Allelopathic effect of the peel of coffee fruit

Scientia Horticulturae 158 (2013) 39–44

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Allelopathic effect of the peel of coffee fruit Regildo M.G. Silva a,∗ , João G.F. Brigatti a , Valter H.M. Santos b , Gustavo F. Mecina a , Luciana P. Silva a a Universidade Estadual Paulista (UNESP), Departamento de Ciências Biológicas – Laboratório de Fitoterápicos, Faculdade de Ciências e Letras de Assis, Avenida Dom Antônio 2100, CEP 19806-900, Assis, São Paulo, Brazil b Universidade Estadual Paulista (UNESP), Instituto de Biociência, Departamento de Botânica, Distrito de Rubião Jr., s/n◦ , CEP 18618-970, Botucatu, São Paulo, Brazil

a r t i c l e

i n f o

Article history: Received 13 July 2012 Received in revised form 24 April 2013 Accepted 25 April 2013 Keywords: Germination Allelopathy Phenolic compounds Mitotic index Antioxidant

a b s t r a c t In coffee (Coffea arabica)-producing areas, particularly in the southeastern region of Brazil, it is part of the agricultural practice to incorporate coffee fruit peels in organic substrates for the production of vegetables, fruit trees, and even in the coffee cultures, for use not only as an organic amendment but also as a way to control weeds. This study aimed to evaluate the allelopathic potential of dry and fresh coffee fruit peel extracts. Therefore, lettuce, Malaysian cabbage and beggar’s tick seeds and seedlings were used as test subjects for the pre-emergence, post-emergence, and mitotic index of meristematic root cell tests. Additionally, the extracts’ contents of phenols, flavonoids and caffeine, in addition to their antioxidant activity, were determined. The development of all the tested seedlings was inferred by the extracts from their roots and hypocotyls. The mitotic index was reduced in comparison to the negative control. A considerable quantity of phenols, flavonoids and caffeine was found in both of the extracts. A progressively growing antioxidant activity of the extracts was observed as their concentrations increased. Through the results obtained in this study, it is possible to conclude that C. arabica has allelopathic compounds. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The maintenance and management of culture residues is an essential part of the no-tillage system, as it guarantees a higher protection against the leaching of nutrients, herbicides and other compounds and lowers the action of the erosive process (Francis and Knight, 1993; Roldán et al., 2003; Schuller et al., 2006). This technique is employed in a large number of crops, such as sugar cane, corn and jack-o-lantern pumpkin (Walters and Young, 2010), in which the cover may interfere with the soil’s chemical, physical and biological properties; decrease the volume of herbicide used; and optimize the conditions for a better yield of plant physiological processes (Sharratt, 1996; Fabrizzi et al., 2004; Kumar and Dey, 2011; Mazurana et al., 2011). The plant residues used under no-tillage conditions may contain allelochemicals capable of interfering with the development and establishment of other vegetable species (Vyvyan, 2002; Macías et al., 2004; Campiglia et al., 2012; Tesio et al., 2011). These compounds originated from the secondary metabolism and/or plant organic material are decomposed by microorganisms (Rizvi and Rizvi, 1992; Gniazdowka and Bogatek, 2005; Zhang et al., 2011).

∗ Corresponding author. Tel.: +55 18 33025848; fax: +55 18 33025802. E-mail addresses: [email protected], [email protected] (R.M.G. Silva). 0304-4238/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2013.04.028

The allelochemicals released in the environment depend on temperature, on decomposition, and, mainly, on the rain action, which leaches the compounds present on plant residues (Ferreira and Aqüila, 2000; Omezzine et al., 2011). Once in the soil, the allelochemicals interfere with neighboring plants (Zhang et al., 2011), may act in stage of pre and post-emergence, and on the seed bank (Aqüila et al., 1999; Han et al., 2008). According to the International Coffee Organization (2012) data of tropical countries producers, coffee is the most commercialized tropical commodity and reached an estimated US $15.4 billion in exports in 2009/2010, when 93.4 million sacks were exported. The Companhia Nacional de Abastecimento (2012) estimates that in 2012, Brazil has approximately 2.3 million hectares of coffee culture and that the production should be approximately 50.61 million sacks, representing a significant part of the global trade. Surveys indicate that there are 388 species of coffee culture invasive plants (Gavilanes et al., 1988) and that the competition between the coffee plants and weeds is one of the factors that lowers Brazilian coffee productivity (Dias et al., 2004; Ronchi and Silva, 2006; Marcolini et al., 2009; Lemes et al., 2010). In the coffee cultures, the coffee peels (Coffea arabica) themselves are used as a soil cover with the finalities previously mentioned, besides serving as a weed control method (Santos et al., 2001). The coffee peel constitutes organic substrates used to produce vegetable crop seedlings and fruit plants with

