Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species

The volatile fractions from fresh inflorescences of naturally growing orchids Anacamptis coriophora (L.) R. M. Bateman, Pridgeon & M. W. Chase subsp. fragrans (Pollini), Anacamptis pyramidalis (L.) R. Ophrys holosericea (Burm.) Greuter and Serapias vomeracea (Burm. f.) B. were isolated by steam distillation and analyzed by GC/FID and GC/MS. Saturated hydrocarbons were quantified as the major constituents of the volatile fraction (47.87–81.57% of the total essential oil), of which long-chain monounsaturated hydrocarbons accounted from 9.20% to 32.04% of the total essential oil. Double bond position in linear alkenes was highlighted by dimethyl disulfide derivatization and MS fragmentation. Aldehydes (from 3.45 to 18.18% of the total essential oil), alcohols (from 0.19% to 13.48%), terpenes (from 0.98 to 2.50%) and acids (0.30 to 2.57%) were also detected. These volatiles compounds may represent a particular feature of these plant species, playing a critical role in the interaction with pollinators. DPPH assay evaluating the antioxidant activity of the essential oils was carried out, showing a dose-dependent antioxidant activity.


Introduction
Pollination of flowers by animals is often influenced by a wide variety of volatile molecules [1,2]. The floral scent in plants has the primary aim to attract and guide pollinators [3,4], playing a critical role both in long-and short-distance attraction [2,5,6]. In fact, bees learn odours easier and more rapidly than colours [3,7]. Furthermore, the floral scent may thus influence/drive pollinator constancy [3,8], which ensures pollen transfer, reduces pollen loss and contributes to maintaining both the plant reproductive fitness and their barriers among species [9,10]. Additional functions of floral volatile chemicals occur as defensive and protective mechanisms vs. biotic and abiotic stresses [11][12][13]. This may explain the wide variety of volatiles fragrances emitted by orchids acting as key characters to drive pollinators when food or sexual deception takes place [2].
Orchidaceae is considered one of the well-represented flowering plant families, worldwide distributed accounting approximately 28,000 species [14]. This abundance leads to a great complexity of floral scents; in fact, orchids can potentially produce almost all the fragrances occurring in nature [15].

Results
The essential oil obtained by steam distillation from fresh inflorescences were evaluated as 1.3 mg for A. coriophora subsp. fragrans, 1.8 mg for A. pyramidalis, 2.6 mg O. holosericea and 3.4 mg for S. vomeracea, respectively. The yields were evaluated as 0.03%, 0.02%, 0.52% and 0.10% (weight/fresh weight basis), respectively. Table 1 shows the results of qualitative and quantitative essential oil analyses on the Elite-5 MS column. The compounds are listed in order of their elution time and reported as percentages of the total essential oil. The total number of peaks for A. coriophora subsp. fragrans was 60 with number of identified peak of 43 (72% identification), A. pyramidalis was 58 with number of identified peak of 45 (78% identification). O. holosericea was 59 with number of identified peak of 49 (83% identification), S. vomeracea was 65 with number of identified peak of 59 (91% identification). As evidenced, the main represented volatiles constituents are saturated hydrocarbons, especially in A. coriophora subsp. fragrans followed by S. vomeracea, O. holosericea and A. pyramidalis, and unsaturated hydrocarbons mainly present in O. holosericea essential oil. Differences in the qualitative and quantitative composition of the volatile essential oils obtained from the four sympatric Italian orchids have been observed.

DPPH Assay
All samples demonstrated a good, dose-dependent, antioxidant activity by the DPPH assay ( Figure 2). ANOVA analysis evidenced that the ROS-scavenging activity is strongly influenced both by the sample and concentrations tested (p < 0.0001). In particular, O. holosericea shows the strongest antioxidant activity, especially at a concentration of 1.5 mg/mL. For S. vomeracea, the concentration did not influence the antioxidant activity (p > 0.05).
Molecules 2019, 24, x FOR PEER REVIEW 6 of 13 and 9-heneicosene the most abundant reaching the 0.84%, 0.77% and 0.71% of the total essential oil, respectively.

