Chemistry and Bioactivity of Croton Essential Oils: Literature Survey and Croton hirtus from Vietnam

Using essential oils to control vectors, intermediate hosts, and disease-causing microorganisms is a promising approach. The genus Croton in the family Euphorbiaceae is a large genus, with many species containing large amounts of essential oils, however, essential oil studies are limited in terms of the number of Croton species investigated. In this work, the aerial parts of C. hirtus growing wild in Vietnam were collected and analyzed by gas chromatography/mass spectrometry (GC/MS). A total of 141 compounds were identified in C. hirtus essential oil, in which sesquiterpenoids dominated, comprising 95.4%, including the main components β-caryophyllene (32.8%), germacrene D (11.6%), β-elemene (9.1%), α-humulene (8.5%), and caryophyllene oxide (5.0%). The essential oil of C. hirtus showed very strong biological activities against the larvae of four mosquito species with 24 h LC50 values in the range of 15.38–78.27 μg/mL, against Physella acuta adults with a 48 h LC50 value of 10.09 μg/mL, and against ATCC microorganisms with MIC values in the range of 8–16 μg/mL. In order to provide a comparison with previous works, a literature survey on the chemical composition, mosquito larvicidal, molluscicidal, antiparasitic, and antimicrobial activities of essential oils of Croton species was conducted. Seventy-two references (seventy articles and one book) out of a total of two hundred and forty-four references related to the chemical composition and bioactivity of essential oils of Croton species were used for this paper. The essential oils of some Croton species were characterized by their phenylpropanoid compounds. The experimental results of this research and the survey of the literature showed that Croton essential oils have the potential to be used to control mosquito-borne and mollusk-borne diseases, as well as microbial infections. Research on unstudied Croton species is needed to search for species with high essential oil contents and excellent biological activities.


Larvicidal Activity
The C. hirtus essential oil showed good larvicidal activity against mosquitoes with LC 50 values in the range of 15.38-78.27 µg/mL ( Table 1). The compounds β-caryophyllene, α-humulene, and caryophyllene oxide were active against the larvae of Aedes albopictus with LC 50 values at 24 h of exposure of 56.87, 43.86, and 20.61 µg/mL, respectively [58]. The compounds germacrene D and β-elemene showed very strong toxicity against the larvae of Ae. aegypti, Ae. albopictus, and Cx. quinquefasciatus [59][60][61][62][63][64]. β-Caryophyllene and its mixtures with caryophyllene oxide and α-humulene (ratio tested relative to the percentage in the essential oil) (Tables 2 and 3) exhibited distinctly different toxicities against Ae. aegypti and Ae. albopictus. This mixture played a major role in the larvicidal activity against Ae. aegypti, but conversely it did not play a role in the larvicidal activity against Ae. albopictus.

Literature Survey
In order to put this current investigation into context, a survey of the literature on Croton essential oils and their biological activities was carried out. A total of two hundred and forty-four references were collected, which included one hundred and seven species of Croton. The distribution of the study samples collected was as follows: Brazil (one hundred and seventy-six), Venezuela (six), South Africa (six), Cuba (five), Colombia (five), Ecuador (five), Madagascar (four), Nigeria (three), Costa Rica (three), Vietnam (two, not included in this study), Kenya (two), India (two), Cameroon (two), Kenya (two), Peru (two), Ethiopia (two), Mexico (two), Korea (one), Laos (one), Central African Republic (one), Gabon (one), Curacao (one), Benin (one), Argentina (one), Ivory Coast (one), Congo-Brazzaville (one), Sudan (one), Guadeloupe (one), China (one), Thailand (one), Island (one) and Malaysia (one). After reviewing the articles based on the set criteria, seventy-one articles and one book satisfied the criteria and were included herein. Most of the studies collected plant material and were conducted in Brazil.
The previously reported larvicidal activities of Croton species against mosquitoes are summarized in Table 4. Most of the essential oils of Croton spp. showed good activity (LC 50 < 100 µg/mL). It is worth noting that C. zehntneri essential oil has been characterized by its main component (E)-anethole, which has shown very good larvicidal activity against Ae. aegypti [52,65]. Furthermore, studies have reported that the yield of essential oil from the leaves of this species is greater than 1%, and it is non-toxic to mice (LD 50 : 3464 mg/kg) [65], so this essential oil may be considered for potential use as a biological pesticide. However, since this essential oil has shown geographical variation in terms of its chemical composition [66], an evaluation of the larvicidal activity of all of its chemotypes has not been performed. Leaves: Nd α-Pinene, trans-β-guaiene, β-pinene, β-gurjunene, β-elemene. 102 129 [73] Leaves: Nd Methyleugenol, α-copaene, β-caryophyllene 84 Nd [73] a : The order of the compounds is sorted by percentage from high to low and greater than 5.0%. Nd = not determined.
The main constituents of the essential oils of Croton spp. were evaluated for their larvicidal activity against mosquito species (Table 5). There were differences between different authors when reporting the activities of the compounds β-caryophyllene, α-pinene, β-pinene, α-terpineol, α-humulene, and α-phellandrene. This difference may have been due to the different health or developmental stages of the larvae between the groups. The larvicidal activity of (E)-anethole was weaker than that of C. zehntneri essential oil [52,65], which demonstrated the important role of components in small concentrations, such as anysyl-acetate and dihydroaromadendrene. Bicyclogermacrene did not have synergistic effects with the compounds spathulenol, β-caryophyllene, camphor, or germacrene D in the larvicidal activity of the essential oils of C. argyrophyllus, C. heliotropiifolius, and C. pulegiodorus. This trend was also observed in the larvicidal activity of Eugenia calycina essential oil [76].

