Chemical characterization of two morphologically related Espeletia (Asteraceae) species and chemometric analysis based on essential oil components

ABSTRACT

In this study, a comprehensive phytochemical characterization of two morphologically related species from the genus Espeletia Mutis ex Bonpl., namely, Espeletia grandiflora Humb. & Bonpl. and Espeletia killipii Cuatrec., Asteraceae, has been performed by gas chromatography coupled to flame ionization detection, gas chromatography coupled to mass spectrometry and ultra-high performance liquid chromatography coupled to ultraviolet and high-resolution mass spectrometry. Analysis of ethanol extracts (70%, v/v) from leaves and concomitant compound dereplication allowed the identification of major peaks, most of them new reports for the genus Espeletia or the subtribe Espeletiinae. Chemical characterization of resins essential oils indicated several similarities and differences between both species and from other members of the subtribe. Chemometric analysis (hierarchical clustering analysis and orthogonal partial least-squares discriminant analysis) applied to the essential oil composition of 31 species from Espeletiinae furthermore allowed the identification of three primary clusters correlated with the taxonomy. Hence, this study underscored qualitative and semiquantitative differences between the chemical composition of leaves and resins of E. grandiflora and E. killipii, provided information on chemotaxonomy and described the presence of different trends in the essential oil composition from species of Espeletiinae.

Keywords:
Asteraceae; Chemotaxonomy; Essential oils; Espeletia; Chemometric analysis

Introduction

The genus Espeletia Mutis ex Bonpl., Asteraceae, constitutes a neotropical group endemic to the northern Andean regions of Venezuela, Colombia, and Ecuador. With ca. 71 species, Espeletia is the most diverse genus within the subtribe Espeletiinae, which also comprises the genera Espeletiopsis Cuatrec., Coespeletia Cuatrec., Ruilopezia Cuatrec., Libanothamnus Ernst, Carramboa Cuatrec., Tamania Cuatrec., and Paramiflos Cuatrec. (Cuatrecasas, 2013; Diazgranados, 2012a).

Espeletia grandiflora Humb. & Bonpl. was the first species in Espeletiinae to be described and continues to be frequently studied in terms of its morphology, ecophysiology, distribution, and taxonomy (Fagua and Gonzalez, 2007; Cuatrecasas, 2013). However, gaps remain in the taxonomic delimitation of this species, which could possibly include different taxonomic entities, given its high polymorphism and broad geographic distribution (Cuatrecasas, 2013). Its shared morphological characteristics with Espeletia killipii Cuatrec. (e.g., leaf shape, plant height, and overall appearance) moreover hinder its unequivocal identification. According to Cuatrecasas (2013), two primary morphological characteristics that distinguish E. grandiflora from E. killipii include the presence of at least one pair of sterile opposite leaves in the proximal part of the synflorescences in the former species—by contrast, E. killipii lacks such sterile leaves—and the length of the synflorescences, which are longer in E. grandiflora. However, the frequent hybridization of the two species in zones where overlapping populations occur (e.g., La Chisacá páramo in Colombia) further complicates their unambiguous taxonomic delimitation (Cuatrecasas, 2013).

The secondary metabolite chemistry of E. grandiflora and E. killipii has been poorly investigated. Nevertheless, classic phytochemical studies of E. grandiflora resins have reported the presence of six ent-kaurane diterpenes—kaur-16-ene, kaur-16-en-19-al, kaur-16-en-19-ol, kaurenoic acid, grandiflorolic acid, and grandiflorenic acid (Piozzi et al., 1968, 1971, 1972)—whereas other chemical classes (e.g. flavonoids, sesquiterpene lactones, and triterpenes) have been reported from the leaves of E. killipii (Torrenegra et al., 1994; Torrenegra and Tellez, 1995, 1996). However, no comprehensive phytochemical characterization that includes the leaves and resins of both species has been performed, and the similarities and differences between the metabolic fingerprints of E. grandiflora and E. killipii remain unknown.

