Arbutus unedo L.: Chemical and Biological Properties

Arbutus unedo L. (strawberry tree) has a circum-Mediterranean distribution, being found in western, central and southern Europe, north-eastern Africa (excluding Egypt and Libya) and the Canary Islands and western Asia. Fruits of the strawberry tree are generally used for preparing alcoholic drinks (wines, liqueurs and brandies), jams, jellies and marmalades, and less frequently eaten as fresh fruit, despite their pleasing appearance. An overview of the chemical composition of different parts of the plant, strawberry tree honey and strawberry tree brandy will be presented. The biological properties of the different parts of A. unedo and strawberry tree honey will be also overviewed.

In the flavonol group there were also some differences between fruits from Portugal and Spain. In this country, the authors [22]    Extracts of strawberry tree fruits, after enzymatic hydrolysis with hesperidinase followed by cellulose, had gallic acid, protocatechuic acid, (+)-catechin, phloroglucinaldehyde, ellagic acid, myricetin and quercetin [35]. The conjugated sugars found by these authors in the extracts were glucoside, galactoside, rutinoside, rhamnoside and arabinoside. The
Strawberry tree fruits from Croatia also had as main fatty acids linoleic and linolenic acids (34.8% and 31.3%, respectively), but the third more important was palmitic acid (19.0%) and not oleic acid (14.9%) [38] as reported above for the other samples from different locations.

Vitamins and Others
Fruits of strawberry tree from Trás-os-Montes (North-eastern Portugal) contained vitamin E. Among the vitamin E vitamers, the most important was γ-tocotrienol. The total free vitamin E content reduced with ripening: from 1369 mg/kg in unripe fruits to 557 mg/kg in ripe ones [29]. α-, β-, γ-and δ-Tocopherols and α-tocotrienol were also present in fruits ( Figure 5). Other studies characterizing the nutrients and phytochemicals with antioxidant properties of strawberry tree fruits collected in the Natural Park of Montesinho territory, in Trás-os-Montes, North-eastern Portugal, also showed the presence of tocopherols (235 mg/kg), being α-tocopherol the most important (235 mg/kg), followed by γ-tocopherol and β-tocopherol [36]. These authors did not find δ-tocopherol in fruits.  The presence of α-tocopherol was also reported by [22], nevertheless in much lower amounts (0.2 mg/kg) than those reported by other authors [29,36] (271 mg/kg in unripe fruits to 32 mg/kg in ripe fruits). Total vitamin E in wild fruits of strawberry fruits from Central and Western Spain was also evaluated by [37]. The concentration (39 mg/kg) found by these authors were similar to those reported by [29] in ripe fruits. Among the vitamin E isomers, the authors found that α-tocopherol predominated (35 mg/kg), followed by γ-tocopherol, β-tocopherol and δ-tocopherol.
Triterpenes were also found in fruits of A. unedo when separated and identified by high pressure liquid chromatography coupled to a mass spectrometer by means of a particle beam interface (HPLC-PBMS). Through this methodology, the authors identified α-and β-amyrin, lupeol as well as a new natural triterpene (olean-12-en-3β,23-diol) ( Figure 6) [41].