Molluscicidal and Antiparasitic Activities
The essential oil of C. hirtus demonstrated molluscicidal activity against adult P. acuta with a 48 h LC 50 value of 10.09 µg/mL (Table 6). Based on the classification by the World Health Organization (WHO) [114], this essential oil is considered as an active plant molluscicide (LC 50 < 20 µg/mL). Croton rudolphianus essential oil, which is characterized by the main components β-caryophyllene, bicyclogermacrene, δ-cadinene, and germacrene D, was active against Biomphalaria glabrata with a 48 h LC 50 of 47.89 µg/mL [115]. Table 6. Molluscicidal activity of Croton hirtus essential oil and its major components against Physella acuta adults (µg/mL). Although only a few studies have been carried out evaluating the toxicity of Croton essential oils against snails as disease vectors, a number of essential oil components have been evaluated for their molluscicidal activity [32]. β-Caryophyllene exhibited strong toxicity against Bulinus truncatus with an LC 50 value of 1.66 µg/mL [116].

Material
In contrast to the molluscicidal activity, the antiparasitic activities of essential oils and single components have been extensively studied and have also been studied at the in-vivo level [117,118] (Tables 7 and 8). Tables 7 and 8 show that essential oils with high concentrations of β-caryophyllene and/or caryophyllene oxide showed a trend of stronger activity than other essential oils.

Material
Gram The antimicrobial activities of the essential oils of Croton species are summarized in Table 10. It is noteworthy that there are reports on the synergism of essential oils and antibiotics, although essential oils alone or antibiotics alone have shown weakness. Synergistic effect with cefepime.

Plant Material and Isolation of Essential Oil
The specimens of Croton hirtus L'Hér. were collected at Phong Dien Nature Reserve, Thua Thien Hue Province (16 • 24 15,84" N 107 • 12 00,01" E, 415 m elevation) in July 2022. The specimen (label: NCXS-H 110) of this species was identified by Van Huong Bui and was deposited in the Vietnam National Museum of Nature (VNMN) herbarium. The fresh aerial parts were chopped and hydrodistilled with a Clevenger apparatus (Witeg Labortechnik, Wertheim, Germany) for 6 h. The EO was dried over anhydrous Na 2 SO 4 and stored at 4 • C until use.

Gas Chromatographic Analysis
Gas chromatography-mass spectral analyses (GC-MS) of C. hirtus essential oil were carried out using previously published instrumentation and protocols [58,114]. A Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) with a ZB-5 ms fused silica capillary column (60 m length, 0.25 mm diameter, and 0.25 µm film thickness) (Phenomenex, Torrance, CA, USA), He carrier gas, 2.0 mL/min flow rate, injection and ion source temperatures of 260 • C, and a GC oven program of 50 to 260 • C at 2.0 • C/min was used. A 0.1 µL amount of a 5% (w/v) sample of essential oil in CH 2 Cl 2 was injected in split mode with a 24.5:1 split ratio. Identification of the essential oil components was carried out with a comparison of MS fragmentation and retention indices (RI) with those available in the databases [189][190][191][192]. Quantification was performed using external standards of representative compounds from each compound class.

Screening for Larvicidal Activity
Two species of Aedes mosquitoes were maintained continuously at Duy Tan University [193]. Egg rafts of Cx. fuscocephala were collected from rice fields in Hoa Vang district, Da Nang (GPS: 16 • 00 49" N, 108 • 06 12" E). Egg rafts of Cx. quinquefasciatus were collected from car tires containing decomposing organic matter in Da Nang City. Each Culex egg raft was hatched separately in plastic trays with tap water overnight to facilitate the precise examination of the species. The larvae were fed a mixture of dog food and yeast (ratio 3:1, w/w).
Essential oil and purified compounds were dissolved with ethanol (Sigma-Aldrich, Ho Chi Minh, Vietnam) to obtain a 1% stock solution. Twenty-five larvae of each mosquito species were transferred into 250 mL beakers containing 150 mL of distilled water. Different volumes of each the stock solution were transferred into the beakers containing larvae to obtain exposure concentrations of 100, 75, 50, 25, 12.5, and 6.25 µg/mL. Each concentration was cloned 4 times, and permethrin (Sigma-Aldrich) was used as a positive control. After 24 h and 48 h of exposure, the larvae were determined for mortality.

