Larvicidal Activity of Essential Oils of Apiaceae Plants against Malaria Vector, Anopheles stephensi.

BACKGROUND: Plant extracts and oils may act as alternatives to conventional pesticides for malaria vector control. The aim of this study was to evaluate the larvicidal activity of essential oils of three plants of Apiaceae family against Anopheles stephensi, the main malaria vector in Iran.
METHODS: Essential oils from Heracleum persicum, Foeniculum vulgare and Coriandrum sativum seeds were hydro distillated, then their larvicidal activity were evaluated against laboratory-reared larvae of An. stephensi according to standard method of WHO. After susceptibility test, results were analysis using Probit program.
RESULTS: Essential oils were separated from H. persicum, F. vulgare and C. sativum plants and their larvicidal activities were tested. Result of this study showed that F. vulgare oil was the most effective against An. stephensi with LC(50) and LC(90) values of 20.10 and 44.51 ppm, respectively.
CONCLUSION: All three plants essential oil can serve as a natural larvicide against An. stephensi. F. vulgare oil exhibited more larvicidal properties.

Introduction

Mosquitoes play an important role in transmission of some human diseases such as malaria, dengue fever, yellow fever and filariasis, which consider them among the greatest health problems across the glob (James 1992). Anopheles species are considered as vectors of human malaria, filariasis and certain arboviruses (Sedaghat and Harbach 2005). Malaria is considered as one of the most important health problem in Iran especially in southern parts. In the south parts of Iran there are six anopheline vectors including Anopheles culicifacies, An. stephensi, An. dthali, An. fluviatilis, An. superpictus, and An. pulcherrimus, (Naddaf et al. 2003, Vatandoost and Moinvaziri 2004, Vatandoost et al. 2004, 2005ab, Hanafi-Bojd et al. 2006, Soltani et al. 2008, Hanafi-Bojd et al. 2010, Vatandoost et al. 2006, 2007, 2009, 2011). Anopheles sacharovi and An. maculipennis can transmit human malaria in northern part of the country (Sedaghat et al. 2003a, 2003b, Oshaghi et al. 2003, Doosti et al. 2007).

Anopheles stephensi, an oriental malaria vector, is distributed in Indo-Persian area from India, Pakistan and Iran, to countries around the Persian Gulf (Nagpal and Sharma 1995). In Iran, it occurs in the southern areas of the country in Khuzestan, Fars, Kerman, Hormozgan, Sistan and Baluchistan and southern Kermanshah Provinces (Manouchehri et al. 1976, Sedaghat and Harbach 2005).

Although there are several methods for control of Anopheles mosquitoes however environmental effect and resistance is a main human concern. Synthetic pyrethroids which considered as the most effective insecticides against anophelines, are still expensive and beyond the financial resources of some countries.

Using botanical insecticides is a technique which can apply as an alternative to synthetic chemical formulations. Most botanicals are rapid acting and breakdown quickly in the environment. The extract of whole leaf and essential oil of some certain plants have been investigated against some public health pests (Saxena and Sumithra 1985, Kumar and Dutta 1987, Chariandy et al. 1999, Hadjiakhoondi et al. 2000 ab, Markouk et al. 2000, Hadjiakhoondi et al. 2003, Tare et al. 2004, Vatandoost et al. 2004, 2008, Hadjiakhoondi et al. 2005–2006, Oshaghi et al. 2008 a).

Apiaceae (Umbelliferae) is one of the best known families of flowering plants, which comprise 300–450 genus and 3000–3700 species (Constance 1971, Pimenov and Leonov 1993). They are aromatic plant and have a distinctive flavor which diverse volatile compounds from the fruits and leaves. This family also encompasses toxic plants that some had used in ancient Athens to execute those sentenced to death (Constance 1971, Pimenov and Leonov 1993). Heracleum persicum Desf., known as Persian Hogweed or “Golpar”, is an annual native plant native to Iran with a wide distribution across the country. It is used in traditional medicine because of its antioxidant and anticonvulsant activity (Parsa 1948, Souri et al. 2000, Sayyah et al. 2005). Foeniculum vulgare Mill., known as Fennel or “Razianeh” in Iran and neighboring countries, has been used by humans since antiquity. It is generally considered indigenous to the shores of the Mediterranean, but now can be found in many parts of the world. (Muckensturm et al. 1997). The bioactivities of this plant have been investigated for its antioxidant activity (Oktaya et al. 2003) fumigant activity (Kim et al. 2003), antimicrobial activity (Ruberto et al. 2000), larvicidal activity (Chantraine et al. 1998, Pitasawat et al. 2007), insecticidal activity (Laurent et al. 1997), and acaricidal activity (Lee 2004).

