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Article

Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches

by
Valeria Cavalloro
1,2,†,
Stefania Pagliari
3,†,
Fabio Gosetti
2,4,
Luca Campone
2,3,
Cristina Sottani
5,
Simona Collina
6,
Emanuela Martino
1,2,* and
Francesco Saverio Robustelli della Cuna
5,6
1
Department of Earth and Environmental Sciences, University of Pavia, Via Ferrata 1, 27100 Pavia, Italy
2
NBFC—National Biodiversity Future Center, Piazza Marina 61, 90133 Palermo, Italy
3
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza Della Scienza 2, 20126 Milan, Italy
4
Department of Earth and Environmental Sciences, University of Milano-Bicocca, Piazza Della Scienza 1, 20126 Milan, Italy
5
Environmental Research Center, ICS Maugeri SPA SB, Institute of Pavia, IRCCS, Via Maugeri 2, 27100 Pavia, Italy
6
Department of Drug Sciences, University of Pavia, Viale Taramelli 12, 27100 Pavia, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2024, 13(8), 1064; https://doi.org/10.3390/plants13081064
Submission received: 11 March 2024 / Revised: 5 April 2024 / Accepted: 8 April 2024 / Published: 10 April 2024
(This article belongs to the Special Issue Spectra Analysis and Plants Research 2.0)
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Versions Notes

Abstract

:
Orchids are experiencing wide success in ornamental, medicinal, and food fields. The reason for their success is correlated with both their morphology and metabolomics, the latter linked to their taste and biological effects. Despite many orchids having already been the subject of chemotaxonomic works, some of them are still untapped, like the case of Orchis purpurea. O. purpurea is one of the most common species of the genus Orchis, present in hedgerows, verges, and light woodland, where it is one of the few herbaceous plants able to be unpleasant to herbivorous animals. Essential oil from roots, stems, leaves, and flowers were analyzed via GC/MS analyses, revealing the presence of 70 compounds, with a clear prevalence of coumarin. The high concentration of this metabolite may explain the resistance of O. purpurea to herbivores, being associated with appetite-suppressing properties and a bitter taste. Non-volatile fractions were analyzed via UHPLC-MS analysis revealing the presence of hydroxycinnamic acid derivatives, polyphenols, and glycosidic compounds, probably responsible for their color and fragrance. Taken together, the herein presented results shed light on both the defensive strategy and the chemotaxonomy of O. purpurea.

1. Introduction

Orchids are well-known ornamental plants, appreciated worldwide, and ranked among the best sellers in the global potted plant trade. Their beautiful flower is the reason for their commercial success, even if orchids are also well-known for other applications [1,2]. Particularly, orchids are the ingredient of traditional products such as Chikanda, Faham, and Salep [1,3]. This latter is a powder obtained from the dried tubers of more than 35 species of terrestrial orchids, including species from the genera Anacamptis, Dactylorhiza, and Orchis, to cite just a few [4]. Its high consumption in the eastern Mediterranean area is causing serious risks to both consumers and orchids [5]. Thus, tubers are often illegally harvested causing conservation concerns [6], while the final product may be adulterated with potential health risks [7].
Among orchids, the genus Orchis has a high diversity with about 20 terrestrial species. They are characterized by two egg-shaped underground tubers and one spike with flowers of different colors, and most species have several narrow leaves at the base [8].
One of the most common species of the genus Orchis is Orchis purpurea Huds (Asparagales: Orchidaceae). This species was first described in 1762 and it is widely spread in Central Asia, Europe, and North Africa. O. purpurea grows on alkaline soils and favors slightly shaded locations such as hedgerows, verges, and light woodland. Its name refers to the purple inflorescence, which is also the reason for its common name Lady Orchid. O. purpurea is one of the tallest and most robust European orchids, being able to reach almost one meter in height (Figure 1). This plant is present on almost all the Italian territory, with few exceptions (i.e., Valle d’Aosta and Sicily regions), and it can be mainly found in mature and luminous forests, and in semi-natural herbaceous vegetation. Interestingly, in these environments, O. purpurea is one of the few herbaceous plants able to resist herbivorous animals.
Despite its long-lasting presence in checklists of different territories and, more generally, the great interest of the scientific community in its belonging genus, O. purpurea remains underexplored. Thus, using “Orchis purpurea” as keywords on Scopus, only 51 articles were retrieved (update January 2024), mainly related to floristic checklists, morphological characterization, or behavior vs. climate change. Its first preliminary metabolomic characterization was published only in 2022, and it referred only to the volatile fraction obtained by the inflorescences [9]. Despite its preliminary nature, this work allowed us to highlight an interesting and peculiar trait of O. purpurea: the main compound identified in the inflorescences’ essential oils was coumarin. Coumarin is a well-known secondary metabolite that can be found in many different genera and species. Its main physiological role is the defense of the producing organism against both biotic and abiotic stress [10,11]. This metabolite is generally stored inside the vacuoles in its glycosylated form, while the aglycone is usually formed after stress.
To the best of our knowledge, coumarin has never been isolated from the genus Orchis thus far and it has been recently considered an underexplored metabolite in the Orchidaceae family [12]. Consistently, in this work, we wanted to deepen its presence in different organs of O. purpurea and to draw the first complete metabolomic fingerprint of the volatile fraction of this underestimated plant. Furthermore, a preliminary investigation of the non-volatile fraction has also been performed.

