Cantinoa althaeifolia essential oil: chemical composition and biological, antioxidant, antimicrobial, and antitumor activities

The species of the Lamiaceae family produce essential oils in the glandular trichomes, leaf surfaces, and in inflorescences, and can also produce many secondary compounds, which indicates great potential for study. The aim of this paper was to describe the chemical composition and the biological, antioxidant, antimicrobial, and antitumor activities of the essential oil of Cantinoa althaeifolia for the first time. The essential oil was obtained from the leaves by hydrodistillation and analyzed by gas chromatography mass spectrometry (GC/MS). The antioxidant activity was established by the phosphomolybdenum method. The minimum inhibitory concentration against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans was determined. The lethality test of Artemia salina was performed. The cytotoxicity against murine fibroblasts (3T3), non-tumor cells (McCoy), murine melanoma (B16F10), and human breast carcinoma (MCF-7) cells was assessed. Five major compounds were isolated: himachalene (11.62%), spathulenol (10.08%), caryophylline oxide (9.93%), (E)-caryophylene (9.21%), and pogostol (6.31%). The phosphomolybdenum complex method showed positive results. The minimum inhibitory concentration was greater than 1000 μg/mL for the tested microorganisms. The mortality rate of Artemia salina was low. The tested Research, Society and Development, v. 10, n. 2, e9910212040, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12040 2 cancer cell lines showed an IC50 of 12.5 and 25 μg/mL. These findings indicate that the essential oil of Cantinoa althaeifolia has antioxidant, antimicrobial, and antitumor activities.


Introduction
The Lamiaceae family comprises about 240 genera and 7200 species, with a cosmopolitan distribution (Raymond Harley & Pastore, 2012). These species grow mainly in open places, in almost all types of habitats and at all altitudes, from the Arctic to the Himalayas, from Southeast Asia to Hawaii, from north to south in the New World, and in Australia and Africa as well. The Mediterranean and Central Asia are the regions with the highest concentration of species (Heywood & Royal Botanic Gardens, 2007). In Brazil, there are 34 native genera and about 496 species (Forzza et al., 2010).
The Lamiaceae family stands out for its economic importance, as it can be used for ornaments, cooking, or even as wood (RM Harley et al., 2009). Some plants can be used for culinary purposes, such as Ocium Basilicum L. Recently, essential oils have been receiving great interest due to their bioactive compounds, especially because of their antimicrobial, medicinal, antioxidant, cytostatic, and insecticidal properties (Tongnuanchan & Benjakul, 2014). These oils are widely used as natural additives in foods and food products, for embalming and preserving food, and in the production of local anesthetics and antimicrobial, analgesic, sedative, anti-inflammatory, and spasmolytic remedies (Bakkali, Averbeck, Averbeck, & Idaomar, 2008).
The species of the Lamiaceae family produce essential oils in the glandular trichomes, leaf surfaces, and in inflorescences, and can also produce many secondary compounds (Raymond Harley et al., 2004), which indicates great potential for study. The aim of this study was to describe the chemical composition of the essential oil of Cantinoa althaeifolia, and to evaluate its antimicrobial, antioxidant, and antitumor activities.

Methodology
This research was organized following the Scientific Research Methodology recommendations (Pereira et al., 2018).

Plant material
The botanical material was collected at the district of Boqueirão (coordinates 25º32'43" S, 49º14'11" W), located in

Essential oil obtention
The essential oil was obtained from 600 g of dried leaves, ground in a knife mil, hydrodistilled for 6 h using modified Clevenger type apparatus. This process was repeated 3 times. The essential oil was stored in a sealed amber glass at -18°C until subsequent analysis. The yield calculation was performed in milliliters (mL/%) of essential oil per 100 g of the drug (ANVISA, 2010).

Identification of essential oil constituents
The characterization of chemical constituents of the essential oil of leaves of Cantinoa althaeifolia was carried out using gas chromatography, composed of gas chromatograph coupled to a Shimadzu® CGEM-QP 2010 Plus mass spectrometer (Shimadzu, Kyoto, Japan), with a Rtx-5MS capillary column (30m x 0.25 mm x 0.25 μm; Bellefonte, PA, USA).
The injector was set in splitless mode at 250°C, and the interface and source of ions were set at 300°C. The mass window was set between m/z 40 and m/z 350, using helium as the entrainment gas with a column pressure of 20 psi. The injection ramp started at 50°C and was kept for 5 min. Then, the temperature was increased at a rate of 5°C/min to a final temperature of 200°C.
The chemical components of the oil were identified by comparing their mass spectra with the reference spectra, and by comparing their Kovat's index with those described in the literature (Adams & Adams, 2001).

