In Vitro Pharmacological Screening of Essential Oils from Baccharis parvidentata and Lippia origanoides Growing in Brazil

In this study, the in vitro antimicrobial, antiparasitic, antiproliferative and cytotoxic activities of essential oil from Baccharis parvidentata Malag. (EO-Bp) and Lippia origanoides Kunth (EO-Lo) were explored. The relevant effects were observed against the parasitic protozoans Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei and Leishmania amazonensis (ranging 0.6 to 39.7 µg/mL) and malignant MCF-7, MCF-7/HT, 22Rv1, and A431 cell lines (ranging 6.1 to 31.5 µg/mL). In parallel, EO-Bp showed better selective indexes in comparison with EO-Lo against peritoneal macrophages from BALB/c mice and MRC-5 cell line. In conclusion, EO-Lo is known to show a wide range of health benefits that could be added as another potential use of this oil with the current study. In the case of EO-Bp, the wide spectrum of its activities against protozoal parasites and malignant cells, as well as its selectivity in comparison with non-malignant cells, could suggest an interesting candidate for further tests as a new therapeutic alternative.


Introduction
The search for biologically active compounds is currently a subject of interest in the food, cosmetic and pharmaceutical industries, among others [1]. Plant-based natural products are a potential source of new agents and/or drugs [2]. The ability of plant species to biosynthesize a broad variety of complex and innovative scaffolds is outstanding and can be utilized or further developed as a major source of leads for new drugs if appropriate in vitro and in vivo assays are established. Numerous plant extracts and pure natural compounds have been discovered [3,4]. Among those, essential oils (EOs) have been shown to display a wide range of therapeutic properties [5,6]. EOs are a mixture of volatile compounds that have been found in different plant organs from several botanical families [5], including
In general, the EOs showed low antimicrobial activity except for the EO-Bp that caused relevant growth inhibition of S. aureus with a median inhibitory concentration (IC 50 ) value < 10 µg/mL (Table 1). Both EOs inhibited all protozoal parasites. EO-Bp displayed the best activity, T. brucei and P. falciparum were the most susceptible to its effect ( Table 2). The protozoal parasites were also susceptible to EO-Lo, although with higher values of IC 50 , except for L. amazonensis equally susceptible to EO-Lo and EO-Bp and for L. infantum, which showed IC 50 > 64 µg/mL. Moreover, both EOs showed antiproliferative activity (Table 3), but EO-Bp, with IC 50 ranging from 6.1 to 15.1 µg/mL, was a better inhibitor of all malignant cell growth than EO-Lo with IC 50 from 9.1 to 31.5 µg/mL.
In the same way, EO-Bp showed higher cytotoxic effects than EO-Lo in the used models (Table 3), according to the obtained median cytotoxic concentration (CC 50 ). However, when the selectivity indices (SI) were obtained, the most promising activity was achieved for EO-Bp in PMM and MRC-5 models with respect to antiprotozoal activities ( Figure 1A,C). In contrast, for the antiproliferative effect, EO-Bp showed better results in the PMM model, while for EO-Lo, better indexes were obtained with respect to the MRC-5 model ( Figure 1B,D). In particular, with regards to the SI values calculated with respect to MCF-10A, both EOs showed unspecific activity in comparison with a malignant sensitive cell line (MCF-7) and a hormone-resistant cell subline (MCF-7/HT) with values around unity.   Brazil is reputed for its floristic diversity; hence its flora should be considered an important reservoir of active molecules with potential industrial applications [15]. Plant species from several botanical families have been described for their EO content and their antimicrobial [16], antiparasitic [17], anticancer [18], anti-oxidant [19], and insecticidal [20] activities. In the present study, the antiproliferative properties of EO from B. parvidentata and L. origanoides, collected in Brazil, against bacteria, fungi, protozoal parasites, malignant and non-malignant cells were investigated.