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R.M.G. Silva et al. / Scientia Horticulturae 158 (2013) 39–44

conventional agriculture (Godoy et al., 2008). The present study had the objective of evaluating the allelopathic potential of different concentrations of the ethanolic extract of dry (DE) and fresh (FE) coffee fruits through bioassays of seed germination and the development of lettuce (Lactuca sativa L. cv. Grand Rapids), Malaysian cabbage (Brassica chinensis var. Parachinensis [Sinskaja]), and beggar’s tick (Bidens pilosa L.) seedlings, as well as evaluating the phytotoxicity through the determination of the mitotic index of meristematic root cells of the plants cited above; quantification of phenols, flavonoids and caffeine; and determination of the antioxidant activity of all extracts. 2. Materials and methods 2.1. Vegetal material and extract preparation Coffee producers donated the peel fruits (fresh and dry) of C. arabica planted in the region of Patrocínio – MG (18◦ 56 39 S and 46◦ 59 35 W, height: 950 m). The ethanolic extract was prepared by the mechanical maceration of the powdered plant material in PA ethanol (IMPEX, Brazil) (at a ratio of 1:10 [w/v]) for 24 h at room temperature. The extract was vacuum filtered. This process was performed three times with the same plant material. The resulting solutions were gathered and concentrated on a rotary evaporator (model: MA120, Marconi, Brazil), at an average temperature of 60 ◦ C. Then, the solutions were heated in a forced convection oven at an average temperature of 40 ◦ C until a dry residue resulted, which was stored in an amber glass bottle at 4 ◦ C.

2.4. Allelopathy bioassay of post-emergence This test was performed according to the methodology proposed by Soares (2000) and Alves et al. (2004). Lettuce, Malaysian cabbage, and beggar’s tick seeds were previously germinated in Petri plates covered with germination paper moistened with distilled water. After 24 h in a BOD greenhouse, the seedlings with an average length of 2 mm were used in this randomized experimental design bioassay. Petri dishes containing germination paper as a substrate moistened with 1 mL of the ethanolic extracts with different concentrations received 25 pre-germinated seedlings. Six replicates were prepared for each treatment and for the negative control (distilled water only). In sequence, the hypocotyls and the primary seedling roots were observed and measured by the use of a digital paquimeter (model: IP65, DIGIMESS® , Brazil), after 24 and 48 h (2 times) of exposure. After this time, the seedlings were dried at 70 ◦ C until constant mass to determine the dry weight of each treatment.

2.5. Osmotic potential, pH and electrical conductivity The osmotic potential was calculated according to Villela et al. (1991). Polyethylene glycol 6000 (PEG 6000) osmotic solutions were used to establish the osmotic potential from −0.01 to −1.0 MPa. These values were compared to those detected in C. arabica extracts. The pH of the concentrated extracts of C. arabica was determined using a pH meter (Tecnopon® MPA 210 model). Similarly, the electrical conductivity was measured with a conductivity meter (Conductivity Meter Instrutherm® , model CD860).

2.2. Powder dilution

2.6. Mitotic index determination

The resulting powder of the extraction was diluted in distilled water at 1, 5, 10 and 20 mg mL−1 concentrations and then used in all of the bioassays and experimental groups of this study. Regarding the negative control group, distilled water was used in all of the assays.

