Molluscicidal and larvicidal activities and essential oil composition of Cymbopogon winterianus

Abstract

Context. Cymbopogon winterianus Jowitt ex Bor (Poaceae), known as citronella grass, is an aromatic herbaceous plant and the essential oil extracted from this grass is used in cosmetics, perfumes, hygiene and cleanliness products worldwide.

Objective: This study investigated the composition and molluscicidal and larvicidal activities of the essential oil of C. winterianus cultivated in North Brazil.

Materials and methods: The oil was obtained by hydrodistillation, analyzed by gas chromatography (GC) and GC-mass spectrometry and then its molluscicidal and larvicidal activities against snails (Biomphalaria glabrata) and hatched larvae of Artemia salina, respectively, were evaluated at concentrations from 10 to 1000 mg/L.

Results: The main constituents of oil were citronellal (26.5%), geraniol (16.2%), elemol (14.5%) and citronellol (7.3%). The molluscicidal test revealed significant lethal concentration (LC) values (LC₉₀ = 97.0 mg/L, LC₅₀ = 54.0 mg/L and LC₂₀ = 22.0 mg/L), indicating the presence of molluscicidal compounds in the oil. In addition, the oil showed moderate larvicidal activity (LC₅₀ = 181.0 mg/L) against the larvae of A. salina, which could justify its use in the aquatic environment without affecting other living organisms.

Discussion and conclusion: The results suggest that the oil of C. winterianus could be an effective alternative to control schistosomiasis, with an average margin of safety to other living organisms that coexist with snails.

• Artemia salina
• Biomphalaria glabrata
• GC/MS analysis
• Poaceae

Introduction

Poaceae comprises about 660 genera and 9000 species (Bassolé et al., 2011), widely distributed worldwide, with about 1300 species present in Brazil (Welker & Longhi-Wagner, 2007). This family is known for its high economic value in the production of wheat, maize, oats, sugar cane and rice, among others (Souza & Lorenzi, 2005). The genus Cymbopogon includes approximately 180 species, subspecies, varieties and subvarieties. Many are aromatic and have commercial importance in the production of essential oils used in perfumery, cosmetics and pharmaceutical applications, such as species with high neral and geranial contents and a mixture of isomeric monoterpene aldehydes (Irkin & Korukluoglu, 2009; Khanuja et al., 2005; Rauber et al., 2005).

The species Cymbopogon winterianus Jowitt ex Bor (Poaceae) is popularly known as citronella grass, citronella or citronella Java (Tanu & Adholeya, 2004). This is an aromatic herb with long caespitose, slender and acute leaves (Weiss, 1997). The grass, native to Southeast Asia, is scattered over the warmer regions of India, Burma, Malaysia and Sri Lanka and was introduced in Brazil in the mid-eighteenth century (Lorenzo et al., 2000). In Brazilian folk medicine, citronella is used as an analgesic, anxiolytic and anticonvulsant (Akhila, 2010).

The essential oil of C. winterianus is rich in citronellal, citronellol and geraniol (Beneti et al., 2011; Silva et al., 2010; Simic et al., 2008). Citronella oil has some scientifically confirmed properties, including antiseptic, antispasmodic, diuretic and febrifugal properties (Lawless, 2002), anticonvulsant activity in rodents in different models of epilepsy (Quintans-Jr et al., 2008), larvicidal activity against Aedes aegypti (Tawatsin et al., 2001), anti-Escherichia coli activity (Duarte et al., 2007), antinociceptive, anti-inflammatory and antioxidant properties (Leite et al., 2010).

Schistosomiasis affects over 200 million people distributed in 54 countries in Africa, Asia and the Americas. In Africa, it is known as “bilharziose” and in Brazil as “barriga-d`água”. Brazil is the country with the largest endemic area of America, with schistosomiasis being present in 19 states. This is a chronic and debilitating disease, characterized by the transmission of the trematode Schistosoma mansoni by snails of the genus Biomphalaria, which are its intermediate hosts and are essential for the development of Schistosoma larvae and the continuity of its lifecycle (Oliveira-Filho et al., 2010).

The use of molluscicidal compounds in water in which snails are known to occur is considered essential in the integrated strategy to control the disease, because the drugs available for treatment do not controls the transmission of schistosomiasis, and also have numerous side effects that hinder the tolerability of patients. The World Health Organization (WHO) recommends only niclosamide to be used as a molluscicidal product in management programs. However, the high cost of this synthetic compound, concerns about its toxicity to non-target organisms and the possible development of resistance in the mollusk have stimulated the search for natural molluscicides of plant origin (WHO, 1993).