Although MIC values of these EOs: 625 to 1250 µg/mL against bacteria (E. coli and S. aureus), 156 to 2500 µg/mL against fungi (Aspergillus niger, Fonsecaea pedrosoi, and Trycophyton rubrum) and 78 to 2500 µg/mL against yeast (C. albicans and Cryptococcus neoformans) have been reported [9], our results confirm that no relevant activity was observed at 64 µg/mL, in both studied EOs except EO-Bp against S. aureus. It is known that S. aureus is a Gram-positive bacterium frequently found in the upper respiratory tract and on the skin as an opportunistic pathogen, being a common cause of sinusitis, abscesses, and food poisoning [21,22]. In this sense, EOs of other Baccharis species have been documented due to their potentialities against S. aureus, such as B. dracunculifolia DC [23] and B. oreophila Malme [24]. Brazil is reputed for its floristic diversity; hence its flora should be considered an important reservoir of active molecules with potential industrial applications [15]. Plant species from several botanical families have been described for their EO content and their antimicrobial [16], antiparasitic [17], anticancer [18], anti-oxidant [19], and insecticidal [20] activities. In the present study, the antiproliferative properties of EO from B. parvidentata and L. origanoides, collected in Brazil, against bacteria, fungi, protozoal parasites, malignant and non-malignant cells were investigated.
Although MIC values of these EOs: 625 to 1250 µg/mL against bacteria (E. coli and S. aureus), 156 to 2500 µg/mL against fungi (Aspergillus niger, Fonsecaea pedrosoi, and Trycophyton rubrum) and 78 to 2500 µg/mL against yeast (C. albicans and Cryptococcus neoformans) have been reported [9], our results confirm that no relevant activity was observed at 64 µg/mL, in both studied EOs except EO-Bp against S. aureus. It is known that S. aureus is a Gram-positive bacterium frequently found in the upper respiratory tract and on the skin as an opportunistic pathogen, being a common cause of sinusitis, abscesses, and food poisoning [21,22]. In this sense, EOs of other Baccharis species have been documented due to their potentialities against S. aureus, such as B. dracunculifolia DC [23] and B. oreophila Malme [24].
A wide inhibitory spectrum was appreciated against protozoal parasites of medical importance and malignant cells. Therefore, our screening strategy suggests the activity of the studied EOs against eukaryotic cells. The mechanisms underlying the antiprotozoal and antiproliferative actions of the tested EOs were not studied. Nevertheless, during the last decade, it has been postulated that the mechanism of action for oils and their A wide inhibitory spectrum was appreciated against protozoal parasites of medical importance and malignant cells. Therefore, our screening strategy suggests the activity of the studied EOs against eukaryotic cells. The mechanisms underlying the antiprotozoal and antiproliferative actions of the tested EOs were not studied. Nevertheless, during the last decade, it has been postulated that the mechanism of action for oils and their constituents is rather complex. Several of these effects are attributable to the lipophilic nature and low molecular weight of the main components that comprise the EOs, which allow them to cross cell membranes, alter membrane composition, and increase membrane fluidity, leading to the leakage of ions and cytoplasmic molecules. Altering membranes leads to reduced ATP production, alteration of the pH gradient, and loss of mitochondrial potential, which can result in cell death. In addition, the induction of cell death by the activation of apoptotic and/or necrotic processes, cell cycle arrest, and loss of function of essential organelles has been observed. In parallel, some EOs may also act as pro-oxidant elements, which can alter the cellular redox state and compromise cellular survival. Nevertheless, the activities of EOs generally result from complex interactions between the different classes of compounds that can result in a great diversity of mechanisms of action and molecular targets [5].
