Chemical Compositions, Mosquito Larvicidal and Antimicrobial Activities of Leaf Essential Oils of Eleven Species of Lauraceae from Vietnam

The Lauraceae is a family rich in aromatic and medicinal plants. Likewise, essential oils derived from members of this family have demonstrated a myriad of biological activities. It is hypothesized that members of the Lauraceae from Vietnam will yield essential oils that may be useful in controlling mosquito populations and treating microbial infections. In this work, the leaf essential oils of eleven species of Lauraceae (Beilschmiedia erythrophloia, B. robusta, B. yunnanensis, Cryptocarya concinna, C. impressa, C. infectoria, Litsea viridis, Machilus balansa, M. grandifolia, Neolitsea ellipsoidea, and Phoebe angustifolia) have been obtained by hydrodistillation and the chemical compositions analyzed by gas chromatography – mass spectrometry (GC-MS) and gas chromatography with flame ionization detection (GC-FID). The essential oils were screened for larvicidal activity against Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus, and for antimicrobial activity against Enterococcus faecalis, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and Candida albicans. The leaf essential oil of N. ellipsoidea, rich in (E)-β-ocimene (87.6%), showed excellent larvicidal activity against Ae. aegypti with a 24 h LC50 of 6.59 μg/mL. The leaf essential oil of C. infectoria, dominated by germacrene D (55.5%) and bicyclogermacrene (11.4%), exhibited remarkable larvicidal activity against Cx. quinquefasciatus (48 h LC50 = 0.40 μg/mL). N. ellipsoidea leaf essential oil also demonstrated notable antibacterial activity against E. faecalis and B. cereus with minimum inhibitory concentration (MIC) values of 16 μg/mL, while the leaf essential oil of C. impressa showed excellent anticandidal with an MIC of 16 μg/mL. Leaf essential oils from the Lauraceae should be considered for utilization as alternative agents for controlling mosquito populations and as antimicrobial agents.

Based on the utility and properties of Lauraceae essential oils, it is hypothesized that members of the Lauraceae found in Vietnam have biologically active essential oils that may be useful in controlling mosquito populations or as antimicrobial agents. Eleven species of Lauraceae from north-central Vietnam have been collected, the essential oils obtained by hydrodistillation, chemical compositions analyzed, and the oils screened for mosquito larvicidal activity and for antimicrobial activity.
The genus Beilschmiedia Nees is comprised of around 250 species of trees and shrubs [9] and are widespread in tropical Africa, Madagascar, Asia, Southeast Asia, Melanesia, Australia, New Zealand, have been isolated from the methanol bark extract of C. infectoria [27,28]. The isoquinoline alkaloids atherosperminine, N-methylisococlaurine, and N-methyllaurotetanine have also been isolated from the bark of C. infectoria [29]. There have been apparently no essential oil analyses on this plant, however.
The genus Litsea Lam. consists of around 300 species distributed in tropical and warm subtropical regions of Asia, Australia, and the Americas [19]. Litsea viridis H. Liu is a small tree, 3-6 m tall, found in south-eastern Yunnan province (China) and Cao Bằng, Nghệ An, Đà Nẵng, and Đắk Lắk provinces (Vietnam) [12,14]. There do not seem to be any previous studies on the phytochemistry of this plant.
The genus Machilus Rumph. ex Nees is comprised of around 100 species distributed in southern and south-eastern Asia [12,14]. Machilus balansae (Airy Shaw) F.N. Wei & S.C. Tang (syn. Persea balansae Airy Shaw) is endemic to Vietnam and is generally found at low elevations in north Vietnam [30]. Machilus grandifolia S.K. Lee & F.N. Wei is now regarded as a new synonym of M. balansae [30]. To our knowledge, there have been no phytochemical studies reported on M. balansae or M. grandifolia.
The genus Neolitsea (Benth.) Merr. Contains around 85 species distributed from Indo-Malaysia to East Asia [12,14]. Neolitsea ellipsoidea K.C. Allen is a tree up to 30 m in height [31]. The species has been recorded in Hainan (China) and Vietnam (Hoà Bình, Quảng Ninh, Hà Tĩnh, and Gia Lai provinces). To our knowledge there have been no reports on the phytochemistry of this species.
There are around 100 species in the genus Phoebe Nees [19], which range from the Neotropics (Mexico, south to Brazil, Bolivia, and Argentina) and Southeast Asia (southern China, Vietnam, Thailand, Myanmar, Cambodia, and Singapore), as well as Indonesia, New Guinea, and India [9].

Results and Discussion
The essential oil collection details and yields are summarized in Table 1.