Before the application of molluscicides to any natural water resource, it is first necessary to determine the possible toxic effects of the active components to aquatic organisms, birds and mammals (Bilia et al., 2000). In this context, a bioassay using larvae of the brine shrimp, a microcrustacean from seawater that has high sensitivity for a broad range of compounds, can be used to indicate the toxicity and larvicidal activity of a potential molluscicide against other organisms that coexist with snails, such as fish and zooplankton (Lima et al., 2002; Meyer et al., 1982; Santos et al., 2007).

Considering the importance of the discovery of new natural compounds with molluscicidal activity, the essential oil of C. winterianus was tested against the snails of Biomphalaria glabrata and its toxicity evaluated against larvae of Artemia salina, with the aim of identifying a selective, powerful and potentially safe molluscicide to control schistosomiasis. In addition, the oil composition was analyzed and its volatile constituents identified.

Materials and methods

Chemicals

Anhydrous sodium sulfate, dimethylsulfoxide (DMSO) and potassium dichromate (K₂Cr₂O₇) were purchased from Merck Chemical Company (Darmstadt, Germany). Distilled pure water was used.

Plant material

The large leaves of C. winterianus were collected in the Medicinal Garden “Berta Lange Morretes”, located at Universidade Federal do Maranhão (UFMA), Campus do Bacanga, São Luís, Maranhão, Brazil, in January 2010 (2°33′21,9′′S; 44°18′3,61′′W). A plant voucher (No. 1383/SLS017213) was deposited in the Herbarium “Atticus Seabra” of UFMA and identified by Dra. Terezinha de J. A. Silva Rêgo, and by comparison with an authentic sample in the Herbarium “Murça Pires” of Museu Paraense Emílio Goeldi, Belém, Pará, Brazil.

Plant processing

The plant material was air-dried, ground and subjected to hydrodistillation using a Clevenger-type apparatus (100 g, 3 h). The moisture content of the sample was calculated after phase separation in a Dean-Stark trap (5 g, 60 min), using toluene. The oil was dried over anhydrous sodium sulfate, its percentage content was calculated on the basis of the plant dry weight, and then it was stored in a dark flask and maintained at refrigeration (+5 °C) before the chemical and biological analyses (Rao et al., 2004).

Oil-composition analysis

The analysis of the oil was carried out on a THERMO DSQ II gas chromatograph-mass spectrometry instrument (GC-MS, Austin, TX), under the following conditions: DB-5 ms (30 m × 0.25 mm; 0.25-µm film thickness) fused-silica capillary column; programmed temperature: 60–240 °C (3 °C/min); injector temperature: 250 °C; carrier gas: helium, adjusted to a linear velocity of 32 cm/s (measured at 100 °C); injection type: splitless (2 µL of a 1:1000 hexane solution); split flow was adjusted to yield a 20:1 ratio; septum sweep was constant at 10 mL/min; EIMS: electron energy, 70 eV; temperature of ion source and connection parts: 200 °C. Quantitative data regarding the volatile constituents were obtained by peak-area normalization using a FOCUS gas chromatograph/flame ionization detector (GC/FID, Austin, TX) operated under conditions similar to those in GC-MS, except for the carrier gas, which was nitrogen. The retention index was calculated for all the volatile constituents using an n-alkane homologous series.

Individual components were identified by comparison of both the mass spectrum and the GC retention data with authentic compounds previously analyzed and stored in the data system and with the aid of commercial libraries containing the retention indices and mass spectra of volatile compounds commonly found in essential oils (Adams, 2007; NIST, 2005).

Molluscicidal activity evaluation

Molluscicidal activity was evaluated according to the standards recommended by the WHO (1983) and Silva et al. (2007). Initially, a preliminary experiment was carried out at a concentration of 100 mg/L (50 mg oil, dissolved in 5 µL of DMSO, and supplemented using ultrapure water to a solution of 500 mL). In the definitive test, carried out under the same conditions, we assayed concentrations of 25, 50, 75 and 100 mg/L. The blank (control) was assayed in parallel without the presence of oil. All assays were performed in triplicate. In each test, groups of 10 wild snails from the species B. glabrata (10–15 mm diameter) were exposed to the solution for a period of 24 h at room temperature. Then, the groups were transferred to Petri dishes separately, and it was checked whether the snails presented responses to stimuli using forceps. With the stimulus response, the groups were transferred to clean containers containing only ultrapure water and food and then observed after 48 and 72 h following the beginning of the exposure. The number of dead individuals was recorded. The snail death was indicated by discoloration, contraction of the hemolymph, absence of muscle contraction, bleeding and decaying of body tissues.