Coriandrum sativum L, known as Coriander, is native to Iran, however it is widely distributed around the world. The seeds contain an essential oil (up to 1%) and the seeds are used in traditional medicine for indigestion, against worms, rheumatism and pain in the joints (Wangensteen 2004). It has also the nematicidal activity (Kim et al. 2008), antibacterial activity (Cantore et al. 2004) and larvicidal activity (Harve and Kamath 2004).

In recent years, much effort has been focused on the exploration of bioactive chemical compounds from indigenous plants for mosquito control in Iran. So, the purpose of this study was to determine bioactivity of three essential oils obtained from the seeds of plants against 4th instar larvae of An. stephensi.

Materials and Methods

Mosquito rearing

The fourth-instars larvae of An. stephensi used for bioassays. The laboratory-reared An. stephensi Bandar-Abbas strain were reared in the Department of Medical Entomology, Tehran University of Medical Sciences, and maintained at 27° C with a photoperiod of 12 hours light and 12 hours e dark in 80±10% relative humidity. A 10% percentage yeast suspension was used as food source.

Plant materials

In this study, the three plants from Apiaceae (Umbellifera) family were collected from three provinces of Iran in 2009 (Table 1). Examined plants were authenticated and identified by the Department of Pharmacognosy, Faculty of Pharmacy, Tehran University of Medical Sciences. The seeds were air-dried at room temperature and kept in an air-tight light-protected container.

Table 1.

Collection locations, physical properties and yields of volatile oils derived from seeds of H. persicum, F. vulgare and C. sativum

Plant species	Location	Harvest date (2009)	Color	Appearance	%Yield (w/w)
H. persicum	Mazandaran	August	Pale Yellow	Liquid	1.6
C. sativum	Chaharmahal and Bakhtiary	September	Pale Yellow	Liquid	0.9
F. vulgare	East Azarbaijan	September	Pale yellow	Liquid	0.8

Dried seeds of H. persicum, F. vulgare and C. sativum were subjected to hydro distillation using a modified Clevenger-type apparatus for 3 hours, dried over anhydrous sodium sulphate, and stored in amber-colored vials at 5°C until required for further work.

Bioassays and larval mortality

Fourth instar larvae of An. stephensi Bandar-Abbas strain was exposed to test concentrations of 5, 10, 20, 40, 80,160 and 320 ppm of essential oil for 24 hours according to standard method described by WHO (1981). As the oils do not dissolve in water, they were first dissolved in ethanol. The test 400 ml glass beaker were used by adding 1 ml of appropriate dilution of essential oil in ethanol and mixed with 249 ml of water to make up 250 ml of test solution (Dharmagadda et al. 2005). In control beakers, ethanol was applied into the water (1%). A minimum of 20 larvae per each concentrations were used for all the experiments. The dead larvae were counted after 24 hour recovery period, and percentage of mortality was reported from the average for the five replicates. The larvae considered dead were those that did not move when touched with a needle.

Statistical analysis

The lethal concentrations (LC50 and LC90) were calculated using Probit analysis (Finney 1971). For all bioassays, the percentages of mortality were adjusted for the mortality in controls by using Abbott's correction (Abbot 1925). Differences between means were considered significant at P≤ 0.05 (SAS Institute 2001).

Results

Hydro distillation of H. persicum, C. sativum and F. vulgare yielded (w/w, dry weight) of volatile oils (Table 1). The highest yield of volatile oil was obtained from H. persicum, whereas that of F. vulgare was the lowest.

