The Chemical and Enantioselective Analysis of a New Essential Oil Produced by the Native Andean Species Aiouea dubia (Kunth) Mez from Ecuador

Abstract

The present study constitutes the first description of an essential oil, distilled from the leaves of the native Andean species Aiouea dubia (Kunth) Mez. The qualitative and quantitative analyses were carried out through GC–MS and GC-FID respectively, using two columns coated with stationary phases of different polarity. The qualitative composition was determined by comparison of linear retention indices and mass spectra with data from literature, whereas the quantitative composition was obtained through external calibration, calculating the relative response factor of each compound according to the respective combustion enthalpy. Major constituents (≥3.0 on at least one column) of A. dubia EO, on a nonpolar and a polar stationary phase respectively, were germacrene D (12.2%–11.7%), γ-muurolene (7.6%–7.2%), limonene (6.8%–6.2%), δ-cadinene (6.4%–5.9%), cyclosativene (6.0%–5.5%), and (E)-β-caryophyllene (4.4%–4.0%). The enantioselective analysis was also conducted on two different columns, based on β-cyclodextrin as chiral selector. A total of 11 chiral terpenes and terpenoids were analyzed, of which (1S,5S)-(−)-α-pinene and (1R,6S)-(−)-3-carene were enantiomerically pure, whereas the others were observed as scalemic mixtures. Cosmeceutical science could be the main application field for this volatile fraction. In fact, the high content of limonene and (E)-β-caryophyllene suggested that this EO could present antibacterial and anti-inflammatory properties, like other volatile fractions of similar composition.

1. Introduction

Since ancient times, plants have been used as drugs and as a source of fragrances, for both cosmetic and ritual purposes. With the introduction of the scientific method, these properties have been attributed to the presence of secondary or specialized metabolites, whose purification and structure elucidation greatly contributed to the progress of medicinal chemistry and cosmetology. Nowadays, most of the European botanical species have been phytochemically investigated, and their metabolic profiles are quite well-known. Therefore, in search for new natural products, chemists are currently focusing on the flora of so-called “megadiverse countries”, whose biodiversity includes a great fraction of all biological species around the world. Among the megadiverse countries, Ecuador is included. In this territory, historical and logistical reasons avoided that most of the native and endemic botanical species could be so far studied for their metabolic content. , For these reasons, our group has been investigating the Ecuadorian biodiversity for more than 20 years, in search for new natural molecules, biologically active compounds, and unprecedented essential oils (EOs). − In this context, some of the authors recently described a volatile fraction from Aiouea montana (Sw.) R. Rohde, whose composition was dominated by a peculiar, sulphurated compound, responsible for the strong unpleasant smell of the leaves. On the other hand, the leaves of the less common Aiouea dubia did not present a similar smell but a pleasant, quite sweet fragrance. From the botanical point of view, the genus Aiouea Aubl. is currently considered, by some botanists, the correct classification for all the Neotropical taxa, that were previously included into the genera Phoebe and Cinnamomum. The taxon Aiouea is therefore native of America, and it includes 73 species with an accepted name, whereas the reported synonyms are more than 200. For what concerns A. dubia, this species is a shrub, treelet or tree, native of the Andes, and diffused from Venezuela to Peru. − In Ecuador, this plant grows in the range between 2000 and 3000 m above the sea level, being described in the provinces of Loja and Zamora-Chinchipe. Some synonyms are also reported for A. dubia, such as Aiouea granatensis Mez, Aiouea jelskii Mez, Aiouea tambillensis Mez, Aiouea truxillensis Kosterm, Cryptocarya dubia Kunth, Endocarpa corymbosa Raf., Laurus hypericifolia Willd. ex Nees, and Persea hypericifolia Nees. So far, to the best of the authors’ knowledge, only the EOs from Aiouea costaricensis, Aiouea maguireana, and A. montana have been investigated within this genus, whereas no literature has been found describing the chemical and the enantiomeric profiles of any volatile fraction from A. dubia or its synonyms. ,, With these premises, the species A. dubia (Kunth) Mez (Lauraceae) was selected to be analyzed for the chemical and enantiomeric compositions of its EO. Main purposes of the present study were contributing to the preservation of the Ecuadorian biodiversity through the phytochemical knowledge and identifying potential sources of new natural products, suitable to be the object of bioeconomic applications.