Consequently, this study aimed to perform a comprehensive phytochemical characterization of E. grandiflora and E. killipii leaves and resins by using modern and complementary analytical techniques such as GC–FID, GC–MS and UHPLC–UV-HRMS, all to determine similarities and differences between the species, as well as among EO from 31 species of Espeletiinae, as performed by chemometric methods involving multivariate statistical analysis.

Materials and methods

Plant material

Leaves and resins of Espeletia grandiflora Humb. & Bonpl. and E. killipii Cuatrec., Asteraceae, were collected in the Cruz Verde (N 4º 34' 48.5" W 73º 59' 45.3" – 3328 m.) and Sumapaz páramos (N 4º 17' 24" W 74º 12' 24.2" – 3717 m.), respectively, in Cundinamarca, Colombia, by Sandra L. Castañeda and Carlos I. Suárez in January 2015. Plant identification was performed by Dr. Mauricio Diazgranados and Carlos I. Suarez from Jardín Botánico de Bogotá (JBB), and a voucher sample of each species was deposited at JBB's herbarium under the collection numbers SLC-192 and SLC-207, respectively. Plant collections were made with JBB collection permits.

Leaves extraction and UHPLC–UV-HRMS analysis

From each species, 20 mg of dry leaves were powdered with liquid nitrogen and extracted with 2 ml of EtOH:H₂O (7:3, v/v, HPLC grade) in an ultrasonic bath for 15 min at 25 ºC. Plant extracts were centrifuged at 19,975 × g for 10 min and partitioned with 0.5 ml of hexane. The aqueous layer was filtered through a 0.2 µm PTFE membrane and analyzed by UHPLC-UV-HRMS in an Accela UHPLC apparatus (Thermo Scientific, Carlsbad, CA, USA) coupled to an 80-Hz photodiode array detector (PDA) (Thermo Scientific) and an Exactive™ Plus Orbitrap mass spectrometer (MS) (Thermo Scientific).

A heated electrospray probe was used as an ionization source in MS analyses along with the following parameters: positive and negative ionization modes (full scan method) over a mass range of 150–2000 Da, resolution of 70,000 full width at half maximum (FWHM) (m/z 200), mass accuracy <3 ppm, scan time of about 2.5 scans/s, spray voltages of +3.6 kV and −3.2 kV for the positive and negative ionization modes, respectively, capillary and heater temperature of 300 ºC, sheath gas of 30 arbitrary units, and S-lens of 55 arbitrary units.

Subsequently, 10 µl of each plant extract were injected and chromatographed in a C18 column (Hypersil Gold, 1.9 µm, 50 mm × 2.1 mm, Thermo Scientific) connected to a C18 Security Guard™ Ultra cartridge (Phenomenex, Torrance, CA, USA). The mobile phase involved purified water with 0.1% formic acid and acetonitrile, and separation was performed at a flow rate of 300 µl/min. The gradient program started with 5% acetonitrile for the first 3 min and increased the amount linearly to 100% in 30 min. Acetonitrile remained constant for 5 min before ultimately decreasing to 5% after 40 min. The column temperature was maintained at 30 ºC, the tray temperature was fixed to 10 ºC, and the PDA detector was set to record in the range of 200–600 nm.

Dereplication of leaf extracts

Extracts dereplication was performed based on spectral (UV and accurate mass) and retention time (Rt) comparisons with reference compounds previously isolated in our laboratory—namely, 3-O-(E)-caffeoylquinic acid, protocatechuic acid, 3,5-di-O-(E)-caffeoylquinic acid, 4,5-di-O-(E)-caffeoylquinic acid, quercetin, 3-methoxy quercetin, and ent-kaurenoic acid. Compounds whose reference standard was unavailable were tentatively identified by accurate mass and UV spectra comparisons with data from the literature, including the Dictionary of Natural Products (DNP) and the Asteraceae Database (AsterDB), the latter of which corresponds to an in-house database (www.asterbiochem.org) containing hundreds of chemical compounds previously reported in the Asteraceae family, including of the subtribe Espeletiinae. In-source fragmentation in the UHPLC–UV-HRMS (Orbitrap) (MS/MS) was additionally considered to propose some of the peak assignments.

