Next Article in Journal
Impact of Nitrogen Fertilizer Levels on Metabolite Profiling of the Lycium barbarum L. Fruit
Previous Article in Journal
Enhanced Reactant Distribution in Redox Flow Cells
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 24
Issue 21
10.3390/molecules24213878
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Francesco Saverio Robustelli della Cuna
1,2,*,
Jacopo Calevo
3,4,
Elia Bari
2,
Annalisa Giovannini
3,
Cinzia Boselli
2 and
Aldo Tava
1
1
CREA Research Centre for Animal Production and Aquaculture, Viale Piacenza 29, 26900 Lodi, Italy
2
DDS Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
3
CREA Research Centre for Vegetable and Ornamental Crops, Corso degli Inglesi 508, 18038 Sanremo (IM), Italy
4
DBIOS Department of Life Sciences and Systems Biology, University of Torino, Viale Mattioli 25, 10125 Torino, Italy
*
Author to whom correspondence should be addressed.
Molecules 2019, 24(21), 3878; https://doi.org/10.3390/molecules24213878
Submission received: 18 September 2019 / Revised: 24 October 2019 / Accepted: 24 October 2019 / Published: 28 October 2019
(This article belongs to the Section Analytical Chemistry)
Browse Figures
Versions Notes

Abstract

:
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.

Graphical Abstract

1. 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]. This wide variety of floral scents is primarily due to the combination of the great number of orchid species and of the evolution of pollination systems. The mechanisms of deception in orchids include generalized food deception, food-deceptive floral mimicry, brood-site imitation, shelter imitation, pseudo-antagonist, rendezvous attraction and sexual deception, where generalized food deception is the most common mechanism (38 genera) followed by sexual deception (18 genera) [16,17]. In the Orchidoideae subfamily, in particular, consisting of 7 subtribes and about 3630 species [18], the sexual deception mechanism can be recognized for orchid genera such as Drakea or Ophrys [18]. Furthermore, orchids using food-deceptive strategy show flowers resembling those of rewarding co-occurring species [19].
Italy hosts about 236 orchid species [20], that frequently co-occur in the same habitat and population [21]. A peculiar pollination strategy is the sexual deception of the genus Ophrys: in this orchid genus the shape of the labellum looks like the female abdomen of the bee pollinator species and the floral scent contains interactive chemicals resembling the sex pheromones of pollinators. These intriguing visual and olfactory signals are, therefore, of critical importance to driving pollinator choices [22]. On the other hand, genera such as Orchis, Dactylorhiza and some Anacamptis employ a generalized food-deceptive strategy [19], in which these flowers provide floral cues indicating a food reward while animal pollination is achieved without providing nectar, pollen or other food rewards. On the contrary species of the genus Serapias employ an unusual pollination strategy called shelter deception. In fact, these flowers, nectarless and not so brightly coloured, form a small tube used by pollinators as a refuge during cold or rainy weather to rest or sleep [23,24,25].
The aim of the present investigation was to isolate and compare the essential oils from inflorescences of four sympatric orchid species (Anacamptis coriophora (L.) R.M. Bateman, Pridgeon & M. W. Chase subsp. fragrans (Pollini), Anacamptis pyramidalis (L.) R., Serapias vomeracea (Burm. f.) B. and Ophrys holosericea (Burm) Greuter), co-occurring in the same natural site in Italy, in order to characterize their chemical composition and antioxidant activity.

2. 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.

2.1. Anacamptis Coriophora subsp. Fragrans

Major constituents of the volatile fractions of this orchid species were found to be saturated hydrocarbons (81.57% of the total essential oil), from which heneicosane (25.10%), nonadecane (20.51%), tricosane (17.16%), pentacosane (9.31%) and heptacosane (3.43%) are the most abundant compounds. A series of unsaturated linear chain hydrocarbons were identified, of which 9-pentacosene and 9-heptacosene represent the 3.39% and 2.30% of the total volatiles, followed by 9-tricosene and 1-hexadecene accounting for 1.42% and 1.21% of the total essential oil, respectively. Aldehydes are present in the percentage of 3.45%, being nonanal (1.61%), phenylacetaldehyde (0.70%) and anisaldehyde (0.68%) the most represented. Alcohols (0.19%) and terpenes (0.98%) consisted of 2,5-dimethoxybenzyl alcohol (0.19%), thymol (0.36%) and α-copaene (0.26%), respectively.