The most sensitive parasite was T. brucei, which is responsible for African trypanosomiasis or sleeping sickness, mostly found in equatorial Africa. Human African trypanosomiasis takes two forms depending on the parasite involved, which are both transmitted by tsetse flies (Glossina spp.). Sleeping sickness in eastern and southern sub-Saharan Africa is an acute form caused by the subspecies T. brucei rhodesiense. Trypanosomiasis in the central and western regions of Africa is a slow-progressing form caused by T. brucei gambiense. Both trypanosomes invade the brain, causing mental deterioration, coma and death if left untreated [25,26].
The studied EOs displayed an important activity against 22Rv1 malignant cells, which are derived from human prostate carcinoma. In men, prostate cancer is the most commonly identified cancer and one of the most important causes of cancer-related deaths; inflammation is also associated with the pathogenesis of this disease [27,28]. The discovery and development of chemotherapeutic agents that can bind specifically prostate tumor cells is crucial to improve the treatment effectiveness and eventually avoid castration in affected patients [28,29].
Another point to consider is that EO-Bp showed the same IC 50 value (p > 0.05) against susceptible MCF-7 and hormone-resistant MCF-7/HT (12.9 and 12.4 µg/mL, respectively). It is known that one of the hallmarks of cancer therapy resides in the ability of malignant cells to accumulate genetic/epigenetic changes until they achieve self-renewal, produce differentiated progeny, and develop resistance to therapy [30]. Thus, these results could represent a potential treatment opportunity for cancers that develop hormone resistance.
Moreover, toxicity on non-malignant cells is an important criterion in the drug development process. In that regard, EOs showed certain cytotoxicity, with CC 50 values < 100 µg/mL. However, EO-Bp exhibited a selective antiparasitic effect on T. brucei, T. cruzi and P. falciparum, with SI values ranging from 14 to 128. Thus, these EOs should be considered for further exploration in animal models.
In Brazil, there are currently 167 species in the Baccharis genus; the country is one of the main centers of diversity of this genus [35,36]. The species of this genus are very relevant in folk medicine, with several species being used for the control and treatment of diseases, beyond the economic and environmental aspects. In this regard, a previous review highlights the antimicrobial and antiprotozoal activity of Baccharis species, which was considered a promising source of biologically active compounds [10]. In particular, the activity of EO-Bp have been scarcely explored; however, knowledge of the chemical structures may explain the observed biological activity and could serve as scaffolds for rational drug design, suggesting chemical modifications to increase activity, bioavailability, and toxicity, among other characteristics, allowing the design of new and more active compounds [17]. Then, to have a general overview of probable bioactive compounds of EO-Bp, reports retrieved from scientific literature related to the assayed pharmacological activities of identified major compounds are summarized in Table 4 [9]. In this sense, reports about the antimicrobial, antiparasitic and antiproliferative activities of the main compounds from EO-Bp were found. In some instances, although different cell targets in comparison with our study have been assayed, the versatility of activities is demonstrated and supports our results. Among these compounds, reports suggest that sabinene demonstrates a wide spectrum of antimicrobial and antiparasitic activities, while himachalol displayed antimicrobial and anticancer actions. In contrast, β-pinene was the most versatile compound and displayed antimicrobial, antiparasitic and anticancer activities. Table 4. In vitro pharmacological activities retrieved from scientific literature on major compounds identified in the essential oils from Baccharis parvidentata growing in Brazil.

