Phytotoxicity of extracts and fractions of Ouratea

South African Journal of Botany 95 (2014) 174–180

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Phytotoxicity of extracts and fractions of Ouratea spectabilis (Mart. ex Engl.) Engl. (Ochnaceae) G.F. Mecina a, V.H.M. Santos b, A.L. Dokkedal c, L.L. Saldanha c, L.P. Silva d, R.M.G. Silva a,⁎ a Universidade Estadual Paulista (UNESP), Faculdade de Ciências e Letras de Assis, Departamento de Ciências Biológicas - Laboratório de Fitoterápicos, Avenida Dom Antônio 2100, CEP: 19806–900, Assis, São Paulo, Brazil b Universidade Estadual Paulista (UNESP), Instituto de Biociências de Botucatu, Departamento de Botânica, Fisiologia Vegetal, Distrito de Rubião Jr., s/n°, CEP: 18618-970, Botucatu, São Paulo, Brazil c Departamento de Ciência Biológica, Faculdade de Ciências, Universidade Estadual Paulista (UNESP), CEP 17033–360, Bauru, São Paulo, Brazil d Fundação Educacional do Município de Assis (FEMA), Assis, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 2 July 2014 Received in revised form 20 August 2014 Accepted 21 October 2014 Available online 19 November 2014 Edited by L Verschaeve Keywords: Phytotoxic Allelopathy Allelochemicals HPLC -PAD Allium cepa DPPH

a b s t r a c t Among the numerous plant species occurring in the Cerrado, Ouratea spectabilis stands out because of the lack of species that grow beneath its canopy. Therefore, this study aimed to evaluate the phytotoxic potential of different extracts and fractions of the hydroethanolic extract from leaves of O. spectabilis through laboratory bioassays of the pre-and post-emergence of seeds of Lactuca sativa L., determination of the mitotic index in root cells of Allium cepa L., antioxidant activity and phytochemical screening of different classes present in extracts and ethyl acetate fractions. It was possible to verify that different extracts and ethyl acetate fractions of O. spectabilis interfered with germination rates, as reduced germination was observed when compared with the control. Similarly, growth and development was affected in lettuce seedlings, as shown by the reduced length of primary roots and hypocotyls compared with the control. In addition, the mitotic index was reduced in treated groups compared with the negative control. HPLC-PAD analysis for both the hydroethanolic extract and its ethyl acetate fraction, showed a predominance of flavonoid compounds belonging to the groups of isoflavones and catechins in ethyl acetate fractions of hydroethanolic extracts. Thus, it was concluded that this species synthesizes phytotoxic compounds capable of interfering in the stabilization and development of other species. © 2014 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction Currently, there is a growing effort in the study of sustainable agriculture, centered on concerns of the adverse effects and extensive use of synthetic chemicals, such as cultivars with increased resistance to herbicides and soil and water contamination (Jinhu et al., 2012; Tigre et al., 2012). Thus, the interest in alternative compounds with phytotoxic properties has grown in recent decades, providing a promising field for the discovery of pesticides of natural origin that act directly on weeds and, most importantly, do not impose adverse effects on the environment or human health (Alves et al., 2003; Haig et al., 2009). Allelopathy is a phytotoxic phenomenon that is observed in nature in various biomes. Such ecological interaction can be defined as the influence of a body over another that may or may not favor the target organism; it can occur directly or indirectly, and these interactions are mediated by biomolecules called allelochemicals (Rice, 1984; Rizvi and Rizvi, 1992; Ferreira and Aqüila, 2000; Silva and Aqüila, 2006; Inderjit et al., 2011). In the Cerrado, allelopathy is responsible for

⁎ Corresponding author. Tel./fax: +55 18 33025848. E-mail address: [email protected] (R.M.G. Silva).

http://dx.doi.org/10.1016/j.sajb.2014.10.002 0254-6299/© 2014 SAAB. Published by Elsevier B.V. All rights reserved.