2. Results and Discussion

2.1. Essential Oil Characterization

After the unambiguous identification of previously collected Orchis purpurea [8], we extracted its main parts by steam distillation and obtain the volatile fraction via liquid/liquid extraction. The yields of O. purpurea essential oil from fresh roots, stems, leaves, and flowers were 0.009%, 0.03%, 0.09%, and 0.02% (weight of essential oils/weight of fresh material × 100), respectively. As can be noticed, leaves allowed us to obtain higher amounts of essential oils, followed by stems, flowers, and finally roots. GC/MS analyses revealed the presence of 70 compounds, listed in their elution order and reported as percentages of the total EO. The qualitative and quantitative results on the Elite-5MS column are reported in Table 1.
Coumarin was confirmed to be the most abundant secondary metabolite present in all the natural matrices except for roots, where the most abundant metabolite is (E)-15-heptadecenal (Table 1). Particularly, it represented almost the only metabolite present in the essential oil from leaves (98.86%), more than a half of the essential oil from stems and flowers (86.10% and 69.72%, respectively), and finally almost one-third of the essential oil from roots (32.30%). Simple coumarin (2H-1-benzopyran-2-one) and coumarin-derived compounds are widespread in the natural kingdom, especially in the Umbelliferae, Rutaceae, Oleaceae, Orchidaceae Moraceae, and Compositae families [13]. Still, it has also been detected in microorganisms, sponges, and animal species [14,15]. Focusing on orchids, these compounds are present in the essential oil derived from flowers and leaves of both epiphytic (i.e., Dendrobium moschatum, and D. amabile) and terrestrial orchids (i.e., Anacamptis morio, and Ophrys sphegodes) [9,16,17]. The wide diffusion of these metabolites in the natural kingdom can be easily explained considering their biological properties. Coumarins exhibit appetite-suppressing properties and a bitter taste able to protect the producing organism from herbivores [18]. Moreover, coumarins are also endowed with antimicrobial agents and can be released in the rhizosphere or accumulated in other organs after stress. All these activities related to coumarins may explain why O. purpurea is particularly able to resist herbivorous animals.
The other classes of compounds present in the different parts of O. purpurea are detailed below.
Roots: the essential oil was characterized by a high content of aldehydes (44.28%), dominated by (E)-15-heptadecenal (43.39%). The second largest class was represented by alcohols (16.12%), from which 1-hexadecanol (14.03%) and p-cresol (1.0%) were the most abundant compounds.
Stems: the major constituents of the essential oil were found to be alcohols (11.91%), from which p-cresol (11.58%) and 2-phenyl-2-propanol (0.11%) were the most representative compounds. Saturated hydrocarbons (1.04%) were represented by pentacosane (0.18%) and heptacosane (0.14%).
Leaves: the essential oil from leaves was dominated by coumarin (98.86%), followed by alcohols (0.61%), from which p-cresol (0.44%) was the most abundant compound.
Flowers: the most abundant class was represented by alcohols (15.13%) from which p-cresol (12.68%), p-vinylphenol (1.20%), and p-methylguaiacol (0.37%) were the most abundant compounds. The second largest class was represented by saturated hydrocarbons (7.13%).
Except for coumarins, flowers resulted in the most diversified oils in terms of chemical composition (Table 2). On the other hand, roots were particularly rich in long chain acids, alcohol, and aldehydes, confirming their storage function. Of particular interest, p-cresol was produced in considerable amounts by both stems (11.94%) and flowers (12.88%). This secondary metabolite could represent a further defense for the plant, being considered a toxin with phytotoxic allelopathic activity [19]. Another valuable hypothesis suggest that p-cresol is produced due to its ability to specifically attract specific pollinators [20,21]. A Venn diagram (Figure 2) was realized to illustrate qualitative similarities and differences in volatile profiles among the different parts of O. purpurea [22].
As highlighted in Figure 2, a core of 12 compounds (18.4% of the total number of compounds detected) was present only in flowers, 3 (4.61%) in stems, and 14 (21.5%) in roots. In contrast, leaves do not show any specific compounds, but share some with flowers, roots, and stems.

