Study of technical CNSL and its main components as new green larvicides

Abstract

Larvicidal activities against Aedes aegypti of technical cashew (Anarcadium Occidentale L.) nut shell liquid (CNSL) and its main constituents, cardanol, cardol and their products of hydrogenation were evaluated. In addition, the structure-activity relationship is also discussed.

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Introduction

Mosquitoes are vectors responsible for spreading serious human diseases like dengue, yellow fever and malaria. Aedes aegypti is one of the mosquito species responsible for the transmission of both dengue and yellow fever, which are endemic to Africa, Asia and South America. In the world, current estimates suggest that up to 50 million dengue cases occur annually, including 500,000 cases of the more serious related illness, dengue hemorrhagic fever (DHF).¹

In the absence of an effective drug or vaccine, the ideal method for controlling mosquito infestation would be prevention of the mosquito breeding through the use of larvicides. Control the mosquito population in the larval stage is much easier compared to adult stage.²

The use of organophosphates, like temephos and fention, for control of mosquito larvae and insect growth regulators, like diflubenzuron and methoprene, has disrupted natural biological control systems and led to outbreaks of insect species showing pesticide resistance. Other undesirable effects include toxicity to nontarget organisms, and this has fostered environmental and human concerns.³ Based on these problems, new strategies for selective mosquito larval control are needed.

Natural occurring larvicides are receiving considerable attention because they constitute a rich source of bioactive compounds that are biodegradable into nontoxic products and are potentially suitable for use in integral pest management programs.⁴

Despite the immense resource presented by the natural flora of Brazil, control of Aedes aegypti still depends basically on the use of synthetic pesticides.

Cashew (Anacardium Occidentale L.) is one of the well-know species of the Anacardiaceae family. The cashew nut shell liquid (CNSL) is a unique natural source of unsaturated long-chain phenols obtained as a byproduct of the cashew industry.

On the basis of the mode of extraction from cashew nut shell, CNSL is classified into two types, solvent-extracted CNSL and technical CNSL. Commercially available technical CNSL is obtained by roasting shells, and contains mainly cardanol (Fig 1, Ia–d) and cardol (Fig 1, IIa–d), both having degrees of saturation of the C15 alkyl side chain varying from complete saturation to partial unsaturation, as shown in Fig. 1.⁵ World-wide CNSL production is estimated to be 300,000–360,000 tons per annum, and as the production of cashew nuts is rising every year the availability of up to 600,000 tons per annum of CNSL should be reached in the near future.⁶

Fig. 1 Main constituents of technical CNSL.

Cashew nut shell liquid is also reported to be used in protecting wood against termites and is especially used in making insecticidal formulations.⁷

Since the international price of CNSL is about $300/ton,⁸ the use of an abundant and cheap source of natural compounds with larvicidal activity would be of great value not only for many emerging countries, but also for other countries looking for naturally occurring larvicides.

The aim of this work is to study the larvicidal activities of technical CNSL and its isolated main constituents against Aedes aegypti, and also discuss the structure-activity relationship of the products of hydrogenation of cardanol and cardol. Since Brazil, Vietnam and India are the largest producers and exporters of cashew kernel in the world and as Africa, Asia and South America presents the highest numbers of dengue cases annually,¹ the use of plants cultivated near the endemic areas could be of great interest and economically feasible for controlling mosquito infestation in developing countries.

Materials and methods

General

The samples obtained were analyzed by GC-MS on a Hewlett–Packard Model 5971 using a (5%-phenyl)-methylpolysiloxane DB-5 capillary column (30 m × 0.25 mm) with film thickness 0.1 μm; carrier gas helium, flow rate 1 mL/min with split mode. The injector temperature and detector temperature were 250 and 200 °C, respectively. NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz) using CDCl₃ as solvent for cardanol and (CD₃)₂CO for cardol. Column chromatography was run using silica gel 60 (70–230 mesh, Vetec), while TLC was conducted on precoated silica gel polyester sheets (Kieselgel 60 F254, 0.20 mm, Merck). Compounds were detected by spraying with vanillin–perchloric acid–EtOH solution, followed by heating at 120 °C.

Cardanol and cardol

Cardanol (11.8 g) and cardol (2.1 g) were isolated from technical CNSL (20.0 g) using a silica gel column (Silica Gel 60) eluted with a stepwise gradient of n-hexane–ethyl acetate (from 9:1 to 7:3 by volume). The fractions obtained in the column chromatography were analyzed through thin layer chromatography (TLC) and then reunited according their retention factors.

Cardanol . brownish oil, ¹H RMN (CDCl₃, δ): 1.03 (t, 3H); 1.08; 1.36; 1.45; 1.50; 1.71; 2.19 (t, 2H); 2.65; 2.95; 5.13; 5.21; 5.53; 5.92; 6.79 (m, 1H); 6.82 (m, 1H); 6.86 (s, 1H); 7.22(t, 1H).

