Larvicidal potential of extracts and isolated compounds from Piper cubeba fruits against Aedes aegypti (Diptera: Culicidae) larvae

Abstract Aedes aegypti is the primary vector of virus transmission that causes dengue, yellow fever, chikungunya and zika. The primary prevention method has been vector control and synthetic insecticides that can cause environmental side effects. Thus, the work aimed to evaluate the larvicidal potential of extracts and isolated compounds from Piper cubeba against A. aegypti larvae. The larvicidal activity method was executed according to the World Health Organization protocol. The larvae were analyzed by scanning electron microscopy (SEM). Through molecular docking, the action mechanism was investigated. The hydroalcoholic and hexane extracts showed similar larvicidal activity with LC50 of 191.1 μg/mL and 185.84 μg/mL, respectively. Between isolated compounds, hinokinin presented LC50= 97.74 μg/mL. The SEM analysis showed structural damage to the larva’s tegument caused by extracts and isolated compounds. Therefore, the results demonstrate the larvicidal action of hinokinin and extracts, which can lead to the development of new natural larvicides. Graphical Abstract


Introduction
Aedes aegypti (Diptera: Culicidae) is the primary vector of virus transmission that causes dengue and diseases such as yellow fever, chikungunya and zika, causing a significant impact on the health system in Brazil (Espinal et al. 2019;Andrioli et al. 2020). The primary method for preventing the arbovirus spread that causes these diseases has been vector control (Rather et al. 2017). However, the most common way of controlling A. aegypti, in the form of larvae or adults, use synthetic insecticides, mainly organochlorines, organophosphates, carbamates and pyrethroids (Rather et al. 2017). The indiscriminate use of these insecticides favors the emergence of generations of resistant mosquitoes and affects the environment and the insects vital to our ecosystem. The environmental alterations caused by synthetic insecticides can occur from deficiency or inhibition of seed germination in food crops and soil contamination. Thus, the disproportionate use of insecticide can inhibit growth and food crop yield (Ndayambaje et al. 2019). In addition, indiscriminate use can cause the death of essential insects for the environment, such as bees (Schechtman et al. 2020). This resistance and toxicity of synthetic insecticides have aroused interest in identifying natural alternatives to synthetic insecticides due to their selectivity, safety, and biodegradability characteristics ( Delgoda and Murray 2017).
The Plants synthesize compounds that act in their defense against pathogens and predatory insects (Erb and Kliebenstein 2020;Castillo-Morales et al. 2021;Ngongang et al. 2022). This characteristic makes plants an essential source of insecticidal compounds Vivekanandhan, Venkatesan, et al. 2018;Silv erio et al. 2020). Recent studies have shown promising effects of plant extracts and isolated metabolites against the various life forms of A. aegypti until adulthood (Wuillda et al. 2019;de Souza et al. 2020;Vivekanandhan et al. 2020;Alievi et al. 2021;Fernandes et al. 2021;Zuharah et al. 2021).
The genus Piper (Piperale order and Piperaceae family) has been investigated against A. aegypti larvae (Marques and Kaplan 2014;Samuel et al. 2016;Gorgani et al. 2017). Piper cubeba has been very prominent in recent decades in the genus Piper due to various studies of biological properties in vitro and in vivo using its extracts and isolated compounds (Philbin et al. 2022). About 34 lignans have been identified in P. cubeba fruits, such as dibenzylbutyrolactolic (cubebin), dibenzylbutyrolactonic (hinokinin and yatein), and dibenzylbutanedioic (dihydrocubebin) (Godoy de Lima et al. 2018). The wide structural variety of these lignans makes this species a promising source of molecules for biological evaluation . Among the various lignans from P. cubeba, hinokinin and cubebin ( Figure S1) have been the most studied (Marcot ulio et al. 2014;Carlis et al. 2019).
Thus, based on the biological properties of P. cubeba lignans, our study aimed to evaluate the larvicidal potential of extracts from P. cubeba fruits against A. aegypti third instar larvae, along with the cubebin and hinokinin lignans isolated from the extracts. In addition to proposing a possible mechanism of action, realizing molecular docking using the protein UDP-N-acetylglucosamine pyrophosphorilase (Ara ujo et al. 2005). This protein is essential for insect survival, cuticle organization, tracheal tube morphogenesis, and protein glycosylation. UDP-N-acetylgalactosamine (UDPGalNAc) is also a necessary precursor in the chitin biosynthesis of Ae. aegypti (Schwerdt et al. 2021).