Essential oil extraction

The resins of E. grandiflora (2.1 g) and E. killipii (16.8 g), both collected during anthesis, were hydrodistilled using a Clevenger-type apparatus for 3 h and stored at −20 ºC for further analysis. Before gas chromatography analysis EOs were dissolved (1%, v/v) in ethyl acetate.

Essential oil analyses

EO from E. grandiflora and E. killipii resins were analyzed using a Hewlett–Packard 6890N Plus GC–FID (USA) apparatus where the percentage composition of individual components was determined. Identification of compounds was performed by comparison of the retention indices of each component relative to a series of n-alkanes, and by mass spectra comparisons with NIST 08 and Willey 7 databases based on GC–MS analyses on a Shimadzu QP-2010 system (Tokyo, Japan). To confirm identifications, the mass spectrum and retention index of each compound was further compared with published data (Adams, 2007).

During GC-FID analyses, 1 µl of each sample was injected (in triplicate) using a split ratio of 1:20 and separated on a HP-5 fused silica column (30 m; 0.32 mm I.D.; 0.25 µm film thickness). The carrier gas was hydrogen at 1.3 ml/min, the oven temperature was programmed from 60 to 240 ºC at 3 ºC/min, and the injector temperature was 240 ºC. Retention indices were calculated relative to C₈–C₄₀ n-alkanes. For GC–MS analyses, an EN5MS column was employed (30 m; 0.25 mm I.D.; 0.25 µm film thickness) with a source temperature of 250 ºC, carrier gas adjusted to 41.6 cm/s, an ionization energy of 70 eV, and a scan range of 40–500 Da. The temperature program and remaining parameters were identical to those in GC–FID analyses.

Chemometric analyses

The relative amount of the EO components of 31 species from the subtribe Espeletiinae reported in the literature and in the present study (Table 1) were used to perform multivariate statistical methods in the software R 3.0.3 (R Foundation for Statistical Computing, Vienna, Austria) and SIMCA P 13.0.3.0 (Umetrics AB, Malmö, Sweden). Prior to analyses, the data matrix was scaled by using the arcsine method, which is appropriate and commonly used for data expressed as percentages. Hierarchical clustering analysis with bootstrap resampling (HCAbp) was performed in the R package pvclust (Suzuki and Shimodaira, 2006), using Ward's method as the clustering algorithm and Euclidian distance. To generate a quantitative measure of accuracy in the clusters obtained, approximately unbiased (AU) p values were considered in the analysis, calculated by multiscale bootstrap, which is more accurate than traditional bootstrapping, as Suzuki and Shimodaira (2006) have confirmed. In multiscale bootstrapping, since the size of the bootstrap sample varies according to the original matrix, it corrects the bias of the traditional bootstrapping value caused by the constant sample size (Suzuki and Shimodaira, 2006).

Table 1
Species from subtribe Espeletiinae used to perform chemometric analyses based on essential oil compositions; species organized according to the three clusters identified by hierarchical clustering analysis with bootstrap resampling.

