Rev Bras Farmacogn 24(2014): 576-583

Original article Extraction of flavonoids from patula: process optimization and screening for biological activity

Vanessa M. Munhoza, Renata Longhinia, José R.P. Souzab, João A.C. Zequic, Eneri V.S. Leite Mellod, Gisely C. Lopesa, João C.P. Melloa,* aPrograma de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Maringá, PR, Brazil bPrograma de Pós-graduação em Agronomia, Universidade Estadual de Londrina, Londrina, PR, Brazil cInstituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil dDepartamento de Ciências Morfológicas, Universidade Estadual de Maringá, Maringá, PR, Brazil

ARTICLE INFO ABSTRACT

Article history: The flowers of Tagetes patula L., , commonly known as French marigold, are used Received 6 June 2014 in folk medicine as an antiseptic, diuretic, blood purifier and repellent. This study was Accepted 2 October 2014 conducted to optimize the extraction process through the biomonitoring of flavonoids, using a statistical mixture simplex-centroid design, to evaluate the effect of the solvents water, Keywords: ethanol and acetone, as well as mixtures of these solvents, assessed by the total flavonoid Tagetes patula L. content. The extracts were tested for dry residue, radical scavenging activity, chromatographic Flavonoids profile, and larvicidal activity. The acetone extract had the highest total flavonoid content,

Statistical mixture design 25.13 ± 1.02% (4.07%); and the best radical scavenging activity, with IC50 of 15.74 µg/ml ± 1.09 Simplex–centroid (6.92%), but with lower dry residue, 6.62 ± 1.33% (20.10%). The water extracts showed higher Larvicidal activity levels of dry residue, but lower total flavonoid content and radical scavenging activity than Aedes aegypti the acetone extract. The positive correlation between the total flavonoid content and radi- cal scavenging activity of the extracts showed that flavonoids contribute significantly to the antioxidant capacity. The statistical mixture design allowed us to optimize the extraction of flavonoids from flowers of T. patula, with acetone as the best extraction solvent. Preliminary studies on the biological activity of the optimized extracts demonstrated a larvicidal effect of the acetone extract on Aedes aegypti mosquitoes. © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved.

flowers and leaves have been used for their antiseptic, diuretic, Introduction depurative and insect repellent activities (Chadha, 1976). Chemical studies with flowers and leaves of T. patula identified Tagetes patula L., Asteraceae, popularly known as French terpenes (Prakash et al., 2012), alkaloids (Faizi and Naz, 2002), marigold, originated in Mexico. It is widely used as an carotenoids (Piccaglia et al., 1998), thiophenes (Szarka et al., ornamental and is sold freely in open markets and 2006), fatty acids (Deineka et al., 2007), and flavonoids (Guinot garden shops (Vasudevan et al., 1997). In folk medicine the et al., 2008; Faizi et al., 2011a) as constituents, some of which

* Corresponding author. E-mail: [email protected] (J.C.P. Mello). 0102-695X/$ - see front matter © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. http://dx.doi.org/10.1016/j.bjp.2014.10.001 Vanessa M. Munhoz et al. / Rev Bras Farmacogn 24(2014): 576-583 577