Isolation and larvicidal activity of the compounds from P. cubeba fruits
Extraction with ethanol 70% of 200 g of the powered P. cubeba fruit furnished 40 g of hydroalcoholic crude extract (20% yield). Partition of the crude extract with hexane provided 29 g de hydroalcoholic extract (HAE) and 11.0 g of hexane extract (HE). Silica gel column chromatography of the HAE (20.0 g) with a mixture of solvent in a crescent polarity (hexane/ethyl acetate) furnished two major compounds, 0.3801 g of hinokinin (1.9%) and 0.4204 g (2.1%) of cubebin. Results of the NMR analysis (Supplementary data) confirmed the structures of the lignans ( Figure S1) and agreed with the literature data (Laurentiz et al. 2015).
The extracts and lignans evaluated in the bioassays had their LC 50 and LC 90 calculated and compared to the negative control dimethylsulfoxide (DMSO 0.5%) and with the positive control pyriproxyfen (0.02 lg/mL). The percentages of mortality of the controls, extract and lignans against A. aegypti third instar larvae are presented in Table S1. The negative control (DMSO 0.5%) did not show activity, while the positive control, pyriproxyfen, showed 100% mortality of the third instar larvae. In the highest evaluated concentrations (500 lg/mL), extracts and hinokinin showed 100% of mortality of the third instar larvae. Hinokinin was active with LC 50 of 97.94 lg/mL; cubebin did not show activity. Values of LC 50 for HAE and HE were, respectively, 191.1 lg/mL and 185.4 lg/mL. Although these extracts have very close LC 50 values, they have different phytochemical profiles. HE is rich in volatile and nonpolar compounds in the essential oil of P. cubeba fruits, as shown by Magalhães et al. (2012). The results obtained with HE and those obtained with Piper beetle's essential oil were also evaluated as a larvicide against A. aegypti, it is possible to observe a lower value of LC 90 for the HE of P. cubeba (324.3 lg/mL) compared to that found for P. beetle, which was 525.0 lg/mL (Martianasari and Hamid 2019). Although there is no World Health Organization (WHO)-specified value on larvicidal activity for extracts and isolated compounds, researchers generally consider an LC 50 < 50 mg/mL to be very active, LC 50 50-100 mg/mL active, and an LC 50 > 100 mg/mL weak/inactive (Silv erio et al. 2020). Therefore, considering these LC 50 values, P. cubeba extracts are weakly active; however, the LC 50 values for the extracts are better than the values found for many species of the genus Piper (Marques and Kaplan 2014).
The structural difference between cubebin and hinokinin is located at carbon 9, wherein hinokinin this carbon is part of the carbonyl group of the lactone. In cubebin, this carbon is linked to a hydroxyl and is part of a ketal group ( Figure S1). The presence of hydroxyl can lead cubebin to make hydrogen bonds (hydrogen donor) with amino acid residues outside the active site of proteins. Not being a hydrogen bond donor, Hinokinin interacts less with residues outside the active site and, therefore, would be able to reach the active site and, once there, would make the interactions that would be responsible for the observed activity.

