
Merck Lichrospher 100RP18 (5 μm, 250 4 mm) column 2.5% acetic acid and acetonitrile, gradient Flow: 1 mL/min, 50 min
Continued
TABLE 5.4 Published values of oleuropein concentration according to extraction method.—Cont’d
Variety Chemlali
Leaves conditions MW dried leaves 1200 W, 70 °C, 10 min
Extraction Method Microfiltration (0.2 μm), Ultrafiltration (5 kDa) and Nanofiltration (300 Da) H2O, 30 °C, 1 h
Oleuropein Quantification/ Purification
Oleuropein Concentration (mg. g21)
Analytical
Observations
References
16.9
Production of an oleuropein concentrate
Khemakhem et al. [178]
10.7 (Alcoholic Extraction) 19.2 (SAE)
Hybrid process Oleuropein degraded at 60 °C
Baldino et al. [153]
HPLC-UV (254 nm) C18 column (4.6 mm 250 mm) 70% acetonitrile/H2O and 0.1% phosphoric acid/H2O 40 °C Flow: 0.6 mL/min
Unknown
Water content
Solid-Liquid Extraction
Analytical
8% (w/w)
EtOH, 20 °C
HPLC-UV/Vis (280 nm)
Supercritical Antisolvent Extraction (SAE)
C18 LiChrospher
SC-CO2, 150 bar, 35 °C Rate: 2.5%
column (150 mm 3.0 mm i. d., 5 μm, Phenomenex) 2.5% acetic acid in water and 10% acetonitrile Gradient Injection volume: 20 μL Flow: 1 mL/min
a
Mass of oleuropein in mg per gram of olive leaves dry weight.
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SCHEME 5.4 Proposed transformations of the oleuropein aglycone in olive leaves during olive extraction and observed in solution (methanol, chloroform and acidic solutions). Solvents are identified in dashed boxes. Adapted from L.A.S. Cavaca, C.A.M. Afonso, Eur. J. Org. Chem. (2018) 581-589.
FIG. 5.7 Monoterpene moiety chemical features. Reactive positions of the monoterpene moiety are represented by arrows; stereocenters represented by (*).
168 Studies in Natural Products Chemistry
This multifunctional structure makes it difficult to be obtained by other means than extraction from natural sources. The seven potential reactive positions available for synthetic chemical modifications facilitate the valorisation of oleuropein towards the synthesis of diverse and synthetically rich building blocks with potential applications (e.g., pharmaceutical, cosmetic, food additives, etc.), Fig. 5.7. The glucose unit confers to oleuropein solubility in water. However, the presence of such polar moiety difficult the synthetic procedures for oleuropein valorisation, mostly accomplished in organic solvent medium. In addition, it might complicate further purification processes. Therefore, to fully take advantage of all oleuropein chemical features it is essential to remove this problematic unit and produce more handleable molecules. Several authors, Scheme 5.5, have reported a variety of synthetic transformations of oleuropein [205]. The cleavage of the glucose moiety in oleuropein is usually performed by using enzymes or by chemical hydrolysis. The endogenous olive tree β-glucosidase and its commercially available counterpart from almonds have been employed for this purpose, leading to the formation of mixtures of aglycone compounds with different structures [143,206,207]. An enzymatic tailoring of oleuropein was performed using commercial available lipase and thermophilic β-glucosidase. Degradation products of oleuropein using β-glucosidase (i.e., oleuropein aglycone and glucose), lipase (i.e., elenolic acid glucoside and hydroxytyrosol) or both enzymes (i.e., glucose, elenolic acid and hydroxytyrosol) were identified by HRMS/MS spectrometry [208]. The acid treatment of oleuropein has also been reported using sulfuric acid, anhydrous hydrochloric acid, and erbium(III) trifluoromethanesulfonate (F-H, Scheme 5.5) [184,185,209]. In general, complex mixtures of oleuropein aglycone derivatives are obtained, including elenolic acid (7) and compound (8). The use of lanthanides (H, Scheme 5.5) leads to the assignment of several products’ molecular structures, revealing the presence of two isomers (i.e., hemiacetal (8) and dialdehyde), as well as subsequent products resulting from the rearrangement of the oleuropein aglycon, possibly through 1,4-addition of the enol form of the aldehyde closer to methyl ester to the double bond (8a and 8b, Scheme 5.5) [186]. The chemical hydrolysis