Chemical Analysis of the Essential Oils from Three Populations of Lippia dulcis Trevir. Grown at Different Locations in Southern Ecuador

In this investigation, we have analyzed for the first time the essential oils (EOs) isolated by steam distillation of the leaves and flowers of Lippia dulcis Trevir., grown at three different locations in southern Ecuador: the Catacocha canton (Ca), the Vilcabamba parish (Vi), and the Chuquiribamba parish (Ch). Around 98.5% of the oils’ constituents were identified by Gas Chromatography-Mass Spectrometry (GC-MS) and Gas Chromatography-Flame Ionization Detector (GC-FID) analysis using a DB-5ms capillary column. Sesquiterpene hydrocarbons were predominant in the EOs (79.77, 78.22, and 76.51%, respectively). The most representative constituents of the sample from the Ca canton were β-cedrene (16.75%), δ-selinene (11.04%), and β-cubebene (12.09%), while the sample from the Vi parish was characterized by the abundant presence of β-cedrene (17.9%), δ-selinene (12.52%), and bicyclogermacrene (11.34%). β-Cedrene (18.89%), δ-selinene (11.78%), and δ-cadinene (11.07%) were the main constituents of the essential oil (EO) from the Ch parish. The likely occurrence of low amounts of thermolabile hernandulcin in the volatile oils was indicated by the presence of the fragmentation products 6-methyl-5-hepten-2-one and 3-methyl-2-ciclohexen-1-one. In summary, the study gave us a clue to the variability of Lippia dulcis chemotypes depending on the collection sites.


