1. Introduction
Varronia curassavica Jacq., also known as Cordia verbenacea DC., is an aromatic plant known as “erva-baleeira” that belongs to the Boraginaceae family [1]. This plant is native to the Brazilian Atlantic Forest, of the restinga vegetation class, with distribution ranges from the east of the Amazon region to the south of the state of Rio Grande do Sul [2]. The shrubs of this species have leaves covered with glandular trichomes (sites of production and storage of essential oil (EO)), white flowers (ranging between four to six petals) in the axis of inflorescence and red fruit, and can reach up to 2 m in height [3,4,5].
The aerial parts of this species are widely used in traditional medicine in many communities, with anti-inflammatory, analgesic and wound-healing properties [6,7,8,9]. Other biological and pharmacological activities have also been attributed to V. curassavica, such as antibacterial [10], antifungal, antiprotozoal [11,12], antitumor [13], antioxidant [14] and antiulcerogenic [15] properties. Recently, Andrade et al. [16] demonstrated the potential of V. curassavica EO for natural pest control, and Oliveira et al. [17] used V. curassavica EO to study bioassays of fumigation toxicity, aiming to control a common species of ant (Dorymyrmex thoracicus), showing that the oil was able to repel and decrease the motility of the ants.
Many of the previously described therapeutic effects of V. curassavica are associated with plant secondary metabolites, whose preferred source of industrial production is the essential oil extracted from the leaves [18]. Acheflan®, an anti-inflammatory phytomedicine produced in Brazil by the Aché Laboratory, approved in 2004 by ANVISA (the Brazilian National Sanitary Surveillance Agency (NSSA)) and used for treatment of trauma, tendinopathy and muscular pain, is manufactured from essential oil from V. curassavica aerial parts (containing 5 mg of EO and approximately 0.130 mg of α-humulene). Currently, Acheflan® is the most prescribed drug in this market segment, with sales of more than $8 million USD in the pharmaceutical market, being exported to countries in the Americas and Japan [3,19,20].
Considering the commercial value of essential oils (up to $5800.00 USD per liter), the best technique for obtaining the essential oil is to improve the quantity and quality of the V. curassavica EO process, including an exhaustion time of 1.5 h for extracting the oil, with faster extraction of monoterpenes compared to sesquiterpenes, using the steam distillation method, which has been demonstrated to be the best method for extraction [20,21]. The contents of the specific plant secondary metabolites and the chemical composition of the essential oils are influenced by both genetic and environmental factors; generally, the main constituents of V. curassavica EO are trans-caryophyllene, β-santalene, α-pinene and α-humulene [16,22,23,24]. The anti-inflammatory activity of α-humulene and trans-caryophyllene was proven in previous studies, and these two compounds in the EO extracted from V. curassavica leaves for Acheflan® production are used for control by ANVISA here in Brazil [3,4].
Considering the expected effect of age on the composition of plant essential oils and the derived industrial products, this study aimed to assess the influence of the age of V. curassavica plants on the chemical composition of the essential oil extracted at the industrial scale. Moreover, we also analyzed the effect of age on the cytotoxicity against murine cells (BALB/c peritoneal macrophages) and on the activity against bacteria (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis) and yeast (Candida albicans).
2. Material and Methods
2.1. Plant Material and Essential Oil Production
The plants were cultivated in an agricultural production area by the Centroflora Group (Parnaíba, Piauí, Brazil). The V. curassavica plantation is located in a central pivot area covering 59.8 hectares, divided into sub-areas that are numbered and differentiated from each other according to their planting date. The exsiccate of the botanical material used was deposited in the Herbarium Delta do Parnaíba (HDELTA, Parnaíba, Piauí, Brazil), under voucher specimen no. 3930. The area selected for the development of the project corresponded to sub-area 13, at a latitude of 3°1.720’ S and longitude of 41°45.021′ W, with dimensions of 3.34 hectares, with the genotype characterized as ID 13 by Lima et al. [25], with an average height of 70.66 cm and an average diameter equal to 14.01 mm after one year of planting (Figure 1).
The aerial parts were harvested at the same time (05:00) at different ages (4, 10, 14 and 18 months-age) in (i) September 2016 and (ii) March, (iii) July and (iv) November 2017. The parts were collected mechanically with the aid of a forage harvester for essential oil production by the Centroflora Group. The leaves were taken to a laboratory for microscopic study. Briefly, the slides were made with freehand paradermal and transversal cuts of the stem and the midrib of the leaf. The samples were clarified with 2% sodium hypochlorite and later stained with 0.5% safranin for viewing under a stereoscopic microscope, through which it was possible to observe the globular trichomes, where the plant stores and secretes essential oil (Figure 1D) [26,27].
The essential oil (EO) was extracted by steam distillation on an industrial scale, at 09:00 on the same day the material was harvested. Briefly, the aerial parts were uniformly distributed and compressed in a stainless-steel vat. Steam distillation was carried out for two hours (Figure 2).
2.2. Phytochemical Characterization of V. curassavica Essential Oil
Samples of essential oil extracted from plants at different ages (4, 10, 14 and 18 months-age) were characterized using a gas chromatography system coupled to a mass spectrometer with electron impact ionization (GC-MS-QP210 SE, Shimadzu, Kyoto, Japan). Separation was achieved using the Equity®-1 Capillary GC Column (L × I.D. 60 m × 0.25 mm, df 1.00 μm; Supelco, Bellefonte, PA, USA), following the temperature programming from 100 to 240 °C (5 °C/min), where it remained for 5 min, followed by heating from 240 °C to 280 °C (10 °C/min) for the same duration. Helium was used as the inert gas at 1 mL/min, with a split ratio of 1:10 and an injected volume of 1 μL. The injector was kept at 250 °C and the detector at 280 °C. The following conditions were used for the mass spectrometer: ion source temperature: 200 °C; interface temperature: 290 °C; 5 min delay time; analysis mode: scan (35–350 m/z). Dibutyl phthalate was used as the internal standard, following the method validated by the Centroflora Group. Compounds were identified by comparing the mass spectra of samples with those in the NIST 14 library; the retention times of samples with those of the analytical standards of α-pinene, trans-caryophyllene and α-humulene; and the calculated Kovats index values of sample constituents with those available in the literature [28].
