Chemical Profiles and Antimicrobial Properties of Essential Oils from Orange, Pummelo, and Tangelo Cultivated in Greece

Abstract

In the framework of our studies on Citrus cultivars in Greece, the chemical composition of the essential oils (EOs) from the peels and leaves of orange, pummelo, and tangelo (mandarin × grapefruit hybrid) cultivated in Greece have been studied. All EOs have been analyzed through GC-MS, and a total of 47 and 87 metabolites were identified in the peels and leaves, respectively. These metabolites are classified into the chemical groups of terpenes, alcohols, aldehydes, esters, ketones, and organic acids. Limonene was the most abundant compound in the peel EOs. Moreover, bioactive polymethoxyflavones (PMFs) were isolated and structurally determined from the peels of orange and tangelo, highlighting them as a good potential source of natural PMFs. All EOs were evaluated for their antimicrobial activity against nine human pathogenic microorganisms (six bacteria and three fungi), showing an interesting profile. The EOs from the peels of all Citrus species exhibited a stronger antimicrobial activity compared to those from the leaves. The susceptibility of the assayed Gram-positive bacteria was observed to be greater than that of Gram-negative bacteria, while the fungi were also relatively less resistant than bacteria. The rootstock choice did not influence the EO profile of the fruit peel but exerted an influence on the chemical profile of the leaves.

1. Introduction

Citrus sp. (Rutaceae) is one of the most important horticultural crops, the fresh fruit and juice of which are well appreciated for their unique flavor and nutritional value. Citrus fruits are among the most abundant fruits in the world, with an annual production that exceeds 140 million tons (metric) [1]. The cultivation of citrus in Greece has had a long history since the plants were imported into the country in the era of Alexander the Great, and the microclimate and soil favor the growth of all citrus species (orange, mandarin, lemon, citron, bergamot, kumquat, pummelo, grapefruit, lime) [2]. Greece is the third EU country in citrus production after Spain and Italy, with an annual production of 1,165,000 tons, cultivated in an area of more than 415,000 ha and 19,000,000 trees [3]. The cultivation of Citrus is a pillar of the Greek agricultural economy since it is among the three most profitable tree crops, along with olive and stone fruits, contributing to the cultural and economic life of the country. Throughout Greece, the most cultivated Citrus species are oranges, mandarins, and lemons, covering more than 97% of the total cultivated area (Crete Island, Peloponnese peninsula in South Greece, Aitoloakarnania Prefecture, Arta area Central West, and Northwest Greece) while grapefruit, citron, bergamot, and lime cultivation is very limited (3%).

Because the main large-scale consumption of these species concerns the flesh or the extracted juice, large quantities of peels, pulps, and seeds are discarded by citrus processing plants [4]. These citrus by-products account for 45–60% of the total unprocessed fruit and in most cases are rejected without reuse in the environment [5]. This latter action can lead to severe environmental pollution since these wastes can raise soil acidity, have an impact on water quality, and have a toxic effect on fauna and flora due to excess levels of EOs [6]. Nevertheless, these citrus by-products or even other non-edible parts of the citrus tree (leaves and flowers) are valuable matrices of macro- and micro-nutrient compounds (proteins, sugars, minerals, fibers, vitamins) and phytochemicals (carotenoids, EOs, phenolic compounds, etc.) that have been related to human health benefits like antioxidant activity, anti-inflammatory properties, the prevention of cardiovascular diseases, and a reduction in cancer risk [7].

Citrus EOs from fruits and peels, due to their pleasant smell and aroma, have obtained increasing attention owing to consumers’ demands for non-synthetic and more natural food additives [8] and have been industrially employed in many products, including foods (dairy desserts, candies, bakery, etc.) and beverages (alcoholic liqueurs and non-alcoholic ones), cosmetic perfumes [9], and as excipients to mask the unpleasant tastes of drugs and herbal medicine formulations [10].

Citrus EOs have been classified as “Generally Recognized as Safe”, and they have also shown a wide spectrum of biological activities, such as antimicrobial, antioxidative, and anti-inflammatory properties [10]. Due to their high commercial, nutraceutical, and economic importance, numerous studies have been conducted regarding the chemical composition of peels, leaves, and flowers from different Citrus species [11,12,13]. Limonene, β-citronellal, β-citronellol, and linalool are among the most abundant volatile compounds in Citrus sp. [10], while α- and β-pinene, nootkatone, sabinene, germacrene-D, and β-myrcene are other components detected in various citrus cultivars [14]. It is estimated that more than 300 volatile metabolites have been identified in citrus EOs [14]. These metabolites are classified in the chemical groups of terpenes, aldehydes, alcohols, esters, and polymethoxyflavones (PMFs), a group of bioactive flavonoids that exist almost exclusively in Citrus sp. peels [C. sinensis (L.) Osb., C. reticulata Blanco] [15]. PMFs are of particular interest because many of them have a broad spectrum of biological properties, such as anti-inflammatory, antimicrobial, antioxidative, and anti-atherogenic ones [16,17]. Moreover, metabolite profiles of Citrus species can be utilized for the taxonomic classification of the genus and can act complementarily to facilitate existing taxonomic evidence, especially for the identification of hybrid citrus species [18]. Recently, the biological activities of citrus EOs against a wide range of pathogenic microbes have drawn the attention of many scientific groups [19]. Elucidating the chemical profile of natural EOs is a feasible approach by which these valuable natural by-products can be utilized to develop novel antimicrobial agents to overcome the natural obstacle of antimicrobial drug resistance [20].

