Mosquito Larvicidal Activities and Chemical Compositions of the Essential Oils of Leucas Zeylanica Growing Wild in Vietnam

Abstract

Dengue fever and dengue hemorrhagic fever are hyperendemic to Vietnam with frequent outbreaks and epidemics. Control of the vectors for dengue viruses, Aedes mosquitoes, is critical to maintain control of this disease. In this work, the essential oil of Leucas zeylanica was examined for larvicidal activity against the Aedes mosquito vector. Leucas zeylanica was collected from three different locations in Vietnam. The essential oil was obtained by hydrodistillation and analyzed by gas chromatography—mass spectrometry. The essential oil from the aerial parts of L. zeylanica was screened for mosquito larvicidal activity against Aedes aegypti and Aedes albopictus. The major components of L. zeylanica essential oil from Vietnam are β-caryophyllene, α-humulene, germacrene D, β-selinene, caryophyllene oxide, 1-tetradecanol, and phytol. Leucas zeylanica essential oil showed good larvicidal activity against both Ae. albopictus and Ae. aegypti with median lethal concentration—LC₅₀ (24 hours) values of 67 and 44 μg/mL, respectively. Thus, L. zeylanica essential oil represents an inexpensive and environmentally benign agent for control of the dengue virus vectors.

Keywords

dengue fever sesquiterpenes

Aedes mosquitoes are one of the most important vectors of arboviruses such as dengue,¹ yellow fever,² Chikungunya,³ Zika,⁴ and many other diseases affecting humans and animals—the cause of more than one million deaths worldwide per year.⁵ The World Health Organization estimates that there are at around 390 million dengue infections annually.⁶ Vietnam is a hyperendemic dengue country with dengue fever epidemics becoming more frequent and widespread. Between 1963 and 1995, there were more than 1.5 million dengue hemorrhagic fever cases and more than 14 000 reported deaths.⁷

Leucas zeylanica (L.) R. Br. (syn. Phlomis zeylanica L., Leucas sericea Elm., and also called Bạch thiệt, Mè hoang, Mè đất, Tổ ong, Phòng phong thảo, Cỏ man màn, Tích tú phòng phong, in Vietnam) is an annual herb belonging to the family Lamiaceae.^8
-10 In Vietnam, L. zeylanica is distributed in Hòa Bình, Hà Nội, Hải Phòng, Nam Định, Ninh Bình, Thanh Hóa, Nghệ An, Thừa Thiên Huế, Đà Nẵng, Quãng Ngải, Khánh Hòa, Đồng Nai, Ho Chi Minh, and Kiên Giang provinces. The plant is also found in China, India, Sri Lanka, Nepal, Bangladesh, Myanmar, Thailand, Cambodia, Malaysia, Indonesia, Philippines, and New Guinea.⁹ The plant is a weed of sunny dry localities, often on sandy soils, paddy dams, waste places, sea shores, and roadsides; at elevations from the sea level up to 1000 m.^8,9 In Vietnam,¹¹ Malaysia,¹² and Sri Lanka,¹³ decoction of the leaves is taken to treat worm infestations, while the leaves are used externally to treat scabies infections of animals.¹⁴ Based on the traditional uses of L. zeylanica to treat scabies and helminth infections, we hypothesized that L. zeylanica essential oil may be an effective larvicidal agent to control Ae. aegypti and Ae. albopictus populations and help alleviate dengue outbreaks in Vietnam. In this work, we present the essential oil composition of L. zeylanica collected in Vietnam and its larvicidal activity against Ae. albopictus and Ae. aegypti.

The essential oils from the aerial parts, leaves, and stems were obtained in 0.015%, 0.012%, 0.027%, and 0.008% yields, respectively. The essential oil compositions are compiled in Table 1. The major components of the essential oil from the aerial parts and leaves of L. zeylanica were the sesquiterpenoids, β-caryophyllene, α-humulene, germacrene D, β-selinene, and caryophyllene oxide, as well as 1-tetradecanol, and phytol. Essential oil from the stem was found to be rich in tetradecanol, palmitic acid, and alkanes.

Table 1

Essential Oil Compositions of Leucas zeylanica from Vietnam.

