RETRACTED: Larvicidal effects of the major essential oil of Pittosporum tobira against Aedes aegypti (L.)

Abstract

RETRACTED

Keywords::

• Aedes aegypti
• essential oils
• larvicidal effects
• Pittosporum tobira
• l-limonene

Introduction

Synthetic insecticides have created a number of ecological problems, such as the development of resistant insect strains, ecological imbalance, and harm to mammals. Natural products are generally preferred because of their less harmful nature to nontarget organisms and due to their innate biodegradability¹. The mosquito Aedes aegypti is the important vector of dengue fever and dengue hemorrhagic fever in many parts of the world. In the absence of effective vaccine and drugs, dengue prevention and control programs have depended on vector control. Management of this disease vector using synthetic organic chemical insecticides has failed because of insecticide resistance developed by the Aedes mosquitoes². In the search for environmentally safe and relatively inexpensive methods for controlling mosquitoes, plant extracts have received much interest as potential bioactive agents against mosquito larvae. Most of the mosquito control programs target the larval stage in their breeding sites with larvicides³, because the adulticides may only reduce the adult population temporarily⁴. Therefore, a more efficient way to reduce the mosquito population is to target the larvae.

Pittosporum tobira (Pittosporaceae) is a small, slender, evergreen tree that grows on the southwestern Pacific coast of Jeiju Island. The seeds undergo a gradual change in color from green to red in late autumn to winter⁵. Nickavar et al. reported the volatile components of the flower and fruit oils from P. tobira grown in Iran, obtained through hydrodistillation and analyzed by gas chromatography-mass spectroscopy (GC-MS)⁶. Rodrigues Frederico et al. reported the volatile components of the leaf, flower, and fruit volatile oils of P. tobira grown in three locations in Portugal⁷. The essential oil from P. tobira (leaves) shows that target sites other than those used by antibiotics will be active against multidrug-resistant microbial pathogens. However, very little information is available on such activity of aromatic herbs⁸. Maoka et al. reported the isolation and structural elucidation of novel carotenoids from the seeds of P. tobira grown in Japan^9,10. Fujiwara et al. reported the isolation and structural elucidation of new carotenoids from the seeds of P. tobira^11–13. D’Acquarica et al. reported the isolation and structural elucidation of four new triterpenoid estersaponins from the fruits of P. tobira¹⁴. Takaoka et al. reported the structures of sesquiterpene glycosides from P. tobira¹⁵. Nozaki et al. reported the structures of three new germacranolide glycosides from P. tobira¹⁶. Ogihara et al. reported the structures of sesquiterpene glycosides and other terpene constituents from the flowers of P. tobira¹⁷. Suga et al. reported the structure of a new sesquiterpene glycoside from the flowers of P. tobira¹⁸. However, the biological activity of P. tobira has not been investigated in detail.

The aim of this study was to investigate the primary chemical composition and larvicidal properties of the essential oil of P. tobira. To the best of our knowledge, this is the first report on the major chemical composition and larvicidal activity of the essential oil of P. tobira.

Materials and methods

Plant materials and essential oil extraction

Fresh leaves of P. tobira were collected in July 2004 at Jeiju Island (South Korea) and subjected to hydrodistillation using a Clevenger-type apparatus for 6 h. The P. tobira essential oil (PTEO) was dried over anhydrous sodium sulfate, and the purified essential oil was stored in an amber-colored vial at 4°C until further use.

Chemicals

l-limonene (≥95.0%), 4-methyl-1,3-pentadiene (≥98.0%), and undecane (≥99.8%) were purchased from Sigma-Aldrich Korea (Yongin City, Kyunggi-Do, South Korea).

Gas chromatography-mass spectroscopy analysis of the essential oil

GC-MS analysis of the essential oil was performed using a GC-MS spectrometer (QP 2010; Shimadzu, Kyoto, Japan), equipped with a splitless injector. The components were separated on a 0.32 mm i.d. × 60 m DB-1 MS capillary column (Agilent Scientific, Santa Clara, CA, USA) with a film thickness of 0.25 μm. The temperature of the injector was set at 300°C. The initial temperature was set at 80°C and held for 5 min, increased by 5.0°C/min to 280°C, and then held for 10 min. Helium was used as a carrier gas at a flow rate of 1.0 mL/min. One microliter of the sample (diluted 1:10 with acetone) was injected with a split ratio of 1:100. The percent composition of the essential oil was calculated by comparing areas of the GC peaks. The temperatures of the ion source and the injector were set at 200°C and 210°C, respectively. The interface was maintained at 280°C, and mass spectra were obtained at 70 eV. The effluent of the capillary column was introduced directly into the ion source of the mass spectrometer. The sector mass analyzer was set to scan from 50 to 500 amu every 0.5 s. The various components of the essential oil were identified by comparing the mass spectrum of each peak with those of authentic samples found in a library of mass spectra (Wiley Registry of Mass Spectral Data, 7th ed.).

