lable at ScienceDirect Microbial Pathogenesis 95 (2016) 15e20 Contents lists avai Microbial Pathogenesis journal homepage: www.elsevier .com/locate/micpath Antifungal, antiradical and cytotoxic activities of extractives obtained from Tagetes patula L. (Asteraceae), a potential acaricide plant species Fl�avio A.S. Politi, Geisiany M. Queiroz-Fernandes, Edvânio R. Rodrigues, Jolindo A. Freitas, Rosemeire C.L.R. Pietro* School of Pharmaceutical Sciences of Araraquara, Department of Drugs and Medicines, Univ. Estadual Paulista, UNESP, Rodovia Araraquara-Jaú, km 01, CEP 14801 902, Araraquara, SP, Brazil a r t i c l e i n f o Article history: Received 24 September 2015 Received in revised form 24 February 2016 Accepted 26 February 2016 Available online 2 March 2016 Keywords: Entomopathogenic fungi Dermatophytes Antioxidant Cytotoxicity Plant extracts Tagetes patula * Corresponding author. E-mail addresses: flaviopoliti@hotmail.com (F.A.S (G.M. Queiroz-Fernandes), edvaniorodrigues@uol jolindoo@yahoo.com.br (J.A. Freitas), pietrorc@fcfar.un http://dx.doi.org/10.1016/j.micpath.2016.02.016 0882-4010/© 2016 Elsevier Ltd. All rights reserved. a b s t r a c t Tagetes patula L. shows a complex chemical composition, ranging from glycosylated flavonoids and thiophenes in extracts until terpenoids in the essential oil. In the present study, due to this rich fla- vonoidic constitution, its antioxidant potential was determined, having shown values of antiradical percentage superior to reference compounds, mainly the extracts prepared with flowers. Previous studies performed emphasized the acaricide potential of T. patula and thus, the present study aimed to verify the action of extractives obtained from aerial parts on growth of entomopathogenic fungi related to biological control of brown dog tick Rhipicephalus sanguineus and the action against pathogenic fungi closely associated with pets. None of the samples inhibited the growth of strains of Beauveria bassiana or Metarhizium anisopliae, enabling feasible future studies of synergism on acaricide activity of formulations containing fungi and extracts. The antimicrobial activity of ethanolic extract of flowers (FlEtOH70%) against Microsporum canis and Trichophyton rubrumwas significant (193.3 mg/mL and 253.9 mg/mL, respectively), as well as ethanolic extract from aerial parts (APEtOH70%) against T. rubrum (312.5 mg/mL). In order to ensure the safety of a topical formulation containing the extractives of T. patula, the cytotoxic potential of these samples were tested in murine macrophages cells. At higher concentrations all extracts were quite lethal, with IC50 ranging from 210.96 mg/mL to 468.75 mg/mL for APEtOH70% and FlEtOH70%, respectively. These results suggest that the application of a product containing T. patula extractives in the control of ticks could be used, at principle, only on the environment. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Researches about the use of plant species in the control of ani- mal parasites are scarce, with a lack of further information regarding the conditions of production, harvest time, plant parts and quantities used in the elaboration of products [1]. The use of plants, either as phytotherapics or a source of prototype substances, shows the importance of scientific research for the development of a new drug. Among the advantages of herbal medicines that justify their use are synergistic effects of its components, the combination of mechanisms for compounds acting on different molecular tar- gets, lower risk of side effects and less costs in research [2]. . Politi), geisyq@hotmail.com .com.br (E.R. Rodrigues), esp.br (R.C.L.R. Pietro). Tagetes patula L. (Asteraceae), popularly known as dwarf mari- gold or Frenchmarigold is an annual plant, 20e30 cm height, native to North America and widely disseminated throughout the world. The genus Tagetes has many biological activities reported against many organisms such as fungi [3e5], Gram positive and Gram negative bacteria [6e8], virus [9], nematodes [10,11], insects [12e14], ticks [15e18] and others. The phytochemical investigation of different parts of T. patula has resulted in the isolation of several chemical constituents of different classes of secondary metabolites, such as benzofurans, carotenoids, flavonoids and thiophenes, the latter being responsible for a variety of biocides properties [19]. Bano et al. (2002) [20] isolated and characterized thiophenes, ste- roid and terpenoids from roots, leaves and flowers of T. patula. Flavonoids, such as quercetin and kaempferol were reported by Ivancheva and Zdravkova (1993) [21]. Politi et al. (2012) [16], using the same ethanolic extract applied in the tests of the present study, identified by HPLC-MS twelve O-glycosylated flavonoids: Delta:1_given name Delta:1_surname Delta:1_given name Delta:1_surname Delta:1_given name mailto:flaviopoliti@hotmail.com mailto:geisyq@hotmail.com mailto:edvaniorodrigues@uol.com.br mailto:jolindoo@yahoo.com.br mailto:pietrorc@fcfar.unesp.br http://crossmark.crossref.org/dialog/?doi=10.1016/j.micpath.2016.02.016&domain=pdf www.sciencedirect.com/science/journal/08824010 http://www.elsevier.com/locate/micpath http://dx.doi.org/10.1016/j.micpath.2016.02.016 http://dx.doi.org/10.1016/j.micpath.2016.02.016 http://dx.doi.org/10.1016/j.micpath.2016.02.016 F.A.S. Politi et al. / Microbial Pathogenesis 95 (2016) 