Abstract
The flowers of Tagetes patula L., Asteraceae, commonly known as French marigold, are used in folk medicine as an antiseptic, diuretic, blood purifier and insect repellent. This study was 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, ethanol and acetone, as well as mixtures of these solvents, assessed by the total flavonoid content. The extracts were tested for dry residue, radical scavenging activity, chromatographic profile, and larvicidal activity. The acetone extract had the highest total flavonoid content, 25.13 ± 1.02% (4.07%); and the best radical scavenging activity, with IC50of 15.74 μg/ml ± 1.09 (6.92%), but with lower dry residue, 6.62 ± 1.33% (20.10%). The water extracts showed higher levels of dry residue, but lower total flavonoid content and radical scavenging activity than the acetone extract. The positive correlation between the total flavonoid content and radical 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 aegyptimosquitoes.
Tagetes patula L. ; Flavonoids; Statistical mixture design; Simplex–centroid; Larvicidal activity; Aedes aegypti
Introduction
Tagetes patula L., Asteraceae, popularly known as French marigold, originated in Mexico. It is widely used as an ornamental plant and is sold freely in open markets and garden shops (Vasudevan et al., 1997Vasudevan, P., Kashyap, S., Sharma, S., 1997. Tagetes: a multipurpose plant. Bioresour. Technol. 62, 29-35.). In folk medicine the flowers and leaves have been used for their antiseptic, diuretic, depurative and insect repellent activities (Chadha, 1976Chadha, Y.R., 1976. Tagetes Linn. (Compositae). In The Wealth of India. CSIR: New Delhi; p. 109-112.). Chemical studies with flowers and leaves of T. patula identified terpenes (Prakash et al., 2012Prakash, O., Rout, P.K., Chanotiya, C.S., Misra, L.N., 2012. Composition of essential oil, concrete, absolute and SPME analysis of Tagetes patula capitula. Ind. Crops Prod. 37, 195-199.), alkaloids (Faizi and Naz, 2002Faizi, S., Naz, A., 2002. Jafrine, a novel and labile β-carboline alkaloid from the flowers of Tagetes patula. Tetrahedron 58, 6185-6197.), carotenoids (Piccaglia et al., 1998Piccaglia, R., Marotti, M., Grandi, S., 1998. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind. Crops Prod. 8, 45-51.), thiophenes (Szarka et al., 2006Szarka, S.Z., Hethelyi, E., Lemberkovics, E., Kuzovkina, I.N., Banyai, P., Szoke, E., 2006. GC and GC-MS studies on the essential oil and thiophenes from Tagetes patula L. Chromatographia 63, S67-S73.), fatty acids (Deineka et al., 2007Deineka, V.I., Sorokopudov, V.N., Deineka, L.A., Tret’yakov, M.Y., 2007. Flowers of marigold (Tagetes) species as a source of xanthophylls. Pharm. Chem. J. 41, 540-542.), and flavonoids (Guinot et al., 2008Guinot, P., Gargadennec, A., Valette, G., Fruchier, A., Andary, C., 2008. Primary flavonoids in marigold dye: extraction, structure and involvement in the dyeing process. Phytochem. Analysis 19, 46-51.; Faizi et al., 2011aFaizi, S., Dar, A., Siddiqi, H., Naqvi, S., Naz, A., Bano, S., Lubna, 2011a. Bioassay-guided isolation of antioxidant agents with analgesic properties from flowers of Tagetes patula. Pharm.. Biol. 49, 516-525.) as constituents, some of which may elicit the biological activities reported to date; these include insecticidal (Wells et al., 1993Wells, C., Bertsch, W., Perich, M., 1993. Insecticidal volatiles from the marigold plant (genus