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

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South African Journal of Botany

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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, Brazilb 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, Brazilc Departamento de Ciência Biológica, Faculdade de Ciências, Universidade Estadual Paulista (UNESP), CEP 17033–360, Bauru, São Paulo, Brazild Fundação Educacional do Município de Assis (FEMA), Assis, São Paulo, Brazil

⁎ Corresponding author. Tel./fax: +55 18 33025848.E-mail address: regildos@yahoo.com.br (R.M.G. Silva).

http://dx.doi.org/10.1016/j.sajb.2014.10.0020254-6299/© 2014 SAAB. Published by Elsevier B.V. All ri

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 July 2014Received in revised form 20 August 2014Accepted 21 October 2014Available online xxxx

Edited by L Verschaeve

Keywords:PhytotoxicAllelopathyAllelochemicalsHPLC -PADAllium cepaDPPH

Among the numerous plant species occurring in the Cerrado, Ouratea spectabilis stands out because of the lack ofspecies that growbeneath its canopy. Therefore, this study aimed to evaluate the phytotoxic potential of differentextracts and fractions of the hydroethanolic extract from leaves of O. spectabilis through laboratory bioassays ofthe pre-and post-emergence of seeds of Lactuca sativa L., determination of themitotic index in root cells of Alliumcepa L., antioxidant activity and phytochemical screening of different classes present in extracts and ethyl acetatefractions. It was possible to verify that different extracts and ethyl acetate fractions ofO. spectabilis interferedwithgermination rates, as reduced germinationwas observedwhen comparedwith the control. Similarly, growth anddevelopment was affected in lettuce seedlings, as shown by the reduced length of primary roots and hypocotylscompared with the control. In addition, the mitotic index was reduced in treated groups compared with thenegative control. HPLC-PAD analysis for both the hydroethanolic extract and its ethyl acetate fraction, showeda predominance of flavonoid compounds belonging to the groups of isoflavones and catechins in ethyl acetatefractions of hydroethanolic extracts. Thus, it was concluded that this species synthesizes phytotoxic compoundscapable 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 agri-culture, centered on concerns of the adverse effects and extensive useof synthetic chemicals, such as cultivars with increased resistance toherbicides and soil and water contamination (Jinhu et al., 2012; Tigreet al., 2012). Thus, the interest in alternative compoundswith phytotoxicproperties has grown in recent decades, providing a promising fieldfor the discovery of pesticides of natural origin that act directly onweeds and, most importantly, do not impose adverse effects on theenvironment or human health (Alves et al., 2003; Haig et al., 2009).

Allelopathy is a phytotoxic phenomenon that is observed in naturein various biomes. Such ecological interaction can be defined as theinfluence of a body over another that may or may not favor the targetorganism; it can occur directly or indirectly, and these interactions aremediated by biomolecules called allelochemicals (Rice, 1984; Rizviand Rizvi, 1992; Ferreira and Aqüila, 2000; Silva and Aqüila, 2006;Inderjit et al., 2011). In the Cerrado, allelopathy is responsible for

ghts reserved.

inter- and intraspecific interactions in the stabilization and mainte-nance of different forms of life in this particular biome (Jeronimoet al., 2005; Aires et al., 2005).

Among numerous plant species that occur in the Cerrado, Ourateaspectabilis (Mart. ex Engl.) Engl, the Ochnaceae family is popularlyknown as "sawblade leaves ", and it stands out in popular accounts ashaving a peculiar ecological characteristic due to of the lack of develop-ment of other plant species beneath its canopy. This species is a decidu-ous plant that is heliophytic and is indifferent to soil conditions, and itoccurs in the Cerrado biomes and Cerrado fields of Brazil. In folk medi-cine, it is used to treat gastric and rheumatic disorders (Paulo et al.,1986). Phytochemical research of extracts of this species revealed thepresence of bigenkanina andmethoxyflavone, demonstrating its poten-tial in the production of secondary metabolites (Felício et al., 1995).

Given the possible synthetic capacity of allelochemical compoundsand ecological characteristics of this species, the present study aimedto evaluate the phytotoxicity of different extracts and fractions ofhydroethanolic extracts from leaves of O. spectabilis through laboratorybioassays, including analysis of pre- and post-emergence of seeds ofLactuca sativa L., determination of the mitotic index in root cells ofAllium cepa L., antioxidant activity and phytochemical screening of ex-tracts and ethyl acetate fractions.

175G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180

2. Material and methods

2.1. Plant material and preparation of extracts

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

2.2. Bioassay of Allelopathy for Pre-emergence

The pre-emergence bioassay was conducted with seeds of Lactucasativa L. cv. Grand Rapids (lettuce) by controlling the germination ofthese plants in Petri dishes (60 mm × 15 mm) and germination paperwith relative humidity, temperature and light artificially controlled ingreenhouses of Germination type BOD (Biological Oxygen Demand)(model: 411/FPD, New Ethics, Brazil). This experiment was set up in acompletely randomized design (CRD), where the Petri dishes weredivided into experimental and control groups containing 50 seeds oflettuce on each plate, with six replicates for each experimental grouptreated with different extracts of O. spectabilis (at concentrations of 5,10 and 20mgmL−1) and a negative control group (water). The protru-sion and geotropic curvature of the radicle was used as germinationcriteria, as indicated by Labouriau (1983). The seeds that showed falsegermination by soaking were not accounted for in the results. The ger-mination of the species was monitored every 6 h over 48 h.

