Chemical Composition, Enantioselective Profile, and Preliminary Screening of Biological Activities of the Essential Oil from Aerial Parts from Lasiocephalus ovatus Schltdl.

Abstract

Traditionally, Lasiocephalus ovatus Schltdl. (Asteraceae) has been used as an aromatic medicinal plant, particularly in the treatment of kidney-related ailments. However, scientific evidence validating its chemical composition and bioactivity remains limited. According to our literature search, there are no previous studies on the in vitro antibacterial, antioxidant, or anti-inflammatory activities of the essential oil from the aerial parts of Lasiocephalus ovatus; therefore, this study provides the first experimental evidence of these biological activities for this species. An essential oil (EO) was steam-distilled from the aerial parts of L. ovatus, grown at 4410 m above sea level in the paramos of Chimborazo Province (Ecuador), and subsequently analyzed. The distillation yield was 0.21% (w/w) based on dry plant material. Gas chromatography was employed for qualitative (GC-MS) and quantitative (GC-FID) analyses, using two different capillary columns, coated with 5% phenyl methyl polysiloxane (non-polar) and polyethylene glycol (polar) stationary phases. Dual stationary phases were required to provide complementary selectivity, which reinforced the identification and quantification of compounds. The major components of the EO were silphinene (3.4–3.5%), δ-selinene (3.6–3.1%), β-cyclogermacrene (18.7–18.1%), kessane (4.5–4.2%), spathulenol (13.3–13.3%), viridiflorol (3.1–3.0%) and neophytadiene (4.8–4.4%), values referred to the non-polar and polar phase respectively. The enantioselective analysis revealed that (1S,5S)-(−)-α-pinene, (1S,5S)-(+)-β-pinene and (R)-(−)-α-phellandrene were enantiomerically pure, whereas germacrene D was present as a scalemic mixture. The essential oil of L. ovatus exhibited a minimum inhibitory concentration (MIC) of 250 µg/mL against Staphylococcus aureus and 500 µg/mL against Escherichia coli. Its antibacterial activity is likely associated with the presence of bioactive sesquiterpenes such as silphinene, δ-selinene, and spathulenol, which are known for their membrane-disruptive properties. Regarding its antioxidant potential, the observed moderate radical scavenging activity (SC₅₀ = of 375.7 µg/mL) can be attributed to its complex mixture, particularly to oxygenated terpenoids like viridiflorol and spathulenol, which are recognized for their radical-neutralizing capacity. In the anti-inflammatory assay, the EO’s moderate potency (IC₅₀ = 165.29 ± 4.75 μg/mL) is also consistent with the anti-inflammatory profile reported for several of its major constituents, including spathulenol and viridiflorol. While significantly lower than that of aspirin (28.85 ± 7.66 μg/mL), this bioactivity is considerable within the context of a plant extract. Overall, the antibacterial, antioxidant, and anti-inflammatory effects are consistent with the EO’s terpene-rich composition, particularly oxygenated sesquiterpenes, while the enantiomeric distribution of chiral monoterpenes may further modulate bioactivity; consequently, future studies should include enantioselective quantification, broader antioxidant assays (e.g., ABTS, FRAP, ORAC, CUPRAC), cytotoxicity at active concentrations, and mechanistic and in vivo validation.

1. Introduction

Ecuador flora is one of the most diverse among countries worldwide, due to the fact that its territory intersects the tropical Andes and the Tumbes-Chocó-Magdalena corridor, which are considered global biodiversity hotspots [1]. Approximately 26% of the total flora is considered endemic [2], where a 17,748 species have been confirmed to be native [3]. This condition has attracted the attention of several research groups, as most of these species remain chemically unexplored [4]. Furthermore, plants and natural products often exhibit important biological activities and have been used for centuries as vegetal drugs or pharmaceutical active principles. Knowledge of these uses is frequently transmitted from generation to generation [5].

In recent years, the study of secondary metabolites has increased in line with the use of traditional, complementary, and integrative medicine worldwide. The World Health Organization reports that most of the world population (80%) uses some form of traditional, complementary, and integrative medicine (T&CM) [6], including essential oils, as an alternative therapeutic approach [7,8,9]. For this reason and considering that Ecuador is a “megadiverse” country [10], the authors have been studying natural products for many years, with a special focus on essential oils [11,12,13].

The European Pharmacopoeia (Ph. Eur.) defines essential oils (EOs) as oily products, obtained by distillation techniques from plant material or by mechanical techniques from the pericarp of Citrus sp. fruits [14]. The EOs are produced and accumulated in specific areas of plants, such as oil cells, glandular trichomes or secretory ducts [15]. Plants synthesize EOs as a protection mechanism against pests and insects [16]. Their chemical composition is complex and diverse. Biosynthetically, EO constituents mainly fall into three categories: terpenoids, phenylpropanoids, and acetate derivatives. These mixtures consist of esters, aldehydes, alcohols, phenols, oxides, ketones and acids [11,12,17]. This chemical diversity supports a broad range of biological effects. It can contribute to antimicrobial activity through membrane disruption, to antioxidant activity through radical scavenging and modulation of oxidative stress, and to anti-inflammatory effects through inhibition of pro-inflammatory enzymes and signaling pathways [18,19,20].

The chemical composition of EOs depends on several factors, including the geographical location of the species, time of collection, and cultivation practices, among others [21,22]. Therefore, the description of new EOs represents a major objective of the research group’s work, with the aim of contributing to the preservation of the Ecuadorian biodiversity through the development of new bioeconomically relevant products. Among the most promising families, Asteraceae must be mentioned, where unprecedented EOs can easily be discovered. This family is generally found in arid and semi-arid lands of tropical areas, widely distributed all around the world [23]. The EOs obtained from Asteraceae are often characterized by promising biological activities, including antioxidant, antimicrobial, anti-inflammatory, hepato-protective, and antidiabetic effects [24,25,26,27,28]. According to the Catalogue of Vascular Plants of Ecuador, this family has 863 species, of which 370 are considered endemic, and widely distributed in the four climatic regions of Ecuador [29].

Many studies have reported the presence of 23 species of the genus Lasiocephalus distributed in the highlands of montane forests and paramos of Perú, Colombia, Venezuela, Bolivia, and Ecuador [30]. In particular, certain species of the genus Aetheolaena also contribute to this classification, because they have been reclassified within the genus Lasiocephalus for presenting key morphological similarities, such as floral structure and leaf arrangement [31,32,33]. Furthermore, the catalog of Vascular Plants of Ecuador shows species of the genus Aetheolaena as synonyms of the genus Lasiocephalus [29]. This genus is characterized by the fact that the species can grow in two forms: as shrubs with small, elongated leaves and as vines with broad leaves [32]. For the present study, the aromatic species Lasiocephalus ovatus Schltdl. (Asteraceae) has been selected, whose volatile and non-volatile fractions have only been partially studied or described in outdated reports [34,35,36]. In Ecuador, L. ovatus is traditionally known as “Arquitecta” or “Arquitecto”, to which medicinal properties are attributed in the relief of kidney ailments or to disinfect liver and gallbladder [37,38]. It is considered a native Andean species, growing at elevations between 2500 and 4500 m above sea level, and distributed in the provinces of Cañar, Chimborazo, Tungurahua, Cotopaxi, and Pichincha [29]. According to the Missouri Botanical Garden (

Table 1. Synonymy of L. ovatus according to the Missouri Botanical Garden (Tropicos) [39].

Name	Status
Lasiocephalus ovatus Schltdl.	Accepted name
Culcitium ovatum (Schltdl.) S.F. Blake	Synonym
Culcitium reflexum Kunth	Synonym
Culcitium sodiroi Hieron	Synonym
Culcitium uniflorum Hieron	Synonym
Gnaphalium uniflorum Lam	Synonym
Lasiocephalus sodiroi (Hieron.) Cuatrec	Synonym
Senecio reflexus (Kunth) Cuatrec	Synonym
Senecio superandinus Cuatrec	Synonym

), the listed names are recognized as synonyms of L. ovatus.

