Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2017 / Article

Research Article | Open Access

Volume 2017 |Article ID 9602368 | https://doi.org/10.1155/2017/9602368

Letícia Maria Krzyzaniak, Tânia Mara Antonelli-Ushirobira, Gean Panizzon, Ana Luiza Sereia, José Roberto Pinto de Souza, João Antonio Cyrino Zequi, Cláudio Roberto Novello, Gisely Cristiny Lopes, Daniela Cristina de Medeiros, Denise Brentan Silva, Eneri Vieira de Souza Leite-Mello, João Carlos Palazzo de Mello, "Larvicidal Activity against Aedes aegypti and Chemical Characterization of the Inflorescences of Tagetes patula", Evidence-Based Complementary and Alternative Medicine, vol. 2017, Article ID 9602368, 8 pages, 2017. https://doi.org/10.1155/2017/9602368

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

Academic Editor: Ghee T. Tan
Received30 Jun 2017
Accepted13 Nov 2017
Published07 Dec 2017

Abstract

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.

1. 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 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].

Among the plants with bioactive substances, there is Tagetes patula L., popularly known as “cravo-francês,” “cravo-de-defunto,” or “botões-de-solteirão” [11]. T. patula belongs to the family Asteraceae, which is one of the oldest groups of higher plants [12], with approximately 300 genera and 3000 species in Brazil [13], and its flavonoids patuletin and patulitrin are considered important taxonomic markers [14].

Its inflorescences have been used in folk medicine for antiseptic, diuretic, blood purifying, and insect repellent purposes. Its leaves have been used for renal problems and muscle pain and its roots and seeds used as purgatives [15]. Some studies on the chemical composition of T. patula up to now indicate that the flowers and leaves are rich in terpenes [16, 17], alkaloids [18], thiophenes [19], and flavonoids [2022]. This plant has shown the following activities: antihypertensive [23], anti-inflammatory [14], hepatoprotective [24], insecticidal [25], nematicidal [26, 27], larvicidal [19], antibacterial [17], antiviral [28], and antifungal [29].

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.

2. Materials and Methods

2.1. Plant Material

The inflorescences of T. patula were collected in November 2011 in the Garden of Medicinal Plants of the Universidade Estadual de Londrina, Londrina, Brazil, where they were organically grown. The plant material was collected under a permit from IBAMA-SISBIO, number 11995-6, May 13, 2016, authentication code 48926652, under the responsibility of J. C. P. Mello. An exsiccate is deposited at the Herbarium of the Universidade Estadual de Maringá (HUEM) under number 21907, and the identification was provided by Professor Dr. Jimi Nakajima at the Institute of Biology of the Universidade Federal de Uberlândia, Uberlândia, Brazil. The flowers were dried in a convection oven at 38°C for 48 h. The dried plant material was macerated using a hammer mill (Tigre ASN-5).

2.2. Preparation of Crude Extract and Semipurified Fractions

The milled inflorescences (1.9 kg) were defatted with n-hexane by dynamic maceration for three days, with subsequent drying of the inflorescences at room temperature. Afterwards, acetone was used as extraction solvent at a proportion of 4% (w/v) in an Ultra-Turrax® (UTC115KT, Ika Works) for 5 min and then subjected to maceration for 15 h. Next, turbo-extraction was performed for 20 min, with intervals of 5 min (°C). The extract was filtered, concentrated under reduced pressure, frozen, and lyophilized (Alpha 1–4, Christ®) to give the crude acetone extract (CAE, 5.86%). CAE was fractionated according to Filho and Yunes [30]. Briefly, 105 g CAE was resuspended in 1 L of methanol : water (2 : 8, v/v) and partitioned with different solvent volume ratios. The yields were n-hexane (HF) 19.27%, dichloromethane (DF) 10.17%, ethyl acetate (EAF) 13.38%, n-butanol (BF) 36.59%, and aqueous (AQF) 15.02%.

2.3. Reversed-Phase Column Chromatography of n-Butanol Fraction

BF (20.0 g) was separated by column chromatography (CC) with a polyamide column (CC6 Korngrobe, 0.05–0.16 mm; Macherey Nagel) according to Degani et al. [31], and the mobile phase was 100% methanol or water or a combination thereof, providing 34 subfractions (BF#1–34). The subfractions BF#6 (25 mg) and BF#11 (5 mg) precipitated during the organic solvent removal process and were analyzed by nuclear magnetic resonance (NMR), MS, and HPLC.

