Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus

The aim of this study was to determine the volatile phytochemical constituents and evaluate the antimicrobial activity of the essential oils of the leaves from Croton adipatus, Croton thurifer, and Croton collinus. Essential oils were extracted by hydro-distillation using the Clevenger extractor and the phytochemical analysis was determined by Gas chromatography-Mass Spectrometry (GC-MS). The antimicrobial activity was assessed using the agar diffusion and colorimetric broth microdilution methods against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Candida albicans ATCC The essential oils from C. adipatus, C. thurifer, and C collinus had 46, 38, and 35 volatile constituents respectively. The main compounds determined in C. adipatus were β-myrcene (18.34%), while in C. collinus was β-caryophyllene (44.7%), and in C. thurifer was an unknown component (C10H16: 22.38%). Essential oil of C. adipatus showed a MIC against B. subtilis (286.4 µg/mL) and C. albicans (572.8 ± 0 µg/mL); C. thurifer against S. aureus (296.1 ± 0 µg/mL) and B. subtilis (148 ± 0 µg/mL); and C. collinus against B. subtilis (72 ± 0 µg/mL) and C. albicans (576.2 ± 0 µg/mL). The three essential oils of Croton species demonstrated in vitro antimicrobial activity against a strain of bacteria or fungi.


Introduction
In recent decades, the emergence of antimicrobial resistance has generated alarm in the health system. Several factors have contributed to the evolution of antimicrobial resistance, including microbial adaptation through selective biochemical processes, self-medication in the community, and the shortage in the development of new antimicrobial molecules which leads to choices of antimicrobial drugs with an inadequately broad spectrum and side effects, making access to essential antimicrobials more expensive [1]. On the other hand, the medicinal properties of plants have been recognized and have generated a great deal of interest due to their easy access, low toxicity, economic viability and easy preparation forms such as macerations, decoctions, infusions, tinctures, and essential oils [2]. Hence, there are many alternative therapies that include the extended use of essential oils, which are considered a set of volatile secondary metabolites synthesized in and others might be decisive in the composition, proportion, and quantity in the yield rate of essential oils [25].
For E. coli, the essential oil from C. gratissimus leaves at 5 mg/mL reported an inhibition halo of 23.0 mm [39], similar results were obtained with C. adipatus at 95% (20.03 mm). On the other hand, other studies reported that the essential oils of the following plant species did not have good activity: C. zehntneri leaves and stems [36], C. hieronymi roots and leaves [24], C. borarium, C. geayi, and C. greveanus leaves [16]; similar results were observed in C. thurifer at 50% (12.03 mm) and C. collinus at 50% (12.7 mm) (Table 2 and Figure 2).
For C. albicans, the following Croton species did not have a significant activity such as C. zehntneri leaves and stems at 5 ug/disc (<8 mm) and 25 ug/disc (<8 mm) [36], C. hieronymi roots and leaves at 1000 µg/mL (7.9 and 0 mm, respectively) [24], and C. cajucara at 100% had ranging from 6-10 mm [18]. Similar results were obtained for C. thurifer and C. collinus with inhibition zones between 10 and 15 mm. Otherwise, in the present study, C. adipatus at 10% reported an inhibition halo of 19.67 mm (Table 2 and Figure 2).