2.3. Cytotoxicity Assay
Murine peritoneal macrophages were isolated from BALB/c mice and treated with essential oils or α-pinene, and their viability was assessed using an MTT (3-(4, 5-dimethylthiazol-2-118 yl)-2,5-diphenyltetrazolium bromide) assay (Sigma-Aldrich, St. Louis, MO, USA) [29]. Briefly, macrophages were obtained from female BALB/c mice (4–5 weeks old) obtained from the Animal Facility of Universidade Federal do Piauí (UFPI, Teresina, Piauí, Brazil) and kept in a controlled environment with a constant temperature of 24 ± 1 °C and a 12 h light/dark cycle. Then, 3.0% thioglycolate (1.5 mL) was intraperitoneally administered, and after three days the sterile phosphate-buffered saline (PBS, pH 7.4) was injected into the abdominal cavity to the remove the cells. This procedure was approved by the Ethics Committee for the Use of Animals from Universidade Federal do Piauí (CEUA-UFPI, Teresina, Piauí, Brazil) under approval n°457/18.
Then, the macrophages were cultured in RPMI 1640 medium supplemented with fetal bovine serum (10% FBS), penicillin (10,000 IU/mL), and streptomycin (10 mg/mL) (Sigma-Aldrich, St Louis, MO, United States) at a concentration of 1.0 × 105 cells per well, where they were exposed to treatments with V. curassavica EO samples and α-pinene at 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/mL in triplicate. Afterwards, 10 µL of MTT (5 mg/mL in PBS (w/v) was applied to each well and incubated for 4 h throughout the course of 48 h. The supernatant was then discarded, and 100 µL of DMSO was immediately added to each well. To completely dissolve the formazan salt, the plate was stirred for 30 min. The absorbances were measured at 540 nm using the ELx800 microplate reader (BioTek Instruments, Winooski, VT, USA). A concentration–response curve was plotted to determine the mean inhibitory concentration (IC50), using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, United States).
2.4. Antimicrobial Assays
The minimum inhibitory concentration (MIC) values, i.e., the lowest concentrations inhibiting visible microbial growth, of the essential oils were determined following Clinical and Laboratory Standards Institute procedures [30,31] against the Gram-negative bacteria Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853) and the Gram-positive bacteria Staphylococcus aureus (ATCC 29213), methicillin-resistant Staphylococcus aureus (ATCC 43300) and Staphylococcus epidermidis (ATCC 12228), in addition to yeast Candida albicans. Briefly, the bacteria were inoculated in Mueller–Hinton broth (Difco™) at a concentration of 5 × 105 CFU/mL in 96-well microplates, where they were exposed to serial dilutions of V. curassavica EO samples (7.8–1000 µg/mL), then were incubated for 24 h at 37 °C in triplicate. For yeast, the essential oils were tested at the same concentrations against 5.0 × 102–2.5 × 103 CFU/mL of C. albicans in 96-well plates with a U-shaped base. After the incubation period (24 h/37 °C), the result was analyzed via visual readings of microbial growth.
2.5. Statistical Analysis
The relative contribution of each compound identified in the essential oil was used for a principal component analysis of extractions at different plant ages using Minitab software v.19 (Minitab, State College, PA, USA). The levels of α-pinene, trans-caryophyllene and α-humulene were compared using a one-way ANOVA followed by Tukey’s multiple comparisons test in GraphPad Prism software v.6.1 (GraphPad, San Diego, CA, USA). The results of the cytotoxicity assay were compared to the respective control untreated group using a one-way ANOVA followed by Dunnett’s multiple comparisons test. At all instances, p values below 0.05 were considered to indicate statistical significance.
3. Results and Discussion
The chemical characterization via GC/MS identified 19 molecules in V. curassavica essential oils at 4, 10, 14 and 18 months-age old, as reported in Table 1. The respective yields of those essential oils were 0.093%, 0.096%, 0.083% and 0.112% (w/w) (Table 2). Regardless of age, the most abundant compounds in the essential oils were α-pinene, trans-caryophyllene, α-santalene, alloaromadendrene and α-humulene (Table 1). The compositions varied according to the plant age, mainly for less abundant molecules, which were detected in some samples and in others could not be accurately identified. α-Pinene was the most abundant compound of V. curassavica EOs obtained from different regions of Brazil (São Paulo, Rio de Janeiro, Minas Gerais, Bahia, Sergipe and Ceará) [3,27,32,33]. On the other hand, plants of the same species cultivated in Minas Gerais and in the southern region of Bahia (Brazil) showed β-pinene, β-caryophyllene and α-humulene as the major chemical compounds, while α-pinene and caryophyllene were also found [33,34,35].
The data on the chemical characterization of essential oils (Table 1) were further analyzed using a principal component analysis and plotted as a score plot (Figure 3). As expected, the three independent essential oil extractions from plants at the same age clustered together (Figure 3); this indicated that variability in the concentrations of the 19 identified compounds is sufficient to separate essential oil samples according to plant age. The essential oil from plants at 18 month-age appeared as the most distinct from the others; the three samples clustered in the negative areas in principal component analyses 1 (x-axis) and 2 (y-axis). The first component (PC1) separated plants harvested at 4 and 18 month-age from those harvested at 10 and 14 month-age. The second component (PC2) isolated the essential oil from plants at 18 month-age from all other groups, except for one sample from plants at 10 month-age. The compounds had a rather balanced contribution for PC1; the highest coefficient was 0.239 for eucalyptol and β-elemene and the lowest was 0.201 (i.e., a narrow range of 0.201–0.239). In the case of PC2, this component was mainly a combination of β-myrcene (−0.529), β-gurjunene (0.336), α-thujene (−0.289), α-pinene (−0.276) and α-humulene (0.271).
α-Pinene and trans-caryophyllene were described as the major constituents of V. curassavica EO from another geographic area [10]. A similar profile was observed in the V. curassavica EO cultivated in Minas Gerais (Brazil), whose main constituents were α-pinene, α-santalene and (E)-caryophyllene [2]. On the other hand, the most abundant compounds in the essential oil of this species vary according to the cultivation site due to the different environmental conditions, directly influencing the chemical composition. For instance, α-pinene and (E)-caryophyllene were the major oil components in plants from Minas Gerais (Brazil) [35], and tricyclene, α-zingiberene, turmerone, camphene and (E)-caryophyllene were the major oil components in plants from Sergipe (Brazil) [12].
