Recent Findings in the Chemistry of Odorants ... - Wiley Online Library

CHEMISTRY & BIODIVERSITY – Vol. 12 (2015)

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Recent Findings in the Chemistry of Odorants from Four Baccharis Species and Their Impact as Chemical Markers by Manuel Minteguiaga a ), Noelia Umpi¦rrez a ), Vanessa Xavier b ), Aline Lucas b ), Claudio Mondin c ), Laura FariÇa d ), Eduardo Cassel b ), and Eduardo Dellacassa* a ) d ) a

) Ctedra de Farmacognosia y Productos Naturales, Facultad de Qumica, Universidad de la Repfflblica (FQ-UdelaR), Av. General Flores 2124, 11800 Montevideo, Uruguay (phone: þ 598-29244068; e-mail: [email protected]) b ) Laboratýrio de OperaÅ"es Unitrias (LOPE), Faculdade de Engenharia, Pontifcia Universidade Catýlica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, CEP: 90619-900, Porto Alegre, RS, Brasil c ) Departamento de Biodiversidade e Ecologia, Pontifcia Universidade Catýlica do Rio Grande do Sul (PUCRS), Av. Ipiranga 6681, CEP: 90619-900, Porto Alegre, RS, Brasil d ) Laboratorio de Biotecnologia de Aromas, Facultad de Qumica, Universidad de la Repfflblica (FQUdelaR), Av. General Flores 2124, 11800 Montevideo, Uruguay

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the ÐCampos de Cima da SerraÏ highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile-extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained a-pinene, b-pinene, limonene, and (E)-bocimene in high amounts.

1. Introduction. – Nowadays, there is an increasing interest in understanding the role of plant volatile metabolism, not only for academic reasons, but also due to the role these compounds play in the health, pharmaceutical, agricultural, food science, and flavor and fragrance industries [1] [2]. In the phytochemical nomenclature, there is a difference between biogenic volatile organic compounds (VOCs) and essential oils. The first ones represent all the compounds that plants might emit to the atmosphere [3] [4], while essential oils respond more to an operational definition that comprises a mixture of compounds that can condense when a plant is distilled [5]. In general, it ought to be seen like all plants emit volatile compounds, but not all plants can produce essential oils. Plants that produce essential oils are usually known as aromatic plants and eventually can be Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

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translated in an economic resource for sub-developed countries, such as countries in Latin America [6]. According to the European Pharmacopoeia, essential oils are obtained by steam distillation using a Clevenger-type apparatus [7]. In spite of this, there are a lot of techniques to extract volatile compounds. Simultaneous distillation extraction (SDE) is proposed as the technique that yields results that are more closely related to volatile compounds found in nature [8]. When compared with the Clevenger steam distillation, the SDE technique has the advantage of retrieving compounds, which otherwise might be lost, such as volatile compounds that are not able to condense and even polar components (i.e., phenolic alcohols) that can be solubilized in H2O at the oil/H2O partition stage [8] [9]. Furthermore, with the SDE procedure, very few solvent is required, reducing problems of artifact build-up as solvents are concentrated [8] [9], allowing to obtain a more representative volatile plant profile than steam distillation techniques [8] [9]. Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas [10 – 12]. Many species have been reported for their use in traditional medicine by local and indigenous populations [12]. Even if studied in detail, the taxonomic classification among species of this genus is not yet fully defined [11] [13]. In some cases, the difficulty in classification is a consequence of the slight morphological differences in traits between the species, e.g., the species of the Caulopterae section, known as ÐcarquejasÏ in South America. Consequently, some authors began to name B. trimera and its related species (B. crispa, B. cylindrica, B. jocheniana, and B. myriocephala) as the B. trimera complex [13] [14]. Besides, some studies state B. trimera and B. genistelloides var. crispa as synonyms [13]. Hence, many authors have looked for alternatives for a more precise taxonomic classification [13 – 16]. In particular, chemotaxonomy, by the study of volatile [16] [17] and non-volatile compounds [11] [18 – 20], has been applied extensively to classify the species of this genus. B. trimera and B. milleflora are two ÐcarquejaÏ species, characterized by three longitudinal wings or striated stems [13]. The former has a widespread distribution in Brazil, Bolivia, Argentina, and Uruguay, while the distribution of the latter is restricted to southern and southeastern Brazil [13]. B. uncinella and B. tridentata, known popularly as ÐvassouraÏ, are 3 m high shrubs growing wild only in southern and southeastern Brazil [21]. As part of our continuous search for plants as new sources of valuable oil/perfumery products, the relative composition of the volatile compounds obtained from the aerial parts of four Baccharis species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) growing wild in the ÐCampos da Cima da SerraÏ highlands was reported here. The ÐCampos da Cima da SerraÏ region is a characteristic highland region of the Brazilian states of Rio Grande do Sul and Santa Catarina, where the typical vegetation is ombrophilus mixed forest (OMF), a scenery dominated by Araucaria trees [21]. The samples were representative of the species and its geographic area of distribution and grew under the same pedoclimatic and collection conditions. Moreover, the extraction conditions were also identical for all samples. Therefore, the influence of environmental and technical parameters on the chemical composition of the volatile composition was avoided.