A review of the literature indicates that research on the genus Lasiocephalus has mainly focused on taxonomic descriptions, revealing an important knowledge gap regarding its secondary metabolites. Likewise, studies on its essential oils remain scarce, being limited to qualitative descriptions of their composition (GC-MS) with occasional antibacterial evaluations [40,41], often without chromatographic profiling using stationary phases of different polarity, quantitative GC-FID data, enantioselective analyses, or broader biological analyses, particularly for L. ovatus.

To the best of our knowledge, there are no previous studies reporting the in vitro antibacterial, antioxidant, or anti-inflammatory activities of the essential oil obtained from the aerial parts of Lasiocephalus ovatus. Therefore, the present work addresses this scientific gap by providing a comprehensive chemical and enantioselective characterization and by experimentally evaluating (and thereby validating) these biological activities.

2. Results

2.1. Chemical Composition of the EO

The aerial parts of L. ovatus were subjected to steam distillation, yielding an essential oil (EO) with a yield of 0.21% (w/w) based on dry plant material. A total of seventy-one compounds were identified and quantified by GC-MS and GC-FID analyses using two capillary columns of different polarity. These compounds accounted for 98.0% and 91.6% of the total EO composition on the nonpolar and polar columns, respectively. The main compounds (>3% on both columns) identified in the volatile fraction were silphinene (19) (3.4–3.5%), δ-selinene (45) (3.6–3.1%), β-cyclogermacrene (46) (18.7–18.1%), kessane (51) (4.5–4.2%), spathulenol (55) (13.3–13.3%), viridiflorol (57) (3.1–3.0%) and neophytadiene (65) (4.8–4.4%).

The composition of the essential oil of L. ovatus was dominated by the presence of sesquiterpenes hydrocarbons (45.5–42.6%), followed by oxygenated sesquiterpenoids (32.0–31.2%). Oxygenated monoterpenes (0.9–0.5%) were present in the lowest proportion on both columns. The chromatograms for the non-polar and the polar phase are shown in Figure 1 and Figure 2, respectively. Figure 3 shows the chemical structures of the EO main constituents.

Figure 1. GC–MS chromatogram of L. ovatus EO in a 5% phenyl-methylpolysiloxane-based column (non-polar). The main components are presented in Table 2, according to the number corresponding to each peak.

Figure 1. GC–MS chromatogram of L. ovatus EO in a 5% phenyl-methylpolysiloxane-based column (non-polar). The main components are presented in Table 2, according to the number corresponding to each peak.

Table 2. Chemical analysis of L. ovatus EO on two stationary phases of different polarity (5%-Phenyl methyl polysiloxane and Polyethylene glycol).

N.	Compounds	5% Phenyl Methyl Polysiloxane					Ref.	Polyethylene Glycol					Ref.
		LRI a	LRI b	%	σ	RSD (%) c		LRI a	LRI b	%	σ	RSD (%) c
1	α-pinene	932	932	2.9	0.32	0.08	[42]	1019	1017	2.7	0.04	0.17	[43]
2	β-pinene	977	974	0.3	0.06	0.06	[42]	1106	1108	0.2	0.01	0.11	[44]
3	trans-3-decene	992	993	0.5	0.10	0.12	[42]	1141	-	0.3	0.02	0.15	§
4	α-phellandrene	1008	1002	0.8	0.09	0.11	[42]	1161	1160	0.7	0.01	0.23	[45]
5	p-cymene	1029	1020	0.5	0.03	0.05	[42]	1269	1270	0.4	0.01	0.05	[46]
6	cis-β-ocimene	1041	1032	0.1	0.01	0.05	[42]	1239	1240	0.1	0.01	0.06	[44]
7	trans-β-ocimene	1051	1044	0.5	0.03	0.05	[42]	1255	1250	0.4	0.01	0.05	[47]
8	1-undecene	1092	1090	2.4	0.31	0.08	[48]	1141	1139	2.2	0.17	0.09	[49]
9	Undecane	1106	1100	0.3	0.01	0.04	[42]	1403	-	Trace	-	0.10	§
10	trans-limonene oxide	1136	1137	0.1	0.01	0.00	[42]	1488	1482	Trace	-	0.09	[50]
11	7-methyl-3-methyleneoct-6-enal	1144	1140	0.1	0.01	0.03	[42]	1101	-	Trace	-	0.11	§
12	(Z)-isocitral	1155	1160	0.2	0.05	0.07	[42]	1594	-	0.1	0.01	0.05	§
13	p-menth-8-en-2-one	1186	1191	0.1	0.02	0.03	[42]	1195	-	Trace	-	0.13	§
14	Myrtenol	1205	1212	0.1	0.03	0.03	[42]	1792	1792	Trace	-	0.02	[51]
15	1-tridecene	1292	1290	0.2	0.01	0.05	[42]	1344	1343	0.1	0.01	0.04	[52]
16	6-hydroxycarvotanacetone	1305	1309	0.3	0.02	0.07	[42]	1410	-	0.2	0.01	0.03	§
17	undec-9-en-1-al	1316	1322	0.1	0.01	0.05	[42]	1818	-	0.3	0.01	0.06	§