may elicit the biological activities reported to date; these include insecticidal (Wells et al., 1993), nematicidal (Chadha, Materials and methods 1976; Buena et al., 2008), larvicidal (Dharmagadda et al., 2005), antifungal (Faizi et al., 2008), and anti-inflammatory (Yasukawa Plant material and Kasahara, 2013) activities. The flowers have been reported to contain up to 5.5% flavonoids (Munhoz et al., 2012). Seeds of Tagetes patula L., Asteraceae, were donated by As Piccaglia and collaborators (1998) found, the flowers Syngenta Flowers Brazil and were organically grown in the of T. patula are a rich source of lutein and its esters. For this Medicinal Plant Garden of the State University of Londrina, reason the genus is widely cultivated in Central America Brazil. The flowers were collected in November 2011, and the as food coloring, which is approved by the European Union material was identified by Professor Dr. Naoki Jimi Nakagima, (Vasudevan et al., 1997). However, after carotenoids are Federal University of Uberlândia, Brazil. A voucher specimen extracted, the residue is discarded or only used as animal was deposited in the Herbarium of the State University of feed or fertilizer (Gong et al., 2012). Gong et al. (2012) observed Maringá under identification number HUEM 21.907. that this discarded residue contained various compounds of The flowers were dried in a kiln with forced-air circulation interest, including flavonoids, a class of secondary metabolites (Sparrow), heated to 38 ± 2ºC and pulverized in a hammermill with high therapeutic potential, including cardioprotective (Tigre ASN5). (Bandy and Akhlaghi, 2008), anti-inflammatory (Kim et al., 2004), antimicrobial (Lamb and Cushnie, 2005), and antitumor Chemicals and reagents (Harborne and Williams, 2000) activities, among others. The extraction method and solvent used to obtain a All solvents and reagents used were analytical grade. Chemicals flavonoid-enriched extract can greatly influence the biological used were: n-hexane, acetone and methanol (Synth®), ethanol activity. A successful extraction technique combines the (Cerealcool®), methanol HPLC grade (J.T. Baker®), DPPH optimal solvent or mixture of solvents with a convenient (2,2-diphenyl-1-picrylhydrazyl) (Sigma Aldrich®), butylated technique. In order to select an extraction method it is hydroxytoluene (BHT), aluminum chloride and potassium necessary to evaluate the efficiency, the stability of the acetate (Synth®), and Quercetin (Acros Organics). extracted substances, the availability of resources and processing costs, leading towards a biological application of Extraction of flowers: general equipment and conditions the extract (Louzada et al., 2001). The optimization of the extraction techniques is important To obtain the extract, the plant material, not separated for a variety of applications, for example to obtain the selected by particle size, was defatted using n-hexane by dynamic compounds of pharmaceutical interest, with a minimum maceration for 6 days. After drying, the defatted flowers were of impurities. In recent years, statistical models have been macerated for 10 min and the material was extracted in an successfully applied to minimize the number of experiments and industrial blender (Skymsen®, LS-04) in a proportion of 2.5% to identify the effect of the interactions between experimental (m/v) (w/v), for a period of 9 min, with 10-min intervals, and variables such as the solvent strength, and the interactions that using as the extraction liquid, mixtures of three solvents: occur between the biological material and the solvents. These (x1) acetone, (x2) ethanol and (x3) water, determined through models have provided satisfactory results for the optimization of a simplex-centroid design (Fig. 1). The proportions of the the extraction procedures (Box et al., 2005; Bruns et al., 2006). Li solvents used are specified in Table 1. and collaborators (2007) used response-surface methodology to The extracts were vacuum-filtered, evaporated under assess the yield and purity of the polysaccharides by ultrasound reduced pressure (Büchi®, R-200) and lyophilized (Christ®, extraction. Their optimized method increased the yield by Alpha 1-4) (CE). All extracts were prepared in triplicate, approximately 20% over the classic extraction method. randomly. The experimental mixture design has been widely applied in various areas, such as the development of new biofilms (Mali et al., 2010) and for the extraction of secondary metabolites Water #3 from natural products (Garcia et al., 2010; Lonni et al., 2012; 0.00 1.00 DiCiaula et al., 2014). The main goal of this method is to 0.25 evaluate how responses are affected by the variation in the 0.75 proportions of the mixture components (Cornell, 2002). In the #6 0.50 0.50 data analysis, a mathematical polynomial model is used to #7 link the different compound proportions to the properties of 0.75 0.25 interest, in order to determine the best system (Audi et al., #1 #4 #2 2001; Campos et al., 2006). 1.00 0.00 0.00 0.25 0.50 0.75 1.00 We used the statistical mixture design with a simplex- Acetone Ethanol centroid model with acetone, ethanol and water as solvents in order to optimize the extraction of flavonoids from the flowers of T. patula. All the extracts obtained were used for a Figure 1 – Simplex-centroid design with three axial mixture preliminary evaluation of larvicidal activity against the larvae points used to investigate the influence of different solvent of the mosquito Aedes aegypti. proportions on extract preparation. 578 Vanessa M. Munhoz et al. / Rev Bras Farmacogn 24(2014): 576-583