Tagetes). Effect of species and sample manipulation. Chromatographia 35, 209-215.), nematicidal (Chadha, 1976Chadha, Y.R., 1976. Tagetes Linn. (Compositae). In The Wealth of India. CSIR: New Delhi; p. 109-112.; Buena et al., 2008Buena, A.P., Dıez-Rojo, M.A., Lopez-Perez, J.A., Robertson, L., Escuer, M., Bello, A., 2008. Screening of Tagetes patula L. on different populations of Meloidogyne. Crop Prot. 27, 96-100.), larvicidal (Dharmagadda et al., 2005Dharmagadda, V.S.S., Naik, S.N., Mittal, P.K., Vasudevan, P., 2005. Larvicidal activity of Tagetes patula essential oil against three mosquito species. Bioresour. Technol. 96, 1235-1240.), antifungal (Faizi et al., 2008Faizi, S., Siddiqi, H., Bano, S., Naz, A., Mazhar, K., Nasim, S., Riaz, T., Kamal, S., Ahmad, A., Khan, A.S., 2008. Antibacterial and antifungal activities of different parts of Tagetes patula: preparation of patuletin derivatives. Pharm. Biol. 46, 309-320.), and anti-inflammatory (Yasukawa and Kasahara, 2013Yasukawa, K., Kasahara, Y., 2013. Effects of flavonoids from French Marigold (florets of Tagetes patula L.) on acute inflammation model. Int. J. Inflam. 2013, 1-5.) activities. The flowers have been reported to contain up to 5.5% flavonoids (Munhoz et al., 2012Munhoz, V.M., Longhini, R., Silva, T.A.P., Lonni, A.A.S.G., Souza, J.R.P., Lopes, G.C., Mello, J.C.P., 2012. Estudo farmacognóstico de flores de Tagetes patula L. (Asteraceae). Rev. Fitos 7, 225-230.).
As Piccaglia and collaborators (1998)Piccaglia, R., Marotti, M., Grandi, S., 1998. Lutein and lutein ester content in different types of Tagetes patula and T. erecta. Ind. Crops Prod. 8, 45-51. found, the flowers of T. patula are a rich source of lutein and its esters. For this reason the genus is widely cultivated in Central America as food coloring, which is approved by the European Union (Vasudevan et al., 1997Vasudevan, P., Kashyap, S., Sharma, S., 1997. Tagetes: a multipurpose plant. Bioresour. Technol. 62, 29-35.). However, after carotenoids are extracted, the residue is discarded or only used as animal feed or fertilizer (Gong et al., 2012Gong, Y., Liu, X., He, W.H., Xu, H.G, Yuan, F., Gao, Y.X., 2012. Investigation into the antioxidant activity and chemical composition of alcoholic extracts from defatted marigold (Tagetes erecta L.) residue. Fitoterapia 83, 481-489.). Gong et al. (2012)Gong, Y., Liu, X., He, W.H., Xu, H.G, Yuan, F., Gao, Y.X., 2012. Investigation into the antioxidant activity and chemical composition of alcoholic extracts from defatted marigold (Tagetes erecta L.) residue. Fitoterapia 83, 481-489. observed that this discarded residue contained various compounds of interest, including flavonoids, a class of secondary metabolites with high therapeutic potential, including cardioprotective (Bandy and Akhlaghi, 2008Bandy, B., Akhlaghi, M., 2008. Mechanisms of flavonoid protection against myocardial ischemia-reperfusion injury. J. Molec. Cell. Cardiol. 46, 309-317.), anti-inflammatory (Kim et al., 2004Kim, H.P., Son, K.H., Chang, H.W., Kang, S.S., 2004. Anti-inflammatory plant flavonoids and cellular actions mechanisms. J. Pharmacol. Sci. 96, 229-245.), antimicrobial (Lamb and Cushnie, 2005Lamb, A.J., Cushnie, T.P.T., 2005. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 26, 343-356.), and antitumor (Harborne and Williams, 2000Harborne, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481-504.) activities, among others.