Introduction
The plant Lippia dulcis Trevir., known under different vernacular names, such as the Spanish orozuz ("sweet grass"), is a medicinal plant belonging to the family of Verbenaceae Jaume St.-Hill) [1].According to a recent classification, this family includes 32 genera and 800 species of perennial herbs, shrubs, and subshrubs of ornamental and medicinal value [2,3].Phylogenetic studies [4,5] have shown that numerous genera traditionally classified as Verbenaceae belong instead to Lamiaceae.The plants are native to the subtropical regions of warm and semi-warm climates in Africa, Mexico, and Central America [6].However, the flora of this taxon has become cosmopolitan thanks to the introduction of 26 genera and 180 species in North and South America, especially in Argentina and Brazil.
The Ecuadorian biodiversity of the family Verbenaceae is represented by 141 species, including 70 endemic ones, 22 genera, and 23 endemic taxons.They are distributed in the foothills along the "Cordillera de Los Andes", from the Pacific coast to the páramo ecosystems.Thus, seven species are found in coastal forests, six on the islands of Galápagos, and two in the Amazon region.Due to the wide distribution, only one species (L.salicifolia Andersson) is protected by the National System of Protected Areas, while the remaining ones are classified as threatened.However, the names of these species are not available online [7].
Since ancient times, most verbenas, including Lippia species, have been known worldwide for their traditional use in the treatment of gastrointestinal disorders and respiratory Plants 2024, 13, 253 2 of 12 problems [3].Some species have also attributed antimalarial, antiviral, sedative, hypotensive, anti-inflammatory, and cytostatic activities.These properties are due to the presence of bioactive phytochemicals with important pharmacological effects, such as phenolic compounds and essential oils [8][9][10][11].According to literature [10,12], 49 species of the family Verbenaceae grown in Ecuador are traditionally used for one or more healing purposes.Moreover, many species of Verbenaceae are of economic importance in agronomy and are utilized as ornamental plants and seasoning for food preservation [5].
According to different authors, the genus Lippia includes from 140 [2] to 200 [3] species widely distributed from the southern United States to the South and Central America regions and Tropical Africa territories [10], with the highest concentration of species in Brazil and Paraguay [13].
The genus includes aromatic herbs, shrubs, and small trees with white flowers, opposite or triple leaves, and entire or serrated with multiple buds.About the inflorescence, Lippia shows a zygomorphic flower, tubular calyx, and pentamerous bilabiate corolla with exalbuminate seeds [6].
L. dulcis is a perennial aromatic herb with leaves and flowers of intense sweetness [14,15].Its synonyms are Phyla scaberrima (Juss.ex Pers.)Moldenke, Zappania scaberrima Juss.ex Pers., Phyla dulcis (Trevir.)Moldenke, L. asperifolia Rchb., and L. dulcis var.mexicana Wehmer [16].The distribution of this plant in the New World extends from México, Panamá, Puerto Rico, Cuba, and other Caribbean and Central American countries to Venezuela and Colombia [15].In Ecuador, the plant is distributed in the provinces of Loja, Azuay, Pichincha, and Zamora Chinchipe [12].Orozuz uses started in the traditional medicine of the Aztecs/Mexico, who used the plant, under the Nahuatl name Tzopelic xihuitl ("sweet herb"), to treat bronchitis, coughs, and colds.Decoctions or infusions of the leaves and flowers are traditionally used as a topical lotion for respiratory diseases, in the treatment of diarrhea, dysentery, and indigestion, as well as an emmenagogue and a remedy to treat menstrual disorders [8,10,14].Bactericidal, antispasmodic, antimalarial, sedative, analgesic, anti-inflammatory, antipyretic, diuretic, hypotensive, antifertility, abortifacient, and antiviral properties are attributed to the leaves and flowers of L. dulcis in many Latin American countries [8,10,14].
Despite the wide uses in folk medicines, it should be noted that the employment of L. dulcis as a low-cariogenic sweetening agent and as an additive to food and formulations of pharmaceutical products and oral hygiene products [17] is often hampered by the presence in the plant of bitter compounds, especially the toxic camphor, and other constituents with a pungent taste [15,17].
The EOs of Lippia species contain limonene, citral, carvacrol, β-myrcene, p-cymene, camphor, linalool, α-pinene, β-caryophyllene, carvone, and thymol as the main chemical components [10,11].Some examples include carvone and limonene (group I), as in the EO of L. alba; citral and β-caryophyllene found in L. citriodora (group II); and carvacrol, thymol, and p-cymene present in L. origanoides and L. micromera (group III).Group IV is characterized by an abundant presence of p-cymene and small amounts of carvacrol and thymol, as in the L. origanoides EO [11].A remarkable characteristic of Verbenaceae EOs, including those isolated from Lippia species, is the variable chemical composition that determines the existence of many chemotypes, especially for species growing in South American countries [11].The composition of L. alba EO well illustrates such variability [11].In fact, the citral, carvone, and limonene chemotypes were found in different regions of Colombia, while the linalool chemotype was identified in Uruguay.On the other hand, L. alba growing in Argentina presents five chemotypes, namely, the linalool chemotype (up to 91%), the citral chemotype (up to 76% with two subtypes, i.e., myrcene and limonene), the piperitone chemotype (up to 37%), the lippione chemotype (up to 50%), and the dihydrocarvone chemotype [11].The composition of L. dulcis oils also varied significantly with the geographic origin, especially as regards the content of camphor and hernandulcin (1) [23], which has suggested the existence of at least two chemotypes [24].For example, traces of camphor were found in the volatile fractions isolated from plant specimens collected in Brazil and Puerto Rico, whereas high amounts (from 21.2 to 53.2%) were determined in the volatile oil isolated from Mexican samples [15,23].On the other hand, variable amounts of camphor (from 0.02 to 32.6%) were detected in volatile oils from the seeds of L. dulcis collected in Panama [23].Compadre et al. identified 86% of nonoxygenated and oxygenated monoterpenoids and 13% of sesquiterpenes in the volatile oil The essential oil of L. dulcis, at the dose of 100 µg/mL, exhibited anti-histaminergic and anti-cholinergic properties [20], whereas 400 mg/kg of the ethanol extract produced significant inhibition of carrageenan-induced paw oedemas and reduced the weight of cotton pellet-induced granuloma; moreover, the topical application of 0.5 mg/ear of the ethanol extract inhibited the oedema induced with 12-O-tetradecanoylphorbol acetate (TPA) by 49.13% [21].Finally, recent molecular docking studies demonstrated that some sesquiterpenes identified among the volatile compounds of L. dulcis from the Colombian Pacific showed very high affinity against the crystallized structures of two proteins, Mpro and PLpro, associated with Severe Acute Respiratory Syndrome COronaVirus 2 (SARS-CoV-2) [22].
The chemical composition of the volatile oil from L. dulcis Trevir.grown in Ecuador (Figure 2a) has not been investigated so far.In this inaugural study that we intend to extend to the entire country, we describe the components of the oil steam distilled from the leaves and flowers of L. dulcis grown in the Loja province in southern Ecuador.To investigate the variability of the oil composition, three samples were obtained at three different locations near the town of Loja: the Catacocha canton (Ca), the Vilcabamba parish (Vi), and the Chuquiribamba parish (Ch) (Figure 2b).In the folk medicine of local communities, infusions of the flowers and roots of L. dulcis are widely used for stopping menstrual bleeding and treating stomach inflammation [12].
The chemical composition of the volatile oil from L. dulcis Trevir.grown in Ecuador (Figure 2a) has not been investigated so far.In this inaugural study that we intend to extend to the entire country, we describe the components of the oil steam distilled from the leaves and flowers of L. dulcis grown in the Loja province in southern Ecuador.To investigate the variability of the oil composition, three samples were obtained at three different locations near the town of Loja: the Catacocha canton (Ca), the Vilcabamba parish (Vi), and the Chuquiribamba parish (Ch) (Figure 2b).In the folk medicine of local communities, infusions of the flowers and roots of L. dulcis are widely used for stopping menstrual bleeding and treating stomach inflammation [12].Moreover, we were highly interested in the determination of the hernandulcin and camphor contents in view of the possible application of the plant as a sweetener.In this regard, the taste of the three samples of L. dulcis examined in this work was characterized by the presence of sesquiterpenes of the bisabolane type, such as hernandulcin.