inter- and intraspecific interactions in the stabilization and maintenance of different forms of life in this particular biome (Jeronimo et al., 2005; Aires et al., 2005). Among numerous plant species that occur in the Cerrado, Ouratea spectabilis (Mart. ex Engl.) Engl, the Ochnaceae family is popularly known as "sawblade leaves ", and it stands out in popular accounts as having a peculiar ecological characteristic due to of the lack of development of other plant species beneath its canopy. This species is a deciduous plant that is heliophytic and is indifferent to soil conditions, and it occurs in the Cerrado biomes and Cerrado fields of Brazil. In folk medicine, it is used to treat gastric and rheumatic disorders (Paulo et al., 1986). Phytochemical research of extracts of this species revealed the presence of bigenkanina and methoxyflavone, demonstrating its potential in the production of secondary metabolites (Felício et al., 1995). Given the possible synthetic capacity of allelochemical compounds and ecological characteristics of this species, the present study aimed to evaluate the phytotoxicity of different extracts and fractions of hydroethanolic extracts from leaves of O. spectabilis through laboratory bioassays, including analysis of pre- and post-emergence of seeds of Lactuca sativa L., determination of the mitotic index in root cells of Allium cepa L., antioxidant activity and phytochemical screening of extracts and ethyl acetate fractions.

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2. Material and methods

2.4. Statistical analysis for Pre and Post-Emergence testing

2.1. Plant material and preparation of extracts

For statistical treatment of pre and post-emergence tests, normality (Shapiro-Wilks) and homogeneity tests (Levene) were performed. The data did not present normality, and its variances were not homogeneous; therefore, the results were analyzed using the Kruskal-Wallis and Dunn test (α =0.05) with the use of BioEstat 5.3 software, according to the model proposed by Santana and Ranal (2004).

Ouratea spectabilis leaves were collected from specimens at Universidade Estadual Paulista - SP (22°39'42'' S and 50°24'44" W, altitude: 546 m). A voucher specimen was deposited in the herbarium of the Forestry Institute of São Paulo (register: SPSF70323). For preparation of extracts, the leaves were washed, dried in an oven (40 °C) and sprayed. The aqueous extract was obtained by mechanical agitation in distilled water [1:10 (w/v) for 24 h at 24 °C]. Soon after vacuum filtration, the samples were frozen and lyophilized (model L101, Líotop, Brazil) to obtain the dry extract. The hydroethanolic extract was obtained by mechanical stirring in a solution of ethanol: water (70:30) at a ratio of 1:10 (w/v) for 24 h, and the process was repeated three times with the same plant material. Then, the extract was filtered and rotary evaporated (model MA120, Marconi, Brazil) at 60 °C to remove the ethanol and was subsequently frozen and lyophilized to obtain the dry extract. Similarly, the ethanol extract was obtained by replacing the ethanol:water solution (70:30) for absolute ethanol (Impex, Brazil), being that the dried extract was obtained after concentration on a rotary evaporator followed by drying at room temperature.

2.5. Fractionation of the extract The hydroethanolic crude extract from the leaves of O. spectabilis was subjected to fractionation because it showed the highest allelopathic activity in pre- and post-emergence trials. For this purpose, a chromatographic column was fitted with approximately 75% silica and 25% Silica Gel 60 (Sigma-Aldrich ®, USA) incorporated with 2.0 g of extract. The sequence of solvents for the elution was n-hexane, dichloromethane, ethyl acetate, ethyl acetate: methanol (70:30), ethyl acetate: methanol (50:50), ethyl acetate: methanol (30:70) and methanol. Changes in solvents were held whenever the fraction remained without evidence of separation. Filtered fractions were concentrated on a rotary evaporator at 40 ± 2 °C. Then, they were subjected to bioassays for both pre-and post-emergence.