2.2. Anacamptis Pyramidalis

Major constituents of the volatile fractions of A. pyramidalis were found to be saturated hydrocarbons accounting for 52.43% of the total essential oil. Tricosane (17.17%), pentacosane (16.24%), heneicosane (7.50%) and heptacosane (6.04%) are the most abundant constituents of this class of compounds. Aldehydes present as 16.19% were basically represented by nonanal (5.44%), heptanal (4.02%) phenylacetaldehyde (3.82%) and octadecanal (1.44%). Alcohols (13.48%) consist of 2-phenylethanol (12.11%) followed by benzyl alcohol (1.13%). A series of unsaturated linear chain hydrocarbons (10.34%) was instead identified with 9-pentacosene and 9-heptacosene as the 4.69% and 3.90%, followed by 7-heptacosene evaluated as 1.38% of the total essential oil. Acids are also detected as 2.57%, being heptanoic acid (1.41%) the most abundant followed by nonanoic acid (1.16%). Terpenes (2.50%) are mainly represented by α-copaene (0.45%), thymol (0.30%) and α-cadinene (0.14%).

2.3. Ophrys Holosericea

Saturated hydrocarbons, accounting for 47.87% of the total essential oil were found to be the major constituents also of O. holosericea volatiles. Tricosane (27.71%), pentacosane (6.84%), heneicosane (4.34%), heptacosane (2.22%) are the most abundant compounds. A series of unsaturated linear chain hydrocarbons (32.04%) was also identified, of which 7-pentacosene (16.60%) was the major represented, followed by 9-tricosene (3.19%), 9-pentacosene (2.95%), 7-tricosene (2.72%), 7-heptacosene (2.36%), 9-heptacosene (2.17%) and 11-pentacosene (1.77%). Aldehydes (10.74% of the total essential oil) consist mainly of nonanal (4.65%), phenylacetaldehyde (2.07%), heptanal (1.39%) and octadecanal (1.00%). Alcohols (3.20% of the total) are represented by benzyl alcohol (2.59%) and terpenes (1.74% of the total) by thymol (0.66%), α-copaene (0.56%) and γ-muurolene (0.48%).

2.4. Serapias Vomeracea

More than 50% of the S. vomeracea volatile fraction consists of saturated hydrocarbons accounting for 53.29% of the total essential oil. Pentacosane (17.59%), tricosane (14.21%), heneicosane (5.68%), heptacosane (4.99%), nonadecane (2.45%), tetracosane (1.90%) and tetradecane (1.68%) were the most representative compounds. Monounsaturated linear chain hydrocarbons (18.63%) were also identified, of which the 9- and 7- isomers were the most represented. In details the rank order is: 9-pentacosene (3.92%) > 7-heptacosene (3.71%) > 9-heptacosene (2.87%) > 7-pentacosene (2.48%) > 9-tricosene (1.19%) > 7-tricosene (1.18%). Aldehydes accounting for 18.18% of the total volatiles consist mainly of nonanal (7.87%), phenylacetaldehyde (3.91%), heptanal (1.57%), undecanal (1.35%) and octadecanal (0.93%). Acids (1.92% of the total) are represented by palmitic acid (0.77%), nonanoic acid (0.71%) and heptanoic acid (0.37%). Terpenes (2.44%) account for trans-β-farnesene (0.84%), γ-muurolene (0.39%) and thymol (0.28%). The only alcohol found in S. vomeracea volatile fraction was diacetone alcohol as 1.03% of the total essential oil.

