Larvicidal and Enzymatic Inhibition Effects of Annona Muricata Seed Extract and Main Constituent Annonacin against Aedes Aegypti and Aedes Albopictus (Diptera: Culicidae)

Abstract

The mosquitoes Aedes aegypti and Aedes albopictus are vectors of arboviruses that cause dengue, zika and chikungunya. Bioactive compounds from plants are environmentally sustainable alternatives to control these vectors and thus the arboviruses transmitted by them. The present study evaluated the larvicidal activity of an acetogenin-rich fraction (ACERF) and its main constituent annonacin obtained from Annona muricata seeds on Ae. aegypti and Ae. albopictus. The larvicidal assays were performed using different concentrations to calculate the LC₅₀ and LC₉₀ values observed 24 h after exposure to the treatment. Annonacin was more active against Ae. aegypti (LC₅₀ 2.65 μg·mL⁻¹) in comparison with Ae. albopictus (LC₅₀ 8.34 μg·mL⁻¹). In contrast, the acetogenin-rich fraction was more active against Ae. albopictus (LC₅₀ 3.41 μg·mL⁻¹) than Ae. aegypti (LC₅₀ 12.41 μg·mL⁻¹). ACERF and annonacin treated larvae of Ae. aegypti and Ae. albopictus showed significant differences in the inhibition of their metabolic enzymes when compared to untreated larvae. The results demonstrate the relevant larvicidal action of the acetogenin-rich fraction and annonacin showing the potential to develop new products for the control of Ae. aegypti and Ae. albopictus.

1. Introduction

Aedes aegypti Linnaeus (Diptera: Culicidae) is the main vector of arboviruses such as those that cause dengue, zika and chikungunya, diseases that affect all continents, although they are more frequent in tropical and subtropical countries [1,2]. At the same time, Aedes albopictus Skuse (Diptera: Culicidae), originating in Southeast Asia, is vector of diseases such as dengue, yellow fever and encephalitis, among other diseases [3]. In Brazil, its presence has been identified in almost all states [4], and it is considered a secondary vector of arboviruses in the country. Although vaccines against dengue serotypes are being developed [5], the presence of other viruses transmitted by Aedes spp. reinforces the need to control their vectors [6]. These mosquitoes have a broad capacity to adapt to the environment [7]. One of the most effective control strategies is the use of synthetic insecticides for the elimination of immature or adult stages (larvicides and insecticides) [8], although their frequent use has triggered the selection of resistant strains [9].

However, many plants contain bioactive compounds, which are environmentally safe for use as alternatives in the control of Ae. aegypti and Ae. albopictus, since these secondary metabolites are biodegradable [10]. This characteristic of natural products is important in the search for new environmentally safe phytopesticides for the control of immature stages of Aedes [11]. Thus, resistance of these disease vectors to current pesticides is a growing problem, and products from plants are eco-sustainable alternatives in this respect [12,13]. Many plant products have been evaluated against Ae. aegypti [14] and those from Annonaceae family show the best action [15]. The Annonaceae family is composed of a diverse group of angiosperms, including 2500 distinct species [16]. Annona muricata L. (Annonaceae), known as graviola or soursop, is a common edible fruit plant grown in Brazil [17]. Its fruits produce a significant number of seeds and its pulp is widely appreciated. Acetogenins (ACGs) are substances often found among the constituents of the Annonaceae family. Such compounds are characterized by the presence of an aliphatic chain with 35 to 38 carbons, attached to an α,β-unsaturated γ-lactone ring substituted with a methyl terminal, in addition to the presence of one or two tetrahydrofuran (THF) and/or tetrahydropyran (THP) rings along the hydrocarbon chain. The chain contains a specific number of oxygenated groups (hydroxyl, acetoxyl, ketone and epoxy) [18,19]. In A. muricata, annonacin is the acetogenin most reported to be present [18,19,20,21], being considered major component of this species.

Then, the objective of this study was to evaluate the larvicidal activity of an acetogenin-rich fraction and the main constituent, annonacin, against Ae. aegypti and Ae. albopictus, major vectors of arboviruses, as well as analyzing changes in the vital protein activity of these mosquitoes after treatment with the natural product.

2. Materials and Methods

2.1. Seed Collection and Obtaining the Ethanolic Extract of Annona muricata

A. muricata fruits were provided by the company Flora Boa, located in the town Limoeiro do Norte, Ceará, Brazil. The seeds (500 g) were sun dried and triturated, then macerated for 7 days with 96% ethanol. After this period, the ethanol solution was filtered and concentrated in a vacuum rotary evaporator, to obtain the ethanol extract of A. muricata (EESAM).