Screening for Molluscicidal Activity
Adult snails about 1.0 cm in size were collected in the wild (GPS: 16 • 01 08" N, 108 • 07 44" E), and they were acclimated to laboratory conditions 24 h prior to testing. The five snail adults were transferred into 200 mL plastic beakers, which were then filled with 195 mL of distilled water. The adults were exposed to concentrations of 50, 25, 12.5, 6.25, and 3.125 µg/mL. After 24 h of exposure, the snails were recovered by transferring them to plastic beakers containing only distilled water. After 24 h of recovery, the number of dead snails at the exposure concentrations was recorded. Copper sulfate (Xilong Chemicals, Shantou, China) was used as a positive control.

Screening for Antimicrobial Activity
The ATCC international standard for control of microorganisms include three Gramnegative bacteria strains (E. coli ATCC25922, P. aeruginosa ATCC27853, and S. enterica ATCC13076), three Gram-positive strains (E. faecalis ATCC299212, S. aureus ATCC25923, and B. cereus ATCC 14579), and a strain of C. albicans ATCC10231, which were provided by the National Institute for Food Control, Vietnam.
The antimicrobial activity was analyzed based on the multi-concentration dilution method. Samples of essential oils or pure compounds were diluted in DMSO at a decreasing concentration range of 256, 128, 64, 32, 16, 4, and 2 µg/mL, with three replicates for each concentration. Microbial solutions were prepared at a concentration of 2 × 10 5 CFU/mL, and antimicrobial assays were carried out in 96-well microtiter plates. A 5.12 µL sample solution with a 10 mg/ml concentration was aspirated into the first row containing 100 µL of LB medium and then diluted successively by concentration into rows containing 50 µL until reaching a concentration of 2 µg/mL. Then, 50 µL of microbial solution was added at a concentration of 2 × 10 5 CFU/ml and incubated at 37 • C. After incubation for 24 hours at 37 • C, the absorbance at 650 nm was measured using a microplate reader (Epoch, BioTek Instruments Inc., Winooski, VT, USA) [194]. Streptomycin, kanamycin, tetracycline, nystatin, and cycloheximide (all compounds were purchased from Sigma-Aldrich) were used as positive controls.

Data Analysis
Mortality data were analyzed by log-probit analysis [195] to acquire LC 50 and LC 90 values as well as 95% confidence limits using Minitab ® version 19.2020.1 (Minitab, LLC, State College, PA, USA).

Literature Survey
The materials used in this literature survey were searched on the databases https:// scholar.google.com, https://pubmed.ncbi.nlm.nih.gov, and https://www.researchgate.net (accessed on 1 January 2023) with a keyword structure including "essential oil" and Croton; bioactive keywords that were searched for included antimicrobial, antifungal, antibacterial, antiparasitic, and "mosquito larvicidal". There were no language restrictions for the selection of articles. The following criteria were included when considering articles: (1) The articles fully reported the chemical composition and mosquito larvicidal activity of the essential oils. (2) The articles fully reported the chemical composition and the molluscicidal and antiparasitic activities of the essential oils. (3) The articles fully reported the chemical composition and antibacterial activity of the essential oils. (4) Articles that reported unreliable GC/MS analysis results, such as chemical compositions that did not match the elution order, retention time, and retention index, were not considered.

Conclusions
This work presented a literature survey of the volatile phytochemistry and biological activities of Croton species and illustrated the potential utility of these essential oils. Furthermore, the essential oil composition, mosquito larvicidal, molluscicidal, and antimicrobial activities of Croton hirtus from Vietnam was included, which adds to our knowledge of the genus Croton. β-Caryophyllene occurred abundantly in the essential oils [196], and it was present in most of the essential oils of the Croton species. Mixtures of β-caryophyllene, α-humulene, and caryophyllene oxide showed synergistic or antagonistic effects against various organisms. Investigations into the bioactivity of combinations of β-caryophyllene with other major compounds in their respective percentages in the essential oils will help in the development of β-caryophyllene-based products. Based on the results of the antiparasitic activity of β-caryophyllene, caryophyllene oxide, and the Croton essential oils containing these two compounds, we suggest that an investigation into the antiparasitic activity of C. hirtus essential oil may provide interesting results.