15e2016 kaempferol, patuletin, quercetin-3-O-pentoside, quercetin-3-O- glucoside (isoquercitrin) and quercetin-3-O-galactoside (hypero- side), patuletin-7-O-glucoside (patulitrin) or 6-O -methyl-quer- cetin-3-O-glucoside, quercetin-3-O-rhamnosyl-O-xyloside, quercetin-3-O-di-rhamnoside, quercetin-3-O-glycosyl-7-O-rham- nosyl, quercetin-3-O-rhamnosyl-7-O-glycosyl, kaempferol-3-O-di- hexoside and quercetin-3-O-galloyl-hexoside. In another study, by GCeMS, Politi et al. (2013) [17] identified in the essential oil of the aerial parts of T. patula 55 compounds, being the main, 4-vinyl guaiacol and gamma-terpinene, appearing also in good pro- portions limonene, (E)-tagetone and spathulenol. Researches with entomopathogenic fungi as biological control- lers have been made in order to assist the establishment of rational and effective strategies against arthropods of commercial interest or pathogenic [22]. Metarhizium anisopliae and Beauveria bassiana are the most well characterized entomopathogenic fungi. Hence, many studies describe its potential for controlling many plagues, including ticks [23e26]. The capacity of production and obtain- ment of formulations from the association of this fungus with different compounds makes it one of the most traded in the world [27]. Based on the phytochemistry previously described and the biocide potential of the T. patula reported on recent studies of anti- tick potential of the 70% ethanolic extract [16,18] and essential oil [17] of this species against the brown dog tick Rhipicephalus san- guineus, the present investigation aimed to verify the action of these plant extractives on growth of entomopathogenic fungi directly related to the biological control of such ixodids. Besides, the investigation of the activity of these plant extractives over derma- tophytes, may also contribute to eliminate pathogenic fungi closely associated with the primary host of these ticks [28]. Furthermore, based on the rich constitution of flavonoids, was analyzed the antioxidant potential of the samples. In order to verify the safe use of these extractives like a possible acaricidal formulation, in a first moment, cytotoxicity assays were conducted in macrophages cells. 2. Material and methods 2.1. Plant material Aerial parts of T. patula (stems, leaves and flowers) were ob- tained from the Collection of Medicinal and Aromatic Plants (CPMA) of the Multidisciplinary Center for Chemical, Biological and Agricultural Research (CPQBA), Universidade Estadual de Campinas (UNICAMP). The planting was done from seeds of Top Seed Garden line (Agristar®). A voucher specimen was deposited in the CPQBA Herbarium (process number 1421/2013). 2.2. Test microorganisms The microbiological tests were conducted with the following strains: Trichophyton rubrum (INCQS 40004 and a clinical isolate of Oswaldo Cruz Foundation - OCF), Trichophyton mentagrophytes (INCQS 40051), Microsporum canis (clinical isolate, Laborat�orio de Micologia Clínica, FCFAR/UNESP), M. anisopliae (ATCC 343 and a clinical isolate of Oswaldo Cruz Foundation - OCF) and B. bassiana (ATCC 507 and 4531). 2.3. Extracts preparation After the stabilization and drying, the aerial parts of the plant were triturated into cutting mill. The powdered drug was used for preparing the extracts by percolation using ethanol 70% (v/v) as solvent, with average flow rate of 40 drops/minute. After complete solvent evaporation, the dry extract was lyophilized and stored in a desiccator. 2.4. Determination of total flavonoids content The total flavonoid content was estimated using a colorimetric method based on the formation of a flavonoidealuminum complex [29]. The values were calculated from a calibration curve obtained with quercetin (95% purity, Merk) at concentrations of 0.25, 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0 mg/mL. Final results were expressed in mil- ligrams per gram of quercetin equivalent (QE), performed at 430 nm in a spectrophotometer Shimadzu UV-1603. The reading was done after 15 min of color reaction in the dark. An 80% methanolic solution (v/v) was used as blank. Samples of 70% ethanolic extract of the aerial parts (APEtOH70%) and flowers (FlE- tOH70%) of T. patula were prepared to a stock concentration of 0.5 mg/mL in 80% MeOH solution (v/v). Aliquots of each stock so- lution were added to 2 mL of hexahydrate aluminum chloride so- lution AlCl3 (6H2O) in 2% MeOH (v/v), adjusting final volume to 4 mL in 80% MeOH solution (v/v). 2.5. Antiradical potential The antioxidant activity assay of extractives was based on free radical scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) solution [30]. Gallic acid, rutin, quercetin and vitamin C were used as standards; dilutions of APEtOH70%, aerial parts without flowers (APWFEtOH70%) and FlEtOH70% were tested. Briefly, 2.5 mL of DPPH solution in 0.004% MeOH was added to 1 mL of different concentrations of plant extracts. A solution in methanol (2.5:1, v/v) was used as negative control and pure MeOH was the blank. The absorbance was read at 517 nm in spectrophotometer (Shimadzu- 1603). The antiradical activity was calculated as the percentage of DPPH discoloration, according to the equation below: DPPH Scavenged ð%Þ ¼ Ac� At Ac � 100 Where: Ac ¼ absorbance of DPPH solution (negative control); At ¼ absorbance of test sample. 