Table 1 (1 mmol/l) was added to each of these solutions. The mixture Mixture composition in extracts with acetone, ethanol and was vortexed for 15 s and left at room temperature for 30 min. water in a simplex-centroid mixture design. The absorbance was read at 517 nm with an OceanOptics USB 2000+ spectrometer. As a blank, a methanol solution of BHT Components (0.5 mg/ml) added to 500 μl of DPPH solution was used; and Extracts x1 x2 x3 the negative control consisted of a solution containing 3 ml of Acetone Ethanol Water methanol and 375 μl of DPPH solution. The radical-scavenging 1 (CEA) 1.00 0.00 0.00 activity (RSA) was determined as %RSA = [(AbsCN – AbsA) × 2 (CEE) 0.00 1.00 0.00 100]/AbsCN, where AbsA is the absorbance of the extract 3 (CEW) 0.00 0.00 1.00 solution; and AbsCN is the negative control absorbance. The 4 (CEAE) 0.50 0.50 0.00 IC50 value, or extract concentration necessary to inhibit 50% of 5 (CEAW) 0.50 0.00 0.50 DPPH, was obtained by plotting the %RSA as a function of the 6 (CEEW) 0.00 0.50 0.50 sample concentration. 7 (CEAEW) 0.33 0.33 0.33 CEA, acetone extract; CEE, ethanol extract; CEW, aqueous extract; Experimental design CEAE, acetone:ethanol extract; CEAW, acetone:water extract; CEEW, ethanol:water extract; CEAEW, acetone:ethanol:water extract. Experiments were designed and analyzed using the software Statistica® version 8.0 (StatSoft, Oklahoma, USA). As the experimental design used was simplex-centroid, we used Characterization of the extracts the special cubic model to describe the response of a ternary mixture (Eq. (1)) (Barros Neto et al., 2010): All assays were performed in three replicates.

ŷ = b x + b x + b x + b x x + b x x + b x x + b x x x Dry residue (DR) 1 1 2 2 3 3 12 1 2 13 2 3 23 2 3 123 1 2 3 (1)

For each extract, 2 ml were measured volumetrically, Where ŷ is the response of interest predicted by the model; transferred to pre-weighed stainless-steel dishes and let to and b , b , b , b , b , b , b are the estimated parameters, evaporate to dryness in a water bath (Fanem®, 120/3). The 1 2 3 12 13 23 123 corresponding to the pure components: acetone (x ), ethanol dishes containing the residue were placed in an infrared 1 (x ) and water (x ) and their binary and ternary interactions moisture analyzer (Ohaus® MB35), at 105°C for 10 min. The 2 3 predicted by the model. Surface charts were constructed. dry residue was calculated as: DR (%) = (mf/mi) × 100, where mf is the final mass of the sample in grams and mi is the initial HPLC analysis mass, thus the results are expressed relative to 100 g of the extraction solution (Anvisa, 2007). The HPLC analysis was conducted using a liquid chromatograph Thermo Model LC Pump Plus equipped with a Finnigan Determination of total flavonoid content (TFC) Surveyor PDA Plus diode array detector and a C18 Gemini Phenomenex HPLC column (250 mm × 4.6 mm), 5 μm particle The total flavonoids content (TFC) was determined by the size, and a guard column (Phenomenex) with a flow rate of 1 aluminum chloride colorimetric method (Chang et al., 2002) with modifications. To 0.5 ml of the sample solution (CEW – ml/min. Each sample was prepared in a 725 μg/ml solution, 800 µg/ml; other extracts – 400 µg/ml) 1.5 ml ethanol, 0.1 ml of according to the experimental design, and filtered with a ® 1 M potassium acetate (w/v), 0.2 ml of 10% aluminum chloride 0.22 μm Millipore filter. A 10 μl aliquot of this solution was (w/v), and 2.7 ml of distilled water were added. After incubation injected into the HPLC column (10 μl loop). The mobile phase at room temperature for 30 min, the absorbance of the solution contained a mixture of water (phase A), and acetonitrile (phase was measured on an OceanOptics USB 2000+ spectrometer at B), in a gradient system, as follows: 0 min, 5% phase B; 35 min, 430 nm. The 10% aluminum chloride was replaced by the same 58% phase B, followed by a 5-min post-time to re-equilibrate amount of distilled water. Quercetin was used to construct the the system with 5% phase B. The temperature was fixed at calibration curve (y = 0.0503× -0.0128), where y is the value for 25°C. A reference standard (quercetin) was used to identify the absorbance of the sample and x is the sample concentration, major component in the crude extracts. Elution was monitored from 3-15 µg/ml (r² = 0.9944). The total flavonoids content was at 210, 254, and 280 nm. expressed as a percentage, i.e., flavonoids g/100 g of extract. Bioassay test Radical-scavenging activity (RSA) The larvicidal activity of the extract was evaluated using The radical-scavenging ability of the extracts was evaluated laboratory-reared larvae of the dengue vector mosquito using the free radical DPPH scavenging method as described by Aedes aegypti. Larvae from Ae. aegypti were obtained from Amarowicz et al. (2004). The extracts were diluted in methanol a permanent colony maintained at a temperature of 25 ± (5- 35 μg/ml for CEA and CEAE; 50-140 μg/ml for CEW; and 2°C and relative humidity of 70 ± 5% kept in the insectary 10-40 μg/ml for other extracts). Next, 375 μl of DPPH solution of the Laboratory of General and Medical Entomology, State Vanessa M. Munhoz et al. / Rev Bras Farmacogn 24(2014): 576-583 579