Cardol . brownish oil, ¹H RMN (acetone-d6, δ): 1.08 (t, 3H); 1.46; 1.51; 1.58; 1.75; 2.25; 2.62; 2.96; 5.38; 6.22 (s, 2H); 6.65; 6.74; 7.08; 7.27 (s, 1H).

Hydrogenation of cardanol and cardol

Cardanol (3.0 g) was dissolved in 10 mL of ethanol, and 10% Pd/C (0.3 g) was added. The mixture was hydrogenated under pressure (2 bar) for 72 h. After removal of the catalyst and recrystallisation in ethyl ether, 2.56 g (85.3%) of a solid material was obtained (Fig. 1. Ia). Cardol (2.0 g) was hydrogenated with the same procedure described above, using 10% Pd/C (0.2 g); after removal of catalyst and solvent evaporation, 1.44 g (72%) of a solid material was obtained (Fig. 1. Ia).

Hydrogenated cardanol. white solid (m.p.: 51.5–52 °C), ¹H RMN (CDCl₃, δ): 0.90 (t, 3H); 1.04; 1.27; 1.32; 1.56; 1.63; 2.56 (t, 2H); 6.64 (m, 1H); 6.67 (m, 1H); 6.78 (d, 1H); 7.17 (s, 1H).

Hydrogenated cardol. white solid (m.p.: 94.5–95 °C), ¹H RMN (acetone-d6, δ): RMN ¹H (δ): 0.88 (t, 3H); 1.28; 1.56; 2.43 (t, 2H); 3.09; 6.18 (s, 2H); 8.09 (s, 1H).

Larvicidal bioassay

Portions of technical CNSL, its constituents cardanol and cardol and their derivatives (12.5 to 500 μg/mL) were placed in a beaker (50 ml) and dissolved in DMSO/H₂O 1.5% (20 ml). 50 instar III larvae of Aedes aegypti were delivered to each beaker. After 24 hours, at room temperature, the number of dead larvae was counted and the lethal percentage calculated. A control using DMSO/H₂O 1.5% was carried out in parallel. For each sample, three independent experiments were run.⁹

Results and discussion

The results obtained from the biological assay are shown in Table 1. Significant differences in larvicidal activity against Aedes aegypti between technical CNSL and its main constituents were observed. Technical CNSL presented a LC₅₀ value of 51.04 ± 0.62 μg/mL, whereas cardol and cardanol showed LC₅₀ values of 14.20 ± 0.62 and 32.90 ± 0.25 μg/mL, respectively. This way, cardol proved to be the main constituent responsible for the activity demonstrated by technical CNSL.

Table 1 LC₅₀ values for larval mortality caused by technical CNSL, cardanol, cardol, cardanol hydrogenated and cardol hydrogenated

	LC50 (μg/mL)
Technical CNSL	51.04 ± 0.62
Cardanol (Fig 1, Ia–d)	32.89 ± 0.25
Cardol (Fig 1, IIa–d)	14.20 ± 0.62
Cardanol hydrogenated (Fig 1, Ia)	68.18 ± 0.50
Cardol hydrogenated (Fig 1, IIa)	>500

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The differences in the LC₅₀ values between cardanol and cardol can be justified by the degree of unsaturation of those molecules. After hydrogenation, cardol completely lost its larvicidal activity, while cardanol's activity was lowered to 68.18 ± 0.50 μg/mL. A plausible explanation for these results is based in the literature,¹⁰ which describes that a large number of hydroxyl groups prevents the substance penetrating the insect cuticle and reaching their targets; in this case the hydrogenation of the side chain unsaturation diminished the lipophilic character of the molecules, restricting their passage through the larvae membrane. Gas chromatography-mass spectrometry analysis showed that cardanol mixture contains about 65% of the monounsaturated cardanol, 11% of diunsaturated cardanol and 3% of saturated cardanol. The cardol mixture contains aproximately 55% of triunsaturated cardol and 44% of diunsaturated cardol.

The larvicidal activity showed by technical CNSL, which is lower than its main constituents, may be explained as an effect of the polymers and other subproducts present in it, produced in the process of roasting shells, that employs high temperatures, by which technical CNSL is obtained.

Conclusions

The present work demonstrated that technical CNSL has a good larvicidal activity against Aedes aegypti. This result is mostly due the excellent effect exhibited by cardol, which demonstrates the importance of the unsaturation on the alkyl side chain, that increases its liposolubility facilitating passage through the cell membrane.

Since a previous work reported the low toxicity of cardol, which was orally tolerated up to a concentration of 5 g/kg body weight rats,¹¹ studies involving modification of the main constituents of CNSL are being executed in our laboratories searching for a more detailed structure-activity relationship and also to improve the larvicidal effect demonstrated by cardol and cardanol.

All those results suggests that the utilization of technical CNSL component cardol as a new green larvicidal may be considered as a new alternative to combat spreading of dengue.

Acknowledgements

The authors wish to thank to Brazilian agencies CNPq, CAPES, FUNCAP, PRONEX for fellowships and financial support and to Laboratório de Entomologia, Núcleo de Endemias da Secretaria de Saúde do Estado do Ceará, Brazil where the bioassays were performed.

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