2.6. Antimicrobial activity The antimicrobial activity was determined by minimum inhib- itory concentration (MIC) according to adapted protocols from CLSI M38-A2 [31]. Extracts were prepared in dimethylsulfoxide (DMSO) and diluted in RPMI 1640 medium to obtain a 5 mg/mL solution. Amphotericin B (16 mg/mL) was the antibiotic used as positive control for fungal strains. The inoculum was obtained by resus- pending fungal cells in 0.9% saline and adjusted to obtain approx- imately 5 � 103 CFU/mL. Briefly, 100 ml of this cell suspension were applied in 96 wells cell culture plates with 100 ml of medium and 100 ml of the plant extracts, performing serial dilutions. The plates were incubated in an orbital shaker at 100 rpm for 7 days at 28 �C and then the visual reading was done. There were a negative growth control constituted by only medium, a negative extract control containing extracts and medium and a growth control containing cells and medium. The MIC was considered as the lowest concentration that inhibited fungal growth. 2.7. Cytotoxicity assay Cytotoxicity assay was adapted from Ahmed et al. (1994) [32]. The murine macrophage strain J774 was maintained in RPMI me- dium (pH 7) supplemented with 10% fetal bovine serum, 0.2% so- dium bicarbonate, 10 mM Hepes, penicillin (100 U/mL) and Table 2 Antiradicalar activity of patterns and ethanolic extracts of T. patula by DPPHmethod. Sample Absorbance (±SD) Antiradicalar Activity % (±SD) Gallic Acid 0.0408 (±0.0023) 93.154a (±0.386) Rutin 0.0532 (±0.0009) 91.075a (±0.157) Quercetin 0.0457 (±0.0020) 92.327a (±0.339) Vitamin C 0.0232 (±0.0010) 96.105a (±0.173) APEtOH70% 0.0563 (±0.0014) 90.556a (±0.243) APWFEtOH70% 0.0593 (±0.0010) 90.047a (±0.171) FlEtOH70% 0.0396 (±0.0017) 93.350a (±0.285) DPPH: 2,2-diphenyl-1-picrylhydrazyl; APEtOH70%: 70% ethanolic extract from aerial parts of T. patula; APWFEtOH70%: 70% ethanolic extract from aerial parts without flowers of T. patula; FlEtOH70%: 70% ethanolic extract from flowers of T. patula. Values with the same superscript letters within a column do not showed statistically sig- nificant differences by Tukey test (p < 0.05). Table 3 Minimum inhibitory concentration (MIC) values of different extracts of T. patula against dermatophytes and entomopathogenic fungi. Microorganisms MIC (mg/mL) APEtOH70% APWFEtOH70% FlEtOH70% Ampho B T. rubrum (40004) 312.50A,a 625.00A,b 410.15A,a 2.0A,c T. rubrum (OCF) 468.75B,a 937.50B,b 253.90B,a 2.0A,c T. mentagrophytes (40051) 625.0C,a 1041.66B,b 572.91A,a 4.8B,c M. canis (CI) 703.13C,a 703.13A,a 195.31B,b 4.0B,c M. anisopliae (343) 1250.0D,a 1250.0C,a 1250.0C,a 16.0C,b M. anisopliae (OCF) 1250.0D,a 625.0A,b 1250.0C,a 16.0C,c B. bassiana (507) 1250.0D,a 1250.0C,a 1041.66C,a 8.0D,b B. bassiana (4531) 1250.0D,a 625.0A,b 1250.0C,a 8.0D,c CI: clinical isolate; APEtOH70%: 70% ethanolic extract from aerial parts of T. patula; APWFEtOH70%: 70% ethanolic extract from aerial parts without flowers of T. patula; FlEtOH70%: 70% ethanolic extract from flowers of T. patula; Ampho B: amphotericin B. Superscript case letters refer to comparisons within the same column; Superscript lower case letters refer to comparisons within the same row. Values followed by the same letter do not show statistically significant differences by Tukey test (p < 0.05). F.A.S. Politi et al. / Microbial Pathogenesis 95 (2016) 15e20 17 streptomycin (100 mg/mL), subsequently incubated at 37 �C under 5% CO2 atmosphere until exponential growth phase. Stock solutions of plant extracts were prepared in DMSO, diluted with the culture medium 1:10 (v/v) and applied to the 96 wells cell culture plate, proceeding to serial dilution in a 1:1 ratio. The con- centration range test varied from 15.625 mg/mL to 2000 mg/mL. The cytotoxicity of the samples was determined by adding resa- zurin (0.1 mg/mL). The reading was performed on luminescence microplate reader Spectra Fluor Plus at 530 and 590 nm [33]. 2.8. Statistical analysis Analyses of variance (ANOVA) were performed, and the aver- ages were compared by parametric test of Tukey (p < 0.05), using the software StatPlus 2009 (Soft Analyst®). Pearson linear correla- tion test (p < 0.05) was applied to indicate the correlation coeffi- cient between total flavonoids content and the antioxidant activity of the extracts. All the tests were executed in triplicate. 3. Results Yields of 44.9% for the APEtOH70%, 31.9% for the APWFEtOH70% and 41.2% for the FlEtOH70% were obtained in the extracts preparation. The average content of total flavonoids in the extract APEtOH70% and FlEtOH70% was approximately 7.27% and 12.45%, respectively (Table 1). The analysis of variance (ANOVA) revealed that the average of total flavonoid content of aerial parts extracts and flowers extract differ significantly (p < 0.05). The content of fla- vonoids (quercetin equivalents) obtained from the extract of flowers of T. patula is almost double the flavonoid content of the extract of the aerial parts. The FlEtOH70% presented the best antiradicalar activity (Table 2), higher than the gallic acid and quercetin standards. There were no statistically significant differences (p < 0.05) by analysis of variance in mean values of antioxidant activity between the samples and the standards. This results show that the hydroalcoholic extracts of T. patula have a high antiradical potential. The analysis of Pearson linear correlation (p < 0.05) indicated a strong correlation between total flavonoids content