2.2. Characterization of the Non-Volatile Constituents

Once we had characterized the volatile portion of O. purpurea, we focused our attention on the characterization of the non-volatile constituents. Thus, to the best of our knowledge, only the non-volatile fraction obtained by the hypogeal part of O. purpurea has already been analyzed in previous work [23]. Consistently, both leaves and flowers were sequentially extracted by exploiting a Soxhlet apparatus with n-hexane, ethyl acetate, and methanol. The three obtained fractions were evaporated under vacuum and the resulting yields are reported in the table hereunder (Table 3).
As expected, the three fractions contained completely different metabolites. Particularly, n-hexane fraction obtained from leaves and flowers, contained almost only coumarin, as demonstrated by GC-MS (Figure 3), while the ethyl acetate fraction allowed us to obtain a very low yield, and its analytical fingerprint was not significant [24,25].
Different results were obtained on the methanolic fraction. Thus, the UHPLC-MS/MS analysis highlighted that both leaves and flowers contain hydroxycinnamic acid derivatives (i.e., p-coumaroyl derivatives) and polyphenols (i.e., quercetin, luteolin, and kaempferol derivatives) as reported in Table 4. Furthermore, Dactylorhin A and Militarine, both glycosidic compounds, are compounds already found in other orchids belonging to the genus Bletilla, Pleione, and Coeloglossum.
Some secondary metabolites are characterizing of only the leaves or flowers.
Compound stored only in the flowers are Coelovirin E, a kaempferol glycoside, saponins, and Cyanidin-3-O-glucoside. Particularly, this last compound belongs to the class of anthocyanins, which are metabolites that cause a broad spectrum of orchid flower coloration.
On the other hand, Koaburaside and Coelovirin D are present only in the leaves. In detail, Coelovirins B, D, and E are tartrate derivatives already identified in another orchid belonging to genus Coeloglossum, while Koaburaside is a glycosilated phenolic compound already identified in other organism as Fallopia multiflora [26].
Table 4. Identified compounds by UHPLC-MS/MS. The retention time, precursor ion (negative or positive), compound name, chemical formula, MS error (ppm), characteristic product ions, and analyzed extract are reported.
Table 4. Identified compounds by UHPLC-MS/MS. The retention time, precursor ion (negative or positive), compound name, chemical formula, MS error (ppm), characteristic product ions, and analyzed extract are reported.
N°Rt (min)[M − H]−[M + H]+CompoundsFormulaMass Error
(ppm)		Product IonsFlowers/LeavesRef.
12.22331.1033 KoaburasideC14H20O9−0.5123.0445/105.0338leaves[27]
23.59205.0855 UnknownC12H13O3−4.9129.0554/115.0761Flowers/leaves-
34.22367.1247 Coelovirins EC14H24O110.3293.1236/143.0710/
131.0708/99.0811		Flowers[28,29]
45.42625.1410627.1832Quercetin-3-O-gentiobiosideC27H30O17−0.1209.0293/191.0190/
463.0866/301.0346/
151.0031		Flowers/leaves[30]
55.49325.0932 p-Coumaroyl-O-
hexoside		C15H18O81.0163.0395/119.0498Flowers/
leaves		[31]
65.80 449.1083Cyanidin-3-O-
glucoside		C21H20O11−0.2287.0549Flowers[32]
75.89609.1460 Luteolin-diglucosideC27H30O16−0.3447.0923/446.0551/
285.0398/283.0244/
151.0030		Flowers/leaves[30]
86.28695.1471 Kaempferol-
malonylhexose- hexose		C30H32O190.5651.1576/531.1151/
489.1046/446.0859/
285.0402		Flowers/leaves[33]
96.55651.1578 2-O-AcetylrutinC29H32O171.7489.1041/446.0857/
285.0401		Flowers/
leaves		[27]
106.56635.2675 Coelovirins DC21H47O21−1.5349.1143/293.1236/
277.1286/143.0707		leaves[28,29]
116.72473.2021 Coelovirin BC21H30O12−1.5115.0750Flowers/
leaves		[28,29]
126.91325.0932 p-Coumaroyl-O-
hexoside		C15H18O81.0163.0395/119.0498Flowers/
leaves		[31]
137.34787.3220 Unknown--473.1696/285.0981Flowers/
leaves		-
147.59593.1507 Luteolin-O-rutinosideC27H30O19−0.4285.0396Flowers/
leaves		[27]
157.65447.0929 Kaempferol-hexosideC21H20O11−0.3284.0323/255.0295/
227.0346		Flowers[34]
167.68457.2059 UnknownC21H30O11-153.0550/127.0758/
99.0809		Flowers/
leaves		-
177.88887.3233 Dactylorhin AC40H55O221.1619.2239/439.1606/
179.0558/153.0553		Flowers/
leaves		[35]
188.15385.1436 Unknown--177.0551/145.0289/
117.0332		leaves-
198.37771.2741 MilitarineC34H46O17−1.0457.1220/285.0979/
153.0555		Flowers/
leaves		[35]
208.57533.1344 UnknownC21H29O172.2390.0738/333.0760Flowers/
leaves		-
218.781033.3541 Unknown saponin--765.2605/436.1606/
619.2237/325.0923		Flowers-
229.21753.2615 Unknown--439.1609/153.0554leaves-
239.491063.3664 Unknown saponin--749.2662/569.2034/
439.1611/153.0554		Flowers-

3. Materials and Methods

3.1. Chemicals

Octyl octanoate (98%), alkane mix (C6–C35), formic acid, and anhydrous sodium sulfate were obtained by Sigma-Aldrich, Inc. (Milan, Italy). Diethyl ether, n-hexane, ethyl acetate, acetonitrile, and methanol were purchased from Merck (Darmstadt, Germany) and used without further purification. For UPLC/MS analyses, acetonitrile and formic acid LC-MS grade were provided by Romil (Cambridge, UK), and ultrapure water was produced using the Milli Q-Milli RO system, Millipore (Burlington, MA, USA).

3.2. Plant Material

Roots, leaves, stems, and inflorescences of O. purpurea were collected in April 2021 in Pianlago Ponzone (Alessandria, Italy, 44°35′21″ N 8°27′37″ E) according to the regional law and with the legal permission of the regional authorities. Plants were identified according to Chase et al. [8]. A voucher specimen of the species is deposited in the living collection of the Department of Drug Sciences (Pavia, Italy) with the accession number Op02. The plant materials were collected and immediately placed in a PVC bag and stored at +4 °C, and subsequently stored in dark conditions at −20 °C until extraction procedures.

3.3. Extraction of O. purpurea

3.3.1. Steam Distillation

Samples of roots, stems, leaves, and flowers of O. purpurea (10.69 g, 58.21 g, 60.96 g, and 60.92, respectively) were spiked with octyl octanoate (35 mg) as an internal standard, and next steam distilled for 3 h. Steam distillation was performed according to De Agostini et al., 2022 [36]. Briefly, the natural matrix was placed over a stainless steel plate inside the body made of heat-resistant glass. The steam passing through the natural matrix was next cooled through a water condenser, thereby producing the essential oil and aqueous plant extract (hydrosol) simultaneously. The hydrosol was extracted with diethyl ether (3 × 100 mL), dried over anhydrous Na2SO4, concentrated under reduced pressure, and finally the solvent was completely evaporated using a gentle N2 stream. The obtained extract was stored at −20 °C until GC/FID and GC/MS analyses.

3.3.2. Soxhlet Extraction

Samples of leaves and flowers of O. purpurea (26.17 g and 5.95 g, respectively) were placed in the Soxhlet apparatus and extracted sequentially with n-hexane, ethyl acetate, and methanol (3 × 500 mL each). The mixture was refluxed for 60 min, filtered, resuspended in fresh solvent, and refluxed for further 60 min. The fractions were collected, and the solvent removed under vacuum. The samples were successively extracted, following the procedure described above, with ethyl acetate and methanol. All dried extracts were stored at −20 °C until UHPLC-MS/MS analysis.

