Larvicidal Activity against Aedes aegypti and Chemical Characterization of the Inflorescences of Tagetes patula

The crude acetone extract (CAE) of defatted inflorescences of Tagetes patula was partitioned into five semipurified fractions: n-hexane (HF), dichloromethane (DF), ethyl acetate (EAF), n-butanol (BF), and aqueous (AQF). BF was fractionated by reversed-phase polyamide column chromatography, obtaining 34 subfractions, which were subjected to HSCCC, where patuletin and patulitrin were isolated. CAE and the fractions BF, EAF, DF, and AQF were analyzed by LC-DAD-MS, and patuletin and patulitrin were determined as the major substances in EAF and BF, respectively. BF was also analyzed by HPLC and capillary electrophoresis (CE), and patulitrin was again determined to be the main substance in this fraction. CAE and the semipurified fractions (750, 500, 300, 100, and 50 mg/L) were assayed for larvicidal activity against Aedes aegypti, with mortality rate expressed as percentage. All fractions except AQF showed insecticidal activity after 24 h exposure of larvae to the highest concentration. However, EAF showed the highest activity with more than 50% reduction in larval population at 50 mg/L. The insecticidal activity observed with EAF might have been due to the higher concentration of patuletin present in this fraction.


Introduction
Aedes aegypti (Linnaeus, 1762) is an anthropophilic and domicile mosquito, and it is the main vector for dengue viruses in the Americas. This mosquito puts half of the world's population at risk with a 30-fold increase in incidence in the past 50 years in more than 100 endemic countries [1,2].
According to data from the World Health Organization, the number of people affected with dengue in 2015 was 3.2 million, with 500,000 people hospitalized per year [3].
Ae. aegypti also carries chikungunya, zika, and yellow fever urban viruses; so its monitoring and control are necessary. Vector control in Brazil currently occurs with the use of growth regulators of immature stages, such as 2 Evidence-Based Complementary and Alternative Medicine diflubenzuron, and the control of adult mosquitoes with alpha-cypermethrin, deltamethrin, malathion, and others according to recommendations of the WHO Pesticide Evaluation Scheme [4], which are nonspecific products that select resistant insects due to their great genetic plasticity [5], with consequent environmental contamination [6].
There is currently a great deal of interest in alternative methods and selective principles for the control of mosquitoes with less environmental damage [7]. In this sense, substances extracted from plants present a great perspective for the control of Ae. aegypti.
The substances of natural origin have some advantages: they are obtained from renewable resources, and the selection of resistant forms occurs at a slower rate than with synthetic insecticides [8,9]. Another advantage is that they show low or no toxicity to mammals and bees [10].
Accordingly, the aim of this work was to isolate and identify compounds from the semipurified n-butanol fraction of T. patula by reversed-phase column chromatography and high-speed countercurrent chromatography (HSCCC) and to evaluate the chemical profile of the crude extract and semipurified fractions using high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and liquid chromatography-mass spectrometry (LC-DAD-MS). In addition, the larvicidal activity of the crude extract and semipurified fractions was evaluated against Ae. aegypti.

High-Speed Countercurrent Chromatography (HSCCC).
The subfractions BF#16, BF#23, and BF#26 were rechromatographed by HSCCC using a PC Ito5 chromatograph (model 001) equipped with a polytetrafluoroethylene (PTFE) column (2.5 mm i.d., total volume capacity of 320 mL), 10-L sample loop, 800 rpm, and double piston solvent pump (Waters model 510), using a flow-rate of 1.0 mL/min. The organic phase (hexane : ethyl acetate : methanol : water; Table 1) was used as the mobile phase, and water was the stationary phase. Only in HSCCC of BF#16 was a gradient system with n-butanol also used. BF#16, BF#23, and BF#26

HPLC-ESI-MS/MS Analysis. Fractions and subfractions
were analyzed with a Waters HPLC system coupled with a triple quadrupole mass spectrometer (Micromass, Quattro micro6 API) equipped with a Z-electrospray ionization (ESI) source (Waters) and processed by MassLynx6 software (version 4.0, Waters). Chromatographic conditions were as follows: column was a Symmetry C-18 (3.5 m, 75 × 4.6 mm, Waters); mobile phase was water with 0.1% formic acid (v/v) (solvent A) and acetonitrile with 0.1% formic acid (v/v) (solvent B). The gradient system employed was as follows: 0-2 min 5% B; 10 min 50% B; 2 min 50%; and 13-15 min 5% B. The flow rate was 0.5 mL/min and the injection volume 10 L. A sample containing 1000 ng/mL of the isolated substances was injected, and identification was performed analyzing the information of the product ion spectra in comparison to a previously published dataset.