In the framework of our studies on the chemical profile and bioactivity of the EOs from Citrus species grown in Greece [2,11,21], an in-depth analysis of EOs obtained via cold-pressed peel and leaf hydrodistillation of three Citrus cultivars (orange, pummelo, tangelo) cultivated in the National germplasm bank of the Arboricultural Station of Poros (South Greece) is reported herein. To the best of our knowledge, all studied plant materials (peels and leaves) of all three Citrus cultivars, grafted upon Poncirus trifoliata or citrumelo rootstocks, are studied phytochemically for the first time. There is also a lack of knowledge regarding the EO profile and antimicrobial activity from various tissues of Citrus species and varieties cultivated in Greek microclimates. Furthermore, the PMF contents in their peels have also been determined, and their antimicrobial activity has been evaluated. The scope of the current work was to evaluate and compare the phytochemical profile and antimicrobial activity of the EOs from three Citrus species cultivated in Greece in order to highlight those metabolites that dominate and possess antimicrobial efficacy against selected Gram-positive and Gram-negative bacteria and also to provide clues regarding the influence of rootstock choice upon the chemical composition of orange EOs, since the latter is the most cultivated citrus in Greece. All observed data could be further evaluated for the potential valorization of such horticultural crops.

2. Materials and Methods

2.1. Plant Material and Extraction of EOs

All fruits and leaves were selected from five (5) random 20-year-old trees grafted upon Poncirus trifioliata orange [Poncirus trifoliata (L.) Raf.] or Swingle citrumelo [Citrus paradisi MacFaden × Poncirus trifoliata (L.) Raf.] rootstock located at the Arboricultural Station of Poros (South Greece) during the 2019–2020 cultivation period. One group of citrus fruits, the orange [Citrus sinensis (L.) Osbeck] cv. Newhall and the Pummelo [Citrus maxima (Burm.) Merr.] cv. Cuban Shaddock, was harvested early in the season (November 2019), while the second group, the tangelo (Citrus paradisi × Citrus reticulata) cv. Minneola and the orange cv. Ovale Porou, was harvested late in the season (April 2020). All trees were healthy, were cultivated under the same pedoclimatic conditions, and received the same cultivation techniques. All fruits (Figure 1) from each cultivar were of similar size and shape. Fifty (50) fruits and leaves (ten fruits and leaves per tree of similar developmental stage) were sampled from the exterior and the interior of the canopy from all four directions. After harvest, the fruits and leaves were randomly divided among three groups for each replication. In the current work, due to the fact that oranges represent one of the most crucial cultivated crops in Greece, samples (fruits and leaves) from the cultivars Newhall and Valencia Oval Porou were collected from trees that were grafted upon two different rootstocks (trifoliata and citrumelo).

Plant materials (peels and leaves) of the selected Citrus cultivars are listed in

Table 1. Citrus cultivars from the National Germplasm Bank of the Arboricultural Station of Poros (South Greece) analyzed in this study.

Citrus Species	Citrus Cultivar/Rootstock	Code Name (Peels/Leaves)
Orange—Citrus sinensis (L.) Osbeck	Newhall/citrumelo	Csp1/Csl1
	Newhall/trifoliata	Csp2/Csl2
	Valencia Oval Porou/citrumelo	Csp3/Csl3
	Valencia Oval Porou/trifoliata	Csp4/Csl4
Pummelo—Citrus maxima (Burm.) Merr.	Cuban shaddock/trifoliata	Cgp1/Cgl1
Mandarin hybrid—Citrus paradisi × Citrus reticulata (Tangelo)	Minneola/trifoliata	Crp1/Crl1

. The fruits were cut into six portions, and the flesh was removed. The fruits’ albedo layer was peeled off carefully and discarded. The peel of fresh fruits was cold pressed, and the EO was separated from the crude extract by centrifugation (10 min at 15,000 rpm). Fresh leaves (approx. 600 g) were subjected to hydrodistillation for 3 h using a Clevenger-type apparatus, and the obtained EO was collected, dried over anhydrous sodium sulfate, and stored at 4–6 °C in dark glass amber vials until further analysis [21]. The yield of the EOs ranged between 0.50 and 1.50 mL/100 g, with a light orange to light yellow color and the characteristic pleasant citrus odor. All analyses were performed within a week of the EO extraction.

2.2. EO Fractionation

A portion of the EOs from the peels of orange Csp3 (30 mg) and tangelo Crp1 (15 mg) cultivars were subjected to silica gel preparative thin-layer chromatography and developed with toluene/ethyl acetate 70:30 (v/v), to yield the PMFs of 5,6,7,3′,4′-pentamethoxyflavone (sinensetin) (5 mg) [22], 5,6,7,8,3′,4′-hexamethoxyflavone (nobiletin) (4 mg) [23], one mixture of 5,6,7,4′-tetramethoxyflavone (tetra-O-methylscutellarein) and 3,5,6,7,3′,4′-hexamethoxyflavone (3-methoxy-sinensetin) (7 mg) [24], and a mixture of 3,5,6,7,8,3′,4′-heptamethoxyflavone (3-methoxy-nobiletin) and 5,6,7,8,4′-pentamethoxyflavone (tangeretin) (6 mg) [24].

2.3. Gas Chromatography–Mass Spectroscopy (GC-MS) Analysis

The analysis was conducted using an Agilent Technologies Gas Chromatograph 7820A paired with an Agilent Technologies 5977B mass spectrometer system (Agilent, Santa Clara, CA, USA) utilizing electron impact (EI) ionization at 70 eV. The gas chromatograph featured a split/splitless injector and a capillary column HP5MS measuring 30 m in length, with an internal diameter of 0.25 mm and a membrane thickness of 0.25 μm. The temperature program started at 60 °C for 5 min, followed by a ramp of 3 °C/min to 130 °C, then a ramp of 2 °C/min to 180 °C, and finally a ramp of 5 °C/min to a final temperature of 240 °C. The total analysis time was 65.33 min. Helium served as a carrier gas at a flow rate of 0.7 mL/min, with an injection volume of 2 μL, a split ratio of 1:10, and an injector temperature of 280 °C [11]. Compound identification was performed using the Wiley Registry of Mass Spectral Data and comparison with existing literature.