Scanning electron microscopy (SEM)
Structural changes induced in the third instar larvae obtained from bioassays performed with the controls, hinokinin and extracts were investigated using SEM. However, the negative control larvae (DMSO 0.5%) did not show morphological or integumentary changes, as shown in Figure S2A-D, indicating the absence of negative control toxicity, as seen in the results of the bioassays (Table S1). Figure S3A-D shows micrographs of larvae exposed to pyriproxyfen used as a positive control. Micrographs indicate damage to the abdomen ( Figure S3A, B), to the cephalic capsule ( Figure S3C, D), and a decrease in palatal brushes ( Figure S3C) when compared to those obtained from the negative control. Figure S3B shows no damage to the larvae's integument despite narrowing the abdomen region.
In the case of the two P. cubeba extracts, the alterations in the larvae are different from those observed in the pyriproxyfen positive control ( Figure S3A-D) because they are mainly linked to the cuticle of the larvae (tegument). In larvae corresponding to treatment with HE ( Figure S4A-D), it is possible to observe thin bristles and the epithelium with a dehydrated appearance compared to the negative control. The most significant damage in these larvae is concentrated in the abdomen region, being less intense in the cephalic capsule than the damage observed in the positive control larvae. In addition, the damage observed in larvae exposed to HE may be due to exposure through contact to volatile and nonpolar compounds (essential oil) present in the extract that can act on the chitin present in the integument of the larvae (Marques and Kaplan 2014). Larvae treated with HAE showed damage to the integument similar to those observed for HE ( Figure S5A-D). However, as seen in the micrographs, hinokinin showed the most significant damage to the larvae ( Figure S6A-D). Figure S6B shows damage to the anal papilla of the larvae and the entire length of the integument. Chest and head damage is shown in Figures S6C and S6D. This result corroborates the in vitro assays where hinokinin had the lowest LD 50 value. The damage observed in the larval integument for both extracts and hinokinin has a contact relationship. In addition, it can affect proteins essential for maintaining the larval cuticle, such as UDP-N-acetylglucosamine pyrophosphorylase, which is necessary for chitin synthesis (Arakane et al. 2011).

Docking molecular
Homology modeling construction from the PDB ID:1JV1 model resulted in a 3D model with a sequence identity of 54.58% and a Qmean of 0.79 ± 0.05. Furthermore, the validation results revealed that 97.29% of the residues are found in the most favorable regions ( Figure S7). All these results ensure the overall satisfactory quality of the 3D structure.
Redocking was performed with the substrate UDPGalNac (Uridine diphosphate Nacetylgalactosamine. The root mean square deviation (RMSD) result from the redocking process was 1.005. According to the literature (Ram ırez and Caballero, 2018), RMSD values below 2.0 guarantee the quality of docking.
Pyriproxyfen ( Figure S10), used in the in vitro assays as a positive control, showed no interaction with amino acids in the protein's active site. Therefore, only interactions with residues neighboring the active site were observed. This result indicates an interaction weak between compound and protein, suggesting that its mechanism of action does not involve the deactivation of this protein, as demonstrated by WHO (2008).
Hinokinin showed hydrophobic interactions with residue Asn223 of the active site, and the other interactions ( Figure S11) occurred with residues neighboring the site. For example, the cubebin ( Figure S12) was inactive in the in vitro assays and showed no interactions with residues from the site. Furthermore, the interactions of hinokinin with neighboring amino acids can limit the access of the natural substrate to the amino acids of the protein's active site, inhibiting chitin synthesis. The observed results and the low toxicity to the environment make hinokinin an exciting target in developing alternatives to the use of insecticides that have lost their effectiveness against A. aegypti. In addition, natural products can serve as an excellent basis of inspiration for developing new agrochemicals (Sparks and Bryant 2022).

Conclusions
The extracts from the fruits of P. cubeba show weak larvicidal activity against A. aegypti larvae, however, the hinokinin lignan extracted from the HAE was active. From the molecular docking and SEM results, it is possible to suggest a contact relationship between the larvae and the lignan. The exposure of larvae to hinokinin alters the tegument, which indicates, as well as the results of molecular docking action on the protein UDP-N-acetylglucosamine pyrophosphorylase, which is essential for chitin synthesis. They show that the hinokinin has larvicidal properties and, according to literature, presents low cellular toxicity, which can be considered a target in the studies for the use of natural products in A. Aegypti control.

Disclosure statement
No potential conflict of interest was reported by the authors.