Insecticidal Activities of the Leaf Oils of Eight Cinnamomum. species Against Aedes aegypti. and Aedes albopictus.

Abstract

The leaf oils of eight Cinnamomum. species (C. rhyncophyllum. Miq., C. microphyllum. Ridl., C. pubescens. Kochummen, C. mollissimum. Hook. f., C. impressicostatum. Kosterm, C. scortechinii. Gamb., C. sintoc. Bl., and C. cordatum. Kosterm) were investigated for their larvicidal and adulticidal activities against Aedes aegypti. (Aedes aegypti Lynn) and Aedes albopictus. (Aedes albopictus Skuse). Acute mortalities of the fourth instar larvae and the adult mosquitoes were determined according to the standard WHO methods. Among the essential oils studied, the leaf oils of C. rhyncophyllum., C. microphyllum., C. pubescens., C. mollissimum., and C. impressicostatum. showed significant effects against the larvae of Ae. aegypti. and Ae. albopictus. with concentrations that caused 50% mortality (LC₅₀) values of less than 12.8 and 11.8 µg ml⁻¹, respectively. The essential oils that showed strong larvicidal effects also demonstrated relatively strong adulticidal effects on the mosquitoes after 3 h exposure with LC₅₀ values ranging from 133.0 to 243.0 µg ml⁻¹ against Ae. aegypti. and from 118.0 to 194.0 µg ml⁻¹ against Ae. albopictus.. The efficacy of the oils toward the larvae and adult mosquitoes of both species was nonselective as the LC₅₀ values showed little variation. The chemical composition of the oils was investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). This study suggested that the essential oils containing high levels of benzyl benzoate and benzyl salicylate exhibited strong insecticidal activities against the larvae and adult mosquitoes.

• KEYWORD
• Adult mosquitoes
• Aedes aegypti
• Aedes albopictus
• Cinnamomum. species
• essential oils
• mosquito larvae

Introduction

There is a renewed interest in the use of natural products to control destructive insects and vectors of diseases due to the prevalent occurrence of vector resistance to synthetic insecticides and the problem of toxic non-biodegradable residues contaminating the environment and adversely affecting nontarget organisms. More than 2000 plant species are already known to have insecticide properties (Sukumar et al., 1991). Pyrethrin-based products have been widely used to protect people from mosquito bites through their repellent effects and knockdown and killing effects. Many researchers have reported on the effectiveness of plant extracts against mosquito larvae, and the recent examples are by Thangam and Kathiresan (1997), Pitasawat et al. (1998), Mohtar et al. (1999), Markouk et al. (2000) and Ciccia et al. (2000).

Cinnamomum., belonging to the family Lauraceae, is a genus of trees found in continental Asia, eastern and southeastern Asia, Australia, the Pacific region, and a few species in Central and South America (Jantan et al., 1995). Twenty-one species of Cinnamomum. have been found on the Malaysian peninsula (Kochummen, 1989). A number of these species are used in traditional medicine and as spices (Burkill, 1966). Systematic chemical studies on the essential oils of some of the Malaysian species have been carried out. The oils were found to contain mainly safrole, eugenol, linalool, camphor, benzyl benzoate, or cinnamaldehyde as major components (Jantan & Goh, 1990, 1992; Jantan et al., 1994a, 2002, 2003). Antimicrobial activities of the essential oils of several Cinnamomum. species from peninsular Malaysia have been reported (Jantan et al., 1994b; Mohtar et al., 1999; Nor Azah et al., 2002).

In this study, the leaf oils of eight Cinnamomum. species (C. rhyncophyllum. Miq., C. microphyllum. Ridl., C. pubescens. Kochummen, C. mollissimum. Hook. f., C. impressicostatum. Kosterm, C. cordatum. Kosterm, C. scortechinii. Gamb., and C. sintoc. Bl.) were investigated for their larvicidal and adulticidal activities against Aedes aegypti. and Aedes albopictus., which are daytime biting nuisance mosquito species and the potential vectors of dengue and yellow fever viruses. The chemical composition of the essential oils was analysed by GC and GC-MS.