18	silphiperfol-5-ene	1322	1326	0.5	0.04	0.02	[42]	1413	1403	0.3	0.02	0.03	[53]
19	Silphinene	1341	1344	3.4	0.35	0.05	[42]	1457	-	3.5	0.07	0.03	§
20	7-epi-silphiperfol-5-ene	1344	1345	0.1	0.01	0.05	[42]	1450	-	0.1	-	0.03	§
21	silphiperfola-4,7(14)-diene	1356	1358	0.4	0.05	0.05	[42]	1846	-	0.3	0.02	0.04	§
22	Cyclosativene	1366	1369	0.8	0.16	0.04	[42]	1465	-	0.5	0.04	0.03	§
23	Ylangene	1374	1373	2.2	0.34	0.04	[42]	1478	1483	2.1	0.14	0.03	[54]
24	unidentified (MW = 204)	1383	-	0.6	0.04	0.04		1491	-	0.6	0.02	0.03	-
25	β-cubebene	1387	1387	0.5	0.04	0.04	[42]	1530	1532	0.3	0.01	0.03	[55]
26	β-elemene	1390	1389	0.4	0.03	0.07	[42]	1560	1560	0.2	0.01	0.03	[56]
27	α-gurgujene	1401	1409	0.9	0.35	0.04	[42]	1509	1511	0.7	0.02	0.03	[57]
28	α-cedrene	1415	1410	0.2	0.01	0.04	[42]	1560	1566	Trace	-	0.03	[58]
29	(E)-β-caryophyllene	1419	1417	1.1	0.15	0.04	[42]	1584	1585	1.0	0.03	0.02	[59]
30	2,5-dimethoxyp-cymene	1423	1424	0.1	0.01	0.06	[42]	1878	1876	0.1	0.02	0.02	[60]
31	β-copaene	1430	1430	0.1	0.01	0.06	[42]	1529	-	0.3	0.01	0.03	§
32	α-guaiene	1438	1437	0.1	0.01	0.06	[42]	1652	1659	Trace	-	0.02	[55]
33	cis-β-farnesene	1440	1439	0.2	0.01	0.04	[42]	1639	1639	0.1	0.03	0.02	[61]
34	myltayl-4(12)-ene	1444	1445	0.7	0.11	0.04	[42]	1605	-	0.7	0.02	0.02	§
35	amorpha-4,11-diene	1445	1449	0.8	0.13	0.04	[42]	1577	-	1.0	0.03	0.07	§
36	unidentified (MW = 218)	1453	-	0.3	0.03	0.06		1602	-	0.1	0.01	0.02	-
37	Humulene	1456	1452	2.4	0.13	0.04	[42]	1655	1656	2.1	0.06	0.07	[62]
38	Alloaromadendrene	1458	1458	0.3	0.03	0.06	[42]	1631	1631	0.4	0.02	0.02	[63]
39	9-epi-β-caryophyllene	1460	1464	0.7	0.10	0.04	[42]	1630	1631	0.5	0.02	0.05	[64]
40	4,5-di-epi-aristolochene	1471	1471	0.2	0.04	0.04	[42]	1668	1665	0.3	0.02	0.02	[65]
41	β-chamigrene	1474	1476	0.6	0.13	0.04	[42]	1709	1702	0.9	0.04	0.02	[66]
42	γ-muurolene	1477	1478	0.4	0.02	0.04	[42]	1679	1681	0.5	0.02	0.02	[67]
43	γ-curcumene	1479	1481	1.0	0.13	0.06	[42]	1686	1683	0.7	0.10	0.04	[68]
44	germacrene D	1482	1480	2.8	0.19	0.04	[42]	1696	1694	3.1	0.13	0.06	[62]
45	δ-selinene	1490	1492	3.6	0.44	0.04	[42]	1699	1707	3.1	0.13	0.02	[60]
46	β-cyclogermacrene	1498	1500	18.7	0.48	0.06	[42]	1721	1720	18.1	0.47	0.06	[62]
47	α-muurolene	1501	1500	0.6	0.03	0.04	[42]	1716	1716	0.8	0.05	0.02	[69]
48	germacrene A	1509	1508	0.3	0.02	0.04	[42]	1705	1700	0.7	0.04	0.02	[70]
49	δ-cadinene	1521	1522	0.7	0.16	0.04	[42]	1679	-	0.5	0.02	0.02	§
50	cis-calamenene	1527	1528	0.3	0.06	0.04	[42]	1825	-	0.2	0.01	0.02	§
51	Kessane	1532	1529	4.5	0.29	0.04	[42]	1769	1779	4.2	0.53	0.02	[71]
52	Isokessane	1539	1530	2.0	0.29	0.05	[72]	1769	1777	2.1	0.05	0.04	[73]
53	α-calacorene	1547	1544	0.4	0.02	0.09	[42]	1907	-	0.2	0.01	0.02	§
54	β-vetivenene	1551	1554	0.4	0.02	0.04	[42]	1847	1836	0.3	0.02	0.04	[74]
55	spathulenol	1585	1577	13.3	0.49	0.02	[42]	2129	2132	13.3	0.15	0.01	[46]
56	Hedycaryol	1592	1589	2.3	0.11	0.05	[42]	2072	2077	2.0	0.08	0.01	[75]
57	Viridiflorol	1601	1592	3.1	0.14	0.05	[42]	2081	2084	3.0	0.42	0.03	[47]
58	cis-isolongifolanone	1611	1612	0.6	0.12	0.03	[42]	2022	-	0.4	0.03	0.02	§
59	humulene II epoxide	1617	1624	0.7	0.05	0.05	[42]	2025	2025	0.5	0.05	0.02	[76]
60	Eremoligenol	1621	1629	0.7	0.13	0.05	[42]	2178	2182	0.8	0.07	0.03	[77]
61	unidentified (MW = 218)	1627	-	0.7	0.06	0.05		2091	-	0.6	0.05	0.04	-
62	epi-α-muurol	1635	1640	0.9	0.14	0.05	[42]	2150	2151	0.8	0.07	0.01	[44]
63	Agarospirol	1639	1646	2.0	0.10	0.03	[42]	2183	-	2.1	0.17	0.01	§
64	Khusinol	1675	1679	0.1	0.02	0.03	[42]	2251	-	0.1	0.01	0.01	§
65	Neophytadiene	1844	1843	4.8	0.37	0.03	[42]	1927	1922	4.4	0.21	0.02	[78]
66	hexahydrofarnesyl acetone	1856	1856	0.6	0.04	0.03	[42]	2132	2131	0.3	0.01	0.01	[79]
67	unidentified (MW = 212)	1866	-	1.8	0.13	0.03		2559	-	1.4	0.12	0.01	-
68	oplopanonyl acetate	1892	1895	0.6	0.05	0.01	[42]	2770	-	0.5	0.04	0.01	§
69	Cyclohexadecanolide	1937	1940	0.3	0.02	0.01	[42]	2239	-	0.3	0.01	0.01	§
70	unidentified (MW = 231)	2234	-	1.8	0.14	0.01		2236	-	1.9	0.05	0.01	-
71	unidentified (MW = 214)	2274	-	0.9	0.07	0.01		3208	-	0.9	0.07	0.01	-
	monoterpene hydrocarbons			5.6						4.8
	oxygenated monoterpenes			0.9						0.5
	sesquiterpene hydrocarbons			45.5						42.6
	oxygenated sesquiterpenes			32.0						31.2
	diterpene			4.8						4.4
	Others			9.2						8.1
	Total			98.0						91.6