Major Compounds Pharmacological Property Target (Results) Reference
Sabinene from EO-Bp were found. In some instances, although different cell targets in comparison with our study have been assayed, the versatility of activities is demonstrated and supports our results. Among these compounds, reports suggest that sabinene demonstrates a wide spectrum of antimicrobial and antiparasitic activities, while himachalol displayed antimicrobial and anticancer actions. In contrast, β-pinene was the most versatile compound and displayed antimicrobial, antiparasitic and anticancer activities. Antiparasitic -Trypanosoma cruzi (% growth inhibition 95.7 ± 0.4 at 100 µg/mL, 89.9 ± 9.6 at 50 µg/mL, 71.6 ± 1.6 at 10 µg/mL and 12.3 ± 0.3 at 5 µg/mL) [43] Antiproliferative -Oral squamous cell carcinoma (IC50~67 µg/mL) [44] 3. Materials and Methods

Essential Oils
EO-Bp and EO-Lo were obtained previously by Perera et al. [9] and were obtained by common hydrodistillation in a Clevenger apparatus for 3 h and dried with anhydrous sodium sulfate. The EOs were chemically characterized using gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to flame ionization detection (GC-FID) analyses (Supplemental Materials; Table S1). For biological assays, each EO was diluted in dimethyl sulfoxide (DMSO) at 20 mg/mL. This work is registered in SISGEN (Sistema Nacional de Gestão do Patrimonio Genetico, Brazil) under the access authorization number AC7DDF5.

Antiproliferative
No reports were found -Himachalol from EO-Bp were found. In some instances, although different cell targets in comparison with our study have been assayed, the versatility of activities is demonstrated and supports our results. Among these compounds, reports suggest that sabinene demonstrates a wide spectrum of antimicrobial and antiparasitic activities, while himachalol displayed antimicrobial and anticancer actions. In contrast, β-pinene was the most versatile compound and displayed antimicrobial, antiparasitic and anticancer activities. Antiparasitic -Trypanosoma cruzi (% growth inhibition 95.7 ± 0.4 at 100 µg/mL, 89.9 ± 9.6 at 50 µg/mL, 71.6 ± 1.6 at 10 µg/mL and 12.3 ± 0.3 at 5 µg/mL) [43] Antiproliferative -Oral squamous cell carcinoma (IC50~67 µg/mL) [44]

Essential Oils
EO-Bp and EO-Lo were obtained previously by Perera et al. [9] and were obtained by common hydrodistillation in a Clevenger apparatus for 3 h and dried with anhydrous sodium sulfate. The EOs were chemically characterized using gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to flame ionization detection (GC-FID) analyses (Supplemental Materials; Table S1). For biological assays, each EO was diluted in dimethyl sulfoxide (DMSO) at 20 mg/mL. This work is registered in SISGEN (Sistema Nacional de Gestão do Patrimonio Genetico, Brazil) under the access authorization number AC7DDF5. β-Pinene from EO-Bp were found. In some instances, although different cell targets in comparison with our study have been assayed, the versatility of activities is demonstrated and supports our results. Among these compounds, reports suggest that sabinene demonstrates a wide spectrum of antimicrobial and antiparasitic activities, while himachalol displayed antimicrobial and anticancer actions. In contrast, β-pinene was the most versatile compound and displayed antimicrobial, antiparasitic and anticancer activities. Antiparasitic -Trypanosoma cruzi (% growth inhibition 95.7 ± 0.4 at 100 µg/mL, 89.9 ± 9.6 at 50 µg/mL, 71.6 ± 1.6 at 10 µg/mL and 12.3 ± 0.3 at 5 µg/mL) [43] Antiproliferative -Oral squamous cell carcinoma (IC50~67 µg/mL) [44]

Essential Oils
EO-Bp and EO-Lo were obtained previously by Perera et al. [9] and were obtained by common hydrodistillation in a Clevenger apparatus for 3 h and dried with anhydrous sodium sulfate. The EOs were chemically characterized using gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to flame ionization detection (GC-FID) analyses (Supplemental Materials; Table S1). For biological assays, each EO was diluted in dimethyl sulfoxide (DMSO) at 20 mg/mL. This work is registered in SISGEN (Sistema Nacional de Gestão do Patrimonio Genetico, Brazil) under the access authorization number AC7DDF5.

Essential Oils
EO-Bp and EO-Lo were obtained previously by Perera et al. [9] and were obtained by common hydrodistillation in a Clevenger apparatus for 3 h and dried with anhydrous sodium sulfate. The EOs were chemically characterized using gas chromatography-mass spectrometry (GC-MS) and gas chromatography coupled to flame ionization detection (GC-FID) analyses (Supplemental Materials; Table S1). For biological assays, each EO was diluted in dimethyl sulfoxide (DMSO) at 20 mg/mL. This work is registered in SISGEN (Sistema Nacional de Gestão do Patrimonio Genetico, Brazil) under the access authorization number AC7DDF5.