2.2. Bioassay of Allelopathy for Pre-emergence 2.6. Determination of osmotic potential, pH and electrical conductivity The pre-emergence bioassay was conducted with seeds of Lactuca sativa L. cv. Grand Rapids (lettuce) by controlling the germination of these plants in Petri dishes (60 mm × 15 mm) and germination paper with relative humidity, temperature and light artificially controlled in greenhouses of Germination type BOD (Biological Oxygen Demand) (model: 411/FPD, New Ethics, Brazil). This experiment was set up in a completely randomized design (CRD), where the Petri dishes were divided into experimental and control groups containing 50 seeds of lettuce on each plate, with six replicates for each experimental group treated with different extracts of O. spectabilis (at concentrations of 5, 10 and 20 mg mL−1) and a negative control group (water). The protrusion and geotropic curvature of the radicle was used as germination criteria, as indicated by Labouriau (1983). The seeds that showed false germination by soaking were not accounted for in the results. The germination of the species was monitored every 6 h over 48 h. From the resulting data obtained in the assay, different indices were calculated: germinability or germination percentage ([∑ ni/A]•100), germination mean time (Tm = [∑ ni•ti]/∑ ni), and germination mean speed (Vm = 1/Tm) in which ni = the number of seeds that germinated in each time gap “ti”; A = the total number of seeds in the test; and ti = the time gap between the beginning of the experiment and the observation time (Santana and Ranal, 2004; Pereira et al., 2009). 2.3. Bioassay of Allelopathy for Post-Emergence The bioassay was performed according to the methodology proposed by Soares and Vieira (2000) and Alves et al. (2004) and adapted to our laboratory conditions. Lettuce seeds were previously germinated in Petri dishes lined with germination paper moistened with distilled water. After 24 h under BOD greenhouse conditions, the seedlings that showed an average of 2 mm in length were used in the bioassay, which was set up in a completely randomized design (CRD) with Petri dishes containing germination paper moistened with 1 mL of the solution from the different extract concentrations of O. spectabilis. These were divided into experimental and control groups, containing 25 seedlings on each plate with four replicates per treatment and for the control (water). The evolution process of the treatments were observed and the measurement of roots and hypocotyls were performed using a digital caliper (model: IP65, DIGIMESS®, Brazil) every 24 h up to 48 h of exposure (Miró et al., 1998; Procópio et al., 2005).

The osmotic potential was determined according to the technique described by Villela et al. (1991). The treatment was evaluated by osmotic solutions obtained using polyethylene glycol 6000 (PEG 6000) in the amounts indicated to establish the osmotic potential of − 0.02 to −1.0 MPa. The values of osmotic potential obtained in PEG6000 solutions were compared with the values found in the different concentrations of the extracts of O. spectabilis. The pH from the different extract concentrations and fraction of ethyl acetate of O. spectabilis was determined using a pH meter (Tecnopon ® model: MPA210). Similarly, the electrical conductivity was measured with a conductivity meter (Conductivity Meter Instrutherm ®, model: CD860). 2.7. Mitotic index in root cells of Allium cepa Seeds of Allium cepa (onion) were previously germinated in Petri dishes. Once the roots of the seedlings reached 1 cm in length, they were exposed to extracts at concentrations that showed greater activity in the pre-and post-emergence experiments for a period of 48 h. After a 48-h period, the roots were replaced into a Petri dish containing distilled water until they reached an average length of 5 cm (recovery period). The entire experiment was conducted in a greenhouse germination type BOD. The roots were fixed in Carnoy (absolute ethyl alcohol and glacial acetic acid, 3:1). For assembly and analysis, the roots were hydrolyzed in hydrochloric acid (HCl) 1 N at 60 °C for 8 min and then they were stained with Schiff Reactive for 2 h protected from light. The roots were placed on slides and a drop of 2% acetic Carmine was added, covered with coverslips and were crushed and fixed. Analyses of 5000 cells/treatment was performed using an optical microscope (100x). Phytotoxic effects of the extracts were determined by analysis of the mitotic index (the total number of dividing cells divided by the total number of cells analyzed, multiplied by 100). Statistical analysis of the results from the A. cepa assay was submitted to the nonparametric tests: Kruskal-Wallis and Mann–Whitney (analysis significance level of 5 % and 1%) according to Leme and Marin-Morales (2009). 2.8. Test of the antioxidant activity The antioxidant activity of the extracts and the ethyl acetate fraction was determined by the H + donor ability to the stable radical 1,1-

