Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti

Toxicon 44 (2004) 829–835 www.elsevier.com/locate/toxicon

Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti Carolina B. Wandscheera, Jonny E. Duqueb, Mario A.N. da Silvab, Yoshiyasu Fukuyamac, Jonathan L. Wohlkea, Juliana Adelmanna, Jose´ D. Fontanaa,* a

Biomass Chemo/Biotechnology Laboratory (LQBB), Department of Pharmacy, Federal University of Parana (UFPR), 80210-170 Curitiba, PR, Brazil b Medical and Veterinary Entomology Laboratory (LEMV), Department of Zoology, Federal University of Parana (UFPR), 80210-170 Curitiba, PR, Brazil c Faculty of Pharmaceutical Sciences, Institute of Pharmacognosy, Tokushima Bunri University, Tokushima 770-8514, Japan Received 7 April 2004; accepted 6 July 2004

Abstract Ethanolic extracts from the kernels of ripe fruits from the Indian Lilac Melia azedarach and from the well-known Neem tree, Azadirachta indica were assayed against larvae of Aedes aegypti, the mosquito vector of dengue fever. The lethality bioassays were carried out according to the recommendations of the World Health Organization. Extracts were tested at doses ranging from 0.0033 to 0.05 g% in an aqueous medium for 24 and 48 h, at 25 or 30 8C, with or without feeding of the larvae. LC50, LC95 and LC99 were determined. Both seed extracts proved lethal for third to fourth instar larvae. Non-fed A. aegypti larvae were more susceptible to Azadirachta extracts at both temperatures. Under a more realistic environmental situation, namely with fed larvae at 25 8C, the death rates caused by the Melia extract were higher, although at 30 8C the extract of Azadirachta had an even higher lethality. Inter allia, the LC50 values for the crude extracts of these two members of the Meliaceae ranged from 0.017 to 0.034 g% while the LC99 values ranged from 0.133 to 0.189 g%. Since no downstream processing was undertaken to purify the active agents in the extracts, our findings seem very promising, suggesting that it may be possible to increase the larvicidal activity further by improving the extraction and the fractionation of the crude limonoids, for instance removing the co-extracted natural fats. q 2004 Elsevier Ltd. All rights reserved. Keywords: Melia azedarach; Azadirachta indica; Aedes aegypti; Limonoids; Lethality; Larvicidal; Phytopesticides

1. Introduction The incidence of dengue fever (DF) has increased dramatically over the last decade. It has become endemic in

* Corresponding author. Biomass Chemo/Biotechnology Laboratory (LQBB), Department of Biochemistry, Federal University of Parana (UFPR), 81531-990 Curitiba, PR, Brazil. Fax: C55-41-2662042. E-mail address: [email protected] (J.D. Fontana). 0041-0101/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2004.07.009

more than 100 countries and more than 2.5 billion people are at risk, mainly in Africa, the Americas, the western Mediterranean, South and East Asia, and the west Pacific. Over the first 4 months of 1998, which is the summer period in the southern hemisphere, Brazil, with 234,828 reported cases, was responsible for 60% of all cases in North, Central, and South America. Brazil recorded 406,206 cases in 2001 and 560,000 cases in 2002. International monitoring organizations indicated 123,948 cases in the first trimester of 2003, as reported by the World Health Organization (WHO) in its Communicable Disease Surveillance