Organic Acids
Fumaric (1.49 mg/g, d.w.), lactic (0.49 mg/g), malic (0.84 mg/g), suberic (0.23 mg/g) and citric (0.01 mg/g) acids were the organic acids detected and quantified by [16] in A. unedo fruits from Samsun, Turkey. In Spain, fruits of strawberry tree had only oxalic acid (96.53 mg/100 g, f.w.); and fumaric acid (0.73 mg/100 g), according to the results reported by [37]. Some variability in the organic acids content of strawberry tree fruits was found in samples gathered in three different seasons (November and December 2007-2009) and from two localities with different environmental conditions (Madrid, center of Spain; and Cáceres, west of Spain): oxalic acid (146.75-48.44 mg/g, f.w.); malic acid (314.94-203.3 mg/g); and fumaric acid (0.919-0.539 mg/g) [31]. In Portugal, fruits harvested in Tapada da Ajuda, Lisboa, had malic and quinic acids (Figure 7), which concentrations depended on the ripening stage of fruits [17]. In contrast to those samples from Turkey, in which fumaric acid predominated, in Portugal only traces were detected mainly in red mature fruits.  ) were the predominant sugars in fruits from Turkey, followed by sucrose (1.80%) and maltose (1.11%) [16]. Similar concentrations of fructose, glucose and sucrose were also reported by Şeker and Toplu [40] in Arbutus fruits collected in North-western Turkey (24.09, 19.09% and 2.65%, d.w., respectively). In the Natural Park of Montesinho territory, in the Trás-os-Montes, North-eastern Portugal, the major sugar was also fructose (24.21%, d.w.) in fruits of strawberry tree, but the glucose amount was much lower (12.14%) and sucrose level was much higher (4.20%) than those reported by [16,40] for Turkish fruits. Alarcão-e-Silva et al. [17] found that the amounts of glucose and fructose were dependent on the ripening stage, whereas sucrose content did not change (8.77 and 8.68%, d.w.) in unripe and red mature fruits, respectively. Glucose amount ranged from 3.95 in unripe fruits to 12.5% in red mature fruits (d.w.). This difference between two ripening stages was even higher for fructose (2.33-20.8, d.w., respectively). A great variability in fructose (12.69%-3.65%, f.w.) and glucose (6.50%-2.34%) amounts were reported by [31] in fruits of A. unedo, depending on the harvest date and location of the orchards (center and west of Spain). For the same samples, the levels of sucrose ranged from 0.48% to absence. The levels of fructose and glucose in strawberry tree fruits collected in Croatia were within the range reported for Portuguese and Spanish samples (12.62% and 5.27%, respectively) [38].

Volatiles
The analysis of the volatiles of strawberry tree fruits performed using headspace-solid phase micro-extraction (HS-SPME) allowed the identification of a total of 41 compounds in Portuguese samples. The fruits were collected at different ripening stages in the region of Trás-os-Montes (north-eastern Portugal). Alcohols [(Z)-3-hexen-1-ol, 1-hexanol] are the main volatile compounds detected in the three ripening stages, followed by aldehydes [hexanal, (E)-2-hexenal], and esters [(Z)-3-hexenyl acetate] [42]. Generally, these volatiles decreased during ripening, maybe due to the reduction of the activity of lipoxygenase [42]. The remaining chemical classes found in fruits were norisoprenoid derivatives, sesquiterpenes and monoterpenes but in very low amounts. In the progress of the fruit ripening, the amounts of monoterpenes decrease from unripe to intermediate maturation, presenting in the ripe stage the highest quantity; whereas sesquiterpenes increase their amount from unripe to intermediate fruits, after which their presence was lower. Norisoprenoids derivatives decrease their presence as the maturation increases [42].
The essential oil isolated from fruits of A. unedo in the north-eastern Turkey by hydrodistillation and analysed by GC-MS was mainly constituted by hexadecanoic acid, ethyl dodecanoate, ethyl linoleate, tetradecanoic acid, and trans-carane [43]. Flower oil had a different chemical profile because the major components were hexadecanoic acid, α-terpineol, nonanal and linalool [43].

Leaves
Arbutin was reported as being present in leaf extracts of A. unedo collected in Montenegro, along with hydroquinone derivatives [46]. Quercitrin, isoquercitrin, hyperoside, and chlorogenic acid are other phenolic compounds identified and quantified by [5,47] in extracts of leaves of A. unedo from Croatia. In this work, the authors also reported that the concentrations of these compounds changed over the year. For example, higher concentrations of hyperoside and quecitrin were found in January, whereas chlorogenic acid was in higher amounts in June, July and October.
Leaf extracts of A. unedo from the Natural Park of Montesinho (Bragança, Northeast of Portugal) after LC-DAD-ESI/MS were shown to have flavanols (catechins, procyanidin dimers and respective gallate esters), flavonols (glucosides of myricetin, quercetin, kampferol), several galloyl (gallotannins) and ellagic (ellagitannins) derivatives. These compounds were also present in fruit extracts; however the number of compounds identified was higher in leaf extracts than in fruit [27].
α-Tocopherol was also found in leaves of A. unedo from Turkey, being the amounts found dependent on the collection season. Leaves collected in March had the highest concentration of α-tocopherol [48].

Stems
Catechin and epicatechin are monomers constituting the proanthocyanidin also known as condensed tannins, a broad family of oligomers and polymers belonging to the flavonoid class. The stems of A. unedo L. from Algeria after extraction using a water/methanol/acetone mixture and structural analysis by 1 H-NMR, 13 C-NMR, IR and mass spectra using an ion-trap spectrometer, operating on an ESI mode showed the presence of (+)-catechin, (+)-afzelechin and (3,4-dihydroxyphenyl)-5,7dihydroxychroman-3-yl 4-hydroxybenzoate [53].