Identification of the Constituents by LC-DAD-MS.
The analyses of CAE and the fractions DF, EAF, BF, and AQF were performed on UFLC Shimadzu Prominence chromatograph coupled to a diode array detector (DAD) and MicrOTOF-Q III mass spectrometer (Bruker Daltonics). A Kinetex C-18 chromatographic column (2.6 m, 150 × 2.1 mm, Phenomenex) was used. Acetonitrile (solvent B) and deionized water (solvent A), both with 0.1% formic acid (v/v), were used as mobile phase. The gradient elution profile was the following: initial 3% B, 2-25 min 25% B, 25-40 min 80% B, and 40-43 min 80% B. The negative and positive ion modes were carried out, and nitrogen was applied as a nebulizer gas (4 bar) and dry gas (9 L/min).

Capillary Electrophoresis (CE)
. CE for BF analysis was carried out using a Beckman P/ACE6 MDQ electrophoresis system equipped with a filter-based UV/Vis detector and 32 Karat6 version 7.0 software. The column used was a fused silica capillary column (Beckman-Coulter) with dimensions of 60.2 cm total length, 50.0 cm effective length, 363 m o.d., and 75 m i.d. The sample was injected hydrodynamically at 0.5 psi for 3 s, 30 kV, and the electropherogram was recorded at 214 nm. The cartridge coolant of the CE was set with a thermostat at 25 ∘ C. The background electrolyte consisted of 80 mmol/L borate buffer (pH 8.80) containing 10 mmol/L methyl--cyclodextrin (Me--CD). The sample solution (500 g/mL) was prepared by dissolving 5 mg BF in 10 mL of 20% methanol and was eluted through the solid-phase extraction (SPE) cartridge (Strata C18-E, Phenomenex), preconditioned with methanol and water. Tp1 and Tp2 were used as standards for peak identification. All solutions were filtered with 0.45 m Millipore filters.
2.9. Evaluation of Larvicidal Activity. CAE, AQF, EAF, HF, BF, DF, and the fatty waste obtained in the preparation of the crude extract were tested for larvicidal activity.
Immature forms of Ae. aegypti were obtained from the insectary of the Malaria and Dengue Laboratory, Instituto Nacional de Pesquisas da Amazônia (INPA), Manaus, Brazil. The insectary began with the collection of eggs in the field by using traps (egg traps). All the procedures for the maintenance of mosquitoes and the use of animals for blood meal were authorized by the Animal Experiment Ethics Committee (CEUA/INPA 04/2013). The bioassay methods were according to Lacey [32], and WHO [33,34], with modifications.
Fourth instar larvae were used for all experiments. Three replicates with 15 immature forms and 50 mL of distilled water per container were assayed. The crude extract and semipurified fractions were diluted in dimethyl sulfoxide (DMSO) at an initial concentration of 30,000 mg/L in a total volume of 10 mL. The samples were solubilized using an ultrasonic bath for 15 min. To determine mortality rates in percent, lethal concentrations (LC 50 and LC 90 ), and their limits, five concentrations were used: 750, 500, 300, 100, and 50 mg/L. DMSO solution at 300 mg/L and distilled water were used as controls. The assay was performed using a photoperiod of 12/12 h, at 26 ± 2 ∘ C. Mortality readings were performed at 24, 48, 72, 96, and 120 h.
Statistical package Spss Inc. 2005 was used for the calculation of the survival curve of the fractions and the lethal time of Ae. aegypti for EAF. (mAU) (  Tp2 (BF#6, BF#11, and BF#16.5) was also obtained as a yellow powder. Its mass spectrum showed an intense ion of / 495 and fragment ions at / 318 and 333. The UV spectrum of Tp2 revealed maxima at 258 nm (band I) and 371 (band II), which was also compatible with flavonols.

