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Article

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

by
Juana Liz Leslie Cucho-Medrano
1,
Sammy Wesley Mendoza-Beingolea
1,
César Máximo Fuertes-Ruitón
2,
María Elena Salazar-Salvatierra
3 and
Oscar Herrera-Calderon
4,*
1
Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima 15001, Peru
2
Institute for Research in Pharmaceutical Sciences and Natural Resources, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima 15001, Peru
3
Research Institute in Biological Chemistry, Microbiology and Biotechnology, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima 15001, Peru
4
Department of Pharmacology, Bromatology and Toxicology, Faculty of Pharmacy and Biochemistry, Universidad Nacional Mayor de San Marcos, Jr. Puno 1002, Lima 15001, Peru
*
Author to whom correspondence should be addressed.
Antibiotics 2021, 10(11), 1387; https://doi.org/10.3390/antibiotics10111387
Submission received: 19 October 2021 / Revised: 4 November 2021 / Accepted: 10 November 2021 / Published: 12 November 2021
(This article belongs to the Special Issue Antimicrobial Activity of Essential Oils)
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Abstract

:
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.

1. 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 different plant organs as a response to adverse environmental conditions, and the attack of herbivores [3]. Several research studies attribute different medicinal properties to essential oils such as antibacterial, antifungal, antiviral, antioxidant, anti-inflammatory, anticancer, antidiabetic, antiparasitic, insecticidal, antimutagenic, antiprotozoal, immunomodulatory, and antitumor [4].
Peru is a megadiverse country due to its geographical location; the Peruvian flora comprises around 25,000 species that are distributed in the different ecological levels. An important part of the flora grows in the inter-Andean valleys and are exposed to a high solar radiation and low temperatures [5]. Among the plants belonging to the Euphorbiaceae family, Peru presents 61 genera, and 323 species of which the Croton genus is the most numerous in endemic species [6]. Additionally, Croton is the second most abundant genus of the Euphorbiaceae family due to its variety and richness, which is why it includes more than 1200 species. The Croton genus presents a cosmopolitan distribution both in the tropics and subtropics throughout the world, primarily in arid and semi-arid zones [7,8]; however, a wide variety are found in Madagascar, Brazil, and the Caribbean [9]. In Peru they are found in different regions between altitudes ranging from 110 to 3200 meters above sea level.
The Croton genus is important locally for its different medicinal properties, i.e., their leaves are used as healing, hemostatic, antidiarrheal, anti-inflammatory, and antiseptic agents. They are used for conditions such as uterine colic, gastrointestinal ulcers, urinary retention, antepartum vaginal baths, postpartum hemorrhages, skin diseases, and for the control of fever associated with gastrointestinal illnesses [10]. Essential oils from Croton species have presented several volatile constituents as fingerprint, which might be related to its antimicrobial activity, i.e., C. ceanothifolius showed bicyclogermacrene (26.3%), germacrene D (14.7%), and E-caryophyllene (11.7%) as major components [11], in C. heliotropiifolius were found β-caryophyllene as the major constituent followed by bicyclogermacrene, germacrene-D, limonene, and 1,8-cineole [12], in C. argyrophyllus had bicyclogermacrene (38.42 %), (Z)-caryophyllene (14.06 %), epi-longipinanol (9.78 %), and germacrene B (9.1 %) [13], in C. zenhtneri, C. nepetaefolius, and C. sonderianus were trans-anethole (94.09%), methyl eugenol (48.47%), and β-phellandrene (18.21%) respectively [14], finally in C. pulegiodorus were identified β-caryophyllene (20.96%), bicyclogermacrene (16.89%), germacrene-D (10.55%), τ-cadinol (4.56%), and β-copaen-4-α-ol (4.35%) [15].
Otherwise, Croton adipatus, Croton thurifer, and Croton collinus are known for their antimicrobial properties; however, such properties have not been scientifically proven as well as their chemical composition. Thus, the main aim in this study was to determine the chemical composition and the in-vitro antimicrobial activity of the essential oils from Croton adipatus, Croton thurifer, and Croton collinus against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 9027, and Candida albicans ATCC 10231.