Seeds
The triacylglycerol characterisation of A. unedo seeds made by [55] revealed low saturated fatty acids, high content of oleic acid, a significant presence of ω6 and ω3 unsaturated fatty acids with a low ω6/ω3 fatty acid ratio. The distribution of fatty acids among the three sn-positions of triacylglycerol is asymmetric, nevertheless with a high incorporation of essential fatty acids in the sn-2-position, which is very important from a nutritional point of view [55].  [56].

Fruits
The extracts of A. unedo fruits revealed to have in vitro antioxidant activity [26,29,36,37,40,[58][59][60][61][62]. The type of extraction of phenols present in fruits of A. unedo influenced the antioxidant activity. The capacity for scavenging free radicals of the ripe fruit extracts of strawberry tree obtained by supercritical fluid extraction in the adequate values of pressure (60 bar), temperature (48 °C), concentration of co-solvent (ethanol 19.7%) by CO2 flow rate of 15 g/min for 60 min, at a solvent/feed ratio of 30 was better when compared to that obtained by solid-liquid extraction (Soxhlet) using ethanol as extraction solvent, but similar when compared to the water extracts. In contrast, the capacity for preventing lipid peroxidation, measured through the β-carotene bleaching method, was better in water extract [58]. According to these authors this extract had the lowest oxidation rate (0.661) and the highest activity coefficient (809) in comparison to supercritical CO2 (0.958 and 492, respectively) and ethanol extracts (1. 101 and 330, respectively).
The antioxidant capacity of fruit extracts reported by [59] was higher when obtained from fully red fruits, except H2O2 scavenging activity which was higher in green fruit. The concentrations of red and yellow fruit extracts providing 50% antioxidant activity (EC50) were 0.409 and 0.499 mg/mL, respectively, in the DPPH (2,2-diphenyl-1-picrylhydrazyl) method. The EC50 values found when the β-carotene bleaching method was used were 0.246 and 0.328 mg/mL in red and yellow fruit extracts, respectively. The H2O2 scavenging capacity was low (27.10% and 25.86% for green and red fruit extracts, respectively). The influence of maturation stage on antioxidant activity measured through the DPPH method was also found by [29]. Intermediate (EC50 = 0.37 mg/mL) and ripe fruits (EC50 = 0.25 mg/mL) of strawberry tree fruits possessed higher antioxidant activity than unripe fruits (EC50 = 0.58 mg/mL). Storage of fresh fruits of A. unedo at 0 °C preserved better the antioxidant activity than at higher temperatures (3 and 6 °C) during the experimental period (15 days) [61], evaluated by the capacity for scavenging peroxyl radicals [Oxygen Radical Absorbance Capacity-ORAC method] and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) cation radicals [Trolox Equivalent Antioxidant Capacity-TEAC method]. In the same experiment, the authors also reported that the type of films covering the fruits (linear low density polyethylene or polyethylene film perforated with holes) did not influence the antioxidant activity of fresh fruits [61].
The antioxidant activity was influenced by the drying fruit processes. Some authors [60] reported that fruits submitted to freeze drying had higher capacity for scavenging DPPH free radicals (EC50 = 2.125 mg/mL) and preventing lipid peroxidation (EC50 = 0.290 mg/mL) than those submitted to hot air drying (EC50 = 6.956 and 0.880 mg/mL, respectively). This loss when using hot air drying would be expectable due to the possible degradation and/or loss of some compounds such as phenols and vitamins [60,63].
Beyond the antioxidant activity of fruit extract of A. unedo found by [62] measured through the capacity of the extract with phenolic acids, flavones/ols, flavan-3-ols and galloyl derivatives to inhibit peroxidation in animal brain homogenates, the same extract had also antitumor potential against NCI-H460 human cell line (non-small lung cancer). The authors obtained two main types of extracts from Arbutus unedo, Prunus spinosa, Rosa micrantha and Rosa canina fruits collected in Portugal: non-anthocyanin phenolic compounds enriched extract and anthocyanins enriched extract. Non-anthocyanin phenolic compounds enriched extract of A. unedo had the highest capacity to inhibit lipid peroxidation in animal brain homogenates (EC50 = 7.21 μg/mL) as well as a high antitumor potential against NCI-H460 human cell line. The authors considered that such activities might be attributed to the presence of galloyl derivatives exclusively found in this species [62].