Cluster	Species	Locality of collection (Country)	Altitude m.a.s.l.	Date of collection	Plant stage	Plant part	Reference
1	Co. moritziana 1	La Culata (VEN)	3750	9/2/1998	Anthesis	Leaves	Aparicio et al. (2002)
1	Co. moritziana 2	Piñago (VEN)	3800	12/9/1999	Anthesis	Leaves	Aparicio et al. (2002)
1	Co. moritziana 3	Los Osos (VEN)	3700	5/25/2004	Anthesis	Leaves	Ibáñez and Usubillaga (2006a)
1	Co. moritziana 4	Pico del Águila (VEN)	4100	9/22/2002	Anthesis	Leaves	Ibáñez and Usubillaga (2006a)
1	Co. spicata 1	La Culata (VEN)	3500	11/22/1998	Anthesis	Leaves	Aparicio et al. (2002)
1	Co. spicata 2	Las Cruces (VEN)	3850	12/9/1999	Anthesis	Leaves	Aparicio et al. (2002)
1	Co. timotensis	Pico del Águila (VEN)	4000	n.r.	Anthesis	Aerial parts	Rojas et al. (1999)
1	E. nana	Paramo de Ortiz (VEN)	3085	5/1/2010	n.r.	Leaves	Peña et al. (2012)
1	E. batata	Piedras Blancas (VEN)	4200	11/1/1998	n.r.	Leaves	Usubillaga et al. (2001b)
1	E. grandiflora	Paramo de Cruz verde (COL)	3328	1/22/2015	Anthesis	Resin	Present study
1	E. killipii	Paramo de Sumapaz (COL)	3717	1/26/2015	Anthesis	Resin	Present study
1	L. occultus ssp humbertii	Laguna Negra (VEN)	3200	12/2/1999	Anthesis	Leaves	Usubillaga et al. (2001a)
2	L. neriifolius	El Batallón (VEN)	2800	11/6/1998	Anthesis	Leaves	Usubillaga et al. (2001a)
2	L. occultus	El Batallón (VEN)	2800	11/6/1998	Anthesis	Leaves	Usubillaga et al. (2001a)
2	L. lucidus	La Aguada (VEN)	3400	11/24/1999	Anthesis	Leaves	Usubillaga et al. (2001a)
2	E. schultzii 1	La Culata (VEN)	2800	9/12/2002	Anthesis	Leaves	Ibáñez and Usubillaga (2006b)
2	E. schultzii 2	Los Osos (VEN)	3700	9/22/2002	Anthesis	Leaves	Ibáñez and Usubillaga (2006b)
2	E. schultzii 3	Pico del Águila (VEN)	4100	10/22/2002	Anthesis	Leaves	Ibáñez and Usubillaga (2006b)
2	E. weddellii	Las Cruces (VEN)	4080	n.r.	Anthesis	Leaves	Khouri et al. (2000)
2	E. semiglobulata	Pico del Águila (VEN)	3800	n.r.	n.r.	Leaves	Usubillaga et al. (1999)
2	E. x aurantia	Pico del Águila (VEN)	n.r.	11/15/2003	Anthesis	Leaves	Ibáñez and Usubillaga (2008)
3	R. marcescens 1	El Batallón (VEN)	3000	6/22/1999	Anthesis	Leaves	Aparicio et al. (2001)
3	R. marcescens 2	El Batallón (VEN)	3000	1/27/2000	Anthesis	Leaves	Aparicio et al. (2001)
3	R. atropurpurea 1	El Batallón (VEN)	3400	11/6/1998	Anthesis	Leaves	Aparicio et al. (2001)
3	R. atropurpurea 2	El Batallón (VEN)	3400	6/22/1999	Anthesis	Leaves	Aparicio et al. (2001)
3	R. lindenii	San José (VEN)	3100	8/10/1999	Anthesis	Leaves	Aparicio et al. (2001)
3	R. floccosa	Pico del Águila (VEN)	3800	5/25/1999	Anthesis	Leaves	Aparicio et al. (2001)
3	Ca. badilloi	La Azulita (VEN)	1700	n.r.	n.r.	Leaves	Cordero et al. (2011)
3	Ca. badilloi var. pittieri	Bailadores (VEN)	1800	n.r.	n.r.	Leaves	Rojas et al. (2008)
3	Co. thyrsiformis	La negra (VEN)	3000	6/22/1999	Anthesis	Leaves	Aparicio et al. (2002)
3	Es. angustifolia	San José (VEN)	2870	6/1/2006	n.r.	Leaves	Meccia et al. (2007)

• n.r., not reported. Genera abbreviations: Co., Coespeletia; E., Espeletia; L., Libanothamnus; R., Ruilopezia; C., Carramboa; Es., Espeletiopsis.VEN, Venezuela; COL, Colombia.

To identify the compounds responsible for species clustering in the three primary clusters observed in the HCAbp, an orthogonal partial least-squares discriminant analysis (OPLS-DA) was performed with SIMCA P. This method separates the systematic variation in X into correlated (predictive) and uncorrelated (orthogonal) variables to Y (Eriksson et al., 2012). The variables responsible for the clustering pattern observed were identified according to a variable importance in the projection (VIP) value >1.0, a significant contribution to the loadings plot, and a high magnitude and high reliability confidence intervals in the coefficients plot.

