(Berg) Burret, commonly known as feijoa, is an evergreen shrub, belonging to the Myrtaceae family with slow growth, originating from the highlands of South America. The most known varieties (apollo, coolidge, gemini, mammoth, moore, and triumph) differ in the time of maturation, size, and quality of the fruits. Feijoa fruits having a length of 4–8 cm, are oval, with a bright waxy green skin and a light, dense, granular, honey-colored, sweet and juicy pulp, with translucent seeds. It possess a peculiar floral aroma and a sweet-acidulous taste [1
The peel flavor is much more resinous than the pulp, so that it is usually discarded. However, in some recipes, such as different types of chutney, it is often used because of its pleasant floral scent. The feijoa aroma is derived largely from volatile esters methyl benzoate, ethyl benzoate, and ethyl butanoate. Although, these esters have been found in other fruits, a unique feature of the feijoa aroma is the high percentage of methyl benzoate, which confer to it the peculiar aroma [2
The fruit is also characterized, like apples, by a very high amount of pectin.
It is common consumed fresh or as a juice, but it is also used by the food industry for the production of several confectionery products, such as sorbet, ice cream, and so on [3
The fruit contains volatile aromatic compounds, such as terpenes, tannins, steroidal saponins, and polyphenols. Furthermore, it is a good source of vitamin C, iodine, calcium, magnesium, and dietary fibres. Feijoa exerts digestive and laxative properties and, given its low calorie content, it is considered a valid diet adjuvant [2
In Brazil and Uruguay, apart from some small crops, the fruits are not cultivated on a commercial scale, although some are harvested from wild or domestic plants. The commercial development of the feijoa occurred mainly outside these countries, starting from Europe [2
], because of Edouard Andrè, a well-known French botanist and horticulturist, who imported the plant as ornamental fruit tree, common in domestic gardens and in warm-temperate regions. In this way, the seeds and plant have been widely distributed, leading to its cultivation and selection of crops in France, Israel, Italy, Russia, California, and New Zealand. The latter holds the greatest production.
In the past, greater importance was attributed to the medicinal properties of the plant than to the fresh fruit. In traditional medicine, the infusion of the plant leaves were used to treat dysentery and cholera, especially in children. Still today, in some countries, homeopathic pharmacies sell feijoa tea for this purpose. However, the most powerful infusion can be obtained from the feijoa fruit-dried peel [2
Although the feijoa fruit has not been extensively investigated, several studies are already available, in particular, on the fruit extract or its juice, which report several interesting health effects, such as antibacterial, antifungal, analgesic, anti-inflammatory, anti-ulcer, anti-tumor, and osteoblastic activity [4
]. Although, the polyphenol extracts of the feijoa fruit are the most investigated, Piscopo et al. [14
] reported an interesting antioxidant and antimicrobial activity of a protein fraction from the feijoa fruit against several Gram-positive and Gram-negative standards, as well as clinically isolated bacterial strains. Moreover, these activities increased 10 fold, and 2–4 fold, respectively, after in vitro gastrointestinal digestion [14
However, to date, no data are available about the biological properties of feijoa fruit peel essential oils (EO).
EOs are complex mixtures of plant secondary metabolites, with well-known antioxidants, cyto-protective, anti-inflammatory, allopathic, and antimicrobial properties [15
In particular, EO antimicrobial activity has been validated by several studies, which often open new research perspectives. Among them, the quantitative activity-composition relationships classification models, based on machine learning algorithms, have been recently developed, in order to investigate the chemical components of the EOs that are mainly involved in the inhibition of Pseudomonas aeruginosa
and Staphylococcus spp.
biofilm production [17
It has been observed, in fact, that often, the antibacterial activity of EOs is not directly related to their bacteriostatic/bactericidal activity, but to the phenotypic change in the bacterial strains [17
In light of this, these screening tools could be very useful in selecting the most effective EOs for preventing infections, especially into healthcare field [17
The aim of the present study was intended to first characterize the micromorphological features of the feijoa fruit peel, by optical and scanning electron microscopy (SEM). Subsequently, the phytochemical profile of the EO, isolated from the fresh fruit peel (Acca sellowiana (Berg) Burret var. coolidge) was characterized by GC-FID and GC-MS analysis. Moreover, different biological properties, such as antioxidant, cytoprotective, and antimicrobial properties, were evaluated by several in-vitro cell-free and cell-based assays.