Larvicidal assay

The F²¹ laboratory strain of A. aegypti Linn was obtained in 2008 from the National Institute of Health, Seoul, South Korea. Adult female mosquitoes were maintained on a 10% sucrose solution, and anesthetized mice were used for blood feeding of the mosquitoes. Larvae were reared in plastic trays and fed a diet of chicken chow and yeast (8:2). Mosquitoes were maintained at 27 ± 2°C, 70 ± 5% relative humidity, and a photoperiod of 16L:8D. The larvicidal activity was analyzed according to the standard procedures recommended by the World Health Organization¹⁹. The essential oil was dissolved in 1 mL acetone, and various concentrations were prepared (0, 12.5, 25, 50, 100, and 200 ppm) using distilled water. Twenty larvae at the early fourth stage were used in the larvicidal assay, and five replicates were maintained for each concentration. Larval mortality was calculated after 24 h of exposure. The lethal concentrations LC₅₀ and LC₉₀ and 95% confidence intervals were calculated using profit analysis (SigmaPlot^® software; Systat Software, Inc., San Jose, CA, USA).

Results and discussion

The Pittosporum tobira leaves yielded 0.1% (v/w) essential oil with a foul odor. Table 1 lists the major chemical constituents, as identified by GC and GC-MS analyses. In order of elution from the column, these major compounds were undecane (31.11%), 4-methyl-1,3-pentadiene (11.34%), (1,3-dimethyl-2-butenyl)benzene (5.45%), and l-limonene (14.08%). The larvicidal effects of PTEO are presented in Table 2. The oil had significant toxic effects against the larvae of A. aegypti, with an LC₅₀ value of 58.92 ppm and an LC₉₀ value of 111.31 ppm. Also, l-limonene (≥95.0%), 4-methyl-1,3-pentadiene (≥98.0%), and undecane (≥99.8%) were tested against the F²¹ laboratory strain of A. aegypti. The current Pittosporum species was tested for the first time against A. aegypti, and l-limonene (≥95.0%), 4-methyl-1,3-pentadiene (≥98.0%), and undecane (≥99.8%) were tested for the first time against mosquito larvae. l-limonene was the most toxic among the three major components (l-limonene, 4-methyl-1,3-pentadiene, and undecane), with an LC₅₀ value near 39.7 ppm. The above indicates that l-limonene may play an important role in the toxicity of essential oil. The larvicidal effects are summarized in Tables 2 and 3. In general, plant essential oils have been recognized as important natural sources of insecticides^20,21. Differences in the toxicity of essential oils against various mosquito species have been well-documented²², and arise because of qualitative and quantitative variations in their components. For example, Cheng et al.²³ reported the larvicidal activity of linalool from Cinnamomum osmophloeum against A. aegypti (LC₅₀ = 50 ppm).

Table 1.  Major constituents of Pittosporum tobira essential oil.

Retention time^a	Component	M^+	Peak area (%)	Fragments
1.13	Undecane	156	31.11	57	43
1.27	l-limonene	136	14.08	93	69
1.56	4-Methyl-1,3-pentadiene	82	11.34	67	43
1.84	(1,3-Dimethyl-2-butenyl)benzene	160	5.45	91	105

^aRetention time relative to that of α-pinene.

Table 2.  Larvicidal activity of Pittosporum tobira essential oil against Aedes aegypti.

Concentration (ppm)	Percent mortality ± SE	LC50 (ppm)	LC90 (ppm)	95% Confidence interval for LC50	95% Confidence interval for LC90
12.5	13 ± 2.1
25	28 ± 4.5
50	49 ± 7.3	58.92	111.31	33.23–89.24	94.16–201.98
100	88 ± 4.9
200	95 ± 7.9

Table 3.  LC₅₀ and LC₉₀ values for undecane, l-limonene, and 4-methyl-1,3-pentadiene.

Compound	LC50 (ppm)	LC90 (ppm)	95% Confidence interval for LC50	95% Confidence interval for LC90
Undecane	NE	NE	NE	NE
l-limonene	39.7	78.11	30.28–40.74	62.34–105.26
4-Methyl-1,3-pentadiene	76.2	109.33	61.34–103.11	83.29–200.24

Note. NE, not effective.

The findings of the present study indicate that essential oil extracted from the leaves of P. tobira could be studied as a potential natural larvicide.

Declaration of interest: This work was supported by the MRC program of MOST/KOSEF (grant #: R13-2005-013-01000-0), Korea. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.