The larvicidal activities of the essential oils against An. stephensi larvae under laboratory conditions are given in Table 2. All plants oil exerted significant larvicidal potential against An. stephensi after exposure for 24 h (Table 2). The lethal dosage of 50% (LC50) ranged between 20.10 and 120.95 ppm.

Table 2.

Parameters of probit regression line Anopheles stephensi to essential oil derived from H. persicum, F. vulgare and C. sativum seeds at different interval concentrations

Specimens	A	B±SE	LC50, 95% C.I.	LC90, 95% C.I.	X2 (df)	P value
C. sativum	−5.25	2.52±0652	47.68	181.62	43.82 (3)	<0.05
			120.95	389.90
			479.9	181172.4
H. persicum	−11.73	5.81±1.725	26.30	114.40	41.85 (3)	<0.05
			104.80	174.22
			336.90	1073788.4
F. vulgare	−4.83	3.71±1.023	6.05	24.30	53.6 (3)	<0.05
			20.10	44.51
			67.62	15839.5

Foeniculum vulgare oil was the most effective against An. stephensi with LC50 and LC90 values of 20.10 and 44.51 ppm, respectively, while C. sativum had the least mortality with LC50 and LC90 values of 120.95 and 389.90 ppm, respectively. This was also 5 times lower than H. persicum, which showed an LC50 of 104.80 ppm.

In regression line a positive correlation were observed between the essential oil concentrations and the percent mortality (Fig. 1).

Fig. 1.

Probit regression line of An. stephensi exposed to different interval concentrations of H. persicum, F. vulgare and C. sativum seed essential oils

Discussion

The use of plant essential oils in vector control is an alternative method for minimizing the side effects of chemical pesticides on the environment (Fatope et al. 1993). In recent surveys, it has been found that some of secondary plant metabolites act as botanical insecticides (Watanabe et al. 1993, Vatandoost et al. 2004, Nathan 2007). According to the biological results of present study, all of three plant oils exerted significant larvicidal potential against An. stephensi and the essential oil of F. vulgare had an appropriate effect. A study in Bolivia reported that essential oil of F. vulgare, was one of the most toxic oils on Ae. aegypti larvae. Also its LC50 and LC90 were 24.3 and 30.8 ppm, respectively (Chantraine et al. 1998). In the other study in Lebanon for assay of repellency and toxicity of aromatic plant extracts against the mosquito Culex pipiens form molestus, results showed that F. vulgare was one of the most significant repellency against this species (52.0 min of protection with concentration of 3%) also the LC50 and LC90 from essential oil of F. vulgare was 24.5 and 34 mg/l respectively (Traboulsi et al. 2005). In several study on chemical compositions of plants by using of gas chromatography and mass spectrometry (GC/MS), results showed that different compounds were present in different parts of the plants. For example, in the study on seeds essential oil of H. persicum it is found that it contains hexyl butyrate (35.5%), octyl acetate (27%) and hexyl isobutyrate (3.2%) (Sefidkon et al. 2004). In the other study on the leaves of C. sativum, Matasyoh et al (2009) reported four main compounds, including: 2E-decenal (15.9%), decanal (14.3%), 2E-decen-1-ol (14.2%) and n-decanol (13.6%). In the two same study on fruit and flower of F. vulgare, the transanethole (85.63%), estragole (5.27%) and D-limonene (3.8%) were present in the fruit (Dadalioglu and Evrendilek 2004), While other report showed limonene (63.6%), anethole (25.5%), fenchyl acetate (2.6%), α-Pinene (0.97%), myrcene (0.98%) and estragole (1.1%) from flower (Traboulsi et al. 2005). It seems that the larviciding effect of these essential oils of plant is attributed to these main compounds. We propose test different components of the plants against mosquitoes.

Results of the current study showed that the essential oil of these plants exhibited the biological effect on the larvae of An. stephensi. The use of botanical pesticide may help in reducing the environmental side effects by the synthetic insecticides. The results obtained suggest that the essential oils of H. persicus, F. vulgare and C. sativum are promising as larvicide against An. stephensi. Moreover, these results could be useful in the search for newer, more selective, and biodegradable larvicidal natural compounds. Further investigations are currently underway to isolate these compounds.