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Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.22 no.4 Curitiba July/Aug. 2012 Epub May 24, 2012
Rainiomar Raimundo da FonsecaI; Fredy A. Ortiz-RamírezII; Diana N. CavalcantiII; Carlos José B. RamosIII Valéria L. Teixeira, *, I, III; Antonio Pedro da Silva Sousa FilhoIV
IPrograma de Pós-graduação em Química, Instituto de Química, Universidade Federal Fluminense, Brazil
IIPrograma de Pós-graduação em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Brazil
IIIDepartamento de Biologia Marinha, Universidade Federal Fluminense, Brazil
IVEmbrapa/CPATU, Belém-PA, Brazil
Four extracts from the marine red alga Plocamium brasiliense (Greville) M.A.Howe & W.R.Taylor were prepared to identify and characterize their potential allelopathic effects on seed germination, radicle elongation and hypocotyl development of the weeds Mimosa pudica L. and Senna obtusifolia (L.) Irwin & Barneby. The four extracts were prepared in a sequence of solvents of increasing polarity: n-hexane, dichloromethane, ethyl acetate and ethanol/water (7:3). The germination bioassay was carried out at 25 °C with a 12 h photoperiod and the radicle elongation and hypocotyl development at 25 °C with a 24 h photoperiod. The dichloromethane extract showed inhibitory effects on seed germination of both plants (35 and 14%, respectively, in M. pudica and S. obtusifolia), radical germination (52 and 41.7%, respectively) and hypocotyl development (17.1 and 25.5%, respectively). Given the high sensitivity of this parameter to the potential allelopathic effects and the insufficient number of references found in the literature, these results are expected to stimulate new tests with other species of marine algae. Given the high sensitivity of the method for the detection of allelopathic potential, the species P. brasiliense emerges as a possible source of allelopathic substances against weed species. The results are attributed to the chemical composition, especially in relation to the presence of halogenated monoterpenes.
Keywords: allelopathy, germination, inhibition, Plocamium brasiliense, halogenated monoterpenes, weeds
The inadequate use of fire and mowing, along with the application of synthetic herbicides, has become a very common practice for controlling weeds in Brazilian pastures. Nonetheless, these procedures have been demonstrated to be inefficient (Souza Filho et al., 2006). The synthetic herbicides currently utilized in agriculture have organochlorine and organophosphate chemicals in their composition. Thus, despite the importance of such herbicides for the control of invasive plants, there has been ample discussion concerning the damage they cause to both man and the environment.
Therefore, the investigation of the allelopathic potential of various living organisms might be a more viable alternative when it comes to controlling invasive plants. In fact, the liberation of chemical substances that have phytotoxic activities can have a significant influence on the population dynamics of plants in the countryside, since these substances may alter the pattern and the density of the vegetation (Souza Filho & Alves, 2000, Souza Filho, 2002, Souza Filho et al., 2006; Souza et al., 2006). Taking this into consideration, our study aims to enable quantitative and qualitative improvements in the production of food on both small and large agricultural properties in Brazil.
In this context, Brazil has a large biological diversity, especially in marine environments, where macroalgae are especially relevant since they represent a natural strategic resource for the development of biotechnology. The genus Plocamium Lamouroux has approximately 42 species and more than 100 monoterpenes have been isolated from twelve species (Teixeira, 2009). P. brasiliense, the only species on the Brazilian coast, is distributed in North America, the Caribbean Islands (Taylor, 1960; Schneider & Searles, 1991), Trinidad & Tobago (Duncan & Lee Lum, 2006) and South America (Taylor, 1930, 1960; Joly, 1957, 1965; Ganesan, 1990; Schneider & Searles, 1991; Díaz-Pulido & Díaz-Ruíz, 2003).
Previous studies of the chemical components isolated from Brazilian P. brasiliense have been performed by our group (Ferreira et al., 2010; Vasconcelos et al., 2010). The halogenated monoterpenes 1, 2 and 3 were isolated from P. brasiliense in a chemotaxonomic analysis (Vasconcelos et al., 2010). In another study, the crude CH2Cl2 extract (containing monoterpenes 1, 2 and 4) and a fraction enriched with the halogenated monoterpene 2 from P. brasiliense were evaluated for cytotoxicity and against the virus HSV-1 (Ferreira et al., 2010).
Studies showed that these metabolites represent a means of defense against marine herbivores, fouling organisms and pathogens, as well as protection against radiation and allopathic agents (e.g., Sakata et al., 1991). Based on these conclusions, the present study assesses the potential for allelopathic activity of four extracts (n-hexane, dichloromethane, ethyl acetate and aqueous ethanol) obtained from the marine alga Plocamium brasiliense (Rhodophyta).
Materials and Methods
Specimens of Plocamium brasiliense (Greville) M.A.Howe & W.R.Taylor were collected in October, 2010, in the sublittoral of Enseada do Forno, Armação de Búzios (RJ) (22º45'S e 41º52'W). The seaweeds were washed with local seawater and separated from sediments, epiphytes and other associated organisms. The algae were dried for twenty days and extracted with the appropriate solvent. Voucher specimens were deposited in the Herbarium of the Universidade do Estado do Rio de Janeiro (HRJ 10331-32).