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For tests with hydroethanolic and ethanolic extracts, a similar pattern was observed. For concentrations of 5 and 10 mg mL−1, significant differences were observed when compared with those treated with 20 mg mL− 1, which showed a reduction in the germination rate (hydroethanolic = 22.67% and ethanolic = 10.00%) and were significantly different from the control (99.33%). For the average time and average speed of germination, seeds treated with 5 mg mL−1 of ethanol extract were significantly different compared with those treated with 10 and 20 mg mL−1. Treatments of 10 and 20 mg mL−1 did not differ between them but were significantly different when compared with the control. Three concentrations showed to be significantly different in the hydroethanolic extracts compared with the control, where a concentration of 5 mg mL−1 was the only concentration that did not differ from control (Table 1). For post-emergence assays, the different concentrations from aqueous, hydroethanolic and ethanol extracts were significantly different from each other and were also significantly different when compared with the control in relation to the average length of the radicle. In relation to hypocotyl length, different concentrations of aqueous and ethanol extract statistically differed when compared with the control, which was not observed for the hydroethanolic extract (Table 1).

diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA), according to the in vitro methodology proposed by Blois (1958). The experiment was performed in triplicate using a solution of 1 mL of acetate buffer (pH 5.5 and 100 mM), 1.25 mL of ethanol P.A., 250 μL of DPPH solution and 50 mL of samples. The extract reacted with DPPH radical for a period of 30 min under low light and was then subjected to a UV–vis spectrophotometer (Femto-600 Plus) at a wavelength of 517 nm (Brand-Williams et al., 1995). The calculation of the antioxidant activity was performed according to the formula: antioxidant activity (%) = [(control-sample)/control]x100. The antioxidant activity of the extract can be seen by the degree of discoloration of the reagent after the 30 min required for the reaction to attain a plateau, beyond the low IC50 value, which means the ability of the extract to inhibit the radical oxidation of 50% (Di Mambro and Fonseca, 2005). Gallic acid (Vetec-Fine Chemicals, Brazil) was used as a standard. 2.9. High Performance Liquid Chromatography (HPLC) Chromatographic separations were performed on high performance liquid chromatography (analytical, quaternary gradient) model: PU2089S Plus (Jasco®) coupled to a photo diode array detector with scan range of 200 to 900 nm, model: MD-2015 Plus (Jasco®), automatic injector model: AS-2055 (Jasco®) with 50 mL loop and column oven model: CO-2060 Plus. The Jasco ChromPass software (version 1.8.1.6) was used during the acquisition and processing of chromatographic data. An immobilized reverse phase column with octadecylsilane was used, model: Luna C18 (2) 100A (Phenomenex®) of 250x4.6 mm i.d, with an average particle size of 5 μM with guard column (Phenomenex®) 4x3 mm i.d. An aliquot of 10 mg from the hydroethanolic extract and ethyl acetate fraction were dissolved in 1 mL of 100% ACN and filtered with a syringe filter with a pore size of 0.45 microns. A PDA detector in the range of 200 to 600 nm was used to monitor the samples. The chromatogram was obtained at 254 nm. Mobile phase: Acetonitrile +0,1% Formic Acid (A) and Water +0.1% Formic Acid. Gradient: 10-30% of A in B in 60 min.

3.2. Test of pre- and post-emergence for the fractions of the hydroethanolic extract of O. spectabilis

3. Results 3.1. Test of pre- and post-emergence with the extracts of O. spectabilis In the pre-emergence trial of Lactuca sativa seeds, the aqueous extract significantly reduced the germination rate after treatment with a concentration of 20 mg mL−1 (53.34%) compared with the control (99.33%). Regarding the average time and average speed of germination, treatments of concentrations of 10 and 20 mg mL−1 showed no significant difference between the two concentrations; however, there was a significant difference when compared with seeds treated with 5 mg mL−1 and the control group (Table 1). There were no significant differences between the 5 mg mL−1 treatment and the control (Table 1).

Table 2 shows the test results of the pre-emergence of seedlings treated with fractions of the hydroethanolic extract. For seeds treated with different fractions of 5 mg mL−1 each, it was found that only the treatment with the ethyl acetate fraction showed a significant reduction in germination rate (66%), differing significantly from the other fractions and the control (98%). Indices of average time and average speed of germination exhibited a similar pattern for the ethyl acetate fraction treatment, which was significantly different from the water only control. As for measuring the mean root length of seedlings treated with 1 mg mL−1 of different fractions of the hydroethanolic extract over 48 h of exposure, significant differences were observed when compared with the control, however they do not show any significant difference between them. For the mean hypocotyl length, no significant difference between the different factions or between them and the water control was observed (Table 3). 3.3. pH, osmotic potential and electrical conductivity of extracts and fractions The physicochemical characterization of different organic extracts and the ethyl acetate fraction from hydroethanolic extracts of O. spectabilis revealed a pH range between 3.67 and 5.09. The water used in the control groups showed pH of 6.06.