of β-glycosidic bond with sulfuric acid in dioxane produced elenolic acid (7) in 7% yield (F, Scheme 5.5), which is of particular interest because of its potent and broad-range antiviral activity. These conditions allowed the removal of the glucose moiety together with the hydrolysis of the hydroxytyrosol ester to a carboxylic acid [185]. Erbium(III) was also applied as a catalyst for the peracetylation of oleuropein under mild conditions, affording a yield of 65% [186]. Peracetylated oleuropein is of additional interest due to its antiproliferative effects [210]. The hydrolysis of the hydroxytyrosol ester to afford the corresponding carboxylic acid (A, Scheme 5.5) was performed under basic conditions with
SCHEME 5.5 Synthetic transformations of oleuropein. Adapted from L.A.S. Cavaca, C.A.B. Rodrigues, S.P. Simeonov, R.F.A. Gomes, J.A.S. Coelho, G.P. Romanelli, A.G. Sathicq, J.J. Martı´nez, C.A.M. Afonso, ChemSusChem 11 (2018) 2300–2305. (The reference numbers correspond to those of article [219]).
170 Studies in Natural Products Chemistry
sodium hydroxide, followed by acetylation to afford the oleoside monomethyl ester peracetate (2, Scheme 5.5). This compound is involved in the stereocontrolled synthesis of the secoiridoid glucosides nudiflosides D and A, natural compounds extracted from the plant Jasminum nudiflorum (Oleaceae) [210]. Transformations of the monoterpene core for the synthesis of different scaffolds with additional importance have been published. The enzymatic acetal cleavage by β-glucosidase formed either pyridine alkaloid jasminine in 20% yield (4; C, Scheme 5.5) [211] or compound 5 in 5% yield (D, Scheme 5.5) [143,207,208], depending on the ammonium salt used. The phytopharmacological profile of Jasminum grandiflorum Linn. (Oleaceae), the natural source of jasminine (4), has been known to be antimicrobial, an angiotensin converting enzyme inhibitor, anti-ulcer and antioxidant [212–214]. Treatment of oleuropein with sodium borohydride in water produces oleuropeinol in 85% yield (3, B, Scheme 5.5), with reduction of both the hydroxytyrosol ester and the methyl ester to their primary alcohols. The acid rearrangement of oleuropeinol in the presence of aqueous hydrochloric acid removes the glucose moiety, and an equilibrium between the closed acetal (3a, 20% yield, Scheme 5.5) and the open dialdehyde (3b, 80% yield, Scheme 5.5) forms is observed. The mixture was further acetylated affording the monoacetyl derivative of the open form (3c, 98% yield, Scheme 5.5). This means that the equilibrium shifts towards the open form, which is further acetylated. The same mixture was also reduced with sodium borohydride in methanol, transforming the aldehyde into alcohol 3d (98% yield, Scheme 5.5) [215]. The only reported modification of oleuropein in methanol has been the transesterification of the hydroxytyrosol ester to a methyl ester. In contrast the acid rearrangement of oleuropein gave two major products. One results from the formation of a cyclic hemiacetal moiety between the primary alcohol function and the aldehyde function, which affords the cyclic acetal. This reaction produces a mixture of compounds 3e and 3f, with compound 3e being formed in larger amounts (40% yield). This suggests the existence of an equilibrium between these two compounds, with compound 3f converting slowly into the more stable compound 3e [216]. An efficient procedure for the semisynthesis of oleacein (6, Ea, Scheme 5.5) from oleuropein through Krapcho demethoxycarbonylation was developed. The possible conversion of oleuropein into oleacein involves three main transformations: cleavage of the glucose moiety, secoiridoid ring-opening followed by formation of two aldehyde groups and demethoxycarbonylation. This reaction was applied in order to perform these three steps in one-pot fashion in 22% yield [95]. Recently, a simple and environment friendly microwave-assisted method (180 °C, 20 min) to produce oleacein (6, Eb, Scheme 5.5) in a good yield (48%) starting from the easily available oleuropein. Oleacein has several reported biological activities, including antioxidant and antimicrobial [216].