2. Results and Discussion

2.1. Chemical Analysis

The dry leaves of A. dubia afforded, after preparative steam-distillation, a clear pale-yellow oil, characterized by a sweet light fragrance. The distillation yield, by weight, was 0.3%. A total of 69 compounds were detected and quantified on at least one of two chromatographic columns. The columns were coated with different stationary phases, being 5%-phenyl-methyl-polysiloxane a nonpolar phase and polyethylene glycol a polar phase. The great difference in polarity among these two phases ensured that chromatographic properties were also different enough to obtain a good separation, for almost all compounds, on at least one column. Furthermore, different chromatographic properties produced different linear retention indices for a same compound, ensuring a more reliable identification through the coincide of both linear retention indices with data from literature. Finally, these specific stationary phases are the most used in EO analysis, ensuring that a lot of literature is available as a source of reference linear retention indices. The total mass of the quantified components corresponded to 85.1%–82.8% of the whole oil mass, expressed as the sum of all quantified compounds on the nonpolar and polar column, respectively. This volatile fraction was dominated by sesquiterpenes and sesquiterpenoids, corresponding together to 65.5%–61.4% of the total amount. Major components of the EO (≥3.0% on at least one column) were germacrene D (12.2%–11.7%, 45), γ-muurolene (7.6%–7.2%, 44), limonene (6.8%–6.2%, 14), δ-cadinene (6.4%–5.9%, 51), cyclosativene (6.0%–5.5%, 32), and (E)-β-caryophyllene (4.4%–4.0%, 36). The chemical structures of the main EO components are represented in Figure , whereas the gas chromatographic (GC) profiles, obtained on a nonpolar and a polar stationary phase, are respectively represented in Figure and Figure . The complete qualitative and quantitative analyses are reported in Table .

1.

Major components (≥3.0% on at least one column) of A. dubia leaf EO. The numbers refer to Table : limonene (14), cyclosativene (32), (E)-β-caryophyllene (36), γ-muurolene (44), germacrene D (45), and δ-cadinene (51).

2.

GC–MS profile of A. dubia EO on a 5%-phenyl-methyl-polysiloxane stationary phase. The peak numbers refer to major compounds (≥3.0% on at least one column) in Table .

3.

GC–MS profile of A. dubia EO on a polyethylene glycol stationary phase. The peak numbers refer to major compounds (≥3.0% on at least one column) in Table .

1. Qualitative (GC–MS) and Quantitative (GC-FID) Chemical Composition of A. dubia EO on 5%-Phenyl-methyl-polysiloxane and Polyethylene Glycol Stationary Phases ^,