RI	Compound	%
RI	Compound	AP-DN^a	AP-QN	L-EO	S-EO
932	α-Pinene	0.9	0.7	1.7	0.1
971	Sabinene	–^b	–	tr^c	–
977	1-Octen-3-ol	0.5	–	–	–
977	β-Pinene	–	–	0.1	–
983	3-Octanone	0.3	–	–	–
988	Myrcene	0.1	0.3	0.2	–
996	3-Octanol	0.1	–	–	–
1024	p-Cymene	–	0.1	–	–
1028	Limonene	0.4	0.3	0.5	–
1099	Linalool	0.1	0.1	–	–
1104	Nonanal	0.1	–	0.1	–
1112	(E)−4,8-Dimethylnona-1,3,7-triene	0.1	–	–	–
1206	Decanal	–	–	–	0.1
1287	Dihydroedulan I	0.1	0.1	–	–
1292	Dihydroedulan II	0.1	0.1	–	–
1300	Tridecane	0.2	0.1	0.1	–
1333	γ-Lonone	0.2	0.1	0.2	–
1375	α-Copaene	1.8	1.2	2.3	0.7
1377	(E)-β-Damascenone	–	–	tr	–
1381	cis-β-Elemene	0.1	–	0.1	–
1383	β-Bourbonene	0.8	0.5	1.2	0.4
1387	β-Cubebene	0.3	0.3	0.4	–
1388	β-Elemene	1.5	1.0	1.2	0.2
1400	Nonyl propanoate	–	0.1	0.2	0.3
1402	Sesquithujene	0.1	–	–	–
1402	Cyperene	–	0.1	–	–
1408	Dodecanal	0.1	–	0.1	0.2
1412	Geosmin	–	0.1	0.2	–
1413	cis-α-Bergamotene	0.1	–	–	–
1419	β-Caryophyllene	25.5	21.4	23.9	5.7
1429	β-Copaene	0.3	0.3	0.4	0.1
1432	trans-α-Bergamotene	0.3	0.1	0.2	0.1
1443	Aromadendrene	0.1	–	0.1	–
1446	epi-β-Santalene	–	0.1	–	–
1451	(E)-β-Farnesene	1.3	0.4	1.0	0.4
1454	α-Humulene	9.8	8.4	9.4	2.7
1460	6-Demethoxyageratochromene (=precocene I)	0.2	–	–	–
1460	4,6,8,10-Tetramethyltridecane	–	–	0.1	–
1461	cis-Muurola-4(14),5-diene	–	–	0.1	–
1467	cis-Cadina-1(6),4-diene	0.1	–	0.1	–
1472	β-Chamigrene	0.6	0.8	1.1	0.5
1475	γ-Muurolene	0.3	0.6	0.4	0.2
1477	(E)-β-Ionone	0.1	–	–	–
1482	Germacrene D	14.6	2.8	15.9	0.9
1482	γ-Himachalene	0.7	0.4	0.2	0.2
1490	β-Selinene	9.3	11.9	15.0	9.3
1491	trans-Muurola-4(14),5-diene	0.1	0.1	0.1	–
1495	α-Selinene	0.7	1.0	1.2	0.8
1497	α-Muurolene	0.2	0.2	0.2	0.1
1500	Pentadecane	0.3	0.3	0.2	0.3
1502	(E,E)-α-Farnesene	0.2	–	0.2	–
1506	β-Bisabolene	0.2	0.1	0.2	0.1
1508	Bicyclogermacrene	0.1	–	0.1	–
1512	δ-Amorphene	0.1	0.1	0.1	-
1514	Cubebol	0.1	0.1	0.1	0.1
1517	δ-Cadinene	1.7	0.7	2.2	0.3
1522	β-Sesquiphellandrene	0.1	–	–	–
1531	trans-Cadina-1,4-diene	–	–	0.1	–
1533	Dodec-5-en-11-olide	–	0.1	–	–
1540	α-Calacorene	–	0.1	–	–
1550	Isocaryophyllene oxide	0.1	0.5	0.1	0.2
1559	(E)-Nerolidol	0.2	0.2	0.1	0.2
1575	Caryophyllenol	–	–	–	0.1
1576	Germacra-1(10),5-dien-4β-ol	0.1	–	0.1	–
1576	Spathulenol	–	0.4	–	–
1577	1-Tridecanol	0.4	0.4	0.3	0.3
