Essential Oils of Five Baccharis Species: Investigations on the Chemical Composition and Biological Activities

This paper provides a comparative account of the essential oil chemical composition and biological activities of five Brazilian species of Baccharis (Asteraceae), namely B. microdonta, B. pauciflosculosa, B. punctulata, B. reticularioides, and B. sphenophylla. The chemical compositions of three species (B. pauciflosculosa, B. reticularioides, and B. sphenophylla) are reported for the first time. Analyses by GC/MS showed notable differences in the essential oil compositions of the five species. α-Pinene was observed in the highest concentration (24.50%) in B. reticularioides. Other major compounds included α-bisabolol (23.63%) in B. punctulata, spathulenol (24.74%) and kongol (22.22%) in B. microdonta, β-pinene (18.33%) and limonene (18.77%) in B. pauciflosculosa, and β-pinene (15.24%), limonene (14.33%), and spathulenol (13.15%) in B. sphenophylla. In vitro analyses for antimalarial, antitrypanosomal, and insecticidal activities were conducted for all of the species. B. microdonta and B. reticularioides showed good antitrypanosomal activities; B. sphenophylla showed insecticidal activities in fumigation bioassay against bed bugs; and B. pauciflosculosa, B. reticularioides, and B. sphenophylla exhibited moderate antimalarial activities. B. microdonta and B. punctulata showed cytotoxicity. The leaves and stems of all five species showed glandular trichomes and ducts as secretory structures. DNA barcoding successfully determined the main DNA sequences of the investigated species and enabled authenticating them.

For B. microdonta, Lago et al. [22] reported 24% of caryophyllene oxide, whereas Sayuri et al. recorded 31% of elemol, 34% of spathulenol, 19% of β-caryophyllene, and 24% of germacrene D as the major compounds. Both of these studies used samples collected from Campos do Jordão, Brazil. None of the earlier studies have reported kongol from any of the five Baccharis species, whereas this compound was found in high quantity (22.22%) in B. microdonta in the present study. Even though the chemical composition of EOs is frequently associated to environmental and phenological influences, it is necessary to investigate whether these variations in B. punctulata and B. microdonta are possibly linked to different chemotypes. Not only the compositions of EOs, but also the quantities of the compounds vary throughout the life of the plant. This is related to the circadian rhythms, seasonal conditions, and environmental influences that impact the development of the species [24]. To avoid some of these factors, in the present work, all five species were grown in the same locality and collected on the same day and at the same time. Additionally, the sample preparation, hydrodistillation, and characterization of the EOs by GC/MS analysis of all of the materials were performed under the same experimental conditions.   Retta et al. [8] analyzed five species of Baccharis, namely B. gaudichaudiana DC., B. microcephala (Less.) DC., B. penningtonii Heering, B. phyteumoides (Less.) DC., and B. spicata (Lam.) Baill., and reported that they were qualitatively, but not quantitatively, similar. In the present study, although the five species presented similar qualitative patterns, some compounds were found only in one specific species. Qualitative similarities in these species were expected, as they belonged to the same genus. However, they were classified into two different taxonomic groups: B. punctulata belonged to subgenus Molina, whereas the other four species belonged to the subgenus Baccharis.
By comparing the GC chromatograms of the EOs of the five species of Baccharis, it is possible to distinguish them by the quality and quantity of their major constituents (Figure 1).