2.2. Obtaining the Acetogenin-Rich Fraction of A. muricata Seeds

The EESAM was subjected to filtration through silica gel column chromatography, eluted with the solvents hexane, chloroform, ethyl acetate and methanol in mixtures of increasing polarity. The fractions eluted with chloroform and ethyl acetate were pooled based on the thin-layer chromatography (TLC) analysis, using Kedde’s reagent (Solution A: 3,5-dinitrobenzoic acid in 3% methanol, and Solution B: 5.7% KOH in water, in the ratio of 1:1), to obtain an acetogenin-rich fraction (ACERF).

2.3. Analysis of the Acetogenin-Rich Fraction by HPLC and Separation of Its Main Constituent

The analyses were performed with Shimadzu high-performance liquid chromatograph in reverse phase, with an SCL-10Avp controller system, SPD-10Avp UV-Vis detector and LC-10Atvp gradient pump, under the following chromatographic conditions: flow rate of 1 ml per minute at a wavelength of 214 nm, mobile phase in the gradient mode of a solution composed of deionized water and methanol (15%:85%) up to 40 min, and (5%:95%) up to 60 min, oven temperature of 40 °C, 20 μL loops, and CLC-ODS M C18 column (250 mm × 4.6 mm).

To separate the constituents, 200 mg of ACERF was dissolved in 20 mL of methanol. An aliquot of 1 mL was withdrawn from that solution and dissolved in 10 mL of methanol for injection in an analytical high-performance liquid chromatograph equipped as follows: SLC-10AVP controller system, SPD-10AVP UV-Vis detector, and LC-10-ATVP isocratic pump. The column used was a Shimpack CLC-ODS M (C18), measuring 250 cm × 4.6 mm D, employing a wavelength of 214 nm, run time of 30 min, flow rate of 1 mL per minute, mobile phase composed of 87% spectroscopic methanol and 13% deionized water, injection of 20 μL, and oven temperature of 40 °C. An aliquot of the ACERF solution (12 mL) was injected and the main fraction was collected separately. This procedure was repeated several times. Then, the fractions collected were concentrated in a rotary evaporator and lyophilized to obtain annonacin, whose chemical structure was characterized by nuclear magnetic resonance spectroscopy.

The purity of the main constituent of the ACERF, identified as annonacin, was determined by injection in a liquid chromatograph in analytical mode. For this purpose, a 0.84 mg/mL solution of fraction 2 in methanol was prepared and the following system was used: Shimadzu preparative high-performance liquid chromatograph with SCL-10Avp controller system, SPD-10Avp UV-Vis detector, and LC-10Atvp gradient pump, under the following chromatographic conditions: flow rate of 1 ml per minute at a wavelength of 214 nm, mobile phase in the gradient mode of a solution composed of deionized water and methanol (15%:85%); up to 40 min and (05%:95%) up to 60 min, oven temperature of 40 °C, 20 μL loop and CLC-ODS M C18 column (250 mm × 4.6 mm).

2.4. Collection and Maintenance of Ae. aegypti and Ae. albopictus Mosquitoes

During the dengue control program in Fortaleza, Ceará, ovitraps are used to collect Ae. aegypti and Ae. albopictus eggs. Positive ovitraps were sent to the Entomology Laboratory (LE) of Federal University of Ceará for eggs to hatch. The larvae were obtained from a colony of successive generations maintained at the insectarium of the LE. These larvae were kept under controlled conditions of temperature (25 ± 2 °C), relative humidity (80 ± 10%) and light and dark photoperiod (12:12) in plastic containers holding 1000 mL of distilled water, and were fed with fish meal until reaching the third and fouth instars.

2.5. Larvicidal Assay

The larvicidal tests were performed at concentrations of 0.5, 1, 5, 10, 20, 25, 50 and 75 μg·mL⁻¹ of ACERF and annonacin from A. muricata, all of which were performed in triplicate. The ACERF and annonacin at different concentrations were diluted in a solution of 5% dimethyl sulfoxide (DMSO) and 95% distilled water, and a negative control was also tested with DMSO and water. Twenty larvae were included in each test and mortality was verified after 24 h of exposure [22].

2.6. Enzymatic Effect of the Acetogenin-Rich Fraction and Annonacin of A. muricata on the Mosquito Larvae

The enzymatic assays were performed using a Bioplus Bio-200 biochemical analyzer. The action of ACERF and annonacin on the larvae of Ae. aegypti and Ae. albopictus exposed at a concentration of 25 μg·mL⁻¹ during 24 h was verified from the analysis of the main enzymes of the metabolism of Culicidae larvae by adaptation of the method of Suryawanshi et al. [23]. The enzymes assayed were alkaline phosphatase, acid phosphatase, proteases, esterases and amylases. All experiments were performed with larval stages.