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and Response (CSR) report ‘Disease Outbreaks’ of June 8, 1998, entitled ‘Dengue in Brazil’, and in fact sheet 117 ‘Dengue and Dengue Haemorrhagic Fever’ of April 2002. The number of reported cases of dengue and dengue hemorrhagic fever (DHF) in regions of the Americas is confirmed by the Pan American Health Organization (PAHO) in its report of 2003 (Halstead et al., 2001). These data are also supported by other organizations such as the Caribbean Epidemiology Center at Tobago (CAREC) and the Center for Disease Control (CDC) of the USA and also in the newsletter of October 2001, TDR News 66, edited by The Special Program for Research and Training in Tropical Diseases. The factors accounting for dengue outbreaks are: uncontrolled demographic increase, poor urban planning, reduced epidemiological surveillance and progressive resistance of the vector mosquito to several insecticides produced by the chemical industry (Rawlins, 1998; Macoris et al., 1999; Wirth and Georghiou, 1999; Rodrı´guez et al., 1999; Hemingway and Ranson, 2000; Campos and Andrade, 2001; Gluber, 2002; Guzma´n and Kouri, 2002). Melia azedarach (known as the Chinaberry tree or Indian Lilac and also known as ‘cinnamon’ or ‘Santa Barbara’ in Brazil) and Azadirachta indica (Neem) are related Meliaceae trees, originally from Asia, which have spread to several tropical and subtropical regions of Africa, America and Australia (Hurst, 1942). The oil and organicsolvent extracts from these trees leaves and fruits display several bioactivities against a wide range of insects and other organisms, governing chemical maturation of molt hormones, chitin biosynthesis and feed deterrence. These biological effects are mainly due to limonoids or tetranortriterpenoids, such as meliacarpinin for Melia and azadirachtin for Azadiracta, although over 50 different bioactive compounds (terpenoids and others) have been reported in both plants. Hence they are promising sources for the control of flying and hematophagous insects (Srivastava, 1986; Champagne et al., 1992; Nakatani et al., 1994; Sharma et al., 1995; Dua et al., 1995; Huang et al., 1996; Mitchell et al., 1997; Karnavar and Dlamini, 1998; Bohnenstenger et al., 1999; Siddiqui et al., 2000). As another example, bitter limonoids and new meliacarpinins were isolated from fruits and roots of M. azedarach, respectively (Fukuyama et al., 1983, 2000). In fact, A. indica (the holy Neem tree of Indian medicine) protects against the bite of Anopheles (Diptera: Culicidae) (Sharma et al., 1995), it repels Culex quinquefasciatus (Diptera: Culicidae) and Aedes spp. (Diptera: Culicidae) (Dua et al., 1995) and it kills larvae from the last instars of the late mosquito (Zebitz, 1984; Monzon et al., 1994; Zarroug et al., 1988; Dzul et al., 2002). Aqueous extracts of Neem cause nymphal death in Bemisia tabaci (Hemiptera: Aleyrodidae) (Souza and Vendramim, 2000) and inhibit feeding in Sesamia nonagrioides (Lepidoptera: Noctuidae) (Juan et al., 2000). Sublethal doses of Neem azadirachtin

deter feeding in Lepidoptera and change their feeding behavior (Martinez and van Endem, 1999). To date there is no report about the use of M. azedarach fruit kernels against the mosquito vector of dengue fever, Aedes aegypti. We decided to investigate this potential use since this tree is very common across Brazil and ethanol is a cheap organic solvent that would be readily available for a large-scale extraction process, with a national production of more than 14 billions liters from 2003 to 2004 sugar cane crop.

2. Materials and methods 2.1. Plant collection Ripe fruits from M. azedarach L (Rutales: Meliaceae) were collected in Curitiba, in the State of Parana, Brazil, between May and June, 2002 (autumn). A. indica A. Juss (Rutales: Meliaceae) seeds (endocarps) were purchased in Campinas, in the State of Sa˜o Paulo, Brazil. 2.2. Extraction Both fruits were processed in the Biomass Chemo/Biotechnology Laboratory of the Department of Pharmacy of the Federal University of Parana (UFPR) Melia, fruits were manually de-pulped and the kernels (endocarps) were thoroughly washed with distilled water, air dried, and triturated in a commercial Waring blender at maximal speed for 5 min in successive intermittent cycles thus avoiding heating of the sample, but ensuring complete rupture of the coarse and hard lignocellulosic seed coat. Since for each Melia stony endocarp, several seed embryos (cotyledons) are encased by a heavily pigmented dark-brown testa, solubilization of this colored material in ethanol was a good indicator of the effectiveness of the grinding of the whole seed. Conversely, the Neem endocarp, resembling the edible pistachio, with a single embryo, was more easily processed since it is encased in a thinner coat that breaks apart with manual pressure. In this case, the release of chlorophyll indicated effective grinding. Ground endocarps from both trees were then extracted with absolute ethanol (10 vol.) at room temperature for 24 h in the dark, using sealed Erlenmeyer flasks that had been flushed with nitrogen and kept under agitation in a rotatory shaker. Extracts were filtered through Whatman No. 3 paper and the residue twice re-extracted with warmed (50 8C) ethanol. The solvent from the combined extracts was removed in a rotary evaporator (Laborota 4000; Heidolph, Germany) and most of the residual moisture eliminated in a vacuum centrifuge (Savant). The extracts were then lyophilized (Freeze Dryer 4.5; Labconco) and stock solutions of each oily extract at 200 mg/ml were prepared using again ethanol as solvent for use in the bioassays. These are referred to as the standardized ethanolic extracts (SEE).