3.4. Gas Chromatographic Analysis

GC-FID analyses were carried out using an Agilent model 5980 GC (Agilent Technologies, Lexington, CA, USA), equipped with Elite-5MS (5% phenyl methyl polysiloxane) capillary column (30 m × 0.32 mm i.d.) and film 0.32 μm thick (Agilent Technologies, Lexington, CA, USA). The carrier gas was He at a flow of 1 mL/min. Aliquots of 1 μL of each essential oil after dilution (1 mg/mL) with dichloromethane were manually injected in “split” mode (30:1) with a column temperature program of 40 °C for 5 min, then increased to 260 °C at 4°C/min, and finally held at this last temperature for 10 min. The injector and detector were set at 250 and 280 °C, respectively. The flow conditions for the FID detector were 40 mL/min for hydrogen and 400 mL/min for air. The relative amount of each component was calculated based on the corresponding FID peak area without response factor correction.
The same conditions were also used for GC-MS analyses using a GC Model 6890 N, coupled to a benchtop MS Agilent 5973 Network (Agilent Technologies, Lexington, CA, USA). 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 positive electron-impact ionization (EI) mode using an ionization voltage of 70 eV.
The identification of the volatile metabolites was performed by their retention indices (RI), their mass spectra, and by comparison with a NIST database mass spectral library, as well as with literature data [37,38,39]. Retention indices were calculated columns using n--C6–C35 alkanes. The quantitative data were obtained from GC/FID analyses by an internal standard method and assuming an equal response factor for all detected compounds.

3.5. UPLC-MS Analysis

Qualitative analyses of flower and leave methanol extracts were performed using a Water ACQUITY UPLC system coupled with the high-resolution mass spectrometer (HRMS/MS) Waters Xevo G2-XS QTof (Waters Corp., Milford, MA, USA). The chromatographic separation was performed using a Biphenyl column (100 × 2.1 mm, 2.6 µm; Phenomenex, Torrance, CA, USA). The mobile phase consisted of water (A) and acetonitrile (B) both acidified to 0.1% (v/v) formic acid. The linear gradient was set at 0.0–10.0 min, 5–95% B, after each run of 5.0 min of column washing (95% B), and 5.0 min of column equilibration (5% B) before the next injection was used, and the flow rate used was 0.4 mL/min. The injected volume was set at 5.0 µL of each sample at a concentration of 0.5 mg/mL. The Xevo GS-XS QTof mass spectrometer was used in both ionization modes (positive and negative) to acquire full-scan MS and HRMS/MS analysis. The calibration of the mass spectrometer used 0.5 M sodium formate and leucine-enkephalin (200 pg/mL) as LockMass (m/z 556.2771 in positive and 554.2615 in negative ionization), infused simultaneously with the flow of column at 2 µL/min and acquired for 0.5 s every 15 s. The following experimental conditions were adopted for the electrospray (ESI) source: capillary voltage of 2.0 kV, source temperature of 150 °C, and desolvation temperature of 500 °C. High-purity nitrogen gas was used as a desolvation gas at a flow rate of 1000 L/h. MS spectra were acquired by full-range acquisition covering a mass range from 50 to 1200 m/z. The HRMS/MS acquisition was performed by data-dependent scan (DDA) experiments where the two most intense ions from the HRMS scan event were selected and subjected to collision-induced dissociation (CID) by applying a minimum signal threshold of 250, an isolation width at 2.0, and collision energy normalized to 30%. A resolving power of 30,000 both in full and in MS/MS scan modes was used. Compounds’ deconvolution was attributed using UNIFI Portal software v1.9 SR4 (Waters Corp., Milford, MA, USA) comparing MS/MS spectra with a proprietary scientific library (Traditional Medicine Library) or ChemSpider, and confirmation with the scientific literature. The MassLynx software (version 4.2, Waters Corp., Milford, MA, USA) was used for instrument control and data acquisition.

4. Conclusions

To conclude, the present work fills the gap related to the characterization of the phytochemical profile of Orchis purpurea, a herbaceous plant characterizing the Italian flora but still underexplored. The high amount of coumarin in all the organs of O. purpurea is of particular interest, this metabolite mainly being present in genera belonging to the Araliales, Rutales, and Asterales orders [40]. In the Orchideaceae family, simple coumarin has already been identified in a few species, mainly belonging to the genus Dendrobium, even if in percentages much lower with respect to the ones reported in the present work [12,16]. Taken together, the herein presented results shed a light on both the defensive strategy and the chemotaxonomy of O. purpurea. However, our results are only descriptive and should not push the reader to consider this organism as a possible source of coumarin, it being a plant protected by many national laws and whose harvesting is strictly regulated.

Author Contributions

Conceptualization, S.C. and F.S.R.d.C.; data curation, E.M.; formal analysis, V.C., S.P., L.C., E.M. and F.S.R.d.C.; methodology, V.C., S.P., F.G. and F.S.R.d.C.; supervision, F.G. and LC.; writing—original draft, V.C.; writing—review and editing, C.S., S.C., E.M. and F.S.R.d.C. All authors have read and agreed to the published version of the manuscript.

Funding

Project funded under the National Recovery and Resilience Plan (NRRP), Mission 4. Component 2 Investment 1.4—Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union—NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022. Adopted by the Italian Ministry of University and Research, CUP, H43C22000530001 Project title “National Biodiversity Future Center—NBFC”.