Microorganisms and Cells
The reference strain of Gram-negative E. coli ATCC8739, Gram-positive S. aureus

Antimicrobial Screening
The integrated antimicrobial screening in 96-well plates was adopted from Cos et al. [45]. Serial dilutions of the EOs were prepared from the DMSO stock solutions in sterile demineralized water in 96-well plates using an automated liquid-handling workstation (Beckman Coulter Biomek 3000), in which the final DMSO concentration was <1%. In all cases, negative (100% growing) and positive (a reference drug) control were included.
The antiprotozoal assessment was carried out using different models, and parasite forms as will be described below. Activity against P. falciparum was measured with parasites cultured in human erythrocytes A+ (with 1% parasitemia and 2% hematocrit) in RPMI medium (with 0.5 (g/v)% AlbumaxTM) at 37 • C in an atmosphere of 3% O 2 , 4% CO 2 , and 93% N 2 [47].  [48]. In the case of trypomastigotes of T. brucei, the assessment of activity was performed in Hirumi-9 medium supplemented with 10% inactivated fetal calf serum (FCSi) at 37 • C and 5% CO 2 [49]. Assays were performed by adding 1.5 × 10 4 trypomastigotes/well to the EOs at different concentrations and incubating them for 72 h under the same conditions. The fluorimetric resazurin method, with an additional incubation for 24 h at 37 • C, was used to measure parasite growth. Evaluation of activity against L. amazonensis promastigotes was carried out in a 96-well plate with 2 × 10 5 parasites/mL and with the different concentrations of EOs. The plates were sealed with Parafilm and incubated at 26 • C for 72 h. Afterward, 20 µL of a solution (5 mg/mL) of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) was added to each well and incubated for an additional 4 h. The supernatant was removed, and the formazan crystals were dissolved with 100 µL DMSO. The absorbance of each well was determined with a plate reader (Molecular Devices, Sunnyvale, CA, USA) with a test wavelength of 560 nm and a reference wavelength of 630 nm [50]. On the other hand, the evaluation of antileishmanial activity against the intracellular amastigote form of L. amazonensis and L. infantum was also performed. In the model of L. amazonensis, a PMM monolayer was used (plated at 10 6 /mL in 24-well plates) and infected with stationary-phase promastigotes at a 4:1 parasite/macrophage ratio for 4 h at 5% CO 2 and 37 • C. The cells were washed to remove free parasites. EOs were added at different concentrations, and the plates were further incubated under the same conditions for 48 h [51]. For L. infantum, 3 × 10 4 PMM were infected with amastigotes obtained from an infected hamster at a ratio of 15 parasites per cell. The plate was incubated for 48 h at 37 • C and 5% CO 2 , and different concentrations of EOs were added. Then, the plate was incubated under the same conditions for 120 h. In both parasite species, the supernatant was discarded after the incubation period with the products; cells were fixed with methanol, stained with 10% Giemsa and microscopically examined (Motic, Japan) under immersion oil. Total parasite burden was determined by the number of infected macrophages and the number of amastigotes inside the macrophages. In the antiprotozoal assays, chloroquine, enznidazole, suramine and miltefosine (donated by Special Programme for Research and Training in Tropical Diseases from the World Health Organization (WHO-TDR)) were used as reference drugs for P. falciparum, T. cruzi, T. brucei, and L. infantum, respectively, while pentamidine (Richet, Buenos Aires, Argentina) was used for L. amazonensis.