The extraction method and solvent used to obtain a flavonoid-enriched extract can greatly influence the biological activity. A successful extraction technique combines the optimal solvent or mixture of solvents with a convenient technique. In order to select an extraction method it is necessary to evaluate the efficiency, the stability of the extracted substances, the availability of resources and processing costs, leading towards a biological application of the extract (Louzada et al., 2001Louzada, A.O., Padilha, C.D., Ortega, G.G., Petrovick, P.R., 2001. Achyroclines satureioides (Lam.) DC. Asteraceae: Comparative evaluation of the vegetal drug and preliminary optimization studies on extraction. Cad. Farm. 17, 33-38.).
The optimization of the extraction techniques is important for a variety of applications, for example to obtain the selected compounds of pharmaceutical interest, with a minimum of impurities. In recent years, statistical models have been successfully applied to minimize the number of experiments and to identify the effect of the interactions between experimental variables such as the solvent strength, and the interactions that occur between the biological material and the solvents. These models have provided satisfactory results for the optimization of the extraction procedures (Box et al., 2005Box, G.E.P., Hunter, J.S., Hunter, W.G., 2005. Statistics for Experimenters (2nd Ed.). Wiley: Hoboken; 664 p.; Bruns et al., 2006Bruns, R.E., Scarminio, I.S., Barros Neto, B., 2006. Statistical Design: Chemometrics (1st Ed.). Elsevier: Amsterdam; 422 p.). Li and collaborators (2007)Li, J.W., Ding, S.D., Ding, X.L., 2007. Optimization of the ultrasonically assisted extraction of polysaccharides from Zizyphus jujuba cv jinsixiaozao. J. Food Eng. 80, 176-183. used response-surface methodology to assess the yield and purity of the polysaccharides by ultrasound extraction. Their optimized method increased the yield by approximately 20% over the classic extraction method.
The experimental mixture design has been widely applied in various areas, such as the development of new biofilms (Mali et al., 2010Mali, S., Debiagi, F., Grossmann, M.V.E., Yamashita, F., 2010. Starch sugarcane bagasse fibre and polyvinyl alcohol effects on extruded foam properties: A mixture design approach. Ind. Crops Prod. 32, 353-359.) and for the extraction of secondary metabolites from natural products (Garcia et al., 2010Garcia, L.M.Z., Oliveira, T.F., Soares, P.K., Bruns, R.E., Scarminio, I.S., 2010. Statistical mixture design - Principal component determination of synergic solvent interactions for natural product extractions. Chemometr. Intell. Lab. 103, 1-7.; Lonni et al., 2012Lonni, A.A.S.G., Longhini, R., Lopes, G.C., Mello, J.C.P., Scarminio, I.S., 2012. Statistical mixture design selective extraction of compounds with antioxidant activity and total polyphenol content from Trichilia catigua. Anal. Chim. Acta 719, 57-60.; DiCiaula et al., 2014DiCiaula, M.C., Lopes, G.C., Scarminio, I.S., Mello, J.C.P., 2014. Statistical mixture design investigation of total extracts and development of a UV/VIS spectrophotometric method for analysis of total polyphenols from bark of Schinus terebinthifolius. Quim. Nova 37, 158-163.). The main goal of this method is to evaluate how responses are affected by the variation in the proportions of the mixture components (Cornell, 2002Cornell, J.A., 2002. Experiments with mixture: designs models, and the analysis of mixtures data (3rd Ed.). Wiley: New York; 680 p.). In the data analysis, a mathematical polynomial model is used to link the different compound proportions to the properties of interest, in order to determine the best system (Audi et al., 2001Audi, E.A., Campos, E.J.V., Rufino, M., Cortez, D.A.G., Bersani-Amado, C.A., Soares, L.A.L., Petrovick, P.R., Mello, J.C.P., 2001. Petiveria alliacea L.: plant drug quality control, hydroalcoholic extract standardization and pharmacological assay of lyophilized extract. Lat. Am. J. Pharm. 20, 225-232.; Campos et al., 2006Campos, L.F.A., Amorim, L.V., Ferreira, H.C., 2006. Experimental design applied to the study of composition effect of bentonite on the rheology of drilling fluids. Part I: binary compositions. Cerâmica 52, 69-75.).