2. Results and Discussion

2.1. Phytochemical Screening of the Volatile Components of the Essential Oils from Croton Species

The extraction yields of the essential oils of Croton adipatus, Croton thurifer, and Croton collinus were 0.47 ± 0.01%, 0.07 ± 0.005%, and 0.06 ± 0.001%, respectively; being the first reports in these species (Supplementary Materials: Table S1). Other studies reported yield percentages in C. borarium leaves and C. geayi leaves values of 0.68% and 0.72%, respectively [16], C. zambesicus (leaves: 0.29%) [17], C. cajucara [18](leaves: 0.4% and 0.65%), C. heterocalyx (leaves: 0.45%) [19], C. pullei (stems: 0.06%, leaves: 0.5%) [20], C. campestris (branches: 0.02%; leaves: 0.04%) [21] C. blanchetianus (leaves: 0.7%) [22], C. oblongifolius (bark: 0.9%) [23], and C. hieronymi (roots: 0.06%; leaves: 0.07%) [24]. In this study, C. adipatus, C. thurifer, and C. collinus showed extraction yields similar to the average reported in other species of the Croton genus. Several factors such as soil composition, climate, geographic location, variety of the species, vegetative state, seasonal variation, organ used for extraction, collection period, storage, drying methods, extraction processes, and others might be decisive in the composition, proportion, and quantity in the yield rate of essential oils [25].
Regarding to the determination of the volatile phytochemicals by GC-MS of the essential oil of C. adipatus, 46 compounds were determined (Table 1; Supplementary Materials: Figures S1 and S2, Table S2), of which monoterpenes represent 72.73% (monoterpenes hydrocarbon 69.76%; oxygenated monoterpenes 2.97%), followed by sesquiterpenes 18.82% (sesquiterpenes hydrocarbon 16.06%; oxygenated sesquiterpenes 2.76%), phenylpropane derivatives 0.24%, and others 8.21%. β-myrcene was the most abundant component with 18.34% followed by α-thujene at 12.69%, D-limonene at 10.94%, α-phellandrene at 8.19%, and β-elemene at 6.47%. These results are the first report of the chemical composition; the concentrations of β-myrcene (18.34%) and α-thujene (12.69%) are the major ones found in regard to other species of the Croton genus. A similar concentration of D-limonene (10.94%) was also found by De Almeida et al. in C. campestris (9.7%) [21], and a lower concentration in C. geayi (22.92%) [16].
For the essential oil of C. thurifer, 35 compounds were determined (Table 1; Supplementary Materials: Figures S3 and S4, Table S3). The composition of sesquiterpenes were predominantly abundant, at 62.26% (sesquiterpenes hydrocarbon 33.88%; oxygenated sesquiterpenes 28.38%), followed by monoterpenes at 35.39% (monoterpenes hydrocarbon 34.88%; oxygenated monoterpenes 0.51%) and other at 2.35%. The unknown monoterpenes component with 22.38% (C10H16; Rt: 18.57) was the most abundant component, followed by another unknown sesquiterpene component 21.8% (C15H26O; Rt: 53.7), β-elemene 11.87%, germacrene D 10.22%. Moreno et al. reported in C. heterocalyx leaves, β-elemene (8.2%), germacrene D (12.5%) [19] and De Araújo reported C. rhamnifolius stems (17.28%) [26]. On the other hand, Dai et al. in C. cascarilloides (6.0%) [27] and Turiel et al. in C. draconoides (9.0%) [28].
In C. collinus essential oil, 38 compounds were determined (Table 1; Supplementary Materials: Figures S5 and S6, Table S4). Sesquiterpenes 70.26% were obtained predominantly (hydrocarbon sesquiterpenes 68.25%; oxygenated sesquiterpenes 2.51%), followed by monoterpenes 28.03% (hydrocarbon monoterpenes 27.44%; oxygenated monoterpenes 0.59%) and others 1.21%. The major component was β-caryophyllene with 44.7%, followed by D-limonene 8.73%, β-thujene 6.96%, β-myrcene 6.79%, and β-elemene 6.7%. β-caryophyllene was the highest abundant volatile component among Croton species. However, other studies reported lower concentrations of this component (β-caryophyllene): in C. antanosiensis leaves (28.23%), C. decaryi leaves (26.65%) [29], C. rhamnifolioides (6.33%) [30], C. huberi (18.3%) [31], C. conduplicatus (7.8%) [32], C. trinitatis (15.3%) [33], C. isabelli (14.3%), and C. pallidulus (11.5%) [34].
Other studies of the Croton genus reported other major compounds according to the organ of the plant such as in C. zambesicus leaves and stems (1,8-cineole 27.07% and cymene 13.80%) [35]; C. zehntneri leaves (estragole 93.61% and 84.7%) [36], C. hieronymi leaves (camphor 13.9%) [24], C. hieronymi root (γ-asarone 25.7%) [24], C. oblongifolius bark (terpinen-4-ol 17.8%) [23]; C. pullei leaves (linalol 24.9%) [20], C. greveanus leaves (1.8 cineol 40.40% and linalol 23.81%); C. borarium leaves (β-phellandrene 39. 72% and α-terpineol 25.12%); and in C. geayi leaves (β-pinene 28.74% and limonene 22.92%) [16]. The diversity of the qualitative and quantitative characteristics of the components of essential oils in C. adipatus, C. thurifer, and C. collinus can be explained due to many factors reported by other authors such as the harvesting season, extraction method, drying method, drying time, geographical conditions, genetic variability, soil composition, plant organ, climate, drought condition, vegetative state cycle, plant nutrition, application of fertilizers, stress during growth, and post-harvest storage [25,37,38].
On the other hand, differences in the qualitative and quantitative compositions of the obtained essential oils have been observed in Table As shown in the Venn diagram (Figure 1), α-thujene, α-pinene, sabinene, β-pinene, α-phellandrene, D-limonene, β-elemene, β-caryophyllene, germacrene D, α-cadinene, and heptadecane were shared by all three species, for a total of 11 compounds. Additionally, 18 compounds were uniquely identified in C. adipatus, 11, and 10 in C thurifer and C. collinus, respectively.