and the respective antioxidant activity of APEtOH70% and FlEtOH70% (r ¼ 0.98 and r ¼ 0.99, respectively). All extracts showed good results against the T. rubrum, high- lighting the best antifungal action of FlEtOH70% against OCF strain (Table 3). Against T. mentagrophytes, the most effective extracts were FlEtOH70% and APEtOH70%, with no statistically significant dif- ference between them (p < 0.05). Significant results against M. canis were exhibited by all extracts, emphasizing the action of FlEtOH70%, the lowest inhibitory concentration of all extract tested. The entomopathogenic fungi, was not sensitive to extracts showing MIC values only at the highest inhibitory concentration applied Table 1 Spectrophotometric quantification (430 nm) of the total flavonoids (equivalent in quercetin) present in the dried extract of T. patula. C (mg/mL) FC (mgEQ/g) APEtOH70% (±SD) FlEtOH70% (±SD) 125.0 73.06a (±0.5351) 123.95b (±1.0480) 156.25 71.98a (±0.9612) 121.90b (±2.4881) 187.5 72.88a (±1.6569) 126.78b (±0.7272) 250.0 73.03a (±0.6638) 125.75b (±2.6054) Mean 72.74a (±0.5012) 124.59b (±0.9680) C: sample concentration; APEtOH70%: 70% ethanolic extract from aerial parts of T. patula; FlEtOH70%: 70% ethanolic extract from flowers of T. patula; FC: flavonoids content, calculated as milligrams of quercetin equivalents per gram of dry extract (mgEQ/g); SD: standard deviation. Values with the same superscript letters within a column do not showed statistically significant differences by Tukey test (p < 0.05). (Table 3). Considering that these microorganisms represent an efficient biological control of ticks, it was satisfactory that the ex- tracts tested presented a low activity against them, thus preserving its acaricidal potential. All extracts were highly cytotoxic, eliminating almost 100% of the macrophage cells, especially the APEtOH70% (96.24% cell lysis) (Fig. 1). The concentrations that destroy 50% of macrophages (IC50) varied from IC50 ¼ 210.93 mg/mL, for the APEtOH70%, to IC50 ¼ 468.75 mg/mL, for FlEtOH70% (Table 4). The IC50 values of the APEtOH70% and APWFEtOH70% showed no statistically significant dif- ferences between them (p < 0.05). Fig. 1. Cell viability of murine macrophage front of ethanolic extracts of T. patula. Table 4 IC50 (mg/mL) of 70% ethanolic extracts of T. patula front of J774 macrophage cells. Sample IC50 (mg/mL) ± SD APEtOH 210.93a (±11.04) APWFEtOH 375.0a (±35.35) FlEtOH 468.75b (±44.19) SD: standard deviation; APEtOH: 70% ethanolic extract from aerial parts of T. patula; FlEtOH: 70% ethanolic extract from aerial parts of T. patula; APWFEtOH: 70% ethanolic extract from aerial parts without flowers of T. patula. Values fol- lowed by the same letter do not show statistically significant differences by Tukey test (p < 0.05). F.A.S. Politi et al. / Microbial Pathogenesis 95 (2016) 15e2018 4. Discussion During the last decades, the health professionals interest have been directed to natural therapies based on phytotherapics, not only in developing countries but also in global economic powers, which has attracted the attention of multinational pharmaceutical companies, encouraging the investment of millions of dollars in this research area. However, despite this expansion, there is still much to be done regarding to standardization, regularization and confirmation of the pharmacological activities of the great majority of natural products marketed. Because the preponderant presence of flavonoids in the extract of T. patula, the quantifying of this important class of secondary metabolites was performed in this study, using a colorimetric assay of complexation of aluminum chloride with flavonoid nucleus of the compounds present in the sample [34]. This assay is accurate and reproducible method, providing very small or no deviations between a test and another with the same samples. In our work, the results obtained with the 70% ethanolic extract of T. patula are similar that found in other studies with plants of the same genus. Our results are quite superior of other plant species, for example, the value of total flavonoid content of 31.15 mg C/g (milligrams of catechin per gram of dry extract) provided by Hajimahmoodi et al. (2008) [35] for Punica granatum (Punicaceae). The search for the discovery of novel antioxidants compounds with natural origin to replace synthetics such as butylhydroxyanisole (BHA) and butyl- ated hydroxytoluene (BHT) e used for preserving the quality and safety of food, drugs and cosmetics e is a scientific hotspot [36]. This demand is due to the fact that these synthetic antioxidants have a high volatility and instability at high temperatures, which present some toxicity and because they are less potent than the natural antioxidant agents [37,38]. In the present study, analysis of variance showed that the ex- tracts tested presented similar values of antiradical activity when compared with each other and compared to pattern compounds (gallic acid, vitamin C, quercetin and rutin), highlighting the FlE- tOH70%, which showed antiradical activity higher than gallic acid, quercetin and rutin. For comparison, Li et al. (2007) [39] obtained with ethanolic extract of Tagetes erecta 93% of antiradical activity for Xinhong cultivar, however, for other cultivars the average ac- tivity was only 75.59%. The great antiradical potential presented by the extracts is related to its high