Materials and Methods

Plant materials

The fresh samples of eight Cinnamomum. species (C. rhyncophyllum., C. microphyllum., C. pubescens., C. mollissimum., C. impressicostatum., C. scortechinii., C. sintoc., and C. cordatum.) were collected from Gunung Berembun, Cameron Highlands, Pahang, in the month of June 2002. The voucher specimens were identified and deposited at the Herbarium of the Forest Research Institute Malaysia, Kepong.

Oil isolation

The plant materials were air-dried, comminuted, and 150 g of each sample was hydrodistilled in a Clevenger-type apparatus for 8 h. The oily layers obtained were separated and dried over anhydrous magnesium sulfate. The yields were averaged over three experiments and calculated based on dry weight of the plant materials.

Analysis of the oils

The oils were analyzed on a Shimadzu (Kyoto, Japan) GC 2000 chromatograph Hewlett-Packard (USA) GC-MSD 5890 series equipped with a flame ionization detector (FID) detector using a DB-5 capillary column (25 m × 0.25 mm, 0.25-µm film thickness). The operation parameters were nitrogen as carrier gas at 50 cm/s, injector and detector temperatures were maintained at 250°C. The column was programmed initially at 75°C for 10 min, then 3°C/min to 210°C and held for 1 min. The oils were also examined using a DB-1 stationary phase column (25 m × 0.25 mm, 0.25 µm film thickness) programmed from 60°C for 10 min, then 3°C/min to 180°C and held for 10 min. Peak areas and retention times were measured by electronic integration. The relative amounts of individual components are based on peak areas obtained, without FID response factor correction. Temperature program linear retention indices of the compounds were also determined relative to n.-alkanes (Kovats, 1965). The oils were also analyzed by GC-MS with a Hewlett-Packard GC-MSD 5890 series 2 mass spectrometer (70-eV direct inlet) on a BPX5 column (30 m × 0.25 mm, 0.25 µm film thickness) initially at 75°C for 10 min, then 3°C/min to 210°C and held for 1 min with helium as carrier gas. The constituents were identified by comparison of their retention indices with literature values and their mass spectral data with those from the Wiley mass spectral database, and in some cases by co-chromatography on the different columns with authentic samples (Adams, 1989; McLafferty & Staufer, 1989; Davies, 1990).

Larvacidal test

The fourth instar larvae of Aedes aegypti. and Ae. albopictus. served as the test organisms. The larvae colonies of the mosquito were collected from the Medical Entomology Insectary of the Institute for Medical Research, Kuala Lumpur. Each oil in 5 ml of ethanol was dissolved in distilled water to prepare a 200 µg ml⁻¹ stock solution from which concentrations of 1–200 µg ml⁻¹ were prepared by dilution. Each solution (250 ml) was placed in a beaker; 25 larvae of the mosquito were transferred into each beaker using a disposable pipette. The treatment was replicated 3 times on each concentration (WHO, 1981). A control was prepared by the addition of 5 ml of ethanol to the distilled water in each beaker that contained 25 larvae. Solutions of Abate dissolved in water at 1–200 µg ml⁻¹ concentrations were used as standard toxicant. Mortality counts were made after 24 h. Larvicidal tests on standard samples, viz., camphor, benzyl benzoate, benzyl salicylate, 1,8-cineole, terpineols, linalool, safrole, eugenol, methyl eugenol, cinnamaldehyde, and methyl cinnamate, which are commonly found in the essential oils of Cinnamomum. species were also carried out.

