First Report on the Chemical Composition, Antioxidant Capacity, and Preliminary Toxicity to Artemia salina L. of Croton campinarensis Secco, A. Rosário & PE Berry (Euphorbiaceae) Essential Oil, and In Silico Study

Croton campinarensis Secco, A. Rosário & PE Berry is an aromatic species recently discovered in the Amazon region. This study first reports the chemical profile, antioxidant capacity, and preliminary toxicity to A. salina Leach of the essential oil (EO) of this species. The phytochemical profile of the essential oil was analyzed by gas chromatography (GC/MS) and (GC-FID). The antioxidant capacity of the EO was measured by its inhibition of ABTS•+ and DPPH• radicals. Molecular modeling was used to evaluate the mode of interaction of the major compounds with acetylcholinesterase (AChE). The results indicate that the EO yield was 0.24%, and germacrene D (26.95%), bicyclogermacrene (17.08%), (E)-caryophyllene (17.06%), and δ-elemene (7.59%) were the major compounds of the EO sample. The EO showed a TEAC of 0.55 ± 0.04 mM·L−1 for the reduction of the ABTS•+ radical and 1.88 ± 0.08 mM·L−1 for the reduction of the DPPH• radical. Regarding preliminary toxicity, the EO was classified as toxic in the bioassay with A. salina (LC50 = 20.84 ± 4.84 µg·mL−1). Through molecular docking, it was found that the majority of the EO components were able to interact with the binding pocket of AChE, a molecular target related to toxicity evaluated in A. salina models; the main interactions were van der Waals and π-alkyl interactions.


Introduction
Natural products, in particular volatile oils isolated from aromatic plants, have been the subject of several studies over the years. This may be related to their complex chemical compositions, which may include a series of classes of chemical compounds such as monoterpenes, sesquiterpenes, hydrocarbons, oxygenated mono-and sesquiterpenes, and phenylpropanoids [1][2][3][4]. In addition, this diverse chemical composition may be related to potential activities, such as antioxidant [5][6][7]. Amazonian flora, for example, include several aromatic plant species that produce EOs [8,9] that can serve as inputs for various sectors of the chemical, pharmaceutical, cosmetic, and food industries; these potential applications show promise in generating development in the region [4,[10][11][12]. Among

Distillation of Essential Oil
The processed botanical material was subjected to hydrodistillation to obtain EO, using a modified Clevenger apparatus for 3 h. After distillation, the EO was centrifuged and dehydrated with anhydrous sodium sulfate (Na 2 SO 4 ). Then, it was stored and preserved in a freezer. The EO yield was calculated on a moisture-free basis [45].

Analysis of the Chemical Composition of the Essential Oil
The analysis of the phytochemical profile of the essential oil of C. campinarensis was carried out following the same protocols of our research group, as well as the brand and model of the equipment [33,[48][49][50]. Masses (GC-MS) and quantification were performed by gas chromatography with a flame ionization detector (GC-FID). The identifications in the GC/MS were performed based on the calculated retention index [51] and compared with the literature [52]. This method was performed to analyze the potential of EO of C. campinarensis to inhibit the 1,1-diphenyl-2-picrylhydrazyl (DPPH • ) radical, a violet chromophore, resulting in the formation of the hydrogenated DPPH product, which is yellow or colorless [53]. Description of the method can be found in [54].

ABTS Method
This method was performed to analyze the potential of C. campinarensis EO to inhibit the 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS; Sigma-Aldrich; A1888, São Paulo, Brazil) radical, according to modification by Re et al. [55] of the experimental method proposed by Miller et al. [56]. Description of the method can be found in [54].

Determination of Preliminary Toxicity against Artemia salina Leach
For toxicity tests on A. salina, the essential oil was prepared at concentrations ranging from 1-100 µg·mL −1 , according to methods described by [45]. A total of ten A. salina larvae were added to each test flask with the aid of automatic micropipettes. In the control group and the positive group with lapachol, the same solvent was used for the samples and larvae, under the same conditions as the bioassay. The counting of A. salina was carried out after a period of 24 h at each concentration used and the IC50 was calculated, with the experiments being carried out in triplicate (n = 3).

Statistical Analysis
In the experimental tests, with the exception of the analysis of the chemical composition, the statistical Student's t-test was applied, and a significance level of 5% (p ≤ 0.05) was considered.
Molecular interactions were performed with the majority compounds and AChE in Molegro Virtual Docker (MVD) 5.5 software (Molexus IVS, Odder, Denmark) [61][62][63][64]; the structure of the protein used in the molecular modeling study can be obtained from the Protein Data Bank (https://www.rcsb.org/, accessed on 1 September 2022), using the ID code 4M0E [65]. The MolDock Score (GRID) was carried out as described by [45].