2.2. Antimicrobial Activity of the Essential Oils of Croton Species

2.2.1. Agar Diffusion Method

Table 2 shows that C. adipatus essential oil at a concentration of 50% inhibited the growth of S aureus (20.0 ± 0 mm), while a concentration of 95% inhibited the growth of B. subtilis (25.0 ± 0 mm) and E. coli (20.03 ± 0.29 mm), while at 10% inhibited C. albicans (19.67 ± 1.47 mm). On the other hand, no significant activity was observed against P. aeruginosa. Additionally, C. thurifer (concentration 10%, 50% and 95%) essential oil presented inhibition halos ranging from 11 to 14 mm for S. aureus, B. subtilis, E. coli, and P. aeruginosa. In C. albicans was observed an inhibition halo of 15.03 ± 0.38 mm at 10%. C. collinus (concentration 10%, 50% and 95%) presented inhibition halos between 11 and 15 mm for S. aureus, B. subtilis, E. coli, and C. albicans (Table 2 and Figure 2).
In other studies of the Croton genus regarding the antimicrobial activity (diffusion method, the following inhibition halos were reported:
In S. aureus: C. borarium leaves at 100% (22.5 mm) [16] and C. gratissimus leaves at 5 mg/mL (21.6 mm) [39]. Similar results were obtained in our study of C. adipatus at 50% (20.0 mm). On the other hand, other authors reported that the essential oils of the following plant species did not have good activity: leaves and stems of C. zehntneri at 50 µg/disc (14.53 ± 0.25 mm) [36] in C. geayi leaves at 100% (10 mm) and C. greveanus leaves at 100% (11.5 mm) [16]; as well as in C. thurifer at 95% (14.07 mm) and C. collinus at 95% (12 mm) (Table 2 and Figure 2).
Regarding B. subtilis; the essential oils of C. zehntneri at 5 µg/disc and 25 µg/disc (<8 mm and 13.33 ± 0.42 mm, respectively) [36], C. borarium (15 mm), C. geayi (9 mm), and C. greveanus leaves (13.2 mm) at 100% [16] did not have a significant activity against this strain. Similar results were evidenced in C. thurifer at 50% (12.53 mm) and C. collinus at 95% (15 mm). However, C. adipatus at 95% showed an inhibition halo of 25.0 mm (Table 2 and Figure 2).
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).
In P. aeruginosa, C. borarium leaves at 100% showed an inhibition halo of 18.3 mm [16]. In contrast, C. zehntneri at 5, 25, and 50 ug/disc (<8 mm) [36], C. geayi leaves at 100 % (3.17 mm), C. greveanus leaves at 100% (8.01 mm) [16], and C. gratissimus leaves at 5 mg/mL (11.0 ± 0.6 mm) [39] did not show antimicrobial activity being similar with our findings, which ranges between 10 ± 0 mm and 13.57 ± 0.85 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 25ug/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).
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 (Table 2 and Table 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].