University of Londrina. The colony was founded with eggs The extracts produced with solvent mixtures including collected on the field, using ovitraps. water contained the highest levels of dry residue, from 39.25 The extracts were evaluated in accordance with the to 41.57% (w/w) (Fig. 2A). However, the Tukey’s test indicated protocol of the World Health Organization (WHO) with slight no significant difference between them. modifications (WHO, 2005). Twenty-five 4th-instar larvae were The results clearly indicate that the extraction is affected transferred to transparent polyethylene pots, 11 cm in diameter by the polarity of the solvents. Prewashing the flowers with and 7 cm deep, containing 150 ml of distilled water. Initially, n-hexane decreased the dry mass of the plant by approximately 100 mg of each extract was solubilized in 1 ml of DMSO and 9 14%. Using GC-MS (gas chromatography-mass spectroscopy), ml of water, and then diluted with distilled water to obtain five Faizi et al. (2011b) identified 63 compounds in the nonpolar concentrations (80, 30, 10, 5, and 1 mg/l) for evaluation. Controls fractions of T. patula flowers, with long-chain fatty acids, were evaluated with DMSO diluted in the same conditions as hydrocarbons and thiophenes being the predominant classes the samples, but using only distilled water. The number of of compounds in the petroleum ether fraction. This suggests dead larvae was recorded after 24 and 48 h of exposure, and that the lower efficiency of the acetone is related to the fact the percentage of mortality was calculated. Each extract was that some medium- and low-polarity substances were initially tested in triplicate and the assay was repeated twice. extracted with n-hexane. The predominant positive effect

Lethal concentration (LC50) values were determined by of water and ethanol may be due to the presence of polar probit analysis using SPSS version 12 software. Results with p substances such as phenolic glycosides. ≥ 0.05 were considered to be statistically significant. The TFC ranged between 4.63 and 25.13% (w/w) (Table 2). In contrast with the results for the dried residue, the CEA showed the largest TFC, 25.13% (w/w). This was confirmed through Results and discussion Equation 3, obtained by the special cubic model (r2 = 0.979),

which showed that acetone (x1) had a strong effect on the TFC The experimental results are presented as the mean ± standard when used either pure or in combination with ethanol (x1x2). deviation and are shown in Table 2. The linear effect of acetone (x1) was positive, while water (x3) The dry residue was determined in order to evaluate the was less effective (TFC = 4.63% (w/w). The presence of water in strength of the extraction solvent used on flowers of T. patula. the ternary mixture (x1x2x3) decreased the extractive capacity The special cubic model gave a satisfactory value for the compared to the pure solvents acetone (x1) and ethanol (x2), as determination coefficient (r2 = 0.949), and all coefficients were shown by a negative coefficient and a TFC of 12.94% (w/w). The significant at the 95% confidence level (Eq. (2): binary interaction (x1x3) was not significant (p ≥ 0.05).

DR = 6.62x1 + 32.22x2 + 39.25x3 - 22.00x1x2 + 74.52x1x3 + 21.84x2x3 + 154.69x1x2x3 TFC = 24.98x1 + 16.77x2 + 4.46x3 + 10.26x1x2 + 10.31x2x3 - 128.15x1x2x3 (2) (3)

The percentage of dried residue was positively and linearly Purification with n-hexane increased the selectivity of the influenced by water (x3) and ethanol (x2), respectively. Of the solvent, especially acetone, for flavonoids. Thus, acetone was the linear trials, acetone (x1) resulted in the lowest coefficient; the most appropriate solvent for extracting these compounds from smallest proportion of dried residue (6.62% w/w) was obtained French marigold flowers, possibly because of the predominance with acetone alone (Table 2). Among the binary interactions, of free and/or methoxylated aglycones in the defatted plant the presence of acetone reduced the power of extraction of material, such as kaempferol, quercetin (Ivancheva and ethanol (x1x2), but did not reduce the power of extraction Zdravkova, 1993), patuletin (Tarpo, 1967; Guinot et al., 2008), of water (x1x3). The ternary interaction (x1x2x3) displayed a quercetagetin (Tarpo, 1969), luteolin and quercetagetin 5-methyl synergistic effect among the components of the mixture. ether (Bhardwaj et al., 1980), which are less soluble in water

Table 2 Dry residues, total flavonoids and antioxidant activity of different extracts.