		5%-phenyl-methyl-polysiloxane						polyethylene glycol
N.	compounds	RT	LRI	LRI	%	σ	reference	RT	LRI	LRI	%	σ	reference
1	thujene	15.02	925	924	0.3	0.01		6.12	1023	1023	0.5	0.01
2	α-pinene	15.48	930	932	0.8	0.08		5.94	1018	1019	0.5	0.08
3	α-fenchene	16.58	940	945	0.1	0.01		7.43	1059	1056	0.1	0.01
4	sabinene	18.66	970	969	0.6	0.03		12.79	1161	1156	0.1	0.01
5	β-pinene	18.83	972	974	0.6	0.03		9.26	1105	1105	0.8	0.03
6	6-methyl-5-hepten-2-one	19.77	984	981	0.1	0.01		25.22	1342	1345	trace
7	β-myrcene	20.25	989	988	0.8	0.04		13.09	1165	1167	1.0	0.04
8	4-methylen-1-(1-methylethyl)-cyclohexene	20.86	997	1002	trace			15.63	1205	1206	trace
9	α-phellandrene	21.13	1000	1002	0.3	0.01		12.80	1161	1160	0.1	0.01
10	δ-3-carene	21.33	1003	1008	0.3	0.04		12.37	1154	1152	0.8	0.02
11	δ-2-carene	21.62	1006	1001	1.7	0.10		11.74	1144	1146	1.9	0.14
12	α-terpinene	22.15	1013	1014	0.1	0.01		13.75	1176	1175	0.1	0.04
13	p-cymene	22.56	1018	1024	0.6	0.04		20.08	1268	1268	0.6	0.02
14	limonene	23.15	1025	1029	6.8	0.28		15.00	1195	1195	6.2	0.16
15	1,8-cineole	23.23	1026	1026	1.8	0.25		15.43	1202	1203	2.9	0.30
16	unidentified (mw = 136)	23.48	1030		1.1	0.08		10.68	1128		1.2	0.09
17	(Z)-β-ocimene	24.07	1037	1032	0.6	0.04		18.00	1238	1239	1.0	0.04
18	(E)-β-ocimene	24.88	1047	1050	0.1	0.01		19.11	1254	1253	trace
19	unidentified (mw = 136)	25.43	1054		0.3	0.05		11.73	1144		0.5	0.02
20	γ-terpinene	25.59	1056	1054	0.6	0.04		18.31	1243	1243	1.0	0.04
21	3-methyl-6-(1-methylethylidene)-cyclohexene	27.77	1083	1085	0.1	0.01		20.55	1274	1272	0.3	0.02
22	1-methyl-4-(1-methylethylidene)-cyclohexene	27.91	1085	1086	0.3	0.08		20.88	1279	1279	0.3	0.02
23	linalool	28.88	1097	1095	0.1	0.02		42.08	1554	1556	0.1	0.01
24	n-nonanal	29.21	1101	1100	0.2	0.02		29.17	1399	1400	0.2	0.01
25	unidentified (mw = 136)	30.26	1115		0.7	0.08		24.46	1331		0.7	0.03
26	borneol	33.71	1160	1165	0.3	0.05		47.93	1705	1705	0.2	0.04
27	terpinen-4-ol	34.61	1172	1174	0.2	0.02		42.10	1604	1605	0.3	0.04
28	α-terpineol	35.61	1186	1186	0.1	0.02
29	α-cubebene	46.89	1348	1348	0.9	0.08		32.46	1449	1449	0.8	0.07
30	isoledene	48.01	1365	1374	0.8	0.09		33.54	1466		0.8	0.04
31	α-ylangene	48.31	1369	1373	0.5	0.06		33.88	1471	1470	0.3	0.05
32	cyclosativene	48.62	1374	1369	6.0	0.15		34.44	1480	1483	5.5	0.17
33	β-bourbonene	49.16	1382	1387	0.3	0.05		36.08	1506	1504	0.1	0.02
34	β-cubebene	49.53	1388	1387	0.7	0.05		37.50	1529	1529	0.9	0.07
35	unidentified (mw = 204)	49.90	1393		1.3	0.06		32.57	1448		1.3	0.07