Antiproliferative Assay on Malignant Cells
MCF-7, MCF-7/HT, 22Rv1 and A431 cancer cells were cultured in standard 4.5 g/L glucose DMEM medium (Gibco-Life Technologies, Paisley, UK), when 22Rv1 were cultivated in RPMI-1640 medium (Gibco-Life Technologies, Paisley, UK) supplemented with RPMI-1640 Vitamins (PanEco, Moscow, Russia). In all cases, the culture was supplemented with 10% fetal calf serum (FCS), antibiotics (50 µg of streptomycin/mL and 50 U of penicillin/mL) and 0.1 mg/mL sodium pyruvate (Santa Cruz Biotechnology, Dallas, TX, USA) and maintained in a NuAir incubator (NuAir, Plymouth, MN, USA) at 37 • C, 5% CO 2 and 80-85% humidity. Then, 100 × 10 3 22Rv1 cells/well, 40 × 10 3 MCF-7 or MCF-7/HT cells/well and 35 × 10 3 A431 cells/well, were seeded into 24-well plates in 900 µL of the medium, and the plates were incubated for 24 h at 37 • C and 5% CO 2 . Subsequently, EOs or reference drug were added at different concentrations and the plates were incubated for 72 h under the same conditions. Cell viability was assessed using MTT at 0.2 mg/mL per well [52]. After additional incubation for 2 h, the supernatant was discarded, the MTT formazan purple crystals were dissolved in DMSO (350 µL per well) and the absorbance was measured at 571 nm and 630 nm as a reference in a MultiScan reader (ThermoFisher, Waltham, MA, USA) after the plates were gently shaken. Cisplatin (Teva Pharmaceutical Industries, Petah Tikva, Israel) was used as a reference drug.

Cytotoxicity Test on Non-Malignant Cells
The cytotoxicity analysis of EOs on 10 4 MRC-5 cells/well was performed in MEMsupplemented medium at 37 • C and 5% CO 2 seeded onto the test plates for 72 h. Cell viability was assessed fluorometrically after the addition of resazurin as described above. Cytotoxicity was also studied using MCF-10A cells cultured in medium supplemented with 5% donor horse serum (BioSera, Nuaille, France), 20 ng/mL epidermal growth factor (PanEco, Moscow, Russia), 0.5 µg/mL hydrocortisone (ChemCruz, Dallas, TX, USA), and 10 µg/mL insulin (PanEco) at 37 • C, 5% CO 2 and 80-85% humidity). Briefly, 60 × 10 3 MCF-10A cells were seeded into 24-well plates in 900 µL of the medium, and then, plates were incubated for 24 h at 37 • C and 5% CO 2 . Then, different concentrations of EOs were added, and the plates were incubated for 48 h under the same conditions. Cell viability was then measured by the MTT method as described above for cancer cell lines. Finally, in the model of PMM, 3 × 10 5 cells/mL was treated with different concentrations of EOs over 48 h in the same conditions. Then, 15 µL of MTT solutions were added to each well, and, after 4 h of additional incubation in the same conditions, the supernatant was discarded, formazan crystals were dissolved with 100 µL of DMSO and the absorbance was obtained as described above.

Statistical Analysis
In each model included in this assessment, three experiments were performed and percentage growth inhibition for each product concentration was calculated compared to the untreated cultures (negative control). The IC 50 for antibacterial, antifungal and antiprotozoal assays were determined, while CC 50 was obtained in mammalian cell assays. In both cases, values were obtained from dose-response curves, and results were expressed as mean with a 95% confidence interval. Finally, SI was calculated as the ratio of the CC 50 for MRC-5/PMM cells and the IC 50 for microorganism or malignant cells.

Conclusions
In conclusion, EO-Lo is known to show a wide range of health benefits. The current study added another potential use of this oil in the area of infectious parasitic and malignant diseases. EO-Bp had antiparasitic and antiproliferative activity with a wide spectrum and selectivity in comparison with non-malignant eukaryotic cells. Thus, these products from a plant that grows in Brazilian regions can be considered interesting candidates for further tests as new therapeutic alternatives.