Results and discussion

Leaves characterization by UHPLC–UV-HRMS

The metabolic fingerprinting of crude ethanol (70% v/v) extracts from E. grandiflora and E. killipii leaves by using UHPLC–UV-HRMS (Fig. 1) and concomitant dereplication allowed the identification of several similarities and differences between the species. As observed in total ion current chromatograms (Fig. 1), both species have highly similar fingerprints with greater semiquantitative instead of qualitative differences. All dereplicated compounds (Fig. 1) were detected in both species, though E. killipii showed a greater relative abundance of peaks 6 and 10–15 (Fig. 1). Four main chemical classes of secondary metabolites were identified in the leaves extracts of both species: trans-cinnamic acid derivatives, flavonoids, diterpenes, and one triterpene.

Fig. 1
Total ion current (TIC) chromatograms of E. grandiflora and E. killipii leaves extracts obtained in negative mode. (1) 3-O-(E)-caffeoylquinic acid, (2) protocatechuic acid, (3) di-caffeoylquinic acid isomer, (4) 3,5-di-O-(E)-caffeoylquinic acid, (5) 4,5-di-O-(E)-caffeoylquinic acid, (6) tri-caffeoylaltraric acid isomer, (7) quercetin, (8) 3-methoxy quercetin, (9) 8,8"-methylene-bisquercetin, (10) ent-12-oxo-kaura-9(11),16-dien-18-oic acid, (11) ent-12-hydroxy-kaura-9(11),16-dien-18-oic acid, (12) ent-15α-acetoxy-kaur-16-en-19-oic acid, (13) grandiflorenic acid, (14) ent-kaurenoic acid, (15) oleane triterpene. * identified by comparison with reference substances.

Among flavonoids, both species produce high quantities of 3-methoxy quercetin (peak 8, Fig. 1). This flavonoid, identified by its Rt, UV, and accurate mass comparison with a reference compound, has been previously reported only in the leaves of E. killipii (Torrenegra et al., 1994) and E. barclayana (Gutierrez et al., 1998). Its presence in E. grandiflora therefore constitutes a new report for this species. Quercetin (peak 7, Fig. 1), also identified in both species by its comparison with a reference substance, constitutes a new report for E. grandiflora as well. Peak 9 (Fig. 1) was tentatively proposed to be 8,8"-methylene-bisquercetin (flavonoid). This compound showed a deprotonated molecule at 615.07815 m/z and a protonated one at 617.09174 m/z for C₃₁H₂₀O₁₄. The mass spectrum in the negative mode also showed an intense peak at 299.01935 m/z, originated by in-source fragmentation of a quercetin unit and a methyl group in accordance with the literature (Martucci et al., 2014). Although this compound has not been previously reported in the subtribe Espeletiinae, Martucci et al. (2014) reported it in the genus Vernonia (Asteraceae).

Six trans-cinnamic acid derivatives were identified (Fig. 1). Among them, 3-O-(E)-caffeoylquinic acid (peak 1, Fig. 1), protocatechuic acid (peak 2, Fig. 1), 3,5-di-O-(E)-caffeoylquinic acid (peak 4, Fig. 1), and 4,5-di-O-(E)-caffeoylquinic acid (peak 5, Fig. 1) were identified based on spectral (UV and accurate mass) and Rt comparisons with reference substances. Peak 3 (Fig. 1) was tentatively proposed to be another dicaffeoylquinic acid isomer. This compound showed a deprotonated molecule at 515.11884 m/z and a protonated one at 517.13385 m/z for C₂₅H₂₄O₁₂. Its mass spectrum in the positive mode showed in-source fragmentation with two main peaks: one at 499.12311 m/z, which corresponds with the loss of a water molecule, and another at 163.03882 m/z, which corresponds with an ionized residue of caffeic acid (Santos et al., 2008). The UV spectrum of this peak exhibited two absorptions at approximately 300 and 325 nm, which suggest chlorogenic acid derivatives.