Several factors, including climatic, altitude, soil, area of cultivation, processing, and genetic variations, explain such differences [2]. Moreover, it should be noted how these variations in content are especially relevant for industrial purposes since these main identified compounds exert biological activity. The assessment of interactions of biologically active compounds represents a first step in the evaluation of the biological activity of a plant [36,37,38]. α-Pinene and trans-caryophyllene present anti-inflammatory properties through reductions in PGE2, COX-2 and inducible NOS levels [39]. Alloaromadendrene presents cytotoxic and antiproliferative activities against cancer cells [40]. α-Santalene has been used in the perfume and flavoring industries, and its analogues present some pharmacological properties such antifungal, antibacterial and antitumor activities [41].
Acheflan® contains α-humulene obtained from V. curassavica essential oil, and the α-humulene and trans-caryophyllene contents are controlled by the Brazilian regulatory agency, ANVISA [3,18]. Therefore, we used specific analytical standards to quantify the contents of three essential oil components, namely α-pinene, trans-caryophyllene and α-humulene. In Figure 4, the concentrations of the selected compounds (α-pinene, α-humulene and trans-caryophyllene) in the essential oil extracted from V. curassavica at different ages (4, 10, 14 and 18 months-age) via steam distillation on an industrial scale are reported. The α-pinene content at 10 month-age increased by 128% compared to the content at 4 month-age. At 14 month-age, the content of α-pinene decreased by 27% compared with that in the essential oil of 10-month-old plants, and at 18 month-age it returned to levels similar to those at 4 month-age (Figure 4A). The α-humulene levels tended to increase as the plants aged, reaching their maximum value at 14 month-age and then decreasing at 18 month-age (Figure 4B).
The essential oil from plants at 14 month-age contained 48%, 15% and 45% more α-humulene than that from plants at 4, 10 and 18 months-age, respectively; the trans-caryophyllene levels followed an inverted-U pattern, reaching their maximum values at 10 month-age (Figure 4C). According to Facanali et al. [42], the average yield of an EO is affected by the season, with higher production rates in spring and lower rates in winter. In addition, the α-humulene content can also be affected by the genotype and season, being produced in greater quantities in spring and summer. At 18 month-age, the trans-caryophyllene content decreased, reaching values that were 29% lower than in plants at 4 month-age.
Ontogenetic effects on the compositions of essential oils have also been observed for other plant species [16,43,44]. As plants age, reductions in the activity of the metabolic pathways can be observed, which can lead to reductions in the synthesis of essential oil compounds [44,45]. On the other hand, studies have also shown increases in oil production with increasing age, demonstrating variable behavior between species, such as for Melissa officinalis L. oil, which has a richer chemical composition at two years of age when compared to oil extracted from younger plants [46,47]. For V. curassavica, the relationship between the plant age and chemical composition was demonstrated for the first time in our study.
When studying the effect of the Artemisia annua harvest age on essential oil production, Damtew et al. [48] demonstrated that there was no change in the essential oil content, but the content of artemisinin, an antimalarial compound, was significantly altered. The same result was found by Rocha et al. [49] when studying the influence of the age of Cymbopogon citratus (lemon grass) on the chemical composition of the essential oil of this species. The authors reported that there was no change in the extracted oil content, only in the chemical composition.
Our cytotoxicity assay results showed significant differences in relation to the control group in all essential oil concentrations tested at 10, 14 and 18 month-age. In contrast, the EO at 4 month-age at a concentration of 6.25 µg/mL did not differ from the control (Figure 5). A very similar pattern was observed when the cells were treated with pure α-pinene (Figure 5E). The essential oil at 18 month-age had the lowest toxicity among the essential oils, with an IC50 of 171.90 µg/mL (Figure 5F). The cell viability tended to increase as the essential oil aged. Another study identified an IC50 of 120 µg/mL for J774 macrophages exposed to essential oil of the same species [11].
Pereira et al. [27] also reported on the cytotoxicity of V. curassavica EO, which is rich in α-pinene, in mammalian cells (L929 fibroblasts) at concentrations below 200 µg/mL (LC50 of approximately 140 µg/mL). An analysis of the cytotoxic effects of crude and supercritical extracts of V. curassavica against tumor cells resulted in IC50 values of 133.96 and 198.40 µg/mL, respectively [13].
The second most abundant compound, trans-caryophyllene, was present regardless of the plant age, and at 100, 200 and 400 μM the survival of L-929 (murine fibrosarcoma) cells significantly decreased by approximately 54, 100 and 100%, respectively [50]. In addition, the high cytotoxicity of the other essential oils could be due to the capacity of this compound to enhance the penetration of other bioactive substances, increasing the permeability of the plasma membrane, thereby increasing the overall cytotoxicity of the oil [51]. These observations suggest that the content of trans-caryophyllene could have a significant influence on the cytotoxicity of the essential oils extracted from young plants.
The V. curassavica EO exerted little antimicrobial activity, regardless of the plant age, in any of the strains of bacteria and yeast of Candida albicans that were tested (Table 3). Studies have previously described the antimicrobial activity against S. epidermidis (500 µg/mL), S. aureus (MIC ranging from 64 to 250 µg/mL), E. coli (MIC ranging from 64 to 2000 µg/mL) and P. aeruginosa (≤1024 µg/mL) and the fungistatic activity against Candida albicans (MIC = 512 µg/mL) [52,53]. Antimicrobial activity is associated with the presence of specific compounds, which are usually tested at a much higher concentration in their pure form than that in the essential oil and with the strain susceptibility used in the different studies [54]. Thus, the discrepancies described above might be attributed to variations in the levels of essential oil components.
4. Conclusions
Understanding the factors that can affect the essential oil contents of species that have commercial value is very important. We observed that the chemical compositions of V. curassavica EOs produced on an industrial scale varied with plant age. Additionally, the cytotoxicity decreased as the plants aged. Our results indicate that the plant age significantly affects the chemical composition of the essential oils, with significant changes in their cell viability. Further studies should aim to unveil whether age also affects the therapeutic effects (e.g., anti-inflammatory) of plant essential oils. This will allow the manufacture of plant-derived formulations that balance production costs, toxicity and therapeutic effects.
