Chemical Composition and Biological Activities of Essential Oils from Fresh Vismia guianensis (Aubl.) Choisy and Vismia cayennensis (Jacq.) Pers. Leaves

The Vismia Vand. genus encompasses many species indigenous to the Amazon rain forest where they are popularly known as “Lacre” bark and leaves are widely employed by locals to treat dermatophytoses. The aim of this study was to investigate the chemical composition of essential oils (EOs) extracted from the aerial parts of the species Vismia guianensis (Aubl.) Choisy and Vismia cayennensis (Jacq.) Pers. and to assess their antimicrobial activity against the bacteria Staphylococcus aureus Rosenbach 1884 and Escherichia coli (Migula 1895) Castellani and Chalmers 1919 as well as the fungi Candida albicans (C.P. Robin) Berkhout 1923 and Candida parapsilosis (Ashford) Langeron & Talice 1932. The analysis of the chemical composition of the essential oil extracted from V. guianensis leaves (EOVg) indicated 46 components, of which three sesquiterpenes predominated, namely: (E)-caryophyllene (10.40%), αcopaene (29.45%), and (E)-nerolidol (24.06%). As to the essential oil from V. cayennensis leaves (EOVc), 61 components were identified, of which two oxygenated sesquiterpenes stood out as the main components, namely, germacrone (25.42%) and curzerene (25.29%). EOVg exhibited Minimum Inhibitory Concentration (MIC) of 1.56 μg/mL against the yeast C. parapsilosis whereas EOVc was active against the bacteria E. coli and S. aureus as well as the yeast C. parapsilosis. The results obtained in this study strongly recommend further research on the essential oils Research, Society and Development, v. 10, n. 8, e37410817440, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17440 2 in question with a view to isolating and identifying the components responsible for their observed antimicrobial activities.

Additionally, Rojas et al. (2011) described the fungicide potential of Vismia essential oils, which supports the scientific community's interest in identifying the chemical components and assessing the pharmacological actions of this genus. In this vein, this study aimed at investigating the chemical profile and antimicrobial potential of essential oils extracted from V. guianensis and V. cayennensis (Jacq.) Pers. leaves against the microorganisms E. coli, S. aureus, Candida albicans (C.P. Robin) Berkhout 1923 and Candida parapsilosis (Ashford) Langeron e Talice 1932.

Extraction of essential oils from Vismia guianensis and V. cayennensis leaves
The essential oils from V. guianensis and V. cayennensis leaves were obtained by hydrodistillation of fresh material in a modified Clevenger-type apparatus coupled to a round bottom flask with distilled water (5 L) for approximately 4 hours.
Next, the EO samples were centrifuged (3500 rpm) for 10 minutes for water/oil separation, which was done with a graduated micropipette. Extractions were performed in triplicate, with 800 g of fresh leaves of both Vismia species in each flask. Then, the EO samples were stored in Eppendorf tubes, sealed, and kept at -4 °C until analysis and testing. The samples of V.
guianensis and V. cayennensis EOs were coded as EOVg and EOVc, respectively.

Gas chromatography coupled to mass spectrometry (GC/MS)
The obtained essential oils underwent analysis in a SHIMADZU gas chromatographer coupled to a SHIMADZU QP2010 mass spectrometer (GC-MS). For component chromatography, a 30 m × 0.25 mm DB-5MS column with 0.25 μm inner film thickness was employed. The chemical components were identified by interpreting their respective mass spectra, calculating their Kovats Indexes (KIs), and matching them up to data found in the literature. The calculated KIs were then compared to those tabulated for isolated compounds by Adams (2007).

Antimicrobial assay
The antimicrobial activity test was carried out at the Microbiology Laboratory of the Exact Sciences and Technology Institute (ICET/UFAM), using the microplate dilution test to analyze the antibacterial and antifungal potential of OEVg and OEVc.