The olive-tree leaves as a source of high-added value molecules Chapter
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A set of oleuropein aglycone derivatives were synthesized by transacetylation under mild conditions in 60–90% yield (12a-i, J, Scheme 5.5). Oleuropein aglycone in closed ring form is obtained by hydrolysis of oleuropein using Lewis acid [Er(OTf)3] catalysis [217]. Recently, the acid-promoted methanolysis of oleuropein was studied using a variety of homogeneous and heterogeneous acid catalysts. Exclusive cleavage of the acetal bond between the glucoside and the monoterpene subunits [(S,S)-9 and (S,R)-9, I, Scheme 5.5] or further hydrolysis of the hydroxytyrosol ester and subsequent intramolecular rearrangement (10, I, Scheme 5.5) were observed upon identification of the most efficient catalyst and experimental conditions. Formation of ()-methyl elenolate (11, Scheme 5.5) was also observed, which is a reported precursor for the synthesis of the antihypertensive drug ()-ajmalicine [218].
Conclusions This chapter gives an overview about the olive-tree leaves exploitation as a cheap and easily available source of polyphenols, mainly the high-added value natural compound oleuropein. An insight is made about the olive tree and related by-products as natural sources of bioactive compounds, in particular about oleuropein. The occurrence, biosynthesis, biodegradation, quantification, extraction and purification, as well as biological activities and synthetic transformations of oleuropein are reviewed in this chapter. Oleuropein is a natural product that can be extracted in quite large amounts from olive tree (O. europaea L.) leaves, which are a cheap and easily available source of this bioactive compound. The chemical features of the monoterpene ring make oleuropein a valuable bio-renewable synthetic building block for the generation of structurally more diverse derivatives, which might allow the identification of new molecules with valuable biological activities or with application in cosmetic and food research areas. The potential of oleuropein should be explored, and studies at the level of synthetic modifications of this valuable compound deserve further development taking advantage of the rich and close functionalities. Additionally, the isolation of pure oleuropein from olive leaf extracts with potential scaleup application without the requirement for chromatographic methods or complex separation processes is still an open issue.
Acknowledgments I.M.L.-C. and G.S. are grateful for the financial support of the Regional Government of Extremadura (Junta de Extremadura) and the European Regional Development Fund through grants GR15094 and IB16167.
172 Studies in Natural Products Chemistry
Abbreviations AFPL AOM CI CID DAD DHPEA-EA DHPEA-EDA DMSO DNA DSS EI ESI FAB FD GAE GC HAD HPEA-EA HPEA-EDA HPLC HRMS IGS ISSR IT LO MAE matK MS NMR PAL PPO RAPD RFLP RNA ROS SFE SHLE SSR TOF UAE UPE
amplified fragment length polymorphism azoxymethane chemical ionization collision-induced dissociation diode array detector oleuropein-aglycone mono-aldehyde oleuropein-aglycone dialdehyde dimethylsulfoxide deoxyribonucleic acid dextran sulfate sodium electron ionization electron spray ionization fast atom bombardment freeze drying gallic acid equivalents gas chromatography hot air drying ligstroside-aglycone mono-aldehyde ligstroside-aglycone dialdehyde high performance liquid chromatography high resolution mass spectrometry inter gene spacer inter simple sequence repeat ion trap lipooxygenase microwave-assisted extraction Maturase K mass spectrometry nuclear magnetic resonance phenylalanine ammonia-lyase polyphenol oxidase randomly-amplified polymorphic DNA restriction fragment length polymorphism ribonucleic acid reactive oxygen species supercritical fluid extraction superheated liquid extraction simple sequence repeat time-of-flight ultrasound-assisted extraction ultrahigh pressure extraction
The olive-tree leaves as a source of high-added value molecules Chapter
UPLC UV/Vis UVB
5 173
ultra performance liquid chromatography ultraviolet/visible ultraviolet B
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