36	(E)-β-caryophyllene	51.37	1416	1415	4.4	0.09		40.83	1583	1583	4.0	0.11
37	β-copaene	51.99	1426	1430	0.3	0.08		40.47	1577	1580	0.7	0.04
38	aromadendrene	52.59	1436	1439	0.2	0.05		43.61	1630	1637	0.3	0.05
39	cis-muurola-3,5-diene	53.06	1444	1448	0.3	0.07		43.44	1627		0.1	0.01
40	trans-muurola-3,5-diene	53.30	1447	1448	0.3	0.06		42.95	1619		0.7	0.01
41	α-humulene	53.49	1451	1452	1.8	0.13		45.08	1656	1657	2.1	0.06
42	cis-muurola-4(14),5-diene	54.08	1460	1465	0.7	0.09		47.66	1700		0.6	0.04
43	dauca-5,8-diene	54.79	1471	1471	0.6	0.10		44.72	1649		0.6	0.03
44	γ-muurolene	55.01	1475	1473	7.6	0.26		46.44	1679	1681	7.2	0.09
45	germacrene D	55.27	1479	1480	12.2	0.48		47.47	1697	1699	11.7	0.10
46	trans-muurola-4(14),5-diene	55.87	1489	1493	0.6	0.11		45.22	1656		0.5	0.09
47	cis-β-guaiene	56.07	1492	1492	2.7	0.52		47.70	1701	1702	2.5	0.50
48	α-muurolene	56.43	1498	1500	0.7	0.12		48.52	1716	1716	0.3	0.03
49	δ-amorphene	56.84	1505	1511	0.6	0.09		50.53	1752		0.6	0.11
50	γ-cadinene	57.24	1511	1513	2.9	0.18		50.31	1748	1752	2.4	0.17
51	δ-cadinene	57.85	1522	1522	6.4	0.32		50.44	1751	1752	5.9	0.06
52	trans-cadina-1,4-diene	58.33	1530	1533	0.7	0.13	17	51.56	1771		0.3	0.07
53	α-cadinene	58.64	1535	1537	0.6	0.06		52.16	1782		0.6	0.09
54	α-calacorene	58.89	1540	1544	0.8	0.07		58.78	1907	1906	0.6	0.04
55	unidentified (mw = 220)	59.20	1545		1.4	0.07		60.37	1939		1.5	0.05
56	β-calacorene	60.12	1561	1564	0.1	0.05		60.98	1951	1954	0.1	0.01
57	spathulenol	60.91	1574	1578	0.6	0.06		69.39	2123	2121	0.6	0.02
58	salvial-4(14)-en-1-one	61.84	1590	1594	0.2	0.06		63.15	1994	1995	0.2	0.01
59	β-atlantol	62.90	1608	1608	0.3	0.04
60	1,10-di-epi-cubenol	63.11	1612	1618	0.3	0.06		66.07	2055	2054	0.2	0.01
61	junenol	63.28	1615	1618	0.8	0.04		65.71	2047	2052	0.7	0.09
62	α-corocalene	63.52	1619	1622	0.6	0.08		66.14	2056		0.7	0.05
63	1-epi-cubenol	63.85	1625	1627	0.6	0.11		66.27	2059		0.6	0.03
64	cubenol	64.06	1629	1619	0.2	0.03		70.30	2152		0.1	0.02
65	epi-α-cadinol	64.58	1638	1638	2.1	0.10		72.23	2201	2204	2.0	0.10
66	α-muurolol	64.85	1643	1644	0.5	0.07		71.33	2178	2177	0.5	0.02
67	α-cadinol	65.30	1652	1652	2.3	0.09		73.04	2238	2235	2.1	0.12
68	cadalene	66.39	1671	1675	0.1	0.01		72.64	2220		0.2	0.05
69	eudesma-4(15),7-dien-1β-ol	67.02	1682	1687	0.5	0.07		75.29	2340	2333	0.5	0.05
	monoterpenes				16.8						17.7
	oxygenated monoterpenoids				2.6						3.5
	sesquiterpenes				55.0						51.6
	oxygenated sesquiterpenoids				10.5						9.8
	others				0.2						0.2
	total				85.1						82.8