Microorganisms
The EO samples underwent antimicrobial susceptibility tests in vitro according to the protocol described in the literature (Vaz et al., 2009), using a panel with ATCC strains (American Type Culture Collection, USA). The antibacterial activity of the essential oils was assessed against the Gram-positive bacterium Staphylococcus aureus (ATCC 25923), Gramnegative bacterium Escherichia coli (ATCC 25922), and fungi Candida albicans (ATCC 10231) and Candida parapsilosis (ATCC 22019).

Preparation and standardization of microbial inoculums
The bacterial and fungal strains were grown in Mueller Hinton broth for 24 h at 37 °C and standardized by adding sterile PBS (pH 7.2) until obtaining turbidity equal to that of the suspension in the 0.5 tube on the McFarland scale (approximately 1.0 × 10 8 CFU/mL). Then, a spectrophotometric reading was performed at 620 nm to confirm the microorganism concentration. Subsequently, small amounts of bacterial and fungal strains were removed, with the aid of a sterile loop, and added to 5 mL of sterile LB broth and 5 mL of YPD broth for the bacteria and fungi in question, respectively.
The microorganism concentration was confirmed by spectrophotometer reading.

Preparation of EO samples
Firstly, a 10% dimethyl-sulfoxide (DMSO) solution was prepared by diluting DMSO (100 µL) in sterile distilled water (900 µL). Then the EOVg and EOVc samples were diluted in 10% DMSO by solubilizing the samples (10 mg) in the previously prepared solvent (100 µL). Through this procedure, stock solutions of each sample were prepared to a concentration of 100 µg/mL. The assays were performed on five 96-well ELISA microplates, which were divided as follows: 1. The wells used validating the method and measurement of results were identified as "positive and negative controls": the positive control comprised the culture medium, bacterial or fungal suspension and the reference antimicrobial standard whereas the negative control consisted of the culture medium and 10% DMSO; 2. Wells identified as "blank" contained the culture medium and the essential oil of each sample in order to eliminate the turbidity caused by its color when evaluating the results; and 3. Wells identified as "assay" contained the culture medium, a mixture of essential oil with DMSO, and the bacterial or fungal suspension.

Microdilution for bacteria
MIC was performed in triplicate at 1:2 concentration. The bacteria under investigation were E. coli and S. aureus.
Chloramphenicol and 10% DMSO were employed as reference standard (positive control) and negative control, respectively.
The 96 microplate wells were filled with the LB broth (100 µL). Then, in the first well, 100 µL of the EO stock solution was added prepared initially in concentration at 100 µg/mL. After a serial dilution was conducted in the seven consecutive wells, removing 100 µL from the highest concentration well, resulting in a solution of up to 0,39 µg/mL. The assay was performed in triplicate for each concentration. Likewise, the LB broth (50 µL) plus Chloramphenicol (50 µL) were added to the positive control whereas the LB broth (50 µL) plus 10% DMSO (50 µL) were added to the negative control, in triplicate. Finally, the microorganism suspensions (10 µL) were added to every well and incubated for 24 hours at 37 °C.

Microdilution for fungi
MIC was performed in triplicate for C. albicans and C. parapsilosis with methanol (positive control) and 10% DMSO (negative control) as reference standard . The microplate wells were filled with the YPD broth (100 µL). Then, in the first well, 100 µL of the EO stock solution was added prepared initially in concentration at 100 µg/mL. After a serial dilution was conducted in the seven consecutive wells, removing 100 µL from the highest concentration well, resulting in a solution of up to Research, Society and Development, v. 10, n. 8, e37410817440, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i8.17440 5 0,39 µg/mL. The YPD broth (50 µL) plus methanol (50 µL) were added to the positive control whereas the YPD broth (50 µL) plus 10% DMSO (50 µL) were added to the negative control, in triplicate. Then, the microorganism suspensions (10 µL) were added to the wells and incubated for 48 h at 37 °C. After incubation, a visual reading and a reading with Resazurin dye (100 µg/mL) of the microbial growth were performed. Table 1 shows the variation in yields of EOVg and EOVc. EOVg yield was higher in September/2019 (0.04%), a period of intense drought, and lower yield in November/2019 (0.03%), the rainy season in the region. EOVc exhibited a yield of 0.54% in the dry season, a satisfactory value as compared to that obtained for EOVg during the same period. The difference between the yield values may be attributed to several factors, e.g., temperature, rainfall, place and time of sample collection, which can have critical effects on both the quantity and quality of essential oils. The lower yields of the essential oils during the rainy season may be due to lixiviation, i.e., continuous rain may result in loss of hydro-soluble substances in leaves and roots. This may apply to plants that produce alkaloids, glycosides, and even volatile oils (Evans, 1996;Walterman & Mole 1994).

