Larvicidal activity of major essential oils from stems of Allium monanthum Maxim. against Aedes aegypti L

Abstract

The stems of Allium monanthum were extracted, and the major essential oil composition and larvicidal effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy revealed that the essential oils of A. monanthum stems. The A. monanthum essential oil yield was 4.25%, and gas chromatography and mass spectroscopy analysis revealed that its major constituents were dimethyl trisulfide (23.21%), dimethyl tetrasulfide (11.24%) and methlyl propyl trisulfide (8.21%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC₅₀ value of 23.14 ppm and an LC₉₀ value of 36.31 ppm. Also, dimethyl trisulfide (≥95.0%), dimethyl tetrasulfide (≥95.0%) and methlyl propyl trisulfide (≥95.0%) were tested against the F₂₁ laboratory strain of A. aegypti. Methlyl propyl trisulfide (≥95.0%) has good activity with an LC₅₀ value of 19.38 ppm. Also, the above indicates that other major compounds may play a more important role in the toxicity of essential oil.

Keywords::

• Aedes aegypti
• Allium monanthum
• essential oils
• larvicidal activity
• methlyl propyl trisulfide

Introduction

Natural products are generally preferred because of their less harmful nature to non-target organisms¹. Dengue virus infection is spread by the mosquito vector Aedes aegypti, and clinical manifestations of dengue fever vary from asymptomatic infection to serious disease. Symptoms include sudden onset of fever, retro-orbital headache, abnormal taste sensation, arthralgia, maculopapular rash, and anorexia¹. They cause serious health problems to humans and present obstacles to the socioeconomic development of developing countries, particularly in the tropical region². In the search for environmentally safe and relatively inexpensive methods to control mosquitoes, plant extracts have received much interest as potential bioactive agents against the mosquito larvae. Most mosquito control programs target the larval stage at their breeding sites with larvicides³. Since adulticides may reduce the adult population only temporarily⁴. Therefore, a more efficient approach to reduce the population of mosquitoes would be to target the larvae. The mosquito A. aegypti is the world’s most important vector of yellow fever and dengue viruses⁵.

Essential oils are natural volatile substances found in a variety of plants. It is well known that plant-derived natural products are extensively used as biologically active compounds. Among them, essential oils were the first preservatives used by man, originally in their natural state within plant tissues and then as oils obtained by water distillation. Essential oils composed by isoprenoid compounds, mainly mono- and sesquiterpenes are the carriers of the smell found in the aromaticplants⁶. During our search for new types of natural products possessing an immunotoxicity activity from wild and cultivate plants, we investigated the essential oils from the leaves of Zingiber officinale Roscoe.

Allium monanthum Maxim (Liliaceae) is a perennial herb wildly distributed in field. Allium constituents have been shown to inhibit the covalent binding of the carcinogen, 7,12-dimethylbenza-anthracene, to DNA, an intracellular event that correlates with decreased mutagenesis and carcinogenesis⁷. Kowara group reported antiplatelet aggregation of Allium species has positive correlation with the content of organosulfuric substance⁸. Wu et al. searched the changes of the organosulfurics contents in shallots by processing⁹. Garlic, one of Allium species, has the activities of antiplatelet aggregation¹⁰, antihepatotoxicity¹¹ and antitumor activity^{12 13}. Some pharmacological properties of A. monanthum have been identified from 70% methanol extracts such as antioxidative activity¹⁴. Although little biological activity has been discovered from A. monanthum, details biological activity has not been fully characterized. The aim of this study was to investigate the immunotoxicity properties of the essential oils of A. monanthum stemes. To the best of our knowledge, this is the first report on the chemical composition and immunotoxicity activity of the essential oils of A. monanthum stems.

Experimental

Plant material and essential oils extraction

The A. monanthum stems provided to Korea local markets in May 2008 at Kyung-Ju, Kyungsangbukdo, South Korea. A voucher specimen (DKU-2008-02315) was deposited at herbarium, Jeonnam Institute of Natural Resources Research, Jangheung, South Korea, and identified by Dr. Hyung-In Moon, and subjected to hydrodistillation using a Clevenger type apparatus for 6 h. The essential oil was dried over anhydrous sodium sulphate and the purified essential oil was stored in an amber-coloured vial at 4°C until further use.

Chemicals

Dimethyl trisulfide (98.0%), dimethyl tetrasulfide (98.0%), allyl methyl disulfide (98.0%), dipropyl trisulfide (98.0%) and methlyl propyl trisulfide (98.0%) were purchased from Chemos GmbH (Regenstauf, Germany).

Gas chromatography–mass spectroscopy (GC–MS) analysis of the essential oils

GC–MS analysis of the essential oil was performed using a GC–MS spectrometer (QP 2010), 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) with a film thickness of 0.25 µm. The temperature program used for the analysis was as follows: The temperature of the injector was set at 300°C. The initial temperature was set at 80°C and held for 5 min, set at 5.0°C/min to reach 280°C, and held for 10 min. Helium was used as a carrier gas at a flow rate of 1.0 ml/min. One microlitre 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 the areas of the GC peaks. The temperature of the ion source and of the injector was set at 200°C and 210°C, respectively. The interface was kept at 280°C and the 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 different components of the essential oils were identified by comparing the mass spectra of each peak with those of authentic samples found in a library of mass spectra (The Wiley Registry of Mass Spectral Data, 7th edn.).