^a Calculated linear retention index; ^b Linear retention index according to Ref.; Trace < 0.1%; MW = molecular weight; § = identification based on MS only. ^c RSD = Relative standard deviation of retention times.

Table 2. Chemical analysis of L. ovatus EO on two stationary phases of different polarity (5%-Phenyl methyl polysiloxane and Polyethylene glycol).

N.	Compounds	5% Phenyl Methyl Polysiloxane					Ref.	Polyethylene Glycol					Ref.
		LRI a	LRI b	%	σ	RSD (%) c		LRI a	LRI b	%	σ	RSD (%) c
1	α-pinene	932	932	2.9	0.32	0.08	[42]	1019	1017	2.7	0.04	0.17	[43]
2	β-pinene	977	974	0.3	0.06	0.06	[42]	1106	1108	0.2	0.01	0.11	[44]
3	trans-3-decene	992	993	0.5	0.10	0.12	[42]	1141	-	0.3	0.02	0.15	§
4	α-phellandrene	1008	1002	0.8	0.09	0.11	[42]	1161	1160	0.7	0.01	0.23	[45]
5	p-cymene	1029	1020	0.5	0.03	0.05	[42]	1269	1270	0.4	0.01	0.05	[46]
6	cis-β-ocimene	1041	1032	0.1	0.01	0.05	[42]	1239	1240	0.1	0.01	0.06	[44]
7	trans-β-ocimene	1051	1044	0.5	0.03	0.05	[42]	1255	1250	0.4	0.01	0.05	[47]
8	1-undecene	1092	1090	2.4	0.31	0.08	[48]	1141	1139	2.2	0.17	0.09	[49]
9	Undecane	1106	1100	0.3	0.01	0.04	[42]	1403	-	Trace	-	0.10	§
10	trans-limonene oxide	1136	1137	0.1	0.01	0.00	[42]	1488	1482	Trace	-	0.09	[50]
11	7-methyl-3-methyleneoct-6-enal	1144	1140	0.1	0.01	0.03	[42]	1101	-	Trace	-	0.11	§
12	(Z)-isocitral	1155	1160	0.2	0.05	0.07	[42]	1594	-	0.1	0.01	0.05	§
13	p-menth-8-en-2-one	1186	1191	0.1	0.02	0.03	[42]	1195	-	Trace	-	0.13	§
14	Myrtenol	1205	1212	0.1	0.03	0.03	[42]	1792	1792	Trace	-	0.02	[51]
15	1-tridecene	1292	1290	0.2	0.01	0.05	[42]	1344	1343	0.1	0.01	0.04	[52]
16	6-hydroxycarvotanacetone	1305	1309	0.3	0.02	0.07	[42]	1410	-	0.2	0.01	0.03	§
17	undec-9-en-1-al	1316	1322	0.1	0.01	0.05	[42]	1818	-	0.3	0.01	0.06	§
18	silphiperfol-5-ene	1322	1326	0.5	0.04	0.02	[42]	1413	1403	0.3	0.02	0.03	[53]
19	Silphinene	1341	1344	3.4	0.35	0.05	[42]	1457	-	3.5	0.07	0.03	§
20	7-epi-silphiperfol-5-ene	1344	1345	0.1	0.01	0.05	[42]	1450	-	0.1	-	0.03	§
21	silphiperfola-4,7(14)-diene	1356	1358	0.4	0.05	0.05	[42]	1846	-	0.3	0.02	0.04	§
22	Cyclosativene	1366	1369	0.8	0.16	0.04	[42]	1465	-	0.5	0.04	0.03	§
23	Ylangene	1374	1373	2.2	0.34	0.04	[42]	1478	1483	2.1	0.14	0.03	[54]
24	unidentified (MW = 204)	1383	-	0.6	0.04	0.04		1491	-	0.6	0.02	0.03	-
25	β-cubebene	1387	1387	0.5	0.04	0.04	[42]	1530	1532	0.3	0.01	0.03	[55]
26	β-elemene	1390	1389	0.4	0.03	0.07	[42]	1560	1560	0.2	0.01	0.03	[56]
27	α-gurgujene	1401	1409	0.9	0.35	0.04	[42]	1509	1511	0.7	0.02	0.03	[57]
28	α-cedrene	1415	1410	0.2	0.01	0.04	[42]	1560	1566	Trace	-	0.03	[58]
29	(E)-β-caryophyllene	1419	1417	1.1	0.15	0.04	[42]	1584	1585	1.0	0.03	0.02	[59]
30	2,5-dimethoxyp-cymene	1423	1424	0.1	0.01	0.06	[42]	1878	1876	0.1	0.02	0.02	[60]
31	β-copaene	1430	1430	0.1	0.01	0.06	[42]	1529	-	0.3	0.01	0.03	§
32	α-guaiene	1438	1437	0.1	0.01	0.06	[42]	1652	1659	Trace	-	0.02	[55]
33	cis-β-farnesene	1440	1439	0.2	0.01	0.04	[42]	1639	1639	0.1	0.03	0.02	[61]
34	myltayl-4(12)-ene	1444	1445	0.7	0.11	0.04	[42]	1605	-	0.7	0.02	0.02	§
35	amorpha-4,11-diene	1445	1449	0.8	0.13	0.04	[42]	1577	-	1.0	0.03	0.07	§
36	unidentified (MW = 218)	1453	-	0.3	0.03	0.06		1602	-	0.1	0.01	0.02	-
37	Humulene	1456	1452	2.4	0.13	0.04	[42]	1655	1656	2.1	0.06	0.07	[62]
38	Alloaromadendrene	1458	1458	0.3	0.03	0.06	[42]	1631	1631	0.4	0.02	0.02	[63]
39	9-epi-β-caryophyllene	1460	1464	0.7	0.10	0.04	[42]	1630	1631	0.5	0.02	0.05	[64]
40	4,5-di-epi-aristolochene	1471	1471	0.2	0.04	0.04	[42]	1668	1665	0.3	0.02	0.02	[65]
41	β-chamigrene	1474	1476	0.6	0.13	0.04	[42]	1709	1702	0.9	0.04	0.02	[66]
42	γ-muurolene	1477	1478	0.4	0.02	0.04	[42]	1679	1681	0.5	0.02	0.02	[67]
43	γ-curcumene	1479	1481	1.0	0.13	0.06	[42]	1686	1683	0.7	0.10	0.04	[68]
44	germacrene D	1482	1480	2.8	0.19	0.04	[42]	1696	1694	3.1	0.13	0.06	[62]
45	δ-selinene	1490	1492	3.6	0.44	0.04	[42]	1699	1707	3.1	0.13	0.02	[60]
46	β-cyclogermacrene	1498	1500	18.7	0.48	0.06	[42]	1721	1720	18.1	0.47	0.06	[62]
47	α-muurolene	1501	1500	0.6	0.03	0.04	[42]	1716	1716	0.8	0.05	0.02	[69]
48	germacrene A	1509	1508	0.3	0.02	0.04	[42]	1705	1700	0.7	0.04	0.02	[70]
49	δ-cadinene	1521	1522	0.7	0.16	0.04	[42]	1679	-	0.5	0.02	0.02	§
50	cis-calamenene	1527	1528	0.3	0.06	0.04	[42]	1825	-	0.2	0.01	0.02	§
51	Kessane	1532	1529	4.5	0.29	0.04	[42]	1769	1779	4.2	0.53	0.02	[71]
52	Isokessane	1539	1530	2.0	0.29	0.05	[72]	1769	1777	2.1	0.05	0.04	[73]
53	α-calacorene	1547	1544	0.4	0.02	0.09	[42]	1907	-	0.2	0.01	0.02	§
54	β-vetivenene	1551	1554	0.4	0.02	0.04	[42]	1847	1836	0.3	0.02	0.04	[74]
55	spathulenol	1585	1577	13.3	0.49	0.02	[42]	2129	2132	13.3	0.15	0.01	[46]
56	Hedycaryol	1592	1589	2.3	0.11	0.05	[42]	2072	2077	2.0	0.08	0.01	[75]
57	Viridiflorol	1601	1592	3.1	0.14	0.05	[42]	2081	2084	3.0	0.42	0.03	[47]
58	cis-isolongifolanone	1611	1612	0.6	0.12	0.03	[42]	2022	-	0.4	0.03	0.02	§
59	humulene II epoxide	1617	1624	0.7	0.05	0.05	[42]	2025	2025	0.5	0.05	0.02	[76]
60	Eremoligenol	1621	1629	0.7	0.13	0.05	[42]	2178	2182	0.8	0.07	0.03	[77]
61	unidentified (MW = 218)	1627	-	0.7	0.06	0.05		2091	-	0.6	0.05	0.04	-
62	epi-α-muurol	1635	1640	0.9	0.14	0.05	[42]	2150	2151	0.8	0.07	0.01	[44]
63	Agarospirol	1639	1646	2.0	0.10	0.03	[42]	2183	-	2.1	0.17	0.01	§
64	Khusinol	1675	1679	0.1	0.02	0.03	[42]	2251	-	0.1	0.01	0.01	§
65	Neophytadiene	1844	1843	4.8	0.37	0.03	[42]	1927	1922	4.4	0.21	0.02	[78]
66	hexahydrofarnesyl acetone	1856	1856	0.6	0.04	0.03	[42]	2132	2131	0.3	0.01	0.01	[79]
67	unidentified (MW = 212)	1866	-	1.8	0.13	0.03		2559	-	1.4	0.12	0.01	-
68	oplopanonyl acetate	1892	1895	0.6	0.05	0.01	[42]	2770	-	0.5	0.04	0.01	§
69	Cyclohexadecanolide	1937	1940	0.3	0.02	0.01	[42]	2239	-	0.3	0.01	0.01	§
70	unidentified (MW = 231)	2234	-	1.8	0.14	0.01		2236	-	1.9	0.05	0.01	-
71	unidentified (MW = 214)	2274	-	0.9	0.07	0.01		3208	-	0.9	0.07	0.01	-
	monoterpene hydrocarbons			5.6						4.8
	oxygenated monoterpenes			0.9						0.5
	sesquiterpene hydrocarbons			45.5						42.6
	oxygenated sesquiterpenes			32.0						31.2
	diterpene			4.8						4.4
	Others			9.2						8.1
	Total			98.0						91.6

^a Calculated linear retention index; ^b Linear retention index according to Ref.; Trace < 0.1%; MW = molecular weight; § = identification based on MS only. ^c RSD = Relative standard deviation of retention times.

Figure 2. GC–MS chromatogram of L. ovatus EO in a polyethylene glycol-based column (polar). The main components are presented in Table 2, according to the number corresponding to each peak.

Figure 2. GC–MS chromatogram of L. ovatus EO in a polyethylene glycol-based column (polar). The main components are presented in Table 2, according to the number corresponding to each peak.

Figure 3. Major constituents (>3% on both columns) of L. ovatus EO according to Table 2: silphinene (19), δ-selinene (45), β-cyclogermacrene (46), kessane (51), spathulenol (55), viridiflorol (57) and neophytadiene (65).

Figure 3. Major constituents (>3% on both columns) of L. ovatus EO according to Table 2: silphinene (19), δ-selinene (45), β-cyclogermacrene (46), kessane (51), spathulenol (55), viridiflorol (57) and neophytadiene (65).

2.2. Enantioselective Analysis

An enantioselective GC-MS analysis was carried out on the essential oil of L. ovatus (Table 2). The biological relevance of stereochemistry lies in the fact that many microbial targets and inflammatory pathways involve chiral recognition. Enzymes, membrane-associated proteins, and other chiral binding environments can distinguish between mirror-image molecules. As a result, enantiomers may differ markedly in binding affinity and in the downstream cellular effects they trigger [80].