Dry residue Total flavonoids Radical scavenging activity Extracts (% w/w) (% w/w) (IC50 µg/ml) 1 (CEA) 6.62 ± 1.33 25.13 ± 1.02 15.74 ± 1.09 2 (CEE) 32.22 ± 3.98 16.77 ± 0.77 24.92 ± 1.68 3 (CEW) 39.25 ± 3.89 4.63 ± 0.47 114.76 ± 3.07 4 (CEAE) 13.92 ± 2.90 23.44 ± 0.96 17.77 ± 2.06 5 (CEAW) 41.57 ± 2.07 14.43 ± 1.28 24.37 ± 2.49 6 (CEEW) 41.20 ± 3.68 13.19 ± 0.86 25.46 ± 1.35 7 (CEAEW) 40.02 ± 2.62 12.94 ± 1.00 21.87 ± 1.18 CEA, acetone extract; CEE, ethanol extract; CEW, aqueous extract; CEAE, acetone:ethanol extract; CEAW, acetone:water extract; CEEW, ethanol:water extract; CEAEW, acetone:ethanol:water extract. 580 Vanessa M. Munhoz et al. / Rev Bras Farmacogn 24(2014): 576-583

(Chebil et al., 2007). This is probably the reason that the TFC The evaluation of the radical scavenger activity of the aqueous extract (CEW) gave the lowest result. extracts generally revealed a similar behavior to that obtained Previous reports indicate that water is not an efficient solvent for flavonoids. The results, described in Table 2, are expressed for extracting phenolic compounds, which are generally more as IC50. CEA had the lowest IC50, in contrast to CEW. In Equation soluble in organic solvents less polar than water (Kim and 4 (special cubic model; r2 = 0.997), among the linear effects,

Lee, 2001). Several studies have reported that the extraction water (x3) had a significant negative effect on the extraction efficiency of phenolic compounds, including flavonoids, is of compounds with antioxidant activity; however, combining increased by the use of a mixture of water and organic solvents water with acetone (x1x3) or ethanol (x2x3) increased the such as acetone, methanol and ethanol; whereas the use of pure extraction efficiency. As seen in Fig. 2C, the lowest IC50 values solvents can reduce the power of extraction (Gong et al., 2012; were obtained with acetone and its binary mixtures, as well Meneses et al., 2013). As it can be seen in Figure 2B, the addition as the mixture of ethanol: water (75:25 v/v). of ethanol or acetone to water increased the TFC. However, pure RSA = 15.74x + 24.93x + 114.76x - 10.25x x - 163.53x x - acetone gave the best results, probably due to the chemical 1 2 3 1 2 1 3 177.55x x + 245.54x x x composition of French marigolds. 2 3 1 2 3 (4) Munhoz et al. (2012) used HPLC to generate a chemical profile for T. patula, and found that quercetin was the major The phenolic compounds present in are known substance in the plant drug. Malwade et al. (2013) evaluated for their antioxidant activity. Flavonoids have this capability the solubility of quercetin in four common solvents, and mainly because their hydroxyls donate an electron (H+) to reported that quercetin is soluble in: acetone > ethanol > radicals as hydroxyl (HO•), superoxide (O2•-), and peroxyl methanol > acetonitrile, in decreasing order. Those reports are (ROO•), neutralizing them (Harborne and Williams, 2000). in accordance with the results presented here for TFC. Previous studies have shown that the antioxidant properties From the chromatographic profiles of the extracts obtained of plants are highly correlated with the content of total phenols by the simplex centroid design, it was possible to determine the and flavonoids (Gong et al., 2012; Meneses et al., 2013), as it was retention time of quercetin (21.05 min) using the spectroscopic further confirmed in this study (Fig. 5). data for the standard, and to analyze the area under the curve. The results suggest that flavonoids contribute significantly Fig. 3 illustrates the chromatograms obtained for the seven to the antioxidant activity of the extracts obtained with extracts. As can be seen, the chromatographic profiles were not acetone and acetone:ethanol. However, the extracts derived affected by the different solvents used to extract the flavonoids. from binary mixtures containing water also inhibited the DPPH The evaluation of the area under the quercetin peak revealed the radical, possibly due to the presence of phenolic acids such positive effect of acetone and ethanol on the extraction (Fig. 4). as gallic, chlorogenic, caffeic, vanillic, p-coumaric, and ferulic