2.2.2. Colorimetric Microdilution Method

According to our results showed in Table 3 and Figure 3, the essential oil of C. adipatus showed antimicrobial activity against B. subtilis (MIC = 286.4 µg/mL) and C. albicans (MIC = 572.8 µg/mL) and did not show activity against S. aureus ATCC 25923 (MIC > 1000 µg/mL), E.coli (MIC = 1000 µg/mL) and P. aeruginosa (MIC > 1000 µg/mL). The essential oil of C. thurifer showed activity against the following bacteria: S. aureus (MIC = 296.1 µg/mL) and B. subtilis (MIC = 148 µg/mL). Furthermore, it did not show activity against E. coli (MIC > 1000 µg/mL), P. aeruginosa (MIC > 1000 µg/mL), and C. albicans (MIC => 1000 µg/mL). The essential oil of C. collinus showed antimicrobial activity against B. subtilis (MIC = 72 µg/mL), and C. albicans (MIC = 576.2 µg/mL). Additionally, it did not show activity against S. aureus (MIC > 1000 µg/mL), E. coli (MIC > 1000 µg/mL), and P. aeruginosa (MIC > 1000 µg/mL) (Table 3 and Figure 3).
In S. aureus, good activity was evidenced in the aerial parts of C. zambesicus (MIC = 16 µg/mL) [17]. C. cajucara leaves (MIC = 33.4 µg/mL) [18], C. campestris (MIC = 128 µg/mL) [32], C. zehntneri stems and leaves (MIC = 145.0 µg/mL) [36], in the aerial parts of C. heliotropiifolius (MIC = 500 µg/mL) [44], C. limae leaves (MIC = 512 µg/mL) [45], and C. gratissimus leaves (MIC = 600 µg/mL) [39]. Similar results were obtained in the present study for C. thurifer leaves (MIC = 296.1 µg/mL). On the other hand, other investigations reported that the essential oils of the following plant species did not have a good activity such as C. borarium (MIC = 1151 µg/mL) [16], C. greveanus (MIC = 1126 µg/mL) [16], and C. geayi (MIC = 2260 µg/mL) [16].
In B. subtilis, other Croton species presented good activity such as: C. zambesicus steam and leaves (MIC = 16 µg/mL) [17], C. heliotropiifolius aerial parts (MIC = 62.5 µg/mL) [44], C. zehntneri steam and leaves (MIC = 58.75 µg/mL) [36], and C. borarium leaves (MIC = 287 µg/mL) [16]. Similar results were obtained for the essential oils of the leaves of the three species of Croton, obtaining better results for C. collinus (MIC = 72 µg/mL), followed by C. thurifer (MIC = 148 µg/mL), and C. adipatus (MIC = 286.4 µg/mL). On the other hand, C. greveanus leaves (MIC = 1126 µg/mL) [16], and C. geayi (MIC = 4520 µg/mL) [16] did not present activity against this microorganism.
For E. coli, the following authors reported a significant activity in C. zambesicus stems and leaves (MIC = 16 µg/mL) [17] and C. hieronymi (MIC = 100 µg/mL) [24]. On the other hand, C. limae leaves (MIC ≥ 1024 µg/mL) [45], C. boraium leaves (MIC = 1151 µg/mL) [45], C. greveanus (MIC = 4505 µg/mL) [16], and C. gratissimus leaves (MIC = 1300 µg/mL) [16]. Similarly, in our study, no activity was found in C. adipatus, C. thurifer and C. collinus.
In P. aeruginosa, C. zambesicus leaves showed an inhibition at 250 µg/mL [17]. Meanwhile C. limae leaves (MIC = 1024 µg/mL) [17], C. boraium leaves (MIC= 1151 µg/mL) [16], C. greveanus (MIC = 4505 µg/mL) [16], and C. gratissimus leaves (MIC=5000 µg/mL) [39] did not show promising results, as well as the studied species of Croton in our study.
For C. albicans, a good activity was reported according to Alviano et al. in C. cajucara leaves (MIC = 13.4 µg/mL) [18], C. zehntneri stem and leaves (MIC = 58.75 µg/mL) [36], and in C. hieronymi roots (MIC = 100 µg/mL) [24]. Similar results were presented in the present study for the species of C. adipatus (MIC = 571 µg/mL) and C. collinus (MIC = 574 µg/mL). On the other hand, C. hieronymi roots (MIC > 1000 µg/mL) [24] did not present activity, in the same way with C. thurifer (MIC > 1000 µg/mL). MIC of standard drugs are shown in Table 3 and in Supplementary Materials Figure S7.
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 Gram-negative 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], β-myrcene [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 Saccharomyces 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.