1581	Caryophyllene oxide	1.7	8.4	1.4	5.2
1584	Isocaryphyllenol	–	0.1	–	–
1600	Hexadecane	0.6	0.8	0.4	1.0
1608	Humulene epoxide II	0.4	1.9	0.4	1.5
1612	Tetradecanal	0.1	–	0.1	0.3
1632	iso-Spathulenol	0.1	–	–	–
1636	Caryophylla-4(12),8(13)-dien-5β-ol	0.1	0.2	–	–
1638	allo-Aromadendrene epoxide	0.1	0.3	0.1	–
1643	τ-Muurolol	0.2	0.2	0.3	0.2
1654	α-Cadinol	0.1	0.3	0.2	–
1654	α-Eudesmol	–	–	–	0.5
1657	Pogostol	0.1	–	–	–
1657	Selin-11-en-4α-ol	–	0.1	–	–
1661	13-nor-Eremophil-1(10)-en-11-one	–	0.1	–	–
1667	(Z,Z)-1,8,11-Heptadecatriene	-	0.4	0.4	0.6
1668	Citronellyl tiglate	0.4	–	–	–
1671	(Z,Z,Z)-1,8,11,14-Heptadecatetraene	0.1	–	0.1	0.3
1676	1-Tetradecanol	7.8	9.3	6.5	15.4
1681	8-Hydroxyisobornyl isobutanoate	–	0.2	–	–
1683	Germacra-4(15),5.10(14)-trien-1α-ol	0.4	0.2	0.1	–
1700	Heptadecane	0.7	0.8	0.5	1.2
1713	Pentadecanal	0.7	0.3	0.8	2.1
1765	Benzyl benzoate	–	0.1	–	0.1
1775	1-Pentadecanol	0.2	0.2	0.1	0.2
1800	Octadecane	0.1	0.1	–	0.2
1815	Hexadecanal	0.1	–	0.1	0.3
1834	Neophytadiene	0.1	0.2	–	0.5
1839	Phytone	0.3	0.8	0.3	1.9
1860	Platambin	0.2	0.5	0.1	0.3
1872	(9E)-Nonadecene	0.1	0.2	0.1	0.3
1877	2,4-Phytadiene	–	0.1	–	0.2
1884	(Z)-Hexadecatrienal	0.5	0.1	0.4	1.3
1889	(E)-Hexadecatrienal	0.8	–	0.5	1.2
1892	(9Z)-Heptadecenal	0.1	–	0.1	0.2
1900	Nonadecane	–	0.1	–	0.1
1910	1,5-Epoxysalvial-4(14)-ene	0.1	0.3	–	0.2
1917	Heptadecanal	0.2	–	0.2	0.6
1943	iso-Phytol	0.2	0.3	–	0.3
1957	Palmitic acid	0.8	0.5	0.9	7.9
2006	Unidentified^d	0.3	0.7	0.2	1.0
2019	(E,E)-Geranyl linalool	–	–	–	0.2
2053	Manool	–	0.1	–	–
2100	γ-Stearolactone	–	0.2	–	–
2105	Phytol	5.3	9.8	1.1	8.4
2125	Linoleic acid	0.1	–	–	0.2
2131	Linolenic acid	0.2	–	0.1	0.3
2136	Palmitaldehyde, diallyl acetal	0.2	1.6	0.1	1.4
2145	Phytol isomer	–	0.1	–	0.1
2159	Stearic acid	0.1	–	–	0.3
2300	Tricosane	0.3	0.3	0.3	4.2
2400	Tetracosane	–	–	1.4	5.6
2500	Pentacosane	0.2	0.3	0.2	0.5
2600	Hexacosane	–	–	–	0.3
2700	Heptacosane	0.7	1.2	0.8	4.8
2800	Octacosane	–	–	0.1	0.4
	Monoterpene hydrocarbons	1.3	1.3	2.5	0.1
	Oxygenated monoterpenoids	0.4	0.3	0.0	0.0
	Sesquiterpene hydrocarbons	70.7	52.6	77.3	23.0
	Oxygenated sesquiterpenoids	3.9	13.8	2.7	8.5
	Diterpenoids	6.0	11.4	1.4	11.6
	Others	17.0	17.8	15.5	52.5
	Total identified	99.3	97.3	99.6	95.6

