Analysis of the Volatile and Enantiomeric Compounds Emitted by Plumeria rubra L. Flowers Using HS-SPME–GC

The volatile components emitted by fresh aromatic flowers of Plumeria rubra L., harvested in southern Ecuador during three different months were determined to evaluate the fluctuation of secondary metabolites. The volatile compounds were analyzed using headspace solid-phase microextraction (HS-SPME) followed by gas chromatography coupled to mass spectrometry (GC–MS) and a flame ionization detector (GC–FID) using two types of columns: a non-polar (DB-5ms) and polar column (HP-INNOWax). The principal chemical groups were hydrocarbon sesquiterpenes (43.5%; 40.0%), oxygenated sesquiterpenes (23.4%; 26.4%), oxygenated monoterpenes (14.0%; 11.2%), and hydrocarbon monoterpenes (12.7%; 9.3%). The most representative constituents were (E,E)-α-Farnesene (40.9–41.2%; 38.5–50.6%), (E)-nerolidol (21.4–32.6%; 23.2–33.0%), (E)-β-ocimene (4.2–12.5%; 4.5–9.1%), (Z)-dihydro-apofarnesol (6.5–9.9%; 7.6–8.6%), linalool (5.6–8.3%; 3.3–7.8%), and perillene (3.1–5.9%; 3.0–3.2%) in DB-5ms and HP-INNOWax, respectively. Finally, we reported for the first time the enantiomeric distribution of P. rubra flowers, where the enantiomers (1R,5R)-(+)-α-pinene, (S)-(−)-limonene, (S)-(+)-Linalool, and (1S,2R,6R,7R,8R)-(+)-α-copaene were present as enantiomerically pure substances, whereas (S)-(+)-(E)-Nerolidol and (R)-(+)-(E)-Nerolidol were observed as scalemic mixtures. This study provides the first comprehensive and comparative aroma profile of Plumeria rubra cultivated in southern Ecuador and gave us a clue to the variability of P. rubra chemotypes depending on the harvesting time, which could be used for future quality control or applications in phytopharmaceutical and food industries.


Introduction
Plants have been used for medicinal purposes by humans since ancient times, and their properties have contributed to the elimination of diseases and thus to survival [1].According to WHO estimates, about 80% of the population uses herbal medicine for primary health care despite the growing technology of organic synthesis [2].Apocynaceae, a family of flowering plants, belongs to the order Gentianales.It is one of the ten largest angiosperm families globally [3][4][5], consisting of approximately 366 genera and around 5100 species [6][7][8].The family is divided into five subfamilies: Rauvolfioideae, Apocynoideae, Periplocoideae, Secamonoideae, and Asclepiadoideae.Apocynaceae has a widespread distribution, with members native to various regions, including Europe, Asia, Africa, Australia, and the Americas [8][9][10].
Ecuador, despite having an area of 283,561 km 2 , is characterized by a diversity of ecosystems with different microclimates and habitats [11].Thus, due to its high biological and cultural diversity, it has become one of the countries with great potential in traditional medicine [12].In this context, the genus Plumeria consists of many species distributed all Plants 2024, 13, 2367 2 of 17 over the world, 11 of which grow in tropical and subtropical regions [13].Both essential oil and the aromatic components of the flowers of many of these species are used in perfumery, cosmetics, and aromatherapy [14].The most popular species are Plumeria obtusa L., Plumeria alba, and Plumeria rubra L. [13].
Plumeria rubra L. is commonly known as "flor de mayo" and belongs to the Apocynaceae family and is native to Mexico [15].However, due to its easy propagation by cutting, it has spread throughout the world, especially in warm regions such as Hawaii, where it is cultivated in abundance [16].It grows as a small tree that can reach a height of two to eight meters [13].In terms of medicinal use, it is reported that the decoction of P. rubra is traditionally used to treat asthma, constipation, to stimulate menstruation, and to reduce fever [17].It is important to note that this species, which does not have a nectary, is pollinated by insects through floral mimicry [18].In our country, it can be found in provinces such as Chimborazo, Los Ríos, El Oro, Manabí, Guayas, Esmeraldas, Imbabura, and the Galapagos Islands [19].
A gas chromatograph coupled to a mass spectrometer (GC-MS) was used to identify the aromatic compounds of the species [22] and extracted using headspace solid-phase microextraction (HS-SPME), which is currently quite dominant due to its simplicity, absence of solvents, high sensitivity, and low cost [23].This method is based on a fiber coated with one or more extraction polymers, which removes the analytes from the sample by adsorption to be subsequently introduced into the GC-MS system for thermal desorption and analysis [24].
The aim of this research was to determine the chemical composition and report, for the first time, the enantiomeric distribution of some terpenes emitted by P. rubra flowers that provides its characteristics such as its odor or therapeutic properties.In this way, it will lay the foundation for future research and, at the same time, contribute to the knowledge of new techniques for extracting compounds, since the technique to be tested (HS-SPME) has not been studied much in this field, although it has advantages over other common methods such as steam distillation or hydrodistillation, such as the time required and the absence of solvents for extracting compounds [25].
Plants 2024, 13, 2367 7 of 17 In order to complement the information on the main compounds identified in this study, the biological activities related to therapeutic effects on the organism is presented in Table 3.
Table 3.Chemical structure, odor, and biological properties of the major compounds identified.