2.5. Venn’s Diagram

Figure 1 shows the Venn’s diagram [27] in which 22 compounds are shared among all the 4 sympatric Italian orchids. Even if just a few, some peculiarities were found to be species-specific. In detail, four compounds were found only in A. coriophora subsp. fragrans, i.e., creosol, anisaldehyde, γ-eudesmol and methyl-p-methoxycinnammate, although poorly represented (<1% of the total essential oil). In A. pyramidalis only one compound, 2-phenylethanol (12.11% of the total essential oil), seems to be species-specific, further characterizing this species. O. holosericea showed to have four peculiar compounds, i.e., heptanol, 3.5-octadien-2-one, 2.3-dimethyldecane and 11-pentacosene. It should be stressed that only the latter one is present with a valuable amount, reaching 1.77% of the total essential oil. On the contrary, S. vomeracea was the species showing eight unique chemicals, being trans-β-farnesene, that represents the 50% of total terpenes, palmitic acid and 9-heneicosene the most abundant reaching the 0.84%, 0.77% and 0.71% of the total essential oil, respectively.

2.6. 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).

3. 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 C9 to C29 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].
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 in its flower’s scent as reported (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].
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].
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.
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

4. Materials and Methods

4.1. 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.

4.2. 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 Na2SO4 concentrated at first with a rotary evaporator and finally using a gentle stream of N2 and then analyzed by GC/FID and GC/MS.

4.3. 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].

4.4. 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.

4.5. 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.

4.6. 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 (C8-C23) 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.

4.7. 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
where A is the absorbance of the negative control and B is the absorbance of the tested sample. Analyses were performed in three replicates.

4.8. 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.

Author Contributions

F.S.R.d.C., J.C., A.G.: conceptualization, investigation and writing original draft preparation, data analysis; E.B.: antioxidant activity; C.B. and A.T.: writing-review & editing.

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.