Antimalarial Activity
In order to explore the antimalarial properties of the five species of Baccharis, their EOs were investigated against chloroquine-sensitive (D6) and chloroquine-resistant (W2) strains of Plasmodium falciparum ( Table 2). The EOs of B. microdonta and B. punctulata were cytotoxic to Vero cells (selectivity control), and because of this result, they were not indicated to be used in cellular media as an antimalarial. These two EOs differed from other studied species due to the presence of the chemical markers spathulenol (22.74%) and kongol (22.22%) for B. microdonta and α-bisabolol for B. punctulata (23.63%). Due to their cytotoxic properties, these EOs can be further explored in other cytotoxicity or anticancer studies, as previously reported by Pereira et al. for B. milleflora DC. [16]. Otherwise, the EO of B. pauciflosculosa showed moderate antimalarial activity against both P. falciparum clones (lower than 15 µg/mL), while B. reticularioides and B. sphenophylla demonstrated discrete antimalarial effects. Significant differences in the quality and quantity of the chemical components of the five EOs can be strongly related to these data. In particular, the variation in the quantities of the main components e.g., monoterpenes β-pinene (18.33%) and limonene (18.77%), might be responsible for the antimalarial effect of the EO of B. pauciflosculosa. In the genus Baccharis, antimalarial studies were carried out for a few species using their plant extracts or isolated compounds. B. dracunculifolia DC. is the most important plant source of the Brazilian green propolis, and showed antimalarial activities against P. falciparum (D6) using crude hydroalcoholic green propolis extract (13 µg/mL) and hautriwaic acid lactone with IC 50 values of 0.8 µg/mL (D6 clone) and 2.2 µg/mL (W2 clone) [39]. The extracts of leaves from B. rufescens Spreng. and B. genistelloides (Lam.) Pers. also showed in vitro antimalarial activity, achieving 100% of inhibition at 100 µg/mL against a P. falciparum chloroquine-resistant strain [40]. In spite of the antiplasmodial activity of plant EOs widely reported in the literature [28], the present work represents the first study involving the antimalarial effect using the EOs of the Baccharis species.
Additionally, the selectivity index (SI) was calculated to predict how toxic the samples were to normal cells. The calculated selectivity indices showed that B. pauciflosculosa had better selectivity to P. falciparum clones than for Vero cells. This EO was safer than other examined samples, making it a candidate for further development as an antimalarial agent, mainly via the inhalation route.

Antitrypanosomal Activity
Trypanosoma brucei is a protozoan that causes human African trypanosomiasis (HAT). There are currently only four drugs available for its treatment, namely pentamidine, melarsoprol, suramin, and eflornithine. Considering the lack of phytochemical and pharmacological data available for plants with efficacy against trypanosomes and aiming at proposing alternative treatments for HAT, an initial screening of the EOs of the five species of Baccharis were carried out against T. brucei (Table 3).

Insecticidal Studies with Bed Bugs
Most of the plant-based insecticides and repellents are derived from plants containing EOs [43]. In addition, receptors responding to DEET (N,N-diethyl-3-methylbenzamide) can also respond to volatile terpenes [44]. Therefore, this exploratory study was aimed at investigating the insecticidal potential of EOs of Baccharis species against bed bugs because of the increasing demands for information about effective control tactics and their public health risks.
The results of fumigation studies involving bed bugs are illustrated in Figure 2. Out of the five EOs analyzed, only B. sphenophylla produced 66.67 ± 3.33% mortality in the insecticide-resistant strain 'Bayonne', while producing 83.33 ± 3.33% mortality in the susceptible strain 'Ft.Dix', 24 h after treatment. All of the other EOs showed less than 15% mortality. In particular, B. sphenophylla EO exhibited a wide range of volatile compounds with no specific chemical markers. In that sense, its fumigation effect could be attributed to the synergic effects of terpenes, as previously reported in the literature [45]. Several monoterpenes were isolated and demonstrated fumigation effects on different insects, e.g., α-pinene, β-pinene, 3-carene, limonene, myrcene, α-terpinene, and camphene [43]. Of these compounds described in the literature, only camphene was not present in the EO of B. sphenophylla, which reinforces the hypothesis that its effect was based on a synergism of various volatile compounds present in the EO.  None of the EOs showed high mortality when applied topically at 50 μg/bug. Only in B. punctulata did the mortality reach 20% seven days after the treatment (Figure 3). In residual study, none of the EOs produced mortality in bed bugs seven days of exposure at 100 μg/cm 2 .   None of the EOs showed high mortality when applied topically at 50 µg/bug. Only in B. punctulata did the mortality reach 20% seven days after the treatment (Figure 3). In residual study, none of the EOs produced mortality in bed bugs seven days of exposure at 100 µg/cm 2 .  None of the EOs showed high mortality when applied topically at 50 μg/bug. Only in B. punctulata did the mortality reach 20% seven days after the treatment (Figure 3). In residual study, none of the EOs produced mortality in bed bugs seven days of exposure at 100 μg/cm 2 .

Secretory Structures
In Asteraceae, EOs are biosynthesized and accumulated in various secretory structures, such as idioblast oil cells, oil cavities, secretory ducts, and glandular trichomes [46]. In Baccharis, EOs can be found in roots, stems, leaves and flowers [35,47,48], and are stored in secretory ducts and glandular trichomes [4].
In the present study, the leaves and stems of all of the Baccharis species showed glandular trichomes, either isolated or in clusters (Figure 4a-d), and frequently inserted in small epidermal depressions. There were three types of glandular trichomes, namely biseriate (Figure 4a,c,d), flagelliform with straight body (Figure 4a,b,d), and flagelliform C-shaped (Figure 4c). Biseriate glandular trichomes were present in all of the species, except for B. pauciflosculosa. The flagelliform trichomes with straight body were found in all of the species except B. punctulata, and only this species had flagelliform C-shaped trichomes (Figure 4c).