DPPH Assay
All samples demonstrated a good, dose-dependent, antioxidant activity by the DPPH assay ( Figure 2). ANOVA analysis evidenced that the ROS-scavenging activity is strongly influenced both by the sample and concentrations tested (p < 0.0001). In particular, O. holosericea shows the strongest antioxidant activity, especially at a concentration of 1.5 mg/mL. For S. vomeracea, the concentration did not influence the antioxidant activity (p > 0.05).    Figure 1. Venn's diagram shows both the number of compounds shared and unshared/peculiar among the four orchid species. Percentages are referred to the total number of compounds found, not to the relative abundance [27].

Discussion
A high percentage of saturated hydrocarbons has been detected in the volatile fraction of all the four Italian sympatric orchid species. The presence of saturated hydrocarbons in higher percentage with respect to other published research [28] is probably due to the different extraction procedures like solid-phase microextraction that work at lower extraction temperatures. In detail, a series of homologous linear chain

Discussion
A high percentage of saturated hydrocarbons has been detected in the volatile fraction of all the four Italian sympatric orchid species. The presence of saturated hydrocarbons in higher percentage with respect to other published research [28] is probably due to the different extraction procedures like solid-phase microextraction that work at lower extraction temperatures. In detail, a series of homologous linear chain compounds ranged from C 9 to C 29 have been reported for all the terrestrial and epiphytic orchids. The presence of hydrocarbons as allelochemicals is associated with epicuticular wax chemistry playing an important role in plant/herbivore interactions. Saturated alkanes exerted an interesting, although limited, activity in pollinator deception in different floral species [25,[29][30][31].
The position of the double bond in linear alkene isomers was determined by GC/MS after iodine-catalyzed reaction with dimethyl disulfide. Derivatization procedure was applied to alkene mixtures, which have chemotaxonomic value for the pollinator populations. Linear chain monounsaturated hydrocarbons were previously identified in several orchid species with a high content of these compounds in flowers [30]. Although these compounds seem not to act are as specific contributors to the aroma of the plant, they might be crucial in modulating plant-herbivore interaction [32]. In fact, several studies demonstrated that this class of compounds is endowed with an interesting, although limited, activity in pollinator deception in Orchidaceae [21,31]. Furthermore, multifactorial mechanisms involved in protection actions from environmental factors such as water loss, thermal-or UV-related stress, seem to be related to the occurrence of hydrocarbons [30,32].
Another class of substances that might be involved in plant-insect interaction is reported to be benzenoids [33,34]. A. pyramidalis, a specialized species and mainly dependent on butterflies for pollination [35][36][37][38], shows a strong presence of 2-phenylethanol (12.11% of the total volatiles) in its flower's scent compared to the other orchis species (see Table 1). It should be stressed that benzenoids play a critical role in pollinator attraction strategy of A. pyramidalis, as well as those of two other terrestrial orchids, Nigritella nigra (today accepted as Gymnadenia nigra) (L) Reichb. f. (2-phenylethanol) [15,39] and Gymnadenia conopsea (L) Br.R., (benzyl acetate) [15]. Given the volatile extract's composition, which included ROS-scavenging compounds, the antioxidant activity for each extract has been evaluated by DPPH method. All samples demonstrated a good antioxidant activity that is probably related to the presence of benzyl alcohol (A. pyramidalis 1.13%, O. holosericea 2.59%) and phenylacetaldehyde (A. coriophora subsp. fragrans 0.70%, A. pyramidalis 3.82%, O. holosericea 2.07% and S. vomeracea 3.91%) as previously reported for essential oils from Laurus nobilis and Fagopyrum species [40,41], to the presence of thymol (A. coriophora subsp. fragrans 0.36%, A. pyramidalis 0.30%, O. holosericea 0.66% and S. vomeracea 0.28%) and α-copaene (A. coriophora subsp. fragrans 0.26%, A. pyramidalis 0.45% and O. holosericea 0.56%) as reported for essential oil from Cinnamodendron dinisii and Siparuna guianensis [42,43]. However, even if benzenoids have been observed to be predominant in specialist butterfly-pollinated flower scents, these compounds have been also found in generalist plants, suggesting that they might be emitted by both specialists and generalists [44]. It should be stressed that differences in floral scents, visual attraction and reward systems are decisive for chemical communication in pollination strategies of sympatric orchids to guarantee pollination efficiency and fidelity.
In detail, Ophrys flowers act as "false female" mimicking her visual, tactile and olfactory stimuli, so male pollinators attempt to copulate with the orchid labellum removing and delivering pollen, a process termed pseudocopulation. It should be noticed that the complex blend of different odours mimicking female pheromones mainly consists of long-chain alkanes and alkenes derivatives and that the relative alkanes and alkenes abundance makes each floral scent unique resulting in different pollinator species attraction [22,30].
As regards S. vomeracea, its flowers form a small tube, which pollinators use basically as nest-replacement or refugee during rainy weather, although the pollination strategy of this orchid relies not only in the floral shape but also in the olfactory attractors to assure sufficient degree of pollinator fidelity; in fact both visual and olfactory signals are of critical importance in pollinator choice. Again, according to the literature [45] alkanes and alkenes have been found to be very important volatile components in this orchid species scent as reported in Table 1. In fact, a solitary bee such as Megachile rotundata has been found to mark its nest with an olfactory trace consisting of a mixture of alkanes and alkenes similar to that present in the Serapias and Ophrys scents [30].
However, even if benzenoids, in this case mainly 2-phenylethanol, have been observed to be predominant in specialist butterfly-pollinated flower scents, these compounds have been also found in generalist plants, suggesting that they might be emitted by both specialists and generalists [44].
Among the species investigated A. coriophora subsp. fragrans can be occasionally pollinated by Lepidoptera (even if they are not the main pollinators), as shown in Table 2 [46]. Table 2. Relationship between each sympatric Italian orchid and its pollinators, according to GIROS [46]. Unlike A. pyramidalis, in A. coriophora subsp. fragrans alkanes and alkenes are the main constituents of the scent. The results reported in the present paper are different from those reported in the scarce literature available data [47][48][49]. In this investigation, we analyzed pre-pollinated flowers of A. coriophora subsp. fragrans chemotype europaeus, while both inflorescences and mature seeds of A. coriophora subsp. fragrans chemotype africanus [47], or inflorescences of A. coriophora subsp. coriophora [48], respectively, were used by other Authors.