We used the statistical mixture design with a simplex-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 preliminary evaluation of larvicidal activity against the larvae of the mosquito Aedes aegypti.
Materials and methods
Plant material
Seeds of Tagetes patula L., Asteraceae, were donated by Syngenta Flowers Brazil and were organically grown in the Medicinal Plant Garden of the State University of Londrina, Brazil. The flowers were collected in November 2011, and the material was identified by Professor Dr. Naoki Jimi Nakagima, Federal University of Uberlândia, Brazil. A voucher specimen was deposited in the Herbarium of the State University of Maringá under identification number HUEM 21.907.
The flowers were dried in a kiln with forced-air circulation (Sparrow), heated to 38 ± 2ºC and pulverized in a hammermill (Tigre ASN5).
Chemicals and reagents
All solvents and reagents used were analytical grade. Chemicals used were: n-hexane, acetone and methanol (Synth®), ethanol (Cerealcool®), methanol HPLC grade (J.T. Baker®), DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma Aldrich®), butylated hydroxytoluene (BHT), aluminum chloride and potassium acetate (Synth®), and Quercetin (Acros Organics).
Extraction of flowers: general equipment and conditions
To obtain the extract, the plant material, not separated by particle size, was defatted using n-hexane by dynamic maceration for 6 days. After drying, the defatted flowers were macerated for 10 min and the material was extracted in an industrial blender (Skymsen®, LS-04) in a proportion of 2.5% (m/v) (w/v), for a period of 9 min, with 10-min intervals, and using as the extraction liquid, mixtures of three solvents: (x1) acetone, (x2) ethanol and (x3) water, determined through a simplex-centroid design (Fig. 1). The proportions of the solvents used are specified in Table 1.
Simplex-centroid design with three axial mixture points used to investigate the influence of different solvent proportions on extract preparation.
Mixture composition in extracts with acetone, ethanol and water in a simplex-centroid mixture design.
The extracts were vacuum-filtered, evaporated under reduced pressure (Büchi®, R-200) and lyophilized (Christ®, Alpha 1-4) (CE). All extracts were prepared in triplicate, randomly.
Characterization of the extracts
All assays were performed in three replicates.
Dry residue (DR)
For each extract, 2 ml were measured volumetrically, transferred to pre-weighed stainless-steel dishes and let to evaporate to dryness in a water bath (Fanem®, 120/3). The dishes containing the residue were placed in an infrared moisture analyzer (Ohaus® MB35), at 105°C for 10 min. The 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 mass, thus the results are expressed relative to 100 g of the extraction solution (Anvisa, 2007Anvisa, 2007. Guia de controle de qualidade de produtos cosméticos. Agência Nacional de Vigilância Sanitária (2ndEd.). Brasília; 120 p.).
Determination of total flavonoid content (TFC)
The total flavonoids content (TFC) was determined by the aluminum chloride colorimetric method (Chang et al., 2002Chang, C.C., Yang, M.H., Wen, H.M., Chern, J.C., 2002. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J. Food Drug Anal. 10, 178-182.) with modifications. To 0.5 ml of the sample solution (CEW – 800 µg/ml; other extracts – 400 µg/ml) 1.5 ml ethanol, 0.1 ml of 1 M potassium acetate (w/v), 0.2 ml of 10% aluminum chloride (w/v), and 2.7 ml of distilled water were added. After incubation at room temperature for 30 min, the absorbance of the solution was measured on an OceanOptics USB 2000+ spectrometer at 430 nm. The 10% aluminum chloride was replaced by the same amount of distilled water. Quercetin was used to construct the calibration curve (y = 0.0503× -0.0128), where y is the value for absorbance of the sample and x is the sample concentration, from 3-15 µg/ml (r2 = 0.9944). The total flavonoids content was expressed as a percentage, i.e., flavonoids g/100 g of extract.