2.4. Nuclear Magnetic Resonance (NMR)

¹H-NMR spectra were obtained on a Bruker Avance III 400 MHz spectrometer (Bruker BioSpin, Rheinstetten, Germany) using CDCl₃ (D007K, Chembiotin S.A., Voula, Greece) as the solvent. Chemical shifts are presented in parts per million (ppm) relative to the solvent employed for recording the spectra [CDCl₃, 99.80%, δ_H 7.26 ppm].

2.5. Antimicrobial Activity

The antimicrobial activity was analyzed as described previously [11,25]. The EOs were dissolved in dimethyl sulfoxide (DMSO) using Muller–Hinton broth or RPMI with MOPS for fungal cultivation. The antimicrobial activity was tested against six strains of human pathogenic bacteria and three fungi using the micro-dilution broth method to determine the minimum inhibitory concentration (MIC) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The experiments were performed on the Gram-positive bacteria Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermis (ATCC 12228) and the Gram-negative bacteria Escherichia coli (ATCC 25922), Enterobacter cloacae (ATCC 13047), Klebsiella pneumoniae (ATCC13883), and Pseudomonas aeruginosa (ATCC 227853), as well as and three human pathogenic fungi, Candida albicans (ATCC 10231), Candida tropicalis (ATCC 13801), and Candida glabrata (ATCC 28838). Sterile 96-well polystyrene microtitrate plates were prepared by dispensing 100 μL per well of the appropriate dilution of the tested EOs in a broth medium using a two-fold dilution series. This resulted in final concentrations of the tested oils ranging from 0.50 to 10 mg/mL. Each EO was dissolved in DMSO to an initial concentration of 200 mg/mL. Inoculums prepared from fresh microbial cultures in sterile 0.85% NaCl adjusted to the turbidity of the 0.5 McFarland standard were added to the wells to achieve a final density of 1.5 × 10⁶ CFU/mL for bacteria and 5 × 10⁴ CFU/mL for yeasts (CFU: colony forming units). The plates have been inoculated at a temperature of 37 °C for 24 h for all bacteria. In the case of inoculated Candida, a similar approach was employed, but with the substitution of RPMI medium in place of MHB for approximately 48 h. After incubation, the MICs were determined as the lowest concentrations that prevented visible turbidity (visual growth) of the reference microbial strains. In addition to the used EOs, standard antibiotics such as netilmicin and amoxicillin (at concentrations of 4–88 μg/mL for bacteria), as well as 5-flucytosine and amphotericin B (at concentrations of 0.5–25 μg/mL for fungi), were tested (Sanofi Paris, Paris, France, Diagnostics Pasteur). For each experiment, a positive control was used to assess microbial growth (consisted of MHB without EO), and negative control (with MHB that was devoid of EO and microbes) was used, while a pure solvent solution was used as the blind control.

3. Results and Discussion

In this study, the peels and leaves of six Citrus cultivar/rootstock combinations (cv. New Hall or cv. Valencia Ovale Porou orange/trifoliata or citrumelo, Cuba Shaddock/trifoliata and tangelo cv. Minneola/trifoliata) were employed to obtain EOs. The peels were extracted through cold pressure, and the leaves through hydrodistillation. The chemical composition of the EOs (Table S1) from peels and leaves was identified by GC-MS analyses (Figures S1–S12). The peels of all studied samples showed qualitatively comparable chemical profiles, with limonene being the most abundant metabolite in all six of the citrus peels analyzed. However, the EOs obtained from the leaves showed different profiles and significantly lower levels of limonene compared with studied peels.

3.1. Chemical Composition of Eos from Citrus Peels

Forty-seven (47) components were identified in the cold-pressed peel EOs (94.01–99.77%) (

Table 2. Chemical composition and relative concentration (area %) of peel EOs from six Citrus cultivar/rootstock combinations.

	Area %
Compounds	Csp1	Csp2	Csp3	Csp4	Cgp1	Crp1
α-Thujene	-	-	-	-	-	0.27
2. α-Pinene	0.51	0.62	0.99	0.87	0.73	1.39
3. Sabinene	0.25	0.40	0.43	1.29	-	0.08
4. β-Pinene	-	-	0.45	1.71	0.51	-
5. Myrcene	2.06	1.88	2.20	0.54	2.51	1.54
6. α-Terpinene	-	-	-	-	-	0.11
7. δ-3-Carene	0.19	-	-	-	-	-
8. β-Terpinene	-	-	0.09	-	-	-
9. Limonene	87.69	95.61	89.10	89.82	88.93	79.86
10. γ-Terpinene	-	-	-	-	-	4.98
11. Terpinolene	-	-	-	-	-	0.31
12. Linalool	0.13	-	1.34	0.86	-	0.73
13. Valencene	1.36	1.26	0.03	0.32	-	1.73
14. trans-β-Ocimene	-	-	-	-	0.66	0.17
15. Terpinen-4-ol	-	-	0.07	-	-	0.32
16. α-Terpineol	-	-	0.11	0.47	-	-
17. Thymol methyl ether	-	-	-	-	-	0.08
18. Thymol	-	-	-	-	-	0.08
19. δ-Elemene	-	-	-	-	0.22	0.33
20. Neryl acetate	-	-	-	-	0.07	-
21. Decanal	-	-	0.16	-	-	-
22. Citronellyl acetate	-	-	-	-	-	0.06
23. α-Copaene	-	-	0.08	0.09	-	0.08
24. β-Elemene	-	-	-	-	-	0.17
25. trans-Caryophyllene	-	-	-	-	-	0.09
26. γ-Elemene	-	-	-	-	-	0.51
27. β-Cubebene	-	-	0.08	0.03	-	0.40
28. Calarene	-	-	-	0.07	-	-
29. β-Caryophyllene	-	-	0.04	-	0.55	-
30. α-Bergamotene	-	-	-	-	1.02	-
31. Germacrene-D	-	-	0.13	0.08	0.34	-
32. β-Bisabolene	-	-	-	-	1.23	-
33. Germacrene B	-	-	-	-	0.18	-
34. δ-Cadinene	-	-	0.10	0.14	-	0.25
35. Nootkatone	-	-	0.03	-	0.74	0.20
36. O-Methylated coumarin (osthole)	-	-	-	-	0.49	-
37. 7-Methoxy-8-(2-oxo-3-methylbutyl)coumarin	-	-	-	-	0.06	-
38. Palmitic acid	0.58	-	0.03	-	-	0.25
39. Linoleic acid	-	-	0.10	0.25	-	-
40. Oleic acid	-	-	-	-	-	0.15
41. Pentamethoxyflavone	-	-	0.13	0.16	-	-
42. Tetramethoxyflavone	1.24	-	0.12	-	-	-
43. Heptamethoxyflavone	-	-	0.14	0.13	-	0.20
44. Hexamethoxyflavone	-	-	0.12	0.28	-	0.18
45. Pentadecanal	-	-	-	-	0.51	-
46. n-Pentacosane	-	-	-	-	0.14	-
47. (z)14-Tricosenyl formate	-	-	-	-	0.36	-
Total %	94.01	99.77	97.13	97.11	97.81	95.66