From the resulting data obtained in the assay, different indices werecalculated: germinability or germination percentage ([∑ni/A]•100),germination mean time (Tm = [∑ni•ti]/∑ni), and germinationmean speed (Vm= 1/Tm) in which ni= the number of seeds that ger-minated 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 theobservation 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 pro-posed by Soares and Vieira (2000) and Alves et al. (2004) and adaptedto our laboratory conditions. Lettuce seeds were previously germinatedin Petri dishes lined with germination paper moistened with distilledwater. After 24 h under BOD greenhouse conditions, the seedlings thatshowed an average of 2 mm in length were used in the bioassay,which was set up in a completely randomized design (CRD) with Petridishes containing germination paper moistened with 1 mL of the solu-tion from the different extract concentrations of O. spectabilis. Thesewere divided into experimental and control groups, containing 25 seed-lings on each platewith four replicates per treatment and for the control(water).

The evolution process of the treatmentswere observed and themea-surement of roots and hypocotylswere 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).

2.4. Statistical analysis for Pre and Post-Emergence testing

For statistical treatment of pre and post-emergence tests, normality(Shapiro-Wilks) and homogeneity tests (Levene) were performed. Thedata did not present normality, and its variances were not homoge-neous; therefore, the results were analyzed using the Kruskal-WallisandDunn test (α=0.05)with theuse of BioEstat 5.3 software, accordingto the model proposed by Santana and Ranal (2004).

2.5. Fractionation of the extract

The hydroethanolic crude extract from the leaves of O. spectabiliswas subjected to fractionation because it showed the highest allelo-pathic activity in pre- and post-emergence trials. For this purpose, achromatographic column was fitted with approximately 75% silica and25% Silica Gel 60 (Sigma-Aldrich ®, USA) incorporated with 2.0 g of ex-tract. The sequence of solvents for the elution was n-hexane, dichloro-methane, ethyl acetate, ethyl acetate: methanol (70:30), ethyl acetate:methanol (50:50), ethyl acetate: methanol (30:70) and methanol.Changes in solventswere heldwhenever the fraction remainedwithoutevidence of separation. Filtered fractions were concentrated on a rotaryevaporator at 40±2 °C. Then, theywere subjected to bioassays for bothpre-and post-emergence.

2.6. Determination of osmotic potential, pH and electrical conductivity

The osmotic potential was determined according to the techniquedescribed by Villela et al. (1991). The treatment was evaluated by os-motic solutions obtained using polyethylene glycol 6000 (PEG 6000)in the amounts indicated to establish the osmotic potential of −0.02to−1.0MPa. The values of osmotic potential obtained in PEG6000 solu-tions were compared with the values found in the different concentra-tions of the extracts of O. spectabilis.

ThepH from the different extract concentrations and fraction of ethylacetate of O. spectabilis was determined using a pH meter (Tecnopon ®model: MPA210). Similarly, the electrical conductivity was measuredwith 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 Petridishes. Once the roots of the seedlings reached 1 cm in length, theywere exposed to extracts at concentrations that showed greater activityin the pre-and post-emergence experiments for a period of 48 h. After a48-h period, the rootswere replaced into a Petridish containing distilledwater until they reached an average length of 5 cm (recovery period).The entire experiment was conducted in a greenhouse germinationtype BOD. The roots were fixed in Carnoy (absolute ethyl alcohol andglacial acetic acid, 3:1). For assembly and analysis, the rootswere hydro-lyzed in hydrochloric acid (HCl) 1 N at 60 °C for 8 min and then theywere stained with Schiff Reactive for 2 h protected from light. Theroots were placed on slides and a drop of 2% acetic Carmine wasadded, covered with coverslips and were crushed and fixed. Analysesof 5000 cells/treatment was performed using an optical microscope(100x). Phytotoxic effects of the extracts were determined by analysisof the mitotic index (the total number of dividing cells divided by thetotal number of cells analyzed, multiplied by 100). Statistical analysisof the results from theA. cepa assaywas submitted to thenonparametrictests: Kruskal-Wallis and Mann–Whitney (analysis significance level of5 % 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 fractionwas determined by the H + donor ability to the stable radical 1,1-

176 G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180

diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA), according to the in vitromethodology proposed by Blois (1958). The experimentwas performedin triplicate using a solution of 1 mL of acetate buffer (pH 5.5 and100 mM), 1.25 mL of ethanol P.A., 250 μL of DPPH solution and 50 mLof samples. The extract reacted with DPPH radical for a period of30min under low light andwas then subjected to a UV–vis spectropho-tometer (Femto-600 Plus) at a wavelength of 517 nm (Brand-Williamset al., 1995). The calculation of the antioxidant activity was performedaccording to the formula: antioxidant activity (%) = [(control-sam-ple)/control]x100. The antioxidant activity of the extract can be seenby the degree of discoloration of the reagent after the 30 min requiredfor the reaction to attain a plateau, beyond the low IC50 value, whichmeans 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 performanceliquid chromatography (analytical, quaternary gradient) model: PU-2089S Plus (Jasco®) coupled to a photo diode array detector with scanrange of 200 to 900 nm,model:MD-2015 Plus (Jasco®), automatic injec-tor 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 usedduring the acquisition and processing of chromatographic data. Animmobilized reverse phase column with octadecylsilane was used,model: Luna C18 (2) 100A (Phenomenex®) of 250x4.6 mm i.d, with anaverage particle size of 5 μM with guard column (Phenomenex®)4x3 mm i.d. An aliquot of 10 mg from the hydroethanolic extract andethyl acetate fraction were dissolved in 1 mL of 100% ACN and filteredwith a syringe filter with a pore size of 0.45 microns. A PDA detector inthe range of 200 to 600 nmwas used to monitor the samples. The chro-matogram was obtained at 254 nm. Mobile phase: Acetonitrile +0,1%Formic Acid (A) and Water +0.1% Formic Acid. Gradient: 10-30% of Ain B in 60 min.