The receptor plants used in the bioassays were two of the main weeds that occur in areas of cultivated pastures in the Amazon region, which are known as malícia (Mimosa pudica) and mata-pasto (Senna obtusiloba). The seeds of these two plants were collected in the municipality of Castanhal-PA. The seeds were cleaned and purged and placed in seed conservation chambers. The seed dormancy was overcome as established by Souza Filho (2002) and Souza Filho et al. (2006).
Preparation of crude extracts
The algae P. brasiliense, collected and dried at room temperature (average of 28 ºC), were triturated in an industrial blender and placed in a plastic tray, yielding 5.16 g of powdered whole algae. The extracts were obtained by an exhaustive extraction using a decanting funnel (1 L). The procedures involved the sequential utilization of solvents of increasing polarity. Therefore, the order in which the solvents were used was: n-hexane, dichloromethane, ethyl acetate and ethanol/water (7:3). In the first step, 5.16 g of dry algae were placed in the decantation funnel with 400 mL of n-hexane for eight days. This process was repeated twice. The extracts were concentrated on a rotary evaporator, obtaining 122 mg of n-hexane extractable material. The sequence of extraction was repeated for the other solvents, which generated the following masses: dichloromethane, 196 mg; ethyl acetate, 42 mg; and ethanol/water, 250 mg.
The seed germination bioassays were developed under controlled conditions of constant temperature (25 °C) and a photoperiod of 12 h. Germination was monitored for fifteen days with daily counts and elimination of the germinated seeds. Germinated seeds were considered to be those that had a root length equal to or greater than 2.00 mm (Souza Filho, 2002, Souza Filho et al., 2006). Each transparent Petri dish, 9.0 cm in diameter, received twenty seeds. The radicle elongation biossays were performed under controlled conditions of constant temperature (25 °C) and a photoperiod of 24 h in a germination chamber with cool white fluorescent lamps and and a luminous flux of approximately 10 µmol.m-2.s-1. We used six seeds pre-germinated for approximately three days in a transparent Petri dish 9.0 cm in diameter. At the end of a period of ten days of growth, we measured the length of the radicles. Each Petri dish was covered with quantitative filter paper (Souza Filho, 2002).
A concentration of 1% m/v of each crude extract was added at the beginning of each bioassay. Since then, only distilled water was added to the system when necessary. The test solutions were added only once, at the beginning of the bioassays, and thereafter distilled water added only when necessary.
The statistical test for all experiments was completely randomized with three repetitions. Data were submitted to the F test for analyses of variance, while the means were compared by the Tukey test (p<5%). All analysis was performed with the program SAS for Windows (Sas, 1989).
Results and Discussion
The results for the effect of the crude extracts on the germination of M. pudica and S. obtusifolia are summarized in Table 1. No seaweed extract promoted a significant allelopathic effect on the seeds of S. obtusifolia, the best result (14% of inhibition) being that of the dichloromethane extract. By comparison, the allelopathic effects of the extracts on M. pudica were more significant than those obtained with S. obtusifolia. The dichloromethane extract was the most active, with the greatest inhibition of the germination of the seeds (25%).
The analysis of the effect of each extract on radicle elongation is presented in Table 2. The results show that the dichloromethane extract of P. brasiliense contains active components that inhibited 52.0 and 41.7%, respectively, of the radicle elongation of M. pudica and S. obtusiloba. However, in the radicle elongation experiments, the ethyl acetate extract also showed 32.0% inhibition of M. pudica and 21.1% for S. obtusiloba. These expressive values in the radicle elongation bioassay demonstrate that the sensitivity to potential allelopathic effects is much greater in the radicle elongation phase than in seed germination.
In the analysis of the effect of each extract on hypocotyl development of the weeds, the ethanol/water and dichloromethane extracts were more effective with S. obtusifolia than with M. pudica (Table 3). The dichloromethane extract was again the greatest inhibitor (17.1% for M. pudica and 25;5% for S. obtusifolia), followed by the ethanol/water extract (13.4% for M. pudica and 14.8% for S. obtusifolia). The crude dichloromethane extract was the most efficient and, therefore, the extract that represents the main source of secondary metabolites with allelopathic activity present in P. brasiliense. In addition, the P. brasiliense extracts tested were more efficient for M. pudica in the germination and radicle development bioassays, whereas S. obtusifolia showed better results in the hypocotyl experiment since it was more sensitive to the allelochemicals present in the dichloromethane and ethyl acetate extracts.
These results also indicate that the dichloromethane extract is more likely to inhibit the development of invasive plants than their germination, because the bioassay for radicle development presented results of greater magnitude.
The results obtained with the extracts point to the importance of a search for the chemical components responsible for these phytotoxic activities. In any event, the red marine alga P. brasiliense can now be considered to be a potential source of herbicides.
We are grateful to the CNPq and FAPERJ for financial support and for Productivity Fellowships to VLT. DNC thanks the CNPq for providing PostDoctoral support and RRF and FAOR thank CAPES for DSc fellowships.
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Received 24 Nov 2011
Accepted 9 Jan 2012
* Correspondence: Valéria Laneuville Teixeira Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense Caixa Postal 100.644, 24097-100 Niterói-RJ, Brazil firstname.lastname@example.org
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