2. Materials and Methods
Commercially available terpene analytical standards were purchased from Extrasynthese (Lyon, France). Dichloromethane was GC-grade, and was purchased from Merck (Darmstadt, Germany). C7-C40 n-alkanes standard mix solution, as well as other analytical grade chemicals and solvents were purchased from Sigma-Aldrich (Milan, Italy).
2.2. Plant Material and Isolation of Essential Oil
Feijoa (Acca sellowiana (Berg) Burret var. coolidge) fruits were harvested on 4 December 2018 by a local farmer in Lamezia Terme (Catanzaro, Italy), and immediately sent to the laboratory. The fresh fruits peel (250 g) was manually peeled off and the EO isolated by hydrodistillation, according to the current European Pharmacopoeia method. The EO was dried on Na2SO4 and stored in a dark-sealed vial with nitrogen headspace until analysis. For chemical characterization, EO was diluted in dichloromethane (10%, v/v), whereas for the evaluation of its biological activities, a stock solution (100 mg/mL) in DMSO was properly diluted in methanol.
2.3. Micromorphological Evaluation
For light microscopy (LM), small pieces of fruit peel were excised from a zone of maximum diameter, and hand cut by a razor blade. Cross sections were processed with 1% phloroglucinol in 20% hydrochloric acid for lignin staining. For scanning electron microscopy (SEM), small pieces of fruit peel (1–1.5 cm2
) were fixed in FineFIX working solution (Milestone s.r.l., Bergamo, Italy) with 70% ethanol, and left for 24 h at 4 °C [19
Subsequently, the tissue samples were dehydrated by increasing the concentrations of ethanol, followed by a critical point drying in carbon dioxide, using a CPD processor (K850 2M Strumenti S.r.l., Rome, Italy). Dried specimens were then mounted on aluminum stubs and sputter-coated with gold. Finally, specimens were observed by a Scanning Electron Microscope (SEM) Vega3 Tescan LMU (Tescan USA Inc., Warrendale, PA, USA) at an accelerating voltage of 20 kV.
2.4. GC-FID and GC-MS Analysis
Gas chromatographic (GC) analysis was performed on an Agilent gas chromatograph 7890A, equipped with a flame ionization detector (FID) and a mass spectrophotometer (MS) 5975C (Agilent Technologies Santa Clara, CA, USA). The elution was carried out by a HP-5MS capillary column (30 mm, 0.25 mm coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 μm film thickness) by using helium as carrier gas (1 mL/min) according to the method reported by Smeriglio et al. [16
]. The injection of EO (1 μL, 10% CH2
) was done in split mode (50:1) setting the injector and detector temperature at 250 °C, and 280 °C, respectively, for GC-FID analysis, and 250 °C, and 180 °C, respectively for GC-MS analysis. In the latter case, the ionization voltage was set at 70 eV, the electron multiplier at 900 V, and the ion source and transfer line temperature at 230 °C, and 260 °C, respectively. Mass spectra data were acquired in scan mode in the m/z
range 45-450 amu. The compounds were identified based on their Kovats retention index (KI), relative to a standard mixture of n-alkanes, the values reported in the literature [15
], and matching the mass spectra data with those of the MS library (NIST 08). Moreover, a comparison of MS fragmentation patterns with those reported in literature, and, whenever possible, co-injection with commercial available standards (purity ≥ 99%) were carried out. The percentages of compounds were determined from their peak areas in the GC-FID profiles.
2.5. Antioxidant and Free-Radical Scavenging Activity
The antioxidant and free-radical scavenging activity of feijoa EO was evaluated by several in vitro assays, based on different mechanisms and reaction environments. The results were expressed as an inhibition percentage (%) of the oxidative/radical activity, calculating the half-maximal inhibitory concentration (IC50) with the respective confident limits at 95%.