To verify the phytotoxic activity of C. arabica extracts, the mitotic index was determined. The seeds that germinated in distilled water and showed an average length of 1 mm were transferred into Petri dishes containing germination paper moistened with 1 mL of the different concentrations of the prepared extracts. Then, these plates were kept in a BOD greenhouse until the primary roots reached a length longer than 5 mm. The seedlings were fixated in Carnoy’s solution (ethylic alcohol PA and glacial acetic acid, 3:1) for 24 h and then stocked in a refrigerator in 70% ethanol. Roots were subsequently hydrolyzed in 1 N hydrochloric acid at 60 ◦ C for 6 min and later colored with 2% acetic carmine for 15 min. Following this procedure, the roots were placed in microscope slides, soaked in 45% acetic acid, and smashed. An optical microscope (100× zoom, with the help of immersion oil) was used for the slide analysis according to Guerra and Souza (2002). Ten roots per treatment were used, and 5000 cells/treatment were examined. The mitotic index calculation was as follows: (no. of cells in the division process/no. of cells analyzed) × 100.

2.3. Allelopathy bioassay of pre-emergence The pre-emergence bioassay was performed with lettuce seeds (Lactuca sativa L. cv. Grand Rapids), Malaysian cabbage (Brassica chinensis var. Parachinensis [Sinskaja]), and beggar’s tick (Bidens pilosa L.) with germination control in Petri plates (60 mm × 15 mm) containing germination paper. The relative humidity, temperature and luminosity were artificially controlled by a BOD (Biological Oxygen Demand) (model: 411/FPD, Nova Ética, Brazil) germination greenhouse. A randomized experimental design was prepared with Petri plates divided into experimental and negative control groups, each one containing 50 seeds. Each group had six replicates. The radicle protrusion and its geotropical curvature were used as evaluation criteria according to Labouriau (1983). The seeds that presented a false germination due to imbibitions were not registered. The germination monitoring was performed every 6 h for 120 h. From the resulting data obtained in the assay, different indexes percentage  were calculated: germinability or germination   ([ 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 (Labouriau, 1983; Santana and Ranal, 2004; Ranal et al., 2009).

2.7. Stable DPPH free radical scavenging activity The stable 1.1-diphenyl-2-picrylhydrazyl (DPPH, Sigma, USA) radical scavenging activity was determined by Blois’s method (1958). The dried ethanolic extracts of each sample were dissolved in ethanol (75%) at different concentrations (1, 5, 10 and 20 mg mL−1 ) and then mixed with 5 mL DPPH solution (1.5 × 10−4 M). The extracts reacted with the DPPH radical for a period of 30 min at a low luminosity and were then submitted to the UV–vis spectrophotometer (Femto-600 Plus) at a 517 nm wave length. The DPPH ratio of inhibition (I%) was calculated according to the formula I% = [(negative control-sample)/negative

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control] × 100. Gallic acid (Vetec-Química Fina, Brazil) was used as the reference. Three repetitions were performed.

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For the mitotic index, the results were submitted to the variance analysis and the means were compared by the Duncan test at a 5% probability (Sampietro et al., 2006).

2.8. Total phenol determination 3. Results The Folin-Ciocalteu method was used to determine the total phenol content of the extracts, with gallic acid as a comparative pattern. To each 0.5 mL sample of the extracts, 5 mL distilled water and 0.25 mL Folin-Ciocalteu reagent (molybdate, tungstate, and phosphoric acid) were added. After 3 min, 1 mL saturated 10% Na2 CO3 was added and the mixture was stocked for 1 h. Then, the absorbance was measured at 725 nm using a UV–vis spectrophotometer. All measurements were performed in triplicate and the results expressed in mg gallic acid/g extract. 2.9. Flavonoid determination The total flavonoid content of the extracts was determined by a UV–vis spectrophotometer, and the samples were prepared according to the methodology proposed by Zhishen et al. (1999), based on the flavonoid complexation with AlCl3 , which dislocates the absorption bands to higher wavelengths. An aliquot of 250 ␮L of the extract at all concentrations was mixed with 1.25 mL distilled water and 75 ␮L 5% NaNO2 . Six minutes later, 150 ␮L 10% AlCl3 solution was added. After 6 min, 0.5 mL 1 M NaOH was added and then the total volume was brought to 2.5 mL with distilled water. The samples were shaken in a vortex shaker and the absorbance measured at 510 nm. All the tests were done in triplicate and the results expressed in mg rutin/g extract. 2.10. Caffeine percentage determination The determination of the caffeine content of the coffee fruit peels was carried out according to a modification of the Adolfo Lutz Institute method used by Yoshida et al. (2005). For the calculation of the caffeine content (Cce ), the caffeine concentration in the solution (Ccs ) was determined by spectrophotometer readings. The following equation was used: Cce (%) = (Ccs ·Vs /Mp )100 in which Vs is the solution volume and Mp represents the total coffee fruit peel mass. 2.11. Statistics The statistical analyses were carried out with the Shapiro–Wilk normality test and the Levene homogeneity test. The transformed data were normal, and the variances were homogenous; thus, these data were analyzed by the parametrical tests ANOVA and Tukey (˛ = 0.5). The analyses were performed with the aid of the SISVAR software according to Santana and Ranal (2004) and Pereira et al. (2009).