Microdilution assay for fungi and bacteria
The literature indicates remarkable antimicrobial activity in essential oils from Vismia spp. According to Pérez and colleagues (2011)   OEVg inhibited fungal growth of C. parapsilosis strains at 1.56 µg /mL concentration. As stated by Holetz et al. (2002), samples with MIC values below 100 µg/mL are classified as very active, i.e., they strongly inhibit microbial growth.
The main components of OEVg were (E)-caryophyllene sesquiterpenes (10.40%), α-copaene (29.45%), and (E)nerolidol (24.06%). According to previous research, sesquiterpenes function as antimicrobial agents (CITÓ et al., 2003). The mechanism is still unclear, but it has been speculated that the lipophilic compounds found in this essential oil bind to and rupture the membrane of some microorganisms (COWAN, 1999). Reinsvold et al. (2011) showed that (E)-caryophyllene acts against microorganisms and, thus, can be used as antibiotic. This compound can also function biochemically as an anesthetic, anti-inflammatory, and spasmolytic drug. Other authors have reported antimicrobial activity of (E)-caryophyllene in kidney cell culture (RC-37), supporting its use as a prospective antimicrobial agent (Astani, 2009;Reichling & Schnitzler 2009).
Another study by Gelinski et al. (2007) indicates that (E)-nerolidol acts as a limited-spectrum antibiotic as it is not effective against some bacteria such as Salmonella Lignieres 1900 sp., E. coli, and Proteus Hauser 1885 sp. This finding corroborates the results obtained in this study, i.e., the inactivity of OEVg against the bacteria E. coli and S. aureus as well as against the yeast C. albicans, probably due this this essential oil having this sesquiterpene as one of its main components.
OEVc exhibited strong inhibition against the bacteria E. coli and S. aureus at concentrations of 50 µg/mL and 25 µg/mL, respectively, and against the yeast C. parapsilosis at 50 µg/mL concentration. The main components identified in this essential oil are the oxygenated sesquiterpenes curzerene (25.29%) and germacrone (25.42%). Zhang and colleagues (2017) found curzerene to be one of the main components of the essential oil extracted from Curcuma phaeocaulis Valeton and attributed its observed antifungal activity (IC50, 153.33-580.09 μg/ml) and inhibition of bacterial growth (IC50, 485.00-778.33 μg /ml) to this sesquiterpene.
Another research conducted by Ogunwande et al. (2005) identified curzerene (19.7%) and germacrone (27.5%) as the main components of essential oils extracted from fruit and leaves of Curcuma phaeocaulis, respectively. The same study reports strong antibacterial activity for the essential oil extracted from Eugenia uniflora L. fruit and leaves against S. aureus and Bacillus cereus, respectively, with MIC equal to 39 µg/mL. In addition to treating cancer and hepatitis, other studies indicate that germacrone can be employed as an antimicrobial agent (Wang et al., 2000).

Conclusion
The findings of this study are relevant in that they show the antimicrobial potential of EOVg and EOVc against some cayennensis, this study has contributed their chemical profiles, indicated their antimicrobial potential, and, thus, provided a basis for future research with a view to isolating and characterizing the compounds responsible for the biological activities of OEVg and EOVc.