3. Materials and Methods

3.1. 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.

3.2. 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:
Y i e l d   p e r c e n t a g e   ( % )   = v o l u m e   o f   E O   i n   m L   W e i g h t   o f   E O   i n   g   × 100
where EO: essential oil.

3.3. 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.

3.4. Evaluation of the Antimicrobial Activity of the Essential Oils of Croton Species

3.4.1. 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 × 108 CFU/mL for bacteria and 1–5 × 106 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 cork-borer. 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].

3.4.2. 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 × 108 CFU/mL for bacteria and 1–5 × 106 CFU/mL for yeasts. In bacteria, dilutions were made with Müeller Hinton broth to obtain an inoculum of 6.6–13.3 × 105 CFU/mL (2x inoculum). For yeast, consecutive dilutions were made with RPMI 1640 medium to obtain an inoculum of 1–5 × 103 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.

3.5. 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.

4. 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 (C10H16) 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.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10111387/s1, Figure S1: GC-MS chromatogram of the essential oil of Croton adipatus Kunth; Figure S2: Spectrum of the main constituents of the essential oil of Croton adipatus Kunth. A: β-myrcene; B: α-thujene; C: D-limonene; Figure S3: GC-MS chromatogram of the essential oil of Croton thurifer Kunth; Figure S4: Spectrum of the main constituents of the essential oil of Croton thurifer Kunth. A: Unknown (C10H16); B: Unknown (C15H26O); C: β-elemene; Figure S5: GC-MS chromatogram of the essential oil of Croton collinus Kunth; Figure S6: Spectrum of the main constituents of the essential oil of Croton collinus Kunth. A: β-caryophyllene B: D-limonene; C: β-thujene; Figure S7: Determination of the Minimum Inhibitory Concentration of the standard drugs; 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.

Author Contributions

Conceptualization, J.L.L.C.-M and S.W.M.-B.; methodology, M.E.S.-S..; software, J.L.L.C.-M; validation, C.M.F.-R, and M.E.S.-S; formal analysis, S.W.M.-B; investigation, M.E.S.-S; resources, M.E.S.-S; writing—original draft preparation, O.H.-C; writing—review and editing, O.H.-C; visualization, O.H.-C.; supervision, C.M.F.-R.; project administration, J.L.L.C.-M. and S.W.M.-B.; funding acquisition, M.E.S.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

The authors thank Biologist Jose Campos for providing us with the taxonomic classification of Croton species.

Conflicts of Interest

The authors declare no conflict of interest.