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A sample of Melia endocarps was dissected to recover the embryos (cotyledons) separately, which were then processed following the same procedure. 2.3. Limonoid spectrometry and chromatography The total limonoid contents of Melia and Azadirachta ethanolic extracts (SEE’s) were spectrophotometrically determined at 577 nm using the optimized vanillin– sulphuric acid assay (Dai et al., 1999), but including blanks for each SEE in the absence of the chromogen to avoid any overestimation arising from the seed testa pigments. HPLC was carried out in a 14.5!0.6 cm Shimpack CLC-ODS (M) column using 40% aqueous acetonitrile at a 0.5 ml/min flow rate (900 psi pressure) in a Shimadzu multi-modular RID/ SPD/SCL/LC-10A(T) apparatus. Since azadirachtin and related limonoids are transparent to visible light, a lmax of 210 nm, read from a Sigma-Aldrich authentic azadirachtin standard, was adopted. Aza-16, an enriched preparation of azadirachtin from Neem, provided by Rym Exports, Mumbai, India, was also tested. Melianin B was isolated at Tokushima Bunri University, Japan. Thin layer chromatography (TLC) was carried out in Merck SG-60 chromatoplates using dichloromethane: methanol as the mobile phase and using vanillin for revelation, but replacing the original acid by a milder one, namely phosphoric acid, in order to render the hot spray less destructive. Afterwards, the sprayed plates were heated on a hot plate (O105 8C) and photographed with a digital Sony DSC P92 CyberShot camera under natural and UV (354 nm) light.

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2.5. Larval toxicity bioassay The larvicidal bioassay was carried out according to the recommendations of WHO (1981), using five different doses of both SEE’s (from 0.0033 to 0.05 g%; dry content). Depending on the concentration of the extract, the final aqueous solution had an opalescent or slightly milky appearance. Twenty healthy Aedes larvae from the late third and early fourth instars were used in each bioassay pot. The larvae were exposed to these solutions for either 24 or 48 h with a 12 h photoperiod, either at 25 8C or 30 8C, in 320 ml plastic pots containing 150 ml of water. Half of the pots were fed with 10 mg Tetraminw. Each treatment was performed in five replicate pots. Blanks and assays (irrespective to the amount of dry matter being added) were normalized to the same final concentration of ethanol (!1%) since it did not affect the development of larvae into adult insects. The dissolution or suspension of the extracts in the pot water was carried out by fast mixing in order to avoid over-exposition of the larvae to the concentrated ethanolic solution, which was added last. 2.6. Statistical analyses The analysis program Probit (Finney, 1971) was used for the determination of LC50, LC95, and the diagnostic concentration at LC99 in 48 h.

3. Results and discussion 3.1. Lethality bioassays

2.4. Insects, eggs, and larvae The A. aegypti Linneaus 1762 (Diptera:Culicidae) ‘Rockfeller’ strain, originally from the CDC (Center of Disease Control; Porto Rico laboratory), was kindly provided by the Department of Zoology of the University of Campinas, Sa˜o Paulo, Brazil Mosquito colonies were maintained in the Laboratory of Medical and Veterinary Entomology of the Department of Zoology of UFPR at 25 8C and 80% relative humidity. The larvae were fed with an artificial diet (Tetraminw). Adult mosquitoes (females) were fed on dried rat blood in order to stimulate egg laying.

Table 1 shows the lethal concentrations of the ethanolic extracts from seed kernels (whole endocarps) of M. azedarach and A. indica in bioassays with A. aegyti larvae, without food at 25 and 30 8C. The corresponding results for fed larvae are shown in Table 2. The observed effects were expressed as LC50, LC 95 and LC99 following Probit analysis. The fighting of hematophagous mosquitoes that are vectors of diseases like dengue, malaria and yellow fever depends on the routine application of progressively larger amounts of synthetic insecticides. Conversely, relatively

Table 1 Lethal concentrations (g%) of ethanolic extracts of M. azedarach and A. indica seed kernels for non-fed larvae of A. aegypti at different temperatures M. azedarach

LC50 LC95 LC99 c2

A. indica

25 8C

30 8C

25 8C

30 8C

0.166(0.093–1.83) 0.604(0.210–59.0) 1.03(0.293–249.8) 0.931292

0.152(0.091–0.758) 0.537(0.212–12.06) 0.907(0.298–38.10) 2.750521

0.044(0.039–0.051) 0.124(0.098–0.173) 0.190(0.142–0.289) 1.850673

0.063(0.042–0.159) 0.217(0.104–1.826) 0.363(0.149–1.826) 9.907842

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Table 2 Lethal concentrations (g%) of ethanolic extracts of M. azedarach and A. indica seed kernels for fed larvae of A. aegypti at different temperatures M. azedarach