By contrast, peak 6 (Fig. 1) was proposed to be a possible tricaffeoylaltraric acid isomer based on accurate mass comparisons with compounds reported in AsterDB and maximum UV absorptions. This compound displayed a deprotonated molecule at 695.12531 m/z for C₃₃H₂₈O₁₇ and two UV maxima at approximately 300 and 329 nm. The presence of the previously reported trans-cinnamic acid derivatives in E. grandiflora and E. killipii constitutes the first report for this chemical class in the subtribe Espeletiinae. However, their presence is unsurprising, for they constitute a highly common class of secondary metabolites widespread in Asteraceae (Chagas-Paula et al., 2011).

In the leaves of E. grandiflora and E. killipii, five ent-kaurane diterpenes were also identified, among which ent-kaurenoic acid (peak 14, Fig. 1) was unambiguously identified by Rt and accurate mass comparison with a reference substance. The identities of the other four (peaks 10–13, Fig. 1) were tentatively proposed based only on accurate mass and chemotaxonomic information. All of these diterpenes have been previously reported in Espeletiinae (Bohlmann et al., 1980; Usubillaga et al., 2003).

Lastly, the identity of an oleane-type triterpene (peak 15, Fig. 1) was tentatively proposed based on accurate mass and database search. This compound showed a deprotonated molecule at 655.42114 m/z and a protonated one at 657.43524 m/z for C₃₉H₆₀O₈. An accurate mass search in DNP afforded three possible compounds: brevenal (CAS: 776331-34-1), olean-12-ene-3,16,21,22,28-pentol, 16,28-diacetate 22-(2-methyl-2-butenoate) (CAS: 124641-09-4), and olean-12-ene-3,16,21,22,28-pentol, 22,28-diacetate 21-[2(or 3)-methyl-2-butenoate] (CAS: 55949-26-3). Among them, only the two oleane-type triterpenes have previously been isolated from species belonging to plant families (Apiaceae and Polemoniaceae), whereas brevenal has been reported only in the marine dinoflagellate Karenia brevis (DNP). Further analyses are therefore necessary to unambiguously identify this compound in Espeletiinae.

Resin characterization by GC–FID and GC–MS

The hydrodistillation of E. grandiflora and E. killipii resins provided yellowish EOs with yields of 14.29% and 14.88% (v/w), respectively, based on their fresh weight. In both species, the identified substances constitute 95–97% of the total oil composition (Table 2).

Table 2
Percentage of essential oil components identified in E. grandiflora and E. killipii resins.

#	Compound name	RI	RI-lit	E. grandiflora	E. killipii
1	Santolina triene	908	906	0.3	0.9
2	α-Thujene	927	924	–	0.3
3	α-Pinene	937	932	69.7	61.8
4	Camphene	950	946	0.4	0.3
5	Thuja-2,4(10)-diene	955	953	1.2	0.5
6	Sabinene	975	969	0.6	6.5
7	β-Pinene	979	974	3.4	4.0
8	o-Cymene	1026	1022	2.1	0.6
9	Limonene	1029	1024	1.0	1.4
10	1,8-Cineole	1033	1026	0.8	2.1
11	γ-Terpinene	1058	1054	t	0.2
12	Fencholenic aldehydea	1092	–	0.6	t
13	Linalool	1101	1095	t	0.1
14	α-Campholenal	1128	1122	2.8	1.2
15	trans-Pinocarveol	1141	1135	2.3	1.2
16	cis-Verbenol	1143	1137	0.8	0.3
17	trans-Verbenol	1147	1140	2.9	1.1
18	trans-Pinocamphone	1162	1158	0.2	t
19	Pinocarvone	1164	1160	0.6	0.2
20	p-Mentha-1,5-dien-8-ol	1169	1166	2.9	2.3
21	Terpinen-4-ol	1178	1174	t	0.8
22	p-Cymen-8-ol	1186	1179	–	0.2
23	α-Terpineol	1187	1186	t	0.4
24	Myrtenal	1188	1193	0.2	t
25	Myrtenol	1199	1194	1.6	1.0
26	Verbenone	1212	1204	1.4	1.2
27	trans-Carveol	1221	1215	0.8	0.2
28	Carvone	1246	1239	0.1	t
29	Linalool acetate	1253	1254	t	0.2
30	Lavandulyl acetate	1293	1288	0.1	t
31	α-Copaene	1376	1374	t	1.7
32	β-Bourbonene	1385	1387	t	0.7
33	γ-Muurolene	1477	1478	t	1.0
34	δ-selinene	1491	1492	t	0.2
35	α-Muurolene	1500	1500	–	0.3
36	δ-Cadinene	1524	1522	0.2	1.5
37	Spathulenol	1577	1577	t	0.3
38	Copaborneol	1604	1613	–	0.9
39	kaurane diterpeneb	1981		t	0.3
40	kaurane diterpeneb	1988		t	0.7
Monoterpene hydrocarbons				78.7	76.5
Oxygenated monoterpenes				18.1	12.5
Sesquiterpene hydrocarbons				0.2	5.4
Oxygenated sesquiterpenes				–	1.2
Total identified				97.0	95.6