References

  1. Dudareva, N. Biochemical and Molecular Genetic Aspects of Floral Scents. Plant Physiol. 2000, 122, 627–634. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  2. Knudsen, J.T.; Eriksson, R.; Gershenzon, J.; Ståhl, B. Diversity and Distribution of Floral Scent. Bot. Rev. 2006, 72, 1–120. [Google Scholar] [CrossRef]
  3. Dobson, H.E.M. Floral volatiles in insect biology. In Insect–Plant Interactions; Bernays, E., Ed.; CRC: Boca Raton, FL, USA, 1994; Volume 5, pp. 47–81. [Google Scholar]
  4. Raguso, R.A. Floral Scent, Olfaction, and Scent-Driven Foraging Behavior, in Cognitive Ecology of Pollination; Chittka, L., Thomson, J.D., Eds.; Cambridge University Press: Cambridge, UK, 2001; p. 83. [Google Scholar]
  5. Dodson, C.H.; Dressler, R.L.; Hills, H.G.; Adams, R.M.; Williams, N.H. Biologically Active Compounds in Orchid Fragrances. Science 1969, 164, 1243–1249. [Google Scholar] [CrossRef] [PubMed]
  6. Dobson, H.E.M.; Danielson, E.M.; Van Wesep, I.D. Pollen odor chemicals as modulators of bumble bee foraging on Rosa rugosa Thunb. (Rosaceae). Plant. Spec. Biol. 1999, 14, 153–166. [Google Scholar] [CrossRef]
  7. Menzel, R. Learning in honey bees in an ecological and behavioral context. In Experimental Behavioral Ecology; Hölldobler, B., Lindauer, M., Eds.; Fischer: Stuttgart, Germany, 1985; pp. 55–74. [Google Scholar]
  8. Waser, N.M. Flower Constancy: Definition, Cause, and Measurement. Am. Nat. 1986, 127, 593–603. [Google Scholar] [CrossRef]
  9. Pellmyr, O. Three pollination morphs in Cimicifuga simplex; incipient speciation due to inferiority in competition. Oecologia 1986, 68, 304–307. [Google Scholar] [CrossRef]
  10. Grant, V. Modes and origins of mechanical and ethological isolation in angiosperms. Proc. Natl. Acad. Sci. USA 1994, 91, 3–10. [Google Scholar] [CrossRef]
  11. Friedman, M.; Henika, P.R.; Mandreil, R.E. Bactericidal activities of plant essential oils and some of their isolated constituents against Campylobacter jejuni, Escherichia coli, Listeria mono-cytogenes, and Salmonella enterica. J. Food Protect. 2002, 65, 1545–1560. [Google Scholar] [CrossRef]
  12. Hammer, K.A.; Carson, C.F.; Riley, T.V. Antifungal activity of the components of Melaleuca alternifolia (tea tree) oil. J. Appl. Microbiol. 2003, 95, 853–860. [Google Scholar] [CrossRef]
  13. Huber, F.K.; Kaiser, R.; Sauter, W.; Schiestl, F.P. Floral scent emission and pollinator attraction in two species of Gymnadenia (Orchidaceae). Oecologia 2005, 142, 564–575. [Google Scholar] [CrossRef]
  14. Christenhusz, M.J.; Byng, J.W. The number of known plants species in the world and its annual increase. Phytotaxa 2016, 261, 201. [Google Scholar] [CrossRef]
  15. Kaiser, R. The Scent of Orchids: Olfactory and Chemical Investigations; Elsevier: Amsterdam, The Netherlands, 1993. [Google Scholar]
  16. Salzmann, C.C.; Nardella, A.M.; Cozzolino, S.; Schiestl, F.P. Variability in floral scent in rewarding and deceptive orchids: The signature of pollinator-imposed selection? Ann. Bot. 2007, 100, 757–765. [Google Scholar] [CrossRef] [PubMed]
  17. Schiestl, F.P.; Peakall, R.; Ibarra, F.; Francke, W.; Mant, J.G.; Schulz, C.; Franke, S. The Chemistry of Sexual Deception in an Orchid-Wasp Pollination System. Science 2003, 302, 437–438. [Google Scholar] [CrossRef] [PubMed]
  18. Pridgeon, A.M.; Cribb, P.J.; Chase, M.W.; Rasmussen, F. Genera Orchidacearum, Vol. II: Orchidoideae (Part I); OUP: Oxford, UK, 2001. [Google Scholar]