-: not detected.

). Limonene, α-pinene, and myrcene were detected in all studied peels (orange, pummelo, and tangelo–mandarin hybrid), which is in accordance with the literature, as they are among the major compounds in almost all previously studied Citrus EOs [10,26,27,28]. Among orange cultivars (Newhall and Valencia Ovale Porou), Valencia Ovale Porou/citrumelo (Csp3) was the cultivar with the richest chemical profile, with twenty-three (23) constituents, followed by Valencia Ovale Porou/trifoliata (Csp4) with seventeen (17), Newhall/citrumelo (Csp1) with nine (9), and Newhall/trifoliata (Csp2) with only five (5). Moreover, all orange cultivar EOs contained the monoterpene hydrocarbon limonene as the major constituent (87.69–95.61%) and lower amounts of myrcene (0.54–2.20%), sabinene (0.25–1.29%), α-pinene (0.51–0.99%), and valencene (traces–1.36%). Linalool and PMFs were identified in all oranges, except Csp2. The results indicated that there was no strong influence of the rootstock (trifoliata or citrumelo) upon the metabolic profile of PMFs in the peel of each orange variety (Newhall or Valencia Ovale Porou). The latter result is in accordance with those published by Zouaghi et al. [29], who stated that the use of rangpur lime or Swingle citrumelo 4475 rootstocks did not influence the chemical composition of the Citrus sinensis peel EOs, since there was no difference in the production of monoterpene hydrocarbons and limonene. The chemical analyses of the EOs from orange peels of orange cultivars, especially the Newhall and Valencia Ovale (Csp1, Csp2, Csp3 and Csp4), could be compared with very recent results [30] that showed that orange peel EO composition was characterized by high percentage of limonene (>90%) and myrcene (>2%), while in the current study, limonene’s percentages were from 87.69 up to 95.61% and myrcene varied from 2.06 to 0.54%. The dominance of limonene in the EO peel from sour orange, orange, and lemon fruit was also proposed by Azghar et al. [31]. Furthermore, it is witnessed that limonene, β-pinene, and sabinene were the most abundant compounds in the EOs of Citrus meyer, Citrus paradise, and C. sinensis [32]. It must be taken into consideration that the literature data are from cultivars from a different geographical region (island of Crete) and also that the EOs were extracted using different method, hydrodistillation, and not through cold pressure as in the present study. The effect of the extraction method on the chemical profile of obtained EOs from sour orange (Citrus aurantium L.) flowers has also been reported by Mohagheghniapour et al. [33], who witnessed variations in the amount of limonene and β-pinene due to the implemented isolation methods.

The characterization of PMFs derived from orange peels has been the subject of numerous studies according to the literature [17,24,34]; due to their broad bioactivities, they could potentially be further used in the cosmetics and pharmaceutical industries.

Furthermore, the EO analysis from the peels of pummelo Cuban Shaddock (Cgp1) and tangelo Minneola (Crp1) identified eighteen (18) and twenty-seven (27) constituents, respectively. Limonene was also predominant in both Cgp1 (88.93%) and Crp1 (79.86%). Additionally, in Cgp1, myrcene, β-bisabolene, and α-bergamotene followed limonene, while nootkatone (0.74%), a characteristic aroma constituent and indicator for identifying pummelos [27,35], was the only ketone that was identified in the Cuban Shaddock cultivar. The identification of nootkatone in the Cuban Shaddock pummelo is of great importance, since the latter compound is a known inhibitor of acetylcholinesterase [36]. The obtained data are largely in agreement with the studies in the literature on pummelo EO composition [37,38].

This work highlighted the fact that PMFs were absent from pummelo Cuban Shaddock (Cgp1) peel EOs, while it is noteworthy that minor fractions of O-methylated coumarin (osthole) (0.49%) and 7-methoxy-8-(2-oxo-3-methylbutyl)-coumarin (0.06%) were also detected. Coumarins have been also detected previously in other pummelo samples in different percentages due to the different geographical origins of the tested samples [27,35].

Moreover, in the tangelo Minneola (Crp1) peel profile, limonene (79.86%) was followed by γ-terpinene, valencene, myrcene, and α-pinene, which is in accordance with the literature [39,40]. Low levels of hexamethoxyflavone and heptamethoxyflavone (0.18 and 0.20%, respectively) were detected and have also been reported previously in tangelo Nova peel [41].

The EOs from all peels revealed the predominance of monoterpene hydrocarbons (88.71–98.51%) and lower amounts of sesquiterpene hydrocarbons (0.46–3.56%) (

Table 3. Chemical categories and relative concentrations (area %) of the studied EOs from peels of six Citrus cultivar/rootstock combinations.