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

National Recovery and Resilience Plan (NRRP), Mission 4. Component 2 Investment 1.3—Call for tender No. 3138, 16 December 2021, rectified by Decree n.341 of 15 March 2022 of Italian Ministry of University and Research funded by the European Union—NextGenerationEU; Project code PE0000003 ON FOODS—CUP:H43C22000820001—Spoke 6, Project title “ON Foods—Research and innovation network on food and nutrition Sustainability, Safety and Security—Working ON Foods”.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hinsley, A.; de Boer, H.J.; Fay, M.F.; Gale, S.W.; Gardiner, L.M.; Gunasekara, R.S.; Kumar, P.; Masters, S.; Metusala, D.; Roberts, D.L.; et al. A review of the trade in orchids and its implications for conservation. Bot. J. Linn. Soc. 2018, 186, 435–455. [Google Scholar] [CrossRef]
  2. Fonge, B.A.; Essomo, S.E.; Bechem, T.E.; Tabot, P.T.; Arrey, B.D.; Afanga, Y.; Assoua, E.M. Market trends and ethnobotany of orchids of Mount Cameroon. J. Ethnobiol. Ethnomed. 2019, 15, 29. [Google Scholar] [CrossRef] [PubMed]
  3. Dhanda, S.; Bullough, L.-A.; Whitehead, D.; Grey, J.; White, K. A Review of the Edible Orchid Trade; Royal Botanic Gardens: London, UK, 2022; Volume 9. [Google Scholar]
  4. De Boer, H.J.; Ghorbani, A.; Manzanilla, V.; Raclariu, A.-C.; Kreziou, A.; Ounjai, S.; Osathanunkul, M.; Gravendeel, B. DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade. Proc. R. Soc. B Biol. Sci. 2017, 284, 20171182. [Google Scholar] [CrossRef] [PubMed]
  5. Tsaballa, A.; Kelesidis, G.; Krigas, N.; Sarropoulou, V.; Bagatzounis, P.; Grigoriadou, K. Taxonomic Identification and Molecular DNA Barcoding of Collected Wild-Growing Orchids Used Traditionally for Salep Production. Plants 2023, 12, 3038. [Google Scholar] [CrossRef] [PubMed]
  6. Masters, S.; Anthoons, B.; Madesis, P.; Saroja, S.G.; Schermer, M.; Gerritsen, W.; Karahan, A.; Verdoes, R.; Schwallier, R.; van Andel, T.; et al. Quantifying an online wildlife trade using a web crawler. Biodivers. Conserv. 2022, 31, 855–869. [Google Scholar] [CrossRef]
  7. Kemsawasd, V.; Jayasena, V.; Karnpanit, W. Incidents and Potential Adverse Health Effects of Serious Food Fraud Cases Originated in Asia. Foods 2023, 12, 3522. [Google Scholar] [CrossRef]
  8. Chase, M.W.; Cameron, K.M.; Freudenstein, J.V.; Pridgeon, A.M.; Salazar, G.; van den Berg, C.; Schuiteman, A. An updated classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef]
  9. Robustelli della Cuna, F.S.; Cortis, P.; Esposito, F.; De Agostini, A.; Sottani, C.; Sanna, C. Chemical Composition of Essential Oil from Four Sympatric Orchids in NW-Italy. Plants 2022, 11, 826. [Google Scholar] [CrossRef]
  10. Duan, Z.; Yan, Q.; Wu, F.; Wang, Y.; Wang, S.; Zong, X.; Zhou, P.; Zhang, J. Genome-Wide Analysis of the UDP-Glycosyltransferase Family Reveals Its Roles in Coumarin Biosynthesis and Abiotic Stress in Melilotus albus. Int. J. Mol. Sci. 2021, 22, 10826. [Google Scholar] [CrossRef]
  11. Martino, E.; Ramaiola, I.; Urbano, M.; Bracco, F.; Collina, S. Microwave-assisted extraction of coumarin and related compounds from Melilotus officinalis (L.) Pallas as an alternative to Soxhlet and ultrasound-assisted extraction. J. Chromatogr. A 2006, 1125, 147–151. [Google Scholar] [CrossRef]
  12. Priyadarsini, S.S.; Kumar, P.R. Coumarins in orchidaceae: A review connecting ethnomedicine and therapeutic expectations. Rasayan J. Chem. 2022, 14, 2591–2597. [Google Scholar] [CrossRef]
  13. Ortiz, A.; Sansinenea, E. Phenylpropanoid Derivatives and Their Role in Plants’ Health and as antimicrobials. Curr. Microbiol. 2023, 80, 380. [Google Scholar] [CrossRef] [PubMed]
  14. Mabry, T.J.; Ulubelen, A. Chemistry and utilization of phenylpropanoids including flavonoids, coumarins, and lignans. J. Agric. Food Chem. 1980, 28, 188–196. [Google Scholar] [CrossRef] [PubMed]
  15. Stringlis, I.A.; de Jonge, R.; Pieterse, C.M.J. The Age of Coumarins in Plant–Microbe Interactions. Plant Cell Physiol. 2019, 60, 1405–1419. [Google Scholar] [CrossRef] [PubMed]
  16. 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, 1934578X1801300. [Google Scholar] [CrossRef]
  17. Robustelli della Cuna, F.S.; Calevo, J.; Bazzicalupo, M.; Sottani, C.; Grignani, E.; Preda, S. Chemical Composition of Essential Oil from Flowers of Five Fragrant Dendrobium (Orchidaceae). Plants 2021, 10, 1718. [Google Scholar] [CrossRef]