Larvicidal activity evaluation

We investigated the larvicidal activity of the oil (Lewan et al., 1992). A saline solution with 60 g of sodium chloride in 1000 mL of ultrapure water was prepared and used for hatching brine shrimp (A. salina). In a glass container, the cysts of A. salina were incubated in the saline solution, with constant aeration provided by an air pump connected to the glass apparatus, as well as a 25 W light to provide artificial lighting. The glass container was divided into two compartments. In the first compartment, with the dark environment, the cysts of A. salina were placed and the second, which was connected to the first compartment, was provided with light for migration of the larvae, thus, showing positive phototropism. About 20 mg of essential oil was dissolved in 20 µL of DMSO and this volume was made up to 2 mL saline. From this solution, dilutions (10, 100 and 1000 mg/L) were prepared, and 10 larvae at the metanauplius stage were added to each dilution. For the negative control, we used saline in association with DMSO (0.01%), while the positive control was carried out with potassium dichromate. After 24 h incubation, the numbers of living and dead larvae were counted.

Statistical analysis

The evaluation of the oil toxicity against B. glabrata and A. salina was performed by calculating the median LC by Probit analysis (Finney, 1971) using the software SPSS 13.0. Data were analyzed by ANOVA followed by the Tukey post-test using the Graph Pad Prism software, version 4.03. Differences between concentrations were considered statistically significant when p < 0.05.

Results

Oil composition

Cymbopogon winterianus presented colorless oil, with a distinct odor and a yield of 1.3%. Using GC and GC-MS we identified 41 constituents, totaling 98% of the oil composition, which are listed in Table 1. Monoterpenes and sesquiterpenes (both hydrocarbons and oxygenated) were present at 64.9 and 30.3%, respectively, among the identified constituents. The main constituents found in the oil were citronellal (26.5%), geraniol (16.2%), elemol (14.5%) and citronellol (7.3%).

Table 1. Oil composition (%) of C. winterianus.

RI	Constituents	Oil (%)
990	Myrcene	3.3
1026	Limonene	2.2
1045	(E)-β-Ocimene	0.7
1095	Linalool	0.7
1128	allo-Ocimene	0.2
1150	Citronellal	26.5
1175	(E)-Isocitral	0.2
1223	Citronellol	7.3
1228	Nerol	0.4
1237	Neral	0.5
1250	Geraniol	16.2
1266	Geranial	0.7
1350	Citronelyl acetate	2.5
1359	Neryl acetate	0.1
1379	Geranyl acetate	3.4
1390	β-Elemene	4.4
1420	β-Ylangene	0.3
1432	β-Gurjunene	0.2
1440	Aromadendrene	0.1
1453	Neryl propanoate	0.1
1455	α-Humulene	0.1
1462	cis-Cadina 1,(6),4- diene	0.1
1468	cis-Muurola- 4,(14),5- diene	0.1
1480	γ-Muurolene	0.1
1484	Germacrene D	1.1
1494	trans-Muurola- 4,(14),5- diene	0.1
1497	Viridiflorene	0.1
1501	α-Muurolene	0.4
1513	γ-Cadinene	0.4
1523	δ-Cadinene	2.5
1528	Zonarene	0.1
1539	α-Cadinene	0.1
1549	Elemol	14.5
1622	10-epi-γ-Eudesmol	0.1
1629	1-epi-Cubenol	0.1
1632	γ-Eudesmol	0.8
1639	epi-α-Cadinol	0.5
1643	epi-α-Muurolol	0.7
1651	β-Eudesmol	0.2
1653	α-Eudesmol	0.2
1654	α-Cadinol	2.7
1714	(2E,6Z)-Farnesol	0.2
	Monoterpene hydrocarbons	6.4
	Oxygenated monoterpenes	58.5
	Sesquiterpene hydrocarbons	10.3
	Oxygenated sesquiterpenes	20.0
	Total	95.2

RI = retention index on DB-5 ms column.

Bold values highlight the main constituents.

Biological activity

Percentage mortality values at the tested concentrations and the lethal concentrations (LC₉₀, LC₅₀ and LC₂₀) obtained by Probit analysis of the bioassay data of the oil against adult snails of B. glabrata are shown in Table 2. These results show toxic activity for the snails at all concentrations tested, between 24 and 72 h after exposure. The lethal concentration values of the oil were LC₉₀ = 569.4 mg/L in 24 h, LC₅₀ = 125.8 mg/L in 48 h and LC₂₀ = 97.4 mg/L in 72 h. In determining the larvicidal activity against brine shrimp larvae, the oil of C. winterianus showed an LC₅₀ value of 181.5 mg/L, in 24 h of exposure, which indicates moderate toxicity (Amarante et al., 2011). The observed activity was concentration dependent and Figure 1 shows the results of larvae mortality at the tested concentrations.