Species
In conclusion keeping in mind that these four orchids colonize the same environment, bloom in the same time and share also some pollinators, the data reported in the present paper strongly suggest that each species may attain a peculiar combination of olfactory, tactile and/or visual floral signs suitable to explain different interactive communication systems between plants and pollinators. Furthermore, it is reasonable to assess that each orchid species is able to gain especially through different signals a sufficient level of pollinator fidelity and thus maintain its genetic identity even in a population of admixed orchid species

Plant Material
Inflorescences of the four orchid species were collected in May 2016 in Pompeiana (Imperia, Italy, 438,547 N 78,909 E) according to the regional law and with the legal permission of Regional Authorities (Region Liguria, Prot. n. PG/2016/104503). Plants were identified according to Chase and colleagues [48]. A type specimen for each species is deposited in the living collection of CREA-OF (Sanremo, Italy) with the accession numbers ANcf01, ANpy01, OPho01, and SEvo01 for A. coriophora subsp. fragrans, A. pyramidalis, O. holosericea and S. vomeracea, respectively. The flowers were cut and immediately placed in a pyrex bottle containing 100 mL of methylene chloride as a preservative agent and stored at −20 • C.

Isolation of Volatile Fraction
Flowers of A. coriophora subsp. fragrans (4.6 dried g), A. pyramidalis (9.3 dried g), O. holosericea (0.5 dried g) and S. vomeracea (3.3 dried g), to which ethyl decanoate was added as internal standard, were steam distilled together with methylene chloride in a Clevenger-type apparatus for 1 h. The distillate, saturated with NaCl, was extracted with freshly distilled diethyl ether (3 × 100 mL), dried over anhydrous Na 2 SO 4 concentrated at first with a rotary evaporator and finally using a gentle stream of N 2 and then analyzed by GC/FID and GC/MS.