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 aqueousextract significantly reduced the germination rate after treatment witha 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 signifi-cant difference between the two concentrations; however, there wasa significant difference when compared with seeds treated with5 mg mL−1 and the control group (Table 1). There were no significantdifferences between the 5mgmL−1 treatment and the control (Table 1).

Table 1Effects of different concentrations of aqueous, ethanolic and hydroethanolic extracts ofO. spectabimean ± standard deviation. Means with the same letter in the column do not differ by Dunn's teand Vm = germination average speed.

Tratament Extract(mg mL−1)

G ± DP (%) Tm ± DP (ho

Water - 99.33 ± 01.03a 14.91 ± 0.79a5 96.33 ± 02.33a 20.51 ± 1.73a

Aqueous 10 93.33 ± 04.67a 33.06 ± 4.33b20 46.66 ± 18.40b 43.12 ± 2.21b5 97.33 ± 02.73a 19.42 ± 2.02a

Hydroethanolic 10 97.00 ± 03.74a 22.32 ± 1.42b20 77.33 ± 09.26b 30.38 ± 2.23c5 98.66 ± 01.63a 17.37 ± 1.51a

Ethanolic 10 97.66 ± 02.94a 22.03 ± 1.50b20 90.00 ± 03.34b 28.69 ± 2.70b

For tests with hydroethanolic and ethanolic extracts, a similar pat-tern was observed. For concentrations of 5 and 10 mgmL−1, significantdifferences were observed when compared with those treated with20 mg mL−1, which showed a reduction in the germination rate(hydroethanolic = 22.67% and ethanolic = 10.00%) and were signifi-cantly different from the control (99.33%). For the average time and av-erage speed of germination, seeds treated with 5 mg mL−1 of ethanolextract were significantly different compared with those treated with10 and 20 mg mL−1. Treatments of 10 and 20 mg mL−1 did not differbetween them but were significantly different when compared withthe control. Three concentrations showed to be significantly differentin the hydroethanolic extracts comparedwith the control, where a con-centration of 5 mgmL−1 was the only concentration that did not differfrom control (Table 1).

For post-emergence assays, the different concentrations from aque-ous, hydroethanolic and ethanol extracts were significantly differentfrom each other and were also significantly different when comparedwith the control in relation to the average length of the radicle. Inrelation to hypocotyl length, different concentrations of aqueous andethanol extract statistically differed when compared with the control,which was not observed for the hydroethanolic extract (Table 1).

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

Table 2 shows the test results of the pre-emergence of seedlingstreated with fractions of the hydroethanolic extract. For seeds treatedwith different fractions of 5 mg mL−1 each, it was found that only thetreatmentwith the ethyl acetate fraction showed a significant reductionin germination rate (66%), differing significantly from the otherfractions and the control (98%). Indices of average time and averagespeed of germination exhibited a similar pattern for the ethyl acetatefraction treatment, which was significantly different from the wateronly control.

As for measuring the mean root length of seedlings treated with1 mg mL−1 of different fractions of the hydroethanolic extract over48 h of exposure, significant differences were observedwhen comparedwith the control, however they do not show any significant differencebetween them. For themean hypocotyl length, no significant differencebetween the different factions or between them and the water controlwas observed (Table 3).

3.3. pH, osmotic potential and electrical conductivity of extractsand fractions

The physicochemical characterization of different organic extracts andthe ethyl acetate fraction from hydroethanolic extracts of O. spectabilisrevealed a pH range between 3.67 and 5.09. Thewater used in the controlgroups showed pH of 6.06.

lis for seed germination and seedling growth of Lactuca sativa (lettuce).Data are presented asst (α=0.05). Legend: G% = germinationmean percentage, Tm = germination mean time

urs) Vm ± DP (seeds/h) Radicle(mm)

Hypocotyl(mm)

0.067 ± 0.0037a 11.75 ± 3.59a 2.88 ± 0.44a0.049 ± 0.0040a 05.73 ± 1.41b 2.68 ± 0.58b0.030 ± 0.0044b 04.14 ± 1.14c 2.37 ± 0.53c0.023 ± 0.0012b 03.09 ± 1.36d 2.39 ± 2.08c0.051 ± 0.0054a 05.47 ± 1.68b 3.03 ± 0.89b0.044 ± 0.0027b 03.20 ± 1.06c 2.86 ± 0.61a0.033 ± 0.0025c 02.45 ± 0.89d 2.88 ± 0.71a0.057 ± 0.0053a 03.32 ± 1.38b 1.53 ± 0.80b0.045 ± 0.0031b 03.28 ± 0.73b 2.54 ± 0.56c0.035 ± 0.0035b 02.47 ± 0.32c 2.03 ± 0.47d

Table 2Effects of different fractions (Ethyl Acetate, Ethyl Acetate/Methanol 70%/30%, Ethyl Acetate/Methanol 50%/50%, Ethyl Acetate/Methanol 30%/70% and Methanol) from hydroethanolicextract (5 mg mL−1) on germination of Lactuca sativa (lettuce).