Secretory Structures
In Asteraceae, EOs are biosynthesized and accumulated in various secretory structures, such as idioblast oil cells, oil cavities, secretory ducts, and glandular trichomes [46]. In Baccharis, EOs can be found in roots, stems, leaves and flowers [35,47,48], and are stored in secretory ducts and glandular trichomes [4].
In the present study, the leaves and stems of all of the Baccharis species showed glandular trichomes, either isolated or in clusters (Figure 4a-d), and frequently inserted in small epidermal depressions. There were three types of glandular trichomes, namely biseriate (Figure 4a,c,d), flagelliform with straight body (Figure 4a,b,d), and flagelliform C-shaped (Figure 4c). Biseriate glandular trichomes were present in all of the species, except for B. pauciflosculosa. The flagelliform trichomes with straight body were found in all of the species except B. punctulata, and only this species had flagelliform C-shaped trichomes (Figure 4c). All the studied Baccharis species presented secretory ducts in the mesophyll (Figure 4e,f) and midrib of the leaves (Figure 4g), and in the cortex of the stems (Figure 4h,i). They showed a uniseriate epithelium formed by four to 20 cells with large nuclei and dense cytoplasm containing EO droplets, and were found next to the parenchyma sheath near the phloem (Figure 4e-i). These secretory ducts could also sometimes release other chemical compounds such as resins and tannins beside EOs [49]. Essential oils in the trichomes and ducts, and lipophilic compounds in the cuticle reacted positively with Sudan III in the histochemical tests (Figure 4i). All the studied Baccharis species presented secretory ducts in the mesophyll (Figure 4e,f) and midrib of the leaves (Figure 4g), and in the cortex of the stems (Figure 4h,i). They showed a uniseriate epithelium formed by four to 20 cells with large nuclei and dense cytoplasm containing EO droplets, and were found next to the parenchyma sheath near the phloem (Figure 4e-i). These secretory ducts could also sometimes release other chemical compounds such as resins and tannins beside EOs [49]. Essential oils in the trichomes and ducts, and lipophilic compounds in the cuticle reacted positively with Sudan III in the histochemical tests (Figure 4i).

Identification of the Samples by DNA
Classical methods for the identification of medicinal plants include organoleptic, macroscopic, and microscopic methods and chemical profiling. Modern techniques, such as DNA barcoding, have emerged recently and are often used in plant identification [50]. Considering the morphological similarities among Baccharis species [4], all four genomic regions, namely ITS, ETS, psbA-trnH, and trnL-trnF were subjected to amplification and sequencing in order to provide molecular data for the differentiation of the species. Only two samples (ECT0000641, ECT0000642) resulted in an ETS PCR product and consequently sequence data. Only a single sequence per authenticated species was available for sequence comparison. Table 4 shows KP2 distances between five Baccharis samples and authenticated species. The KP2 value determines the genetic distance between samples and the lowest KP2 value of 0.000 indicates a 100% match. The psbA-trnH sequences of samples B. reticularioides (ECT0000642) and B. sphenophylla (ECT0000647) matched 100% to three different species, indicating that the genomic region has not much variation in its sequence to be helpful to distinguish between species.
Kongol ( Figure S1) and spathulenol ( Figure S2) were isolated from EO of B. microdonta and identified by NMR spectroscopy in the present study. Briefly, 180 mg of EO of B. microdonta was subjected to a Biotage ZIP KP-SIL 45-g cartridge, and the isolation was performed on a Biotage Isolera TM system (Biotage, Charlotte, NC). Hexanes-ethyl acetate was used for eluting with increasing proportions of ethyl acetate from 0% to 20%. The eluted fractions (12 mL each) were collected and detected by using thin layer chromatography. Kongol (23.8 mg) and spathulenol (25.3 mg) were obtained from the fractions 102-106 and 95-99, respectively. The proton and carbon NMR spectra of the two isolates were recorded using an Agilent DD2-500 NMR spectrometer (Agilent, Santa Clara, CA) equipped with a One NMR probe operating at 499.79 MHz for 1 H and 125.67 MHz for 13 C. The spectrum of spathulenol was identical to that of the reference standard. The spectral data of isolated kongol was also in agreement with that reported in the literature [52].