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The SDE technique was applied, to obtain the full volatile profile of those species. In parallel, the most relevant compounds of the volatile extracts were determined, to use them as markers for a better comprehension of the species behavior and for future applications in the commercial evaluation of volatile extracts and essential oils. 2. Results and Discussion. – 2.1. Volatile Composition. The composition of the volatile extracts obtained from the leaves of the four Baccharis species studied are reported in the Table. Our research efforts, i.e., the comparison of mass spectra and the determination of two independent retention indices (RIs), allowed the identification of 180 peaks with many coelutions. To be concise, the Table only presents the compounds present at concentrations higher than 1% of the total oil composition in at least one species. The detailed composition of the samples is available from the authors upon request 1). There are no previous studies of the composition of Baccharis volatile extracts that report this many components and coelutions. This is a sign of the high level of complexity that might be faced when working with plants that have an intricate volatile profile such as Baccharis species. 2.2.1. Variability Associated with the Volatile Composition. Usually, Baccharis essential oils are largely known for their content of sesquiterpene cyclic alcohols (spathulenol, globulol, palustrol, ledol, and viridiflorol), which were previously shown to represent a high percentage of the volatile composition, as was the case of B. trimera, B. milleflora, and B. uncinella in the present study. In those volatile extracts, the sesquiterpene fraction accounted for 50 to 80% of the total extracts, while the B. tridentata extract had a low sesquiterpene fraction ( < 8%) and high contents of apinene, b-pinene, limonene, and bornyl acetate (Table). Hence, the composition of the volatile fraction allowed distinguishing B. tridentata from the other species under study. This is important for taxonomic proposes and perfumery applications, because these kinds of sesquiterpene compounds have pleasant aromatic notes. What about the composition of each of the particular species with relation to previous studies? Only two previous studies related with the essential-oil composition of Baccharis tridentata could be found [25] [26], and for both of them, the oil was obtained by hydrodistillation using a Clevenger-type apparatus. In the first study, the oil composition showed a high proportion of spathulenol (21.20%), d-cadinene (7.68%), and globulol (5.86%), while the second one reported a-thujene (22.93%), b-pinene (20.33%), and b-phellandrene (16.15%) as the main components. Compared to these previous reports, although some common essential-oil characteristics could be observed, it is surprising that the volatile compositions in the present study revealed such great differences. It is supposed that they might depend on the habitat conditions, as edaphic factors have been reported to influence the essentialoil composition in general [27] and the vegetal material of the previous studies had been obtained from the southeastern region of Brazil (state of Minas Gerais). Concerning B. milleflora, only one previous study was published, i.e., the one reported by Sim"es-Pires et al. [16]. They analyzed essential oils from plant material collected in the same place as the one of the present study. Sim"es-Pires et al. [16] 1)

A table with the detailed composition of the volatile extracts of the four Baccharis species can be obtained as Supplementary Material from the authors.

LRIn b ) 922 928 941 969 972 990 1028 1049 1284 1418 1437 1453 1477 1481 1485 1486 1497 1505 1516 1527 1570 1582 1585 1606 1609 1614 1641 1645 1655 1659

Compound name and class a )

a-Thujene a-Pinene Camphene Sabinene b-Pinene Myrcene þ b-phellandrene Limonene (E )-b-Ocimene Bornyl acetate (E )-Caryophyllene trans-a-Bergamotene þ aromadendrene a-Humulene g-Muurolene a-Amorphene þ germacrene D ar-Curcumene b-Selinene Bicyclogermacrene (E,E )-a-Farnesene d-Amorphene d-Cadinene Palustrol Spathulenol Caryophyllene oxide þ globulol Ledol Tetradecanal Humulene epoxide II Isospathulenol t-Muurolol t-Cadinol a-Cadinol

1020 1012 1050 1108 1093 1155, 1193 1185 1240 – 1575 1566, 1615 1645 1671 1671, 1685 1749 – 1709 1700 1736 1735 1903 2095 1952, 2050 2001 1913 2007 – – 2143 –

LRIp b )

0.09 – 0.02 0.26 0.12 3.24 0.07 – 2.07 1.61 3.1 0.47 4.2 7.97 – 4.45 1.87 1.51 0.44 0.52 19.19 4.11 0.55 – 4.36 1.93 1.14 1.3 1.96

–

B. milleflora

Content [%] c ) 0.07 0.91 – 0.15 3.42 0.62 1.15 0.17 0.03 4.11 – 1.12 1.15 3.00 – 1.3 2.87 – – 3.46 20.0 8.53 7.14 2.34 1.28 – 0.81 1.36 1.71 –