LC50 LC95 LC99 c2

A. indica

25 8C

30 8C

25 8C

30 8C

0.034(0.029–0.04) 0.175(0.122–0.29) 0.345(0.217–0.68) 2.091790

0.038(0.024–0.210) 0.119(0.055–22.78) 0.189(0.073–168.0) 26.410502

0.056(0.029–0.268) 0.277(0.096–8.992) 0.536(0.151–40.07) 21.607275

0.017(0.011–0.027) 0.073(0.041–0.343) 0.133(0.062–1.087) 14.311704

minor amounts of biological products are currently being utilized for the same purpose. Concerning the insectiostatic or insecticidal action of M. azedarach, its leaves and fruits significantly reduced growth of Spodoptera littoralis (Lepidoptera: Noctuidae) (Schmidt et al., 1998). Feed deterrence by fruit extracts was also observed for Triatoma infestans (Hemiptera: Reduviidae) nimphae (Valladares et al., 1999). Lymnaea cubensis (Pulmonata: Lymnaeidae), a Cuban vector for fasciolosis, is lethally affected by extracts of M. azedarach seeds (Perez et al., 1998). The values of LC50, LC95 and LC99 within the larval population of A. aegypti that was not fed during the bioassays were lower for ethanolic extracts of the seed kernels of A. indica, indicating that it is more effective than extracts of M. azedarach. This was true at both 25 and 30 8C. However, when the same experiment was done with fed larvae, the extract of Melia proved to be 1.6 times more efficient than that of Azadirachta at 25 8C for all measured dose-response experiments (LC50, LC95 and LC99). When the bioassay temperature was increased to 30 8C, the Azadirachta extracts performed better, possibly due to an improved solubilization or biodisponibilization of its bioactive principles or their higher stability at more elevated temperatures. Inspection of the data from Tables 1 and 2 shows that the ratio of LC95 to LC50 was between 3.6 and 5.2 for Melia at 25 8C and between 3.4 and 4.3 for Azadirachta at 30 8C. Therefore, there was no marked difference in this ratio between the two seed kernel extracts, irrespective of temperature and feeding. Also it is worthy of note that the Melia extract was more effective than the Azadirachta extract on fed larvae at the more moderate temperature of 25 8C. Overall, Azadirachta extracts are more effective than Melia at killing Aedes larvae, but the LC(s) of the former fall within in the confidence interval of the latter. It may be that native compounds from Azadirachta display a greater feed deterrence effect or bind in a special way to the offered food. Note that more than 50 bioactive compounds have been identified in Azadirachta, while identification of the bioactive compounds in Melia has received much less attention. So, for the purpose of formulation of a phyto-pesticide, the possibility of mixing it with a food material or other support could be considered. Melia is readily available in Brazil while recently

established plantations of Azadirachta are restricted to a few growers and the availability of seeds is restricted, and those that are available suffer from elevated prices. Further, increase of the extract concentrations to reach LC99 confirmed, as seen in Table 1, that Azadirachta is more lethal to Aedes when assaying unfed larvae at 30 8C. Conversely, the opposite occurred in the assay at 25 8C with fed larvae (Table 2). In some experimental sets, c2 was higher and, according to Preisler (1988) and Preisler et al. (1990), this may be due to unaccountable variations amongst the repetitions such as individuals competing for the same resources. Thus, the mortality values can change without the risk of invalidating the results. This was evident in our bioassays in which food was added. The use of plant extracts is less explored than the use of synthetic chemicals, except for countries where some plants are historically known for multi-purpose control of harmful insects. Incidentally, India is the native country of the ‘magic tree’ Neem (A. indica), a source of the limonoid azadirachtin and other related and unrelated compounds. Neem has several uses, which encompass specific applications against insects, particularly hematophagous flying insects. The taxonomically related tree M. azedarach is also native to Asia. Contrary to Neem, Melia is quite widespread, although not always as dense plantations. Neem was introduced into Brazil as early as 1986 (Martinez, 2002), but its intensive cultivation started only in 2002 and this in just a few States. The differences observed in the present work between the larvicidal action of extracts of Azadirachta and Melia might be related to anatomical aspects of the seeds, the extraction procedure, or to the nature of the solvent used, since any of these parameters might, either singly or in combination, affect the mortality rates (Zebitz, 1984). Since the observed larvicidal effect is presumably due to antichitinogenic limonoids, spectrophotometric and chromatographic analyses were carried out on both ethanolic extracts. The chromogen vanillin–sulphuric acid generated the expected deep violet derivatives of limonoids and indicated that the total limonoids, expressed as azadirachtin, represented 11.2 and 8.8% of the total sample mass, for Azadirachta and Melia extracts, respectively. HPLC analyses (Fig. 1), further corroborated by TLC (Fig. 2), confirmed azadirachtin (RtZ7.2 min) as a major component in the Neem preparations. Concerning Melia, other