Gas Chromatography-Mass Spectrometry (GC/MS) Analysis
The EOs of B. microdonta, B. pauciflosculosa, B. punctulata, B. reticularioides, and B. sphenophylla were analyzed by GC/MS using an Agilent 7890A GC system equipped with a 5975C quadrupole mass spectrometer and a 7693 autosampler (Agilent Technologies, Santa Clara, CA, USA). Ten microliters of EOs were dissolved in 1 mL of n-hexane for each oil sample, and 1 µL of the sample solution was injected. Helium was used as the carrier gas at a flow rate of 1 mL/min. The inlet temperature was set to 250 • C with a split injection mode for a split ratio of 50:1. Separation was performed on two columns with different polarity, non-polar DB-5MS (column 1) and polar DB-WAX (column 2) capillary columns (Agilent J&W Scientific, Folsom, CA, USA) with the same dimensions of 30 m × 0.25 mm i.d. × 0.25 µm film thickness. The oven temperature program was as follows: (1) Column 1: the initial temperature was 45 • C (held for 2 min); it then increased to 130 • C at a rate of 2 • C/min (held for 10 min), to 150 • C at a rate of 2 • C/min, and finally to 250 • C at a rate of 2 • C/min and isothermal for 10 min at 280 • C with a total experiment time of 70 min; (2) Column 2: the initial temperature was 40 • C (held for 4 min); it then increased to 200 • C at a rate of 3 • C/min, and to 240 • C at a rate of 20 • C/min. Triplicate injections were made for each sample.
Mass spectra were recorded at 70 eV at a scan mode from m/z 35 to 500. The transfer line temperature was 260 • C. The ion source and quadrupole temperatures were 230 • C and 130 • C, respectively. Data acquisition was performed with Agilent MSD Chemstation (F.01.03.2357).
Compound identification involved the comparison of the mass spectra with the databases (Wiley and the National Institute of Standards and Technology (NIST) using a probability-based matching algorithm. Further identification was based on the relative retention indices compared with the literature [38] and the reference standards purchased from commercial sources or isolated in-house.
The raw percentage from the peak area of each compound was obtained in full-scan GC/MS analyses (DB-5MS and DB-WAX columns). Further standardization was not carried out, since our aim was focused on identifying the essential oil compounds for species differentiation.

Antimalarial Activity
The antimalarial activity of EOs from Baccharis species was determined using a colorimetric assay based on plasmodial lactate dehydrogenase (LDH) activity as described by Kumar et al. [53]. A suspension of red blood cells infected with D6 or W2 strains of P. falciparum was added to the wells of a 96-well plate containing test samples diluted in medium at several concentrations. Parasitic LDH activity was determined according to the method described by Makler and Hinrichs [54]. Chloroquine and artemisinin were included as the drug controls. IC 50 values were calculated from the dose-response curves using Excelfit ® . DMSO (0.25%) was used as the vehicle control. For calculating the selectivity index of the antimalarial activity of EOs, their toxicity to Vero cells (monkey kidney fibroblasts) was also determined. Essential oils at different concentrations were added, and plates were again incubated for 48 h. The number of viable cells was determined using a vital dye (WST-8). Doxorubicin was used as a positive control.

Antitrypanosomal Activity
The screening that was employed to test the antitrypanosomal activity of the EOs of Baccharis species against T. brucei was detailed in a previous paper by Jain et al. [55]. Briefly, the samples were tested against trypomastigotes cultures of T. brucei. The cell cultures of T. brucei were treated with varying concentrations of the samples, and the growth of the parasite cells were monitored with Alamar blue assay. The results were analyzed with ExcelFit ® to determine the IC 50 and IC 90 values.