Radical-scavenging activity (RSA)
The radical-scavenging ability of the extracts was evaluated using the free radical DPPH scavenging method as described by Amarowicz et al. (2004)Amarowicz, R., Pegg, R.B., Rahimi-Moghaddam, P., Barl, B., Weil, J.A., 2004. Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies. Food Chem. 84, 551-562.. The extracts were diluted in methanol (5- 35 μg/ml for CEA and CEAE; 50-140 μg/ml for CEW; and 10-40 μg/ml for other extracts). Next, 375 μl of DPPH solution (1 mmol/l) was added to each of these solutions. The mixture was vortexed for 15 s and left at room temperature for 30 min. The absorbance was read at 517 nm with an OceanOptics USB 2000+ spectrometer. As a blank, a methanol solution of BHT (0.5 mg/ml) added to 500 μl of DPPH solution was used; and the negative control consisted of a solution containing 3 ml of methanol and 375 μl of DPPH solution. The radical-scavenging activity (RSA) was determined as %RSA = [(AbsCN – AbsA) × 100]/AbsCN, where AbsA is the absorbance of the extract solution; and AbsCN is the negative control absorbance. The IC50 value, or extract concentration necessary to inhibit 50% of DPPH, was obtained by plotting the %RSA as a function of the sample concentration.
Experimental design
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 the special cubic model to describe the response of a ternary mixture (Eq. (1)) (Barros Neto et al., 2010Barros Neto, B., Scarminio, I.S., Bruns, R.E., 2010. Como fazer experimentos: pesquisa e desenvolvimento na ciência e na indústria(4th Ed.). Bookman: Porto Alegre; 414 p.):
Where ŷ is the response of interest predicted by the model; and b1, b2, b3, b12, b13, b23, b123 are the estimated parameters, corresponding to the pure components: acetone (x1), ethanol (x2) and water (x3) and their binary and ternary interactions predicted by the model. Surface charts were constructed.
HPLC analysis
The HPLC analysis was conducted using a liquid chromatograph Thermo Model LC Pump Plus equipped with a Finnigan Surveyor PDA Plus diode array detector and a C18 Gemini Phenomenex HPLC column (250 mm × 4.6 mm), 5 μm particle size, and a guard column (Phenomenex) with a flow rate of 1 ml/min. Each sample was prepared in a 725 μg/ml solution, according to the experimental design, and filtered with a 0.22 μm Millipore® filter. A 10 μl aliquot of this solution was injected into the HPLC column (10 μl loop). The mobile phase contained a mixture of water (phase A), and acetonitrile (phase B), in a gradient system, as follows: 0 min, 5% phase B; 35 min, 58% phase B, followed by a 5-min post-time to re-equilibrate the system with 5% phase B. The temperature was fixed at 25°C. A reference standard (quercetin) was used to identify the major component in the crude extracts. Elution was monitored at 210, 254, and 280 nm.
Bioassay test
The larvicidal activity of the extract was evaluated using laboratory-reared larvae of the dengue vector mosquito Aedes aegypti. Larvae from Ae. aegypti were obtained from a permanent colony maintained at a temperature of 25 ± 2°C and relative humidity of 70 ± 5% kept in the insectary of the Laboratory of General and Medical Entomology, State University of Londrina. The colony was founded with eggs collected on the field, using ovitraps.
The extracts were evaluated in accordance with the protocol of the World Health Organization (WHO) with slight modifications (WHO, 2005WHO, 2005. World Health Organization Guidelines for Laboratory and Field Testing of Mosquito Larvicides. World Health Organization, Geneva.). Twenty-five 4th-instar larvae were transferred to transparent polyethylene pots, 11 cm in diameter and 7 cm deep, containing 150 ml of distilled water. Initially, 100 mg of each extract was solubilized in 1 ml of DMSO and 9 ml of water, and then diluted with distilled water to obtain five concentrations (80, 30, 10, 5, and 1 mg/l) for evaluation. Controls were evaluated with DMSO diluted in the same conditions as the samples, but using only distilled water. The number of dead larvae was recorded after 24 and 48 h of exposure, and the percentage of mortality was calculated. Each extract was tested in triplicate and the assay was repeated twice.