2.4. High-Speed Countercurrent Chromatography (HSCCC)

The subfractions BF#16, BF#23, and BF#26 were rechromatographed by HSCCC using a PC Ito® chromatograph (model 001) equipped with a polytetrafluoroethylene (PTFE) column (2.5 mm i.d., total volume capacity of 320 mL), 10-μL 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 yielded 8, 15, and 18 subfractions, respectively. The subfractions BF#16.5 (7 mg), BF#23.4 (13 mg), BF#26.6 (4.5 mg), and BF#26.15 (5 mg) were selected and analyzed by NMR and MS.


SubfractionEluent systems (v/v)

FB#16hexane : ethyl acetate : methanol : water (2 : 2 : 2.5 : 2)
Gradient elution with n-butanol:
0–400 mL – 0 mL n-butanol
400–800 mL – 20 mL n-butanol
800–1200 mL – 30 mL n-butanol
1200–1400 mL – 40 mL n-butanol

FB#23hexane : ethyl acetate : methanol : water (1 : 5 : 1 : 5)

FB#26hexane : ethyl acetate : methanol : water (2 : 2 : 2.5 : 2)

2.5. NMR Analysis

The subfractions BF#6, BF#11, BF#16.5, BF#23.4, BF#26.6, and BF#26.15 were analyzed by NMR spectroscopic methods 1D (1H and 13C) and 2D (1H/1H-COSY and HMBC), with a Varian Mercury Plus 300 (75 MHz for 13C and 300 MHz for 1H), using deuterated solvents and TMS as internal reference. The spectra of the subfractions were related to the compounds Tp1 (BF#23.4, #26.6, and #26.15) and Tp2 (BF#6, #11, and #16.5), which were analyzed and compared to literature data.

Patuletin (Tp1) H-NMR (, 300 MHz). 6.48 (H-8), 7.72 (d, J 2.0 Hz, H-2′), 6.88 (d, J 8.08 Hz, H-5′), 7.63 (dd, J 8.6 Hz; 2.0 Hz, H-6′), 3.88 (OCH3-6). 13C-NMR (CD3OD, 75 MHz): 146.9 (C-2), 135.6 (C-3), 176.2 (C-4), 151.6 (C-5), 130.8 (C-6), 157.1 (C-7), 93.4 (C-8), 152.3 (C-9), 103.5 (C-10), 120.3 (C-1′), 114.6 (C-2′), 144.8 (C-3′), 147.4 (C-4′), 114.8 (C-5′), 122.8 (C-6′), 59.60 (CH3-6).

Patulitrin (Tp2) H-NMR (, 300 MHz). 6.93 (s) (H-8), 7.72 (d, J 2.2 Hz, H-2′), 6.89 (d, J 8.5 Hz, H-5′), 7.54 (dd, J 8.5 Hz; 2.1 Hz, H-6′), 5.13 (d; J 7.2, H-1′′), 3.32 (d; J 2,2, H-2′′) 3.45 (m) (H-3′′), 3.17 (m) (H-4′′), 3.48 (m) (H-5′′), 3.72 (m) (H-6′′), 3.78 (s) CH3O-6. 13C-NMR (CD3OD, 75 MHz): 147.9 (C-2), 135.8 (C-3), 176.2 (C-4), 151.1 (C-5), 131.8 (C-6), 156.4 (C-7), 93.8 (C-8), 151.4 (C-9), 105.0 (C-10), 120.0 (C-1′), 115.5 (C-2′), 145.0 (C3′), 147.7 (C-4′), 115.4 (C-5′), 121.8 (C-6′), 100.1 (C-1′′), 73.2 (C-2′′), 77.2 (C-3′′), 69.5 (C-4′′), 76.7 (C-5′′), 60.6 (C-6′′), 60.3 (CH3O-6).

2.6. HPLC-ESI-MS/MS Analysis

Fractions and subfractions were analyzed with a Waters HPLC system coupled with a triple quadrupole mass spectrometer (Micromass, Quattro micro™ API) equipped with a Z-electrospray ionization (ESI) source (Waters) and processed by MassLynx™ software (version 4.0, Waters). Chromatographic conditions were as follows: column was a Symmetry C-18 (3.5 μm, 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 μL. 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.

2.7. 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 μm, 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).