B. trimera 1.31 46.72 1.36 3.09 11.36 1.84 14.06 3.63 6.01 0.81 0.04 0.03 0.02 1.49 – 0.06 3.4 0.03 – 0.28 – 0.93 0.2 – – – – – 0.12 –

B. tridentata

3.37 11.68 0.08 2.48 7.56 0.98 8.63 0.3 – 3.3 0.7 0.96 0.67 4.25 1.54 – 4.61 0.19 0.37 2.74 1.45 10.23 4.26 1.22 0.63 – – – 1.18 –

B. uncinella

Table 1. Qualitative and Quantitative Composition of the Volatile Compounds Obtained by Simultaneous Distillation Extraction of the Aerial Parts of the Four Baccharis Species Studied

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7.32 3.93 28.87 47.17 5.97

93.26

– 1.23 0.58 2.6

B. trimera

84.21 7.15 6.36 1.5 0.12

99.34

– – – –

B. tridentata

36.83 4.44 28.4 24.37 1.39

95.43

0.44 – 1.33 –

B. uncinella

) Compounds are listed in the order of elution from the nonpolar SE-52 column; peak identification was based on the comparison of LRI values and mass spectra with those of pure standards or reported in the literature [22 – 24]. b ) LRIn and LRIp : Linear retention indices determined on the nonpolar SE-52 and the polar Rt Ô-CW20M capillary columns, respectively. c ) Relative contents of the compounds determined on the SE-52 column, obtained by peak-area normalization (relative response factors were taken as one), and expressed as percentages; for each compound reported, the relative contents for the same species were not significantly different between sample replicates (p > 0.05).

a

3.95 2.28 36.65 43.88 3.92

1.11 1.42 1.25 1.01 90.68

– 1771 –

–

B. milleflora

Content [%] c )

Monoterpenes Hydrocarbons Oxygenated Monoterpenes Sesquiterpenes Hydrocarbons Oxygenated Sesquiterpenes Others

1667 1674 1690 1849

Eudesma-4(15),7-dien-1b-ol Aromadendrene oxide Germacra-4(15),5,10(14)-trien-1a-ol Neophytadiene

LRIp b )

Total Identified

LRIn b )

Compound name and class a )

Table 1 (cont.)

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identified (E)-b-caryophyllene (19.7%), g-gurjunene (23.6%), and a-selinene (20.7%) as main oil compounds. On the other hand, a more complex profile, with spathulenol as the main component (19.19%), followed by ar-curcumene (7.97%) and many compounds reaching contents from 1 to 5%, was observed here (Table). In addition, Sim"es-Pires et al. [16] presented carquejyl acetate as a chemomarker for B. trimera, but in the present study, this compound was found in the volatile extract of B. milleflora and not of B. trimera. Moreover, carquejyl acetate has been found in Eupatorium buniifolium (Asteraceae) oil [28], which undoubtedly indicates that this compound cannot be considered as a chemical marker for B. trimera. The oil composition of B. uncinella and B. trimera has been previously studied by many authors [16] [29 – 37], who obtained similar results to those reported here. As chemical variation related to phenological and environmental factors was frequently observed in aromatic plants [38], the small differences found between the present and published data could be due to experimental factors as the sampling material, population homogeneity, extraction process, and phenotypic behavior. Under the present experimental conditions, the samples of volatile extracts analyzed showed the same major components that had been found for representative samples of the respective species over its geographical area of distribution, with no remarkable variations in composition (Table). Therefore, it can be concluded that the present investigation of the volatile composition of B. uncinella and B. trimera allowed identifying the same compounds characterizing these extracts as those reported by other authors. 2.2.2. Volatile-Profile Behavior during the Flowering Period. Over the full flowering growth stage (September to November), the volatile composition of the aerial parts of each of the four Baccharis species remained almost constant, within the range of analytical variation and for the samples analyzed (Table). Indeed, for each compound reported, the relative contents for the same species were not significantly different between sample replicates (p > 0.05). Hence, the great differences observed between the volatile compositions of the four Baccharis species were species dependent. It is also important to note the high chemical variety observed among the volatile compounds, including green leaf volatile compounds (GLVs), hemi-, mono-, and sesquiterpene compounds, shikimic acid derived compounds, norisoprenoids, and many aliphatic hydrocarbons. 2.3. Multivariate Statistical Analyses. To evaluate whether the identified constituents may be useful in reflecting relationships among the Baccharis species studied, all the components reported in the Table with relative contents exceeding 1% of the total volatile-extract composition in at least one Baccharis species were subjected to cluster analysis (CA) and principal component analysis (PCA). The resulting dendrogram and PCA plots are shown in Figs. 1 and 2, respectively. The results obtained for both multivariate analyses suggest that the four populations of Baccharis form two separate groups. In the PCA (Fig. 2), the horizontal axis accounted for 53.8% of the total variation, while the vertical axis explained a further 33.2%.The first cluster comprised the volatile extracts of B. milleflora, B. trimera, and B. uncinella, characterized by a high degree of similarity in the volatile composition and the presence of high contents of sesquiterpene compounds, particularly of spathulenol. This is relevant, because spathulenol has