Minimum inhibitory concentration in broth microdilution
Microorganisms were seeded in potato medium and incubated at 37°C for 48 h. The suspension of Candida albicans was prepared in physiological saline at a concentration of 1 -5.0 x 106 CFU/mL. Then, a 1:100 dilution was made in RPMI-1640 with glucose 2% (Gibco/Invitrogen, Carlsbad, CA, USA) in order to achieve a concentration of 103 CFU/mL, as recommended by The Clinical & Laboratory Standards Institute (CLSI).
A stock solution of all the extracts, fractions, and sub-fractions was prepared at a concentration of 16,000 μg/5 mL in polysorbate. Aliquots of 100 μL of RPMI-1640 with glucose 2% were added to the 96-well microplates. The following concentrations of the extracts, fractions, and sub-fractions were evaluated: 2000 μg/mL, 1000 μg/mL, 500 μg/mL, 250 μg/mL, 125 μg/mL, and 62.5 μg/mL. The plates were incubated at 37°C for 24 h.

Lethality test of Artemia salina
The toxicity assay designed for the lethality tests uses the brine shrimp Artemia salina. A saline solution at the concentration of 30 g/L of sea salt at a pH of 9 was used for the hatching of Artemia salina cysts and for the preparation of dilutions (Meyer et al., 1982). The cysts were placed in the saline solution for 48 h, under continuous aeration and exposure to daylight. The temperature was maintained between 27 and 30°C and the pH between 8 and 9. The recipient was kept under illumination during the first hour.
After the hatching of the cysts, about 10 nauplii of Artemia salina were transferred to tubes containing the essential oil diluted in 0.5% of polysorbate 80 and saline solution at the concentrations of 75, 200, 500, 750 or 1000 μg/mL. A positive control was prepared with saline solution and quinidine sulphate, and a saline solution with polysorbate 80 was used as a negative control. The assay was performed in triplicate. The tubes were incubated at 27-30°C for 24 h, and the live and dead nauplii were counted by visual inspection.

Cytotoxicity assay
The cytotoxicity of the essential oil was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetratozolium bromide] colorimetric assay to determine cell viability through mitochondrial activity. MTT is a water-soluble tetrazolium salt, which is converted to an insoluble purple compound known as formazan after cleavage of the tetrazolium salt by mitochondrial dehydrogenases (Mosmann, 1983).
McCoy, B16F10, and MCF7 cells (3 x 103 cells/well) were plated 24 h prior to exposure to Cantinoa althaeifolia oil for adherence, scattering, and stabilization of the cells on the plaque. The cells were then exposed to increasing oil concentrations (0.84 μg/mL to 8.4 mg/mL in RPMI medium, supplemented with fetal bovine serum 10% and antibiotics for 72 h). Immediately prior to treatment, the solutions containing the oil and the culture medium were vigorously shaken using a vortex-type stirrer for at least 1 min.
After the treatment period, the medium was replaced by a solution of MTT (0.5 μg/μL) for 2 h at 37°C. The MTT solution was then replaced by isopropyl alcohol (0.1 M HCl) for solubilization of the formazan crystals, and the absorbance was read at 570 nm. A sample with no treatment was used as a control and the cell viability of other groups was expressed as the percentages of the control.

Statistical analysis
The MIC values were submitted to ANOVA and Tukey's test at the 5% significance level (p <0.05). The results of the lethality test of Artemia salina were analyzed using the PROBIT test, which provided LC50 values with reliability of 95%. The degree of toxicity was based on three classifications: low toxicity, LC50 > 500 μg/mL; moderate toxicity, LC50 between 100 μg/mL and 500 μg/mL; and high toxicity, LC50.
The concentration that inhibited the mitochondrial dehydrogenases activity by 50% (IC50) was calculated using the