Insecticidal Studies against Bed Bugs
The bed bug strains (Bayonne 'Insecticide resistant' and Ft. Dix 'Susceptible') were provided by Dr. Changlu Wang, Department of Entomology, Rutgers University, New Brunswick, NJ, and their colony was raised as explained by Montes et al. [56] using blood feeders (CG-1836-75 ChemGlass). The insecticidal activity of EOs against bed bugs was evaluated by fumigation, topical application, and residual studies. For fumigation test, the bed bugs were subjected to vapor toxicity in 125-mL clear glass jars using two microliter aliquots of 125 µg/µL EO stock solution that was injected directly onto inner bottle wall~4 cm from the bottom. The jars were covered immediately with a screw cap and then sealed with parafilm 'M'. The jars were then placed in the growth chamber, and data for mortality was recorded 24 h after treatment. Solutions were made in acetone, and the control treatment received acetone only. 2,2-Diclorovinil-dimetilfosfato (DDVP) was used as the standard.
Studies in topical application were performed with adult bugs, which were separated in the Petri dishes and anesthetized with CO 2 . Using a hand-held repeating dispenser, 1 µL of treatment solution (50 µg/bug) in acetone was delivered onto the dorsal surface of the abdomen. Control bugs received 1 µL of acetone alone. Data for the mortality of the bed bugs was recorded for seven days after treatment. There were three replicates with 10 bugs (mixed sex)/replicate. Deltamethrin was used as the standard (2.4 ng/bug).
For residual studies, the method described by Campbell and Miller [57] was used with minor modifications. A 100-µL aliquot of treatment (diluted in acetone) was applied on 20-cm 2 Whatman #1 filter paper achieving 100 µg/cm 2 of residues. The treated filter papers were then placed in the Petri dish. Control treatments received only acetone. Ten adult bugs were released on the filter paper and mortality was recorded as mentioned in topical application. Deltamethrin was used as standard. Data of insecticidal investigations were analyzed for means, standard error, and one-way ANOVA in JMP 10.0.

Microscopic Procedure
The methods employed for light and scanning electron microscopy analysis of leaves and stems of Baccharis species are fully detailed in a previous paper by Budel et al. [4].

DNA Extraction, PCR, Sequencing
To extract genomic DNA from Baccharis, 100 mg of freeze-dried leaves were ground to fine powder. Genomic DNA from Baccharis samples was extracted using the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, Spain). Four genomic regions-namely ITS, ETS, psbA-trnH, and trnL-trnF-were amplified in 25-µL reactions.
The PCR consisted of a 25-µL reaction mixture containing 2 µL of the DNA solution, 1x PCR reaction buffer, 0.2 mM of dNTP mixture, 0.2 µM of each forward and reverse primers (Table 5), 1.5 mM of MgCl 2 and 1 U of Platinum Taq DNA Polymerase (Invitrogen, Carlsbad, CA, USA). The program comprised of one initial denaturation step at 94 • C for 3 min, followed by 35 cycles at 94 • C for 30 s, X • C for 30 s, and 72 • C for X s (see Table 5 for annealing temperature and extension time). After amplification, each PCR reaction was analyzed by electrophoresis on a 1.5% borate agarose gel and visualized under UV light. The sizes of the PCR products were compared to the molecular size standard 1 kb plus DNA ladder (cat no.: 10787-018, Invitrogen, Carlsbad, CA, USA).
Successfully amplified PCR products were isolated with NucleoSpin ® Gel and a PCR Clean-up kit (MACHEREY-NAGEL, cat no. 740609.50) and eluted with 30 µL of Buffer AE from the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, Spain). PCR products were sequenced in both directions at GeneWiz (South Plainfield, NJ, USA). Sequences were analyzed with DNASTAR (DNASTAR, Madison, WI, USA) and Clone manager 9 (Scientific and Educational Software, Cary, NC, USA) and visually inspected. Contiguous sequences were screened against previously sequenced authenticated samples.

Conclusions
In the present work, profiles of EOs from five species of Baccharis were analyzed and compared. The chemical compositions of EOs of B. pauciflosculosa, B. reticularioides, and B. sphenophylla are reported for the first time. Although the qualitative compositions of the EOs of these species were more or less similar, they showed distinctive differences in the quantity of the components. Some compounds were unique to these species, and hence can be used as chemical markers for species identification and authentication. B. microdonta differed from the other species by having kongol and spathulenol in high concentrations. B. pauciflosculosa showed β-pinene and limonene as major compounds. α-Bisabolol was found only in B. punctulata. B. reticularioides showed α-pinene, while B. sphenophylla presented α-pinene, β-pinene, limonene, and spathulenol as major compounds.
The leaves and stems of all five Baccharis species possessed glandular trichomes and ducts as secretory structures. All three types of glandular trichomes that were observed in this study contained EOs. DNA barcoding using ITS and trnL-trnF sequences were useful for the authentication of the studied Baccharis species.