Results and Discussion
On the basis of 1 H and 13 C-NMR data obtained and comparison with literature values [35][36][37], Tp1 and Tp2 were identified as the flavonols patuletin and patuletin-7-O-glycoside (patulitrin), respectively, which was confirmed by HMBC, HSQC, and COSY data.
CAE and AQF, EAF, DF, and BF from T. patula were analyzed by LC-DAD-MS, and the compounds were identified on the basis of UV and accurate mass and fragmentation data, which were compared with the literature data. From the samples, eleven compounds were detected and identified ( Figure 1, Table 2). The higher peak intensity was compound 4 (patulitrin) for BF and AQF, compounds 4, 5 (patulitrin isomer), and 8 (patuletin) for EAF, compound 8 for DF, and compounds 4 and 8 for CAE (Figure 1).

CE Fingerprint of BF of T. patula.
In this work, BF of T. patula was evaluated by CE. The major peaks were identified by addition of the isolated substances of this work. Peak 1 was identified as Tp2 and peak 2 as Tp1 (Figure 2). This fingerprint shows that the major substance was Tp2, and the same profile was observed by LC-DAD-MS analysis (Figure 1).
Some studies with T. patula have been performed using thin layer chromatography (TLC), HPLC, and HPLC-MS [19,38]. However, CE was employed here for the first time to identify the compounds obtained from T. patula.
Comparing the HPLC and CE methods developed for evaluation of BF of T. patula, CE was more efficient, being almost four times faster. In addition, in the CE method, organic solvents are not used to separate the analytes, and the volume of electrolytic solution used for analyses is small, making the technique less costly and polluting [39][40][41].

Larvicidal Activity.
All the fractions of T. patula evaluated showed insecticidal activity against Ae. aegypti after 24 h exposure of the larvae to a concentration of 750 mg/L, with exception of AQF.
After 120 h (5 days) of exposure, the following mortality rates were observed at a concentration of 300 mg/L for the different samples evaluated: CAE (31.0%), AQF (17.8%), EAF (53.0%), HF (13.0%), BF (15.6%), DF (8.9%), and fatty waste (31.0%). No deaths occurred during a four-day observation period for the DMSO control, but at the end of the fifth day, mortality was 6.7%. The distilled water control did not cause any mortality during the whole experiment period (Table 3).
EAF and fatty waste showed the best time-dependent results ( Figure 3)   with the exception of AQF, showed notable larvicidal activity in the present study. Among the fractions analyzed, EAF was the most promising, where a concentration as low as 50 mg/L reduced the larval population by more than half, and where 750 mg/L caused the death of all larvae within 96 h. Faizi et al. [27] carried out a nematicidal study with flowers of T. patula, which were first subjected to a defatting process with petroleum ether and then extracted with methanol, finally resulting in aqueous, dichloromethane, ethyl acetate, and butanol fractions. In that study, EAF had a higher concentration of patuletin, while the BF showed a lower amount of this substance. The authors reported that patuletin is generally more potent than patulitrin in other biological assays, such as antimicrobial and antioxidant.
Comparing our results of larvicidal activity against Ae. aegypti with those for nematicidal activity against Heterodera zeae reported by Faizi et al. [27], it is observed that, in both studies, the fraction with better activity was that with a higher concentration of patuletin and lower level of patulitrin. Thus, it is suggested that the larvicidal activity observed in EAF may  be due to the higher concentration of patuletin seen in this fraction.

Conclusion
Among the semipurified fractions obtained from CAE of the inflorescences of T. patula, BF showed a higher yield of the flavonoids patuletin and patuletin-7-O--glycoside (patulitrin).
LC-DAD-MS analysis of CAE and the fractions DF, EAF, BF, and AQF confirmed that the main substance in EAF was patuletin and patulitrin in BF.
EAF showed the highest larvicidal activity against Ae. aegypti with more than 50% decrease in larval population at a concentration of 50 mg/L. This high insecticidal activity observed in EAF may be due to the higher concentration of patuletin in this fraction.

Conflicts of Interest
The authors have declared no conflicts of interest.