Chemical composition and insecticidal activity of the essential oil from Helichrysum faradifani endemic to Madagascar

Abstract Helichrysum faradifani (Asteraceae) is a perennial shrub growing in rocky and sandy places of Madagascar. The plant is used in the Malagasy traditional medicine as a wound-healing agent, disinfectant and for the treatment of syphilis, diarrhea, cough and headache. In the present work, we analysed the chemical composition of the essential oil distilled from the aerial parts of H. faradifani by GC-MS and evaluated its insecticidal activity against 2nd, 3rd and 4th instar larvae of the lymphatic filariasis vector Culex quinquefasciatus by acute toxicity assays. The most sensitive were 2nd instar (LC50 = 85.7 μL L−1) larvae. For the 3rd and 4th instar larvae, the estimated LC50 were 156.8 and 134.1 μL L−1, respectively. Monoterpene hydrocarbons (51.6%) were the major fraction of the essential oil, with the bicyclic α-fenchene (35.6%) as the predominant component. Sesquiterpene hydrocarbons (34.0%) were the second major group characterising the oil, with γ-curcumene (17.7%) as the most abundant component.


Introduction
Helichrysum Mill. is a large genus of the Asteraceae family (Gnaphalieae tribe) with a cosmopolitan distribution (Giuliani et al. 2016 their secondary metabolites such as flavonoids, heterodimeric pyrones, sesquiterpene lactones and terpene-containing essential oils (Appendino et al. 2015;Giuliani et al. 2016). Helichrysum species enjoys a good reputation in perfumery and cosmetics, being used in a wide array of glamorous fragrances and personal care products (Appendino et al. 2015). Furthermore, extracts from Helichrysum species exert notable pharmacological activities such antioxidant, antimicrobial and anti-inflammatory ones (Bremner 2009;Mancini et al. 2011).
The Helichrysum genus is represented in Madagascar by 115 species, among which most are endemic. Helichrysum faradifani Scott-Elliot is a perennial shrub spread all over Madagascar where it is found in rocky and sandy places, along rivers and uncultivated lands from sea level to 1800 m a.s.l. (Debray et al. 1971). The plant is known under several Malagasy vernacular names, namely 'ahibalala' , 'aferombohitra' and 'alamanitra' . In the Malagasy traditional medicine, the juice from crushes leaves is used for healing wounds and as disinfectant (Pernet and Meyer 1957;Boiteau et al. 1999) and for the treatment of opened boils and wounds (Beaujard 1988). The leaf decoction is used in the treatment of syphilis, diarrhea, cough and headache (Ramisiray 1901;Beaujard 1988).
Mosquito vector-borne diseases including malaria, dengue, yellow fever, chikungunya, St. Louis encephalitis, Japanese encephalitis, filariasis and, more recently, outbreaks of Zika virus Benelli and Romano 2017) still represent a burden for developing countries such as Madagascar, due to political and economic instability, and lack of conventional medical treatments (Benelli and Beier 2017;Benelli et al. 2017a). Culicidae may also infect animals with parasites and key pathogens causing dog heartworm, West Nile virus and Eastern equine encephalitis (WHO 2012;Nicoletti et al. 2016). Furthermore, in the last 20-30 years, most synthetic pesticides and antibiotics used contributed to increase the resistance phenomenon and the risks for human health and environment (Benelli and Mehlhorn 2016;Pavela and Benelli 2016a;Benelli et al. 2016bBenelli et al. , 2017a. Culex quinquefasciatus Say is a key vector of lymphatic filariasis, Rift Valley fever, West Nile, St. Louis encephalitis and Western equine encephalitis virus (Vadivalagan et al. 2017). Recently, several researches stressed the importance of the eco-friendly control of C. quinquefasciatus young instar populations using plant-borne pesticides, including nanoformuled ones (Benelli 2015a(Benelli , 2015b(Benelli , 2016a(Benelli , 2016bGovindarajan and Benelli 2016;Benelli et al. 2017b;Benelli 2018).
In Madagascar, local people already used alternative natural treatments to control mosquito vectors and related diseases (Benelli et al. 2016c(Benelli et al. , 2017a. Indeed, the outstanding and extremely diverse flora of this land and the occurrence of popular treatments against mosquito-borne diseases make Madagascar the ideal territory where new plant-borne compounds can be experimented against mosquito vectors of public health importance. Thus, continuing our studies on the chemical and biological characterisation of essential oils from aromatic and medicinal plants growing in Madagascar (Nicoletti et al. 2012;Randrianarivo et al. 2013;Rasoanaivo et al. 2013;Benelli et al. 2016c;Rakotosaona et al. 2016;, in this work, we present the chemical composition of H. faradifani essential oil and its insecticidal activity against various larval instars of the filariasis vector mosquito C. quinquefasciatus.

Insecticidal activity
The effect of the essential oil obtained from H. faradifani aerial parts on mortality of different larval instars of C. quinquefasciatus is shown in Table 2. The results indicate that the most sensitive were the 2nd instar larvae, with an estimated LC 50 of 85.7 μL L −1 , i.e. with the significantly lower lethal concentration than the ones estimated for the 3rd and 4th instar larvae (LC 50 = 156.8 and 134.1 μL L −1 , respectively). Although there was a certain difference between sensitivity for the 3rd and 4th instar larvae, the reliability intervals of LC 50 overlapped and therefore the difference cannot be considered significant (P ≤ 0.05). Moreover, there was nearly no difference between the LC 90 values that were estimated at 251.1 and 260.2 μL L −1 for the 3rd and 4th instar larvae, respectively.
As demonstrated by a number of scientific studies (Pavela 2015a), mosquito larvae are sensitive to essential oils and substances contained therein. Nevertheless, larvicidal effectiveness of essential oils is often varied because it depends not only on the mosquito species, the size and age of larvae but, particularly, on the chemical composition of essential oils and on the mutual synergic and antagonistic effects between substances contained therein (Pavela 2015b;Benelli et al. 2017bBenelli et al. , 2017c. Although the essential oil from H. faradifani used for our tests demonstrated worse effectiveness than, e.g., thymol, carvacrol or trans-anethole, it was more effective than, e.g., menthone or a-terpineol (Pavela 2015b). Therefore, the oil can be considered promising for development of environmentally-friendly botanical larvicides.
Nevertheless, even though essential oils are often seen as relatively safe for non-target aquatic organisms (Pavela 2014(Pavela , 2016Pavela and Benelli 2016b;Pavela and Govindarajan 2017), more studies will be needed to investigate environmental safety of potential larvicidal formulations based on the essential oil from H. faradifani.

Experimental
See Supplementary Material.

Conclusions
Overall, the essential oil from H. faradifani showed a unique chemical composition, which was characterised by the abundance of the bicyclic monoterpene α-fenchene. This profile may result from geographical isolation of the species. Our results allow to conclude that the essential oil composition of H. faradifani is particularly sensitive to the variance of microclimate and geographical factors occurring in Madagascar (Ralijerson et al. 2005). The essential oil was moderately toxic to larvae of C. quinquefasciatus, thus encouraging further studies for the development of sustainable and effective formulations (e.g. microencapsulated ones) to be used against mosquito larvae.