J.P.F., A.L.A.N.B., A.R.d.A.-N., E.P.C.S.-J. and M.M.d.M.A. contributed to the acquisition, analysis, interpretation of the data and drafting of the paper. F.A.d.A.C., K.A.d.F.R., I.M.d.A., F.A.e.S.-F., D.C.M., D.F.L. and D.D.R.A. contributed to the acquisition, analysis and interpretation of the data. M.L. and A.D. contributed to the critical review. J.R.d.S.d.A.L., as an advisor, contributed to the research design and analysis and interpretation of the data. All authors have read and agreed to the published version of the manuscript.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee on the Use of Animals (CEUA/UFPI) of UNIVERSIDADEFEDERAL DO PIAUÍ (protocol code 457/18 and date of approval of 18 May 2018).
Not applicable.
The raw data and detailed results of the PCA analysis are available under request to corresponding authors.
J.P. Farias thanks CAPES for awarding the scholarship under the master’s degree course. The authors thank the logistical and financial support of Phytobios LTDA, Aché (Pharmaceutical Laboratories), and the Centroflora Group.
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. Images of V. curassavica. (A) at the place of cultivation. (B) The leaves, flowers and fruit of the plant. (C) The leaf details. (D) A leaf under microscopy showing the epidermis and the essential oil storage location (black arrows).
Enlarge this image.Figure 2. Representative diagram of essential oil extraction process of V. curassavica on an industrial scale.
Enlarge this image.Figure 3. The principal component analysis (PCA) of three independent essential oil samples extracted from V. curassavica at different ages (4, 10, 14 and 18 months-age) via steam distillation on an industrial scale. The relative concentrations of the 19 identified compounds (detailed in Table 1) were used to perform the analysis.
Enlarge this image.Figure 4. Concentrations of selected compounds in the essential oils extracted from V. curassavica at different ages (4, 10, 14 and 18 months-age) via steam distillation on an industrial scale: (A) α-pinene; (B) α-humulene; (C) trans-caryophyllene. Different letters (i.e., groups not sharing any letter) denote statistically significant differences (one-way ANOVA followed by Tukey’s multiple comparisons test). Data are presented as means ± SDs.
Enlarge this image.Figure 5. Effects of essential oils extracted from V. curassavica on cell viability as assessed using an MTT assay. Murine peritoneal macrophages were treated with 6.25–800 µg/mL essential oil from plants at 4 (A), 10 (B), 14 (C) and 18 (D) month-age. Cells were also treated with 6.25–800 µg/mL α-pinene (E) for comparison. The data in panels (A–D) were used to calculate the IC50 values plotted in panel (F). The asterisks denote statistically significant differences in comparison to the untreated control cells (one-way ANOVA followed by Dunnett’s multiple comparisons test).
Chemical composition of the essential oils from V. curassavica at different ages extracted by steam distillation on an industrial scale. Data were obtained from three independent GC-MS chromatograms and are presented as (%) percentages of area of each substance relative to the total chromatogram.
| Compound | RT (min) | KIlit | KIc | Sample A (%) | Sample B (%) | Sample C (%) | Sample D (%) |
|---|---|---|---|---|---|---|---|
| α-tujene | 10.74 | 930 | 941.98 | - | - | 1.35 | 1.56 |
| Sabinene | 10.78 | 975 | 943.24 | 1.20 | 1.49 | 1.05 | 0.95 |
| α-pinene | 11.17 | 961 | 955.50 | 33.08 | 34.60 | 33.45 | 37.55 |
| Myrcene | 12.08 | 990 | 984.12 | 1.1 | 1.22 | - | - |
| β-pinene | 12.33 | 979 | 991.98 | 1.34 | 1.30 | 1.13 | 1.42 |
| D-limonene | 13.60 | 1040 | 1031.93 | - | - | 0.45 | - |
| 1, 8-Cineole | 13.72 | 1031 | 1035.70 | 1.79 | 1.74 | 1.56 | 1.65 |
| Bornyl acetate | 21.25 | 1288 | 1272.52 | 0.78 | 0.56 | - | - |
| δ-elemene | 23.25 | 1338 | 1335.42 | 3.35 | 4.13 | 3.66 | 4.03 |
| Copaene | 24.55 | 1376 | 1376.30 | 0.73 | 0.56 | - | 0.58 |
| β-elemene | 24.84 | 1390 | 1385.42 | 4.15 | 5.66 | 5.26 | 4.62 |
| Trans-α-bergamotene | 25.24 | 1434 | 1398.00 | 1.09 | 1.10 | 1.03 | 0.96 |
| α-santalene | 25.65 | 1417 | 1410.90 | 8.29 | 8.67 | 7.74 | 7.34 |
| Trans-caryophyllene | 25.95 | 1419 | 1420.33 | 13.05 | 16.25 | 18.25 | 12.52 |
| β- cedrene | 26.10 | 1420 | 1425.05 | 1.74 | - | 1.54 | 1.42 |
| β-funebrene | 26.22 | 1429 | 1428.82 | - | 1.75 | - | - |
| β-santalene | 26.58 | 1447 | 1440.15 | 0.77 | - | - | - |
| α-humulene | 26.98 | 1454 | 1452.73 | 2.69 | 3.32 | 4.17 | 2.62 |
| Alloaromadendreno | 27.13 | 1452 | 1457.44 | 9.51 | 6.85 | 7.04 | 7.37 |
| 9-Epi-E-cariofilene | 27.28 | 1466 | 1462.16 | 2.91 | 2.14 | 2.27 | 2.16 |
| β-bisabolene | 27.59 | 1528 | 1471.91 | - | 0.59 | 0.56 | 0.53 |
| β-sesquifilandrene | 28.05 | 1522 | 1486.38 | 0.81 | - | 0.69 | 0.66 |
| δ-cadinene | 28.24 | 1523 | 1492.35 | - | - | - | 1.99 |
| Amorpha-4, 7 (11)-diene | 28.33 | 1481 | 1495.18 | 2.38 | 1.98 | 1.99 | - |
| Bergamotol | 32.59 | 1690 | 1629.16 | - | - | 0.86 | - |
| α-santalona | 32.78 | 1577 | 1635.14 | 0.96 | 0.96 | - | 1.23 |
| Santalol (Z)-α | 33.17 | 1675 | 1647.40 | 1.20 | 1.24 | 1.06 | 1.46 |
| Total | 93 | 96.37 | 95.19 | 92.75 | |||
| Unknown | 7 | 3.63 | 4.81 | 7.25 |
Legend: Sample A = 4 month-age; sample B = 10 month-age; sample C = 14 month-age; sample D = 18 month-age.