Chemical Categories	Area %
	Csp1	Csp2	Csp3	Csp4	Cgp1	Crp1
Monoterpenes	90.51	98.51	93.26	94.23	93.34	88.71
Sesquiterpenes	1.55	1.26	0.46	0.73	3.54	3.56
Aldehydes	-	-	0.16	-	0.51	-
Ketones	-	-	0.03	-	0.74	0.20
Alcohols	0.13	-	1.52	1.33	-	1.21
Esters	-	-	0.10	-	0.07	0.06

), which could be explained by their involvement in fruits’ defense cascades, which are mostly used to prevent uncontrolled proliferation due to pathogenic bacteria and/or attacks by insects [42]. This chemical profile is also in accordance with other reported studies on Citrus fruits [28,38,43]. Limonene, which is the most abundant metabolite in all studied peels, has attracted the attention of many researchers, as its presence in Citrus spp. contributes to their bioactivities due to their potential anti-inflammatory, antioxidant, analgesic, and anticoagulant properties [44].

3.2. Chemical Composition of EOs from Citrus Leaves

Eighty-seven (87) compounds were identified in total from leaf EOs of the studied Citrus cultivars, with a notable chemical variability, especially between the examined orange variety/rootstock combinations (

Table 4. Chemical composition and relative concentration (area %) of leaf EOs from six Citrus cultivar/rootstock combinations.

Compounds	Area %
	Csl1	Csl2	Csl3	Csl4	Cgl1	Crl1
α-Thujene	0.86	-	-	0.18	-	-
2. α-Pinene	-	-	3.27	-	-	-
3. Sabinene	3.19	0.58	43.74	0.56	0.34	-
4. Myrcene	-	-	3.69	-	-	-
5. α-Terpinene	1.12	-	-	0.22	-	-
6. Phellandrene	-	-	-	0.50	-	-
7. δ-3-Carene	-	-	3.96	-	-	-
8. p-Cymene	2.36	-	-	-	-	-
9. Limonene	17.34	1.32	18.84	0.19	0.45	32.25
10. 1,8-Cineole	1.78	1.10	-	-	-	10.31
11. trans-β-Ocimene	-	-	4.86	-	-	-
12. γ-Terpinene	1.89	-	1.86	0.36	-	-
13. cis-Cabinehydrate	-	2.18	0.94	0.59	-	-
14. Terpinolene	-	-	0.51	-	-	-
15. Linalool	2.40	3.93	1.02	-	0.52	2.31
16. α-Thujone	-	-	-	2.48	-	-
17. Verbenol	-	-	-	0.14	-	-
18. Sabina ketone	2.99	2.18	-	6.97	-	-
19. Citronellal	-	-	0.63	-	-	-
20. Terpinen-4-ol	0.50	0.80	1.02	-	0.35	-
21. p-Cymen-8-ol	-	2.04	-	3.49	-	-
22. Myrtenol	1.22	-	-	2.92	1.69	-
23. trans-Carveol	-	-	-	1.71	-	-
24. Cuminal	-	-	-	0.67	-	-
25. Linalyl acetate	1.77	2.77	0.95	0.83	-	7.70
26. Estragole	2.17	-	-	-	-	-
27. trans-Anethole	-	1.21	0.49	-	-	-
28. Carvacrol	-	2.05	-	-	-	-
29. Car-3-en-2-one	-	1.21	-	-	-	-
30. cis-Limonene oxide	-	0.95	-	-	2.07	-
31. Citronellyl propionate	-	-	0.18	-	-	-
32. Neryl acetate	-	-	0.15	-	-	-
33. 3-Methylcamphor	-	-	-	1.23	-	-
34. Isocyclocitral	-	-	-	0.50	-	-
35. 2-Caren-10-al	-	-	-	0.62	-	-
36. Cuminol	-	-	-	2.10	-	-
37. Isofenchol	-	-	-	15.71	-	-
38. p-Mentha-1,4 dien-7-ol	-	-	-	1.55	-	-
39. Sabinol	-	-	-	6.61	-	-
40. β-Elemene	1.23	2.39	7.23	1.87	-	7.48
41. Citral B (neral)	-	-	-	2.05	-	-
42. 2-Pinen-4-ol	-	0.82	-	-	1.37	-
43. cis-Carveol	-	-	-	-	1.41	-
44. Alloaromadendrene	-	0.83	-	-	-	-
45. Elemol	-	0.25	-	-	-	-
46. β-Caryophyllene	-	-	0.86	-	-	-
47. α-Humulene	-	-	0.57	-	-	-
48. trans-β-Farnesene	-	-	0.61	-	-	-
49. β-Sinensal	-	-	3.10	-	-	-
50. α-Sinensal	-	-	0.88	-	-	-
51. Caryophyllene oxide	1.10	7.88	-	3.93	3.74	-
52. Spiro (4,5) decane	-	-	-	1.81	-	-
53. β-Selinene	-	1.73	-	-	-	-
54. Globulol	-	1.45	-	-	-	-
55. Nootkatone	-	1.59	-	-	-	-
56. Germacrene B	-	-	-	0.94	-	-
57. Ledene	-	-	-	1.10	-	-
58. Isospathulenol	-	3.54	-	0.93	-	-
59. Spathulenol	-	-	-	0.95	3.27	8.20
60. Valerenol	-	-	-	0.81	-	-
61. Linalool oxide	-	-	-	-	9.39	-
62. Isolongifolol	-	-	-	-	1.88	-
63. β-Oplopenone	-	-	-	-	1.94	-
64. Caryophylla-3,8(13)-dien-5-β-ol	-	-	-	-	2.43	-
65. Caryophylla-4(12),8(13)-dien-5-β-ol	-	-	-	-	-	1.95
66. Hexahydrofarnesyl acetone	-	1.43	-	3.53	12.00	5.80
67. Aromadendrenepoxide	-	-	-	2.66	-	-
68. Longifolenaldehyde	-	-	-	1.89	-	-
69. Aromadendrene	-	-	-	0.72	-	-
70. Ledenoxide	-	1.27	-	-	-	-
71. Phytosterol	-	-	-	-	-	3.90
72. Phytol	-	1.45	0.27	1.45	2.73	5.36
73. cis-Piperitol	-	1.67	-	-	-	-
74. Neophytadiene	-	2.44	-	2.29	-	-
75. Pentadecane	-	3.23	-	-	-	-
76. Eicosane	-	0.61	-	1.15	-	-
77. Docosane	-	-	-	-	10.54	-
78. Tricosane	2.26	-	-	-	1.95	-
79. Tetracosane	0.82	-	-	-	-	-
80. Pentacosane	3.41	3.09	-	-	4.20	-
81. Hexacosane	1.83	0.80	-	-	-	-
82. Heptacosane	5.04	5.39	-	-	5.86	-
83. Octacosane	3.10	1.33	-	-	-	-
84. Nonacosane	10.49	6.02	-	-	-	-
85. Triacontane	3.19	-	-	-	-	-
86. Hentriacontane	15.25	8.27	-	-	18.87	-
87. Unknown	5.20	8.20	-	14.52	4.80	5.63
Total (%)	92.60	88.06	99.63	95.28	91.55	90.93