  19. Pellegrino, G.; Bellusci, F.; Musacchio, A. Double floral mimicry and the magnet species effect in dimorphic co-flowering species, the deceptive orchid Dactylorhiza sambucina and rewarding Viola aethnensis. Preslia 2008, 80, 411–422. [Google Scholar]
  20. Bartolucci, F.; Peruzzi, L.; Galasso, G.; Albano, A.; Alessandrini, A.; Ardenghi, N.M.G.; Astuti, G.; Bacchetta, G.; Ballelli, S.; Banfi, E.; et al. An updated checklist of the vascular flora native to Italy. Plant Biosyst. 2018, 152, 179–303. [Google Scholar] [CrossRef]
  21. Cozzolino, S.; Schiestl, F.P.; Müller, A.; De Castro, O.; Nardella, A.M.; Widmer, A. Evidence for pollinator sharing in Mediterranean nectar-mimic orchids: Absence of premating barriers? Proc. R. Soc. B Boil. Sci. 2005, 272, 1271–1278. [Google Scholar] [CrossRef]
  22. Schiestl, F.P.; Ayasse, M.; Paulus, H.F.; Löfstedt, C.; Hansson, B.S.; Ibarra, F.; Francke, W. Orchid pollination by sexual swindle. Nature 1999, 399, 421. [Google Scholar] [CrossRef]
  23. Dafni, A.; Ivri, Y.; Brantjes, N.B.M. Pollination of Serapias vomeracea Briq. (Orch.) by imitation of holes for sleeping solitary male bees (Hym.). Acta Bot. Neerl. 1981, 30, 69–73. [Google Scholar] [CrossRef]
  24. van der Cingel, N.A. An Atlas of Orchid Pollination: European Orchids; A.A. Balkema Publishers: Rotterdam, The Netherlands, 1995. [Google Scholar]
  25. Pellegrino, G.; Luca, A.; Bellusci, F.; Musacchio, A. Comparative analysis of floral scents in four sympatric species of Serapias L. (Orchidaceae): Clues on their pollination strategies. Plant Syst. Evol. 2012, 298, 1837–1843. [Google Scholar] [CrossRef]
  26. Adams, R. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing Corporation: Carol Stream, IL, USA, 2007. [Google Scholar]
  27. Oliveros, J.C. (2007–2015) Venn’sy. An Interactive Tool for Comparing Lists with Venn’s Diagrams. Available online: http://bioinfogp.cnb.csic.es/tools/Venn’sy/index.html (accessed on 19 June 2019).
  28. Manzo, A.; Panseri, S.; Vagge, I.; Giorgi, A. Volatile Fingerprint of Italian Populations of Orchids Using Solid Phase Microextraction and Gas Chromatography Coupled with Mass Spectrometry. Molecules 2014, 19, 7913–7936. [Google Scholar] [CrossRef] [Green Version]
  29. Robustelli della Cuna, F.S.; Boselli, C.; Papetti, A.; Mannucci, B.; Calevo, J.; Tava, A. Composition of Volatile Fraction from Inflorescences and Leaves of Dendrobium moschatum (Orchidaceae). Nat. Prod. Commun. 2018, 13, 93–96. [Google Scholar] [CrossRef]
  30. Schiestl, F.P.; Ayasse, M.; Paulus, H.F.; Löfstedt, C.; Hansson, B.S.; Ibarra, F.; Francke, W. Sex pheromone mimicry in the early spider orchid (Ophrys sphegodes): Patterns of hydrocarbons as the key mechanism for pollination by sexual deception. J. Comp. Physiol. A 2000, 186, 567–574. [Google Scholar] [CrossRef] [PubMed]
  31. Mant, J.; Brändli, C.; Vereecken, N.J.; Schulz, C.M.; Francke, W.; Schiestl, F.P. Cuticular hydrocarbons as sex pheromone of the bee Colletes cunicularius and the key to its mimicry by the sexually deceptive orchid, Ophrys exaltata. J. Chem. Ecol. 2005, 31, 1765–1787. [Google Scholar] [CrossRef] [PubMed]
  32. Eigenbrode, S.D.; Espelie, K.E. Effects of Plant Epicuticular Lipids on Insect Herbivores. Annu. Rev. Entomol. 1995, 40, 171–194. [Google Scholar] [CrossRef]