2.7. Statistical Analysis

The LC₅₀ values (concentration that kills 50% of larvae and pupae) and LC₉₀ (concentration that kills 90% of larvae) were obtained from probit analysis using the SPSS statistical software. Analysis of variance (ANOVA) was used to investigate the existence of significant differences in the parameters of the enzymatic activities of treated and untreated mosquito larvae after ACERF and annonacin application (p < 0.05). The normality criterion was applied to the variables and the Tukey test was used to verify where the differences occurred in the studied groups.

3. Results and Discussion

3.1. Analysis of the Constituents of the Acetogenin-Rich Fraction (ACERF)

In the TLC analysis of the various ACERF fractions obtained in the chromatographic column (those that revealed a characteristic pink color with Kedde’s reagent) were collected and the chromatogram of HPLC analysis (Figure 1) presented several peaks with similar UV spectra (Figure 2). The ACERF was subjected to semi-preparative HPLC fractionation to separate the main acetogenin constituent with a retention time of 9.6 min. The major substance was separated and analyzed by nuclear magnetic resonance, and was revealed to be annonacin (Figure 3) by comparison with spectral data reported in the literature for this substance [24].

3.2. Efficacy of the ACERF and Annonacin of A. muricata Seeds on Ae. aegypti and Ae. albopictus

The larvicidal action of the acetogenin-rich fraction of A. muricata seeds is shown in

Table 1. Lethal concentration of the fraction rich in acetogenins of A. muricata against fourth instar larvae of Ae. aegypti and Ae. Albopictus.

Mosquito Strains	LC50 (CL at 95%)	LC90 (CL at 95%)
Ae. aegypti	12.41 (9.85–14.86)	30.21 (26.17–36.52)
Ae. albopictus	3.41 (2.83–4.00)	6.17 (5.37–7.43)
Ae. aegypti (Rockefeller strain)	26.75 (21.36–34.00)	54.46 (44.78–71.01)

Control: no mortality. As the level of significance is greater than 0.05, no heterogeneity factor was used in the calculation of confidence limits. LC₅₀: concentration that kills 50% of the exposed larvae (in μg·mL⁻¹) with confidence limit at 95%. LC₉₀: concentration that kills 90% of the exposed larvae (in μg·mL⁻¹) with confidence limit at 95%.

. The LC₅₀ of ACERF for Ae. albopictus was 3.41 μg·mL⁻¹—the best result in relation to other strains. The Ae. aegypti (Rockefeller strain) showed highest resistance to treatment with ACERF of A. muricata, with LC₅₀ and LC₉₀ values, respectively, of 26.75 and 54.46 μg·mL⁻¹, as determined by the probit analysis. Concentrations higher than 50 μg·mL⁻¹ were responsible for 100% mortality of Ae. albopictus and concentrations higher than 75 μg·mL⁻¹ were totally lethal to the other strains.

A study of the leaf extract from other Annonaceae plants, such as A. reticulata, reported effective action against Ae. aegypti larvae, with LC₅₀ and LC₉₀ values of 95.24 and 262.64 µg·mL⁻¹, respectively [25]. The hexane extract of the leaves of A. muricata gathered in Tarlac City (Philippines) showed the highest toxicity, with LC₅₀ value of 25.99 μg·mL⁻¹and LC₉₀ value of 49.47 μg·mL⁻¹, while the ethanol extract yielded LC₅₀ of 46.36 µg·mL⁻¹ and LC₉₀ of 195.39 µg·mL⁻¹. Characterization of the ethanol extract showed the presence of flavonoids (leucoanthocyanidins), condensed tannins, unsaturated steroids, triterpenoids, and fats and oils.

Regarding the strain of Ae. albopictus, Kempraj and Bhat [26] reported the larvicidal action of the acetone soluble fraction of the ethanolic extract of A. squamosa seeds, with respective LC₅₀ and LC₉₀ values of 5.26 and 38.37 μg·mL⁻¹, on fourth instar larvae. The authors also performed tests of ovicidal and adulticide activity, as well as tests to verify the effect of the A. squamosa extracts on oviposition of females, with toxicity observed in all trials.