-: not detected.

). The occurrence of limonene, which was the principal monoterpene in Citrus fruit peel EOs, was detected at a high range in the EOs of orange leaves of the cultivars Newhall (Csl1, 17.34%) and Valencia Ovale Porou (Csl3, 18.84%) when grafted upon citrumelo rootstock, in contrast with very low ranges in the same cultivars when grafted upon Poncirus trifoliata rootstock (Csl2, 1.32%; Csl4, 0.19%). The latter fact shows that the use of Poncirus trifoliata rootstock directly influences the petitgrain (leaf) EO composition. It has also been reported that Poncirus trifoliata rootstock produces rather low quantities of limonene and γ-terpinene in the leaf EOs [45]. Rootstock choice influences nutrient uptake, water use, and tree vigor, factors that can impact the biosynthesis and composition of EO compounds in the leaves of the scion [46]. The direct influence of the rootstock upon the leaf or even flower EO profile has been proposed [9,45]. The dominance of limonene is in accordance with the data of Ferrer et al. [47], who reported more than 90% and also stated that the composition of the EOs from the fruits and peels of 43 orange cultivars is very similar and thus aromatic variability cannot be used as a selection criterion in orange trees. In addition, sabinene, linalyl acetate, and β-elemene were identified in all the studied orange leaves. It is noteworthy that monoterpene sabinene, which is considered one of the major components in the leaf EO of navel oranges, Valencia oranges, and grapefruit [48,49], dominated in the leaves of the Valencia Ovale Porou/citrumelo combination (Csl3, 43.74%) but was detected at very low ranges (0.56–3.19%) in the other orange combinations (Csl1,2,4) and tangelo leaves (Cgl1). The alkanes hentriacontane (15.25%) and nonacosane (10.49%) were detected in high concentrations in the Newhall/citrumelo (Csl1), while caryophyllene oxide (7.88%) was one of the main compounds in Newhall/trifoliata (Csl2). Isofenchol was found only in Valencia Ovale Porou/trifoliata (Csl4) in a high percentage (15.71%). Several other monoterpenes, alcohols, ketones, and alkanes have been identified in almost all Citrus/rootstock leave EOs: γ-terpinene (0.36–1.89%), linalool (1.02–3.93%), sabina ketone (2.18–6.97%), terpinen-4-ol (0.50–1.02%), cis-sabinehydrate (0.59–2.18%), p-cymen-8-ol (2.04–3.49%), myrtenol (1.22–2.92%), isospathulenol (0.93–3.54%), hexahydrofarnesyl acetone (1.43–3.53%), nonacosane (6.02–10.49%), octacosane (1.33–3.10%), and heptacosane (5.04–5.39%).

Furthermore, in the pummelo Cuban Shaddock leaf EO (Cgl1), twenty-two (22) compounds were identified, among which hentriacontane was the major one (18%). According to the literature, hentriacontane is a dominant alkane between leaf wax hydrocarbons in Citrus spp. [50], while it has been also identified in Citrus maxima (Burm.) Merr. [35]. Additionally, hexahydrofarnesyl acetone (12%), as well as linalool oxide (9.39%) and caryophyllene oxide (3.74%), were also detected, while very low levels of the monoterpenes limonene and sabinene have been observed in the leaves of the Cuban Shaddock pummelo, a result which is in accordance with a previous study [49], which also demonstrated rather low levels of limonene and increased E-phytol in the leaf EO of the pummelo Cuban Shaddock cultivar.

In the tangelo Minneola leaf EO (Crl1), eleven (11) components were identified, with high limonene content (32.35%). Other metabolites, found in lower percentages were: 1,8-cineole, linalool, spathulenol, linalyl acetate, and β-elemene. Sabinene, which is a common component with high variability in Citrus leaves [49], was absent from the tangelo Minneola cultivar. To our knowledge, this is the first report regarding the leaf EO profile of tangelo Minneola leaves when grafted upon Poncirus trifoliata rootstock.

Regarding the classification of chemical categories, a significant percentage of alkanes was detected in all six Citrus cultivar/rootstock leaf EOs (

Table 5. Chemical categories and relative concentrations (area %) of the studied EOs from leaves of six Citrus cultivar/rootstock combinations.

Chemical Categories	Area (%)
	Csl1	Csl2	Csl3	Csl4	Cgl1	Crl1
Monoterpenes	28.56	5.19	77.71	3.27	0.79	42.57
Sesquiterpenes	1.23	4.97	13.23	4.64	-	7.48
Aldehydes	-	-	4.61	1.13	-	5.63
Ketones	2.99	6.42	-	10.50	13.94	5.80
Alcohols	6.31	16.29	2.80	41.14	14.81	19.78
Esters/oxides	2.87	12.89	1.28	10.96	15.21	7.70
Alkanes	45.43	28.76	-	2.97	30.44	1.95

-: not detected.