Compounds
Odor Biological Properties Ref.
(E,E)-α-Farnesene In order to complement the information on the main compounds identified in this study, the biological activities related to therapeutic effects on the organism is presented in Table 3.In order to complement the information on the main compounds identified in this study, the biological activities related to therapeutic effects on the organism is presented in Table 3.

Enantiomeric Distribution
The enantioselective analysis permitted the identification of three enantiomerically

Enantiomeric Distribution
The enantioselective analysis permitted the identification of three enantiomerically pure compounds in P. rubra flowers.They were (1R,5R)-(+)-α-pinene; (S)-(−)-limonene; (S)-(+)-Linalool; (1S,2R,6R,7R,8R)-(+)-α-copaene; and (E)-nerolidol.Detailed results of the enantioselective analysis are given in Table 4, Figure 3.The PCA of the chemical composition of the volatile compounds of flowers using the DB-5ms column (Figure 4) showed different compounds found in the different months of collection.After the PCA analysis, it was possible to determine the dispersion of ical composition obtained after chromatographic analysis on the DB-5ms column.The first component accounted for 95.41% of the total variance in the data set, characterized by the compounds (Z)-β-farnesene, cedrol, δ-decalactone, perillene, and junenol, among others, while the second component accounted for 4.58% of the variance, characterized by the compounds (E)-β-ocimene, (E,E)-α-farnesene, (E)-nerolidol, and linalool, among others.In terms of similarity, the months of March and May showed greater variability than The PCA of the chemical composition of the volatile compounds of flowers using the DB-5ms column (Figure 4) showed different compounds found in the different months of collection.After the PCA analysis, it was possible to determine the dispersion of the chemical composition obtained after chromatographic analysis on the DB-5ms column.The first component accounted for 95.41% of the total variance in the data set, characterized by the compounds (Z)-β-farnesene, cedrol, δ-decalactone, perillene, and junenol, among others, while the second component accounted for 4.58% of the variance, characterized by the compounds (E)-β-ocimene, (E,E)-α-farnesene, (E)-nerolidol, and linalool, among others.In terms of similarity, the months of March and May showed greater variability than July.In the PCA analysis of HP-INNOWax column, the first component accounted for 85.24% of the total variance in the data set, characterized by the compounds 1-hexanol (E)-tridecen-1-ol, (2E)-hexenal, hexanal, and (E,E)-α-farnesene, among others, while the second component accounted for 15.75% of the variance in the data set, characterized by the compounds linalool, (E)-β-ocimene, perillene, and (E)-nerolidol.In terms of similarity the months of March and May showed a higher variance (Figure 5).In the PCA analysis of HP-INNOWax column, the first component accounted for 85.24% of the total variance in the data set, characterized by the compounds 1-hexanol, (E)-tridecen-1-ol, (2E)-hexenal, hexanal, and (E,E)-α-farnesene, among others, while the second component accounted for 15.75% of the variance in the data set, characterized by the compounds linalool, (E)-β-ocimene, perillene, and (E)-nerolidol.In terms of similarity, the months of March and May showed a higher variance (Figure 5).