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Antibiotics 10 01387 g001 550
Figure 1. Venn diagram showing both the number of compounds shared and unshared/peculiar among the three Croton essential oils.
Figure 1. Venn diagram showing both the number of compounds shared and unshared/peculiar among the three Croton essential oils.
Antibiotics 10 01387 g001
Antibiotics 10 01387 g002 550
Figure 2. Antimicrobial activity of Croton species at different concentrations 10%, 50% and 95% by the agar diffusion method. The white acronym represents the analyzed plant species: Ca (C. adipatus); Ct (C. thurifer), Cc (C. collinus); while the yellow acronym represents microorganisms such as Sa (S. aureus), Bs (B. subtilis), Pa (P. aeruginosa), and Ca (C. albicans); the yellow letters represent the concentration percentages of each essential oil.
Figure 2. Antimicrobial activity of Croton species at different concentrations 10%, 50% and 95% by the agar diffusion method. The white acronym represents the analyzed plant species: Ca (C. adipatus); Ct (C. thurifer), Cc (C. collinus); while the yellow acronym represents microorganisms such as Sa (S. aureus), Bs (B. subtilis), Pa (P. aeruginosa), and Ca (C. albicans); the yellow letters represent the concentration percentages of each essential oil.
Antibiotics 10 01387 g002
Antibiotics 10 01387 g003 550
Figure 3. Determination of the minimum inhibitory concentration of the essential oil and standard drugs. SC (Sterility Control), GC (Growth Control), Sa (S. aureus), Bs (B. subtilis), Ec (E. coli), Pa (P. aeruginosa), Ca (C. albicans).
Figure 3. Determination of the minimum inhibitory concentration of the essential oil and standard drugs. SC (Sterility Control), GC (Growth Control), Sa (S. aureus), Bs (B. subtilis), Ec (E. coli), Pa (P. aeruginosa), Ca (C. albicans).
Antibiotics 10 01387 g003
Table
Table 1. Chemical composition of C. adipatus, C. thurifer, and C. collinus essential oil.
Table 1. Chemical composition of C. adipatus, C. thurifer, and C. collinus essential oil.
N°ChemicalsC. adipatusC. thuriferC. collinusChemical Group
%%%
1Isobutyl isobutyrate0.2 Carboxylic acid esters
2α-Thujene12.691.70.17Monoterpene hydrocarbon
3α-Pinene3.640.960.41Monoterpene hydrocarbon
4Sabinene3.171.20.31Monoterpene hydrocarbon
5β-Pinene1.550.380.36Monoterpene hydrocarbon
6β-Myrcene18.34 6.79Monoterpene hydrocarbon
7Unknown (C10H16) 22.38 Monoterpene hydrocarbon
8α-Phellandrene8.193.30.61Monoterpene hydrocarbon
9Isoamyl isobutyrate0.530.99 Fatty acid ester
10α-Terpinene0.56 Monoterpene hydrocarbon
11p-Cymene 0.360.29Monoterpene hydrocarbon
12o-Cymene1.38 Monoterpene hydrocarbon
13D-Limonene10.941.948.73Monoterpene hydrocarbon
14β- Thujene 6.96Monoterpene hydrocarbon
15β-Phellandrene4.941.44 Monoterpene hydrocarbon
16Eucalyptol0.70.51 Oxygenated monoterpenes
17γ-Terpinene 0.882.1Monoterpene hydrocarbon
18cis-Ocimene0.43 0.35Monoterpene hydrocarbon
19τ−Τerpinene3.21 Monoterpene hydrocarbon