Three compounds, (1S,5S)-(−)-α-pinene, (1S,5S)-(+)-β-pinene and (R)-(−)-α-phellandrene, were enantiomerically pure, whereas germacrene D was present as both the enantiomeric forms in a scalemic mixture. The enantiomeric purity of α-pinene was determined on a 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin-based column, because its enantiomers are inseparable on the other chiral selector. For all the others chiral compounds, a 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin-based column was used, carrying out a better separation. The enantiomeric peak corresponding to germacrene D enantiomers showed good separation, with a resolution of Rs = 1.43. The complete results are detailed in

Table 3. Enantiomeric analysis of the components of L. ovatus EO.

Chiral Selector	Enantiomer	LRI a	LRI b	Enantiomeric Distribution %	e.e. %	Rs c
DAC	(1S,5S)-(−)-α-pinene	915	915	100.0	100.0	-
DAC	(1S,5S)-(+)-α-pinene	917	918	-
DET	(1S,5S)-(−)-β-pinene	950	950	-	100.0	-
DET	(1S,5S)-(+)-β-pinene	965	965	100.0
DET	(R)-(−)-α-phellandrene	1020	1021	100.0	100.0	-
DET	(S)-(+)-α-phellandrene	1023	1025	-
DET	(R)-(+)-germacrene D	1460	1460	32.9	34.3	1.43
DET	(S)-(−)-germacrene D	1466	1467	67.1

DAC = 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin; DET = 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin; LRI = linear retention index; ^a values from pure standards [81]; ^b calculated linear retention index; e.e. = enantiomeric excess; ^c Resolution between enantiomeric peaks was calculated as $R_s=2(t_{R2}-t_{R1})/(w_1+w_2)$, where $t_R$ is the retention time and $w$ is the peak width at baseline.

.

2.3. Antimicrobial Activity

The antibacterial activity of the EO from the aerial parts from L. ovatus was evaluated using the broth microdilution method.

Table 4. Antibacterial activity of the EO from L. ovatus.

Microorganism Strains	Essential Oil	Positive Control	Negative Control OD600
	MIC (µg/mL)
Staphylococcus aureus ATCC 25923	250	0.35	2.377
Escherichia coli ATCC 25922	500	1.62	2.512

MIC: Minimum Inhibitory Concentration. Positive controls: ampicillin for S. aureus, ciprofloxacin for E. coli. Negative control: Sterile growth medium inoculated with the bacterial suspension, showing normal growth (optical density at 600 nm, OD600, >2.0 after 24 h incubation).

shows the tested microorganisms and minimum inhibitory concentration (MIC) values of both the EO and positive control. The values of the negative control are also shown. Ampicillin was used as a positive control for Staphylococcus aureus, and ciprofloxacin was used as a positive control for Escherichia coli. The L. ovatus EO reported MIC values of 250 µg/mL against S. aureus and 500 µg/mL against E. coli. This preliminary screening, employing two well-characterized reference strains (S. aureus ATCC 25923 and E. coli ATCC 25922) as standard Gram-positive and Gram-negative models, provides an initial assessment of the EO’s antibacterial potential and establishes a benchmark for its spectrum of activity.

2.4. Radical Scavenging Capacity

The free radical scavenging capacity of the EO was assessed using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH).

Table 5. Antioxidant activity of the EO from L. ovatus.

Sample	DPPH
	SC50 (µg/mL) ± SD
L. ovatus essential oil	375.7 ± 2.1
Trolox	33.0 ± 1.5

SC₅₀: Half-maximal scavenging concentration. Values represent the mean ± standard deviation (SD) of n = 3 independent experiments.

shows the scavenging capacity (SC₅₀) of EO and positive control. The maximum evaluated concentration was 1000 µg/mL. L. ovatus EO showed a SC₅₀ of 375.7 µg/mL.

2.5. Anti-Inflammatory Activity

The inhibition of superoxide production by neutrophils (oxidative burst) by L. ovatus is summarized in

Table 6. Anti-inflammatory effect of the EO from L. ovatus.

Concentration (µg/mL)	L. ovatus Essential Oil	Aspirin
3.1	ND	13.26 ± 2.21
6.2	2.77 ± 0.43	24.74 ± 3.63 **
12.5	5.63 ± 1.07	33.28 ± 3.09 **
25.0	19.94 ± 1.76	47.62 ± 5.11 *
50.0	27.88 ± 1.28	58.39 ± 2.44 **
100.0	34.61 ± 1.05	70.84 ± 4.38 **
200.0	49.12 ± 0.81	86.35 ± 5.67 **

The values represent the mean ± SD, n = 3. ND: Not Detected (inhibition below the limit of quantification). Significant values, ** p < 0.01, * p < 0.05, using Student’s t-test; L. ovatus essential oil versus aspirin.

: Superoxide generation was measured using the water tetrazolium salt (WST-1) assay.

A comparative analysis of the IC50 values revealed that the anti-inflammatory potency of the EO (165.29 ± 4.75 μg/mL) was significantly inferior to that of the reference drug aspirin (28.85 ± 7.66 μg/mL). Despite the difference in efficacy, the essential oil showed a clear dose–response relationship across concentration of 6.2–200 μg/mL, and its anti-inflammatory activity is reported here for the first time.

3. Discussion

3.1. Chemical Composition and Main Components

The essential oil yield obtained in this study (0.21% w/w) is within the average values reported for the Asteraceae family (0.1–2.0%) [17,82,83], which agrees with the values observed in congeneric species [36].

In 1990, De Bernardi et al. studied for the first time the volatile fraction obtained from the stems of L. ovatus by hexane extraction and subsequent GC-MS analysis, identifying only sesquiterpene compounds: copaene, alloaromadendrene, humulene, eremophilene, α-curcumene [35]. Although the technique used to obtain the volatile fraction of L. ovatus differs from the technique used in our work, the qualitative analysis of the chemical profile shows that the common components are: alloaromadendrene, humulene, β-cubebene, β-elemene, δ-selinene, α-muurolene.

Years later, Araujo et al. obtained the essential oil from the aerial parts of L. ovatus collected in Sangay National Park. Chemical analysis identified a total of 27 compounds, with 1,2,5,5-tetramethyl-1,3-cyclopentaadiene and camphor as the major constituents, representing 11.90% and 40.48% of the oil, respectively. However, this study was conducted using only a single chromatographic separation column, and the percentage composition was not determined using a quantitative method. In addition, oxygenated monoterpene compounds predominated in percentage over sesquiterpene hydrocarbons [36].

The two studies distinct chemical profiles. In our study, the essential oil was dominated by sesquiterpene hydrocarbons, whereas the only compound common to both studies was a α-pinene, present at 1.9% in the previous report compared with 2.9% in our oil.

Regarding other studies on Lasiocephalus species, the essential oil obtained from the aerial parts of L. longipenicillatus, collected in Piñago, Mérida State, Venezuela (2300 m above sea level), was reported to contain only 11 compounds. The oil was predominantly composed of α-pinene, α-humulene, and germacrene D, accounting for 48.3%, 15.8% and 15.5% of the total composition, respectively. Monoterpenes were the predominant class of compounds, whereas sesquiterpene hydrocarbons were present in lower proportions [41]. These three major constituents were also detected in our EO; however, they occurred at much lower levels (2.9%, 2.4%, and 2.8%, respectively). The collection of this species in two different seasons of the year showed that the chemical profile of the essential oil changes evidencing that the sesquiterpene compounds are in higher percentage when L. longipenicillatus is collected at the beginning of the year, while the monoterpenes are present in higher percentage when the plant is collected in the middle of the year [40]. A comparison among the major constituents (>3.0% in at least one oil) of L. ovatus (Sangay Park), L. ovatus (Chimborazo Province) and L. longipenicillatus is represented in Figure 4.

Although some species of the genus Senecio are classified in Lasiocephalus Willd. ex Schltdl, and most are endemic to Ecuador; the volatile fraction of this group has not been studied [84].