Tratament G ± DP (%) Tm ± DP (hours) Vm ± DP (seeds/h)

Water 98.00 ± 02.00a 17.37 ± 0.76a 0.057 ± 0.0026aAE 34.00 ± 10.58b 41.95 ± 2.67b 0.023 ± 0.0016bAE/Me(70/30) 96.00 ± 02.00a 19.66 ± 0.34a 0.050 ± 0.0009aAE/Me(50/50) 96.00 ± 02.00a 18.45 ± 0.50a 0.054 ± 0.0015aAE/Me(30/70) 94.66 ± 04.16a 18.47 ± 0.63a 0.054 ± 0.0019aMe 99.33 ± 01.15a 19.12 ± 0.72a 0.052 ± 0.0020a

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

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

Tratament Extract(mg mL−1)

pH OsmoticPotential(MPa)

ElectricConductivity(mS cm−1)

5 5.09 −0.0004 0.336Aqueous 10 5.06 −0.0081 0.490

20 5.01 −0.0284 0.5505 4.67 −0.0004 0.166

Hydroethanolic 10 4.58 −0.0088 0.30820 4.50 −0.0250 0.5645 4.06 −0.0018 0.081

Ethanolic 10 3.83 −0.0109 0.14220 3.67 −0.0299 0.207

Ethyl Acetate Fraction 5 4.83 −0.0004 0.029Water - 6.06 0.0 0.004

177G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180

The osmotic potential ranged from−0.0004 to−0.0299MPa for thedifferent extracts and fractions. The average values of electrical conduc-tivity ranged from 0.029 to 0.564mS cm−1 for the different extracts andfractions (Table 4).

3.4. Mitotic index of root meristem cells of Allium cepa

The mitotic index of root meristem cells of Allium cepa treated withthe aqueous and hydroethanolic extracts ofO. spectabiliswith a concen-tration of 20 mg mL−1 was 06.96 and 07.08, respectively, and wassignificantly different from the negative (14.52) and positive controlMMS (9.74), but did not show significant differences when comparedbetween them. Treatment of the ethanol extract at this concentrationcould not be performed due to recurrent necrosis of the taproot followedby the emergence of adventitious roots (Table 5).

The number of cells in prophase, metaphase, anaphase or telophaseas a result of treatment of the different extracts at a concentration of20 mg mL−1 was significantly reduced compared with the negativecontrol, and no significant differences were observed when comparedwith the positive control or between treatments (Table 5).

3.5. Antioxidant Activity

The antioxidant activity became progressively greater with increas-ing concentrations for different aqueous, hydroethanolic and ethanolextracts and ethyl acetate fractions of the hydroethanolic extract.The highest antioxidant activity was observed for concentrations of1000 μgmL−1 presenting 88.91%, 86.45%, 84.50% and 79.28%, respective-ly, and an EC50% of 282.99 μg mL−1, 148.24 μg mL−1, 145.33 μg mL−1, e512.35 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 ofO. spectabilis andethyl acetate fractions (Fig. 1A andB) showed chromato-graphic profiles ofmetabolites when using a C18 column (250x4.6mm idof particles with average size of 5 μm). With the aid of the PAD detectorperforming scanning in the spectral range of 200 to 600 nm, peaks were

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

Tratament Radicle(mm)

Hypocotyl(mm)

Water 20.30 ± 6.76a 3.36 ± 0.74aAE 15.13 ± 3.33b 3.80 ± 1.46aAE/Me(70/30) 15.93 ± 3.61b 3.25 ± 0.79aAE/Me(50/50) 15.83 ± 5.05b 3.79 ± 0.78aAE/Me(30/70) 12.29 ± 3.33b 3.08 ± 0.53aMe 13.45 ± 4.05b 3.39 ± 0.73a

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

obtained in the UV spectra where the typical absorption of flavonoidsoccurs (Fig. 2A), which are recognized by the present Band II area wasobserved with a maximum absorbance in the spectral range of 240 to290 nm, the A-ring and band Iwas assignedwith amaximumabsorbancein the spectral range of 300 to 390nm, the B-ringwas assigned to a higherincidence of molecules from flavones and flavonols, among these thepresence of flavonoid glycosides and metabolites was identified, possiblybelonging to the group of isoflavones and catechins (Fig. 2B and C).

4. Discussion

Cerrado plants are exposed to high temperatures, nutritionally poorand acidic soils and intense competition for nutrients. These factorsstimulate the production of bioactive compounds that influence differ-ent interactions within this environment (Klink and Machado, 2005;Fine et al., 2006; Haridasan, 2008). O. spectabilis, a characteristic speciesfrom Cerrado, has been highlighted by the fact of its capacity to restrictthe development of other species around its stem coupled to the factthat there are very few studies on the potential of bioactive compoundsfrom this plant.