Fractionation and Alkylthiolation of Alkenes
A portion of the essential oil from each sample was placed onto a glass column (7 × 30 mm) of silica gel 60, 230-400 mesh (Merck, Milano, Italy), preconditioned with pentane [29]. The non-polar fraction was eluted with 2 mL of pentane and used for the determination of double bond position in alkenes by alkylthiolation according to reported method [50].

GC/FID Analysis
The analyses were carried out using a Hewlett Packard model 5890 GC, equipped with Elite-5MS (5% phenyl methyl polysiloxane, Supelco, Sigma Aldrich, Milano, Italy) capillary column of (30 m × 0.32 mm i.d.) and film 0.32 µm thick. The carrier gas was He at a flow of 1 mL/min. One µL aliquots of each essential oil were manually injected in "split" mode (30:1). The oven temperature program included an initial isotherm of 40 • C for 5 min, followed by a temperature ramp to 260 • C at 4 • C/min, and a final isotherm at this temperature for 10 min. Injector and detector temperatures were set at 250 and 280 • C, respectively.

GC/MS Analysis
The analyses were carried out using a GC Model 6890 N, coupled to a benchtop MS Agilent 5973 Network, equipped with the same capillary column and following the same chromatographic conditions used for the GC/FID analyses. The carrier gas was He at a constant flow of 1.0 mL/min. The essential oils were diluted prior to analysis (1mg/10mL in n-hexane), and 1.0 µl of the diluted solution was manually injected into the GC system with a split ratio of 30:1. The ion source temperature was set at 200 • C, while the transfer line was at 300 • C. The acquisition range was 40-500 amu in electron-impact (EI) positive ionization mode using an ionization voltage of 70 eV.

Identification and Quantification of the Essential Oil Components
The identification of the essential oil volatile components was performed by their retention indices (RI) and their mass spectra, and by comparison with a NIST 98 and Wiley 5 MS libraries, as well as with literature data [26]. Retention indices were calculated by an Elite-5MS capillary column using a series of n-alkanes (C 8 -C 23 ) under the same GC conditions as for samples [51]. The relative amount of each individual component of the essential oil was expressed as percent peak area relative to the total peak area from GC/FID analyses of the whole extract.

DPPH Assay
The ROS-scavenging activity of the essential oils was evaluated by the DPPH (2,2-diphenyl-2 picrylhydrazyl hydrate) method according to the previously described method with slight modifications [52][53][54]. At first, the essential oils were solubilized in dimethyl sulfoxide and then diluted in methanol at final concentrations of 1.5 and 0.75 mg/mL. 270 µL of DPPH (0.028% w/v in methanol) was mixed with 30 µL of each sample. Reaction mixtures were incubated in the dark for 20 min at room temperature before measuring the absorbance at 517 nm using a microplate reader (Synergy HT, BioTek, Swindon, United Kingdom). Ascorbic acid (1.25 mg/mL) was used as a positive control, while the reaction mixture without any sample was used as a negative control. ROS scavenging activity percentage was calculated as follows: % activity = (A − B)/A × 100 (1) where A is the absorbance of the negative control and B is the absorbance of the tested sample. Analyses were performed in three replicates.

Statistical Analysis
Results of DPPH assay are reported as mean ± standard deviation for the values with a normal distribution (or interquartile range and median for the values that did not adhere to the Gaussian distribution). For the data with normal distribution, an analysis of variance (ANOVA) was performed, considering the compound and its concentration as fixed factors, while the inhibition percentage as the dependent variable. The significance criterion was set to p < 0.05. Funding: Research partially funded by the project "Implementation of the FAO International Treaty on Plant Genetic Resources for Food and Agriculture" of the Ministry of Agriculture, Alimentation and Forestry Policies, aimed at research and experimentation supporting the collection, characterization and evaluation of plant genetic resources.

Conflicts of Interest:
The authors declare no conflict of interest.