The plants of the Annonaceae family show high toxicity to Aedes spp. larvae. The larvicidal activities against Ae. aegypti have been determined in the ethanolic extracts obtained from 51 Brazilian medicinal plants. Eleven of the 84 extracts studied showed significant (LC₅₀ < 100 μg·mL⁻¹) activities against larvae, with extracts from A. crassiflora (root bark, LC₅₀ = 0.71 μg·mL⁻¹; root wood, LC₅₀ = 8.94 µg·mL⁻¹ and A. glabra seed, LC₅₀ = 0.06 μg·mL⁻¹, showing the highest activities [27].

Natural products from plants of other families (Myrtaceae, Rutaceae, Euphorbiaceae, Piperaceae, Asteraceae and Liliaceae) in general are less active. The essential oils of Eucalyptus cinerea had LC₅₀ of 380 μg·mL⁻¹ [28], Citrus sinensis had LC₅₀ of 446.84 µg·mL⁻¹ [29] and Piper gaudichaudianum had LC₅₀ of 121 µg·mL⁻¹ [30]. Various solvent extracts of stems, roots and leaves of Parthenium hysterophorus showed low efficacy, with LC₅₀ above 400 μg·mL⁻¹ [31], and Aloe vera, with LC₅₀ of 300.06 µg·mL⁻¹ [32]. The LC₅₀ values of petroleum ether extracts of Jatropha curcas, Piper tithymaloides, Phylantus amarus, Euphorbia hirta, and E. tirucalli were 8.79, 55.26, 90.92, 272.36, and 4.25 μg·mL⁻¹, respectively, against Ae. aegypti and 11.34, 76.61, 113.40, 424.94, and 5.52 μg·mL⁻¹, respectively, against C quinquefasciatus [33].

Annonacin is considered an environmental neurotoxin and is found in the pulp of several fruits of the Annonaceae family, whose consumption has been linked to the occurrence of sporadic atypical Parkinsonism with dementia. A method for its quantification in rat brain homogenates by UPLC-MS/MS was developed and validated. This method was applied to the quantitation of annonacin in rat brains after intravenous (0.5 mg/kg) and oral (10 mg/kg, 100 mg/kg) administration. Nevertheless, annonacin appeared to have very low distribution in rat brains by both routes [34].

Annonacin was shown to be significantly active against Ae. aegypti (Rockefeller strain), presenting lower lethal concentrations than those observed for the acetogenin-rich fraction (LC₅₀ 2.65 μg·mL⁻¹; LC₉₀ 4.83 µg·mL⁻¹). The larvae of Ae. albopictus presented LC₅₀ of 8.34 μg·mL⁻¹ and LC₉₀ of 16.30 μg·mL⁻¹ (

Table 2. Lethal concentration of annonacin from A. muricata against fourth instar larvae of Aedes spp.

Mosquito Strains	LC50 (CL at 95%)	LC90 (CL at 95%)
Ae. aegypti (Rockefeller strain)	2.65 (1.87–3.64)	4.83 (3.80–7.35)
Ae. albopictus	8.34 (7.10–10.26)	16.30 (13.56–21.13)

Control: no mortality. As the level of significance is greater than 0.05, no heterogeneity factor was used in the calculation of confidence limits. LC₅₀: concentration that kills 50% of the exposed larvae (in μg·mL⁻¹) with confidence limit at 95%. LC₉₀: concentration that kills 90% of the exposed larvae (in μg·mL⁻¹) with confidence limit at 95%.

).

According to the literature, the larvicidal action against mosquitoes of Annonaceae plant extracts is comparable to that reported for pure acetogenins [27]. The ethanolic extract of A. glabra had LC₅₀ of 27 μg·mL⁻¹ against Ae. aegypti larvae [35], in contrast to isolated acetogenins such as goniothalamin, gigantriocin and longimicin, with LC₅₀ values of 13.3 μg·mL⁻¹, 18.5 μg·mL⁻¹ and 27.3 μg·mL⁻¹, respectively [36].

The high activity of ACERF and annonacin against larvae of Ae. aegypti and Ae. albopictus enables the use of low amounts. The small distribution of this substance in the rat brain, conferring low toxicity, justifies the use in phytotherapic preparations as larvicidal agents.

3.3. Changes in the Enzymatic Level of Ae. aegypti and Ae. albopictus Treated with ACERF and Annonacin

The mechanism of action of natural products on Aedes spp. larvae has been investigated in recent studies [1,37]. Among the explanations of this mechanism are the ultrastructural alterations of organs such as anal papillae [38], intestinal and tracheal system damage [1], inhibition of enzymatic activity [39], and morphological aberrations [40]. In this study, we investigated the effect of ACERF and annonacin on the inhibition of proteins important for the development of Aedes spp. larvae.