Discussion
The use of orthogonal columns confirms the identification of a greater variety of compounds [57].According to the results, approximately 53-59 different compounds were identified in both columns; only 14 of them were found in both phases, including the main constituents.This affinity of the stationary phase results in a different elution order.In the non-polar column, the stationary phase (5% phenylpolydimethylsiloxane) presents an affinity for the less polar compounds and will therefore retain them until the end of the chromatographic run, eluting the non-polar compounds first.This contrasts the stationary phase (polyethylene glycol) of the polar column, which has a higher affinity for the polar compounds, eluting the polar compound first [57].

Discussion
The use of orthogonal columns confirms the identification of a greater variety of compounds [57].According to the results, approximately 53-59 different compounds were identified in both columns; only 14 of them were found in both phases, including the main constituents.This affinity of the stationary phase results in a different elution order.In the non-polar column, the stationary phase (5% phenylpolydimethylsiloxane) presents an affinity for the less polar compounds and will therefore retain them until the end of the chromatographic run, eluting the non-polar compounds first.This contrasts the stationary phase (polyethylene glycol) of the polar column, which has a higher affinity for the polar compounds, eluting the polar compound first [57].
The chemical compound (E)-β-ocimene, one of the main compounds reported in our study, was also mentioned in the study by Barreto et al. (2014) [18], but as a minority.For the majority of reported compounds, including (Z)-dihydro-apofarnesol and perillene, no reports were found, even in other analyses performed on species of the same genus.
Chemical variability is associated with primary causal factors, including genetic differences between plants; the growing environment (humidity, sunlight, soil, and nutrient bioavailability); the life cycle stage of the plants, as their composition may vary at early or late stages; biological interactions with animal species such as pollinators [61]; and the time between collection and extraction, as a longer interval may lead to a decrease or change in the composition of the volatile analytes present [63].
For the first time, as a contribution of new knowledge, it is reported the enantioseparation of some terpenes, and for one of them, i.e., E-nerolidol, the presence of the distomer R has been detected, although at a very low level (0.14%), in P. rubra flowers.It is well known that enantiomers are chiral compounds with identical physical and chemical properties, except for their optical activity, and can also exhibit different biological effects, and some species use their stereochemical properties for communication [64].
The HS-SPME analysis differs from the hydrodistillation used in the studies by Barreto et al. (2014) [18], ElZanaty et al. (2022) [58], and Goswami et al. (2016) [14].In addition, both techniques aim to extract volatile compounds, with the difference that hydrodistillation is applied more to essential oils and extracts a wide range of volatile compounds, some of which may be difficult to capture with HS-SPME [64].However, considering that our study works with flowers of a cultivated species, the sample size is limited, since the application of HS-SPME is more selective and faster due to its automated facility, short analysis time, and was used with different types of biological samples [25] and a small amount of sample [65].

Plant Material
Plumeria rubra L. fresh flowers were collected during the morning in the sector of San Antonio, San Pablo de Tenta, Saraguro, Loja, with the coordinates 3 • 30 59.1 S 79 • 17 36.9W (Figure 6).The collection was authorized by the Ministry of Environment, Water and Ecological Transition (MAATE) with authorization code MAATE-ARSFC-2022-2839.The authenticity of the species was verified by Ing.Jorge Armijos, curator of the HUTPL herbarium, who registered with voucher number 14,778.Helium (99.999% purity) (Indura, Guayaquil, Ecuador) was used as carrier gas at a constant flow rate of 1 mL/min [68].The injection mode was split (10:1) with a temperature of 250 • C at the injector.The ion source temperature was set to 230 • C and 150 • C quadrupled.The chromatography oven was programmed from 40 to 150 • C (3 • C/min), then to 180 • C (5 • C/min) for 5 min, and finally to 230 • C (7 • C/min) for a total run time of 67 min.

Analysis of Volatile Compounds GC-FID
For quantitative analysis, the same equipment was used as for the GC-MS analysis, except that it was coupled to a flame ionization detector (GC-FID).Samples were injected under the same conditions as described above.The injector temperature was 270 • C and the injector gas mixture was UHP hydrogen (30 mL/min), zero grade air (300 mL/min), and UHP nitrogen (45 mL/min) [69] using a Parker gas generator hydrogen generator (UK-UK).The content (%) of each identified oil component was calculated as the % of the area of the corresponding peak in the gas chromatography-flame ionization detector (GC-FID) chromatogram compared to the sum of the areas of all identified peaks.No correction factor was applied.