  18. Matos, M.J.; Santana, L.; Uriarte, E.; Abreu, O.A.; Molina, E.; Yordi, E.G. Coumarins—An important class of phytochemicals. In Phytochemicals—Isolation, Characterisation and Role in Human Health; InTech: Rijeka, Croatia, 2015. [Google Scholar]
  19. Tena, C.; Santiago, A.d.R.; Osuna, D.; Sosa, T. Phytotoxic Activity of p-Cresol, 2-Phenylethanol and 3-Phenyl-1-Propanol, Phenolic Compounds Present in Cistus ladanifer L. Plants 2021, 10, 1136. [Google Scholar] [CrossRef]
  20. Johnson, S.D.; Jürgens, A. Convergent evolution of carrion and faecal scent mimicry in fly-pollinated angiosperm flowers and a stinkhorn fungus. South African J. Bot. 2010, 76, 796–807. [Google Scholar] [CrossRef]
  21. Kyte, G.C.; Hetterscheid, W.L.A.; Lewis, M.J.; Boyce, P.C.; Ollerton, J.; Cocklin, E.; Diaz, A.; Simmonds, M.S.J. Inflorescence odours & pollinators Arum & Amoprphophallus. In Reproductive Biology; Owens, S.J., Rudall, P.J., Eds.; Royal Botanic Gardens: London, UK, 1998; pp. 295–315. [Google Scholar]
  22. Heberle, H.; Meirelles, G.V.; da Silva, F.R.; Telles, G.P.; Minghim, R. InteractiVenn: A web-based tool for the analysis of sets through Venn diagrams. BMC Bioinform. 2015, 16, 169. [Google Scholar] [CrossRef]
  23. Ertürk, Ö.; Ayvaz, M.Ç.; Çil, E.; Bağdatlı, E. Gas Chromatography-Mass Spectrometry Analysis and Antimicrobial and Antioxidant Activities of Some Orchid (Orchidaceae) Species Growing in Turkey. Braz. Arch. Biol. Technol. 2023, 66, e23210265. [Google Scholar] [CrossRef]
  24. Miranda, M.; Cuellar, A. Farmacognosia y Productos Naturales; La Habana Félix Varela: Havana, Cuba, 2001. [Google Scholar]
  25. Zeitler, K.; Rose, C.A. An Efficient Carbene-Catalyzed Access to 3,4-Dihydrocoumarins. J. Org. Chem. 2009, 74, 1759–1762. [Google Scholar] [CrossRef] [PubMed]
  26. Zhang, D.; Zhang, Z.; Wu, G.; Sun, Y.; Jiang, Y.; Zhang, H.; Song, X.; Wang, W.; Li, Y. Phenolic derivatives with cytotoxic activities from the roots of Fallopia multiflora var. ciliinervis. Phytochem. Lett. 2022, 52, 72–75. [Google Scholar] [CrossRef]
  27. ChemSpider. Available online: https://www.chemspider.com (accessed on 12 February 2024).
  28. Wang, X.; Zhong, X.-J.; Zhou, N.; Cai, N.; Xu, J.-H.; Wang, Q.-B.; Li, J.-J.; Liu, Q.; Lin, P.-C.; Shang, X.-Y. Rapid Characterizaiton of Chemical Constituents of the Tubers of Gymnadenia conopsea by UPLC–Orbitrap–MS/MS Analysis. Molecules 2020, 25, 898. [Google Scholar] [CrossRef] [PubMed]
  29. Huang, S.-Y.; Li, G.-Q.; Shi, J.-G.; Mo, S.-Y.; Wang, S.-J.; Yang, Y.-C. Chemical constituents of the rhizomes of Coeloglossum viride var. bracteatum. J. Asian Nat. Prod. Res. 2004, 6, 49–61. [Google Scholar] [CrossRef] [PubMed]
  30. Schütz, K.; Kammerer, D.R.; Carle, R.; Schieber, A. Characterization of phenolic acids and flavonoids in dandelion (Taraxacum officinale WEB. ex WIGG) root and herb by high-performance liquid chromatography/electrospray ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2005, 19, 179–186. [Google Scholar] [CrossRef] [PubMed]
  31. Cadahía, E.; Fernández de Simón, B.; Aranda, I.; Sanz, M.; Sánchez-Gómez, D.; Pinto, E. Non-targeted Metabolomic Profile of Fagus sylvatica L. Leaves using Liquid Chromatography with Mass Spectrometry and Gas Chromatography with Mass Spectrometry. Phytochem. Anal. 2015, 26, 171–182. [Google Scholar] [CrossRef] [PubMed]
  32. Gecchele, E.; Negri, S.; Cauzzi, A.; Cuccurullo, A.; Commisso, M.; Patrucco, A.; Anceschi, A.; Zaffani, G.; Avesani, L. Optimization of a Sustainable Protocol for the Extraction of Anthocyanins as Textile Dyes from Plant Materials. Molecules 2021, 26, 6775. [Google Scholar] [CrossRef] [PubMed]
  33. Loizzo, M.R.; Napolitano, A.; Bruno, M.; Geraci, A.; Schicchi, R.; Leporini, M.; Tundis, R.; Piacente, S. LC-ESI-HRMS analysis of glucosinolates, oxylipins and phenols in Italian rocket salad (Diplotaxis erucoides subsp. erucoides (L.) DC.) and evaluation of its healthy potential. J. Sci. Food Agric. 2021, 101, 5872–5879. [Google Scholar] [CrossRef] [PubMed]
  34. Neilson, A.P.; Hopf, A.S.; Cooper, B.R.; Pereira, M.A.; Bomser, J.A.; Ferruzzi, M.G. Catechin Degradation with Concurrent Formation of Homo- and Heterocatechin Dimers during In Vitro Digestion. J. Agric. Food Chem. 2007, 55, 8941–8949. [Google Scholar] [CrossRef]