• – not detected; t, traces.
• ^a
  Identification based only on mass spectra comparison with Willey 7 database.
• ^b
  Tentative identification based on fragmentation pattern comparison with Adams (2007) and Usubillaga and Nakano (1979); The two most abundant compounds in E. grandiflora and E. killipii are emphasized in bold.

A comparison of the EO composition of E. grandiflora and E. killipii resins revealed no significant differences; both species were characterized by a predominance of monoterpene hydrocarbons with α-pinene as the major component (Table 2), which represented more than 61% of the total oil composition (Table 2). However, important differences were observed. For example, β-pinene represented the second major compound (3.4%) in E. grandiflora resin EO, whereas sabinene constituted the second major peak in E. killipii (6.5%), as Table 2 shows. E. grandiflora possesses a greater proportion of oxygenated monoterpenes (18.1%) than E. killipii (12.5%), but a lesser proportion of sesquiterpene hydrocarbons (Table 2). Remarkably, only one sesquiterpene hydrocarbon was detected in E. grandiflora resin EO—namely, δ-cadinene—whereas several others, including α-copaene, β-bourbonene, δ-selinene, and γ-muurolene, were detected in E. killipii resin EO (Table 2). In general, E. killipii presents a more complex EO composition than E. grandiflora, since more compounds and different chemical classes were detected in this species (Table 2).

Monoterpene hydrocarbons constitute the main EO components from all species of Espeletiinae investigated thus far (Table 1). Among them, α-pinene usually constitutes the chief EO component in numerous species (Aparicio et al., 2002; Ibáñez and Usubillaga, 2006a, 2008; Meccia et al., 2007; Peña et al., 2012). However, some species produce other compounds such as limonene and α-phellandrene as the major peaks (Aparicio et al., 2001; Ibáñez and Usubillaga, 2006b). Based on this observation of different terpenoids predominance in EOs from Espeletiinae, chemometric analyses using GC data has been performed to identify trends in the EO composition of 31 species from Espeletiinae, as discussed below.

Chemometric analyses of essential oils

Percentage EO compositions of 31 species from Espeletiinae were used as input data to perform chemometric analyses (HCAbp and OPLS-DA). Table 1 shows the species names and their locality, altitude and date of collection, as well as the plant part and reproductive stage. In this study, we considered only species collected in the anthesis period, including a few cases in which that information was not specified (Table 1).

Results from HCAbp (Fig. 2) demonstrated trends in EO composition of species from Espeletiinae, and three primary clusters were determined based on chemical similarities and high support values (≥83%). As Fig. 2 also shows, intraspecies variation displayed by some species collected in the same or different localities (e.g., Coespeletia moritziana and Espeletia schultzii) was not significant enough to promote species grouping in a different cluster, which provides support for the proposed groups.

Fig. 2
Hierarchical clustering analysis with bootstrap resampling of 31 species of the subtribe Espeletiinae based on percentage of essential oil compositions; dotted boxes correspond to the three primary clusters.