content of flavonoids. It is generally assumed that the antioxidant or antiradical maximum activity of flavonoids is mainly due to the occurrence of the 2, 3 double bond in combination with the 4-keto group in the C ring, and the addi- tional presence of hydroxyl groups at positions 30, 40 on B ring and hydroxy group in position 7 of the B ring [40]. Such interactions were observed in the compounds identified by Politi et al. (2012) [16] in ethanolic extracts of T. patula. Considering the interest in use of T. patula extracts for prepa- ration of acaricide topical formulations, it became imperative to determine the cytotoxicity. The results presented here indicate that the FlEtOH70% had the highest IC50, however, at the highest con- centration tested (2000 mg/mL) was as cytotoxic as the others. On average, up to 250 mg/mL, the only extract that showed cytotoxicity index higher than 50%was APEtOH70%, showing 58% of cell lysis, with the lowest IC50, characterizing it as the most cytotoxic sample. If we imagine the use of a product based on the T. patula extractives as an insecticide, to be sprinkled on the environment, significant results, as those mentioned by Dharmagadda et al. (2005) [41], could be achieved. However, if we develop a topical formulation to use in domestic animals, further studies with other cell strains would be necessary in order to ensure the security of your application. For centuries, formulations containing flavonoids as the main pharmacological active constituents have been used to treat dis- eases. Antibacterial activity of flavonoids has been reported in several studies [42]. In the present work, the best results against the filamentous fungi were obtained with the FlEtOH70%, probably due to its higher concentration of flavonoids, with MIC values ranging from 195.3 mg/mL to 572.9 mg/mL against M. canis and T. mentagrophytes, respectively. The APEtOH70% showed good anti- fungal activity against T. rubrum (40004), whereas Lima et al. (2009) [43], using methanolic extract of aerial parts of Tagetes mendocina found MIC values greater than 1000 mg/mL against Mycrosporum gypseum, T. rubrum and T. mentagrophytes strains. According to Rios and Recio (2005) [44], the presence of activity is very interesting in the case of concentrations below 100 mg/mL for extracts and 10 mg/mL for isolated compounds. In addition, the antimicrobial activity of 70% ethanolic extracts of T. patulawas evaluated against two strains of B. bassiana and two strains of M. anisopliae, with the objective of obtaining high MIC values, enabling, in further studies, a possible synergistic action of these samples with fungal suspensions. The pathogenicity of M. anisopliae fungus was observed in eggs and larvae of Rhipicephalus microplus [45], which was found high mortality rates. The in vitro action of the fungus B. bassiana for eggs of the same species of tick was also evaluated by Bittencourt et al. (1996) [46]. The authors observed that the percentage of hatching observed in the treated groups was lower than that observed in the control group. Garcia et al. (2004) [47] described the events involved in themechanism of adhesion, penetration and colonization of adult engorged females of R. sanguineus byM. anisopliae. Prette et al. (2005) [24] found that B. bassiana were pathogenic for eggs, larvae and engorged nymphs of R. sanguineus, with higher efficiency pathogenic action as the concentration of spore suspension used to infect the different stages of the life cycle of the tick was increased. The results of our study are very promising, since, with the exception of the APWFE- tOH70% that showed MIC value of 625 mg/mL against M. anisopliae (OCF) and B. bassiana (4531), all other extracts had MIC values higher than 1250 mg/mL. 5. Conclusions The high antioxidant potential presented by the samples, with percentages of antiradical activity similar to analyzed patterns, may be explained due to the high concentration of total flavonoids present in T. patula, especially in the flowers. No significant results against dermatophytes or against entomopathogenic fungi were obtained. In the second case, the result is interesting from the point of view of seeking a synergy of strains of B. bassiana and M. anisopliae with compounds of T. patula, in order to intensify the anti-tick potential of both, constituting a first step on this way. The high cytotoxic activity observed for these extractives in assays with murine macrophages suggest the application of products devel- oped from these samples against ticks only on the environment, until further studies would be performed with other cell lines F.A.S. Politi et al. / Microbial Pathogenesis 95 (2016) 15e20 19 especially dermal strains. Acknowledgments We thank to S~ao Paulo Research Foundation (FAPESP) for finan- cial support and University of Campinas (UNICAMP) by the partnership. Glossary Dermatophyte a fungus belonging to the genera Epidermophyton, Microsporum, or Trichophyton with the ability to utilize keratin to infect hair, nail and skin. Entomopathogenic fungi a fungus able of causing disease or kill insects. Flavonoids secondarymetabolites found in plants, includingmany different compounds, such as flavonols, flavones, isoflavones, catechins and anthocyanidins, which has antioxidant potential. Ixodid a tick belonging to the family Ixodidae, also known as hard tick. Macrophage