Physical Properties of the EOs
The three EOs were obtained in yields ranging from 0.08 to 0.20%, depending on the plant samples.They had a pale-yellow color, a fresh aroma, and a pleasant, sweet taste.The relative density, refractive index, and optical rotation of the oils are reported in Table 1.

Chemical Composition of the EOs
A total of 76 compounds with percentages >0.05% were identified in the three EOs steam distilled from fresh leaves and flowers of L. dulcis.They accounted for 98.23, 97.39, and 99.89%, respectively, of the Ca, Vi, and Ch EOs analyzed on a DB-5ms (dimethylsilicone phase with 5% phenyl groups) capillary column (Figure 3).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.The values of percentage and standard deviation were the means of three determinations.Each EOs component was identified by comparing the calculated linear retention index (LRI) on the DB-5ms column and/or the GC-MS spectrum with the PubChem (nih.gov)database (https://pubchem.ncbi.nlm.nih.gov,accessed on 1 September 2023) and the references [27][28][29].The LRIs were calculated according to the method of Van Den Dool and Kratz [30], using a mixture of n-alkanes (C 9 -C 25 ) injected under the same chromatographic conditions as the EOs.The qualitative and quantitative compositions of the oils are reported in Table 2.
, 69 (53), 67 (22), 55 (31), 53 (7), 43 (10), 41 (55), 40 (100); Unknown B: m/z (%) 204 (15, M + ), Indeed, the analysis of the three EOs was tentatively also carried out on a capillary column containing a polar polyethylene glycol stationary phase (INNOWax).We observed, however, that the peaks were generally more overlapped than on the DB-5ms column; moreover, we found that the LRIs reported in the literature for the EO constituents are often greatly different, making compound identification rather uncertain.Therefore, the data from the GC analysis on the polar column have not been considered.
The components occurring in the three EOs with percentages >3% on the DB-5ms column are listed in Table 3, while their chemical structures are depicted in Figure 3.Only the relative stereochemistry is shown.