AP-DN, aerial part essential oil from Da Nang; AP-QN, aerial part essential oil from Quang Nam; L-EO, leaf essential oil; S-EO, stem essential oil.

- : Not detected.

tr: Trace (<0.05%).

Mass spectrum (MS): 320(15%), 260(8%), 245(8%), 230(9%), 217(16%), 189(17%), 175(15%), 135(80%), 122(100%), 119(44%), 109(71%), 95(30%), 81(20%), 69(20%), 55(22%), 43(60%), 41(23%).

Several essential oils of Leucas species, including L. aspera, L. deflexa, L. indica, L. martinicensis, and L. milanjiana, are dominated by the presence of sesquiterpenoids, often with β-caryophyllene as a major component (Table 2). Some Leucas essential oils (e.g., L. glabrata and L. virgata), however, have shown the oxygenated monoterpenoids to be dominant.

Table 2. Major Components of Leucas Essential Oils.

Essential oil	Origin	Major components	Ref.
L. aspera leaf	India	α-Thujene (12.6%), menthol (11.3%), α-farnesene (26.4%)	¹⁵
L. aspera aerial parts	India	1-Octen-3-ol (14.8%), β-caryophyllene (34.2%), α-humulene (6.3%)	¹⁶
L. aspera aerial parts	Nepal	1-Octen-3-ol (30.6%), β-caryophyllene (23.4%), α-humulene (6.4%), (E)-nerolidol (9.4%), caryophyllene oxide (24.4%), β-eudesmol (5.7%)	¹⁷
L. deflexa leaf	Cameroon	β-Caryophyllene (24.1%), α-humulene (9.9%), germacrene D (27.8%)	¹⁸
L. glabrata aerial parts	Tanzania	iso-Menthone (31.8%), pulegone (11.4%), piperitone (10.6%), piperitenone (6.7%)	¹⁹
L. indica aerial parts	India	β-Caryophyllene (51.1%), α-humulene (10.2%)	²⁰
L. martinicensis leaf	Rwanda	1-Hepten-3-ol (7.6-21.5%), germacrene D (30.3–39.9%)	²¹
L. milanjiana leaf	Zimbabwe	α-Pinene (10.7%), limonene (5.2%), terpinolene (5.3%), β-cubebene (38.2%), β-caryophyllene (7.4%), β-selinene (5.1%)	²²
L. virgata aerial parts	Soqotra (Yemen)	Fenchone (5.4%), camphor (20.5%), caryophyllene oxide (5.1%), β-eudesmol (6.1%)	²³

The mosquito larvicidal activities of the essential oil from the aerial parts of L. zeylanica are summarized in Table 3. The essential oil showed notable activity against both Ae. albopictus and Ae. aegypti. The median lethal concentration (LC₅₀) values, ranging from 37 to 67 µg/mL, compare favorably with other larvicidal essential oils.^24,25 Interestingly, the major component of L. zeylanica essential oil, β-caryophyllene, has been shown to be inactive against Ae. aegypti (LC₅₀ >1000 µg/mL),^26,27 but relatively active against Ae. albopictus (LC₅₀ = 44.8 µg/mL).²⁸ α-Humulene²⁹ and caryophyllene oxide³⁰ have also shown larvicidal activity against Ae. albopictus (LC₅₀ = 6.82 and 65.6 µg/mL, respectively), while germacrene D (LC₅₀ = 63.6 µg/mL)³¹ and caryophyllene oxide (LC₅₀ = 125 µg/mL)²⁶are active against Ae. aegypti. The larvicidal activity of L. zeylanicum essential oil can be attributed to the relatively abundant presence of sesquiterpenoids; many sesquiterpene-rich essential oils have demonstrated excellent larvicidal activities.^24,25 In addition to their larvicidal properties, β-caryophyllene and α-humulene have also demonstrated oviposition deterrent activity on Ae. aegypti.³² An ethanol extract of L. zeylanica has also shown oviposition deterrence against Callosobruchus spp. (bean weevils), which are important pests of stored grains.³³

Table 3. Mosquito Larvicidal Activities of Leucas zeylanica Essential Oil Against Aedes albopictus and Aedes aegypti.