  33. Mohd-Hairul, A.R.; Namasivayam, P.; Lian, G.E.C.; Abdullah, J.O. Terpenoid, Benzenoid, and Phenylpropanoid Compounds in the Floral Scent of Vanda Mimi Palmer. J. Plant Boil. 2010, 53, 358–366. [Google Scholar] [CrossRef] [Green Version]
  34. Steiner, K.E.; Kaiser, R.; Dötterl, S. Strong phylogenetic effects on floral scent variation of oil-secreting orchids in South Africa. Am. J. Bot. 2011, 98, 1663–1679. [Google Scholar] [CrossRef]
  35. Lind, H.; Lindeborg, M. Lepidopterans as presumptive pollinators of Anacamptis pyramidalis. Entomol. Tidskr. 1989, 110, 156–160. [Google Scholar]
  36. Lind, H. Occurrence, population and fruit setting in Anacamptis pyramidalis on OÖland, Sweden. Sven. Bot. Tidskr. 1992, 86, 329–336. [Google Scholar]
  37. Lind, H. Lepidoptera—Important Long-Distance Pollinators for Plants in Fragmented Habitats. Sven. Bot. Tidskr. 1994, 88, 185–187. [Google Scholar]
  38. Proctor, M.; Yeo, P.; Lack, A. The Natural History of Pollination, 1st ed.; HarperCollins: London, UK, 1996. [Google Scholar]
  39. Tava, A.; Cecotti, R.; Confalonieri, M. Characterization of the volatile fraction of Nigritella nigra (L.) Rchb. F. (Orchidaceae), a rare species from the Central Alps. J. Essent. Oil Res. 2012, 24, 39–44. [Google Scholar] [CrossRef]
  40. Politeo, O.; Jukic, M.; Milos, M. Chemical composition and antioxidant activity of free volatile aglycones from Laurel (Laurus nobilis L.) compared to its essential oil. Croat. Chem. Acta 2007, 80, 121–126. [Google Scholar]
  41. Zhao, J.; Jiang, L.; Tang, X.; Peng, L.; Li, X.; Zhao, G.; Zhong, L. Chemical Composition, Antimicrobial and Antioxidant Activities of the Flower Volatile Oils of Fagopyrum esculentum, Fagopyrum tataricum and Fagopyrum Cymosum. Molecules 2018, 23, 182. [Google Scholar] [CrossRef] [PubMed]
  42. Yanishlieva, N.V.; Marinova, E.M.; Gordon, M.H.; Raneva, V.G. Antioxidant activity and mechanism of action of thymol and carvacrol in two lipid systems. Food Chem. 1999, 64, 59–66. [Google Scholar] [CrossRef]
  43. Andrade, M.A.; das Graças Cardoso, M.; de Andrade, J.; Silva, L.F.; Teixeira, M.L.; Valério Resende, J.M.; da Silva Figueiredo, A.C.; Barroso, J.G. Chemical composition and antioxidant activity of essential oils from Cinnamodendron dinisii Schwacke and Siparuna guianensis Aublet. Antioxidants 2013, 2, 384–397. [Google Scholar] [CrossRef]
  44. Andersson, S.; Nilsson, L.A.; Groth, I.; Bergström, G. Floral scents in butterfly-pollinated plants: Possible convergence in chemical composition. Bot. J. Linn. Soc. 2002, 140, 129–153. [Google Scholar] [CrossRef]
  45. Schiestl, F.P.; Cozzolino, S. Evolution of sexual mimicry in the orchid subtribe orchidinae: The role of preadaptations in the attraction of male bees as pollinators. BMC Evol. Boil. 2008, 8, 27. [Google Scholar] [CrossRef]
  46. G.I.R.O.S. Gruppo Italiano Ricerca Orchidee Spontanee. Available online: http://www.giros.it/forum/viewtopic.php?f=7&t=1498 (accessed on 9 January 2019).
  47. El Mokni, R.; Hammami, S.; Dall’Acqua, S.; Peron, G.; Faidi, K.; Braude, J.P.; Sebei, H.; El Aouni, M.H. Chemical composition, antioxidant and cytotoxic activities of essential oil of the inflorescence of Anacamptis coriophora subsp. fragrans (Orchidaceae) from Tunisia. Nat. Prod. Commun. 2016, 11, 857–860. [Google Scholar] [CrossRef]
  48. Dormont, L.; Delle-Vedove, R.; Bessiere, J.-M.; Schatz, B. Floral scent emitted by white and coloured morphs in orchids. Phytochemistry 2014, 100, 51–59. [Google Scholar] [CrossRef]