Vegetal raw material, resulting mass and yield of essential oils produced from V. curassavica at different ages (area of 3.34 ha).
| Plant Age (Month-Age) | Vegetal Raw Material (kg) | Essential Oil (kg) | Yield (%) |
|---|---|---|---|
| 4 | 1252.00 | 1.160 | 0.093% |
| 10 | 1300.00 | 1.250 | 0.096% |
| 14 | 1200.00 | 0.996 | 0.083% |
| 18 | 889.43 | 1.000 | 0.112% |
Minimum inhibitory concentration (MIC, µg/mL) values of essential oil samples produced from V. curassavica at different ages against bacteria and yeast.
| Microorganism | ATCC® ID | Plant Age (Month-Age) | |||
|---|---|---|---|---|---|
| 4 | 10 | 14 | 18 | ||
| Bacteria | |||||
| Escherichia coli | 25922™ | >1000 | >1000 | >1000 | >1000 |
| Pseudomonas aeruginosa | 27853™ | >1000 | >1000 | >1000 | >1000 |
| Staphylococcus aureus | 29213™ | >1000 | >1000 | >1000 | >1000 |
| Staphylococcus aureus (MRSA) | 43300™ | >1000 | >1000 | >1000 | >1000 |
| Staphylococcus epidermidis | 12228™ | >1000 | >1000 | >1000 | >1000 |
| Yeast | |||||
| Candida albicans | 1000 | 1000 | 1000 | 1000 | |
References
1. Matias, E.F.F.; Alves, E.F.; Silva, M.K.d.N.; Carvalho, V.R.d.A.; Coutinho, H.D.M.; Costa, J.G.M.d. The genus Cordia: Botanists, ethno, chemical and pharmacological aspects. Rev. Bras. Farm.; 2015; 25, pp. 542-552. [DOI: https://dx.doi.org/10.1016/j.bjp.2015.05.012]
2. Sciarrone, D.; Giuffrida, D.; Rotondo, A.; Micalizzi, G.; Zoccali, M.; Panto, S.; Donato, P.; Rodrigues-Das-Dores, R.G.; Mondello, L. Quali-quantitative characterization of the volatile constituents in Cordia verbe-nacea D.C. essential oil exploiting advanced chromatographic approaches and nuclear magnetic resonance analysis. J. Chromatogr. A; 2017; 1524, pp. 246-253. [DOI: https://dx.doi.org/10.1016/j.chroma.2017.10.007] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29030035]
3. Marques, A.P.; Bonfim, F.P.; Dantas, W.F.; Puppi, R.J.; Marques, M.O. Chemical composition of essential oil from Varronia curassavica Jacq. accessions in different seasons of the year. Ind. Crops Prod.; 2019; 15, 111656. [DOI: https://dx.doi.org/10.1016/j.indcrop.2019.111656]
4. Hoeltgebaum, M.P.; Montagna, T.; Lando, A.P.; Puttkammer, C.; Orth, A.I.; Guerra, M.P.; Reis, M.S. Reproductive Biology of Varronia curassavica Jacq.(Boraginaceae). An. Acad. Bras. Cienc.; 2018; 1, pp. 59-71. [DOI: https://dx.doi.org/10.1590/0001-3765201820160273] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/29424392]
5. Feijó, E.V.; Oliveira, R.A.; Costa, L.C. Light affects Varronia curassavica es-sential oil yield by increasing trichomes frequency. Rev. Bras. Farmacogn.; 2014; 24, pp. 516-523. [DOI: https://dx.doi.org/10.1016/j.bjp.2014.10.005]
6. Fernandes, E.S.; Passos, G.F.; Medeiros, R.; da Cunha, F.M.; Ferreira, J.; Campos, M.M.; Pianowski, L.F.; Calixto, J.B. Anti-inflammatory effects of compounds alpha-humulene and (-)-trans-caryophyllene isolated from the essential oil of Cordia verbenacea. Eur. J. Pharm.; 2007; 569, pp. 228-236. [DOI: https://dx.doi.org/10.1016/j.ejphar.2007.04.059] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17559833]
7. Rogerio, A.P.; Andrade, E.L.; Leite, D.F.; Figueiredo, C.P.; Calixto, J.B. Pre-ventive and therapeutic anti-inflammatory properties of the sesquiterpene al-pha-humulene in experimental airways allergic inflammation. Br. J. Pharm.; 2009; 158, pp. 1074-1087. [DOI: https://dx.doi.org/10.1111/j.1476-5381.2009.00177.x]
8. Sertie, J.A.; Woisky, R.G.; Wiezel, G.; Rodrigues, M. Pharmacological assay of Cordia verbenacea V: Oral and topical anti-inflammatory activity, analgesic effect and fetus toxicity of a crude leaf extract. Phytomedicine; 2005; 12, pp. 338-344. [DOI: https://dx.doi.org/10.1016/j.phymed.2003.09.013] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/15957367]
9. Perini, J.A.; Angeli-Gamba, T.; Alessandra-Perini, J.; Ferreira, L.C.; Nasciutti, L.E.; Machado, D.E. Topical application of Acheflan on rat skin injury accelerates wound healing: A histopathological, immunohistochemical and biochemical study. BMC Complement. Med.; 2015; 15, 203. [DOI: https://dx.doi.org/10.1186/s12906-015-0745-x]
10. de Carvalho, P.M.; Rodrigues, R.F.O.; Sawaya, A.C.H.F.; Marques, M.O.M.; Shimizu, M.T. Chemical composition and antimicrobial activity of the essential oil of Cordia verbenacea D.C. J. Ethnopharmacol.; 2004; 95, pp. 297-301. [DOI: https://dx.doi.org/10.1016/j.jep.2004.07.028]
11. Braga, F.G.; Bouzada, M.L.; Fabri, R.L.; de, O.M.M.; Moreira, F.O.; Scio, E.; Coimbra, E.S. Antileishmanial and antifungal activity of plants used in traditional medicine in Brazil. J. Ethnopharmacol.; 2007; 111, pp. 396-402. [DOI: https://dx.doi.org/10.1016/j.jep.2006.12.006] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/17234373]