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Fig. 1. Dendrogram obtained by hierarchical cluster analysis of the composition of the volatile extracts of the four Baccharis species studied

been suggested as a good candidate to be used in combination with chemotherapy for the treatment of multidrug-resistant diseases and, therefore, is worthy for further in vivo studies [39]. The second cluster was formed by the volatile extract of B. tridentata, which contained a-pinene, b-pinene, limonene, and (E)-b-ocimene in high amounts. In spite of the correlation obtained for the four Baccharis species, there is an outstanding percentage of variability in the composition of the volatile extracts that should be the subject of subsequent genetic studies. 3. Conclusions. – Four different Baccharis species growing in ombrophilus mixed forest in the area of Rio Grande do Sul and Santa Catarina, sharing the same environmental, soil, and cultural conditions, were used as an experimental model to obtain more information about the chemical variability of the volatile extracts of these species. In addition, the extraction of volatiles and their subsequent characterization by GC/MS analysis was carried out under the same experimental conditions. Consequently, it was assumed that the differences of the chemical composition among the species were not significantly influenced by climatic factors and crop techniques. Thus, the results of the comparative analysis among them is thought to reflect a genetic basis. The discriminating power of the chemical composition of the volatile extracts and its contribution to taxonomy at the varietal level showed the usefulness of phytochemical compounds for this aim. According to the results, the separation of the four species into two groups was proposed: the first group was represented by B. milleflora, B. trimera, and B. uncinella, and the second one comprised B. tridentata. This study, however, was based on a single sampling period and not on the monitoring of the volatile compositions during the whole vegetation period. Hence, in addition to the present results, the genetic implications should be tested by means of analysis of genetic molecular markers.

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Fig. 2. a) Score plot and b) loading plot obtained by principal component analysis of the contents of the main volatile-extract compounds of the four Baccharis species studied


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Moreover, the differences in the volatile extracts among the four species provided fundamental information for an improved taxonomic understanding and a future improvement in the development of innovative strategies aimed at the optimization of the volatile-extract composition and to manage valuable components. This provides appreciable information for local producers and purchasers. The authors wish to acknowledge Prof. Eduardo Alonso Paz (FQ-UdelaR) for herbarium support and CAPES (Brazil) and ANII (Uruguay) for financial support. Experimental Part Plant Material. In consideration of the possible variation in the chemical characteristics of a plant in response to seasonal factors, samples of fresh leaves and stems, representing the entire population of Baccharis trimera, B. milleflora, B. tridentata, and B. uncinella, were collected randomly during the fullflowering period (September to November) in 2011 and 2012 in the Nature Preservative and Research Centre (CPCN-Prý Mata-PUCRS, S29829’, W50811’, S¼o Francisco de Paula, RS, Brazil), a highland site of ombrophilus mixed forest (OMF). The samples were identified by Prof. C. Mondin and dried. Voucher specimens have been deposited with the herbariums of the PUCRS (MPUC) and the Faculty of Chemistry, UdelaR (MVFQ) with the following sample codes: B. tridentata, C. Mondin 3570 (MPUC); B. milleflora, C. Mondin 3571 (MPUC); B. uncinella, M. Minteguiaga 4231 (MVFQ); and B. trimera, M. Minteguiaga 4232 (MVFQ). Simultaneous Distillation Extraction (SDE). For the SDE, 120 g of dried and crushed plant material were placed inside a glass vessel attached to a supplier H2O steam boiler, allowing the steam to pass through the plant material, to pull volatile components up the SDE apparatus. The solvent employed for the extraction was hexane (Merck, NJ, USA), and the total time of extraction was 1 h. GC/MS Analysis. GC/MS Analyses were carried out using a Shimadzu QP 5050 apparatus following the experimental conditions described by Lorenzo et al. [28]. Identification and Quantification of Volatile Components. The volatile components were identified based on the comparison of their linear retention indices (LRIs), determined on two columns rel. to a homologous series of n-alkanes, with those of pure standards or reported in the literature [22] [23] and of mass-spectral fragmentation patterns with those stored in GC/MS databases [22] [24]. The relative contents of each component were reported as raw percentages without standardization. The repeatability of the analyses showed variation coefficients below 5% for all the components reported in the Table. Statistical Analysis. All statistical analyses were performed using Statistica software (StatSoft, Tulsa, OK, 1984 – 2005).

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