Results and Discussion
The extract of the essential oil of Cantinoa althaeifolia was made from the leaves of the dried and crushed plant. The oil obtained had a yellowish color and a strong odor, similar to other species of the Lamiaceae family. The calculated yield on the dry material mass was 2.21% (v/m), with a final volume of 3.2 mL of essential oil.
The chemical composition of the essential oil of the leaves is shown in Table 1. The major chemical constituents in order of abundance were himachalene (11.62%), spathulenol (10.08%), caryophyllene oxide (9.93%), (E)-caryophyllene (9.21%), and pogostol (6.31%), as presented in Figure 1. Himachalene is of extreme interest because it has not yet been identified in the Lamiaceae family, and there is little information about this compound in the literature. Spathulenol is found in alcoholic beverages and reported in several families, especially in Lamiaceae. It has antimicrobial (Tadić et al., 2017), toxicity and antitumor (Rolim et al., 2017), antinociceptive and anti-inflammatory activities (Barreto et al., 2016). Caryophyllene oxide is widely used as a flavoring agent and is found in several families, including the one of interest. This compound has antiinflammatory, antioxidant, antibiotic, cytotoxic (Côté et al., 2017)   Research, Society andDevelopment, v. 10, n. 2, e9910212040, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i2.12040 8 The antioxidant activity of the essential oil was calculated in relation to BHT, ascorbic acid, and rutin. Table 2 presents the results of the antioxidant activity by the phosphomolybdenum complexation method. The sample showed a characteristic green color, which demonstrates its antioxidant activity. Table 2. Antioxidant activity of the Cantinoa althaeifolia essential oil. 472.66 ± 3.36 h 243.93 ± 1.14 g 524.98 ± 2.46 g BHTbutylated hydroxytoluene; AAantioxidant activity; SD -standard deviation; AACascorbic acid; and EOessential oil. Data followed by same lower-case letters indicate not significant differences within columns (p>0.05, ANOVA and Tukey's test). Source: Authors (2020).

Sample
The essential oil of Cantinoa althaeifolia showed potential antimicrobial activity for the tested microorganisms. S. aureus, P. aeruginosa and C. albicans presented a MIC of 1000 mg/mL, while E. coli presented a MIC of 500 mg/mL. The bactericidal activity of sesquiterpenes like δ-cadinene, α-humulene, and karyophylene has been previously described (Pereira et al., 2017). However, when these substances were tested together, this activity were either reduced by competitive inhibition or improved because of the synergistic effect (Damasceno et al., 2018).
Artemia salina is a micro-crustacean easily found in specialized aquarium stores, that can used as live food for fish and to estimate toxicity by the average lethal concentration (LC50). This bioassay has been used in previous studies of plant extracts with possible antitumor activity and can be used for the initial assessment of a large number of samples for the evaluation of toxicity and phototoxicity. Several studies have attempted to correlate crustacean toxicity with antifungal, antimicrobial, parasiticidal, virucidal, and trypanosomicidal activities (Meyer et al., 1982). For the calculation of the LC50, the results were subjected to the Probit statistical test (Finney, 1971) and are presented in Table 3. The cytotoxicity test results are presented in Figure 2. As shown in the first graph (1)   . This is the first study to evaluate the cytotoxic activity of this essential oil.
The root extract of Prunella vulgaris, a species also from the Lamiaceae family, reportedly has potent anticancer effects in MCF7 human BC cells both in vitro and in vivo, being able to induce apoptosis, inhibit angiogenesis, arrest cell cycle, and modulate the PI3K/AKT signaling pathway (Gao & Xu, 2019).     The findings from the lethality test of Artemia salina combined with the results from the cytotoxicity test with MTT are promising because they confirm that the oil is only toxic against the tumor cell lines. These findings corroborate with reports that oils with terpenes and sesquiterpenes in their chemical composition have antioxidant and antitumor activities (Morshedloo, Quassinti, Bramucci, Lupidi, & Maggi, 2017).
The literature reports that some constituents found in the essential oil of Turkish Origanum onites, also from the Lamiaceae family, have activity against parasitic diseases (Tasdemir, Kaiser, Demirci, Demirci, & Baser, 2019). According to Batisttini et al. (2019), the chemical constituents of the essential oils of several fruits from the Lamiaceae family have activity against the hepatitis A virus, and several of these constituents were found in the present study (Battistini et al., 2019).

Conclusion
Although there is no marked bactericidal activity, the Cantinoa althaeifolia essential oil has shown considerable antimicrobial activity against all the tested microorganisms, indicating that this plant can be a promising treatment for various infections.
The Cantinoa althaeifolia essential oil has cytotoxic activity against the tested cancer cell lines, especially against melanoma cells. The decrease in the number of cells, morphological changes, and increase in the sub-G0 phase may suggest that the mechanism involved in this effect may be related to apoptosis. The selectivity index showed that the tested samples were less toxic to blood cells, suggesting that this compound could be a promising drug in the treatment of melanoma and human breast cancer.
Further studies, especially in vivo, are necessary to confirm that this plant can be an alternative for enhancing antineoplastic therapy. The susceptivity of different microorganisms and different strains could also be an interesting field to explore in the future.