A B Water Water 0.00 1.00 0.00 1.00

0.25 0.75 0.25 0.75

0.50 0.50 0.50 0.50

45 0.75 0.25 0.75 0.25 40 35 30 24 25 20 1.00 0.00 20 1.00 0.00 16 15 0.00 0.25 0.50 0.75 1.00 12 0.00 0.25 0.50 0.75 1.00 10 * Acetone Ethanol Acetone Ethanol

C Water 0.00 1.00

0.25 0.75

0.50 0.50

0.75 0.25

100 80 1.00 0.00 60 40 0.00 0.25 0.50 0.75 1.00 20 Acetone Ethanol

Figure 2 – Response curves of DR (A), TFC (B) and RSA (C) as a function of proportions of acetone (x1), ethanol (x2) and water (x3). Vanessa M. Munhoz et al. / Rev Bras Farmacogn 24(2014): 576-583 581

Figure 5 – Correlation (simple regression analysis) between antioxidant activity by the DPPH method and total flavonoid content in the extracts obtained by the statistical mixture design (CEA; ∆CEE; CEW; ◊CEAE; CEAW; CEEW; CEAEW).

Figure 3 – Chromatographic system for the extracts described the extraction of flavonoids with antioxidant activity from T. in the optimized conditions by the simplex-centroid design use. patula flowers. The favorable effects of acetone on the extraction results were maximized to select for quercetin. Values less than

18 µg/ml (IC50) were obtained with pure acetone and a binary mixture of acetone: ethanol, to evaluate antioxidant capacity. This study demonstrated the potential of T. patula flowers as a source of flavonoids with antioxidant and larvicidal activities, using acetone and a binary mixture of acetone as solvents.

Authors’ contributions

VMM (MSc student) collected the plant samples, confection of Figure 4 – Effect of different solvents on extraction of quercetin. herbarium, performed the laboratory work, analyzed the data and drafted the paper. RL performed the chromatographic analysis. JRPS grew the plants and collected and dried the flowers. JACZ acid, among others, which have been found in members of the contributed to larvicidal activity analysis. EVSLM contributed to genus Tagetes (Kaisoon et al., 2012). designing the study, discussed the results, and critically read the The mosquito Aedes aegypti is the principal vector of dengue. manuscript. GCL contributed to designing the study, supervised To date there is no vaccine for dengue fever, and the best the laboratory work, and critically read the manuscript. JCPM procedure to combat the disease is to attack the vector, mainly contributed to designing the study, supervised the laboratory by eliminating the sites of oviposition and larval development. work, and helped write the manuscript. All the authors have read Currently, mosquito populations are controlled with the use the final manuscript and approved the submission. of organophosphate insecticides, in increasingly larger doses, a process that has allowed the selection of resistant mosquito populations (Fontoura et al., 2012). In view of the increasing Conflicts of interest interest in insecticide development of plant origin as an alternative to chemical insecticides (Cappiello et al., 2012), The authors declare no conflicts of interest. this study was undertaken to assess the larvicidal potential of the flavonoid-enriched extracts, obtained by the simplex- centroid statistical model. The bioassay test revealed larvicidal Acknowledgments activity in only two extracts. No larval mortality was observed in controls. Of the active extracts, the acetone extract showed Thanks are due to Dr. Janet W. Reid, JWR Associates, a higher larvicidal activity (100%) at a concentration of 1 ppm, Trumansburg, New York, for English revision and to PPSUS- followed by the 50% ethanol extract (100%) in a concentration Fundação Araucária (Process #20576/2010), Capes, Finep, CNPq, of 10 ppm, both after 48 h. In view of the promising response of INCT_if, Pronex for financial support. the acetone extract, further studies will be conducted towards chemical fractionation, in search of a new bioactive molecule. REFERENCES

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