^aCalculated linear retention index (LRI).

^bReference linear retention index (LRI); % = percent by weight of EO. σ = standard deviation; Reference = reference literature for LRIs values. δ = identification by MS only; trace = < 0.1%.

^cN. = progressive number; RT = retention time (min.).

^dMajor components (≥3.0% on at least one column) are reported in bold.

If the chemical composition of A. dubia EO is compared with the ones described for the leaves of other Aiouea spp., very different chemical profiles can be observed. ,, The comparison among the main components (≥3.0% in at least one oil) of these species is represented in Figure . As it can be observed, A. montana EO shown the most peculiar composition, dominated by S-methyl-O-phenylethyl carbonothioate (33.3%)a sulphurated metabolite, completely unprecedented in nature until the description of that volatile fraction. This metabolite is responsible for the unpleasant odor of A. montana and it is absent in all the other EOs. Further important components in A. montana EO are α-copaene (15.7%) and α-phellandrene (14.5%), that are in contrast minor constituents in the remaining volatile fractions. Another peculiar EO is the one from the leaves of A. maguireana, that was dominated by spathulenol (23.0%) and where (5E,9E)-farnesylacetone (3.3%) was exclusively detected. Finally, A. costaricensis EO shown two monoterpenes, α-pinene (14.5%) and β-pinene (14.0%), as main components, that marked a difference within a group of sesquiterpene-based EOs. None of these profiles seemed to present a clear relationship one another or with A. dubia EO. This evidence suggested that the so far poorly studied genus Aiouea is possibly inhomogeneous from the metabolic point of view, i.e. the species belonging to this taxon could possibly be quite diverse from a biochemical point of view.

4.

Compared amounts of major compounds (≥3.0% in at least one oil) in the leaf EOs of A. dubia (red) and A. montana (green), A. maguireana (black), and A. costaricensis (cyan). The compound marked with * corresponds to S-methyl-O-phenylethyl carbonothioate.

As it can be observed in Table , the elution order of the EO components depended on the nature of the stationary phase and on the characteristics of each compound. In fact, 5%-phenyl-methyl-polysiloxane is a nonpolar phase, where chromatographic retention approximatively increases with boiling point. On the other hand, polyethylene glycol is a polar stationary phase, where retention depends not only on volatility but also on dipolar interactions and on the formation of hydrogen bonds. For these reasons, it can be observed that the elution order on the nonpolar phase is sometimes not respected on the polar one, with alcohol compounds such as linalool, borneol and terpinene-4-ol eluting on polyethylene glycol with a delay of about 10 min respect to what it is observed on 5%-phenyl-methyl-polysiloxane.

2.2. Enantioselective Analysis

The enantioselective analyses of A. dubia EO permitted to analyze 11 chiral terpenes and terpenoids, of which (1S,5S)-(−)-α-pinene and (1R,6S)-(−)-3-carene were enantiomerically pure, whereas the other metabolites were present as scalemic mixtures. On the one hand, among the chiral compounds that were observed as enantiomeric pairs, (S)-(+)-α-phellandrene, (1R,2S,4R)-(+)-borneol, and (S)-(−)-germacrene D shown a very high enantiomeric excess (>90%). On the other hand, sabinene was almost racemic. The detailed results of the enantioselective analyses are shown in Table , based on two different chiral selectors (2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin), that were chosen depending on their separative properties on each enantiomeric pair.

2. Enantioselective Analysis of Some Chiral Terpenes from A. dubia EO .

chiral selector	enantiomer	LRI	LRI	E.D. (%)	e.e. (%)
DAC	(1S,5S)-(−)-α-pinene	915	914	100.0	100.0
DAC	(1R,5R)-(+)-α-pinene	917	916
DET	(1R,5R)-(+)-β-pinene	950	950	70.3	40.6
DET	(1S,5S)-(−)-β-pinene	960	961	29.7
DET	(1R,5R)-(+)-sabinene	978	977	49.4	1.2
DET	(1S,5S)-(−)-sabinene	992	991	50.6
DET	(R)-(−)-α-phellandrene	1020	1018	3.6	92.8
DET	(S)-(+)-α-phellandrene	1023	1021	96.4
DET	(1S,6R)-(+)-3-carene	1020	1018		100.0
DET	(1R,6S)-(−)-3-carene	1029	1027	100.0
DET	(S)-(−)-limonene	1059	1060	5.9	88.2
DET	(R)-(+)-limonene	1073	1076	94.1
DET	(R)-(−)-linalool	1182	1182	28.5	43.0
DET	(S)-(+)-linalool	1195	1196	71.5
DET	(1S,2R,4S)-(−)-borneol	1205	1205	0.9	98.2
DET	(1R,2S,4R)-(+)-borneol	1212	1213	99.1
DAC	(R)-(−)-terpinen-4-ol	1292	1291	39.6	20.8
DAC	(S)-(+)-terpinen-4-ol	1297	1297	60.4
DET	(S)-(−)-α-terpineol	1301	1302	35.4	29.2
DET	(R)-(+)-α-terpineol	1314	1314	64.6
DET	(R)-(+)-germacrene D	1460	1461	3.5	93.0
DET	(S)-(−)-germacrene D	1466	1467	96.5

^aValues from the EO.