12. Nizio, D.A.d.C.; Brito, F.d.A.; Sampaio, T.S.; Melo, J.d.O.; Silva, F.L.S.d.; Gagliardi, P.R.; Arrigoni-Blank, M.d.F.; dos Anjos, C.S.; Alves, P.B.; Wisniewski Junior, A. et al. Chemical diversity of native populations of Varronia curassavica Jacq. and antifungal activity against Lasiodoplodia theobromae. Ind. Crops Prod.; 2015; 76, pp. 437-448. [DOI: https://dx.doi.org/10.1016/j.indcrop.2015.07.026]
13. Parisotto, E.B.; Michielin, E.M.Z.; Biscaro, F.; Ferreira, S.R.S.; Filho, D.W.; Pedrosa, R.C. The antitumor activity of extracts from Cordia verbenacea D.C. obtained by supercritical fluid extraction. J. Supercrit. Fluids; 2012; 61, pp. 101-107. [DOI: https://dx.doi.org/10.1016/j.supflu.2011.08.016]
14. Michielin, E.M.Z.; de Lemos Wiese, L.P.; Ferreira, E.A.; Pedrosa, R.C.; Ferreira, S.R.S. Radical-scavenging activity of extracts from Cordia verbenacea DC ob-tained by different methods. J. Supercrit. Fluids; 2011; 56, pp. 89-96. [DOI: https://dx.doi.org/10.1016/j.supflu.2010.11.006]
15. Roldao, E.d.F.; Witaicenis, A.; Seito, L.N.; Hiruma-Lima, C.A.; Di Stasi, L.C. Evaluation of the antiulcerogenic and analgesic activities of Cordia verbenacea DC. (Boraginaceae). J. Ethnopharmacol.; 2008; 119, pp. 94-98. [DOI: https://dx.doi.org/10.1016/j.jep.2008.06.001] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/18588967]
16. Oliveira, A.R.M.F.d.; Jezler, C.N.; Oliveira, R.A.; Costa, L.C.d.B. Influência da idade da planta na produção de óleo essencial de alevante. Rev. Ceres; 2012; 59, pp. 241-245. [DOI: https://dx.doi.org/10.1590/S0034-737X2012000200013]
17. Oliveira, B.M.; Melo, C.R.; Santos, A.C.; Nascimento, L.F.; Nízio, D.A.; Cristaldo, P.F.; Blank, A.F.; Bacci, L. Essential oils from Varronia curassavica (Cordiaceae) accessions and their compounds (E)-caryophyllene and α-humulene as an alternative to control Dorymyrmex thoracius (Formicidae: Dolichoderinae). Environ. Sci. Pollut. Res.; 2019; 26, pp. 6602-6612. [DOI: https://dx.doi.org/10.1007/s11356-018-4044-1] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30623333]
18. Calixto, J.B. Twenty-five years of research on medicinal plants in Latin America: A personal view. J. Ethnopharmacol; 2005; 100, pp. 131-134. [DOI: https://dx.doi.org/10.1016/j.jep.2005.06.004]
19. Sharifi-Rad, J.; Sureda, A.; Tenore, G.C.; Daglia, M.; Sharifi-Rad, M.; Valussi, M.; Tundis, R.; Sharifi-Rad, M.; Loizzo, M.R.; Ademiluyi, A.O. et al. Biological activities of essential oils: From plant chemoecology to traditional healing systems. Molecules; 2017; 22, 70. [DOI: https://dx.doi.org/10.3390/molecules22010070]
20. Andrade, F.P.; Venzon, M.; das Dôres, R.G.; Franzin, M.L.; Martins, E.F.; de Araújo, G.J.; Fonseca, M.C. Toxicity of Varronia curassavica Jacq. Essential Oil to Two Arthropod Pests and Their Natural Enemy. Neotrop. Entomol.; 2021; 50, pp. 835-845. [DOI: https://dx.doi.org/10.1007/s13744-021-00906-x]
21. Tonial, C.H.; Rodrigues, M.F.; Bosse, M.A.; Sousa, I.M.; de Lima, J.D.; da Cunha, M.A.; Foglio, M.A.; Marques, M.O.; Marchese, J.A. Technical and economic evaluation of cultivation and obtaining of Varronia curassavica Jacq. essential oil. Ind. Crops Prod.; 2020; 15, 112650. [DOI: https://dx.doi.org/10.1016/j.indcrop.2020.112650]
22. Nascimento, R.V.; Alves, M.D.; Pinto, T.D.; de Souza Menezes, R.; Damasceno Junior, P.C.; Chaves, D.S.; de Souza, M.A. Hydrodistillation extraction kinetics of volatile oils from Varronia curassavica and Laurus nobilis. Rev. Bras. Farmacogn.; 2020; 30, pp. 503-509. [DOI: https://dx.doi.org/10.1007/s43450-020-00067-9]
23. Barra, A. Factors affecting chemical variability of essential oils: A review of recent developments. Nat. Prod. Commun.; 2009; 4, pp. 1147-1154. [DOI: https://dx.doi.org/10.1177/1934578X0900400827] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/19769002]
24. Pagare, S.; Bhatia, M.; Tripathi, N.; Bansal, Y.K. Secondary metabolites of plants and their role: Overview. Curr. Trends Biotechnol. Pharm.; 2015; 9, pp. 293-304.
25. Lima, F.J.A.; de Sousa, R.O.; Artur, F.; de Araújo Mendes, M.R.; do Val, A.D.B. Caracterização do crescimento e produção de óleo essencial da erva baleeira (Varronia curassavica Jaqc). Res. Soc. Dev.; 2021; 10, e5810716204. [DOI: https://dx.doi.org/10.33448/rsd-v10i7.16204]
26. Oliveira, V.D.; Sajo, M.D. Anatomia foliar de espécies epífitas de Orchidaceae. Rev. Bras. Bot.; 1999; 22, pp. 365-374. [DOI: https://dx.doi.org/10.1590/S0100-84041999000300003]
27. Pereira, P.S.; Oliveira, C.V.; Maia, A.J.; Tintino, S.R.; Oliveira-Tintino, C.D.; Vega-Gomez, M.C.; Rolón, M.; Coronel, C.; Duarte, A.E.; Barros, L.M. et al. Cytotoxicity of Essential Oil Cordia verbenaceae against Leishmania brasiliensis and Trypanosoma cruzi. Molecules; 2021; 26, 4485. [DOI: https://dx.doi.org/10.3390/molecules26154485]
28. Adams, R. Identification of Essential oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy; 4th ed. Allured Pub Corp: Carol Stream, IL, USA, 2007; Volume 456, pp. 544-545.