  35. Lin, B.; Guo, S.; Hong, X.; Jiang, X.; Li, H.; Li, J.; Guo, L.; Li, M.; Chen, J.; Huang, B.; et al. Chemical Characterization and Metabolic Profiling of the Compounds in the Chinese Herbal Formula Li Chang Decoction by UPLC-QTOF/MS. Evid.-Based Complement. Altern. Med. 2022, 2022, 1322751. [Google Scholar] [CrossRef]
  36. De Agostini, A.; Robustelli della Cuna, F.S.; Cortis, P.; Cogoni, A.; Sottani, C.; Soddu, F.; Sanna, C. Volatile Organic Compounds (VOCs) Diversity in the Orchid Himantoglossum robertianum (Loisel.) P. Delforge from Sardinia (Italy). Diversity 2022, 14, 1125. [Google Scholar] [CrossRef]
  37. Adams, R. Identification of Essential Oil Components by Gas Chromatography/Mass Spectrometry, 4th ed.; Allured Publishing Cor-poration: Carol Stream, IL, USA, 2007. [Google Scholar]
  38. Stein, S.E. NIST/EPA/NIH Mass Spectral Database, Version 2.1; Perkin-Elmer Instrument LLC: Waltham, MA, USA, 2000. [Google Scholar]
  39. Joulain, D.; Konig, W.A. The Atlas of Spectral Data of Sesquiterpene Hydrocarbons; E. B. Verlag: Hamburg, Germany, 1998. [Google Scholar]
  40. Ribeiro, C.V.C.; Kaplan, M.A.C. Tendências evolutivas de famílias produtoras de cumarinas em angiospermae. Quim. Nova 2002, 25, 533–538. [Google Scholar] [CrossRef]
Plants 13 01064 g001
Figure 1. Orchis purpurea in its natural habitat in Pianlago Ponzone (Italy). Photo courtesy of Carlo Ugolotti.
Figure 1. Orchis purpurea in its natural habitat in Pianlago Ponzone (Italy). Photo courtesy of Carlo Ugolotti.
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Figure 2. Venn diagram with the number of compounds shared between different parts of O. purpurea.
Figure 2. Venn diagram with the number of compounds shared between different parts of O. purpurea.
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Figure 3. GC-MS chromatographic profile of n-hexane extract of leaves of O. purpurea and GC-MS EI spectrum of the main peak.
Figure 3. GC-MS chromatographic profile of n-hexane extract of leaves of O. purpurea and GC-MS EI spectrum of the main peak.
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Table 1. Chemical composition of essential oils from roots, stems, leaves, and flowers of Orchis purpurea.
Table 1. Chemical composition of essential oils from roots, stems, leaves, and flowers of Orchis purpurea.
CompoundCAS RootsStemsLeavesFlowersIdentification d
AI aAI b% c%%%
Octane111-65-98008000.07 ± 0.44--0.06 ± 0.04STD, MS, RI
Furfural98-01-1836831--0.09 ± 0.070.08 ± 0.05MS, RI
Diacetone alcohol123-42-28418401.11 ± 0.240.08 ± 0.07--MS, RI
Furfuryl alcohol98-00-0855855---0.21 ± 0.06MS, RI
1-Hexanol111-27-38718700.08 ± 0.06--0.08 ± 0.06MS, RI
Heptanal111-71-7902902---0.05 ± 0.03MS, RI
Unidentified--907--0.07 ± 0.060.22 ± 0.06-
Benzaldehyde100-52-79609580.07 ± 0.08 -0.07 ± 0.060.10 ± 0.04MS, RI
Phenol108-95-29859850.07 ± 0.06---MS, RI
1-Decene872-05-99909910.11 ± 0.08---MS, RI
Octanal124-13-099910030.08 ± 0.07---MS, RI
2,4-Heptadienal4313-03-510101010---0.07 ± 0.05MS, RI
2- Ethylhexanol104-76-7103110310.07 ± 0.06-0.05 ± 0.040.11 ± 0.07MS, RI
Benzyl alcohol100-51-6103210340.08 ± 0.08-0.05 ± 0.04-MS, RI
Phenylacetaldehyde122-78-1104210420.14 ± 0.11--0.09 ± 0.05MS, RI
Acetophenone98-86-2106510650.14 ± 0.110.13 ± 0.100.05 ± 0.03-MS, RI
p-Cresol106-44-5107610791.00 ± 0.2011.58 ± 0.320.44 ± 0.1112.68 ± 0.20MS, RI
2-Phenyl-2-propanol617-94-7108910860.13 ± 0.070.11 ± 0.09--MS, RI
Nonanal124-19-6110011050.21 ± 0.140.13 ± 0.110.05 ± 0.040.64 ± 0.06MS, RI
(2E)-2-Nonen-1-al 2463-53-811621161---0.09 ± 0.04MS, RI
1-nonanol143-08-8116911730.13 ± 0.100.07 ± 0.06--MS, RI
Unidentified--1185-0.12 ± 0.120.07 ± 0.060.10 ± 0.06-
1-Dodecene112-41-4119011920.18 ± 0.12---MS, RI
p-Methylguaiacol93-51-611921193-0.08 ± 0.070.04 ± 0.030.37 ± 0.09MS, RI
Decanal112-31-2120012060.17 ± 0.120.11 ± 0.090.04 ± 0.040.09 ± 0.05MS, RI
p-vinylphenol2628-17-3122112200.17 ± 0.12--1.20 ± 0.06MS, RI
2,3-Dihydro-benzofuran496-16-212211221-0.06 ± 0.06--MS, RI
3-(1-Methylethyl) phenol618-45-1122812290.09 ± 0.09---MS, RI
3,5-Dimethoxy-toluene4179-19-512641267---0.19 ± 0.06MS, RI
Nonanoic acid112-05-0127112760.08 ± 0.06--0.67 ± 0.05MS, RI
Unidentified--1308---0.41 ± 0.08-
2-Methoxy-4-vinylphenol7786-61-0131513150.16 ± 0.130.08 ± 0.06-0.08 ± 0.06MS, RI
(2E,4E)-2,4-Decadienal 25152-84-5131713170.21 ± 0.20--0.08 ± 0.07MS, RI
p-Hydroxybenzyl alcohol623-05-2135713560.11 ± 0.13--0.11 ± 0.06MS, RI