20β-Terpineol0.44 Alcohol monoterpene
21Terpinolene0.16 0.17Monoterpene hydrocarbon
22β-Linalool0.4 Oxygenated monoterpenes
231,6-Dimethylhepta-1,3,5-triene0.47 Alkene
244-Terpineol0.74 Oxygenated monoterpenes
252-Decanone0.17 ketone
26α-Terpineol0.12 Oxygenated monoterpenes
272-Undecanone2.16 0.72ketone
282-Dodecanone4.56 ketone
292-Carene0.56 Monoterpene hydrocarbon
301,5,5-Trimethyl-6-methylene-cyclohexene 0.19Monoterpene hydrocarbon
31Copaene 0.440.28Monoterpene hydrocarbon
32Unknown I (C15H24) 0.15 0.23Sesquiterpene hydrocarbon
331-Ethenyl-1-methyl-2,4-bis-(1-methylethenyl)-cyclohexane 0.34 Sesquiterpene hydrocarbon
34Unknown II (C15H24) 0.23Sesquiterpene hydrocarbon
35β-Elemene6.4711.876.7Sesquiterpene hydrocarbon
362-Ethyl-1,3-dimethyl-Benzene0.24 Benzene
37Cyperene 0.691.03Sesquiterpene hydrocarbon
38β-Caryophyllene0.281.9544.7Sesquiterpene hydrocarbon
39δ−Εlemene0.71 Sesquiterpene hydrocarbon
40Geranyl acetone0.16 0.59Monoterpene ketone
41Unknown III (C15H24) 0.74 Sesquiterpene hydrocarbon
42Aromadendrene 1.250.23Sesquiterpene hydrocarbon
43α-Caryophyllene 0.33.73Sesquiterpene hydrocarbon
44Valencene 0.53Sesquiterpene hydrocarbon
45α-Elemene 0.52 Sesquiterpene hydrocarbon
46Unknown IV (C15H24) 1.43 Sesquiterpene hydrocarbon
47α-Curcumene 0.39Sesquiterpene hydrocarbon
48α-Muurolene0.45 Sesquiterpene hydrocarbon
49Unknown V (C15H24) 0.59 Sesquiterpene hydrocarbon
50Germacrene D4.7810.222.51Sesquiterpene hydrocarbon
51α-Selinene0.39 0.21Sesquiterpene hydrocarbon
52Eremophilene 0.67 Sesquiterpene hydrocarbon
53β-Cubebene 0.22Sesquiterpene hydrocarbon
54Bicyclogermacrene1.24 3.88Sesquiterpene hydrocarbon
55Geranil isobutyrate0.41 Carboxylic ester monoterpene
56Unknown I (C15H26O) 3.73 Sesquiterpene hydrocarbon
57Unknown II (C15H26O) 1.62 Sesquiterpene hydrocarbon
58δ-Cadinene0.481.261.96Sesquiterpene hydrocarbon
59α-Muurolene 0.51Sesquiterpene hydrocarbon
60Unknown VII (C15H24) 0.31 0.76Sesquiterpene hydrocarbon
61Elemol1.61 Oxygenated sesquiterpene
62Unknown VIII (C15H24) 0.69 Sesquiterpene hydrocarbon
63(-)-Spatulenol 0.58Oxygenated sesquiterpene
64Unknown (C15H24O) 0.89n.d.
65Unknown III (C15H26O)0.510.64 n.d.
66Unknown (C15H22O) 0.31 n.d.
67β-Maaliene0.11 Sesquiterpene hydrocarbon
68Unknown (C9H14O) 1.15 n.d.
69τ-Muurolene 0.15Sesquiterpene hydrocarbon
70Unknown IV (C15H26O) 0.121.8 n.d.
71τ-Muurolol0.16 0.34Oxygenated sesquiterpene
72α-Cadinol 0.73Oxygenated sesquiterpene
73Unknown V (C15H26O)0.380.28 n.d.
74Heptadecane0.120.260.49Alkane
Total of identified compounds (%) 100.00100.00100.00
n.d.; not determined.
Table
Table 2. Comparative table of the antimicrobial activity of essential oils and standards by the agar diffusion method.
Table 2. Comparative table of the antimicrobial activity of essential oils and standards by the agar diffusion method.
MicroorganismZone of Inhibition (mm)
Essential OilPositive Control
Croton adipatusCroton thuriferCroton collinusCIP [0.05 mg/mL]KET [0.2 mg/mL]