). The alkane distribution model can be strongly influenced by several factors, such as seasonal variations, as well as the type and the age of the plant [51]. Regarding the terpenes, similarities with the above-mentioned peels were observed, as leaves also contain monoterpenes and sesquiterpenes. There are also noteworthy differences compared with peels, as oxides and a great amount of alcohol are common chemical categories in all studied leaves.

3.3. Isolation and Determination of Selected Metabolites

The PMFs (Figure 2) (tetramethoxy-, pentamethoxy-, hexamethoxy-, heptamethoxy-flavone), in the EOs obtained from the peels of orange (cv. Newhall and Valencia Ovale Porou) and tangelo Minneola exhibit interesting chemical profiles with promising biological activity according to the available pharmacological data on PMFs [16,17,34]. The compounds were isolated through preparative chromatography and structurally determined by NMR spectroscopy (Figures S13–S18), and such metabolites have been isolated here, to our knowledge, for the first time from Greek cultivars.

PMFs are usually isolated as mixtures, as difficulties in their separation have been reported due to their structural similarities [23]. They are characterized by a more lipophilic skeleton compared to polyhydroxylated flavonoids (such as quercetin, luteolin, etc.) due to the hydrophobic nature of their methoxy groups. Therefore, PMFs can easily permeate the small intestine and be readily absorbed into the human circulatory system [52]. Among their pharmacological properties, PMFs have been shown to block adhesion molecule biosynthesis by cytokine-induced endothelial cells, inhibit the expression of tumor necrosis factor-R, and also suppress proliferation, while promoting apoptosis and suppressing ethanol-induced gastric hemorrhagic lesions [53]. All the above-isolated PMFs are among the appreciated chemotaxonomic markers of the Citrus genus [54], with well-known bioactivities (antioxidant, anti-inflammatory, antibacterial, cytotoxic) [41,55].

3.4. Antimicrobial Activity

The in vitro antimicrobial activity of EOs from the peels and leaves of six (6) Citrus cultivar/rootstock combinations were assayed against nine (9) human pathogenic microorganisms (two Gram-positive strains, four Gram-negative bacterial strains, and three fungal strains are shown in

Table 6. Antimicrobial activity expressed as minimum inhibitory concentration (MIC mg/mL) of EOs produced from the peel and leaves of six Citrus cultivar/rootstock combinations against certain tested microorganisms.

		S. aureus	S. epidermidis	P. aeruginosa	K. pneumoniae	E. cloacae	E. coli	C. albicans	C. tropicalis	C. glabrata
Peel	Csp1	1.82	1.45	1.32	1.25	1.20	0.95	1.20	1.00	0.95
	Csp2	1.75	1.28	1.40	1.0	0.98	0.80	1.00	0.89	0.77
	Csp3	1.88	1.40	1.17	1.20	0.95	0.82	1.12	0.97	0.80
	Csp4	1.85	1.42	1.35	1.27	0.95	0.84	1.15	0.99	0.80
	Cgp1	1.80	1.40	1.20	1.00	0.94	0.85	1.17	1.00	0.82
	Crp1	1.97	1.35	1.10	1.15	1.37	0.97	1.25	1.16	0.98
Leaves	Csl1	2.20	1.96	2.50	2.64	3.2	2.7	2.95	2.77	2.50
	Csl2	2.52	2.38	3.20	3.15	3.5	3.00	3.20	3.00	2.92
	Csl3	2.00	1.85	2.23	2.25	2.5	2.5	2.50	2.44	2.35
	Csl4	2.70	2.65	3.00	2.79	3.35	2.98	3.00	2.92	2.87
	Cgl1	2.37	2.30	2.94	3.00	3.42	2.90	2.90	2.77	2.60
	Crl1	2.10	2.00	2.50	2.94	2.9	2.75	2.80	2.65	2.50
Netilmicin		3.7·10−3	3.9·10−3	7.5·10−3	8·10−3	7·10−3	3.5·10−3	-	-	-
Amoxicillin		2·10−3	1.8·10−3	2·10−3	2·10−3	2.5·10−3	2·10−3	-	-	-
5-Flucytosine		-	-	-	-	-	-	0.1·10−3	0.9·10−3	9.8·10−3
Amphotericin B		-	-	-	-	-	-	1.2·10−3	0.45·10−3	0.4·10−3

-: not detected.