^bValues from enantiomerically pure standards. E.D. = enantiomer distribution; e.e. = enantiomeric excess; DAC = 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin; DET = 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin.

^cLRI = linear retention index.

Due to the lack of enantioselective analyses for A. maguireana and A. costaricensis EOs, the present volatile fraction can be compared, within its genus, only with the EO from the dry leaves of A. montana. The compared results are represented in Figure .

5.

Compared enantiomeric compositions of some chiral compounds in the leaf EOs of A. dubia (red) and A. montana (green).

As for the chemical composition, also in the case of the enantiomeric distribution a common pattern apparently did not appear. In fact, among the chiral compounds present in both species, only α-phellandrene shown a similar enantiomeric distribution in A. dubia and A. montana EOs. Furthermore, analyzing the enantiomeric composition of the volatile fraction, an interesting phenomenon can be observed. Some chiral compounds, that share the same chiral precursor in their biosynthesis, actually presented a different enantiomeric distribution. This is for instance the case of α-pinene and β-pinene, that derive from the pinyl cation, from which both receive the configuration of their stereogenic centers. Consequently, they are expected to show the same enantiomeric distribution, with a similar enantiomeric excess. However, it can be observed that (1S,5S)-(−)-α-pinene was enantiomerically pure, whereas β-pinene was a scalemic mixture, with an enantiomeric excess of its dextrorotatory form. A similar observation can be done about limonene and α-terpineol, both deriving from the α-terpinyl cation. The different enantiomeric distributions of the pairs α-pinene/β-pinene and limonene/α-terpineol could be explained hypothesizing enantiospecific reactions that occurred, after their biosynthesis, on the two compounds of each pair. On the other hand, the similar enantiomeric excess of the same optical form for terpinen-4-ol and α-terpineol was possibly accidental, since these terpenoids derive from different chiral precursors. A similar profile, where the enantiomer distribution of chiral metabolites is not the one biosynthetically expected, is quite common in nature and it has already been observed in EOs by these authors in previous studies, as well as by other scientists. This is for instance the case of Gynoxys szyszylowiczii, Steiractinia sodiroi, Gynoxys cuicochensis, Gynoxys sancti-antonii, and Abies concolor for the pair α-pinene/β-pinene; S. sodiroi, G. sancti-antonii, Pinus edulis, and P. monophylla for the pair limonene/α-terpineol; G. cuicochensis and A. montana for the pair α-phellandrene/β-phellandrene. In addition to the hypothesis of postsynthetic enantiospecific reactions, this phenomenon could also be partially explained through the theory of racemization. In fact, it has been demonstrated that chiral alcohols, presenting a OH group connected to a quaternary stereogenic atom, easily produce a stable carbocation in acidic conditions, leading to racemization. This is for instance the case of terpinen-4-ol and linalool, especially at the temperature of atmospheric pressure steam-distillation. − With chiral hydrocarbons, such as pinenes, this phenomenon should be less important, so that the hypothesis of enantiospecific postsynthetic reactions cannot be completely discarded. Nevertheless, a partial racemization for limonene has been observed. On the other hand, in the case of α-terpineol, the OH group is connected to a quaternary nonstereogenic carbon atom, adjacent to the only tertiary asymmetric carbon of the molecule. In such a situation, acidic conditions would lead to the formation of the α-terpinyl cation, that can be in equilibrium with the equally stable terpinen-4-yl cation through a 1,2-hydride shift, eventually leading to racemization (see Figure ).

6.

Proposed mechanism for a possible racemization of α-terpineol in A. dubia leaf EO.