For the first time, a comprehensive qualitative and quantitative analysis of the essential oil obtained from aerial parts of L. ovatus was conducted using GC-MS and GC-FID with different stationary phases (a nonpolar 5% phenylmethylpolysiloxane phase and polar polyethylene glycol phase), ensuring effective separation of all components present in the oil. The resulting chemical profile revealed the presence of compounds distinct from those reported in previous studies. Moreover, the lack of standardized studies on the genus Lasiocephalus highlights the importance of these findings, which provide a valuable reference for future investigations of unexplored species within this taxon.

In this study, two major (>1%) peaks could not be reliably identified and were therefore reported as unidentified constituents. The compound at RI 1866 (67) showed prominent EI ions at m/z 212 and 197 (base peak), with fragments at m/z 182, 169, 152, 141, 115, and 91, suggesting a low-volatility terpenoid-like molecule, potentially an oxygenated sesquiterpene or a related high-boiling derivative. The second unidentified compound (71) at RI 2234 exhibited a spectrum dominated by aliphatic fragments (base peak m/z 83, intense m/z 55, and ions at m/z 172, 185, 199, and 214), which is more consistent with a high-boiling, non-polar long-chain aliphatic or lipid-like structure than with typical mono–or sesquiterpenes. In both cases, library matching was inconclusive, and no authentic standards were available, so no tentative structures were assigned. Future work should confirm their identities using improved chromatographic separation, high-resolution MS to determine elemental composition, and, where feasible, isolation followed by NMR, together with solvent and procedural blanks to rule out exogenous contamination for compound (71).

3.2. Chiral GC-MS Analysis for Enantiomeric Distribution

Enantiomeric analysis of essential oils serves as a critical determinant for validating their authenticity, quality and biological properties, as the characteristic presence of specific enantiomers can reveal both botanical origin and potential adulteration [85,86,87]. Advanced chromatographic techniques, in particular chiral column gas chromatography-mass spectrometry (chiral GC-MS), allow the separation, identification and quantification of enantiomers through diastereomeric interactions with specialized stationary phases such as modified cyclodextrins [13,88,89].

The enantioselective analysis of the EO of L. ovatus (Table 3) revealed that (−)-α-pinene, (+)-β-pinene, and (−)-α-phellandrene were present as enantiomerically pure compounds. This result is consistent with their biosynthetic origin since both terpenes α-pinene and β-pinene share a common chiral precursor: the pinyl cation. It is known that the configuration of their stereogenic centers is inherited from this precursor, which explains the high stereospecificity observed in their synthesis [81]. On the other hand, germacrene D showed a scalemic mixture, with a slight enantiomeric excess of 34.3% in favor of the (S)-(−)-germacrene D enantiomer. In particular, this unsaturated sesquiterpene could undergo protonation under aqueous and potentially acidic conditions (e.g., due to organic acids present in the plant material) at the high temperatures of the process [90].

3.3. Antimicrobial Activity

The need to identify novel antimicrobial agents is highlighted by the global rise in antimicrobial resistance. Natural products, especially EOs, are considered valuable resources because they often exhibit mechanisms of action that differ from conventional antibiotics and may help combat resistant strains [91]. By providing preliminary evidence on the antibacterial activity of the EO extracted from the aerial parts of L. ovatus, our findings contribute to this field.

Data from the broth microdilution assay demonstrated measurable antibacterial activity for L. ovatus EO. As shown in Table 4, the oil inhibited the Gram-positive reference strain Staphylococcus aureus ATCC 25923 at 250 μg/mL, whereas a higher MIC (500 µg/mL) was required to inhibit the Gram-negative bacterium Escherichia coli ATCC 25922. As expected, the MICs of the reference antibiotics (ampicillin and ciprofloxacin) were considerably lower than those observed by L. ovatus EO. Nevertheless, the relevance of EOs lies in their chemical complexity. This complexity provides a starting point for future work to identify the constituents driving the observed activity and to test, in a targeted way, whether synergistic or antagonistic interactions occur among components. In this study, synergy is only hypothesized and should be confirmed in future assays.

The difference in susceptibility between the two tested microorganisms is consistent with the well-documented trend that Gram-negative bacteria are generally less susceptible to EO’s. The outer membrane of Gram-negative organisms, rich in lipopolysaccharides (LPS), acts as a hydrophilic barrier, limiting the diffusion of the hydrophobic constituents typical of EOs [92]. In contrast, the more accessible peptidoglycan layer in the cell wall of Gram-positive bacteria like S. aureus allows for easier penetration of these bioactive compounds, leading to membrane disruption and cell lysis [93].

The MIC values presented here are in line with those of numerous other EOs that have been identified as having antimicrobial potential, locating L. ovatus in a category that shows promise for additional research [94]. Rather than serving as a direct replacement for conventional drugs, the practical application of this EO may be found in synergy. A compelling area of future research would be to investigate whether L. ovatus oil can act synergistically with traditional antibiotics, potentially lowering required doses and mitigating resistance development [95]. Furthermore, because of its antimicrobial profile, it can be used in situations where pure antibiotics are not needed, like in natural disinfectants, food packaging materials, or natural cosmetic preservatives, where its moderate activity is adequate and frequently preferred [96].

The biological activity of an essential oil is largely determined by chemical composition. The profile of L. ovatus EO, detailed in a preceding section, is dominated by compounds such as silphinene, δ-selinene, β-cyclogermacrene, kessane, spathulenol, viridiflorol and neophytadiene, which have been reported to exhibit antibacterial properties in the scientific literature [97]. It is most probable that the overall effect is not due to a single molecule but develops from the complex synergistic or additive interactions between its major and minor chemical constituents. This multifaceted nature is a hallmark of essential oils and is thought to be a key reason behind the low incidence of resistance reported against them [98].

3.4. Radical Scavenging Capacity

The search for natural antioxidants has increased, driven by their critical role in mitigating oxidative stress—a key contributor to chronic diseases and food spoilage. While synthetic antioxidants like BHT and BHA are effective, their use is increasingly analyzed due to potential health concerns, creating a significant market for natural alternatives. Within this context, our study provides a first evaluation of the radical scavenging potential of the EO derived from L. ovatus.

The results from the DPPH assay, a typical method for assessing antioxidant activity, reveal that L. ovatus EO possesses a measurable ability to neutralize free radicals. The oil achieved a 50% scavenging capacity (SC₅₀) at a concentration of 375.7 µg/mL. The antioxidant Trolox, a water-soluble analog of vitamin E, exhibited a much lower SC₅₀ of 33 µg/mL, confirming its superior efficacy.

Although the oil’s activity is modest compared to the pure standard, the significance of an SC₅₀ in the hundreds of µg/mL range becomes clear when contextualized within the extensive literature on EOs. Numerous EOs derived from well-known aromatic plants have SC₅₀ values that fall into the same or even higher range (e.g., 300–1000 µg/mL) [99]. Therefore, the activity demonstrated by L. ovatus EO is comparable to that of numerous plant-derived EOs, aligning it as a moderately active natural antioxidant.

In the DPPH assay, radical-scavenging is largely driven by hydrogen atom transfer and or single-electron transfer processes. Compounds bearing oxygenated functionalities often contribute more strongly than terpene hydrocarbons because they can more readily participate in these reactions. In this context, the oxygenated sesquiterpenes detected in our EO, particularly spathulenol and viridiflorol, provide a plausible chemical basis for the observed DPPH activity. Spathulenol has been experimentally evaluated as an isolated constituent and showed relevant radical-scavenging capacity in DPPH, and other oxidative systems (do Nascimento et al., 2018) [100]. Likewise, viridiflorol, reported as a major constituent in Allophylus edulis essential oil, displayed measurable antioxidant activity in both DPPH and ABTS assays (Trevizan et al., 2016) [101].

This finding has a variety of practical ramifications that go beyond its numerical value. The oil’s moderate activity is highly relevant for certain industries, even though it might not be appropriate for applications requiring strong antioxidant power at very low concentrations. For instance, in the food industry, this oil could be explored as a natural preservative in products like meats or oils, where its combined antioxidant and previously demonstrated antibacterial properties could work in tandem to extend shelf life [102]. In a similar vein, such an oil could be used in cosmetic formulations not only for its fragrance but also to prevent rancidity and possibly provide mild skin protection [103].