Discussion
Transparent greenish EOs were steam distilled from fresh samples of L. dulcis collected at three different locations in southern Ecuador.The EO from Catacocha showed the highest isolated yield (0.20%, w/w).However, it should be optimized according to the season and extraction technique.In fact, this value must be compared with the 0.6% and 1.7-3.4% yields obtained by hydrodistillation and supercritical fluid extraction, respectively, of dried L. dulcis collected in Brazil [19].On the other hand, the relative density and the refractive index of the oils (Table 1) resembled those of L. alba collected in Brazil [31].Around 98.5% of the chemical constituents of L. dulcis EOs from the three Ecuadorian locations were identified.The GC-FID and GC-MS analyses on the DB-5ms column (Table 2) indicated that the EOs consisted mainly of sesquiterpene hydrocarbons (79.77, 78.22, and 76.51%, respectively), followed by oxygenated sesquiterpenoids (12.69, 15.67, and 20.38%, respectively).Oxygenated monoterpenoids (2.18, 0.74, and 0.27%, respectively) and other compounds (3.59, 2.76, and 2.73%, respectively) occurred in the EOs as minor components.Noteworthy, monoterpene hydrocarbons were not detected in the three EOs, and the percentages of only nine compounds were >3% (Table 3).
In striking contrast to the EOs isolated from other Lippia species, in which monoterpenoids predominated [11], and to the oils from L. dulcis collected in Mexico, which were rich in camphor [15,25], the high content of sesquiterpene hydrocarbons characterized the EOs from Ecuadorian specimens.This feature also characterized the EOs from L. dulcis collected in Puerto Rico [23], Colombia [22,24], and Brazil [25], which, however, contained different sesquiterpenes as the most abundant components, while hernandulcin (1) and 6-mehyl-5-hepten-2-one occurred in the volatile oils in various amounts (see the Introduction above).These findings, in particular the apparently minimal accumulation of hernandulcin and the absence of camphor in the volatile oils, indicate that Lippia dulcis grown in southern Ecuador may constitute a new chemotype of It should be noted that camphor and hernandulcin (1) were not detected in any of the three volatile oils.Even the products formed by thermal degradation of hernandulcin [15], 6-methyl-5-hepten-2-one (2), and 3-methyl-2-ciclohexen-1-one were found in low amounts.
In striking contrast to the EOs isolated from other Lippia species, in which monoterpenoids predominated [11], and to the oils from L. dulcis collected in Mexico, which were rich in camphor [15,25], the high content of sesquiterpene hydrocarbons characterized the EOs from Ecuadorian specimens.This feature also characterized the EOs from L. dulcis collected in Puerto Rico [23], Colombia [22,24], and Brazil [25], which, however, contained different sesquiterpenes as the most abundant components, while hernandulcin (1) and 6-mehyl-5-hepten-2-one occurred the volatile oils in various amounts (see the Introduction above).These findings, in particular the apparently minimal accumulation of hernandulcin and the absence of camphor in the volatile oils, indicate that Lippia dulcis grown in southern Ecuador may constitute a new chemotype of this species.

Essential Oil Isolation
The three EOs of Lippia dulcis Trevir.were obtained from fresh leaves and flowers (approximately 2.15 kg of plant from each location) by steam distillation for 3 h using Clevenger-type equipment.After decantation, each supernatant oily layer was separated from the condensed aqueous phase using a pipette; subsequently, each oil (3.5, 0.85, and 1.22 mL of Ca, Vi, and Ch, respectively) was dried over anhydrous Na 2 SO 4 , filtered, and stored in an amber vial at 4 • C until use.The procedure was performed in triplicate.

Physical Properties
The relative density of the EOs from L. dulcis was determined according to the international standard AFNOR NF T75-111 (ISO 279:1998) [32] guidelines.The refractive index was determined using an ABBE refractometer, manufactured by Boeco (Hamburg-Germany), according to the international standard AFNOR NF 75-112 (ISO 280:1998) [33] procedure.
The specific optical rotation a] 20 D was measured according to the ISO 592:1998 [34] procedure using an automatic polarimeter Hanon P 810, manufactured by Hanon Advanced Technology Group (Jinan-China).For each physical property, the mean value and the SD were determined from three replicates performed at 20 • C. The L. dulcis EOs were qualitatively analyzed by GC-MS using an Agilent Technologies Chromatograph 6890N coupled to an Agilent mass spectrometer, the 5973 Inert series (Santa Clara, CA, USA), which operated in electron impact mode at 70 eV.The mass spectrometer was operated in SCAN mode (range 35-350 m/z), had a scan rate of 0.2 scan/s, and was controlled by MSD-Chemstation D.01.00SP1.
The reported results of the EOs analysis were obtained on a DB-5ms (5% phenylpolymethylsiloxane; 30 m × 0.25 mm × 0.25 µm, Agilent Technologies; J; W Scientific, Folsom, CA, USA) capillary column; helium (1.00 mL/min) was used as the carrier gas.The injection system operated in split mode (40:1) at 220 • C. The program for the GC oven temperature was as follows: initial temperature of 60 • C, held for 5 min, followed by a ramp of 3 • C/min until 250 • C, which was finally held for 10 min.Each EO was diluted with CH 2 Cl 2 (1:100 v/v), and 1 µL of the solution was injected into each analysis.
To calculate LRIs, a homologous series of n-alkanes C 9 -C 25 (C 9 , BHD purity 99%, and C 10 -C 25 , Fluka purity 99%) were injected after each EO under identical chromatographic conditions.The EOs were quantitatively analyzed by GC-FID on an Agilent Technologies 6890N series gas chromatograph coupled to a flame ionization detector (FID), using the DB-5ms column mentioned in Section 4.4.1.The analytical gas-chromatographic conditions were the same as those used for the GC-MS analysis.