The antioxidant ability of strawberry tree fruits has led to the application of their phenolic-rich extracts as functional ingredients in processed meat products [33,[64][65][66]. For example, the combination of fruits extracts together with sodium ascorbate and sodium nitrite enhance the oxidative stability of frankfurters without modifying their colour and texture properties. The utilization of fruit extracts also minimized the deterioration of quality during the refrigerated storage [64]. The addition of extracts from A. unedo fruits in porcine burger patties protected polyunsaturated fatty acids from oxidative degradation, inhibited the formation of thiobarbituric acid reactive substances and volatiles compounds; and the oxidation of proteins preventing the formation of carbonyl compounds [33,65,66]. The utilization of these extracts as antioxidants enhanced the nutritional, safety and sensory properties of porcine burger patties [33,65,66].
Other applications of fruit extracts in food technology have been reported, such as the enrichment of yogurts with fruit extract of A. unedo which improved the antioxidant activity and the survival of its microbial community, not affecting the chemical and microbiological characteristics of yogurts [67]. Fruit essential oil of A. unedo showed a moderate antibacterial activity against Listeria monocitogenes and Enterococcus faecalis [43].
The collection zone and genotype also influenced the antioxidant properties of A. unedo extracts of leaves [30,69]. Samples collected in Greece showed significantly higher spasmolytic activity (IC50 = 1.66 mg/mL) and DPPH free radical scavenging capacity (IC50 = 4.77 μg/mL) than the samples collected in Montenegro (IC50 = 5.26 mg/mL and 7.14 μg/mL, respectively). The inhibition of lipid peroxidation in liposomes induced by Fe 2+ /ascorbate and determined through thiobarbituric test activity was also higher in sample extracts from Greece, particularly when phenol content was lower [30]. The reducing power and the capacity of scavenging DPPH free radicals were evaluated in 19 genotypes of A. unedo collected in the Trás-os-Montes region of Portugal [69]. In reducing power and DPPH methods, the strongest (EC50 = 0.234 mg/mL and 0.089 mg/mL, respectively) and the weakest activities (EC50 = 0.378 mg/mL and 0.142 mg/mL, respectively) were found in samples collected in Vila Verde and Vila Boa 2, respectively [69].
The human erythrocyte as a cell-based model system was used to elucidate the antioxidant activity of A. unedo extracts [27]. In such model, the peroxidative injury in human erythrocytes is induced by AAPH [2,2'-azobis(2-amidinopropane) dihydrochloride]. The results obtained by the authors showed that leaf extracts were better to prevent peroxidation than fruit extracts. The IC50 values found for leaf and fruit extracts were 0.062 and 0.430 mg of extract/mL, respectively, after 3 h of incubation, but significantly lower than that obtained with ascorbic acid, used as standard (0.031 mg/mL).
According to some authors [23], leaf extracts of A. unedo may be used for the treatment and/or prevention of cardiovascular diseases because their studies demonstrated that such samples reduced store-operated Ca 2+ entry induced by thrombin or by selective depletion of the two Ca 2+ stores in platelets, the dense tubular system and the acidic stores. The same extracts were also able to reduce both basal and thrombin-stimulated protein tyrosine phosphorylation. Therefore, the extracts have in vitro anti-aggregant effects in human platelets. An anti-aggregant effect was also reported by [76] but in rat platelets. The authors reported that tannins isolated from the methanol extract presented a strong anti-platelet effect and therefore responsible for such action.
Ethanol extracts of A. unedo leaves collected in Greece and Montenegro possessed spasmolytic activities in rat ileum, probably mediated via the inhibition of Ca channels. The authors attributed this action to the relative high contents of phenols, tannins, arbutin and flavonoids [30]. Extracts rich in oligomeric condensed tannins and catechin gallates possessed an endothelium-dependent, vasorelaxant activity. Pre-contracted aortic rings, previously removed from male Wistar rats, with noradrenaline, relaxed strongly in the presence of A. unedo extracts [77].