Table 1 Effects of different concentrations of aqueous, ethanolic and hydroethanolic extracts of O. spectabilis for seed germination and seedling growth of Lactuca sativa (lettuce).Data are presented as mean ± standard deviation. Means with the same letter in the column do not differ by Dunn's test (α =0.05). Legend: G% = germination mean percentage, Tm = germination mean time and Vm = germination average speed. Tratament

Extract (mg mL−1)

G ± DP (%)

Tm ± DP (hours)

Vm ± DP (seeds/h)

Radicle (mm)

Water

5 10 20 5 10 20 5 10 20

99.33 96.33 93.33 46.66 97.33 97.00 77.33 98.66 97.66 90.00

14.91 20.51 33.06 43.12 19.42 22.32 30.38 17.37 22.03 28.69

0.067 0.049 0.030 0.023 0.051 0.044 0.033 0.057 0.045 0.035

11.75 05.73 04.14 03.09 05.47 03.20 02.45 03.32 03.28 02.47

Aqueous

Hydroethanolic

Ethanolic

± ± ± ± ± ± ± ± ± ±

01.03a 02.33a 04.67a 18.40b 02.73a 03.74a 09.26b 01.63a 02.94a 03.34b

± ± ± ± ± ± ± ± ± ±

0.79a 1.73a 4.33b 2.21b 2.02a 1.42b 2.23c 1.51a 1.50b 2.70b

± ± ± ± ± ± ± ± ± ±

0.0037a 0.0040a 0.0044b 0.0012b 0.0054a 0.0027b 0.0025c 0.0053a 0.0031b 0.0035b

± ± ± ± ± ± ± ± ± ±

Hypocotyl (mm) 3.59a 1.41b 1.14c 1.36d 1.68b 1.06c 0.89d 1.38b 0.73b 0.32c

2.88 2.68 2.37 2.39 3.03 2.86 2.88 1.53 2.54 2.03

± ± ± ± ± ± ± ± ± ±

0.44a 0.58b 0.53c 2.08c 0.89b 0.61a 0.71a 0.80b 0.56c 0.47d

G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180 Table 2 Effects of different fractions (Ethyl Acetate, Ethyl Acetate/Methanol 70%/30%, Ethyl Acetate/ Methanol 50%/50%, Ethyl Acetate/Methanol 30%/70% and Methanol) from hydroethanolic extract (5 mg mL−1) on germination of Lactuca sativa (lettuce). Tratament

G ± DP (%)

Tm ± DP (hours)

Vm ± DP (seeds/h)

Water AE AE/Me(70/30) AE/Me(50/50) AE/Me(30/70) Me

98.00 34.00 96.00 96.00 94.66 99.33

17.37 41.95 19.66 18.45 18.47 19.12

0.057 0.023 0.050 0.054 0.054 0.052

± ± ± ± ± ±

02.00a 10.58b 02.00a 02.00a 04.16a 01.15a

± ± ± ± ± ±

0.76a 2.67b 0.34a 0.50a 0.63a 0.72a

± ± ± ± ± ±

0.0026a 0.0016b 0.0009a 0.0015a 0.0019a 0.0020a

Means with the same letter in the column do not differ by Dunn's test (α =0.05). Legend: G = germination mean percentage, Tm = germination mean time and Vm = germination average speed.