Mosquito larvae	LC₅₀ (24 h), μg/mL	LC₅₀ (48 h), μg/mL
Aedes albopictus	67 ± 8	38 ± 7
Aedes aegypti	44 ± 4	37 ± 2

Leucas zeylanica is a weedy member of the Lamiaceae, and its essential oil demonstrated good larvicidal activity against both species of Aedes mosquito. The essential oil, therefore, represents an inexpensive, readily available, and environmentally benign vector control agent to prevent the spread of the dengue fever virus.

Experimental

Plant Material

Mature leaves, stems, or aerial parts of L. zeylanica were harvested from plants growing in Dai Loc district, Quang Nam Province (15°53ʹ16ʺN, 107°59ʹ38ʺE; sample AP-QN), Hoa Vang district, Da Nang city (16°00ʹ26.7ʺN, 108°06ʹ30.4ʺE; sample AP-DN), and Hoa Vang district, Da Nang city (15°58ʹ16ʺN, 108°01ʹ38ʺE; samples L-EO and S-EO), in April 2018. The plants were identified by Dr. Do Ngoc Dai, and a voucher specimen (LTH 126) has been deposited in the Pedagogical Institute of Science, Vinh University. Plant materials were air-dried at room temperature (≈25°C), 5 kg samples of each of the plant materials were shredded and hydrodistilled for 4 hours using a Clevenger-type apparatus.

Gas Chromatographic–Mass Spectral Analysis

Each of the essential oils of Leucas zeylanicum was analyzed by gas chromatographic (GC)–mass spectrometry (MS) using a Shimadzu GCMS-QP2010 Ultra (Shimadzu Scientific Instruments, Columbia, MD, USA) operated in the electron impact mode (electron energy = 70 eV, scan range = 40–400 atomic mass units, scan rate = 3.0 scans/s), and using GC-MS solution software. The GC column was a ZB-5 fused silica capillary column (Phenomenex®, Torrance, CA, USA) with a (5% phenyl)-polymethylsiloxane stationary phase and a film thickness of 0.25 µm. The carrier gas was helium with a column head pressure of 552 kPa and a flow rate of 1.37 mL/min. Injector temperature was 250°C and the ion source temperature was 200°C. The GC oven temperature program was programmed for 50°C initial temperature, and the temperature was increased at a rate of 2 °C/min to 260°C. A 5% w/v solution of the sample in CH₂Cl₂ was prepared, and 0.1 µL of the sample was injected using a splitting mode (30:1). Identification of the oil components was based on their retention indices determined by reference to a homologous series of n-alkanes and by comparison of their mass spectral fragmentation patterns with those reported in the literature,³⁴ and stored in our in-house Sat-Set library.³⁵

Mosquito Larvicidal Assay

Larvae of Ae. aegypti were collected from a mosquito colony maintained at the Laboratory of Parasitology and Entomology of Duy Tan University, Da Nang Vietnam. For the assay, aliquots of the essential oil of L. zeylanica (sample AP-DN) dissolved in dimethylsulfoxide (DMSO) (1% stock solution) were placed in a 500 mL beaker and added to water that contained 25 larvae (third and early fourth instar). With each experiment, a set of controls using DMSO was also run for comparison. Mortality was recorded after 24 hours and again after 48 hours of exposure during which no nutritional supplement was added. The experiments were carried out 25 ± 2°C. Each test was conducted with four replicates of five different concentrations (150, 100, 80, 50, and 25 µg/mL). LC₅₀ were determined using the Reed–Muench method.³⁶ Larvicidal activity against Ae. albopictus was also determined in a similar way.

Footnotes

Acknowledgments

P.S. and W.N.S. participated in this work as part of the activities of the Aromatic Plant Research Center (APRC, ).

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The authors are grateful to dōTERRA International () for financial support of the APRC and to Duy Tan University for financial support of this research project.

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