Conclusions
The chemical composition and the physical properties of the steam distilled EOs from L. dulcis collected at three locations in southern Ecuador (Catacocha, Vilcabamba, and Chuquiribamba) were determined for the first time.The profiles of the three oils markedly differed from the EOs of L. dulcis collected in other Central and South American countries, confirming that the chemical composition of L. dulcis volatile oils can vary significantly with the geographic origin of the plant.Instead, the EOs isolated from the three samples collected in Ecuador were similar qualitatively, and this finding may depend on the proximity of the three locations, even if the altitudes varied from 1460 to 2600 m a.s.l.The minimal concentration of hernandulcin (1) in the EOs must be confirmed by collecting the plant in other locations of Ecuador, in different periods of the year, at different stages of growth, and using different extraction and analysis conditions [19].Moreover, the absence of camphor, if confirmed, is promising for the sustainable use of L. dulcis as a natural, non-cariogenic sweetener.

Figure 1 .
Figure 1.Structures of the main compounds isolated from extracts of Lippia dulcis.

Figure 1 .
Figure 1.Structures of the main compounds isolated from extracts of Lippia dulcis.

Figure 2 .
Figure 2. (a) L. dulcis (b) Collection locations of L. dulcis in the Loja province in southern Ecuador.Figure 2. (a) L. dulcis (b) Collection locations of L. dulcis in the Loja province in southern Ecuador.

Figure 2 .
Figure 2. (a) L. dulcis (b) Collection locations of L. dulcis in the Loja province in southern Ecuador.Figure 2. (a) L. dulcis (b) Collection locations of L. dulcis in the Loja province in southern Ecuador.

Figure 4 .
Figure 4.Chemical structures of the main components of the EOs of Lippia dulcis from Ecuador.

Figure 4 .
Figure 4.Chemical structures of the main components of the EOs of Lippia dulcis from Ecuador.

Fresh
leaves and flowers of Lippia dulcis Trevir.were purchased from April to June 2022 from medicinal plant vendors at three different locations in the province of Loja, southern Ecuador: (i) at the Cangonama parish of the Catacocha canton, at an altitude of 1460 m a.s.l.(GPS coordinates: 3 • 58 0 S, 79 • 42 0 W); (ii) at the San Francisco neighborhood of the Vilcabamba parish, at 1700 m a.s.l.(GPS coordinates: 4 • 15 0 S and 79 • 15 0 W); (iii) at the Carmelo sector of the Chuquiribamba parish, at 2600 m a.s.l.(GPS coordinates: 03 • 75 8 S, 79 • 15 00 W).The UTPL botanist Jorge Armijos identified the botanical material, and vouchers of the three plant samples have been deposited at the Herbarium of the Universidad Técnica Particular de Loja (HUTPL) with the codes HUTPL 14610-14612.

Table 1 .
Physical properties of EOs from Lippia dulcis Trevir.collected in southern Ecuador.

Table 2 .
Chemical composition of L. dulcis essential oil from fresh plant samples collected at Catacocha, Vilcabamba, and Chuquiribamba.
RT = retention time; a LRIcalc = calculated linear retention index; b LRI lit = linear retention index taken from literature; c tentatively identified by the MS spectrum; %, percentage; SD, standard deviation.Both values were calculated as the mean of three determinations; * EIMS data for unknown compounds.Unknown A: m/z (%) 168 (~0.1, M + ), 156