The osmotic potential ranged from −0.0004 to −0.0299 MPa for the different extracts and fractions. The average values of electrical conductivity ranged from 0.029 to 0.564 mS cm−1 for the different extracts and fractions (Table 4). 3.4. Mitotic index of root meristem cells of Allium cepa The mitotic index of root meristem cells of Allium cepa treated with the aqueous and hydroethanolic extracts of O. spectabilis with a concentration of 20 mg mL− 1 was 06.96 and 07.08, respectively, and was significantly different from the negative (14.52) and positive control MMS (9.74), but did not show significant differences when compared between them. Treatment of the ethanol extract at this concentration could not be performed due to recurrent necrosis of the taproot followed by the emergence of adventitious roots (Table 5). The number of cells in prophase, metaphase, anaphase or telophase as a result of treatment of the different extracts at a concentration of 20 mg mL− 1 was significantly reduced compared with the negative control, and no significant differences were observed when compared with the positive control or between treatments (Table 5). 3.5. Antioxidant Activity The antioxidant activity became progressively greater with increasing concentrations for different aqueous, hydroethanolic and ethanol extracts and ethyl acetate fractions of the hydroethanolic extract. The highest antioxidant activity was observed for concentrations of 1000 μg mL−1 presenting 88.91%, 86.45%, 84.50% and 79.28%, respectively, and an EC50% of 282.99 μg mL−1, 148.24 μg mL−1, 145.33 μg mL−1, e 512.35 μg mL−1, respectively (Table 6). 3.6. Analysis by high performance liquid chromatography (HPLC-PAD) The screening by HPLC-PAD of the hydroethanolic extract of O. spectabilis and ethyl acetate fractions (Fig. 1A and B) showed chromatographic profiles of metabolites when using a C18 column (250x4.6 mm id of particles with average size of 5 μm). With the aid of the PAD detector performing scanning in the spectral range of 200 to 600 nm, peaks were

Table 3 Effects of different fractions (Ethyl Acetate, Ethyl Acetate/Methanol 70%/30%, Ethyl Acetate/ Methanol 50%/50%, Ethyl Acetate/Methanol 30%/70% and Methanol) of hydroethanolic extract (1.0 mg mL−1) in seedling growth of Lactuca sativa (lettuce) after 48 hours. Tratament

Radicle (mm)

Water AE AE/Me(70/30) AE/Me(50/50) AE/Me(30/70) Me

20.30 15.13 15.93 15.83 12.29 13.45

± ± ± ± ± ±

Hypocotyl (mm) 6.76a 3.33b 3.61b 5.05b 3.33b 4.05b

3.36 3.80 3.25 3.79 3.08 3.39

± ± ± ± ± ±

0.74a 1.46a 0.79a 0.78a 0.53a 0.73a

Means with the same letter in the column do not differ by Dunn's test (α =0.05).

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Table 4 PH , osmotic potential and electrical conductivity of organic extracts and ethyl acetate fraction from hydroethanolic extract of O. spectabilis. Tratament

Aqueous

Hydroethanolic

Ethanolic Ethyl Acetate Fraction Water

Extract (mg mL−1)

pH

Osmotic Potential (MPa)

Electric Conductivity (mS cm−1)

5 10 20 5 10 20 5 10 20 5 -

5.09 5.06 5.01 4.67 4.58 4.50 4.06 3.83 3.67 4.83 6.06

−0.0004 −0.0081 −0.0284 −0.0004 −0.0088 −0.0250 −0.0018 −0.0109 −0.0299 −0.0004 0.0