Within this context, the test for pre-emergencewas observed for thedifferent extracts at concentrations of 20 mg mL−1 and for the ethylacetate fraction of the hydroethanolic extract in a concentration of5 mgmL−1 and were shown to have significant effects in all indices an-alyzed for the target plant (Tables 1 and 2). These results corroboratestudies by Reigosa et al. (1999), Inderjit and Callaway (2003) andBlanco (2007), which highlighted the potential that the allelochemicalspresent can cause changes in several physiological processes, includinggermination, and can be directly related to the concentration and theexposure time to allelochemicals.

For the tests of post-emergence, significant reductions were ob-served in seedling development, as evidenced by root and hypocotylgrowth inhibition. Only treatmentwith hydroethanolic extracts showedno inhibition of hypocotyl growth (Tables 1 and 3). Ferreira and Aqüila(2000) demonstrated that allelochemicals could influence variousaspects of seedling development and that possible effects of specificorgans should also be evaluated. Golisz et al. (2008) observed thatsome allelochemicals induce an increased production of reactive oxygenspecies, which can cause death of root cells, thus reducing their growth.

The factors of pH, electrical conductivity and osmotic potential of thetested extracts and fractions were also evaluated in this study becausewhen these factors are altered, they can interfere with fundamentalcellular processes, causing changes in seed germination and seedlingdevelopment that could be misinterpreted as a possible phytotoxiceffect. However, different organic extracts and ethyl acetate fractionsshowed 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 demonstrat-ed by Baskin and Baskin (1998) and Carmo et al. (2007). Electrical con-ductivity was within that established by Souza et al. (2003), who found

Table 5Mitotic Index of rootmeristem cells ofAllium cepa treatedwith the (hydroethanolic andAqueous) extract ofO. spectabilis in the concentration of 20 mg mL−1, negative control (NC) treated aswater and positive control treated with 0.0077 μl mL−1 of metilmetanosulfanado (MMS).

Tratament Cell Division Mitotic Indexa

Interphase Prophase Metaphase Anaphase Telophase

CN 3387a 386a 70a 39a 86a 14.52 ± 02.17aHydroethanolic 4588b 222b 46b 19b 61b 06.96 ± 01.45cAqueous 4595b 217b 46b 33ba 58b 07.08 ± 01.69cMMS 4353b 322b 65b 23b 77b 09.74 ± 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 -Wallistest ( p b0.05).

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that values below 20 mS cm−1 are not harmful to the germination oflettuce seeds. Observations of the osmotic potential gave valueranges from−0.0004 to−0.0299 MPa of different extracts and frac-tions and did not exceed−0.2 MPa, as reported by Gatti et al. (2004)(Table 4).

Regarding the mitotic index, a mitodepressive effect was observedfor treatments with the aqueous and hydroethanolic extracts, and thereduction observed for these treatmentswas greater than that observedfor the positive control (MMS), possibly being related to the presence ofphytotoxic compounds. Treatment with ethanolic extracts at a concen-tration of 20mgmL−1 caused necrosis in primary roots of seedlings ex-posed, thus suggesting an effective phytotoxic effect on the root tissue(Table 5). In this sense, it is known that some allelochemicals have theability to control the production and accumulation of reactive oxygenspecies (ROS),which accumulates in cells in response to allelochemicalsthat cause cellular damage such as lipid peroxidation, thereby alteringthe membrane permeability and hence leading to cell death (Testa,1995; Mori and Schroeder, 2004; Weir et al., 2004). The data obtainedin this test were similar to those found by Silva et al. (2012) andPawlowski et al. (2013).

Another factor that was evaluated was the antioxidant activity ofdifferent extracts and the ethyl acetate fraction of hydroethanolicextracts, verifying an increase in the concentration-dependentactivity, with the highest activities observed at a concentration of1000 μg mL−1. Huckelhoven and Kogel (2003) demonstrated that dif-ferent allelochemicals with antioxidant potential are not only involvedin the defense mechanisms of plants, but that they can also interferewith the germination and seedling development process.

The analysis of possible compounds involved in the phytotoxic po-tential was performed using HPLC-PAD on both hydroethanolic extractand its ethyl acetate fraction (Fig. 1A and B). A scan was performed inthe UV region in which absorption peaks typical of flavonoids could beobserved (Fig. 2A) which are recognized by presenting the Band II,

Table 6Free radical scavenging activity (DPPH) of organic extracts and ethyl acetate fraction of thehydroethanolic extract of O. spectabilis.

Concentration(μg mL−1)

AqueousExtract

HydroethanolicExtract

EthanolicExtract

Ethyl AcetateFraction

% Antioxidantactivity

% Antioxidantactivity

% Antioxidantactivity

% Antioxidantactivity

25 10.04 09.70 10.56 -50 12.13 18.73 20.89 -75 18.20 25.50 32.15 -100 19.66 34.53 41.31 -250 42.88 61.62 78.63 21.33500 85.56 84.19 84.03 48.741000 88.91 86.45 84.50 79.28EC50% 282.99 148.24 145.33 512.35Quercetin 63.72Gallic Acid 43.80

amg 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, assignedto A-ring and Band I, with maximum spectral range of 300-390 nm,assigned to the B-ring. Being that a higher incidence of moleculesfrom the group of flavones was observed, as shown by spectral bandswith peaks corresponding to band II approximately 240–280 nm andpeaks to corresponding to band I approximately 300–380 nm, thepresence of glycosylated flavonoids can also be identified, accordingto studies by Mabry et al. (1970), Merken and Beecher (2000) andSaldanha (2013). Additionally, peaks were identified at typicalwavelengths corresponding to isoflavones and catechins (260 nmand 278 nm, respectively) (Fig. 2B and C) (Khokhar et al., 1997;Garrett et al., 1999).