Adulticidal test

Five- to 6-day-old sugar-fed adult female mosquitoes of Aedes albopictus. and Ae. aegypti. used in the test were obtained from colonies maintained in the insectary of Entomology Division, Institute for Medical Research, Kuala Lumpur. The essential oils were diluted with ethanol to achieve concentrations of 125, 250, 500, and 1000 µg ml⁻¹, respectively. DDT (an organochlorine) at similar concentrations and at diagnostic concentration of 4% (w/w) was used as a positive control for comparison purposes. The diluted oils and DDT were then separately impregnated on filter papers (140 × 120 mm). A blank paper consisting of only ethanol was used as control. The papers were left to dry at room temperature to evaporate off the ethanol overnight. Impregnated papers were prepared fresh prior to testing. The bioassay was conducted in an experimental kit consisting of two cylindrical plastic tubes both measuring 125 × 44 mm following the WHO method (1976). One tube served to expose the mosquitoes to the oils, and another tube was used to hold the mosquitoes before and after the exposure periods. The impregnated papers were rolled and placed in the exposure tube. Each tube was closed at one end with a 16 mesh size wire screen. Sucrose-fed mosquitoes (15) were released into the tube, and the mortality effects of the extracts were observed every 10 min for 3 h exposure period. At the end of 1-, 2-, and 3-h exposure periods, the mosquitoes were placed in the holding tube. Cotton pads soaked in 10% sugar solution with vitamin B complex was placed in the tube during the holding period of 24 h. Mortality of the mosquitoes was recorded after 24 h. The above procedures were carried out in triplicate for each oil concentration. Adulticidal tests on standard samples, viz., camphor, benzyl benzoate, benzyl salicylate, 1,8-cineole, terpineols, safrole, eugenol, methyl eugenol, cinnamaldehyde, linalool, and methyl cinnamate were also carried out.

Statistical analysis

The concentration mortality data were analysed by using the probit analysis method as described by Raymond (1985). The concentrations that caused 50% mortality (LC₅₀) were determined with 95% confidence intervals for statistically significant comparisons. The percentage mortality were corrected by Abbott's formula (1925) if the control mortality was between 5% and 20%. When the mortality in the control test was over 20%, the tests were discarded. The significance of the differences between the tests and control studies was established by Student's t.-test. p values < 0.05 were considered significant.

Results and Discussion

Larvicidal activity

A total of eight essential oils of Cinnamomum. species were investigated for their larvicidal and adulticidal activities against Aedes aegypti. and Ae. albopictus.. The bioassay techniques were employed to stimulate real ecological parameters that exist in nature to a certain extent. Table 1 shows the results of the larval susceptibility to the essential oils and standard samples. Among the samples studied, the leaf oils of C. microphyllum, C. rhyncophyllum., C. pubescens., C. mollissimum., and C. impressicostatum. showed significant effects against the larvae of Ae. aegypti. and Ae. albopictus. with LC₅₀ values of less than 12.8 and 11.8 µg ml⁻¹, respectively. The leaf oil of C. microphyllum. was the most effective with LC₅₀ values of 6.7 and 6.2 µg ml⁻¹ against Ae. aegypti. and Ae. albopictus., respectively. Except for C. cordatum., all the other oils studied showed moderate activity with LC₅₀ values less than 41.1 µg ml⁻¹. Larvicidal assay on the standard samples, which are commonly found as major components in the essential oils of various Cinnamomum. species, indicated the following order of potency against both mosquitoes: benzyl salicylate > benzyl benzoate > methyl cinnamate > methyl eugenol > cinnamaldehyde > eugenol > linalool > terpineols > terpinen-4-ol > 1,8-cineole.

Table 1 LC₅₀ (µg ml⁻¹) values of the leaf oils of Cinnamomum. species and standard samples against Aedes aegypti. and Ae. albopictus. fourth instar larvae