0.336 0.490 0.550 0.166 0.308 0.564 0.081 0.142 0.207 0.029 0.004

obtained in the UV spectra where the typical absorption of flavonoids occurs (Fig. 2A), which are recognized by the present Band II area was observed with a maximum absorbance in the spectral range of 240 to 290 nm, the A-ring and band I was assigned with a maximum absorbance in the spectral range of 300 to 390 nm, the B-ring was assigned to a higher incidence of molecules from flavones and flavonols, among these the presence of flavonoid glycosides and metabolites was identified, possibly belonging to the group of isoflavones and catechins (Fig. 2B and C). 4. Discussion Cerrado plants are exposed to high temperatures, nutritionally poor and acidic soils and intense competition for nutrients. These factors stimulate the production of bioactive compounds that influence different interactions within this environment (Klink and Machado, 2005; Fine et al., 2006; Haridasan, 2008). O. spectabilis, a characteristic species from Cerrado, has been highlighted by the fact of its capacity to restrict the development of other species around its stem coupled to the fact that there are very few studies on the potential of bioactive compounds from this plant. Within this context, the test for pre-emergence was observed for the different extracts at concentrations of 20 mg mL−1 and for the ethyl acetate fraction of the hydroethanolic extract in a concentration of 5 mg mL−1 and were shown to have significant effects in all indices analyzed for the target plant (Tables 1 and 2). These results corroborate studies by Reigosa et al. (1999), Inderjit and Callaway (2003) and Blanco (2007), which highlighted the potential that the allelochemicals present can cause changes in several physiological processes, including germination, and can be directly related to the concentration and the exposure time to allelochemicals. For the tests of post-emergence, significant reductions were observed in seedling development, as evidenced by root and hypocotyl growth inhibition. Only treatment with hydroethanolic extracts showed no inhibition of hypocotyl growth (Tables 1 and 3). Ferreira and Aqüila (2000) demonstrated that allelochemicals could influence various aspects of seedling development and that possible effects of specific organs should also be evaluated. Golisz et al. (2008) observed that some allelochemicals induce an increased production of reactive oxygen species, which can cause death of root cells, thus reducing their growth. The factors of pH, electrical conductivity and osmotic potential of the tested extracts and fractions were also evaluated in this study because when these factors are altered, they can interfere with fundamental cellular processes, causing changes in seed germination and seedling development that could be misinterpreted as a possible phytotoxic effect. However, different organic extracts and ethyl acetate fractions showed a variation in pH (3.67 to 5.09) within the range (3.0 to 7.0) that does not influence the lettuce germination process, as demonstrated by Baskin and Baskin (1998) and Carmo et al. (2007). Electrical conductivity was within that established by Souza et al. (2003), who found

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Table 5 Mitotic Index of root meristem cells of Allium cepa treated with the (hydroethanolic and Aqueous) extract of O. spectabilis in the concentration of 20 mg mL−1, negative control (NC) treated as water and positive control treated with 0.0077 μl mL−1 of metilmetanosulfanado (MMS). Tratament

Mitotic Indexa

Cell Division

CN Hydroethanolic Aqueous MMS

Interphase

Prophase

Metaphase

Anaphase

Telophase

3387a 4588b 4595b 4353b

386a 222b 217b 322b

70a 46b 46b 65b

39a 19b 33ba 23b

86a 61b 58b 77b

14.52 06.96 07.08 09.74

± ± ± ±

02.17a 01.45c 01.69c 02.50b

a Mitotic Index = (total number of dividing cells /total number of analysed cells x100), Same letters in columns do not differ statistically averages evaluated with the Kruskal -Wallis test ( p b0.05).

that values below 20 mS cm− 1 are not harmful to the germination of lettuce seeds. Observations of the osmotic potential gave value ranges from − 0.0004 to − 0.0299 MPa of different extracts and fractions and did not exceed − 0.2 MPa, as reported by Gatti et al. (2004) (Table 4). Regarding the mitotic index, a mitodepressive effect was observed for treatments with the aqueous and hydroethanolic extracts, and the reduction observed for these treatments was greater than that observed for the positive control (MMS), possibly being related to the presence of phytotoxic compounds. Treatment with ethanolic extracts at a concentration of 20 mg mL−1 caused necrosis in primary roots of seedlings exposed, thus suggesting an effective phytotoxic effect on the root tissue (Table 5). In this sense, it is known that some allelochemicals have the ability to control the production and accumulation of reactive oxygen species (ROS), which accumulates in cells in response to allelochemicals that cause cellular damage such as lipid peroxidation, thereby altering the membrane permeability and hence leading to cell death (Testa, 1995; Mori and Schroeder, 2004; Weir et al., 2004). The data obtained in this test were similar to those found by Silva et al. (2012) and Pawlowski et al. (2013). Another factor that was evaluated was the antioxidant activity of different extracts and the ethyl acetate fraction of hydroethanolic extracts, verifying an increase in the concentration-dependent activity, with the highest activities observed at a concentration of 1000 μg mL−1. Huckelhoven and Kogel (2003) demonstrated that different allelochemicals with antioxidant potential are not only involved in the defense mechanisms of plants, but that they can also interfere with the germination and seedling development process. The analysis of possible compounds involved in the phytotoxic potential was performed using HPLC-PAD on both hydroethanolic extract and its ethyl acetate fraction (Fig. 1A and B). A scan was performed in the UV region in which absorption peaks typical of flavonoids could be observed (Fig. 2A) which are recognized by presenting the Band II,