Among allelochemical compounds, flavonoids are recognized topossess the ability to cause ion efflux that affectmembrane permeabilityand eventually lead to cell death as demonstrated by Yu et al. (2003).Similarly, Weir et al. (2003) demonstrate the potential of molecules inthe catechins family interfere with seed germination. As Perry et al.(2005) have demonstrated, these compounds have the ability to causevariable effects on different species of plant.

The data obtained from the HPLC-PAD analysis support previousstudies such as Felício et al. (1995), who noted the presence ofbigenkanina and methoxyflavone, two biflavonoids, in extracts ofO. spectabilis. Similarly, Moreira et al. (1994, 1999) identified the pres-ence of flavone and isoflavones in leaves of O. hexasperma. Monacheet al. (1967) isolated and identified of the presence of catechin andproanthocyanidin in a study with Ouratea sp.

The presence of biflavonoids and flavonoids were also observedin 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 ofbiflavonoids makes them useful as chemotaxonomic markers for theOuratea genus.

According to Cantrell et al. (2012), the increasing number of researchapplied to allelopathic activity and identification of allelochemicals indifferent species has assisted in the development of new control strate-gies and new models of natural herbicides tailored to be more specificand less harmful to the environment when compared with syntheticherbicides widely used today. In this sense, the results of this study indi-cated thatO. spectabilis has phytotoxic compounds capable of interferingwith the germination and the growth and development of other species.The predominance of flavonoid compounds was indicated by phyto-chemical characterization of hydroethanolic extracts and ethyl acetatefractions and can be directly related to biological activity observed, andthus having the potential to be used in the development of biologicalherbicides.

Acknowledgments

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

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

179G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180

References

Aires, S.S., Ferreira, A.G., Borghetti, F., 2005. Efeito Alelopatico de folhas e frutos de Sola-num lycocarpum St. Hill. (Solanaceae) na germinação e crescimento de Sesamumindicum L. (Pedaliaceae) em solo sob três temperaturas. Acta Botânica Brasílica 19,339–344.

Alves, C.C.F., Alves, J.M., Silva, T.M.S., Carvalho, M.G., Neto, J., 2003. Atividadealelopática de alcalóides glicosilados de Solanum crinitum Lam. Revista Floresta10, 93–97.

Alves, M.C.S., Filho, S.M., Innecco, R., Torres, S.B., 2004. Alelopatia de extratos voláteis nagerminação de sementes e no comprimento da raiz de alface. Pesquisa AgropecuáriaBrasileira 39, 1083–1086.

180 G.F. Mecina et al. / South African Journal of Botany 95 (2014) 174–180

Baskin, C.C., Baskin, J.M., 1998. Seeds – Ecology, Biogeography and Evolution of Dormancyand Germination. Academic Press, San Diego.

Blanco, J.A., 2007. The representation of allelopathy in ecosystem-level forestmodels.Ecological Modelling 209, 65–77.

Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature181, 1199–1200.

Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a Free Radical Method toEvaluate Antioxidant Activity. LebensmittelWissenschaft und Technologie 28, 25–30.

Cantrell, C.L., Dayan, F.E., Duke, S.O., 2012. Natural products as sources for new pesticides.Journal of Natural Products 75, 1231–1242.

Carmo, F.M.S., Borges, E.E.L., Takaki, M., 2007. Allelopathy of Brazilian sassafras(Ocotea odorifera (Vell.)) Rohwer aqueous extracts. Acta Botânica Brasílica 21,697–705.

Carvalho, M.G., Carvalho, G.J.A., Braz-Filho, R., 2000. Chemical Constituents from Ourateafloribunda: Complete 1H and 13C NMR Assignments of Atranorin and its New AcetylDerivative. Journal of the Brazilian Chemical Society 11, 143–147.

Di Mambro, V.M., Fonseca, M.J.V., 2005. Assays of physical stability and antioxidantactivity of a topical formulation added with different plant extracts. Journal of Phar-maceutical and Biomedical Analysis 37, 287–295.

Estevam, C.S., Oliveira, F.M., Conserva, L.M., Lima, L.F.C.O., Barros, E.C.P., Barros, A.C.P.,Rocha, E.M.M., Andrade, E.H.A., 2005. Constituintes químicos e avaliação preliminarin vivo da atividade antimalárica de Ouratea nitida Aubl (Ochnaceae). RevistaBrasileira de Farmacognosia 15, 195–198.

Felício, J.D., Gonçalez, E., Braggio, M.M., Costantino, L., Albasini, A., Lins, A.P., 1995. Inhibi-tion of lens aldose reductase by biflavones from Ouratea spectabilis. Planta Medica 61,217–220.

Felício, J.D., Rossi, M.H., Braggio, M.M., Gonzalez, E., PAK, A., Cordeiro, I., Felício, R.C., 2004.Chemical constituents from Ouratea parviflora. Biochemical Systematics and Ecology32, 79–81.