Sample	Ae. aegypti.	Ae. albopictus.
C. microphyllum.	6.7 (6.1–7.5)	6.2 (5.5–6.9)
C. mollissimum.	10.2 (9.3–11.2)	8.8 (8.0–9.7)
C. pubescen.	12.8 (10.9–14.9)	7.9 (7.0–8.9)
C. rhyncophyllum.	6.0 (5.5–6.5)	11.8 (10.5–13.3)
C. impressicostatum.	10.7 (9.5–12.2)	9.3 (8.4–10.5)
C. scortechinii.	21.5 (18.9–24.5)	16.7 (15.1–18.4)
C. sintoc.	41.1 (35.9–46.5)	36.5 (31.8–41.2)
C. cordatum.	183.6 (156.3–229.6)	160.8 (138.5–195.3)
Camphor	271.5 (237.8–314.1)	201.3 (181.2–224.2)
Benzyl benzoate	6.8 (6.3–7.5)	6.5 (6.0–7.1)
Benzyl salicylate	6.8 (6.4–7.4)	5.5 (5.1–6.0)
1,8-Cineole	642.4 (577.2–740.4)	216.9 (194.8–240.4)
Eugenol	89.9 (82.4–98.1)	80.8 (74.4–87.9)
Methyl eugenol	350.9 (316.8–383.3)	328.3 (298.0–359.2)
Methyl cinnamate	16.8 (14.9–18.7)	12.7 (11.5–14.1)
Cinnamaldehyde	52.8 (47.2–57.9)	35.5 (32.2–38.9)
Linalool	157.4 (145.3–170.5)	143.0 (132.7–153.9)
Safrole	32.1 (26.3–45.8)	28.0 (23.3–37.1)
Terpineols	183.6 (160.5–215.20)	155.5 (138.5–176.2)
Terpinen-4-ol	220.5 (196.2–246.4)	178.1 (157.6–199.6)
Abate	0.00124	0.00131

Confidence limits 95% between parentheses.

Adulticidal activity

The percent mortality of the mosquitoes when exposed to the various concentrations of the eight essential oils for 1, 2, and 3 h are presented in Table 2. The essential oils showed dose-dependent and time-dependent percent mortality values (i.e., increased the concentration of the oils impregnated in the papers, and exposure period increased the percent of mosquitoes killed). There were no mortalities recorded during the 3-h exposure period in the control group (i.e., when a blank paper was used). Doses of 500 and 1000 µg ml⁻¹ for C. microphyllum, C. rhyncophyllum, C. pubescens, C. mollissimum., and C. impressicostatum. oils caused a highly significant adulticidal effects at 3 h (p < 0.005). The oils that showed strong larvicidal effects also exhibited relatively strong adulticidal effects after 3-h exposure period with LC₅₀ values ranging from 133.0 to 243.0 µg ml⁻¹ against Ae. aegypti. and from 118.0 to 194.0 µg ml⁻¹ against Ae. albopictus.. The leaf oil of C. microphyllum. was the most effective with LC₅₀ values of 133.0 µg ml⁻¹ against Ae. aegypti. and 118.0 µg ml⁻¹ against Ae. albopictus. (Table 3). The mortality effects of the standard samples against the adult mosquitoes of both species after 3-h exposure showed the following order of potency: benzyl benzoate > benzyl salicylate > methyl cinnamate > methyl eugenol > cinnamaldehyde > 1,8-cineole > camphor > eugenol > linalool.

Table 2 Adulticidal effect of the leaf oils of Cinnamomum. species on adult mosquitoes exposed continuously for 1, 2, and 3 h.

	% Mortality
	1 h		2 h		3 h
Concentration (µg ml^−1)	Ae. aegypti.	Ae. albopictus.	Ae. aegypti.	Ae. albopictus.	Ae. aegypti.	Ae. albopictus.
C. microphyllum.
1000	4.4	4.4	31.1	33.3	100	100**
500	0	0	20.0	26.7	75.6	77.8**
250	0	0	8.9	11.1	62.2	64.4
125	0	0	2.2	2.2	53.3	55.6
Control	0	0	0	0	0	0
C. mollissimum.
1000	4.4	4.4	31.1	37.8	100	100**
500	0	0	15.6	20.0	100	100**
250	0	0	6.7	8.9	77.8	80.0
150	0	0	2.2	4.4	42.2	48.9
Control	0	0	0	0	0	0
C. pubescens.
1000	2.2	2.2	31.1	33.3	100	100**
500	0	0	11.1	15.6	73.3	77.8**
250	0	0	6.7	6.7	53.3	55.6
150	0	0	2.2	2.2	44.4	48.9
Control	0	0	0	0	0	0
C. scortechinii.
1000	0	0	20.0	22.2	86.7	88.9
500	0	0	11.1	15.6.	60.0	68.9
250	0	0	4.4	4.4	40.0	46.7
150	0	0	2.2	0	28.9	35.6
Control	0	0	0	0	0	0
C. impressicostatum.
1000	0	0	33.3	33.3	100	100**
500	0	0	15.6	20.0	71.1	77.8**
250	0	0	8.9	15.6	57.8	62.2
150	0	0	2.2	2.2	44.4	48.9
Control	0	0	0	0	0	0
C. rhyncophyllum.
1000	0	0	24.4	28.9	71.1	84.4**
500	0	0	8.9	11.1	66.7	75.6**
250	0	0	6.7	6.7	55.6	60.0
150	0	0	2.2	4.4	33.3	35.6
Control	0	0	0	0	0	0
C. cordatum.
1000	0	0	11.1	15.6	53.3	55.6
500	0	0	4.4	4.4	20.0	17.8
250	0	0	0	2.2	4.4	4.4
150	0	0	0	0	0	0
Control	0	0	0	0	0	0
4% DDT	16.0	45.0	100	100	100	100