2.5.1. Total Phenolic Compounds
The total phenols content was evaluated according to Smeriglio et al [20
]. Briefly, 50 μL of EO solution (1.25–10 μg/mL) was added to 500 μL of Folin-Ciocalteu reagent and 450 μL of deionized water. After incubation for 3 min, 500 μL of sodium carbonate (10% w/v
) was added. The samples were left in the dark at room temperature (RT) for 1 h, vortexing every 10 min, and the absorbance was recorded at 785 nm, using an UV-Vis spectrophotometer (Shimadzu UV-1601, Kyoto, Japan).
2.5.2. DPPH Assay
The free radical scavenging activity of EO against DPPH radical was evaluated, according to Smeriglio et al. [21
]. Freshly DPPH methanol solution (10−4
M), was mixed with 37.5 µL of EO solution (range 12.5–100 µg/mL) vortexing for 10 s. The decrease in absorbance at 517 nm, against a blank, was measured after 20 min by UV-Vis Spectrophotometer (Shimadzu UV-1601, Kyoto, Japan).
2.5.3. Trolox Equivalent Antioxidant Capacity (TEAC) Assay
The free-radical scavenging activity against ABTS radical was carried out, according to Smeriglio et al. [15
]. The reagent solution (4.3 mM potassium sulfate and 1.7 mM ABTS Solution 1:5 v/v
) was incubated, in the dark at RT, for at least 12 hours and used within 16 h, by dilution with phosphate buffer (pH 7.4) in order to reach an absorbance of 0.7 ± 0.02 at 734 nm. Fifty microliters of EO solution (1–8 µg/mL) was added into 1 mL of the reagent solution and incubated in the dark at RT for 6 min. An UV-VIS Spectrophotometer (Shimadzu UV-1601, Kyoto, Japan) recorded the absorbance at 734 nm.
2.5.4. Ferric Reducing Antioxidant Power (FRAP) Assay
The assay was carried out according to Smeriglio et al. [20
]. Freshly working FRAP reagent was warmed at 37 °C. Then, 50 µL of EO solution (1–8 µg/mL) were added into 1.5 mL of reagent, and the absorbance was measured after 4 min at 593 nm, by UV-VIS Spectrophotometer (Shimadzu UV-1601, Kyoto, Japan).
2.5.5. Oxygen Radical Absorbance Capacity (ORAC) Assay
The free radical scavenging activity against AAPH radical was tested, according to Bellocco et al. [22
]. Briefly, 20 µL of EO solution (2–16 µg/mL) were dissolved in 75 mM phosphate buffer (pH 7.4) and pre-incubated for 15 min at 37 °C with 120 µL of fresh daily fluorescein solution (117 nM). After that, 60 µL of fresh daily 40 mM AAPH solution was added monitoring the reaction every 30 s for 90 min (λex
: 485; λem
: 520) by a fluorescence plate reader (Fluostar Omega, BMG Labtech, Ortenberg, Germany).
2.5.6. β-Carotene Bleaching Assay
β-Carotene bleaching assay was carried out according to Smeriglio et al. [23
] by mixing fresh daily β-carotene emulsion with EO solution (5–40 μg/mL). A blank emulsion (without β-carotene) was used as negative control. The absorbance was recorded at the starting time (T = 0) at 470 nm and then monitored every 20 min, incubating the sample solution at 50°C in a water bath. The reaction was stopped at 120 min. Butylated hydroxytoluene (BHT) 1 mg/mL was used as a reference compound. The results were expressed as kinetic curves, and showed the capacity for Feijoa EO to counteract the heat-induced β-carotene bleaching.
2.5.7. Iron-Chelating Activity
The iron-chelating activity of EO was evaluated, according to Smeriglio et al. [20
]. Briefly, 50 μL of FeCl2
O solution (1.8 mM) was added to 100 μL of EO solution (1–8 μg/mL) and incubated at RT for 5 min. After that, 100 μL of ferrozine solution (4 mM) was added to the reaction mixture, that was diluted to 3 mL with deionized water, mixed and incubated for 10 min at RT. The absorbance was read at 562 nm, using a UV-VIS spectrophotometer (Shimadzu UV-1601, Kyoto, Japan). The results were expressed as inhibition (%) of the of Fe2+
chelating capacity, by calculating the half-maximal inhibitory concentration (IC50
), with the respective confident limits at 95%.