any mononuclear phagocytic cell arising from monocytic stem cells in the bone marrow. Minimum inhibitory concentration the smallest concentration of an antibiotic or other product that regularly inhibits growth of a microorganism in vitro. Pearson linear correlation is a measure of the strength of the linear relationship between two variables. It can range from �1 to 1, indicate by r coefficient. Percolation procedure of extraction of a plant material using a special apparatus through which a solvent is gradually released. References [1] A. Heimerdinger, C.J. Olivo, M.B. Molento, C.A. Agnolin, M.F. Ziech, L.F.B. Scaravelli, et al., Extrato alco�olico de capim-cidreira no controle do Boophilus microplus em bovinos, Rev. Bras. Parasitol. Vet. 15 (1) (2006) 37e39. [2] R.A. Yunes, R.C. Pedrosa, V. Cechinel Filho, F�armacos e fitoter�apicos: a necessidade do desenvolvimento da indústria de fitoter�apicos no Brasil, Quim. Nova 24 (1) (2001) 147e152. [3] C. Romagnoli, D. Mares, M.P. Fasulo, A. Bruni, Antifungal effects of a-terthienyl from Tagetes patula on five dermatophytes, Phytother. Res. 8 (1994) 332e336. [4] D. Mares, B. Tosi, F. Poli, E. Andreotii, C. Romagnoli, Antifungal activity of Tagetes patula extract on some phytopathogenic fungi: ultrastructural evi- dence on Phytium ultimum, Microbiol. Res. 159 (2004) 295e304. [5] C. Romagnoli, R. Bruni, E. Andreotti, M.K. Rai, C.B. Viventi, D. Mares, Chemical characterization and antifungal activity of essential oil of capitula from wild Indian Tagetes patula L, Protoplasma 225 (2005) 57e65. [6] M.L. Tereschuk, M.D. Baigori, L.R. Abdala, Antibacterial activity of Tagetes terniflora, Fitoterapia 74 (2003) 404e406. [7] M. Rond�on, J. Velasco, J. Hernand�ez, M. Pecheneda, J. Rojas, A. Morales, et al., Chemical composition and antibacterial activity of the essential oil of Tagetes patula L. (Asteraceae) collected from the Venezuela Andes, Rev. Latinoamer Quím. 34 (2006) 1e3. [8] N. Dasgupta, S. Ranjan, P. Saha, R. Jain, S. Malhotra, M.A.A.M. Saleh, Antibac- terial activity of leaf extract of Mexican marigold (Tagetes erecta) against different gram positive and gram negative bacterial strains, J. Pharm. Res. 5 (8) (2012) 4201e4203. [9] M.J. Abad, P. Bermejo, P.S. Sanchez, X. Chirigota, L. Carrasco, Antiviral activity of some South American medicinal plants, Phytother. Res. 13 (1999) 142e146. [10] F.J. Gommers, J. Bakker, Physiological changes induced by plant responses or products, in: G.O. Poinar-Júnior, H.B. Jansson (Eds.), Diseases of Nematodes, Vol. 1, CRC Press Inc, Boca Raton, 1988. [11] I.T.F. Macedo, C.M.L. Bevilaqua, L.M.B. Oliveira, A.L.F. Camurça-Vasconcelos, S.M. Morais, L.K.A. Machado, et al., In vitro activity of Lantana camara, Alpinia zerumbet, Mentha villosa and Tagetes minuta decoctions on Haemonchus con- tortus eggs and larvae, Vet. Parasitol. 190 (2012) 504e509. [12] D. Laurent, L.A. Vilaseca, J.M. Chantraine, C. Ballivian, G. Saavedra, R. Ibanez, Insecticidal activity of essential oils on Triatoma infestans, Phytother. Res. 11 (1997) 285e290. [13] J.M. Chantraine, D. Laurent, C. Ballivan, G. Saavedra, R.V.S. Ibanez, Insecticidal activity of essential oil on Aedes aegypti larvae, Phytother. Res. 12 (5) (1998) 350e354. [14] S. Faizi, S. Fayyaz, S. Bano, E.Y. Iqbal, Siddiqi H. Lubna, et al., Isolation of nematicidal compounds from Tagetes patula L. yellow flowers: structure- activity relationship studies against cyst nematode Heterodera zeae infective stage larvae, J. Agric. Food Chem. 59 (17) (2011) 9080e9093. [15] M.V. Garcia, J. Matias, J.C. Barros, D.P. De Lima, R.S. Lopes, R. Andreotti, Chemical identification of Tagetes minuta Linnaeus (Asteraceae) essential oil and its acaricidal effect on ticks, Rev. Bras. Parasitol. Vet. 21 (4) (2012) 405e411. [16] F.A.S. Politi, G.M.F. Figueira, A.M. Araújo, B.R. Sampieri, M.I. Camargo-Mathias, M.P.J. Szab�o, et al., Acaricidal activity of ethanolic extract from aerial parts of Tagetes patula L. (Asteraceae) against larvae and engorged adult females of Rhipicephalus sanguineus (Latreille, 1806), Paras. Vect. 5 (2012) 295e306. [17] F.A.S. Politi, T.M.S. Moreira, E.R. Rodrigues, G.M. Queiroz, G.M. Figueira, A.H. Janu�ario, et al., Chemical characterization and acaricide potential of essential oil from aerial parts of Tagetes patula L. (Asteraceae) against engorged adult females of Rhipicephalus sanguineus (Latreille, 1806), Parasitol. Res. 112 (6) (2013) 2261e2268. [18] F.A.S. Politi, G.M. Figueira, M.I. Camargo-Mathias, J.M. Berenger, P. Parola, R.C.L.R. Pietro, Action of ethanolic extract from aerial parts of Tagetes patula L. (Asteraceae) on hatchability and embryogenesis of Rhipicephalus sanguineus eggs (Acari: Ixodidae), Ind. Crop Prod. 67 (2015) 55e61. [19] P.E. Massera, T. Rodriguez, A.M. Giulietti, Thiophene production in trans- formed roots cultures of Tagetes filifolia, Biotec. Lett. 20 (6) (1998) 573e577. [20] H. Bano, S.W. Ahmed, I. Azhar, M.S. Ali, N. Alam, Chemical constituents of Tagetes patula L, Pak. J. Pharm. Sci. 15 (2) (2002) 1e12. [21] S. Ivancheva, M. Zdravkova, Flavonoids in Tagetes patula, Fitoterapia 64 (1993) 555e558. [22] D. Chandler, G. Davidson, J.K. Pell, B.V. Ball, K. Shaw, K.D. Sunderland, Fungal biocontrol of Acari, Biocontrol Sci. Technol. 