Ca
19.67 ± 1.47 # 12.5 ± 0 # 11 ± 0 # 15.03 ± 0.38 # 11 ± 0 # 10 ± 0 # 15 ± 0 # 10.53 ± 0.55 # 10 ± 0 # n.  Results for this type of preliminary antimicrobial test showed a certain degree of variability, which could be explained by different factors that influence the solubility of the essential oils in the medium to be diffused, the polarity of the essential oil, and the molecular size of the components that comprise the essential oil, among others; therefore, they would not necessarily correlate with the results of other specific tests such as microdilution or dilution in broth. Regarding the biochemical mechanisms involved in the antimicrobial activity of essential oils, some compounds like p-cymene at 2500 µg/mL have shown alterations in the membrane potential of S. aureus, and also γ-terpinene at 2500 and 3400 µg/mL [40]. β-caryophyllene, the major component in C. collinus was able to alter membrane permeability and integrity of B. cereus, leading possibly to cell death [41]. This finding is related to our results against B. subtilis, which only was selective for this strain (Tables 2 and 3). Limonene, the third abundant component in C. adipatus, is linked to the respiratory metabolism of S. aureus [42]. Otherwise, terpenes are also related to disorders of mitochondrial membrane in Candida species but with low antifungal activity. Moreover, oxygenated sesquiterpenes demonstrated a negative effect on Saccharomyces and Candida strains such as caryophyllene oxide, humulene epoxide, spathulenol, and α-Muurolol in high concentrations [43].
As is evidenced in the present study, the species of C. adipatus, C. thurifer, and C. collinus showed antimicrobial activity according to the microdilution test against Gram-positive and yeast. Other studies of the Croton species revealed diverse activity on the microorganisms under study, which means the high variability of the components present and associated with this activity. Various studies mention a slight sensitivity of Gram positive compared to Gram-negative. The main reason is the presence of the extracellular membrane in Gramnegative microorganisms, which regulate the passage of hydrophobic components and thus prevents the alteration of the membrane's permeability [46]. Likewise, various studies attribute the antimicrobial activity in essential oils to the following components: terpinen-4-ol [4], α-terpinene [47], 1,8-cineole [48], δ-cadinene, γ-muurolene, α-muurolene [49], β-caryophyllene [50], β-elemene [51], germacrene [52], p-cymene [53], γ-terpinene [54], βmyrcene [55], α-pinene [56], β-pinene [56], linalool [57], α-phellandrene [58], among others. All these components were found in the essential oils of Croton in our study. Furthermore, the observed activity could also be explained by the presence of the components in higher proportion or by the synergistic action between them.
Based on our results, we might suppose that the three essential oils had a selective effect against a specific bacterium and on C. albicans. Each metabolite determined could be acting by different mechanisms and according to our chemical analysis, each Croton species showed a different phytochemical marker, which contributes to the antimicrobial activity. Hydrocarbon monoterpenes were the most abundant chemical groups in C. adipatus. However, in C. thurifer and C. collinus were the sesquiterpenes, which might be linked to this difference in the results showed in Table 2 and Table Therefore, the volatile chemicals would be responsible for the antimicrobial effect on S. aureus, B. subtilis, and C. albicans, and not the major phytochemical determined in the GC-MS analysis. Some studies reported that terpenes do not constitute a high inherent antimicrobial effect i.e., some terpenes like p-cymene, the major compound in thyme, had no effect against several Gram-negative bacteria. Also, other chemicals, which were found in Croton essential oils such as α-pinene, limonene, δ-3-carene, sabinene, α-pinene, β-pinene, and α-terpinene revealed no or low effect against different pathogen bacteria, whether Gram-negative or Gram-positive. Additionally, α-pinene, β-pinene, p-cymene, β-myrcene, β-caryophyllene, limonene, and γ-terpinene had low or absent activity against Escherichia coli, Staphylococcus aureus, and Bacillus cereus and p-cymene and γ-terpinene were ineffective against Saccha-romyces cerevisiae [40]. Thus, these reports in vitro indicate that single terpene compounds are inefficient as antimicrobials, which supports our study to understand the relationship between the chemical composition of each Croton species with antimicrobial activity.

Collection of the Plant Material
Croton adipatus Kunth and Croton thurifer Kunth were collected in the locality El Almendral in the district of Jaen of the province of Jaen, Departament of Cajamarca, at an altitude of 650 meters above sea level, and Croton collinus Kunth was collected in Jahuanga, district of Bagua Grande in the province of Utcubamba of the department of Amazonas, at an altitude of 700 meters above sea level, in June 2017 during the dry season. Leaves were separated, selected and dried in open air, avoiding exposure to direct sunlight in order to preserve the volatile components and also to achieve a longer storage time. The identification and taxonomic classification of plant species were developed by the botanical consultant José Campos de la Cruz.