Notably, the three clusters identified should not be considered to be different chemotypes, since we analyzed different species, and by definition, a chemotype corresponds to “organisms categorized under same species, subspecies or varieties having differences in quantity and quality of their components in their whole chemical fingerprint that is related to genetic or genetic expression differences” (Polatoglu, 2013).

Supervised OPLS-DA displayed a good separation (R ² Ycum = 0.985; Q ²cum = 0.789), in which 22% of the variation in X relates to the separation between the three clusters (Fig. 3) and afforded the identification of discriminating substances. Cluster 1 was characterized by high α-pinene, β-pinene, and β-phellandrene contents, cluster 2 by high α-phellandrene, α-thujene, p-cymene, and sabinene contents, and cluster 3 mainly by high germacrene D, limonene, and β-caryophyllene contents (Fig. 3).

Fig. 3
Orthogonal partial least-squares discriminant analysis score plot of 31 species of the subtribe Espeletiinae based on percentage of essential oil compositions; Clusters 1–3, determined by hierarchical clustering analysis with bootstrap resampling, as the Y variable. Discriminating variables of each cluster in order of relevance as follows, Cluster 1: α-pinene, β-pinene, β-phellandrene; cluster 2: α-phellandrene α-thujene, p-cymene, and sabinene; cluster 3: germacrene D limonene, β-caryophyllene and ent-kaur-16-en-al.

Additional OPLS-DA analyses considering species locality or elevation range as Y variables (Table 1) revealed no correlation with EO compositions (data not shown, but are evident from Table 1). This result suggests that intraspecies variation due to geography or elevation, as reported by Aparicio et al. (2002) and Ibáñez and Usubillaga (2006b), is less than interspecies variation in Espeletiinae. Therefore, when considering several species from different genera in a single analysis, even species collected at different localities cluster according to their taxonomic relatedness rather than their geographic origin (Fig. 2). As shown in Table 1, a certain correlation between species EOs and their generic classifications was evident. For example, cluster 1 is composed primarily of species belonging to the genus Coespeletia and a few species of Espeletia, whereas cluster 2 presents species mostly of Espeletia and a few of Libanothamnus. By contrast, cluster 3 is primarily composed of species of Ruilopezia and a few of Carramboa (Table 1). Nevertheless, the clustering of Espeletiinae species based on their EO components does not follow strict generic boundaries (based on morphology), since no evident correlation was observed when considering the six genera as Y variable in OPLS-DA (data not shown).

Conclusions

This research represents the first comprehensive study to report the chemical composition of a Espeletia species by modern and complementary analytical techniques, followed by proper chemometric analyses. The similarities observed between the chemical fingerprints of E. grandiflora and E. killipii agree with their morphological and phylogenetic relatedness. However, important qualitative and quantitative differences were identified in the leaves and resins of both species, which suggest that chemical markers can be potentially used to assist in their taxonomy. Among the fifteen dereplicated compounds, ten correspond to new reports for either or both species, whereas eight correspond to new reports for the genus Espeletia and subtribe Espeletiinae, indicating that the secondary metabolite chemistry of this group of plants remains largely unexplored. This study moreover afforded the identification of different trends in the accumulation of EOs from 31 species of Espeletiinae, which seem to correlate with their taxonomy and thus offer a basis for future studies.

Acknowledgments

The authors gratefully acknowledge CAPES and FAPESP (grant # 2014/17702-0) for financial support. GFPG thanks Dr. Mauricio Diazgranados, M.Sc. Sandra L. Castañeda and Mr. Carlos I. Suarez from JBB for their valuable help collecting plant material and during botanical identifications and Mrs. Nubia Parra for her technical assistance in confection of herbarium vouchers. Special thanks to Prof. Dr. Norberto P. Lopes (FCFRP-USP), for experimental laboratory facilities to perform GC-MS analyses with technical support of Mrs. Izabel C.C. Turatti, and to M.Sc. Gari Ccana-Ccapatinta (AsterBioChem, FCFRP-USP) for enlightening discussions.

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Publication Dates

• Publication in this collection
  Nov-Dec 2016

History

• Received
  3 Feb 2016
• Accepted
  5 May 2016