10 (2000) 357e384. [23] L.M. Basso, A.C. Monteiro, M.A.A. Belo, V.E. Soares, M.V. Garcia, D.A. Mochi, Controle de larvas de Boophilus microplus por Metarhizium anisopliae em pastagens infestadas artificialmente, Pesq. Agropec. Bras. 40 (6) (2005) 595e600. [24] N. Prette, A.C. Monteiro, M.V. Garcia, V.E. Soares, Patogenicidade de isolados de Beauveria bassiana para ovos, larvas e ninfas ingurgitadas de Rhipicephalus sanguineus, Cienc Rural 35 (4) (2005) 855e861. [25] In P. Kaeng, N. Simasatikul, K. Nganvongpanit, M. Thungrabeab (Eds.), Path- ogenicity of Beauveria Bassiana to Engorged Female, Eggs and Larvae Cattle Tick (Boophilus Microplus, Canestrini). Proceedings of the International Con- ference on the Role of Universities in Hands-on Education, Rajamangala University of Technology Lanna, Chiang-Mai, Thailand, 2009 Aug 23-29, pp. 376e381. [26] L.A.G. Barci, J.E.M. Almeida, A.H.C. Nogueira, L.O. Zappelini, A.P. Prado, Seleç~ao de isolados do fungo entomopatogênico Beauveria bassiana (Ascomycetes: Clavicipitaceae) para o controle de Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), Rev. Bras. Parasitol. Vet. 18 (1) (2009) 7e13. [27] X. Jin, D.A. Streett, C.A. Dunlap, M.E. Lyn, Application of hydrophilic-lipophilic balance (HLB) number to optimize a compatible non-ionic surfactant for dried aerial conidia of Beauveria bassiana, Biol. Control 46 (2008) 226e233. [28] C. Cafarchia, D. Romito, G. Capelli, J. Guillot, D. Otranto, Isolation of Micro- sporum canis from the coat of pet dogs and cats belonging to owners diag- nosed with M. canis tea corporis, Veter. Dermatol. 17 (5) (2006) 327e331. [29] A. Djeridane, M. Yousfi, B. Nadjemi, D. Boutassouna, P. Stocker, N. Vidal, Antioxidant activity of some Algerian medicinal plants extracts containing phenolic compounds, Food Chem. 97 (2006) 654e660. [30] S. Bashir, A.H. Gilani, Studies on the antioxidant and analgesic activities of Aztec marigold (Tagetes erecta) flowers, Phytother. Res. 22 (2008) 1692e1694. [31] National Committee for Clinical Laboratory Standards, Reference Method for Broth Dilution Antifungal Susceptilibity Testing of Filamentous Fungi: Approved Standard M-38A2, NCCLS, Wayne, Pennsylvania, 2008. [32] S.A. Ahmed, R.M.J. Cogal, J.E. Walsch, A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alter- native to [3H] thymidine incorporation assay, J. Immun. Meth. 170 (1994) 211e222. [33] J. O'Brien, I. Wilson, T. Orton, F. Pognan, Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity, Eur. J. Biochem. 267 (2000) 5421e5426. [34] R.G.R. Woisky, A. Salatino, Analysis of propolis: some parameters and pro- cedures for chemical quality control, J. Apic. Res. 37 (1998) 99e105. [35] M. Hajimahmoodi, M.R. Oveisi, N. Sadeghi, B. Jannat, M. Hadjibabaie, E. Farahani, et al., Antioxidant properties of peel and pulp hydro extract in tem Persian pomegranate cultivars, Pak. J. Biol. Sci. 11 (12) (2008) 1600e1604. [36] G.M. Williams, M.J. Iatropoulos, J. Whysner, Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives, J. Food Chem. Toxicol. 37 (9e10) (1999) 1027e1038. [37] H. Shi, N. Noguchi, E. Niky, Natural antioxidants, in: J. Pokorny, N. Yanishlieva, M. Gordon (Eds.), Antioxidants in Food Practical Application, CRC, Cambridge, 2001, pp. 147e148. [38] R.M. Seabra, P.B. Andrade, P. Valent~ao, E. Fernandes, F. Carvalho, M.L. Bastos, Antioxidant compounds extracted from several plant materials, in: M. Fingerman, R. Nagabhushanam (Eds.), Biomaterials from Aquatic and http://refhub.elsevier.com/S0882-4010(15)30123-6/sref1 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref1 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref1 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref1 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref1 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref2 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref3 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref3 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref3 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref4 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref4 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http://refhub.elsevier.com/S0882-4010(15)30123-6/sref24 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref24 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref24 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref25 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref26 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref27 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref27 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref27 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref27 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref28 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref28 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref28 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref28 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref29 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref29 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref29 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref29 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref30 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref30 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref30 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref31 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref31 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref31 