Extraction of Essential Oils of Croton Species
The extraction was developed by hydrodistillation using a Clevenger apparatus for two hours. Milligrams of anhydrous sulfate were added to remove residual water. Then it was stored at 4 • C until further use [59]. The following formula was used to calculate the yield percentage: where EO: essential oil.

Chemical Analysis by Gas Chromatography Coupled to Mass Spectrometry (GC-MS) of the Essential Oils of Croton Species
For the analysis of each Croton species, 20 µL of the essential oil was used in 980 µL of dichloromethane, which were injected into the Agilent Technologies 7890A gas chromatograph coupled to an Agilent Technologies 5975C mass selective detector. The separation of the compounds from the mixture was carried out using a DB-5MS apolar capillary column (60 m × 250 µm × 0.25 µm) (J&W of 5% phenyl methylpolysiloxane). The injector temperature was kept at 250 • C and the injection was carried out in split mode (20: 1). The oven temperature program was as follows: initial temperature 50 • C; subsequently, it was increased to 2.5 • C/min up to 180 • C, 10 • C/min up to 200 • C, and finally at 20 • C/min up to 240 • C. The run time was of 56 min, using helium as a stripping gas at a constant flow of 1mL/min. The constituents of essential oils were identified by comparing the mass spectra of each peak with those of the mass spectra library of the Flavor 2 databases and that of the National Institute of Standards and Technology (NIST, 08). This analysis was carried out in the laboratory of the Natural Products Research Unit of the Faculty of Sciences and Philosophy "Alberto Cazorla Talleri", Universidad Peruana Cayetano Heredia.

Agar Diffusion Method
The antibacterial activity of C. adipatus, C. thurifer, and C. collinus essential oils were tested against the following species: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027 and the yeast Candida albicans ATCC Bacteria and yeast were sub-cultured on Trypticase Soy agar and Dextrose Sabouraud agar, respectively, 24 h before the test. The suspension of the microorganisms was prepared in sterile 0.9% saline solution, and the turbidity was adjusted according to the 0.5 tube of the McFarland scale, which corresponds to an approximate concentration of 1-2 × 10 8 CFU/mL for bacteria and 1-5 × 10 6 CFU/mL for yeast.
Petri dishes were prepared as follows: Müeller Hinton agar was used for bacteria and Sabouraud Dextrose agar for yeast, previously reconstituted with distilled water, sterilized by autoclaving, cooled and kept between 45 • C and 50 • C. These were inoculated with 0.4 mL of inoculum suspension for every 100 mL of culture medium, then it was homogenized and distributed in sterile glass Petri dishes of 90 mm in diameter, at a rate of 25 mL per plate. It was allowed to solidify, and wells were made using a sterile corkborer. Then, 100 µL of the concentrations of the essential oils (10%, 50%, and 95%) were incorporated in each well, and finally were kept for 30 min under refrigeration to enhance the diffusion of the essential oils and to avoid the proliferation of microorganisms, then it was incubated in an oven at 37 • C for 24 h. Ciprofloxacin (0.05 mg/mL) and Ketoconazole (0.2 mg/mL) dissolved in water and dimethylsulfoxide (DMSO), respectively, were used as positive controls. Solvent controls (DMSO) were included in each experiment as negative controls. Tests were carried out in triplicate. After incubation, the presence of growth inhibition zones was observed, and the diameters were measured in mm. It was considered a significant antimicrobial activity to a perfectly clear area with a diameter greater than 18 mm [60].

