Chemical Compositions, Mosquito Larvicidal and Antimicrobial Activities of Essential Oils from Five Species of Cinnamomum Growing Wild in North Central Vietnam

Members of the genus Cinnamomum (Lauraceae) have aromatic volatiles in their leaves and bark and some species are commercially important herbs and spices. In this work, the essential oils from five species of Cinnamomum (C. damhaensis, C. longipetiolatum, C. ovatum, C. polyadelphum and C. tonkinense) growing wild in north central Vietnam were obtained by hydrodistillation, analyzed by gas chromatography and screened for antimicrobial and mosquito larvicidal activity. The leaf essential oil of C. tonkinense, rich in β-phellandrene (23.1%) and linalool (32.2%), showed excellent antimicrobial activity (MIC of 32 μg/mL against Enterococcus faecalis and Candida albicans) and larvicidal activity (24 h LC50 of 17.4 μg/mL on Aedes aegypti and 14.1 μg/mL against Culex quinquefasciatus). Cinnamomum polyadelphum leaf essential oil also showed notable antimicrobial activity against Gram-positive bacteria and mosquito larvicidal activity, attributable to relatively high concentrations of neral (11.7%) and geranial (16.6%). Thus, members of the genus Cinnamomum from Vietnam have shown promise as antimicrobial agents and as potential vector control agents for mosquitoes.


Introduction
The Lauraceae is a large family of tropical and subtropical trees and shrubs [1]. In this family, the genus Cinnamomum is comprised of around 250 species with concentrations in east and southeast Asia [1]. Vietnam is home to 45 species of Cinnamomum [2,3], many of which are used in traditional medicine, for essential oils, as well as for timber [4,5].
Mosquitoes have been and continue to be the deadliest animals on earth. Aedes aegypti (L.) (Diptera: Culicidae) and Ae. albopictus (Skuse) are vectors for the arboviral diseases dengue, Zika, chikungunya and yellow fever and Ae. aegypti is also a vector for the emerging Rift Valley fever virus [20]. Culex quinquefasciatus (Say) is a vector of West Nile virus, Saint Louis encephalitis virus and lymphatic filariasis [21]. Culex quinquefasciatus may also serve as a vector in emerging viral diseases such as Zika virus [22], Sindbis virus [23] and Usutu virus [24]. Unfortunately, insecticidal resistance of these mosquito species is increasing leading to failure of vector control programs in many locations [25]. Furthermore, populations of Ae. aegypti [26], Ae. albopictus [27] and Cx. quinquefasciatus [28] are showing widespread resistance to commonly used larvicidal agents. It has been suggested that essential oils may serve as alternative and more ecologically benign mosquito larvicidal agents [29][30][31].
Because of the biological activities and traditional uses of members of the Cinnamomum genus, we hypothesize that Cinnamomum species from Vietnam may also exhibit potentially useful biological activities. As part of our ongoing investigations into the essential oils of Vietnamese Cinnamomum [32][33][34]

Essential Oil Collection and Analysis
Plant materials were collected from mature Cinnamomum trees from different locations in north central Vietnam. The collection details and essential oil yields of the Cinnamomum species are summarized in Table 1. The essential oils were analyzed by gas-chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detector (GC-FID). The chemical compositions of the Cinnamomum species are presented in Table 2.

Antimicrobial Screening
The Cinnamomum essential oils were screened for antimicrobial activity against Gram-positive (Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica) bacteria and a yeast (Candida albicans). Minimum inhibitory concentrations and IC 50 values were determined using the microbroth dilution assay (Table 3).

Larvicidal Screening
The Cinnamomum essential oils were screened for mosquito larvicidal activity against Aedes aegypti, Aedes albopictus and Culex quinquefasciatus. The 24 h and 48 h LC 50 and LC 90 values are summarized in Tables 4 and 5.

Cinnamomum ovatum
The leaf and stem bark essential oils of C. ovatum demonstrated broad antimicrobial activity against the organisms tested with MIC values ranging from 16 to 128 µg/mL ( Table 3). The major components of the leaf and stem essential oils were eugenol (70.5% and 71.2%, respectively), eugenyl acetate (9.5% and 9.3%, respectively) and linalool (5.9% and 8.3%, respectively) ( Table 2). The high concentration of eugenol in these two essential oils is likely responsible for the observed antimicrobial effects. Eugenol has shown broad spectrum antibacterial [35,36] and antifungal [37][38][39] activities. Likewise, the mosquito larvicidal activity of C. ovatum leaf essential oil is likely due to eugenol; that compound has shown larvicidal activity against Ae. aegypti [40], Ae. albopictus [41] and Cx. quinquefasciatus [42].

Plant Material
Leaves or stem bark of the Cinnamomum species were collected from locations in north central Vietnam (see Table 1). Plants were identified by Do N. Dai and voucher specimens (Table 1) have been deposited in the plant specimen room, Faculty Agriculture, Forestry and Fishery, Nghe An, College of Economics. The fresh plant materials (2.0 kg each) were shredded and hydrodistilled using a Clevenger apparatus for 4 h to give the essential oils. The essential oil yields are summarized in Table 1.