Decanoic acid334-48-513721372---0.05 ± 0.04MS, RI
Unidentified--13790.17 ± 0.16--0.11 ± 0.06-
3,4-dihydro-coumarin119-84-613781384-0.12 ± 0.080.07 ± 0.030.08 ± 0.07MS, RI
(E)-damascenone 23726-93-413851386---0.12 ± 0.06MS, RI
2-Tetradecene26952-13-6138913930.22 ± 0.17---MS, RI
Tetradecane629-59-414001400---0.05 ± 0.04STD, MS, RI
Coumarin91-64-51445145532.30 ± 0.3285.98 ± 0.2798.79 ± 0.3469.64 ± 0.22MS, RI
2,6-Di-tert-butyl-p-benzoquinone719-22-214691469---0.11 ± 0.06MS, RI
Unidentified--1560---0.31 ± 0.12-
Dodecanoic acid143-07-7156515660.26 ± 0.17--0.23 ± 0.12MS, RI
1-Hexadecene629-73-2159015920.21 ± 0.15--0.20 ± 0.09MS, RI
Hexadecane544-76-3160016000.10 ± 0.08---STD, MS, RI
Methyl dihydro jasmonate24851-98-7165616570.16 ± 0.15---MS, RI
Tetradecanoic acid544-63-8178017650.16 ± 0.14--0.24 ± 0.10MS, RI
1-Octadecene112-88-9179017940.19 ± 0.20---MS, RI
Octadecane593-45-3180018000.15 ± 0.11---MS, RI
1-Methylethyl tetradecanoate110-27-0182818280.14 ± 0.10---MS, RI
Cyclohexadecane295-65-818811881---0.46 ± 0.07MS, RI
1-Hexadecanol36653-82-41876188714.03 ± 0.21---MS, RI
Nonadecane629-92-5190019000.12 ± 0.11--0.10 ± 0.05STD, MS, RI
7,9-Di-tert-butyl-1-oxaspiro-(4,5)-deca-6,9-diene82304-66-3192919230.06 ± 0.06---MS, RI
Hexadecanoic acid57-10-3197219723.51 ± 0.12--2.54 ± 0.12MS, RI
Ethyl hexadecanoate628-97-7199519950.09 ± 0.09--0.15 ± 0.07MS, RI
Isopropyl palmitate142-91-6202620260.09 ± 0.06---MS, RI
(E)-15-heptadecenal700381-35-72085208543.39 ± 0.400.20 ± 0.11-0.86 ± 0.08MS, RI
Heneicosane629-94-721002100-0.12 ± 0.06-0.88 ± 0.08STD, MS, RI
Unidentified------0.16 ± 0.04-
Docosane629-97-022002200-0.10 ± 0.06--STD, MS, RI
Unidentified--2271-0.12 ± 0.11---
Tricosane638-67-523002300-0.10 ± 0.07-0.73 ± 0.11STD, MS, RI
Tetracosane646-31-124002400-0.21 ± 0.11--STD, MS, RI
9-pentacosene51865-00-024742475---0.07 ± 0.05MS, RI
1-Docosanol661-19-824932493--0.03 ± 0.020.27 ± 0.06MS, RI
Pentacosane629-99-225002500-0.18 ± 0.150.04 ± 0.032.53 ± 0.05STD, MS, RI
Hexacosane630-01-326002600-0.19 ± 0.15-0.22 ± 0.07STD, MS, RI
Heptacosane593-49-727002700-0.14 ± 0.110.04 ± 0.032.10 ± 0.07STD, MS, RI
a Kovats RI according to Adams [ibidem], b RI determined on an Elite-5 column using a homologous series of n-alkanes, c results are the mean of three experiments ± SD. d Method of identification: STD, standard; MS, mass spectrum in comparison with library [ibidem]; RI, retention indices in agreement with literature values.
Table 2. Chemical classes and their relative abundance in the essential oils from O. purpurea.
Table 2. Chemical classes and their relative abundance in the essential oils from O. purpurea.
ClassRootsStemsLeavesFlowers
Acids4.00--3.74
Alcohols16.1211.910.6115.13
Aldeydes44.280.430.252.15
Esters (among which coumarin)32.77 (32.30)86.10 (86.10)98.86 (98.86)69.87 (69.72)
Ketones1.110.080.050.52
Saturated hydrocarbons0.441.040.087.13
Unsaturated hydrocarbons0.90--0.26
Unidentified0.310.370.140.91
Miscellanea0.06--0.19
Oxygenated monoterpenes---0.12
Table 3. Yields (%) of Soxhlet extraction.
Table 3. Yields (%) of Soxhlet extraction.
Extractive Solvent
n-HexaneEthyl AcetateMethanol
Leaves0.33%0.16%3.83%
Flowers0.95%2.05%12.77%
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Cavalloro, V.; Pagliari, S.; Gosetti, F.; Campone, L.; Sottani, C.; Collina, S.; Martino, E.; Robustelli della Cuna, F.S. Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches. Plants 2024, 13, 1064. https://doi.org/10.3390/plants13081064

AMA Style

Cavalloro V, Pagliari S, Gosetti F, Campone L, Sottani C, Collina S, Martino E, Robustelli della Cuna FS. Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches. Plants. 2024; 13(8):1064. https://doi.org/10.3390/plants13081064

Chicago/Turabian Style

Cavalloro, Valeria, Stefania Pagliari, Fabio Gosetti, Luca Campone, Cristina Sottani, Simona Collina, Emanuela Martino, and Francesco Saverio Robustelli della Cuna. 2024. "Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches" Plants 13, no. 8: 1064. https://doi.org/10.3390/plants13081064

APA Style

Cavalloro, V., Pagliari, S., Gosetti, F., Campone, L., Sottani, C., Collina, S., Martino, E., & Robustelli della Cuna, F. S. (2024). Qualitative Metabolite Profiling of Orchis purpurea Huds. by GC and UHPLC/MS Approaches. Plants, 13(8), 1064. https://doi.org/10.3390/plants13081064

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