Yield and Chemical Composition of the Essential Oil
The EO yield for the studied sample was 0.24%, we can observe in Table 1. There are no records in the literature regarding the yield and volatile composition of the EO of C. campinarensis. However, the yield found in the present study is lower than that reported by Turiel et al. [66] for four Croton species from the Amazon (0.50% ± 1.10%). Another notable finding is the high concentration of sesquiterpene hydrocarbons found in both the present sample and the species analyzed by Turiel et al. [66], which had levels between 55.30% and 83.00%. Regarding the chemical composition, we can observe in Table 1, 32 volatile constituents were identified, accounting for 99.86% of the total, and sesquiterpene hydrocarbons dominated (87.95%), mainly germacrene D (26.95%), bicyclogermacrene (17.08%), (E)-caryophyllene (17.06%), and δ-elemene (7.59%). In addition, the monoterpene hydrocarbon γ-terpinene (8.99%) had a high content. Turiel et al. [66] reported that (E)-caryophyllene was the major component of the EOs of C. campestris (23.90%) and C. eriocladus (24.10%), bicyclogermacrene was the main volatile compound of the EO of C. chaetocalyx (13.90%), and spatulenol was the component with the highest content in C. glandulosus EO (19.70%). The authors also found germacrene D at high levels in the EOs of C. campestris (13.70%), C. eriocladus (9.70%), and C. chaetocalyx (17.90%); (E)-caryophyllene in the EOs of C. eriocladus (7.10%) and C. glandulosus (8.90%); bicyclogermacrene in the EO of C. glandulosus (9.60%); and δ-elemene in the EOs of C. chaetocalyx (13.50%) and C. glandulosus (8.00%). The authors also noted that γ-terpinene was identified only in C. campestris EO, with a low content (0.70%). These results indicate that the present sample of C. campinarensis EO has a chemical composition similar to those of other species of this genus present in the Amazon region.
Germacrene D, the major constituent of the sample, shows larvicidal activity against the mosquito Aedes aegypti [68]. EOs containing this compound as a major component also have anti-inflammatory and anti-AChE properties [69]. (E)-Caryophyllene also has potential anti-inflammatory activity and can be used to treat central nervous system diseases, cancer, and dental caries infections caused by etiological agents [70].
Franco et al. [33] reported that germacrene D and (E)-caryophyllene have antioxidant activity, and bicyclogermacrene has been associated with larvicidal and antiviral activity. Figueiredo et al. [71] reported that EOs with high levels of γ-terpinene have moderate activity against food pathogens. According to Dang et al. [72], δ-elemene has anticancer activity against HeLa cells.
Da Silva Júnior et al. [73] reported that germacrene D, bicyclogermacrene and δ-elemene are directly related to plant defense mechanisms and have higher concentrations in the rainy season in the Amazon region. According to the authors, this concentration trend may be directly related to a plant strategy for attracting pollinating agents, especially bees and flies, which are common in the rainy season.