*p < 0.05,

**p < 0.005 with respect to control.

Table 3 LC₅₀ values (µg ml⁻¹) of the leaf oils of Cinnamomum. species and standard samples against adult mosquitoes after 3-h exposure period

Sample	Ae. aegypti.	Ae. albopictus.
C. microphyllum.	133.0	118.0
	(110.0–152.0)	(96.0 – 136.0)
C. mollissimum.	146.0	132.0
	(127.0–162.0)	(112.0–150.0)
C. pubescen.	178.0	150.0
	(113.0–232.0)	(87.0–197.0)
	(113.0–233.0)
C. impressicostatum.	167.0	135.0
	(94.0–203)	(74.0–181.0)
C. rhyncophyllum.	243.0	194.0
	(168.0–318.0)	(146.0–239.0)
C. scortechinii.	283.0	237.0
	(233.0–337.0)	(191.0–283.0)
C. cordatum.	934.0	917.0
	(811.0–1130.0)	(802.0–1094.0)
Camphor	433.0	314.0
	(379.0–500.0)	(280.0–351.0)
Benzyl benzoate	109.0	97.0
	(30.0–164.0)	(24.0–149.0)
Benzyl salicylate	119.0	113.0
	(35.0–345.0)	(33.0–320.0)
1,8-Cineole	357.0	310.0
	(313.0–406.0)	(185.0–516.0)
Eugenol	488.0	471.0
	(435.0–552.0)	(264.0–852.0)
Methyl eugenol	190.0	181.0
	(175.0–205)	(167.0–196)
Methyl cinnamate	190.0	181.0
	(175.0–205.0)	(167.0–196.0)
Linalool	554.0	528.0
	(488.0–639.0)	(463.0–612.0)
Cinnamaldehyde	288.0	288.0
	(182.0–456.0)	182.0–456.0)
Safrole	228.0	218.0
	(183.0–278.0)	(179.0–260.0)
Terpineols	645.0	606.0
	(555.0–775.0)	(524.0–721.0)
Terpinen-4-ol	698.0	645.0
	(586.0–875.0)	(543.0–801.0)
DDT	0.06	0.05
	(0.009–0.421)	(0.008–0.377)

Confidence limits 95% between parentheses.

The efficacy of the oils toward the larvae and adult mosquitoes of both species was nonselective, as the LC₅₀ values showed little variation (Tables 1 and 3). The observed toxicities of the essential oils toward the mosquitoes were due to the nature and proportion of their individual constituents.