Lethal concentration (LC50) values were determined by probit analysis using SPSS version 12 software. Results with p ≥ 0.05 were considered to be statistically significant.
Results and discussion
The experimental results are presented as the mean ± standard deviation and are shown in Table 2.
The dry residue was determined in order to evaluate the strength of the extraction solvent used on flowers of T. patula. The special cubic model gave a satisfactory value for the determination coefficient (r2 = 0.949), and all coefficients were significant at the 95% confidence level (Eq. (2):
The percentage of dried residue was positively and linearly influenced by water (x3) and ethanol (x2), respectively. Of the linear trials, acetone (x1) resulted in the lowest coefficient; the smallest proportion of dried residue (6.62% w/w) was obtained with acetone alone (Table 2). Among the binary interactions, the presence of acetone reduced the power of extraction of ethanol (x1x2), but did not reduce the power of extraction of water (x1x3). The ternary interaction (x1x2x3) displayed a synergistic effect among the components of the mixture.
The extracts produced with solvent mixtures including water contained the highest levels of dry residue, from 39.25 to 41.57% (w/w) (Fig. 2A). However, the Tukey’s test indicated no significant difference between them.
Response curves of DR (A), TFC (B) and RSA (C) as a function of proportions of acetone (x1), ethanol (x2) and water (x3).
The results clearly indicate that the extraction is affected by the polarity of the solvents. Prewashing the flowers with n-hexane decreased the dry mass of the plant by approximately 14%. Using GC-MS (gas chromatography-mass spectroscopy), Faizi et al. (2011b)Faizi, S., Fayyaz, S., Bano, S., Iqbal, E.Y., Lubna, Siddiqi, H., Naz, A., 2011b. Isolation of nematicidal compounds from Tagetes patula L. yellow flowers: structure-activity relationship studies against cyst nematode Heterodera zeae infective stage larvae. J. Agr. Food Chem. 59, 9080-9093. identified 63 compounds in the nonpolar fractions of T. patula flowers, with long-chain fatty acids, hydrocarbons and thiophenes being the predominant classes of compounds in the petroleum ether fraction. This suggests that the lower efficiency of the acetone is related to the fact that some medium- and low-polarity substances were initially extracted with n-hexane. The predominant positive effect of water and ethanol may be due to the presence of polar substances such as phenolic glycosides.
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 Equation 3, obtained by the special cubic model (r2 = 0.979), which showed that acetone (x1) had a strong effect on the TFC when used either pure or in combination with ethanol (x1x2). The linear effect of acetone (x1) was positive, while water (x3) was less effective (TFC = 4.63% (w/w). The presence of water in the ternary mixture (x1x2x3) decreased the extractive capacity compared to the pure solvents acetone (x1) and ethanol (x2), as shown by a negative coefficient and a TFC of 12.94% (w/w). The binary interaction (x1x3) was not significant (p ≥ 0.05).
Purification with n-hexane increased the selectivity of the solvent, especially acetone, for flavonoids. Thus, acetone was the most appropriate solvent for extracting these compounds from French marigold flowers, possibly because of the predominance of free and/or methoxylated aglycones in the defatted plant material, such as kaempferol, quercetin (Ivancheva and Zdravkova, 1993Ivancheva, S., Zdravkova, M., 1993. Flavonoids in Tagetes patula. Fitoterapia 64, 555-558.), patuletin (Tarpo, 1967Tarpo, E., 1967. Patuletin isolation form Tagetes patula flowers. Farmacia (Bucharest) 15, 685-688.; Guinot et al., 2008Guinot, P., Gargadennec, A., Valette, G., Fruchier, A., Andary, C., 2008. Primary flavonoids in marigold dye: extraction, structure and involvement in the dyeing process. Phytochem. Analysis 19, 46-51.), quercetagetin (Tarpo, 1969Tarpo, E., 1969. Isolation of quercetagetin from Tagetes patula flowers. Farmacia (Bucharest) 17, 739-744.), luteolin and quercetagetin 5-methyl ether (Bhardwaj et al., 1980Bhardwaj, D.K., Bisht, M.S., Uain, S.C., Metha, C.K., Sharma, G.C., 1980. Quercetagetin 5-methyl ether from the petals of Tagetes patula. Phytochemistry 19, 713-714.), which are less soluble in water (Chebil et al., 2007Chebil, L., Humeau, C., Anthoni, J., Dehez, F., Engasser, J.M., Ghoul, M., 2007. Solubility of flavonoids in organic solvents. J. Chem. Eng. Data 52, 1552-1556.). This is probably the reason that the TFC aqueous extract (CEW) gave the lowest result.