3.1. The effects of the extracts on the seed germination and on the growth of lettuce, Malaysian cabbage, and beggar’s tick seedlings The allelopathic potential of the DE and FE was evaluated through germination, radicle and hypocotyl length, seedling dry weight tests, as well as the mitotic index of lettuce, Malaysian cabbage and beggar’s tick. The results of the germination test on lettuce seeds exposed to DE and FE were similar, showing a germination reduction for all concentrations; the 20 mg mL−1 had the lowest mean value (DE = 12.83%; FE = 4.54%) compared to the negative control (100%). With regard to the germination mean time and speed, the seeds treated with DE and FE were significantly different compared to the negative control. However, with regard to the DE and FE treatments, only 5 mg mL−1 and 10 mg mL−1 did not differ between them. For the mean speed, all treatments were significantly different for both DE and EF. All the DE and FE concentrations inhibited the radicle length, with a higher concentration producing a greater inhibition. The 20 mg mL−1 concentration in both treatments reduced the radicle length by 85.12% (DE) and 85.86% (FE). A similar reduction was observed for the hypocotyl length. The analysis of the dry weight of the seedlings showed a mass reduction when submitted to all the concentrations of the extracts, with an emphasis on the 20 mg mL−1 treatment, which presented a reduction of 91.86% for DE and 97.67% for FE. The treated groups with 1, 5, 10, and 20 mg mL−1 of DE and FE showed, respectively, a mitotic index of 19.98, 15.70, 10.08, and 08.09 for DE and 15.09, 11.09, 07.43, and 2.16 for FE. When compared to the negative control group index (21.28), the treatments differed statistically (Table 1). The effects of DE and FE on the germination, radicle and hypocotyl length, dry weight, and mitotic index on Malaysian cabbage and beggar’s tick were similar to those on lettuce (Tables 2 and 3). All of the parameters analyzed showed a reduction as the extract concentrations increased, with an emphasis on the action of the 20 mg mL−1 concentration. 3.2. pH, osmotic potential and electrical conductivity The physical–chemical characterization of the DE and FE revealed a minor pH variation, between 6.2 and 6.4 for DE and 6.4 and 6.6 for FE. The osmotic potential varied from −0.0027 to −0.0065 MPa (DE) and from −0.0045 to −0.345 MPa (FE). The

Table 1 Effects of coffee fruit extract on seed germination and seedling growth of lettuce. Coffee fruit

Dry

Fresh

Extract (mg mL−1 )

Germination (%)

0 1 5 10 20 1 5 10 20

100 97.43 69.62 49.22 12.83 89.64 57.46 13.62 4.54

± ± ± ± ± ± ± ± ±

0.000a 0.096a 0.078b 0.125c 0.129d 0.070e 0.122f 0.136d 0.106 g

Mean time (h) 23.62 32.24 49.12 54.52 81.44 41.64 67.42 71.32 80.10

± ± ± ± ± ± ± ± ±

0.169a 0.063b 0.071c 0.077c 0.072d 0.018e 0.052f 0.059f 0.179d

Mean speed (h) 0.042 0.027 0.022 0.017 0.009 0.023 0.019 0.012 0.007

± ± ± ± ± ± ± ± ±

0.000a 0.000b 0.000c 0.000d 0.000ef 0.000c 0.000d 0.000e 0.000f

Radicle length (mm) 24.20 15.12 9.62 7.54 3.60 11.92 9.32 5.54 3.42

Data are presented as the mean ± standard error. Means sharing the same letter in a column do not differ significantly by Tukey’s test (˛ = 0.05).