29. Arcanjo, D.D.R.; Mafud, A.C.; Vasconcelos, A.G.; Silva-Filho, J.C.; Amaral, M.P.M.; Brito, L.M.; Bemquerer, M.P.; Kückelhaus, S.A.S.; Plácido, A.; Delerue-Matos, C. et al. In Silico, In Vitro and In Vivo Toxicological Assessment of BPP-BrachyNH2, A Vasoactive Proline-Rich Oligopeptide from Brachycephalus ephippium. Int. J. Pept. Res.; 2017; 23, pp. 323-331. [DOI: https://dx.doi.org/10.1007/s10989-016-9564-2]
30. CLSI. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition. CLSI Document M07-A9; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2012.
31. CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Third Edition. CLSI Document M27-A3; Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2008.
32. Silva, K.P.; de Carvalho Santos, T.A.; Moutinho, B.L.; da Silva, R.S.; dos Santos Pinto, V.; Blank, A.F.; Corrêa, C.B.; Scher, R.; Fernandes, R.P. Using Varronia curassavica (Cordiaceae) essential oil for the biocontrol of Phytomonas serpens. Ind. Crops Prod.; 2019; 139, 111523. [DOI: https://dx.doi.org/10.1016/j.indcrop.2019.111523]
33. Queiroz, T.B.; Fonseca, F.S.; Mendes, A.D.; Azevedo, A.M.; Martins, E.R. Chemical diversity of accessions of the in vivo germplasm bank of Varronia curassavica (Jacq.). Acta Scientiarum. Agronomy; 2019; 42, [DOI: https://dx.doi.org/10.4025/actasciagron.v42i1.42726]
34. Fernandes, T.O.; Araújo, E.O.; Santos, A.P.; Silva, J.C.; Fonseca, F.S.; Azevedo, A.M.; Martins, E.R. Variability of individuals from a population Varronia curassavica Jacq. considering volatile compounds. Ciência Rural; 2022; 52, [DOI: https://dx.doi.org/10.1590/0103-8478cr20210040]
35. Zotti-Sperotto, N.C.; Melo, E.d.C.; de Souza, M.I.L.; Fonseca, M.C.M.; Gon-zaga, D.A.; de Ávila, M.B.R.; Demuner, A.J.; Ventrella, M.C.; Lelis, A.C.V. Effect of drying with ultrasonic pretreatment on the yield and quality of the essential oil of Varronia curassavica Jacq. and Ocimum gratis-simum Linn. Ind. Crops Prod.; 2020; 147, 112211. [DOI: https://dx.doi.org/10.1016/j.indcrop.2020.112211]
36. Durazzo, A.; D’Addezio, L.; Camilli, E.; Piccinelli, R.; Turrini, A.; Marletta, L.; Marconi, S.; Lucarini, M.; Lisciani, S.; Gabrielli, P. et al. From Plant Compounds to Botanicals and Back: A Current Snapshot. Molecules; 2018; 23, 1844. [DOI: https://dx.doi.org/10.3390/molecules23081844] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30042375]
37. Durazzo, A.; Lucarini, M. Editorial: The State of Science and Innovation of Bioactive Research and Applications, Health, and Diseases. Front Nutr.; 2019; 6, 178. [DOI: https://dx.doi.org/10.3389/fnut.2019.00178] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31824957]
38. Ramadan, M.F.; Durazzo, A.; Lucarini, M. Advances in Research on Food Bio-active Molecules and Health. Molecules; 2021; 26, 7678. [DOI: https://dx.doi.org/10.3390/molecules26247678]
39. Gomes, M.V.d.S.; Silva, J.D.d.; Ribeiro, A.F.; Cabral, L.M.; de Sousa, V.P. Development and validation of a quantification method for α-humulene and trans-caryophyllene in Cordia verbenacea by high performance liquid chromatog-raphy. Rev. Bras. Farm.; 2019; 29, pp. 182-190. [DOI: https://dx.doi.org/10.1016/j.bjp.2019.01.009]
40. Al-Lihaibi, S.S.; Alarif, W.M.; Abdel-Lateff, A.; Ayyad, S.E.N.; Abdel-Naim, A.B.; El-Senduny, F.F.; Badria, F.A. Three new cembranoid-type diterpenes from Red Sea soft coral Sarcophyton glaucum: Isolation and antiproliferative activity against HepG2 cells. Eur. J. Med. Chem.; 2014; 81, pp. 314-322. [DOI: https://dx.doi.org/10.1016/j.ejmech.2014.05.016]
41. Lomba, L.; Afarinkia, K.; Vinader, V. A new route to tricyclane sesquiterpenoids: Total synthesis of alpha-ekasantalic acid. Org. Biomol. Chem.; 2019; 17, pp. 4456-4459. [DOI: https://dx.doi.org/10.1039/C9OB00630C]
42. Facanali, R.; Marques, M.O.; Hantao, L.W. Metabolic profiling of Varronia curassavica Jacq. terpenoids by flow modulated two-dimensional gas chromatography coupled to mass spectrometry. Separations; 2020; 7, 18. [DOI: https://dx.doi.org/10.3390/separations7010018]
43. Ozkan, G.; Baydar, H.; Erbas, S. The influence of harvest time on essential oil composition, phenolic constituents and antioxidant properties of Turkish oregano (Origanum onites L.). J. Sci. Food Agric.; 2010; 90, pp. 205-209. [DOI: https://dx.doi.org/10.1002/jsfa.3788]
44. Pandu Sastry, K.; Srinivas, K.V.N.; Kotesh Kumar, J.; Arigari, N.; Kumar, A. Composition of herb and seed oil and antimicrobial activity of the essential oil of two varieties of Ocimum basilicum harvested at short time intervals. J. Plant Dev.; 2015; 22, pp. 59-76.