Roots
The aqueous extract of roots had antibacterial activity on Escherichia coli comparable to that of piperacilline (diamter of inhibition zone = 30 mm in both cases) [54]. Moderate antibacterial activity was reported by [78] for water extract and phenolic fractions of A. unedo collected in Tlemcen (Algeria) against E. coli (MIC = 200 μg/mL) and Staphylococcus aureus (MIC = 200 μg/mL for a fraction obtained by column chromatography after elution with 50% methanol/water (v/v) containing 0.1% (v/v) acetic acid. Such activity could be attributed to the secondary metabolites detected in samples such as quinones, anthraquinones, flavonoids, tannins and anthocyanins [78].
The biological properties of root extracts from diverse locations are largely referred. Hypoglycemiant effect of aqueous extracts of A. unedo from Morocco in neonatal streptozotocin-induced diabetic rats under chronic treatment [79]. Chronic treatment with aqueous extracts of A. unedo roots reduced hypertension development; prevented the myocardial hypertrophy; ameliorated vascular reactivity and renal functional parameters caused by L-NG-Nitroarginine methyl ester (L-NAME) of male Wistar rats [80]. The extracts also improved the sensitivity of the arterial baroreceptor controlling the heart rate to acute increases of arterial pressure [80].

Spirit Beverages
The spirit beverage that comes from the distillation of fermented fruits of A. unedo has also been target of chemical surveys. In Portugal, this distilled beverage is known as "aguardente de medronho" [92]; in Italy as "Corbezzolo" [93], and in Greece as "Koumaro" [18]. In Portugal, two denominations of origin distillates are already recognized: Medronho do Algarve e Medronho do Buçaco [94].
In Portugal, "aguardente de medronho" has as main constituents methanol and ethyl acetate, nevertheless the levels of methanol never exceeded the concentrations prescribed by law [94]. Alcohols, esters, acids and aromatic compounds constituted the main group of compounds in "koumaro" [18]. Methanol, ethyl acetate, isovaleric acid and trans-anethole were the main components in every group, respectively [18]. In all samples studied by the authors, two of them had concentrations of methanol higher than those permitted by law (1000 d/hL absolute alcohol) [94]. Calcium was the main mineral in the same samples [18].
Methanol and ethyl acetate also predominated in distilled alcoholic beverages obtained from solid-state fermentation of strawberry tree fruits from Spain [95]. In the same work, the authors concluded that the addition of the ethanol-producing yeast (Saccharomyces cerevisiae IFI83) led to a more efficient utilization of the reducing sugars for ethanol production than did the indigenous microbiota of the fruits in the spontaneous fermentations [96].
Sardinian strawberry tree distillate is characterised by a lower content of C7 to C9 primary alcohols and in general by a higher content of C6 to C10 ethyl esters and C12 to C18 fatty esters when compared to those from Greece and Portugal [18,94,95].
The quality of distilled product may be affected by off-flavours when uncontrolled fermentations occur, therefore the nature of yeasts present in musts contribute to the sensory characteristics [93]. These authors studied the diversity of the yeast population and the killer activity of S. cerevisiae isolates obtained during the fermentation period of arbutus fruits from Portugal. They found a diversity of autochthonous yeasts that fermented arbutus fruits at room temperature through a solid state process. As fermentation progressed, a microbial succession is observed, with the final prevalence of S. cerevisiae [93].
New aromatic pomegranate liquor obtained by maceration of pomegranate juice and arils from Portuguese origins ('Assaria') in A. unedo distillate was performed by [97]. The authors found that anthocyanins of pomegranate juice undergo degradation during the maceration, particularly the monoglucosides, while ellagitannin compounds remained stable [97]. Most strawberry tree fruit spirit volatiles (ethanal, ethyl acetate and 1-hexanol) were also detected in the liquor but in lower amounts than in the spirit and the volatiles of pomegranate origin (limonene, 1-hexenol and trans-caryophyllene) also had little contribution to the volatile profile of the liquor [97].

Conclusions
From ancient times, leaves and fruits of A. unedo L. have been used in folk medicine. Fruits have also been used for making brandies, jams, jellies and marmalades, and less frequently eaten as fresh fruit. The chemical composition of fruits and leaves, in which phenolic acids, flavonoids, tannins, anthocyanins, vitamins are present, may be responsible for the reported biological properties. More recently, the antioxidant activity of fruit extracts has also been used in meat industry to preserve the quality and prevent oxidation of lipids and proteins. Arbutus honey has also been chemically characterized and specific floral origin markers have been tentatively found. The biological properties of honey, particularly the antioxidant ability, have been attributed to their phenols and even to homogentisic acid, one of the possible chemical markers. The chemical characterization of distilled beverage obtained from fruits of A. unedo has been established and regulated. Studies regarding the increased shelf life periods of fruits started recently. All of these approaches have as main goal to increase the utilization of fruits and/or derivatives in human nutrition due to their beneficial properties to human health.