± ± ± ± ± ± ± ± ±

0.171a 0.051b 0.018c 0.077d 0.054e 0.034c 0.051c 0.026de 0.019e

Hypocotyl length (mm) 19.22 14.70 10.62 7.94 3.65 13.92 7.52 4.92 3.14

± ± ± ± ± ± ± ± ±

0.099a 0.082b 0.028c 0.028d 0.051e 0.021b 0.046d 0.015e 0.028e

Dry weight per seedling (mg) 0.86 0.79 0.64 0.47 0.07 0.72 0.56 0.11 0.02

± ± ± ± ± ± ± ± ±

0.000a 0.001b 0.000c 0.002d 0.000e 0.001b 0.000f 0.001e 0.000 g

Mitotic index 21.28 19.98 15.70 10.08 8.09 15.09 11.02 7.43 2.16

± ± ± ± ± ± ± ± ±

0.044a 0.040a 0.033b 0.028c 0.003c 0.017b 0.029b 0.002c 0.001d

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Table 2 Effects of coffee fruit extract on seed germination and seedling growth of Malaysian cabbage. Coffee fruit

Dry

Fresh

Extract (mg mL−1 )

Germination (%)

0 1 5 10 20 1 5 10 20

98.82 87.42 73.76 69.64 49.44 79.24 47.72 21.22 10.46

± ± ± ± ± ± ± ± ±

0.109a 0.036b 0.244ce 0.354c 0.311d 0.157e 0.367d 0.182f 0.164 g

Mean time (h) 25.90 29.32 38.13 41.34 59.43 31.95 47.43 53.34 60.13

± ± ± ± ± ± ± ± ±

Radicle length (mm)

Mean speed (h)

0.099a 0.007a 0.123b 0.173b 0.013c 0.076a 0.122d 0.040c 0.181c

0.072 0.047 0.032 0.026 0.015 0.034 0.027 0.016 0.011

± ± ± ± ± ± ± ± ±

0.000a 0.000b 0.000c 0.000d 0.000e 0.000c 0.000d 0.000e 0.000e

39.03 27.12 18.24 10.52 9.62 26.13 19.22 12.66 10.24

± ± ± ± ± ± ± ± ±

0.009a 0.058b 0.011c 0.038d 0.042d 0.036b 0.000c 0.087d 0.058d

Hypocotyl length (mm) 27.42 24.73 20.12 14.32 9.53 17.60 11.42 9.22 6.02

± ± ± ± ± ± ± ± ±

0.125a 0.069b 0.035c 0.034d 0.098e 0.099f 0.068c 0.043e 0.009 g

Dry weight per seedling (mg) 1.36 0.97 0.85 0.69 0.47 0.82 0.61 0.31 0.13

± ± ± ± ± ± ± ± ±

0.001a 0.001b 0.001c 0.001e 0.001f 0.002c 0.001e 0.001 g 0.001 h

Mitotic index 28.31 21.98 18.34 9.34 6.11 17.26 10.34 8.22 4.37

± ± ± ± ± ± ± ± ±

0.046a 0.028b 0.016c 0.029d 0.013e 0.036c 0.045d 0.011d 0.003e

Data are presented as the mean ± standard error. Means sharing the same letter in a column do not differ significantly by Tukey’s test (˛ = 0.05).