Qualitative and quantitative analysis of the essential oils

The yields of oils based on dry weight, obtained by water distillation, from each species are shown in Table 4. The chemical composition of the essential oils was investigated in an effort to correlate the constituents of the oils and their insecticidal activities. The lists of constituents identified in the oils are shown in order of elution on a DB-5 type column in Table 5. Benzyl benzoate was the most abundant component in the leaf oils of C. impressicostatum. (50.9%), C. pubescens. (50.2%), C.. rhyncophyllum. (70.0%), C. mollissimum. (87.6%), and C. microphyllum. (87.8%). Previous study indicated that essential oils containing high levels of benzyl benzoate show high toxicity values against brine shrimp (Jantan et al., 1994b). Based on the results of the assay on standard samples (Tables 1 and 3), it may be that the high levels of benzyl benzoate in the oils and in combination with the monoterpenoids and sesquiterpenoids could be responsible for the high insecticidal activity of the oils. The high activity of the leaf oils of C. impressicostatum. and C. pubescens. may also be due to significant contribution from benzyl salicylate, which was present at 7.3% and 23.4%, respectively. It is interesting to note that except for benzyl benzoate and benzyl salicylate that exhibited LC₅₀ values ranged from 5.5 to 6.8 µg ml⁻¹ against the larvae, the other standard samples showed LC₅₀ values higher than the active oils. The overall activity of the essential oils was due to the nature and proportion of the individual constituents in the oils. The active constituents of the oils might be acting synergistically by different modes of action such as by paralyzing physiology and osmoregulation system of the organisms.

Table 4 Essential oil yield from the leaves of eight Cinnamomum. species

Species	% (on a dry weight basis)
C. rhyncophyllum.	3.5 ± 0.4
C. cordatum.	0.8 ± 0.2
C. mollisimum.	2.8 ± 0.5
C. microphyllum.	3.3 ± 0.2
C. impressicostatum.	7.2 ± 0.5
C. pubescens.	5.4 ± 0.3
C. scortechinii.	0.5 ± 0.1
C. sintoc.	0.5 ± 0.3

Data represent the means ± SE obtained from three experiments.

Table 5 Percentage composition of the essential oils of Cinnamomum rhyncophyllum., C. cordatum., C. microphyllum., C. scortechinii., C. pubescens., C. impressicostatum., C. mollissimum., and C. sintoc.