Previous reports indicate that water is not an efficient solvent for extracting phenolic compounds, which are generally more soluble in organic solvents less polar than water (Kim and Lee, 2001Kim, D.O., Lee, C.Y., 2001. Extraction and isolation of polyphenolics. Curr. Protoc. Food Anal. Chem. 6, I121-I1212.). Several studies have reported that the extraction efficiency of phenolic compounds, including flavonoids, is increased by the use of a mixture of water and organic solvents such as acetone, methanol and ethanol; whereas the use of pure solvents can reduce the power of extraction (Gong et al., 2012Gong, Y., Liu, X., He, W.H., Xu, H.G, Yuan, F., Gao, Y.X., 2012. Investigation into the antioxidant activity and chemical composition of alcoholic extracts from defatted marigold (Tagetes erecta L.) residue. Fitoterapia 83, 481-489.; Meneses et al., 2013Meneses, N.G.T., Martins, S., Teixeira, J.A., Mussatto, S.I., 2013. Influence of extraction solvents on the recovery of antioxidant phenolic compounds from brewer’s spent grains. Sep. Purif. Technol. 108, 152-158.). As it can be seen in Figure 2B, the addition of ethanol or acetone to water increased the TFC. However, pure acetone gave the best results, probably due to the chemical composition of French marigolds.
Munhoz et al. (2012)Munhoz, V.M., Longhini, R., Silva, T.A.P., Lonni, A.A.S.G., Souza, J.R.P., Lopes, G.C., Mello, J.C.P., 2012. Estudo farmacognóstico de flores de Tagetes patula L. (Asteraceae). Rev. Fitos 7, 225-230. used HPLC to generate a chemical profile for T. patula, and found that quercetin was the major substance in the plant drug. Malwade et al. (2013)Malwade, C.R., Qu, H., Rong, B.G., Christensen, L.P., 2013. Purification of artemisinin from quercetin by anti-solvent crystallization. Front. Chem. Sci. Eng. 7, 72-78. evaluated the solubility of quercetin in four common solvents, and reported that quercetin is soluble in: acetone > ethanol > methanol > acetonitrile, in decreasing order. Those reports are in accordance with the results presented here for TFC.
From the chromatographic profiles of the extracts obtained by the simplex centroid design, it was possible to determine the retention time of quercetin (21.05 min) using the spectroscopic data for the standard, and to analyze the area under the curve. Fig. 3 illustrates the chromatograms obtained for the seven extracts. As can be seen, the chromatographic profiles were not affected by the different solvents used to extract the flavonoids. The evaluation of the area under the quercetin peak revealed the positive effect of acetone and ethanol on the extraction (Fig. 4).
Chromatographic system for the extracts described in the optimized conditions by the simplex-centroid design use.
The evaluation of the radical scavenger activity of the extracts generally revealed a similar behavior to that obtained for flavonoids. The results, described in Table 2, are expressed as IC50. CEA had the lowest IC50, in contrast to CEW. In Equation 4 (special cubic model; r2 = 0.997), among the linear effects, water (x3) had a significant negative effect on the extraction of compounds with antioxidant activity; however, combining water with acetone (x1x3) or ethanol (x2x3) increased the extraction efficiency. As seen in Fig. 2C, the lowest IC50 values were obtained with acetone and its binary mixtures, as well as the mixture of ethanol: water (75:25 v/v).