3.5. Anti-Inflammatory Activity

The oxidative burst, the rapid release of reactive oxygen species (ROS) from activated neutrophils, serves as a critical driver in the pathogenesis of numerous chronic inflammatory conditions [104]. Consequently, therapeutic strategies aimed at tempering excessive neutrophil hold promise. Although conventional non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, provide clinical efficacy, their prolonged administration is linked to considerable adverse effects [105]. This has increased interest in safer, naturally derived agents. In this context, and to the best of our knowledge, we report the first investigation the ability of L. ovatus EO to inhibit the neutrophil oxidative burst, assessed by superoxide anion production, in isolated human neutrophils.

The results show that L. ovatus EO has a dose-dependent inhibitory effect on superoxide anion production. A measurable suppression was observed starting at 6.2 µg/mL, with inhibition increasing to nearly 50% at the highest tested concentration (200 µg/mL). The calculated IC₅₀ value for the essential oil was 165.29 µg/mL. As expected, the reference drug aspirin exhibited a clearly lower IC₅₀ of 28.85 µg/mL, consistent with its established anti-inflammatory activity.

Although aspirin was more potent, the biological relevance of the EO lies in its consistent, dose-dependent suppression of a key ROS-mediated inflammatory process by a complex natural mixture. This activity is not isolated; the potency of L. ovatus EO is comparable to that reported for other medicinal plant Eos in similar neutrophil-based models [106]. Thus, its effect falls within a recognized spectrum of natural anti-inflammatory candidates.

The underlying mechanism likely differs from that of aspirin. Whereas NSAIDs as aspirin directly targets COX enzymes, the anti-inflammatory effects of essential oils are often multifactorial and may involve interference with signaling pathways that activate neutrophils (e.g., NF-κB or MAPK pathways), or direct scavenging of already-formed ROS [107]. The EOs composition, rich in terpenoid constituents such as δ-selinene, β-cyclogermacrene, kessane, spathulenol, viridiflorol and neophytadiene, offers a plausible chemical basis, as several of these compound classes have been associated with an anti-inflammatory activity [108]. The observed effect may reflect of synergistic interactions among various constituents, a commonly reported feature of complex natural extracts [109].

The practical implications of this moderate activity should not be underestimated. Not all therapeutic applications require the high potency of a pharmaceutical drug. There is a growing market for natural products that offer gentle modulation of the immune response. The concentration range at which L. ovatus EO is active is of interest for potential topical applications. For instance, it could be explored as a candidate for formulations such as creams, gels, or balms aimed at mitigating oxidative stress and inflammation in conditions like arthritis, muscle soreness, or mild skin irritations [110]. Future studies assessing its cytotoxicity and skin irritation potential are necessary to confirm its safety profile for dermal use. In such applications, a balanced, multi-mechanistic approach with a favorable safety profile is often more desirable than extreme potency alone.

4. Materials and Methods

4.1. Plant Material

The leaves of L. ovatus were collected on 12 April 2025 in the Sendero de los Hieleros area of San Andrés (Guano Canton, Chimborazo Province, Ecuador) at 4410 m above sea level. Plant material was obtained from numerous shrubs scattered around the coordinates 01°30′04′′ S and 78°47′22′′ W, within an approximate 200 m radius; a composite average sample was obtained and mixed for analysis. Samples were immediately transported in sterile containers to the Natural Products Laboratory at the Escuela Superior Politécnica de Chimborazo (ESPOCH) for processing. Botanical identification was carried out by Professor Jorge Caranqui, head of the ESPOCH Herbarium, who compared the collected sample with samples stored in the Herbarium, finding they matched in morphological aspects. The leaves were dried in an oven at 35 °C for 48 h to minimize evaporation, reduce moisture, and prevent the thermal degradation of volatile components prior to distillation.

A voucher specimen was deposited under the code Ofc.No.027.CHEP.2025 and deposited in the ESPOCH Herbarium. This study was conducted under research permit No MAATE-ARSFC-2025-0167 granted by the Ministerium de Ambiente, Agua y Transición Ecológica del Ecuador (MAATE). The botanical sample was transported to the laboratories of the Universidad Técnica Particular de Loja under the mobilization guide 02964 issued by the Ministerio de Ambiente, Agua y Transición Ecológica del Ecuador (MAATE).

4.2. EO Distillation and Sample Preparation

Steam distillation process was performed using a modified Dean-Stark apparatus [111]. Briefly, the entire dried leaves of L. ovatus (700 g) were distilled for 5 h according to the literature [112,113]. This process was carried out only once using all the collected plant material, due to the difficult access to the collection site. In order to remove excess water, the essential oil was subjected to treatment with anhydrous sodium sulfate (Sigma-Aldrich, St. Louis, MO, USA) and permanently stored at a temperature of −15 °C until use. For chromatographic analyses (GC), four samples were prepared by weighing 10 µL aliquots and diluting them with 1 mL of cyclohexane, which contained n-nonane as an internal standard at a concentration of 0.70 mg/mL.

4.3. Qualitative Chemical Analysis

The chemical profile of the volatile components of L. ovatus EO was qualitatively characterized by gas chromatography-mass spectrometry (GC-MS) using a system composed of a Trace 1310 chromatograph coupled to an ISQ 7000 single-quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). In order to guarantee sufficient separation and identification of the compounds. A volume of 1 µL of cyclohexane solution in split mode (40:1 ratio), with helium (Indura S.A., Guayaquil, Ecuador) serving as the carrier gas, while maintaining a constant flow rate of 1 mL/min. The injector was maintained at 250 °C, and finally, ionization was carried out by electron impact at 70 eV, with a mass range of 40–400 m/z in SCAN mode.

Separations were performed on two columns of different polarity: a non-polar DB-5ms (5%phenylmethylpolysiloxane) and a polar TR-WAX (polyethylene glycol), both 30 m × 0.25 mm i.d., with a film thickness of 0.25 µm (Thermo Fisher Scientific, Waltham, MA, USA). The oven temperature program for the DB-5ms column was: 60 °C (5 min), ramp to 100 °C, then 3 °C/min to 150 °C, followed by 5 °C/min to 200 °C, and finally 15 °C/min to 250 °C (15 min hold). The same program was applied to the TR-WAX column, except that the final temperature was 230 °C (15 min hold).

The identification of compounds present in the essential oil of L. ovatus was based on the comparison of their mass spectra and linear retention indices (LRI) with reference libraries. Linear retention indices (LRIs) were calculated using a series of n-alkanes (C₉–C₂₅) according to the method of Van Den Dool and Kratz [114]. The n-alkanes C₉–C₂₅ were purchased from Sigma-Aldrich, St. Louis, MO, USA. For each identified compound, the mean retention time was calculated, and the relative standard deviation (RSD) of the retention times was determined.

4.4. GC-FID Quantitative Analyses

The quantification of the constituents of the essential oil of L. ovatus was performed by gas chromatography with an ionizable flame detector (GC-FID), using the same chromatographic equipment used in the qualitative analysis. Identical instrumental conditions were maintained in terms of thermal programming and the same stationary phases of different polarity (DB-5ms and TR-WAX), carrier gas flow (He, 1 mL/min) and injector temperature (250 °C), and the split was reduced to 10:1 (versus 40:1 used in the qualitative analysis) to adjust the sensitivity of the minor compounds in their quantification. Furthermore, the relative response factor (RRF) of each compound was calculated according to the enthalpy of combustion [115], the integration areas were correlated with the RRFs, and two calibration curves (one for each column) were constructed using isopropyl caproate as the calibration standard (synthesized and purified in this work, it presented a purity of 98.8%, confirmed by gas chromatography analysis) and nonane as the internal standard (Sigma-Aldrich, St. Louis, MO, USA). The correlation coefficient was >0.999 in both curves, as the solutions with the standards were prepared according to the literature [13].