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Fig. 1. High pressure liquid chromatography (HPLC) from the ethanolic extracts of whole seed kernels of A. indica (B) and from Melia azedarach (C) as compared to an azadirachtin standard (A).

non-identified limonoids may be responsible for the observed larvicidal activity and the positive vanillin chromogenic test since azadirachtin was not detected by either chromatographic procedure. We decided to adapt the colorimetric procedure (Dai et al., 1999) to the revelation of the thin layer chromatographic plates by replacing sulphuric acid with phosphoric acid, but keeping the original chromogen:acid ratio at 4 mg:0.3 ml in the revelation spray. Although inspection of the plate under natural light proved to be less sensitive than use of the usual p-anisaldehyde revelation agent, examination under a long

Fig. 2. Thin layer chromatography (TLC) of ethanolic extracts of M. azedarach (Ma) and A. indica (Ai) seed kernels as compared to a commercial Neem preparation (a16) and to azadirachtin (a) and melianin B (m) standards. (A) Natural light; (B) 354 nm UV light.

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wavelength allowed the detection of 14 bands for Azadirachta and seven for Melia (Fig. 2B, third and last lanes). This band profile did not correspond with the heterogeneous profile from HPLC (Fig. 1B; O20 peaks for Azadirachta), but confirmed that the liminoids of Melia are less polar than azadirachtin, as indicated by their higher Rfs in TLC (Fig. 2B; third lane). Curiously, water extracts from A. indica leaves resulted in no activity against Aedes larvae (Zarroug et al., 1988), which opposes the findings of mutagenicity assays of two A. indica meliacins (Siddiqui et al., 1986) as well as LC50 and LC90 determinations, for which Monzon et al. (1994), comparing five different Philippine plants, obtained values larger than those reported here for Neem. This could be explained by the use of different parts of the plants, since fruits are typically more bioactive than leaves (Schmutterer, 1990) and from the 48 h assays as compared to shorter assays (24 h) carried out in previous experiments (Dzul et al., 2002). The finding of Monzon was further corroborated when fourth instar larvae were tested (Siddiqui et al., 2000). Four compounds from A. indica seeds blocked the chemical maturation of the molting hormone through the enzyme ecdysone 20-monooxygenase (Mitchell et al., 1997). It is quite probable that similarly active compounds are also present in M. azedarach seeds. In other words, meliacarpinins and related bioactive compounds from Melia (Huang et al., 1996; Nakatani et al., 1995) could confer antichitinogenic properties similar to azadirachtin from A. indica (Cassier and Papillon, 1991), since the respective limonoids have very similar chemical structures that are related to ecdysone, the insect molting hormone. Repeated bioassays using only isolated embryos (cotyledons) or seed coat-free seeds as the starting material for limonoid extraction indicated that most, if not all, of the larvicidal action originates from these more intimate parts of the whole endocarps (results not shown). To the best of our knowledge the results obtained herein with respect to the lethality of ethanolic extracts of M. azedarach seed kernels for larvae of A. aegypti have not been previously reported, although this is not surprising since this member of the Meliaceae is by far less studied than the taxonomically related A. indica. Normal chitin biosynthesis is of central importance in the full development of the morphogenetic cycle in mosquitoes, as previously demonstrated by direct inhibition of in vitro chitin synthesis by the limonoid azadirachtin (Cassier and Papillon, 1991) and by the reversion of the inhibitory effect of azadirachtin by the pupation triggering hormone, ecdysterone (Jagannadh and Nair, 1992). The present findings therefore suggest that it is worthwhile to purify the limonoids from the ethanolic extracts of M. azedarach and undertake broader bioassays, including antifeeding and feed deterrence studies, in order to elucidate the role of these limonoids in the larvidical effects described in the present work.

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Acknowledgements The authors thank the National Research Council for Scientific and Technological Development (CNPq, Brası´liaDF) and Fundac¸a˜o Arauca´ria-SETI-PR (Curitiba-PR) for financial support. The collaboration of M. Passos, S.M.O. Lyng, J. Adelmann, and T. Bendlin in the laboratory work is gratefully recognized. Thanks are also due to Prof. David A. Mitchell for help with the English expression in the manuscript.

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