Table 3 Effects of coffee fruit extract on seed germination and seedling growth of beggar’s tick. Coffee fruit

Dry

Fresh

Extract (mg mL−1 )

Germination (%)

0 1 5 10 20 1 5 10 20

97.91 73.12 48.34 31.25 16.41 68.70 33.84 11.21 7.32

± ± ± ± ± ± ± ± ±

0.109a 0.036b 0.244c 0.354d 0.312e 0.157b 0.367d 0.182e 0.164f

Mean time (h) 46.23 49.29 58.98 67.27 91.75 51.45 63.16 85.53 97.47

± ± ± ± ± ± ± ± ±

Radicle length (mm)

Mean speed (h)

0.099a 0.007a 0.123b 0.017c 0.013d 0.076b 0.121c 0.040f 0.181 g

0.032 0.023 0.019 0.016 0.010 0.019 0.009 0.006 0.001

± ± ± ± ± ± ± ± ±

0.000a 0.000b 0.000c 0.000d 0.000e 0.000c 0.000e 0.000f 0.000 g

48.60 35.41 19.63 13.15 9.12 20.21 16.41 9.51 4.23

± ± ± ± ± ± ± ± ±

0.009a 0.059b 0.011c 0.038e 0.042f 0.036c 0.001e 0.865f 0.579 g

Hypocotyl length (mm) 36.22 22.13 19.01 7.51 4.62 18.91 9.92 5.14 3.02

± ± ± ± ± ± ± ± ±

0.125a 0.069b 0.035b 0.034c 0.009d 0.099b 0.069c 0.043d 0.009d

Dry weight per seedling (mg) 2.23 1.65 0.94 0.73 0.46 0.61 0.56 0.22 0.10

± ± ± ± ± ± ± ± ±

0.001a 0.001b 0.001c 0.002d 0.001e 0.002f 0.001f 0.001 g 0.001 h

Mitotic index 34.14 20.65 11.22 6.12 4.09 9.10 2.72 1.41 0.27

± ± ± ± ± ± ± ± ±

0.046a 0.028b 0.016c 0.029d 0.013d 0.363c 0.045e 0.011e 0.003f

Data are presented as the mean ± standard error. Means sharing the same letter in a column do not differ significantly by Tukey’s test (˛ = 0.05).

Table 4 pH, osmotic potential and electrical conductivity of the coffee fruit extract (dry and fresh) in different concentrations (1, 5, 10 and 20 mg mL−1 ). Coffee fruit

Extract (mg mL−1 )

pH

Osmotic potential (MPa)

Electrical conductivity (mS cm−1 )

Dry

0 1 5 10 20

6.2 6.1 6.0 6.3 6.4

−0.0027 −0.0031 −0.0035 −0.0049 −0.0065

0.0 1.3 1.6 1.9 2.1

Fresh

1 05 10 20

6.4 6.5 6.5 6.6

−0.0045 −0.0097 −0.0123 −0.0345

1.5 1.8 2.2 2.6

electric conductivity mean values varied from 1.3 to 2.1 mS cm−1 (DE) and from 1.5 to 2.6 mS cm−1 (FE), which were higher than the negative control mean value (0.0 mS cm−1 ) (Table 4). 3.3. Antioxidant activity and total phenol, flavonoids and caffeine The mean concentration of phenols and flavonoids and the caffeine percentage became progressively greater as both the DE and FE concentrations increased. The highest values of phenols were observed at 20 mg mL−1 , which presented 23.11 gallic acid equivalents (GAE) mg/g of extract for DE and 42.96 GAE mg/g of extract for FE. The total flavonoids of DE were 19.03 equivalents quercetin (EQQ) mg/g of extract and of FE were 31.24 EQQ mg/g of extract. The caffeine percentages of DE and FE were 0.36% and 0.53%, respectively. The highest antioxidant activity observed was at 20 mg mL−1 extract concentration, 65.27% (DE) and 72.45% (FE) (Table 5). 4. Discussion Studies of Santos et al. (2001) demonstrated that the incorporation of coffee fruit peels in the soil is capable of hindering the germination and growth of weeds. This information was essential