2.6. Cell-based Assays
2.6.1. Lymphocyte Isolation
The isolation of lymphocytes was performed according to Barreca et al. [24
], with few changes. Heparinized whole blood from healthy volunteers (who have provided their written medical histories by a standardized questionnaire) has been a starting point for the lymphocytes isolation. The samples were diluted with equal volumes of PBS, layered over Histopaque-1077 in centrifuge tubes, and underwent centrifugation at 400 g for 30–40 min at 25 °C. The obtained cells was collected with a pipette, washed by centrifugation, and diluted with PBS. The peripheral blood mononuclear cells (PBMCs) were separated through a Percoll gradient, according to Repnik et al. [25
]. They were then counted with a haemocytometer and suspended in Roswell Park Memorial Institute (RPMI) 1640 medium, added with 2 mM glutamine, 10% fetal calf serum, 100 units/ml streptomycin and penicillin G.
2.6.2. Cytotoxicity and Cytoprotective Assays
The cytotoxicity assay was performed on cells (1 × 105 /mL) incubated in complete medium with, or without, 150, 125, 100, 75, 50, 25, 10, 5, 2.5 and 1.25 μg/mL of feijoia EO for 24 h. The cyto-protective assay were performed on cells (1 × 105 /mL) incubated in complete medium with, or without, 40, 20, 10, 5, 2.5, and 1.25 μg/mL of feijoia EO for 24 h, in the presence of 100 μM tert-butyl hydroperoxide (t-BOOH). The stock solution of feijoia EO was conveniently diluted with PBS to obtain, in the final reaction mixture, a concentration of the solvent below 0.1%.
Trypan blue staining was utilized to test the viability of the completed incubation time, utilizing a haemocytometer and diluting an aliquot of the cell suspension (1:1, v/v) with 0.4% trypan blue.
Cytotoxicity has been also tested by lactate dehydrogenase (LDH) release into a culture medium from damaged cells. A commercially available kit from BioSystems S.A was utilized to analyze LDH activity in the medium, where the total amounts of the enzyme are present in the cells, after lysis by sonication [22
]. Feijoia EO, at the final concentrations used, did not show any interference with the performed tests.
2.6.3. Red Blood Isolation
The isolation of erythrocytes was performed according to Barreca et al. [27
] with few changes. Heparinized whole blood from healthy volunteers (who have provided written medical histories by a standardized questionnaire) has been the starting point for the red blood cells isolation. Erythrocytes were separated by centrifugation (2500 rpm for 5 min) from the plasma and buffy coat, then washed three times with 10 volumes of 0.9% NaCl. Finally, the packed cells were diluted in 10 volumes of PBS and utilized for the following experiments.
2.6.4. Assay for Erythrocyte Hemolysis
The erythrocyte hemolysis assay was carried out according to Barreca et al. [27
]. Different amount of feijoia EO (150, 125, 100, 75, 50, 25, 10, 5, 2.5 and 1.25 μg/mL) were joined in a final volume of 1.0 mL, with erythrocytes (10% in PBS, pH = 7.4). The reaction mixes were incubated for 2 hours at 37 °C in a water bath, diluted with PBS (1:9, v/v
), and centrifuged. The complete haemolysis in the reference sample was achieved by adding 8 volumes of distilled water centrifuging. Another sample without additives was diluted with the same volume of PBS, rather than distilled water, in order to eliminate any interference of spontaneous haemolysis. The absorbance of supernatant, after centrifugation, was analyzed at 540 nm and expressed as a percentage of complete hemolysis.
2.6.5. Quantification of Intracellular Reactive Oxygen Species (ROS)
The ROS quantification was carried out according to Peter et al. [28
]. The compound 2’,7’-dichlorodihydrofluorescein diacetate has been utilized as a dye, in order to detect the amount of intracellular ROS. The cells were incubated with t-BOOH (10 mM) for 2 h in presence/absence of different concentration of feijoa EO (40, 20, 10, 5, 2.5 and 1.25 μg/ml final concentration). The EO was added to the culture medium 30 min before the t-BOOH treatment. Then, the cells were washed twice with PBS (pH = 7.4) and incubated with 2’,7’-dichlorodihydrofluorescein diacetate for 30 min at 37 °C. The incubation time was completed and the fluorescence data were acquired at 525 nm following excitation at 488 nm. The ROS formation has been expressed as the maximum amount of radicals obtained in the samples treated with t-BOOH.