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref32 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref33 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref33 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref33 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref33 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref34 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref34 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref34 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref35 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref35 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref35 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref35 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref36 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref36 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref36 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref36 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref36 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref37 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref37 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref37 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref37 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 F.A.S. Politi et al. / Microbial Pathogenesis 95 (2016) 15e2020 Terrestrial Organisms, Science, Enfield (NH) USA, 2006, pp. 115e174. [39] W. Li, Y. Gao, J. Zhao, Q. Wang, Phenolic, flavonoid, and lutein ester content and antioxidant activity of 11 cultivars of Chinese Marigold, J. Agric. Food Chem. 55 (21) (2007) 8478e8484. [40] C.A. Rice-Evans, N.J. Miller, P.G. Bolwell, P.M. Bramley, J.B. Pridham, The relative antioxidant activities of plant-derived polyphenolic flavonoids, Free Radic. Res. 22 (1995) 375e383. [41] V. Dharmagadda, S. Naik, P. Mittal, P. Vasudevan, Larvicidal activity of Tagetes patula essential oil against three mosquito species, Biores. Technol. 96 (2005) 1235e1240. [42] T.P. Cushnie, A.J. Lamb, Antimicrobial activity of flavonoids, Int. J. Antimicrob. Agents 26 (5) (2006) 343e356. [43] B. Lima, M.B. Agüero, J. Zygadlo, A. Tapia, C. Solis, A.R. Aarias, et al., Antimi- crobial activity of extracts, essential oil and metabolites obtained from Tagetes mendocina, J. Chil. Chem. Soc. 54 (2009) 68e72. [44] J.L. Rios, M.C. Recio, Medicinal plants and antimicrobial activity, J. Ethnopharmacol. 100 (2005) 80e84. [45] V.R.E.P. Bittencourt, C.L. Massard, A.F. Lima, Aç~ao do fungo Metarhizium ani- sopliae (Metschnikoff, 1879) Sorokin, 1883, em ovos e larvas do carrapato Boophilus microplus (Canestrini, 1887), Rev. Univ. Rural. 16 (1994) 32e38. [46] V.R.E.P. Bittencourt, S.L.F.S. Peralva, E.C. Viegas, S.B. Alves, Avaliaç~ao dos efeitos do contato de Beauveria bassiana (Bals.) Vuill. com ovos e larvas de Boophilus microplus (Canestrini, 1887) (Acari: Ixodidae), Rev. Bras. Parasitol. Vet. 5 (1996) 81e84. [47] M.V. Garcia, A.C. Monteiro, M.P.J. Szab�o, Colonizaç~ao e les~ao em fêmeas ingurgitadas do carrapato Rhipicephalus sanguineus causadas pelo fungo Metarhizium anisopliae, Cienc. Rural. 34 (5) (2004) 1513e1518. http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref38 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref39 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref39 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref39 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref39 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref40 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref40 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref40 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref40 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref41 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref41 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref41 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref41 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref42 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref42 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref42 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref43 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref43 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref43 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref43 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref44 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref44 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref44 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref45 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref45 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref45 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref45 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref45 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref46 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 http://refhub.elsevier.com/S0882-4010(15)30123-6/sref47 Antifungal, antiradical and cytotoxic activities of extractives obtained from Tagetes patula L. (Asteraceae), a potential a ... 1. Introduction 2. Material and methods 2.1. Plant material 2.2. Test microorganisms 2.3. Extracts preparation 2.4. Determination of total flavonoids content 2.5. Antiradical potential 2.6. Antimicrobial activity 2.7. Cytotoxicity assay 2.8. Statistical analysis 3. Results 4. Discussion 5. Conclusions Acknowledgments Glossary References