Ferreira, A.G., Aqüila, M.E.A., 2000. Alellopathy: an Emerging Topic in Ecophysiology.Revista Brasileira de Fisiologia Vegetal 12, 175–204.

Fine, P.V.A., Miller, Z.J., Mesones, I., Irazuzta, S., Appel, H.M., Stevens, M.H.H., Sääksjärvi, I.,Schultz, J.C., Coley, P.D., 2006. The growth-defense trade-off and habitat specializationby plants in Amazonian forests. Ecology 87, 150–162.

Garrett, S.D., Lee, H.A., Friar, P.M.K., Morgan, M.R.A., 1999. Validation of a novel estrogenreceptor-based microtitration plate assay for the determination of phytoestrogensin soybased foods. Journal of Agricultural and Food Chemistry 47, 4106–4111.

Gatti, A.B., Perez, S.C.J.G.A., Lima, M.I.S., 2004. Allelopathic activity of aqueous extracts ofAristolochia esperanzae O. Kuntze in the germination and growth of Lactuca sativa L.and Raphanus sativus L. Acta Botânica Brasílica 18, 459–472.

Golisz, A., Sugano, M., Fujii, Y., 2008. Microarray expression profiling of Arabidopsisthaliana L. in response to allelochemicals identified in buckwheat. Journal of Experi-mental Botany 59, 3099–3109.

Haig, T.J., Haig, T.J., Seal, A.N., Pratley, J.E., An, M., Wu, H., 2009. Lavender as a source ofnovel plant compounds for the development of a natural herbicide. Journal ofChemical Ecology 35, 1129–1136.

Haridasan, M., 2008. Nutritional adaptations of native plants of the cerrado biome in acidsoils. Brazilian Journal of Plant Physiology 20, 183–195.

Huckelhoven, R., Kogel, K.H., 2003. Reactive oxygen intermediates in plant–microbe inter-actions: who is who in powdery mildew resistance? Planta 216, 891–902.

Inderjit, Callaway, R.M., 2003. Experimental designs for the study of allelopathy. Plant andSoil 256, 1–11.

Inderjit, Wardle, D.A., Karban, R., Callaway, R.M., 2011. The ecosystem andevolutionarycontexts of allelopathy. Trends in Ecology and Evolution 26, 655–662.

Jeronimo, C.A., Borghetti, F., Sá, C.M., 2005. In: Harper, J.D.I., An, M., Kent, J.H., WaggaWagga, N.S.W. (Eds.), Allelopathic effects of Solanum licocarpum leaf extracts onprotein synthesis during the growth of sesame seedlings. International AllelopathySociety, Australia, pp. 473–476.

Jinhu, M., Guofang, X., Wenxiu, Y., Leilei, M., Mei, G., Yuguo, W., Yuanhuai, H., 2012. Inhib-itory effects of leachate from Eupatorium adenophorum on germination and growth ofAmaranthus retroflexus and Chenopodium glaucum. Acta Ecologica Sinica 32, 50–56.

Khokhar, S., Venema, D., Hollman, P.C.H., Dekker, M., Jongen,W., 1997. A RP-HPLCmethodfor the determination of tea catechins. Cancer Letters 114, 171–172.

Klink, C.C., Machado, R.B., 2005. Conservation of the Brazilian cerrado. Conservation Biology19, 707–713.

Labouriau, L.F.G., 1983. A germinação das sementes. Série Biologia, monografia, n. 24Departamento de Assuntos Científicos e Tecnológicos da Secretaria Geral daOrganizaçãodos Estados Americanos, Washington, p. 174.

Leme, D.M., Marin-Morales, M.A., 2009. Allium cepa test in environmental monitoring: Areview on its application. Mutation Research 682, 71–81.

Mabry, T.J., Markham, K.R., Thomas, M.B., 1970. The Systematic Identification of Flavo-noids. Springer-Verlag, New York.

Mbing, J.N., Gueiffier, C.E., Atchadé, A.T., Allouchi, H., Piéboji, J.G., Mbafor, J.T., Tih, R.G.,Pothier, J., Pegnyemb, D.E., Gueiffier, A., 2006. Two biflavonoids from Ourateanigroviolacea. Phytochemistry 67, 2666–2670.

Merken, H.M., Beecher, G.R., 2000. Measurement of food flavonoids by high-performanceliquid chromatography: a review. Journal of Agricultural and Food Chemistry 28 (3),577–599.

Miró, C.P., Ferreira, A.G., Áquila, M.E.A., 1998. Alelopatia de frutos de erva-mate (Ilesxparaguariensis) no desenvolvimento do milho. Pesquisa Agropecuária Brasileira 33,1261–1270.

Monache, F.D., Albuquerque, I.L., Eerrari, F., Bettólo, G.B.M., 1967. A new catechin andadimeric proantrocyanidin from OURATEA SP. Tetrahedron Lettere 439, 4211–4214.

Moreira, I.C., Sobrinho, D.C., De Carvalho, M.G., Braz-Filho, R., 1994. Isoflavone dimershexaspermone A, B and C from Ouratea hexasperma. Phytochemistry 35, 1567–1572.