Colorimetric Microdilution Method and Determination of MIC
The antibacterial activity was performed by colorimetric microdilution method utilizing 96-well microtiter plates to determine the minimum inhibitory concentration (MIC). The following microorganisms were used: Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Candida albicans ATCC The microorganisms were kept for 24 h before the test on Trypticase Soya agar for bacteria and Dextrose Sabouraud agar for yeast and incubated at aerobic conditions at 37 • C. Later, a few colonies of the microorganisms in sterile 0.9% saline solution was prepared with the help of a Kolle handle and the turbidity of the bacterial suspension was adjusted according to the 0.5 tube of the McFarland scale, which corresponds to an approximate concentration of 1-2 × 10 8 CFU/mL for bacteria and 1-5 × 10 6 CFU/mL for yeasts. In bacteria, dilutions were made with Müeller Hinton broth to obtain an inoculum of 6.6-13.3 × 10 5 CFU/mL (2x inoculum). For yeast, consecutive dilutions were made with RPMI 1640 medium to obtain an inoculum of 1-5 × 10 3 CFU/mL (2X inoculum). For each 20 mL of the suspensions of the 2x inocula (bacteria or yeast), 0.1 mL of 20 mg/mL resazurin solution was added, which was previously prepared under aseptic conditions using a 0.22 µm filter. The stock solutions of the essential oils were prepared at a concentration of 800 µL/mL [essential oil (80): polysorbate 20 (50): Müeller Hinton broth (870)], for testing in bacteria and yeast. The final concentrations (x) of the samples ranged from 0.079 to 40 µL/mL. The positive controls used for the tests were Ciprofloxacin and Ketoconazole, following the CLSI recommendations [61]. The final concentrations (x) of the controls ranging from 0.125 to 64 µg/mL and from 0.0313 to 16 µg/mL for Ciprofloxacin and Ketoconazole respectively. The Müeller Hinton broth with resazurin was used as a sterility control in the case of Ciprofloxacin and the RPMI 1640 medium with resazurin according to the protocol of Liu M et al. [62] in Ketoconazole.
100 µL (essential oil or positive control) of the 2x inoculum was mixed with resazurin indicator. On the other hand, sterility control wells contained Müeller Hinton broth or RPMI 1640 medium, containing resazurin and growth control wells had Müeller Hinton broth for bacteria or RPMI 1640 for Candida albicans. At the end, 96-well microtiter plates were labeled and covered in polyethylene bags to avoid volatilization of the samples. The microtiter plates were incubated at 37 • C for 24 h under aerobic conditions. It was performed visually and any change in color from purple (resazurin) to pink resorufin was considered a positive result. MIC values are defined as the lowest concentration of essential oil that prevents a color change of resazurin. The average of three values were calculated and reported as the MIC. For the result interpretation, the parameters of Holetz et al [63] were considered as follows: Inactive > 1000 µg/mL, Weak activity 500 to 1000 µg/mL, moderate activity 100 to <500 µg/mL, good activity < 100 µg/mL.

Statistical Treatment
The results were subjected to an analysis of variance (ANOVA) followed by the Dunnet test. It is considered a significant difference when P values is less than 0.05.

Conclusions
Based on the obtained results, it can be concluded that the three essential oils from Croton are richest in hydrocarbons, monoterpenes, and hydrocarbons and oxygenated sesquiterpenes. In addition to the main bioactive compounds, C adipatus is rich in β-Myrcene (18.34%), C thurifer in an unknown monoterpene (C 10 H 16 ) with 22.38% and C. collinus in β-caryophyllene (44.7%). Our research showed C. adipatus was selective for B. subtilis and C. albicans, C. thurifer for S. aureus. while C. collinus exhibited their antimicrobial activity towards B. subtilis and C. albicans. Although results showed some potential in the in vitro assay, these still may not be applied in vivo. Further research in in vivo models is necessary to evaluate the antimicrobial activity of these essential oils.  Table S1: Physicochemical properties of the essential oils of Croton species. Table S1: Chemical composition of Croton adipatus Kunth essential oil; Table S2: Chemical composition of Croton thurifer Kunth essential oil. Table S3: Chemical composition of Croton collinus Kunth essential oil.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.