Immunotoxicity assay

The F²¹ laboratory strain of A. aegypti was obtained in 2008 from the National Institute of Health, Seoul, South Korea. Adult female mosquitoes were maintained on a 10% sucrose solution, and anaesthetized mice were used for blood feeding 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 immunotoxicity activity was analyzed according to the standard procedures recommended by the World Health Organisation¹⁵. The essential oil was dissolved in 1 ml of acetone and different concentrations were prepared (0, 25, 50, 75, and 100 ppm) using distilled water. Twenty larvae at the early fourth-stage were used in the immunotoxicity assay and five replicates were maintained for each concentration. The larval mortality was calculated after 24 h of exposure. The lethal concentrations LC₅₀ and LC₉₀, the 95% confidence intervals, and the upper and lower confidence levels were calculated using profit analysis (SigmaPlot^® software).

Results

The A. monanthum essential oil yielded 4.25% (v/w) essential oil with a foul odour. Table 1 lists its major chemical constituents, as identified by GC and GC/MS analyses. In their order of elution from the column, these compounds were dimethyl trisulfide (23.21%), dimethyl tetrasulfide (11.24%) and methlyl propyl trisulfide (8.21%). The immunotoxicity effects of the essential oil of the stems of A. monanthum are presented in Table 2. The oil had significant toxic effects against the larvae of A. aegypti with an LC₅₀ value of 23.14 ppm and an LC₉₀ value of 36.31. The control substance caused no mortality for the larvae. Also, dimethyl trisulfide (≥95.0%), dimethyl tetrasulfide (≥95.0%) and methlyl propyl trisulfide (≥95.0%) were tested against the F₂₁ laboratory strain of A. aegypti. The current A. monanthum were tested for the first time against A. aegypti. However, dimethyl trisulfide (≥95.0%) and dimethyl tetrasulfide (≥95.0%) have no activity. Methlyl propyl trisulfide (≥95.0%) has good activity. Methlyl propyl trisulfide (≥95.0%) was the most toxic among the major components, with an LC₅₀ value near 19.38 ppm. 1,8-cineole (negative control) revealed no toxicity. The immunotoxicity effects are summarized in Table 2.

Table 1.  Major essential oil constituents from stems of Allium monanthum.

No	Components	Peak area (%)
1	Allyl methyl disulfide	4.52
2	Dimethyl trisulfide	23.21
3	Allyl cis-1-propenyl disulfide	2.32
4	Allyl methyl trisulfide	3.21
5	Dimethyl tetrasulfide	11.24
6	Methlyl propyl trisulfide	8.21
7	Dipropyl trisulfide	3.02

Table 2.   LC₅₀ and LC₉₀ values for major components against Aedes aegypti.

Essential oils/compounds	LC50 (ppm)	LC90 (ppm)
Allium monanthum	23.14	36.31
Dimethyl trisulfide	>200	>200
Dimethyl tetrasulfide	>200	>200
Methlyl propyl trisulfide	19.38	58.42
Allyl methyl disulfide	>200	>200
Dipropyl trisulfide	>200	>200
1,8-cineole^a	>200	>200

^aNegative control.

Discussion

In general, plant essential oils have been recognized as an important natural source for insecticides^{16 17}. The differences in the toxicity of essential oils against different mosquito species are well known¹⁸ and are due to qualitative and quantitative variations of the components. Recently the clinical use of essential oils has expanded worldwide also including therapy against various kinds of inflammatory diseases, such as allergy, immuntoxicity, rheumatism and arthritis. These activities have mainly been recognized through clinical experience, but there have been relatively little scientific studies on biological actions of these natural essential oil extracts. For instance, Lee et al. described immunotoxicity activity of 2,6,10,15-tetrame-heptadecane from the essential oils of Clerodendrum trichotomum Thunb. against A. aegypti L. The chemical components in oil extracted from the leaves of C. trichotomum after identification of the chemical constituents with the help of GC, GC–MS are recently reported¹⁹ Lee et al. reported that Filipendula glaberrima growing in the middle region in South Korea contained beta-farnesol (2.96%), l-alpha-terpineol (2.43%), benzenemethanol (2.87%), (Z)-3-hexen-1-ol (5.23%), and 2,6-bis(1,1-dimethylethyl)-4-methylphenol (1.91%) as major components. The immunotoxicity activities of the oil from F. glaberrima obviously increase with increasing concentration of essential oils²⁰. Park et al. described composition and immunotoxicity activity of the major essential oil of Angelica purpuraefolia Chung against A. aegypti L²¹. The findings of the present study indicate that the essential oil extracted from the leaves of Allium victorialis could be studied as a potential natural immunotoxicity effects. A. monanthum essential oils are present only in minute amounts. They have immunotoxicity activity in vitro, which has not been verified in animals or humans. Because minute amounts of single compounds within A. monanthum has been shown to account for its clinical effects, most herbalists now conclude that it is a combination of ingredients, rather than a sufficient single ingredient, that accounts for A. monanthum medicinal effects. So, field evaluation and further investigations on the effects on non-target organisms are necessary.

Declaration of interest

This study was supported by research funds from Dong-A University.