Antioxidant Capacity and Preliminary Toxicity of the Essential Oil
The Table 2, below shows the TEAC of the EO of C. campinarensis, measured through the inhibition of ABTS •+ and DPPH • radicals and its preliminary toxicity against A. salina. The TEAC of the EO to inhibit the ABTS •+ radical was 0.55 ± 0.04 mM·L −1 (p = 0.557). In the DPPH assay, the TEAC was 1.88 ± 0.08 mM·L −1 (p = 0.001). These results indicate that the ABTS •+ radical capture potential of the EO of C. campinarensis was lower than that presented by the Trolox standard (1 mM·L −1 ). On the other hand, for the inhibition of DPPH • radicals, the TEAC of the EO was almost double that of the Trolox standard (1 mM·L −1 ). Regarding the preliminary toxicity against A. salina, the EO had an LC 50 of 20.84 ± 4.84 µg·mL −1 . According to Ramos et al. [74], EOs with LC 50 values lower than 80 µg·mL −1 are classified as toxic.
There are no reports in the literature regarding the antioxidant capacity and preliminary toxicity of C. campinarensis EO. However, other Croton species do have literature data on these properties. Morais et al. [75] reported that the evaluation of antioxidant activity by the DPPH method showed that C. campinarensis EOs have moderate antioxidant activity.
According to the authors, the Croton EOs did not contain phenolic compounds, which is the main cause of their lower antioxidant activity than the phenolic compound thymol and the commercial antioxidant BHT. In addition, the authors identified high levels of oxygenated sesquiterpenes in the samples, especially caryophyllene oxide and spathulenol. Pino et al. [76] found that the EO of Croton wagneri from Ecuador had a moderate elimination effect in the DPPH and ferric reducing antioxidant power (FRAP) assays. According to the authors, cis-chrysanthenol (27.5%) and myrcene (19.2%) were the major components of the sample. Do Vale et al. [77] analyzed the antioxidant capacity of the EO of Croton piauhiensis, characterized by compounds such as (E)-caryophyllene (21.58%), γ-terpinene (10.08%), and germacrene D (9.56%). The authors indicated that this EO showed high antioxidant capacity in the DPPH test, with higher results than the positive control (quercetin).
Regarding preliminary toxicity, Ribeiro et al. [78] evaluated the preliminary toxicity of the EO of Croton rudolphianus leaves by bioassays with A. salina. According to the authors, this EO exhibited high toxicity to microcrustaceans (LC 50 = 68.33 µg·mL −1 ). In addition, the authors identified chemical constituents consistent with those found in the present sample, such as (E)-caryophyllene (17.33%), bicyclogermacrene (7.1%), and germacrene D (5.38%).
Regarding the toxicity of major compounds, Judzentienė et al. [88] reported that the EO of Artemisia vulgaris, containing high levels of germacrene D (10.60-30.50%), showed high toxicity against A. salina. The authors directly attributed the results to the presence of germacrene D in the sample. Machado et al. [89] reported that (E)-caryophyllene showed toxicity against A. salina only at high concentrations (3 mM). Schmitt et al. [90] showed that this sesquiterpene had no significant toxicity against rats.
The biological properties of an EO may be associated with the major constituent of the sample and/or the synergistic and antagonistic effects exerted by all components present in the mixture [95]. Sesquiterpenes have higher toxicity than monoterpenes and phenylpropanoids [96]. Regarding free radical capture ability, sesquiterpenes generally have a lower antioxidant capacity than monoterpenes and phenylpropanoids [97]. These attributes may explain the antioxidant profile and preliminary toxicity exhibited by the EO of C. campinarensis.

Analysis of the Interactions of Major Compounds with AChE
In silico methods have been successfully used to evaluate the interaction between molecules of natural origin and molecular targets of pharmacological interest [98][99][100][101].
In this study, molecular docking was used to evaluate how the major compounds of C. campinarensis interact with the binding pocket of AChE, a molecular target related to toxicity that is investigated in A. salina models [45]. The energy values obtained for the interactions of the compounds with the target enzyme are summarized in Table 3. Table 3. Moldock scores obtained from the docking protocol using MVD 5.5.

Conclusions
The yield of the C. campinarensis EO analyzed in this study was 0.24%. Terpenes characterized the aromatic profile of the EO, with a predominance of sesquiterpene hydrocarbons (87.95%), mainly germacrene D (26.95%), bicyclogermacrene (17.08%), (E)caryophyllene (17.06%), and δ-elemene (7.59%). Regarding the antioxidant capacity of the EO, the evaluation of TEAC by the ABTS method showed that the EO has moderate

Conclusions
The yield of the C. campinarensis EO analyzed in this study was 0.24%. Terpenes characterized the aromatic profile of the EO, with a predominance of sesquiterpene hydrocarbons (87.95%), mainly germacrene D (26.95%), bicyclogermacrene (17.08%), (E)-caryophyllene (17.06%), and δ-elemene (7.59%). Regarding the antioxidant capacity of the EO, the evaluation of TEAC by the ABTS method showed that the EO has moderate antioxidant activity. However, the TEAC evaluation showed important inhibition of DPPH • radicals. The preliminary cytotoxicity test against A. salina indicated that the EO of C. campinarensis can be classified as toxic, with an LC 50 of 20.84 ± 4.84 µg·mL −1 . The energy calculations in Table 1 showed that complex formation was favorable. Hydrophobic interactions dominated the interactions between the major compounds of C. campinarensis EO and AChE. These results may indicate that the chemical components with higher contents in the sample may be related to the high toxicity demonstrated by the EO against the microcrustacean A. salina. This study presents the first report on the chemical composition, antioxidant capacity, and preliminary toxicity of the EO of C. campinarensis, contributing to the knowledge of the phytochemistry of species of the genus Croton from the Amazon region.