10%50%95%10%50%95%10%50%95%
Sa13.13 ± 0.44 *20 ± 0 *14.2 ± 0.7 *11.47 ± 0.5 *12 ± 0 *14.07 ± 0.41 *10.53 ± 0.55 *11.03 ± 0.52 *12 ± 0 *39 ± 0n.d.
Bs13.1 ± 1.32 *17.13 ± 0.34 *25 ± 0 *11.5 ± 0 *12.53 ± 0.46 *12.17 ± 1.25 *12.6 ± 0.8 *14.53 ± 0.4 *15 ± 0 *52.3 ± 1.1n.d.
Ec14.03 ± 0.4116 ± 020.03 ± 0.29 *10 ± 0 *12.03 ± 0.48 *11.53 ± 0.5 *11 ± 0 *11.57 ± 0.5 *10.6 ± 1.63 *15 ± 0n.d.
Pa11.27 ± 1.02 *11.53 ± 0.5 *13.57 ± 0.85 *10 ± 0 *12.5 ± 0 *11 ± 0 *11.63 ± 1.31 *12.7 ± 0.79 *11.03 ± 0.52 *34 ± 0n.d.
Ca19.67 ± 1.47 #12.5 ± 0 #11 ± 0#15.03 ± 0.38#11 ± 0 #10 ± 0 #15 ± 0 #10.53 ± 0.55 #10 ± 0 #n.d.35 ± 0
CIP: Ciprofloxacin; KET: ketoconazole; Sa: S. aureus; Bs: B. subtilis; Ec: E. coli; Pa: P. aeruginosa; Ca: C. albicans; n.d.: non-determined. * P < 0.05; Test Dunnet, mean values were compared to Ciprofloxacin group. # P < 0.05; Test Dunnet, mean values were compared to Ketoconazole group.
Table
Table 3. Comparative table of the minimum inhibitory concentration (MIC) of the essential oils and standards by the colorimetric microdilution method.
Table 3. Comparative table of the minimum inhibitory concentration (MIC) of the essential oils and standards by the colorimetric microdilution method.
MicroorganismMinimum Inhibitory Concentration (MIC)
Essential Oil (µg/mL)Positive Control (µg/mL)
Croton adipatusCroton thuriferCroton collinusCiprofloxacinKetoconazole
Sa>1000296.1 ± 0>10000.5 ± 0n.d.
Bs286.4 ± 0148 ± 072 ± 00.13 ± 0n.d.
Ec>1000>1000>10004 ± 0n.d.
Pa>1000>1000>100032 ± 0n.d.
Ca572.8 ± 0>1000576.2 ± 0n.d.0.03 ± 0
Sa: S. aureus; Bs: B. subtilis; Ec: E. coli; Pa: P. aeruginosa; Ca: C. albicans; n.d.: non-determined.
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Cucho-Medrano, J.L.L.; Mendoza-Beingolea, S.W.; Fuertes-Ruitón, C.M.; Salazar-Salvatierra, M.E.; Herrera-Calderon, O. Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus. Antibiotics 2021, 10, 1387. https://doi.org/10.3390/antibiotics10111387

AMA Style

Cucho-Medrano JLL, Mendoza-Beingolea SW, Fuertes-Ruitón CM, Salazar-Salvatierra ME, Herrera-Calderon O. Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus. Antibiotics. 2021; 10(11):1387. https://doi.org/10.3390/antibiotics10111387

Chicago/Turabian Style

Cucho-Medrano, Juana Liz Leslie, Sammy Wesley Mendoza-Beingolea, César Máximo Fuertes-Ruitón, María Elena Salazar-Salvatierra, and Oscar Herrera-Calderon. 2021. "Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus" Antibiotics 10, no. 11: 1387. https://doi.org/10.3390/antibiotics10111387

APA Style

Cucho-Medrano, J. L. L., Mendoza-Beingolea, S. W., Fuertes-Ruitón, C. M., Salazar-Salvatierra, M. E., & Herrera-Calderon, O. (2021). Chemical Profile of the Volatile Constituents and Antimicrobial Activity of the Essential Oils from Croton adipatus, Croton thurifer, and Croton collinus. Antibiotics, 10(11), 1387. https://doi.org/10.3390/antibiotics10111387

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