  49. Chase, M.W.; Cameron, K.M.; Freudenstein, J.V.; Pridgeon, A.M.; Salazar, G.; Berg, C.V.D.; Schuiteman, A. An updated classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef] [Green Version]
  50. Carlson, D.A.; Roan, C.S.; Yost, R.A.; Hector, J. Dimethyl disulphide derivatives of long chain alkenes, alkadienes and alkatrienes for gas chromatography/mass spectrometry. Anal. Chem. 1989, 61, 1564–1571. [Google Scholar] [CrossRef]
  51. Dool, H.V.D.; Kratz, P.D. A generalization of the retention index system including linear temperature programmed gas—Liquid partition chromatography. J. Chromatogr. A 1963, 11, 463–471. [Google Scholar] [CrossRef]
  52. Bari, E.; Arciola, C.R.; Vigani, B.; Crivelli, B.; Moro, P.; Marrubini, G.; Sorrenti, M.; Catenacci, L.; Bruni, G.; Chlapanidas, T.; et al. In Vitro Effectiveness of Microspheres Based on Silk Sericin and Chlorella vulgaris or Arthrospira platensis for Wound Healing Applications. Materials 2017, 10, 983. [Google Scholar] [CrossRef] [PubMed]
  53. Bari, E.; Perteghella, S.; Marrubini, G.; Sorrenti, M.; Catenacci, L.; Tripodo, G.; Mastrogiacomo, M.; Mandracchia, D.; Trapani, A.; Faragò, S.; et al. In vitro efficacy of silk sericin microparticles and platelet lysate for intervertebral disk regeneration. Int. J. Boil. Macromol. 2018, 118, 792–799. [Google Scholar] [CrossRef]
  54. Chlapanidas, T.; Faragò, S.; Lucconi, G.; Perteghella, S.; Galuzzi, M.; Mantelli, M.; Avanzini, M.A.; Tosca, M.C.; Marazzi, M.; Vigo, D.; et al. Sericins exhibit ROS-scavenging, anti-tyrosinase, anti-elastase, and in vitro immunomodulatory activities. Int. J. Boil. Macromol. 2013, 58, 47–56. [Google Scholar] [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
Molecules 24 03878 g001 550
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].
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].
Molecules 24 03878 g001
Molecules 24 03878 g002 550
Figure 2. ROS-scavenging activity (%) of the tested extracts.
Figure 2. ROS-scavenging activity (%) of the tested extracts.
Molecules 24 03878 g002
Table
Table 1. Percentage composition of the volatile fraction from inflorescences of A. coriophora subsp. fragrans, A. pyramidalis, O. holosericea and S. vomeracea.
Table 1. Percentage composition of the volatile fraction from inflorescences of A. coriophora subsp. fragrans, A. pyramidalis, O. holosericea and S. vomeracea.
Percentage Peak Area
CompoundRI aRI bA. coriophora subsp. FragransA. pyramidalisO. holosericeaS. vomeracea
Hexanal801799tr0.30tr0.21
2,4-Dimethyl heptane820819-0.130.210.19
2-Methyl-2-pentenal821829-0.160.300.24
Diacetone alcohol841844-0.240.371.03
Heptanal9019010.174.021.391.57
Benzaldehyde9619570.100.590.180.13
Heptanol972971trtr0.10tr
Nonane10009990.45-0.080.06
Octanal10011003--0.070.22
Benzyl alcohol10321033tr1.132.59tr
Phenylacetaldehyde104210420.703.822.073.91
2,4-Dimethyldecane106710680.560.551.240.57
3,5-Octadien-2-one10721071--0.08-
Heptanoic acid108310830.141.41-0.37
Undecane11001100-0.31-0.18
Nonanal110211041.615.444.657.87
2-Phenylethanol11071112-12.11trtr
p-Cresol115811580.87---
Octanoic acid11731173---0.07
1-Dodecene11921192---0.19
Dodecane120012000.470.420.450.26
Decanal12041206-0.170.150.49
Anisaldehyde125112530.68---
Nonanoic acid127512720.161.16tr0.71
Thymol127712810.360.300.660.28
Undecanal13071307-tr0.361.35
2,5-Dimethoxy benzyl alcohol132813270.19-0.14-
α-Copaene137613760.260.450.56tr
1-Tetradecene13901392-tr-0.39
Tetradecane140014000.450.540.501.68
Dodecanal14121409-0.050.260.38