2.8. Capillary Electrophoresis (CE)

CE for BF analysis was carried out using a Beckman P/ACE™ MDQ electrophoresis system equipped with a filter-based UV/Vis detector and 32 Karat™ 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 μm o.d., and 75 μm 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-β-cyclodextrin (Me-β-CD). The sample solution (500 μg/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 μm 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 (LC50 and LC90), 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 °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.

3. Results and Discussion

3.1. Structural Analysis

The structural analysis of subfractions BF#6, BF#11, BF#16.5, BF#23.4, BF#26.6, and BF#26.15 was performed by NMR, HPLC-IES-MS/MS and LC-DAD-MS and resulted in the identification of the compounds Tp1 and Tp2.

Tp1 (BF#23.4, BF#26.6, and BF#26.15) was obtained as a yellow powder. The mass spectrum obtained by ESI showed an intense ion peak at 333, corresponding to the protonated ion and fragment ions of 288 and 318. The UV spectrum of Tp1 revealed two absorption maxima in the region of 257 nm (band I) and 372 nm (band II), compatible with the UV spectrum of flavonols.

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.

On the basis of 1H and 13C-NMR data obtained and comparison with literature values [3537], Tp1 and Tp2 were identified as the flavonols patuletin and patuletin-7-O-β-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).


PeakRT (min)CompoundUV (nm)MFNegative mode ()Positive mode ()
MS MS/MSMS MS/MS

(1)10.6NI274
(2)15.0Quercetagetin O-hexoside270, 355C21H20O13479.0822317, 195, 167481.0969319, 273, 199, 181, 169
(3)15.5Ellagic acid290, 360C14H6O8300.9990284, 245, 229303.0117285, 275, 257, 247
(4)18.5Patulitrin257, 369C22H22O13493.0978331, 316, 287, 271, 181, 166495.1142333, 318, 301, 273
(5)18.8Patulitrin isomer260, 351C22H22O13493.0975330, 315, 287495.1106333, 318
(6)21.2Isorhamnetin O-hexoside270, 360C22H22O12477.1041314, 299, 271, 181, 166479.1203317, 302
(7)25.2Kaempferol267, 345C15H10O6285.0391175287.0547241, 161, 153
(8)25.5Patuletin257, 369C16H12O8331.0462316, 287, 271, 181, 166333.0613318, 290, 273
(9)29.5O-Methyl kaempferol270, 355C16H12O7315.0502300, 271, 255, 243, 166317.0655302, 274, 257, 245, 169
(10)29.9Tricoumaroyl spermidine299, 310C34H37N3O6582.2591584.2775438, 420, 292, 275, 218, 204, 146
(11)31.2Coumaroyl spermidine derivative296, 306C41H50N6O10785.3517787.3690641, 623, 495, 477, 275, 204

RT: retention time; MF: molecular formula; error lower than 8 ppm; confirmed by authentic standard.
3.2. 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 [3941].

3.3. 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).


Sample24 h48 h72 h96 h120 h

Crude acetone extract04.422.026.731.0
Fatty waste04.416.024.431.0
Aqueous fraction000017.8
Ethyl acetate fraction22.031.038.049.053.0
n-Hexane fraction2.04.44.44.413.0
n-Butanol fraction011.013.013.315.6
Dichloromethane fraction4.44.48.98.98.9
DMSO00006.7
Distilled water00000

Numbers followed by same letters in a column do not differ according to Tukey test ().

EAF and fatty waste showed the best time-dependent results (Figure 3). The lethal time for 50 percent mortality (LT50) of Ae. aegypti with EAF was 96.7 h (range: 78.4–134.8 h).

Komalasmisra et al. [42] demonstrated that plants with a LC50 lower than 750 mg/L for larvicidal activity are effective against Ae. aegypti. Thus, CAE and all fractions evaluated, 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.

4. 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-β-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.

Acknowledgments

The authors thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia para Inovação Farmacêutica (INCT_if), and Fundação Araucária and Programa de Pós-Graduação em Ciências Farmacêuticas for their financial support. Dr. A. Leyva (USA) helped with English editing of the manuscript.

Supplementary Materials

Figure S1. High performance liquid chromatography fingerprint of CAE (A) and BF (B) from the inflorescences of Tagetes patula. Peaks: (1) Tp2 (patulitrin) and (2) Tp1 (patuletin). (Supplementary Materials)

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Copyright © 2017 Letícia Maria Krzyzaniak et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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