Table 6 Free radical scavenging activity (DPPH) of organic extracts and ethyl acetate fraction of the hydroethanolic extract of O. spectabilis. Concentration (μg mL−1)

25 50 75 100 250 500 1000 EC50% Quercetin 63.72 Gallic Acid 43.80 a

Aqueous Extract

Hydroethanolic Extract

Ethanolic Extract

Ethyl Acetate Fraction

% Antioxidant activity

% Antioxidant activity

% Antioxidant % Antioxidant activity activity

10.04 12.13 18.20 19.66 42.88 85.56 88.91 282.99

09.70 18.73 25.50 34.53 61.62 84.19 86.45 148.24

10.56 20.89 32.15 41.31 78.63 84.03 84.50 145.33

21.33 48.74 79.28 512.35

mg of equivalent gallic acid/g of extract, bmg of equivalent quercetin/g of extract.

with a maximum in the spectral range of 240-290 nm can be, assigned to A-ring and Band I, with maximum spectral range of 300-390 nm, assigned to the B-ring. Being that a higher incidence of molecules from the group of flavones was observed, as shown by spectral bands with peaks corresponding to band II approximately 240–280 nm and peaks to corresponding to band I approximately 300–380 nm, the presence of glycosylated flavonoids can also be identified, according to studies by Mabry et al. (1970), Merken and Beecher (2000) and Saldanha (2013). Additionally, peaks were identified at typical wavelengths corresponding to isoflavones and catechins (260 nm and 278 nm, respectively) (Fig. 2B and C) (Khokhar et al., 1997; Garrett et al., 1999). Among allelochemical compounds, flavonoids are recognized to possess the ability to cause ion efflux that affect membrane permeability and eventually lead to cell death as demonstrated by Yu et al. (2003). Similarly, Weir et al. (2003) demonstrate the potential of molecules in the catechins family interfere with seed germination. As Perry et al. (2005) have demonstrated, these compounds have the ability to cause variable effects on different species of plant. The data obtained from the HPLC-PAD analysis support previous studies such as Felício et al. (1995), who noted the presence of bigenkanina and methoxyflavone, two biflavonoids, in extracts of O. spectabilis. Similarly, Moreira et al. (1994, 1999) identified the presence of flavone and isoflavones in leaves of O. hexasperma. Monache et al. (1967) isolated and identified of the presence of catechin and proanthocyanidin in a study with Ouratea sp. The presence of biflavonoids and flavonoids were also observed in other species of this genus, as reported by Carvalho et al. (2000) in prepared extracts of O. staudtii, by Felício et al. (2004) in O. parviflora, by Estevam et al. (2005) in O. floribunda, by Mbing et al. (2006) in O. nigroviolacea and by Zintchem et al. (2007) in O. nitida. Thus, Zintchem et al. (2007) believe that the constant presence of biflavonoids makes them useful as chemotaxonomic markers for the Ouratea genus. According to Cantrell et al. (2012), the increasing number of research applied to allelopathic activity and identification of allelochemicals in different species has assisted in the development of new control strategies and new models of natural herbicides tailored to be more specific and less harmful to the environment when compared with synthetic herbicides widely used today. In this sense, the results of this study indicated that O. spectabilis has phytotoxic compounds capable of interfering with the germination and the growth and development of other species. The predominance of flavonoid compounds was indicated by phytochemical characterization of hydroethanolic extracts and ethyl acetate fractions and can be directly related to biological activity observed, and thus having the potential to be used in the development of biological herbicides.

Acknowledgments The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the scholarship granted.

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Fig. 1. (A) Chromatographic profile of the hydroethanolic extract obtained by HPLC-PAD. (B) Chromatographic profile of the ethyl acetate fraction from the hydroethanolic extract obtained by HPLC -PAD.

Fig. 2. (A) Maximum absorption bands in the UV region illustrated for flavonoids, (B) structure of a possible isoflavone and (C) a possible structure of catechin.

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