Finally, some considerations could be added about the properties of (S)-(−)-germacrene D, the almost only enantiomer (e.e. = 93.0%) detected for the main component of A. dubia EO (12.0%). According to literature, the biological properties of germacrene D have not been so far exhaustively studied, however its levorotatory form has been demonstrated to act as a semiochemical, exerting an attractant effect toward the mated females of moth Heliotis virescens, of which additionally enhances oviposition. The receptor neurons for (S)-(−)-germacrene D have also been identified in other insects of the same genus, such as H. armigera and H. assulta. , In consideration of its relative abundance and the high enantiomeric excess of its levorotatory form, the same semiochemical property could also be hypothesized for A. dubia EO. About other possible biological activities of this EO, antibacterial and anti-inflammatory capacities are the main properties shown, according to literature, by some major components such as (R)-(+)-limonene, (S)-(−)-germacrene D, and (E)-β-caryophyllene or, in the case of δ-cadinene, by an EO where this compound is relatively abundant. −

3. Conclusions

The steam-distilled dry leaves of A. dubia produced an EO, with the non-negligible yield of 0.3% by weight. This volatile fraction was characterized by a pleasant, sweet smell, similar to the one of other sesquiterpene-based oils. Being dominated by germacrene D and due to the high enantiomeric excess of its levorotatory enantiomer, this EO could act as an attractant for the insects of the genus Heliotis. Despite A. dubia currently is an only wild species, its leaf EO could be considered commercially promising, due to its high distillation yield. Cosmeceutical science could be the main application field for this volatile fraction. In fact, the high content of limonene and (E)-β-caryophyllene suggested that A. dubia EO could present antibacterial and anti-inflammatory properties, like other oils of similar composition. Future research should experimentally investigate the biological activities of this EO, with a special focus on the two main properties deduced since literature. In fact, for cosmeceutical applications, antibacterial and anti-inflammatory capacities are possibly more interesting than other biological activities, whose actions imply a systemic mechanism instead of a topic effect.

4. Methods

4.1. Plant Material

The leaves of A. dubia were collected on 13 December 2023 from the slopes of Mount Villonaco, rising on the western side of the town of Loja, Ecuador. The collection point corresponded to coordinates 4°00′11″S 79°15′25″W, located at the altitude of 2580 m above the sea level. The plant material was collected in equal amounts from different treelets, growing within the range of 200 m from the previously described collection site. Finally, due to the relatively low amount, all the leaves were combined, producing a unique mean sample. The botanical species was identified by one of the authors (N.C.), based on the sample with code MO-257748/A:4059160 of the Missouri Botanical Garden (St. Louis, MO, USA). A botanical voucher was deposited at the herbarium of the Universidad Técnica Particular de Loja (UTPL), with code 15415. The same day of collection, the leaves were dried at 35 °C for 48 h and then stored in a fresh, dark place until use. Both collection and investigation were carried out according to the law, by appointment of the Ministry of Environment, Water, and Ecological Transition of Ecuador (MAATE), with permit code MAATE-DBI-CM-2022–0248.

4.2. Distillation and Sample Preparation

The dry leaves of A. dubia (250.5 g) were preparatively steam-distilled in a modified Dean–Stark apparatus, structurally similar to the one previously described in literature. A pale yellow, sweet-smelling oil was produced, with a yield of 0.3% by weight of plant material. After distillation, the EO was dried over anhydrous Na₂SO₄, filtered, and permanently stored in the darkness, at −15 °C until use. In the gas chromatographic (GC) analyses, four samples were prepared by weighting volumes of 10 μL and diluting them by adding 1 mL of cyclohexane, containing n-nonane as internal standard, at the concentration of 0.7 mg/mL. All the quantitative results were expressed as average value and standard deviation of four injections, each one conducted with the volume of 1 μL. Cyclohexane, n-nonane, and Na₂SO₄ were analytical grade, and purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA).