Moreira, I.C., Carvalhoa, M.G., Bastosa, A.B.F.O., Braz-Filho, R., 1999. A favone dimer fromOuratea hexasperma. Phytochemistry 51, 833–838.

Mori, I.C., Schroeder, J.I., 2004. Reactive oxygen species activation of plant Ca2+ channels.a signaling mechanism in polar growth, hormone transduction, stress signaling, andhypothetically mechanotransduction. Plant Physiology 135, 702–708.

Paulo, M.Q., Lima, E.O., Maia, R.F., Xavier Filho, L., 1986. Atividade antimicrobiana do óleodos frutos de Ouratea parviflora Baill (Ocnaceae) 8. CCS, João Pessoa, p. 1921.

Pawlowski, Â., Kaltchuk-Santos, E., Brasil, M.C., Caramão, E.B., Zini, C.A., Soares, G.L.G.,2013. Chemical composition of Schinus lentiscifolius March. essential oil and its phy-totoxic and cytotoxic effects on lettuce and onion. South African Journal of Botany88, 198–203.

Pereira, R.S., Santana, D.G., Ranal, M.A., 2009. Seedling emergence from newly-collectedand storage seeds of copaifera langsdorffii Desf. (caesalpinoideae), triângulo mineiro,Brazil. Revista Árvore 33, 643–652.

Perry, L.G., Johnson, C., Alford, E.R., Vivanco, J.M., Paschke, M.W., 2005. Screening of grass-land plants for restoration after spotted knapweed invasion. Restoration Ecology 13,725–735.

Procópio, S.O., Santos, J.B., Silva, A.A., Pires, F.R., Ribeiro, J.I.J., Santos, E.A., 2005. Potencialde espécies vegetais para a remediação do herbicida Trifloxysulfuron-Sodium. PlantasDaninhas 23, 9–16.

Reigosa, M.J., Sánchez-Moreiras, A., Gonzáles, L., 1999. Ecophysiological approach inallelopathy. Critical Reviews in Plant Sciences 18, 577–608.

Rice, E.L., 1984. Allelopathy. Academic Press, Orlando, p. 422.Rizvi, S.G.H., Rizvi, V. (Eds.), 1992. Allelopathy: basic and applied aspects. Chapman and

Hall, London, p. 480.Saldanha, L.L., 2013. Prospecção química e avaliação das atividades antioxidante e

alelopática de Myrcia bella CambessTese (Mestrado em Botânica) UniversidadeEstadual Paulista, Instituto de Biociências de Botucatu, p. 161.

Santana, D.G., Ranal, M.A., 2004. Analysis of Germination: A Statistical Approach. UNB,Brasília (247 pp.).

Silva, F.M., Aqüila, M.E.A., 2006. Contribuição ao estudo do potencial alelopático deespécies nativas. Revista Árvore 30, 547–555.

Silva, R.M.G., Livio, A.A., Santos, V.H.M., Mecina, G.F., Silva, L.P., 2012. Allelopathy andphytotoxicity of Zanthoxylum rhoifolium Lam. Allelopathy Journal 30, 221–234.

Soares, G.L.G., Vieira, T.R., 2000. Inibição da germinação e do crescimento radicular dealface (cv. “Grand Rapids”) por extratos aquosos de cinco espécies de Gleicheniaceae.Floresta e Ambiente 7, 180–197.

Souza, L.S., Velini, E.D., Maiomoni-Rodella, R.C.S., 2003. Allelopathic effect of weeds andconcentrations of Brachiaria decumbens on the initial development of eucalyptus(Eucalyptus grandis). Planta Daninha 21, 343–354.

Testa, B., 1995. The Metabolism of Drugs and Other Xenobiotics. Academic Press, New York,p. 475.

Tigre, R.C., Silva, N.H., Santos, M.G., Honda, N.K., Falcão, E.P.S., Pereira, E.C., 2012. Allelo-pathic and bioherbicidal potential of Cladonia verticillaris on the germination andgrowth of Lactuca sativa. Ecotoxicology and Environmental Safety 84, 125–132.

Villela, F.A., Doni Filho, L., Sequeira, E.L., 1991. Tabela de potencial osmótico em função daconcentração de polietileno glicol 6.000 e da temperatura. Pesquisa AgropecuáriaBrasileira 26, 1957–1968.

Weir, T.L., Bais, H.P., Vivanco, J.M., 2003. Intraspecific and interspecific interactionsmediated by a phytotoxin, (S)-catechin, secreted by the roots of Centaurea maculosa(spotted knapweed). Journal of Chemical Ecology 29, 2397–2412.

Weir, T.L., Park, S.W., Vivanco, J.V., 2004. Biochemical and physiological mechanismsmediated by allelochemicals. Current Opinion in Plant Biology 7, 472–479.

Yu, J.Q., Ye, S.F., Zhang, M.F., Hu, W.H., 2003. Effects of root exudates and aqueous rootextracts of cucumber (Cucumis sativus), and allelochemicals on photosynthesis andantioxidant enzymes in cucumber. Biochemical Systematics and Ecology 31, 129–139.

Zintchem, A.A., Atchadé, A.T., Tih, R.G., Mbafor, J.T., Blond, A., Pegnyemb, D.E., Bodo, B.,2007. Flavonoids from Ouratea staudtii Van Tiegh. (ex Keay) (Ochnaceae). BiochemicalSystematics and Ecology 35, 255–256.