Compound	RI	RHY	COR	MIC	SCO	PUB	IMP	MOL	SIN
2-Hexenal	860	—	—	—	—	—	—	—	0.1
α.-Thujene	931	0.2	0.2	—	0.7	0.2	0.2	—	—
α.-Pinene	937	3.5	1.7	—	1.7	1.8	3.9	—	—
Benzaldehyde	961	—	—	1.0	—	—	—	0.2	—
Sabinene	976	1.5	1.3	—	2.4	1.7	—	—	—
β.-Pinene	979	2.7	1.1	—	1.6	1.2	3.4	0.1	0.1
Myrcene	990	1.1	1.2	—	2.4	0.8	1.4	—	t
α.-Phellandrene	1001	0.5	2.6	—	0.8	1.7	12.3	—	0.4
α.-Terpinene	1016	0.1	0.6	—	—	—	0.2	—	—
p.-Cymene	1024	0.7	1.5	—	6.8	11.7	7.0	1.9	0.2
Limonene	1029	0.3	2.4	—	—	—	4.8	—	—
β.-Phellandrene	1030	10.0	9.0	—	17.3	1.1	2.2	—	—
1,8-Cineole	1032	—	—	—	—	—	—	1.0	—
δ.-3-Carene	1032	t	—	—	—	—	—	—	—
Benzyl alcohol	1033	—	—	2.1	—	—	—	—	—
(Z.)-β.-Ocimene	1040	—	—	—	—	0.1	0.2	—	—
(E.)-β.-Ocimene	1051	—	—	—	—	—	0.2	—	—
γ.-Terpinene	1062	0.3	1.5	—	0.3	—	0.3	—	—
trans.-Linalool oxide	1071	—	—	—	0.2	—	—	—	—
cis.-Linalool oxide	1075	—	—	—	0.2	—	—	—	—
Terpinolene	1089	t	0.5	—	0.2	—	0.1	—	—
Linalool	1098	0.4	17.3	1.4	16.4	—	0.1	0.6	6.5
Camphor	1144	—	0.4	—	—	—	—	—	—
Isopulegol	1152	—	—	—	0.3	—	—	—	—
Citronellal	1159	—	0.3	—	—	—	—	—	—
Terpinen-4-ol	1178	1.1	7.0	t	6.6	0.6	0.8	0.4	5.3
α.-Terpineol	1188	0.4	3.6	0.1	2.3	0.2	0.4	0.3	3.3
cis.-Piperitol	1198	—	0.2	—	0.3	—	—	—	—
Nerol	1232	—	0.2	—	0.3	—	—	—	—
Geraniol	1260	—	0.8	0.3	0.6	—	—	—	—
Safrole	1289	0.7	0.1	—	0.2	—	0.2	—	23.4
Eugenol	1357	0.1	0.1	—	—	—	—	—	7.6
Methyl cinnamate	1380	4.3	17.1	—	—	1.8	1.0	—	—
β.-Bourbonene	1377	—	—	—	0.3	—	—	—	—
β.-Elemene	1390	—	—	0.1	0.2	—	—	—	1.9
Methyl eugenol	1402	0.2	4.4	—	—	—	—	0.4	—
cis.-α.-Bergamotene	1415	—	—	—	—	—	0.3	0.6	—
β.-Caryophyllene	1419	0.4	0.3	1.2	0.2	—	0.3	—	2.6
trans.-α.-Bergamotene	1435	—	—	—	—	—	0.1	0.1	—
γ.-Elemene	1437	—	—	—	—	—	—	—	1.2
Aromadendrene	1440	—	0.3	0.1	0.4	—	—	—	0.2
α.-Humulene	1454	1.1	0.2	0.6	0.1	0.4	1.0	1.5	1.8
β.-(E.)-Farnesene	1456	—	—	0.2	0.2	0.1	—	—	—
ar-Curcumene	1480	—	—	—	—	—	—	0.1	—
γ.-Muurolene	1480	—	0.2	—	—	—	—	—	13.5
β.-Selinene	1485	0.1	—	0.6	—	—	—	—	—
cis.-β.-Guaiene	1490	0.1	t	—	—	—	—	—	—
β.-Bisabolene	1505	—	—	—	—	—	—	0.2	—
γ.-Cadinene	1514	—	0.1	—	—	—	—	—	0.7
δ.-Cadinene	1522	0.1	0.9	—	0.3	—	—	—	2.7
(Z.)-Nerolidol	1535	—	0.7	—	—	—	—	—	—
Elemol	1552	—	—	—	—	—	—	—	1.0
Spathulenol	1575	0.1	1.6	0.8	1.7	—	—	—	—
Caryophyllne oxide	1583	—	—	—	—	—	—	0.5	1.1
Globulol	1586	—	1.6	1.7	1.2	—	—	—	—
Viridiflorol	1603	—	1.4	—	—	—	—	—	—
β.-Eudesmol	1651	—	0.5	0.4	0.6	—	—	—	—
α.-Eudesmol	1655	—	0.3	—	0.3	—	—	—	—
(E.,Z.)-Farnesol	1746	—	—	0.1	0.3	—	—	—	—
(E.)-Asarone	1678	—	0.1	—	—	—	—	—	—
Benzyl benzoate	1762	70.0	7.6	87.8	13.5	50.2	50.9	87.6	0.2
Benzyl salicylate	1860	—	4.7	—	9.4	23.4	7.5	0.2	—

Percentages were obtained by peak-area normalization on column DB-5, all relative response factors being taken as one; the retention index of compounds on the DB-1 was also determined; RI, retention index; t, trace; RHY, C. rhyncophyllum.; COR, C. cordatum.; MIC, C. microphyllum.; SCO, C. scortechinii.; PUB, C. pubescens.; IMP, C. impressicostatum.; MOL, C. mollissimum.; SIN, C. sintoc..

Acknowledgments

The work was supported by IRPA project 09-02-02-0052-EA148. The authors are grateful to the Institute for Medical Research for providing the larvae and adult mosquitoes used in this study and to Abu Said Ahmad of the Forest Research Institute Malaysia for running the GC-MS spectra.