trans-Caryophyllene14181421-0.090.04-
trans-β-Farnesene14521458---0.84
γ-Muurolene147114680.080.050.480.39
Pentadecane150015000.150.190.120.36
α-Cadinene153715380.160.14-0.09
1-Hexadecene159115921.21--0.51
Hexadecane160016000.190.140.190.15
Tetradecanal16131613-0.090.310.60
γ-Eudesmol162716350.12---
Methyl-p-methoxycinnamate161716140.58---
Heptadecane170017000.240.240.450.65
Pentadecanal17131713-0.11-0.28
1-Heptadecene175517480.06--0.40
Octadecane18001800---0.08
Nonadecane1900190020.512.260.562.45
Isophytol19441949-1.47tr0.84
Palmitic acid19501960---0.77
Eicosane200019991.560.14-0.17
Octadecanal202120200.191.441.000.93
9-Heneicosene20712073---0.71
Heneicosane2100210025.107.504.345.68
Docosane220021990.96-1.010.80
11-Tricosene226122650.17-0.150.65
9-Tricosene227922741.42-3.191.19
7-Tricosene22872280--2.721.18
Tricosane2300230017.1617.1727.7114.21
Tetracosane240024000.76-1.581.90
11-Pentacosene24692469--1.77-
9-Pentacosene247424743.394.692.953.92
7-Pentacosene24832482-tr16.602.48
Pentacosane250025009.3116.246.8417.59
Hexacosane260026000.270.47-0.85
9-Heptacosene267626752.303.902.172.87
7-Heptacosene268326810.341.382.363.71
Heptacosane270026993.436.042.224.99
9-Nonacosene287628740.310.370.130.43
Nonacosane29002902-0.090.370.47
Saturated hydrocarbons 81.5752.4347.8753.29
Unsaturated hydrocarbons 9.2010.3432.0418.63
Aldehydes 3.4516.1910.7418.18
Alcohols 0.1913.483.201.03
Terpenes 0.982.501.742.44
Acids 0.302.57tr1.92
Miscellaneous 1.45-0.08-
RI a: Retention Indices from literature [26]. RI b: Retention Indices calculated by GC/FID using n-alkane series (from C8 to C23) under the same analytical conditions as for samples. tr, traces (<0.01%); >0.01% quoted to nearest 0.01%.
Table
Table 2. Relationship between each sympatric Italian orchid and its pollinators, according to GIROS [46].
Table 2. Relationship between each sympatric Italian orchid and its pollinators, according to GIROS [46].
SpeciesLepidotperaDipteraHymenopteraColeoptera
Anacamptis coriophora subsp. FragansNymphalidae
Zygaenidae		Tachinidae
Bombyliidae		Apidae
Halictidae
Vespidae		Oedemeridae
Anacamptis pyramidalArctiidae
Crambidae
Hesperidae
Lycaenidae
Nymphalidae
Noctuidae		Bombyliidae
Conopidae
Empididae		ApidaeOedemeridae
Ophrys holosericea--Apidae
Formicidae		Rutelidae
Serapias vomeracea--Apidae
Crabronidae
Eumenidae
Halictidae
Megachilidae		Scarabaeidae

Share and Cite

MDPI and ACS Style

Robustelli della Cuna, F.S.; Calevo, J.; Bari, E.; Giovannini, A.; Boselli, C.; Tava, A. Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species. Molecules 2019, 24, 3878. https://doi.org/10.3390/molecules24213878

AMA Style

Robustelli della Cuna FS, Calevo J, Bari E, Giovannini A, Boselli C, Tava A. Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species. Molecules. 2019; 24(21):3878. https://doi.org/10.3390/molecules24213878

Chicago/Turabian Style

Robustelli della Cuna, Francesco Saverio, Jacopo Calevo, Elia Bari, Annalisa Giovannini, Cinzia Boselli, and Aldo Tava. 2019. "Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species" Molecules 24, no. 21: 3878. https://doi.org/10.3390/molecules24213878

APA Style

Robustelli della Cuna, F. S., Calevo, J., Bari, E., Giovannini, A., Boselli, C., & Tava, A. (2019). Characterization and Antioxidant Activity of Essential Oil of Four Sympatric Orchid Species. Molecules, 24(21), 3878. https://doi.org/10.3390/molecules24213878

Article Metrics

Back to TopTop