4.3. Qualitative (GC–MS) Chemical Analyses

The qualitative chemical composition of A. dubia EO was carried out through gas chromatography, using mass spectrometry as a detection technique (GC–MS). The GC instrument was a gas chromatograph model Trace 1310, purchased from Thermo Fisher Scientific (Waltham, MA, USA). The GC was coupled with a single quadrupole mass spectrometer (MS) model ISQ 7000, purchased from the same provider. The qualitative analyses were conducted with two capillary columns, based on stationary phases of different polarity: 5%-phenyl-methyl-polysiloxane (TR-5 ms, nonpolar) and polyethylene glycol (TR-Wax, polar), purchased form Thermo Fisher Scientific (Waltham, MA, USA). Both columns were characterized by the following dimensions: 30 m in length, internal diameter of 0.25 mm, and film thickness of 0.25 μm. The thermal program was 50 °C for 10 min, followed by an initial gradient of 2 °C/min up to 170 °C, then a second gradient of 10 °C/min up to 230 °C, which was finally maintained for 20 min. All the injections were conducted in split mode (40:1), with helium as a carrier gas, provided by Indura s.a. (Guayaquil, Ecuador), and maintained at the constant flow of 1 mL/min. Injector and transfer line were set at the temperature of 250 °C. The mass spectrometer detector was equipped with an electron ionization source, set at the ionization energy of 70 eV. The detector was operated in SCAN mode, detecting ions within the mass range of 40–400 amu. Both ion source and quadrupole temperatures were 250 °C. All the EO constituents were identified by comparison of each MS spectrum and linear retention index (LRI) with data from literature. The LRIs were calculated according to Van den Dool and Kratz, based on a series of homologous n-alkanes in the range C₉–C₂₄. The alkanes were provided by Merck (Sigma-Aldrich, St. Louis, MO, USA).

4.4. Quantitative (GC-FID) Chemical Analyses

The quantitative chemical analyses were conducted with the same GC instrument and the same columns described for the qualitative ones, but with a flame ionization detector (FID), set at 280 °C, instead of the mass spectrometer. Carrier gas, constant flow, injection temperature and mode, and oven thermal program were the same as the qualitative analyses. All the detected compounds were quantified calculating the relative response factor (RRF) of each constituent according to the respective combustion enthalpy, as described by Chaintreau. , The integration areas, corrected by the RRFs, were applied to two six-point calibration curves (one for each column), that were traced using n-nonane as internal standard and isopropyl caproate as calibration standard. The internal standard was provided by Merck (Sigma-Aldrich, St. Louis, MO, USA), whereas the calibration standard was synthesized in the authors’ laboratory and purified to 98.8% (GC-FID purity). Both curves produced coefficients of determination R ² > 0.998, with standard dilutions prepared as previously described in literature.

4.5. Enantioselective Analyses

The enantioselective analyses were carried out by means of two GC enantioselective columns, whose stationary phases were based on 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin and 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin as chiral selectors. Both columns were 25 m in length, 0.25 mm in internal diameter, and 0.25 μm in phase thickness, purchased from Mega s.r.l, Milan, Italy. The analyses were conducted in the same GC–MS instrument previously described for the qualitative analyses, applying the following thermal program: 50 °C for 1 min, followed by a gradient of 2 °C/min until 220 °C, which was maintained for 10 min. The carrier gas was helium, at the constant pressure of 70 kPa; all the other parameters were the same as the qualitative analyses, except for the injector and transfer line temperatures, that were set at 220 °C. All the enantiomers were identified based on their mass spectrum and linear retention indices, calculated according to Van den Dool and Kratz as described in Section , and compared to data obtained from the injection of enantiomerically pure standards. Since the mass spectra of two enantiomers are identical, they can be used to identify to which compound peaks correspond, but they cannot be used to recognize each enantiomer. To assign a peak to a specific optical isomer, the injection of an enantiomerically pure standard is necessary. After injection, retention times or linear retention indices can be used. Some of the standards were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA), whereas others were available from the University of Turin, Italy.

The need for using two different chiral selectors depended on the different separative properties of each stationary phase toward each enantiomeric pair. For instance, α-pinene and terpinen-4-ol enantiomers are better resolved with 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin, whereas the optical isomers of limonene and germacrene D are separated by 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin but completely unresolved with the other chiral selector.

The authors declare no competing financial interest.