). There was a difference in microbial susceptibility between the Citrus peel and leaf EOs when tested on bacteria and fungi. Regarding the Citrus peel EOs, data showed that fungi were relatively more sensitive than bacteria, a result which is in agreement with those of previous studies [38,56]. Furthermore, it was observed that Gram-positive bacteria were more susceptible to Citrus peel EOs than Gram-negative bacteria, which has also been reported [57,58]. This latter result is directly linked to the different anatomical structures of the outer membrane of the cell wall between Gram-negative and Gram-positive bacteria [59]. It is well established that the lack of hydrophilic lipopolysaccharide (LPS) molecules scattered upon the surface of the outer membrane of Gram-positive bacteria renders them susceptible to the penetration of the components of EOs [38]. This penetration facilitates the combination of EO compounds with site-specific targets of the cell wall, causing a rupture of the cytoplasmic membrane and eventual leakage of chemicals, ions, and metabolic substrates, leading finally to bacterial death [60]. In this work, the EO from the peel of the cv. Newhall orange grafted on trifoliata rootstock exhibited the strongest antimicrobial activity, while the EO from the tangelo Minneola, also grafted on trifoliata rootstock, exhibited the weakest antimicrobial activity against Gram-positive bacteria and fungi among the tested EOs. These results could be attributed to the fact that in the EO from the peel of the Newhall orange (Csp 2) grafted upon trifoliata rootstock, the dominant components were limonene (95.61%), myrcene (1.88%), α-pinene (0.62%), and sabinene (0.4%), compounds with known antimicrobial properties [38,48]. Recent data highlight the fact that the EO antimicrobial activity is strengthened when specific compounds, like limonene, α-pinene, β-pinene, myrcene, and ocimene, co-exist and are dominant compounds in EOs [38]. Additionally, it was reported that the antimicrobial and antioxidant activity of citrus EO extracts can be attributed to the synergistic effect of major monoterpenoid components viz. geranial, neral, citronellal, nerol, geraniol, and geranyl formate [61]. Furthermore, the relatively inferior antimicrobial activity of EO from tangelo Minneola (Crp1) could be related to the fact that the limonene content was only 79.86%, followed by minor amounts of the other 26 compounds. Furthermore, it was witnessed that the EOs the peel of Valencia Ovale Porou (Csp3; Csp4) grafted upon both rootstocks and of Pummelo (Cgp1) exhibited similar antimicrobial activity against the tested microorganisms, a fact that could be related to the presence of similar limonene content (89.1%, 89.82%, 88.93%) and the presence of other minor compounds (22, 16, and 17, in total respectively). It is well established that the antimicrobial effect is also influenced by the presence of minor compounds that pose an antagonistic or synergistic effect against the implementation of the antimicrobial activity by the dominant components, a fact that was witnessed in the EO from the peel of tangelo Minneola (Crp1), Valencia Ovale Porou (Csp3; Csp4), and pummelo (Cgp1) [62,63]. Recent data also highlight the fact that the antifungal activity of EOs from citrus (mandarin) enclaved into nanocapsules could be enhanced via the addition of EOs from other plant species like cinnamon and clove, justifying the synergistic effect between EOs [64]. The proposed synergistic effect was also proposed by Azghar et al. [31], who demonstrated that the EOs from the peel of sour orange, orange, and lemon exert antibacterial activity against multidrug-resistant bacteria due to synergistic interactions between various natural chemical compounds.

Furthermore, in the current work, it was witnessed that the antimicrobial activity of the EOs from the leaves was somewhat weaker than that obtained from the peel of the tested citrus (Table 6). This observation can be attributed to the higher proportion of oxygenated compounds in the citrus peel EOs, a finding that is supported by other research groups. [48]. The collected data pinpoint that the EOs from the leaves of orange cv. Newhall (Csl1) and cv. Valencia Ovale Porou (Csl3) grafted upon citrumelo rootstock, as well as from tangelo cv. Minneola (Crl1), had a stronger antimicrobial activity against all tested microorganisms. This fact could be linked with the elevated levels of limonene that were detected (Csl1, 17.34%; Csl3, 18.84%; Crl1, 32.25%) and, in the case of cv. Valencia Ovale Porou/citrumelo (Csl3), also to the presence of sabinene, a compound with well-documented antimicrobial action [65]. These latter findings strengthen the fact that rootstock choice can influence the antimicrobial activity of the EOs produced from the leaves of citrus, as has been suggested by other research groups. Hamdan et al. [32] pinpointed the synergistic effect of combined Citrus species (C. meyer, C. paradise, C. sinensis) leaf EOs against microbial growth. Also, the findings support the use of citrus EOs for medicinal purposes and as antibacterial agents in food, beverages, and cosmetics products as substitutes for synthetic chemicals [8].

4. Conclusions

In this study, the chemical compositions of EOs from the peels and leaves of six Citrus cultivar/rootstock combinations were evaluated. In all six citrus peel EOs, a large percentage of terpenes (monoterpenes and sesquiterpenes) was identified, with monoterpenes accounting for 88–98% of the total and limonene, which is known to contribute to Citrus bioactivities due to its potential properties, as their main representative (80–95%). Chemotaxonomic markers, such as PMFs in the peels of C. sinensis and Citrus paradisi × Citrus tangerina and nootkatone in Citrus maxima (Burm.) Merr, were identified, confirming the bibliographic data. It was also noted that leaf EOs differed significantly from those of the peels. High percentages of alkanes were identified, while the monoterpene sabinene, which has known antimicrobial activity, showed a wide variation, from total absence (tangelo Minneola/trifoliata, Crl1) up to 44% (Valencia Ovale Porou/citrumelo Csl3) of the total peel oil. Also, it was proposed that the rootstock choice did not influence the EO profile of the fruit peel but exerted an influence upon the chemical profile of the leaves. The EOs from the peel of Newhall oranges grafted on Poncirus trifoliata rootstock exhibited the strongest antimicrobial activity, while those from the peel of tangelo Minneola were the weakest of all tested cultivar/rootstock combinations. The use of citrumelo rootstock favored the antimicrobial activity of leaf EOs from the studied orange cultivars. The leaf EO from tangelo Minneola exhibited strong antimicrobial activity.

Deeper knowledge of the chemical profile and the way the cultivation methods pose an impact upon it can lead to the optimization of the isolation of EOs and PMFs (identified in almost all orange and tangelo cultivar peels). Citrus peels or leaves, which were considered high-added-value by-products, could be recovered and serve as natural alternatives to synthetic preservatives for food and valuable sources of bioactive metabolites for further applications in cosmetics and pharmacy, and they could have great economic importance to these industries. Moreover, the use of nano-emulsion systems can protect citrus EOs from deterioration by external factors and maintain their functional properties (antioxidant, antimicrobial, antimutagenic) so as to be used in various applications like foods, pharmaceuticals, and cosmetics [66].

Due to the increased production of orange fruits (60% among all Citrus fruits), more than 95 million tons in 2021, approx. 100 million tons of industrial Citrus waste will end up in the environment annually, while the Citrus EO demand in the worldwide market reaches 500 billion dollars [30]. The use of EOs could be of high interest at a national level in Greece, as until now, the use of peels and/or Citrus spp. leaves has been ignored. Meanwhile, more experiments are needed to evaluate the antimicrobial and antioxidant activity in real food matrices in order to present all data regarding the use of such sustainable material management or “circular economy” and to promote such wastes becoming promising resources.