2.7. Antimicrobial Activity
The following strains were obtained from an in-house culture collection (University of Messina, Messina, Italy): Staphylococcus aureus
ATCC 6538P, S. aureus
ATCC 43300, three clinical strains of S. aureus
obtained from the pharynges (strains 526, 530, 808), two clinical strains of S. aureus
obtained from duodenal ulcers (strains 8, 14), two clinical strains of S. aureus
obtained from hip prostheses (strains 6, 84); Staphylococcus epidermidis
ATCC 35984; Pseudomonas aeruginosa
ATCC 9027; Escherichia coli
ATCC 10536; Candida albicans
ATCC 10531, three clinical strains of Candida albicans
, two clinical strains of Candida glabrata
, two clinical strains of Candida parapsilosis
. S. aureus
strains, were recently characterized in terms of lipid profiles, and their correlation with antibiotic resistance and hydrophobicity [29
]. Candida sp.
clinical strains were identified by species-specific PCR-based methods [30
All bacterial strains were cultured in Muller Hinton Broth (MHB, Oxoid, CM0405) at 37 °C (24 h). Candida strains were grown in RPMI 1640 (Sigma, Italy) at 30 °C for 24 h.
The minimum inhibitory concentrations (MIC), the minimum bactericidal concentrations (MBC), and the minimum fungicidal concentrations (MFC) of the extract, were determined using a broth microdilution, in accordance with the Clinical and Laboratory Standards Institute [31
]. MIC values were defined as the lowest feijoa EO concentrations showing no bacterial/fungal growth after incubation. The MBC and MFC were defined as the lowest feijoa EO concentration, which killed 99.9% after 24 h incubation at 37 °C, and 24–48 h incubation at 30 °C, respectively. All assays were performed in triplicate.
2.8. Statistical Analysis
The results were expressed as the average ± standard deviation (S.D.) of three independent experiments (n = 3). A one-way analysis of variance (ANOVA), followed by Bonferroni’s post-hoc comparisons tests, using a SigmaPlot 12.0 and GraphPad prism 5.0 software, were carried out. Statistical significance was considered at p < 0.05.
This is the first study which investigates the phytochemical profile, as well as the different biological activities of an EO, obtained from the fruit peel of feijoa var. coolidge. This tasty and functional fruit, less known than other exotic fruits, such as mango and papaya, has recently become more popular in the Italian market. In particular, the tested EO was obtained from fruits harvested in Lametino area (North of Calabria, Italy).
GC-FID and GC-MS analysis lead to the identification of forty compounds, mainly belonged to sesquiterpenes, monoterpene hydrocarbons, and oxygenated monoterpenes. The most abundant compounds were γ-Selinene (17.39%), α-Caryophillene (16.74%), β-Caryophillene (10.37%), and Germacrene D (5.32%).
Feijoa EO showed a strong and dose-dependent antioxidant activity, which was corroborated by the cytoprotective results, that were observed on lymphocytes pre-treated with t-BOOH, as well as on erythrocytes. These markedly decreased the t-BOOH-induced alterations of the redox status. The antimicrobial screening showed that feijoa EO possesses a selective antibacterial and anti-fungal activity towards the Gram-positive bacteria S. aureus, and S. epidermidis, as well as the fungi C. albicans, respectively.
These promising biological activities could not be ascribed exclusively to its major components. It has been demonstrated, indeed, that often EOs show a much stronger biological activity than the mainly present compounds, due to their synergic and antagonistic effects, which characterize the plant complexes [21
In conclusion, the feijoa EO is an important source of natural antioxidants, with cytoprotective and antimicrobial properties, which are useful in various food, nutraceutical, and pharmaceutical fields.
In light of this, further investigations are need to validate the potential use of this EO alone, or in combination with synthetic drugs, as topical agents against both bacterial and fungal skin infections.