Essential Oil Compositions
The essential oil compositions of B. erythrophloia, B. robusta, and B. yunnanensis are compiled in Table 2. All three of the Beilschmiedia leaf essential oils were dominated by sesquiterpene hydrocarbons. A preponderance of sesquiterpene hydrocarbons has been previously seen in Beilschmiedia leaf essential oils from Malaysia [33] and from Costa Rica [34]. The major components in B. erythrophloia essential oil were bicyclogermacrene (30.5%), (Z)-β-ocimene (26.1%), and (E)-caryophyllene (18.3%). While qualitatively similar, there are notable differences between the essential oil from Vietnam in this work and that reported by Su and Ho from Taiwan [17]; the sample from Taiwan was rich in α-humulene (21.9%) compared to that from Vietnam (only 2.6%), but poor in bicyclogermacrene (1.2%) compared to that from Vietnam.
The leaf essential compositions of C. concinna (from two locations), C. impressa, and C. infectoria are listed in Table 3. Sesquiterpene hydrocarbons were abundant in both C. impressa and C. infectoria leaf essential oils, while oxygenated sesquiterpenoids were abundant in C. concinna essential oil from Nam Dong and monoterpene hydrocarbons dominated the leaf oil of C. concinna from Pu Hoat. 1046   1601 The leaf essential oils of C. concinna from two different collection sites were qualitatively similar, but quantitatively different. That is, the abundant components in the Nam Dong sample were also observed in the Pu Hoat sample, and vice versa. Thus, for example, α-pinene, β-pinene, and myrcene were abundant in the Pu Hoat sample (26.7%, 31.3%, and 11.1%, respectively) but were found in lower concentrations in the sample from Nam Dong (8.2%, 9.0%, and 3.9%). Conversely, the sesquiterpenoids, (E)-caryophyllene, spathulenol, and caryophyllene oxide were abundant in the sample from Nam Dong (12.2%, 12.3%, and 21.2%, respectively), but less concentrated in the Pu Hoat sample (5.3%, 1.1%, and 0.4%).
Although M. balansae and M. grandifolia are considered conspecific, the essential oil compositions showed pronounced differences. The leaf oil of M. balansae was dominated by bicyclogermacrene (41.5%), which was not detected in the essential oil of M. grandifolia. Likewise, the sesquiterpene alcohols (E)-nerolidol and globulol were abundant constituents in M. grandifolia (22.7% and 10.2%, respectively), but (E)-nerolidol was much lower in M. balansae (8.7%) and globulol was not detected. The taxonomy of these two plants deserves closer scrutiny.

Larvicidal Activity
The 24-h and 48-h larvicidal activities of Lauraceae leaf essential oils from Vietnam are summarized in Tables 5 and 6. Note that several essential oils were not tested due to lack of sufficient essential oil.

Antimicrobial Activity
Several of the leaf essential oils of the Lauraceae were screened for antimicrobial activity ( Table 7).
All of the essential oils tested showed good antibacterial activities against the Gram-positive organisms. Both L. viridis and N. ellipsoidea leaf essential oils demonstrated particularly notable activities against E. faecalis and B. cereus with minimum inhibitory concentration (MIC) values of 16 µg/mL. The leaf essential oil of C. impressa also showed excellent anticandidal activity against C. albicans with an MIC of 16 µg/mL.
(E)-β-Ocimene dominated the leaf essential oil of N. ellipsoidea, but this compound has demonstrated relatively marginal antibacterial activity [55]. Synergistic interactions of (E)-β-ocimene with minor essential oil components may play a role in the antibacterial activity of N. ellipsoidea leaf oil.
The components responsible for the anticandidal activity of C. impressa leaf essential oil are not obvious. Neither (E)-caryophyllene nor α-humulene have shown strong anti-Candida albicans activity [54,56]. The anticandidal activity of bicyclogermacrene itself has apparently not been determined. However, essential oils rich in both bicyclogermacrene and (E)-caryophyllene do not exhibit notable activity against Candida spp. [59,60]. Dodecanal, however, has shown activity against C. albicans with an MIC of 125 µg/mL [61].

Plant Collection
Leaves were collected from wild-growing trees in north-central Vietnam. Plants were identified by Do Ngoc Dai and voucher specimens (Table 1) have been deposited in the plant specimen room, Faculty Agriculture, Forestry and Fishery, Nghe An, College of Economics. In each case, the fresh leaves were chopped and 2.0 kg was subjected to hydrodistillation using a Clevenger-type apparatus.

Analysis of the Oils
Gas chromatographic (GC) analysis was performed on an Agilent Technologies HP 7890A Plus Gas chromatograph equipped with a FID and fitted with HP-5ms column (30 m × 0.25 mm, film thickness 0.25 µm, Agilent Technologies, Santa Clara, CA, USA). The analytical conditions were: carrier gas H 2 (1 mL/min), injector temperature (PTV: programmable temperature vaporization) 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 using a split mode with a split ratio of 10:1. The volume injected was 1.0 µL. Inlet pressure was 6.1 kPa.
An Agilent Technologies (Santa Clara, CA, USA) HP 7890A Plus Chromatograph fitted with a fused silica capillary HP-5ms column (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 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. Compound identification was carried out by comparison of the MS fragmentation patterns and calculated retention indices with those available in the databases [35][36][37] and, when available, with standard substances.

Conclusions
Of the eleven species of Lauraceae examined in this work, the leaf essential oil of Neolitsea ellipsoidea, dominated by (E)-β-ocimene, showed excellent larvicidal activity against Aedes aegypti and antibacterial activity against Enterococcus faecalis and Bacillus cereus; Cryptocarya infectoria leaf essential oil, rich in germacrene D and bicyclogermacrene, showed excellent larvicidal activity on Culex quinquefasciatus and anticandidal activity against Candida albicans. The leaf essential oil of Litsea viridis, which was rich in bicyclogermacrene, also showed good antibacterial properties. The biological properties of these Lauraceae essential oils suggest that they may serve as potential "green" alternatives, as also described for Lamiaceae family plants [63], for use as insect control or antimicrobial agents.