The one-way ANOVA showed that ACERF has an effect on the total proteins between the groups (p < 0.05). The Tukey test showed the yield of total proteins of Ae. albopictus treated with ACERF was significantly different from the control and Ae. aegypti. However, there was no difference between the control group and the treated Ae. aegypti (Figure 4A).

Alkaline phosphatase activity was altered by ACERF treatment (p < 0.05). The Tukey test indicated no difference between the control group and Ae. aegypti strain, however, both groups were significantly different from Ae. albopictus (Figure 4B). Similarly, there was a change in the enzymatic effect of acid phosphatase (p < 0.05), but it was the only significant difference between Ae. albopictus and control (Figure 4C).

Among the proteases, there was a difference between the groups (p < 0.05). This difference, when verified by the post-hoc Tukey test, was significant between Ae. albopictus and the control and Ae. aegypti (Figure 4D). Differences were also observed between groups in relation to esterase activity (p < 0.05), with a significant increase in the activity of these enzymes in strain Ae. albopictus compared to the other enzymes—which were similar to each other—as verified by the Tukey test (Figure 4E).

The ACERF also elicited significant changes between groups with respect to amylase activity (p < 0.05), and the larval strain Ae. albopictus showed a significant reduction compared to the control and to Ae. aegypti (Figure 4F).

The consistent changes in the enzymatic activity of Ae. albopictus (LC₅₀ = 3. 41 μg·mL⁻¹) treated with ACERF corroborate the greater larvicidal effect of this fraction in this species when compared to Ae. aegypti (LC₅₀ = 12.41 μg·mL⁻¹).

In relation to the treatment of Aedes larvae with annonacin, one-way ANOVA showed a significant difference between groups in activity of total protein (p < 0.05), alkaline phosphatase (p < 0.05) and esterase (p < 0.05). The Tukey post-hoc test confirmed that the activities of the total proteins, alkaline phosphatase and esterase of the control group were significantly different against Ae. aegypti (Rockefeller strain) and Ae. albopictus (Figure 5A,B,E).

There was no significant difference in the activity of acid phosphatase (Figure 5C) (p > 0.05) and protease (Figure 5D) (p > 0.05) between the control and treated groups. The amylase activity showed significant differences between the groups (p < 0.05). While the Tukey post-hoc confirmed significant differences only between the control and Ae. albopictus groups, the Ae. aegypti (Rockefeller strain) group did not present significant differences between these two groups (Figure 5F).

Similarly, studies of enzymatic changes in larvae and pupae of Ae. aegypti treated with the aqueous extract of Sapindus emarginatus Vahl. (Sapindaceae) showed that the activity of esterases and acid phosphatase were affected, and more noticeably in larvae than pupae [41].

Another study correlated the inhibitory capacity of a protease inhibitor from garlic (Allium sativum) with its larvicidal activity [42]. These authors considered that the reduction of 50% in the presence of digestive proteases by the A. sativum protease has biotechnological potential for the control of Ae. aegypti. In this respect, ACERF of A. muricata caused a reduction in proteases of both treated strains and contributed to the inactivation of those proteins, which is detrimental for the survival of Aedes spp.

The larvicidal effect of ACERF was greater on larvae of Ae. albopictus than on other treated strains, whereas annonacin had a larvicidal activity greater in Ae. aegypti (Rockefeller strain). Regarding the inhibitory effect on the enzymes, differences were observed between ACERF and annonacin on total proteins, acid phosphatase and alkaline phosphatase in strains with greater susceptibility to natural products. These differences most likely result from the fact that in ACERF there are other acetogenins besides its major constituent—annonacin. These acetogenins are substances that act synergistically in the enzymatic inhibitory effect and in the larvicidal action, whereas in the isolated annonacin, we have the presence of a single substance that conditions specific effects on the larvae and their enzymes.

4. Conclusions

Ae. aegypti and Ae. albopictus exhibit different susceptibilities to the fraction rich in acetogenins and annonacin—the main constituent. In addition, ACERF-treated larvae of Ae. albopictus showed a significant difference in the activity of all the enzymes studied compared to control larvae, a fact that corroborates the relevant LC₅₀ observed in the larvicidal tests of this lineage of the natural product in question. Regarding the treatment with annonacin, there were significant differences between treated and untreated larvae, however only for the activity of total proteins, alkaline phosphatase, esterase and amylase. This study indicates the promising potential of A. muricata as an alternative source of sustainable, biodegradable and environmentally safe natural product for the control of the dengue vectors Ae. aegypti and Ae. albopictus.