Figure 1. Effects of the oil of C. winterianus (10, 100 and 1000 mg/L) or potassium dichromate (10 mg/L) on brine shrimp larvae. Values are the mean ± SEM (n = 10 per group). ^ap < 0.001 compared to control; ^bp < 0.001 compared to 10 mg/L; ^cp < 0.001 compared to 100 mg/L; ^dp < 0.001 compared to 1000 mg/L.

Table 2. Mortality of snails resulting from the molluscicide action of the oil of C. winterianus.

Observation time	Oil concentration (mg/L)	Mortality %	LC values (mg/L)
24 h	100	50	LC20 = 177.41
	75	30	LC50 = 271.4
	50	10	LC90 = 569.39
	25	0
48 h	100	70	LC20 = 44.38
	75	60	LC50 = 72.15
	50	40	LC90 = 125.84
	25	10
72 h	100	90	LC20 = 22.19
	75	70	LC50 = 54.44
	50	50	LC90 = 97.4
	25	20

Discussion

There are several studies on the essential oil composition of C. winterianus; these reveal high variability in its chemical profile. The identification of citronellal, citronellol and geraniol as being among the main constituents of the oil in our analysis can be confirmed in other specimens of C. winterianus previously reported (Lorenzo et al., 2000; Rao et al., 2004; Simic et al., 2008). A variety of these species occurring in Sergipe in Northeastern Brazil showed a higher percentage of geraniol (40.1%) than citronellal (27.4%), this inversion in the composition of the oil may have been influenced by genetic, environmental or eco-physiological parameters (Quintans-Jr et al., 2008; Sangwan et al., 2001).

Although the essential oil of C. winterianus has already been reported along with some of its biological activities (Oliveira et al., 2011), its molluscicidal activity had not yet been evaluated. Moreover, this is the first time that a species belonging to Poaceae has been evaluated with respect to molluscicidal activity against adult snails of the Biomphalaria genus. Citronellal, citronellol and geraniol, the main monoterpenes identified in the oil of C. winterianus, showed molluscicidal activity as previously reported; among these, geraniol exhibited the greater effect on the snails, with a lethal concentration of 0.15 ppm (Lahlou & Berrada, 2001; Radwan et al., 2007).

The quest to identify novel natural products to control negligible diseases such as schistosomiasis is a promising area of scientific research in Third World countries, due to poor sanitation conditions (Santos et al., 2007). A phytochemical product with molluscicidal property, easily accessible to the population, is a way to reduce the risk of transmission of schistosomiasis, if it can act directly on the parasite’s life cycle, eliminating the intermediate host and constituting a low-cost alternative (Luna et al., 2005).

According to the WHO, molluscicidal activity is considered significant when the lethal concentration is less than 100 mg/L (Singh & Singh, 2009; WHO, 1965, 1983; Yang et al., 2008). Based on our results, the essential oil of C. winterianus with a lethal concentration of 97.4 mg/L, in 72 h, could be developed as a significant molluscicidal product, as an alternative to control schistosomiasis in the endemic areas of North and Northeast Brazil.

The lethality test against brine shrimp larvae to determine the toxicity of the oil, compared to other living organisms that coexist with snails, was used as a bio-indicator. We aimed primarily to determine the safety of the oil for its potential use as a molluscicide, in order to determine the existence of risks within the range required by the WHO (Devappa et al., 2012; Jaki et al., 1999; Santos et al., 2007). Moreover, the term moderate toxicity has a precise meaning, considering that crude extracts with lethal concentrations less than 1000 mg/L show significant correlations with acute oral toxicity in mice, as well as with antitumor, anti-chagasic, and insecticidal activities (McLaughlin et al., 1991; Meyer et al., 1982; Parra et al., 2001).

Conclusion

In the essential oil of C. winterianus the terpenes citronellal, geraniol, citronellol and elemol were the main constituents. The oil was active at the dose recommended by the WHO, with a lethal concentration of 97.4 mg/L, representing an alternative to control B. glabrata infestation, and reduce the level of schistosomiasis transmission. When subjected to larvicidal activity against the brine shrimp larvae the oil showed moderate toxicity, with a lethal concentration of 181.5 mg/L, indicating an average margin of safety to other living organisms that coexist with the snails.

Further investigations and seasonal studies should be performed using the plant and its oil, because the oil composition and consequent molluscicidal activity could be influenced by environmental conditions. In addition, further studies are required to confirm that the molluscicidal activity of the oil is because of the presence of the monoterpenes citronellal, citronellol and geraniol. It is also necessary to evaluate the toxicity to fishes (ictiotoxicity) to guarantee the safe of this oil.

Declaration of interest

The authors are grateful to Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA), grant No APP-Universal-00371/11, for their financial support. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.