45. Santos, C.P.D.; Oliveira, T.C.d.; Pinto, J.A.O.; Fontes, S.S.; Cruz, E.M.O.; de Fátima Arrigoni-Blank, M.; Andrade, T.M.; de Matos, I.L.; Alves, P.B.; Innecco, R. et al. Chemical diversity and influence of plant age on the essential oil from Lippia sidoides Cham. germplasm. Ind. Crops Prod.; 2015; 76, pp. 416-421. [DOI: https://dx.doi.org/10.1016/j.indcrop.2015.07.017]
46. Franz, C.; Voemel, A.; Hoelzl, J. Variation in the essential oil of Matricaria chamomilla L. depending on plant age and stage of development. InI Int. Symp. Spices Med. Plants; 1977; 73, pp. 229-238. [DOI: https://dx.doi.org/10.17660/ActaHortic.1978.73.28]
47. Nurzyńska-Wierdak, R.; Bogucka-Kocka, A.; Szymczak, G. Volatile constituents of Melissa officinalis leaves determined by plant age. Nat. Prod. Commun.; 2014; 9, pp. 703-706. [DOI: https://dx.doi.org/10.1177/1934578X1400900531] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/25026727]
48. Damtew, Z.; Tesfaye, B.; Bisrat, D. Leaf, essential oil and artemisinin yield of artemisia (Artemisia annua L.) as influenced by harvesting age and plant population density. World J. Agric. Sci.; 2011; 7, pp. 404-412.
49. Rocha, R.P.; de Castro Melo, E.; Barbosa, L.C.; dos Santos, R.H.; Cecon, P.R.; Dallacort, R.; Santi, A. Influence of plant age on the content and composition of essential oil of Cymbopogon citratus (DC.) Stapf. J. Med. Plants Res.; 2014; 8, pp. 1121-1126.
50. Hajlaoui, H.; Mighri, H.; Aouni, M.; Gharsallah, N.; Kadri, A. Chemical composition and in vitro evaluation of antioxidant, antimicrobial, cytotoxicity and an-ti-acetylcholinesterase properties of Tunisian Origanum majorana L. essential oil. Microb. Pathog.; 2016; 95, pp. 86-94. [DOI: https://dx.doi.org/10.1016/j.micpath.2016.03.003]
51. Legault, J.; Pichette, A. Potentiating effect of beta-caryophyllene on anti-cancer activity of alpha-humulene, isocaryophyllene and paclitaxel. J. Pharm. Pharmacol.; 2007; 59, pp. 1643-1647. [DOI: https://dx.doi.org/10.1211/jpp.59.12.0005]
52. Hernandez, T.; Canales, M.; Teran, B.; Avila, O.; Duran, A.; Garcia, A.M.; Hernandez, H.; Angeles-Lopez, O.; Fernandez-Araiza, M.; Avila, G. Antimicrobial activity of the essential oil and extracts of Cordia curassavica (Bo-raginaceae). J. Ethnopharmacol.; 2007; 111, pp. 137-141. [DOI: https://dx.doi.org/10.1016/j.jep.2006.11.002]
53. Rodrigues, F.F.; Oliveira, L.G.; Rodrigues, F.F.; Saraiva, M.E.; Almeida, S.C.; Cabral, M.E.; Campos, A.R.; Costa, J.G.M. Chemical composition, antibacterial and antifungal activities of es-sential oil from Cordia verbenacea DC leaves. Pharmacogn. Res.; 2012; 4, pp. 161-165.
54. Swamy, M.K.; Akhtar, M.S.; Sinniah, U.R. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evid.-Based Complement. Altern. Med.; 2016; 2016, 3012462. [DOI: https://dx.doi.org/10.1155/2016/3012462] [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/28090211]
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; Alyne Rodrigues de Araújo-Nobre 1 ; Enoque Pereira Costa Sobrinho-Júnior 3 ; Michel Muálem de Moraes Alves 3 ; Fernando Aécio de Amorim Carvalho 3 ; Klinger Antonio da Franca Rodrigues 1
; Ivanilza Moreira de Andrade 1 ; Silva-Filho, Francisco Artur e 4
; Daniel Carneiro Moreira 5
; David Fernandes Lima 6 ; Lucarini, Massimo 7 ; Durazzo, Alessandra 7
; Daniel Dias Rufino Arcanjo 8
; José Roberto de Souza de Almeida Leite 9
1 Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Biotec, Campus Ministro Reis Velloso, Universidade Federal do Delta do Parnaíba, Parnaíba 64202-020, Brazil
2 Núcleo de Pesquisa em Morfologia e Imunologia Aplicada, Faculdade de Medicina, Campus DarcyRibeiro, Universidade de Brasília, Brasília 70910-900, Brazil; Programa de Pós-graduação em Medicina Tropical, Núcleo de Medicina Tropical, Faculdade de Medicina, Campus Darcy Ribeiro, Universidade de Brasília, Brasília 70910-900, Brazil
3 Laboratório de Atividade Antileishmania, Núcleo de Pesquisas em Plantas Medicinais, Campus Ministro Petrônio Portella, Universidade Federal do Piauí, Ininga, Teresina 64049-550, Brazil
4 Programa de Pós-graduação em Química, Universidade Estadual do Piauí, Parnaíba 64002-150, Brazil
5 Núcleo de Pesquisa em Morfologia e Imunologia Aplicada, Faculdade de Medicina, Campus DarcyRibeiro, Universidade de Brasília, Brasília 70910-900, Brazil
6 Faculdade de Medicina, Universidade Federal do Vale do São Francisco, Paulo Afonso 48605-560, Brazil
7 CREA-Research Centre for Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy
8 Laboratório de Atividade Antileishmania, Núcleo de Pesquisas em Plantas Medicinais, Campus Ministro Petrônio Portella, Universidade Federal do Piauí, Ininga, Teresina 64049-550, Brazil; Laboratório de Estudos Funcionais e Moleculares em Fisiofarmacologia, LAFMOL, Departamento de Biofísica e Fisiologia, Universidade Federal do Piauí, Teresina 64049-550, Brazil
9 Núcleo de Pesquisa em Biodiversidade e Biotecnologia, Biotec, Campus Ministro Reis Velloso, Universidade Federal do Delta do Parnaíba, Parnaíba 64202-020, Brazil; Núcleo de Pesquisa em Morfologia e Imunologia Aplicada, Faculdade de Medicina, Campus DarcyRibeiro, Universidade de Brasília, Brasília 70910-900, Brazil; Programa de Pós-graduação em Medicina Tropical, Núcleo de Medicina Tropical, Faculdade de Medicina, Campus Darcy Ribeiro, Universidade de Brasília, Brasília 70910-900, Brazil