to delineate the present work, through which it was possible to observe, under laboratory conditions, that the extracts of the dry and fresh coffee fruit peels have the ability to interfere with the germination process and the development of tested plants. Regarding the germination, it was possible to observe alterations on the analyzed indexes of lettuce, Malaysian cabbage and beggar’s tick germination (Tables 1–3), and these results are in agreement with those of Fritz et al. (2007) in which it is demonstrated that the allelochemicals act in metabolic reactions, resulting in an interference with the germination process. Both the pre- and post-emergence tests showed an inhibition increase as the extract concentrations increased. The FE presented a higher activity for the evaluated parameters compared to the DE (Tables 1–3). According to Rice (1984) and Inderjit and Keating (1999), this phenomenon can be related to its higher compound concentration because the drying process of the vegetal material degrades chemical compounds, even allelochemicals, resulting in a minor interference with the germination and development of the seedlings. The reduction of the hypocotyls, roots, dry weight, and mitotic index observed in this assay may be directly related to the allelochemical action on the development of the tested plants

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Table 5 Antioxidant activity and total phenol and flavonoid contents and caffeine of the coffee fruit extract (dry and fresh) in different concentrations (1, 5, 10 and 20 mg mL−1 ). Coffee fruit

Extract (mg mL−1 )

Phenol contenta

Dry

1 5 10 20

18.23 19.15 21.07 23.11

± ± ± ±

1.43 1.17 1.65 1.87

6.23 12.01 16.45 19.03

± ± ± ±

0.15 0.21 1.21 1.34

0.26 0.29 0.31 0.36

± ± ± ±

0.01 0.02 0.01 0.03

8.25 18.21 23.34 65.27

± ± ± ±

0.19 0.11 0.13 0.23

Fresh

1 5 10 20

27.32 32.45 36.98 42.96

± ± ± ±

2.19 3.11 3.44 3.94

13.41 16.08 24.36 31.24

± ± ± ±

1.09 1.01 1.79 2.01

0.32 0.38 0.42 0.53

± ± ± ±

0.02 0.04 0.06 0.12

12.98 27.32 46.54 72.45

± ± ± ±

0.32 0.17 0.23 0.31

Flavonoids contenta

% caffeine contenta

% Antioxidant activityb

a Values are the mean ± standard deviation of three replicates for total phenol contents (mg gallic acid equivalent/g of extract); total flavonoid contents (mg quercetin equivalent/g of extract) and % caffeine content. b Values are the mean ± standard deviation of three replicates for DPPH radical scavenging activity test.

(Tables 1–3), as Kato-Noguchi et al. (2002) and Kato-Noguchi (2004) associate the reduction of the hypocotyl–root axis size and of the dry weight as evidence of the allelochemicals action. Reigosa et al. (1999), Inderjit and Nilsen (2003) and Inderjit et al. (2006) related the direct action of allelochemicals on cellular and metabolic alterations, including modifications in the expression and synthesis of DNA and RNA, which could justify the decrease of the mitotic index observed in this assay. This effect demonstrates the relationship of the allelopathic effects with the cytogenetics, as observed in the assays led by Duke et al. (2000) and Charoenyinga and Chamroon (2010). The mitotic index data obtained in the present study were similar to those reported by Pires et al. (2001) and Iganci et al. (2006), who demonstrated that the extracts interfered with the mitotic index of the meristematic root cells, resulting in the decrease in the elongation of the normal tested plants. The physical–chemical characterization tests are a major issue for the allelopathic studies because alterations of the osmotic potential, pH, and electric conductivity provided by the extracts can interfere directly with the germination and the development of the tested plants and, by mistake, be characterized as an allelopathic effect. Gatti et al. (2004) recommend that the osmotic potential of the extracts involved in the germination tests not exceed a value of −0.2 MPa (Table 4). The evaluated concentrations of the extracts showed little pH variation, from 6.0 to 6.6 (Table 4). These data are inside the range of values that do not influence the germination process, as demonstrated by Baskin and Baskin (1998) and Carmo et al. (2007), who evaluated the germination of test plants (lettuce and beggar’s tick) in a wide range of pH values, from 3.0 to 7.0. The conductivity mean values of the treatments were between 0.0 and 2.6 mS cm−1 (Table 4). According to Souza et al. (2003), values below 20 mS cm−1 are not detrimental to the seed germination. Thus, it can be concluded that the osmotic potential, pH, and electrical conductivity did not influence the germination because all of the results were inside the range of the presented patterns. For the investigation of the phytochemical classes present in the extracts, the total phenol and flavonoid concentrations were determined; besides these compounds, the presence and percentage of caffeine were verified (Table 5). Rice (1984) and Inderjit et al. (2006) suggested that the substances with allelopathic potential can be divided into phenolic and alkaloid compounds. Their presence in the phytochemical tests may be one of the factors responsible for the allelopathic activity observed in the germination and radicular growth tests, as well as in the phytotoxic activity (Pietta and Simonetti, 1999). The alkaloid class is known for its bacterial growth inhibitory capacity and for its influence on the growth and development of some plant species, besides being toxic for some invertebrates (Petroski et al., 1990; Inderjit and Keating, 1999). Caffeine, in particular, is known for its allelopathic potential, as the studies performed by Mazzafera et al. (1996) and Rosa et al. (2006) have demonstrated

