Premna Species in Vietnam: Essential Oil Compositions and Mosquito Larvicidal Activities

Essential oils have emerged as viable alternatives to synthetic insecticides for control of mosquito-borne pathogens. The leaf essential oils of eight species of Premna (Lamiaceae) growing in central Vietnam have been obtained by hydrodistillation and analyzed by gas chromatography–mass spectrometry. Sesquiterpene hydrocarbons dominated most of the Premna essential oils, with the notable exception of Premna mekongensis from Ngoc Linh Nature Reserve, which had α-pinene as the major component. Larvicidal activities against Aedes aegypti have been determined and all of the Premna essential oils showed larvicidal activity with 24-h LC50 < 65 μg/mL. The leaf essential oils of Premna cambodiana from Chu Mom Ray National Park and Premna mekongensis from Ngoc Linh Nature Reserve showed the best larvicidal activities with 24-h LC50 of 16.8 and 18.0 μg/mL, respectively. The essential oil compositions and larvicidal activities of P. cambodiana, Premna flavescens, Premna maclurei, P. mekongensis, and Premna puberula are reported for the first time. Although the larvicidal activities of Premna leaf essential oils are promising, the essential oil yields are relatively low (0.10–0.25%).


Introduction
Mosquito-borne infectious diseases have been a persistent problem in Vietnam. Dengue fever and dengue hemorrhagic fever are especially problematic and chikungunya fever is an emerging threat in the country [1,2]. Aedes aegypti (L.) (Diptera: Culicidae), the yellow fever mosquito, and Aedes albopictus (Skuse) (Diptera: Culicidae), the Asian tiger mosquito, are important vectors of several viral pathogens, including dengue fever virus [3], yellow fever virus [4], chikungunya fever virus [5], and possibly Zika virus [6]. Culex quinquefasciatus Say (Diptera: Culicidae), the southern house mosquito, is a vector of

Essential Oil Compositions
The Premna leaf essential oils were analyzed by gas chromatography-mass spectrometry and the chemical compositions are summarized in Table 3.

Premna cambodiana
A total of 72 compounds were tentatively identified in the leaf essential oil of P. cambodiana, accounting for 97.4% of the total composition (Table 3). Sesquiterpene hydrocarbons dominated P. cambodiana leaf essential oil with α-copaene (23.3%), α-gurjunene (11.3%), (E)-caryophyllene (12.8%), and δ-cadinene (5.5%) as the major sesquiterpene components. There have been no previous phytochemical investigations on P. cambodiana reported in the literature; this is the first report on its essential oil composition.

Premna chevalieri
Eighty-five components (99.8% of the composition) were tentatively identified in P. chevalieri essential oil. The major components in the leaf essential oil of P. chevalieri were the sesquiterpenes (E)-caryophyllene (31.5%) and α-humulene (7.5%) and the monoterpenes α-pinene (12.2%) and β-pinene (16.8%) ( Table 3). There have been no previous phytochemical investigations on P. chevalieri reported in the literature; this is the first report on the leaf essential oil composition of this plant.

Premna mekongensis
Essential oils were obtained from leaves of P. mekongensis from two different locations, Ngoc Linh Nature Reserve in Quang Nam Province, and Chu Mom Ray National Park. The leaf essential oil compositions are listed in Table 3. The two samples showed very different compositions. The Ngoc Linh sample was dominated by α-pinene (66.9%) and (E)-caryophyllene (14.7%). The leaf essential oil from Chu Mom Ray, on the other hand, had relatively low concentrations of α-pinene (1.5%) and (E)-caryophyllene (3.9%). In addition, the Chu Mom Ray essential oil was much more complex with 95 identified components compared to only 37 in the Ngoc Linh sample. The high concentration of α-pinene in P. mekongensis leaf essential oil from Ngoc Linh was unexpected and uncharacteristic of Premna leaf essential oils, which are generally low in monoterpene hydrocarbon concentrations (see below). To our knowledge, there have been no previous studies on the essential oil composition of P. mekongensis.

Premna tomentosa
The leaf essential oil composition of P. tomentosa is shown in Table 3. A total of 82 compounds were tentatively identified in the essential oil accounting for 99.8% of the composition, which was dominated by sesquiterpene hydrocarbons, especially (E)-caryophyllene (22.0%) and germacrene D (11.4%). The only previous examination of the essential oil of P. tomentosa is a relatively old work by Narayan and Muthana in 1953 [48]. These workers identified limonene (57.8%), (E)-caryophyllene (17.2%), an unidentified cadinane sesquiterpene (7.8%), an unidentified sesquiterpene alcohol (5.6%), and an unidentified diterpene hydrocarbon (5.5%) in the leaf essential oil from southern India.

Mosquito Larvicidal Activity
The Premna leaf essential oils have been screened for mosquito larvicidal activity against Aedes aegypti and, if sufficient mosquito larvae were available, also against Ae. albopictus and Culex quinquefasciatus. The 24-h and 48-h larvicidal activities are shown in Tables 4 and 5, respectively. Considering larvicidal activities against Ae. aegypti, the most active Premna leaf essential oils were P. cambodiana (24-h LC 50 = 16.8 µg/mL) and P. mekongensis from Nghe An (24-h LC 50 = 16.8 µg/mL).

Plant Material
Leaves of Premna species were collected from several different locations in central Vietnam ( Table 1). The plants were identified by Dr. Do Ngoc Dai, and voucher specimens (see Table 2) have been deposited in the plant specimen room, Faculty Agriculture, Forestry, and Fishery, Nghe An, College of Economics. The fresh leaves (2.0 kg each), immediately after collection, were shredded and hydrodistilled for 4 h using a Clevenger type apparatus (Witeg Labortechnik, Wertheim, Germany). Essential oil yields are summarized in Table 2. Essential oils were dried over anhydrous Na 2 SO 4 and stored in sealed glass vials at 4 • C until analyzed.

Gas Chromatography-Mass Spectrometry
The Premna leaf essential oils were analyzed by gas chromatography-mass spectrometry (GC-MS) as described previously [56]: Shimadzu GCMS-QP2010 Ultra, electron impact (EI) mode (electron energy = 70 eV), scan range = 40-400 atomic mass units, scan rate = 3.0 scans/s; ZB-5ms column (30 m length × 0.25 mm inner diameter × 0.25 µm film thickness); He carrier gas, head pressure of 552 kPa, flow rate of 1.37 mL/min; injector temperature was 250 • C, ion source temperature was 200 • C; GC oven temperature program: 50 • C initial temperature, increased 2 • C/min to 260 • C; 5% solution of essential oil in CH 2 Cl 2 , 0.1 µL injection, splitting ratio 30:1. Putative identification of the essential oil components was based on their calculated retention indices (RI), based on a homologous series of n-alkanes (C 8 -C 40 ), and their mass spectral fragmentation patterns compared with those reported in the databases [42][43][44][45], with RI values within ±10 units and with matching factors >80%. The concentrations of the essential oil components were calculated from raw peak areas, normalized to 100%, without standardization.

Mosquito Larvicidal Assay
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 Premna essential oils were determined following the protocol previously reported [62]: 250-mL beakers, 150 mL of water, and 20 larvae (fourth instar), aliquots of the Premna essential oils dissolved in EtOH (1% stock solution) were added to give final concentrations of 100, 50, 25, 12.5, and 6 µg/mL; EtOH only (negative control) and permethrin (positive control), mortality recorded after 24 h and 48 h of exposure, experiments were carried out at 25 ± 2 • C, each test was conducted with four replicates. The data obtained were subjected to log-probit analysis [63] to obtain