The phenolic compounds present in plants are known for their antioxidant activity. Flavonoids have this capability mainly because their hydroxyls donate an electron (H+) to radicals as hydroxyl (HO•), superoxide (O2•-), and peroxyl (ROO•), neutralizing them (Harborne and Williams, 2000Harborne, J.B., Williams, C.A., 2000. Advances in flavonoid research since 1992. Phytochemistry 55, 481-504.). Previous studies have shown that the antioxidant properties of plants are highly correlated with the content of total phenols and flavonoids (Gong et al., 2012Gong, Y., Liu, X., He, W.H., Xu, H.G, Yuan, F., Gao, Y.X., 2012. Investigation into the antioxidant activity and chemical composition of alcoholic extracts from defatted marigold (Tagetes erecta L.) residue. Fitoterapia 83, 481-489.; Meneses et al., 2013Meneses, N.G.T., Martins, S., Teixeira, J.A., Mussatto, S.I., 2013. Influence of extraction solvents on the recovery of antioxidant phenolic compounds from brewer’s spent grains. Sep. Purif. Technol. 108, 152-158.), as it was further confirmed in this study (Fig. 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).
The results suggest that flavonoids contribute significantly to the antioxidant activity of the extracts obtained with acetone and acetone:ethanol. However, the extracts derived from binary mixtures containing water also inhibited the DPPH radical, possibly due to the presence of phenolic acids such as gallic, chlorogenic, caffeic, vanillic, p-coumaric, and ferulic acid, among others, which have been found in members of the genus Tagetes (Kaisoon et al., 2012Kaisoon, O., Konczak, I., Siriamornpun, S., 2012. Potential health enhancing properties of edible flowers from Thailand. Food Res. Int. 46, 563-571.).
The mosquito Aedes aegypti is the principal vector of dengue. To date there is no vaccine for dengue fever, and the best procedure to combat the disease is to attack the vector, mainly by eliminating the sites of oviposition and larval development. Currently, mosquito populations are controlled with the use of organophosphate insecticides, in increasingly larger doses, a process that has allowed the selection of resistant mosquito populations (Fontoura et al., 2012Fontoura, N.G., Bellinato, D.F., Valle, D., Lima, J.B.P., 2012. The efficacy of a chitin synthesis inhibitor against field populations of organophosphate-resistant Aedes aegypti in Brazil. Mem. I. Oswaldo Cruz 107, 387-395.). In view of the increasing interest in insecticide development of plant origin as an alternative to chemical insecticides (Cappiello et al., 2012Cappiello, A., Tirillini, B., Famiglini, G., Trufelli, H., Termopoli, V., Flender, C., 2012. Determination of natural pyrethrins by liquid chromatography-electron ionization-mass spectrometry. Phytochem. Analysis 23, 191-196.), this study was undertaken to assess the larvicidal potential of the flavonoid-enriched extracts, obtained by the simplexcentroid statistical model. The bioassay test revealed larvicidal activity in only two extracts. No larval mortality was observed in controls. Of the active extracts, the acetone extract showed a higher larvicidal activity (100%) at a concentration of 1 ppm, followed by the 50% ethanol extract (100%) in a concentration of 10 ppm, both after 48 h. In view of the promising response of the acetone extract, further studies will be conducted towards chemical fractionation, in search of a new bioactive molecule.
Conclusions
The statistical mixture design was successfully applied to obtain an optimized set of extraction conditions to maximize the extraction of flavonoids with antioxidant activity from T. 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.
Acknowledgment
Thanks are due to Dr. Janet W. Reid, JWR Associates, Trumansburg, New York, for English revision and to PPSUS-Fundação Araucária (Process #20576/2010), Capes, Finep, CNPq, INCT_if, Pronex for financial support.
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Publication Dates
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Publication in this collection
Sep-Oct 2014
History
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Received
06 June 2014 -
Accepted
02 Oct 2014