Compound Identification
The components of the flowers were identified by comparing the linear retention indices (LRIs), calculated according to Van den Dool and Kratz (70), and mass spectra with data from the literature.A mixture of n-alkanes C 9 -C 24 (ChemService, West Chester, PA, USA) [70] was used.For compounds analyzed on the DB-5ms column, peaks were identified by comparing mass spectra with their LRIs using the ADAMS book [26].For the HP-INNOWax column, the NIST library (NIST Libraries, National Institute of Standards and Technology, Gaithersburg, MD, USA) was used [71].The compound was considered identified if the calculated retention index did not differ by ±25 from the reference values [22].

Enantiomeric Analysis
The chromatographic conditions to achieve the enantioseparation of some chiral terpenes present in P. rubra flowers involved the use of a chiral capillary column MEGA-DEX-DAC based on 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin (25 m, 0.25 mm film thickness, 0.25 µm, purchased from MEGA S.r.l.(Legnano, MI, Italy)).The temperature programme for the gas chromatography (GC) oven was as follows: an initial temperature of 50 • C for 1 min, followed by a gradient increase of 2 • C per minute until 220 • C; finally, it was maintained for 10 min.In addition, the enantiomers were identified and compared by their MS spectrum, linear retention indices from the bibliography, and by the injection of enantiomerically pure standards (Sigma-Aldrich, St. Louis, MO).Using the formula originally proposed by van Den Dool and Kratz, we calculated linear (arithmetic) retention indices [68,72].

Statistical Analysis
The analysis of volatile compounds in relation to the harvest time and the type of column used was carried out using principal component analysis (PCA), a multivariate analysis technique that makes it possible to visualize the similarities or differences between a group of data, which in this study refers to the compounds found [73].All these analyses were carried out using the statistical software PAST 4.10 [74].

Conclusions
In this study, we reported for the first time the volatile composition of Plumeria rubra L. flowers using a HS-SPME-GC from Ecuador, and it was found that there was a significant difference between collection times of the species.The chemical groups more representative were hydrocarbon sesquiterpenes (43.48%), followed by oxygen sesquiterpenes (26.92%), oxygen monoterpenes (10.95%), alcohols (8.25%), and hydrocar-

Figure 3 .
Figure 3. Enantiomeric analysis of the Plumeria rubra L. flowers using the β-cyclodextrin column.

Figure 3 .
Figure 3. Enantiomeric analysis of the Plumeria rubra L. flowers using the β-cyclodextrin column.

Figure 4 .
Figure 4. PCA analysis of the Plumeria rubra L. flowers using the DB-5ms column.

Figure 4 .
Figure 4. PCA analysis of the Plumeria rubra L. flowers using the DB-5ms column.

Figure 5 .
Figure 5. PCA analysis of the Plumeria rubra L. flowers using the HP-INNOWax column.

Figure 5 .
Figure 5. PCA analysis of the Plumeria rubra L. flowers using the HP-INNOWax column.

Table 1 .
Volatile chemical compounds of Plumeria rubra L. species detected using polar DB-5ms column.

Table 2 .
Volatile chemical compounds of Plumeria rubra L. species detected using polar HP-INNOWax column.
b : linear (arithmetic) calculated retention index; LRI a : linear (arithmetic) retention index according to references; % ± SD: area percentage and standard deviation of triplicate injections; NI: not identified.

Table 2 .
Volatile chemical compounds of Plumeria rubra L. species detected using polar HP-INNOWax column.Figure 1. Gas chromatogram of the Plumeria rubra L. flowers, obtained using DB-5ms column.

Table 3 .
Chemical structure, odor, and biological properties of the major compounds identified.

Table 3 .
Chemical structure, odor, and biological properties of the major compounds identified.

Table 3 .
Chemical structure, odor, and biological properties of the major compounds identified.

Table 4 .
Enantiomeric distribution of Plumeria rubra L. flowers from Ecuador on β-cyclodextrin column.