4.5. Enantioselective Analysis

An enantioselective analysis of the four pairs of enantiomers presented by the essential oil of L. ovatus was carried out using gas chromatography coupled to mass spectrometry (GC-MS). The analysis was performed using enantioselective capillary columns of 25 m length, 0.25 mm internal diameter and 0.25 μm in phase thickness, with stationary phases based on β-cyclodextrin derivatives: 2,3-diacetyl-6-tert-butyldimethylsilyl-β-cyclodextrin (DAC) and 2,3-diethyl-6-tert-butyldimethylsilyl-β-cyclodextrin (DET) (Mega, Milan, Italy). The instrumental conditions were as follows: a split ratio of 50:1, a constant helium carrier gas pressure of 70 kPa, and a temperature program commencing at 60 °C for 2 min, increasing at 2 °C/min until reaching 220 °C, and maintaining this final temperature for 2 min. Mass spectra and linear retention indices (LRI), calculated using a series of C₉–C₂₅ n-alkanes (Sigma-Aldrich, St. Louis, MO, USA), were used to compare the detected enantiomers with data from the injection of enantiomerically pure standards. This was done according to the method of Van den Dool and Kratz. The selection of the DAC and DET columns was based on their observed efficiency in resolving the specific enantiomeric pairs present in the sample.

4.6. Antimicrobial Activity Assay

4.6.1. Bacterial Strains and Culture Conditions

Staphylococcus aureus (ATCC 25923; Gram-positive) and Escherichia coli (ATCC 25922; Gram-negative) were used in this study. Strains were cultured overnight at 37 °C in Mueller-Hinton broth (MHB) under aerobic conditions. Prior to testing, bacterial suspensions were adjusted to 0.5 McFarland (approximately 1–2 × 10⁸ CFU/mL) using sterile saline solution (0.85% NaCl).

4.6.2. Determination of Minimum Inhibitory Concentration (MIC)

The Minimum Inhibitory Concentration (MIC) was determined by the broth microdilution method in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [116] with minor modifications. Briefly, a two-fold serial dilution of the EO was prepared in MHB (Mueller–Hinton broth (MHB) was purchased from TM Media (Delhi, India), code TM 325) in a 96-well microtiter plate, resulting in a concentration range from 1000 to 0.98 µg/mL. Each well was then inoculated with the standardized bacterial suspension to a final concentration of approximately 5 × 10⁵ CFU/mL. Wells containing only broth and inoculum (growth control) and broth with inoculum and a known antibiotic (ampicillin or ciprofloxacin, obtained from Sigma-Aldrich /Merck, St. Louis, MO, USA, as a positive control) were included. The negative control consisted of sterile growth medium inoculated with the bacterial suspension, which showed normal growth (OD600 > 2.0 after 24 h of incubation).

The plates were incubated at 37 °C for 24 h. After incubation, 40 µL of a 0.2 mg/mL nitrobluetetrazolium chloride (NBT, ca. 98% TLC, Merck Sigma-Aldrich, St. Louis, MO, USA) solution was added to each well and incubated for a further 30 min. The MIC was defined as the lowest concentration of the extract that prevented the visible bacterial growth and the color change in NBT (from yellow to blue).

4.7. Antioxidant Activity Assay (DPPH Free Radical Scavenging)

The free radical scavenging activity of the EO was evaluated using the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma-Aldrich, St. Louis, MO, USA) radical method, as described by Brand-Williams et al. (1995) [117] with minor modifications. Briefly, a 0.1 mM solution of DPPH in absolute methanol (obtained from Pharmco by Greenfield Global, Palo Alto, CA, USA) was prepared. The EO was diluted in methanol to obtain concentrations ranging from 10 to 1000 µg/mL. In a 96-well microplate, 50 µL of each dilution was mixed with 150 µg of the DPPH solution. The mixture was incubated for 30 min at room temperature in the dark. Absorbance was then recorded at 517 nm using a microplate reader. A negative control (DPPH solution with methanol instead of sample) and a blank (methanol without DPPH) were included for baseline correction. The radical scavenging activity was calculated as a percentage of inhibition using the following formula:

$$\%Inhibition= {\displaystyle \frac{A_{control}-A_{sample}}{A_{control}}}\times 100$$

where A_control is the absorbance of the negative control and A_sample is the absorbance of the test sample. The results were expressed as the half-maximal inhibitory concentration (IC₅₀), calculated from the dose–response curve using non-linear regression analysis. Trolox was used as a positive control.

4.8. Anti-Inflammatory Activity (Oxidative-Burst Assay)

The anti-inflammatory activity of L. ovatus essential oil was evaluated by the oxidative burst assay developed by Tan and Berridge [118], with modifications reported by Vinueza et al. [119]. Human neutrophils were isolated from heparinized venous blood obtained from one healthy adult donor (male; age 40 years) using density gradient centrifugation on Ficoll-Paque™ PLUS (from Cityva, Uppsala, Sweden). The polymorphonuclear cell fraction was collected, and residual erythrocytes were lysed by a brief hypotonic shock (NH₄Cl from J.T. Baker, Mexico City, México, 0.83% w/v). The isolated neutrophils were then washed twice with Hank’s Balanced Salt Solution from Sigma-Aldrich, St. Louis, MO, USA (HBSS, pH 7.4) and finally resuspended in HBSS at a density of 1 × 10⁷ cells/mL for immediate use in the assays.

Anti-inflammatory activity was determined as a function of the reduction in water-soluble tetrazolium salt (WST-1 obtained from Roche, Manheim, Germany) in the presence of activated neutrophils. The assay was carried out in a total volume of 250 µL of HBSS (pH 7.4) containing 107 neutrophils/mL, 500 µM WST-1, and various concentrations of essential oil (3.12, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL) or aspirin (obtained from Sigma-Aldrich/Merck, St. Louis, MO, USA), which was used as the reference compound, both dissolved with HBSS enrichment with DMSO, such that the final concentration of DMSO in each well was 0.05%. The control contained HBSS, a neutrophil suspension, and WST-1. All compounds were equilibrated at 37 °C and the reaction was initiated by adding opsonized Zymosan A from Sigma-Aldrich, St. Louis, MO, USA (15 mg/mL), which was prepared by mixing it with human pooled serum, followed by centrifugation at 3000 rpm, and the pellet was suspended in HBSS. Absorbance was measured at 450 nm. DMSO Sigma-Aldrich/Merck, St. Louis, MO, USA (0.05% v/v) together with the neutrophil suspension and opsonized Zymosan A aliquot were used as a blank, and the anti-inflammatory activity was expressed as the produced superoxide anion inhibition percent [13].

This test was conducted in compliance with ethical standards and upon approval by the Comité de Bioética en Investigación-CBIESPOCH, approval code CIBE-2025-0087.

5. Conclusions

The EO obtained from dried leaves of L. ovatus had a yield of 0.21%, the chemical profile showed that silphinene, δ-selinene, β-cyclogermacrene, kessane, spathulenol, viridiflorol and neophytadiene are the major constituents (>3.0%). The EO exhibited measurable biological activities, including antibacterial activity—being more effective against Staphylococcus aureus (MIC = 250 µg/mL) than against Escherichia coli (MIC = 500 µg/mL)—as well as antioxidant activity, with an SC₅₀ of 375.7 µg/mL in the DPPH assay. In addition, the EO showed anti-inflammatory potential in the oxidative burst model, reaching a maximum inhibition of 49.12% at the highest concentration tested (200 µg/mL), although it was markedly less potent than the positive control (aspirin).

Overall, the observed activities are consistent with the EO diverse terpene profile, particularly oxygenated sesquiterpenes that may support antibacterial and antioxidant effects. In parallel, monoterpene bioactivity can depend strongly on enantiomeric composition because chiral targets may respond differently to stereoisomers. Therefore, determining the enantiomeric distribution of key constituents would strengthen structure–activity interpretation and comparisons across essential oils.

In summary, these findings support the pharmacological potential of L. ovatus EO and warrant further studies to isolate and characterize the major constituents, evaluate component interactions, elucidate mechanisms through cytotoxicity testing and inflammatory marker analysis, and confirm efficacy in relevant in vivo models. A key limitation is the lack of cytotoxicity data at the active concentration ranges, which is necessary for a robust safety assessment in future applications, and antioxidant capacity was evaluated only by the DPPH assay without additional datasets, so complementary methods such as ABTS, FRAP, ORAC, or CUPRAC should be included in future work to strengthen and contextualize these findings.