that this alkaloid negatively influenced the growth and establishment of the tested plants. The antioxidant activities of the phenolic compounds and caffeine were determined. It was found that an increase in the activity depended on the evaluated concentrations, reaching higher values at 20 mg mL−1 , which were 65.27% (DE) and 72.45% (FE). The se data suggest a possible mechanism of action, as Huckelhoven and Kogel (2003) demonstrated, in which different allelochemicals with antioxidant potential are not only involved in the signal transduction and in the defense mechanisms of the plant, but they also accumulate and damage the cells of the plant, frequently leading to cellular death and interfering in the germination process and the development of seedlings. According to the results and in agreement with the studies found in the literature, it is possible to suggest that the free radical scavenging activity, as well as the high polyphenol and caffeine content of the extracts, can be included in a mechanism of action, characterizing the allelopathic effect performed by these extracts. Considering the results of the allelopathy tests and the physical–chemical evaluation carried out in this assay, as well as the chemical characteristics of the coffee fruit peel extracts, it is possible to conclude that this species contains allelochemical compounds capable of directly interfering with the germination and development of other species, as previously reported in studies where residues of coffee were incorporated and maintained in the culture. Acknowledgements The authors thank the Cooperativa dos Cafeicultores do Cerrado (EXPOCACCER) from Patrocínio, Minas Gerais, Brazil, for their aid in obtaining the coffee fruits. References Alves, M.C.S., Medeiros Filho, S., Innecco, R., Torres, S.B., 2004. Allelopathy of plant volatile extracts on seed germination and radicle length of lettuce. Pesqui. Agropecu. Bras. 39, 1083–1086. Aqüila, M.E.A., Ungaretti, J.A.C., Michelin, A., 1999. Preliminary observation on allelopathic activity in Achyrocline satureioides (Lam.) DC. Acta Hortic. 502, 383–388. Baskin, C.C., Baskin, J.M., 1998. Seeds – Ecology, Biogeography and Evolution of Dormancy and Germination. Academic Press, San Diego. Blois, M.S., 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199–1200. Campiglia, E., Radicetti, E., Mancinelli, R., 2012. Weed negative control strategies and yield response in a pepper crop (Capsicum annuum L.) mulched with hairy vetch (Vicia villosa Roth.) and oat (Avena sativa L.) residues. Crop Prot. 33, 65–73. Carmo, F.M.S., Borges, E.E.L., Takaki, M., 2007. Allelopathy of Brazilian sassafras (Ocotea odorifera (Vell.)) Rohwer aqueous extracts. Acta Bot. Bras. 21, 697–705. Charoenyinga, M.T., Chamroon, L., 2010. An allelopathic substance isolated from Zanthoxylum limonella Alston fruit Patchanee. Sci. Hortic. 125, 411–416. Companhia Nacional de Abastecimento – CONAB. Available in: http://www.conab.gov.br/ (accessed 12.03.12). Dias, G.F.S., Alves, P.L.C.A., Dias, T.C.S., 2004. Brachiaria decumbens supresses the initial growth of Coffea arabica. Sci. Agric. (Piracicaba, Braz.) 61, 579–583.

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