Gas Chromatographic Analysis
Gas chromatography (GC) analysis was performed on an Agilent Technologies (Santa Clara, CA, USA) HP 7890A Plus Gas chromatograph equipped with a flame ionization detector (FID) and fitted with HP-5ms column (30 m × 0.25 mm, film thickness 0.25 µm, Agilent Technologies). The analytical conditions were-carrier gas H 2 (1 mL/min), injector temperature (PTV) 250 • C, detector temperature 260 • C, column temperature programmed from 60 • C (2 min hold) to 220 • C (10 min hold) at 4 • C/min. Samples were injected by splitting and the split ratio was 10:1. The volume injected was 1.0 µL. Inlet pressure was 6.1 kPa.
An Agilent Technologies (Santa Clara, California, USA) HP 7890A Plus Chromatograph fitted with a fused silica capillary HP-5ms (30 m × 0.25 mm, film thickness 0.25 µm) and interfaced with a mass spectrometer (HP 5973 MSD) was used for the GC-MS analysis, under the same conditions as those used for GC-FID analysis. The conditions were the same as described above with He (1 mL/min) as carrier gas. The MS conditions were as follows-ionization voltage 70 eV; emission current 40 mA; acquisitions scan mass range of 35-350 amu at a sampling rate of 1.0 scan/s. The identification of constituents was performed on the basis of retention indices (RI) determined with reference to a homologous series of n-alkanes, under identical experimental conditions, co-injection with standards (Sigma-Aldrich, St. Louis, MO, USA) or known essential oil constituents, MS library search (NIST 08 and Wiley 9 th Version) and by comparing with MS literature data [64]. The relative amounts of individual components were calculated based on the GC peak area (FID response) without using correction factors.
Minimum inhibitory concentration (MIC) and median inhibitory concentration (IC 50 ) values were measured by the microdilution broth susceptibility assay [65]. Stock solutions of the oil were prepared in dimethylsulfoxide. Dilution series were prepared from 16,384 to 2 µg/mL (2 14 , 2 13 , 2 12 , 2 11 , 2 10 , 2 9 , 2 7 , 2 5 , 2 3 and 2 1 µg/mL) in sterile distilled water in micro-test tubes from where they were transferred to 96-well microtiter plates. Bacteria grown in double-strength Mueller-Hinton broth or double-strength tryptic soy broth and fungi grown in double-strength Sabouraud dextrose broth were standardized to 5 × 10 5 and 1 × 10 3 CFU/mL, respectively. The last row, containing only the serial dilutions of sample without microorganisms, was used as a positive (no growth) control. Sterile distilled water and medium served as a negative (no antimicrobial agent) control. Streptomycin was used as the antibacterial standard, nystatin and cycloheximide were used as antifungal standards. After incubation at 37 • C for 24 h, the MIC values were determined to be the well with the lowest concentration of agents completely inhibiting the growth of microorganisms. The IC 50 values were determined by the percentage of microorganisms that inhibited growth based on the turbidity measurement data of EPOCH2C spectrophotometer (BioTeK Instruments, Inc Highland Park Winooski, VT, USA) and Rawdata computer software (Brussels, Belgium) according to the following equations: where OD is the optical density, control(-) are the cells with medium but without antimicrobial agent, test agent corresponds to a known concentration of antimicrobial agent, control(+) is the culture medium without cells, High conc /Low conc is the concentration of test agent at high concentration/low concentration and High inh% /Low inh% is the % inhibition at high concentration/% inhibition at low concentration). Each of the antimicrobial screens were carried out in triplicate.

Larvicidal Screening
Eggs of Aedes aegypti were purchased from Institute of Biotechnology, Vietnam Academy of Science and Technology and maintained at the Laboratory of Department of Pharmacy of Duy Tan University, Da Nang, Vietnam. Adults of Culex quinquefasciatus and Aedes albopictus collected in Hoa Khanh Nam ward, Lien Chieu district, Da Nang city (16 • 03 14.9"N, 108 • 09 31.2"E) and were identified by National institute of Malariology, Parasitology and Entomology, Ho Chi Minh City. Adult mosquitoes were maintained in entomological cages (40 × 40 × 40 cm) and fed a 10% sucrose solution and were allowed to blood feed on 1-week-old chicks and mice, respectively. Egg hatchings were induced with tap water. Larvae were reared in plastic trays (24 × 35 × 5 cm). The larvae were fed on Koi fish food. All developmental stages were maintained at 25 ± 2 • C, 65-75% relative humidity and a 12:12 h light:dark cycle at the Laboratory of the Faculty of Environmental and Chemical Engineering of Duy Tan University, Da Nang, Vietnam.
Larvicidal activities of the Cinnamomum essential oils were evaluated according to the protocol Liu and co-workers [66] with slight modifications. For the assay, 150 mL of water that contained 20 larvae (fourth instar) was placed in 250-mL beakers and aliquots of the Cinnamomum essential oils dissolved in EtOH (1% stock solution) were then added. With each experiment, a set of controls using EtOH only (negative control) and permethrin (positive control) were also run for comparison. Mortality was recorded after 24 h and again after 48 h of exposure during which no nutritional supplement was added. The experiments were carried out at 25 ± 2 • C. Each test was conducted with four replicates with five concentrations (100, 50, 25, 12.5 and 6 µg/mL). The data obtained were subjected to log-probit analysis [67] to obtain LC 50 values, LC 90 values and 95% confidence limits using Minitab ® 19 (Minitab, LLC, State College, PA, USA).

Conclusions
The essential oils of five species of Cinnamomum were collected from north central Vietnam and screened for antimicrobial and mosquito larvicidal activities. According to Duarte and co-workers [68], essential oils with MIC values between 50 and 500 µg/mL can be considered to have strong antimicrobial activity. Similarly, Dias and Moraes have concluded that essential oils with LC 50 < 100 µg/mL are considered to be active [69]. Therefore, all of the Cinnamomum essential oils in this study can be considered to be active and show promise as antimicrobial agents and as alternative insecticidal agents against mosquito larvae.