Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz. Avda. República Saharaui, s/n, 11510 Puerto Real (Cádiz), Spain. *firstname.lastname@example.org
Rice is one of the most interesting crops in the world from social and economic points of view. In order to carry out a study that guides us to the isolation of the most active compounds from rice, different extracts were achieved and their activities evaluated. The activity of these extracts was evaluated on wheat etiolated coleoptiles and on Echinochloa crus-galli, a threatening weed in rice. The bioactive extracts were fractionated and 15 compounds were isolated and identified by spectroscopic methods. Eight of these compounds were isolated for the first time from Oryza sativa. The most active compounds were ergosterol peroxide and 7-oxo-stigmasterol. In the case of ergosterol peroxide the activity was higher than that of the commercial herbicide Logran.
The bioactivity of different rice extracts was evaluated on wheat etiolated coleoptiles bioassay and Echinochloa crus-galli. The most active compounds were ergosterol peroxide and 7-oxo-stigmasterol.
Steroid, Allelopathy, ergosterol peroxide, 7-oxo-stigmasterol, Echinochloa crus-galli, rice.
Rice is one of the most interesting crops in the world both from social and economic points of view. In many cases, the salinity, quality, and other characteristics of the soils in those areas where is predominant allow a reduced number of other crop choices (López-Bellido 1991). Thus, monoculture becomes a much extended practice. The monoculture practices along with the heavy use of herbicides are characteristic of modern agriculture and are inducing the appearance of tolerant and/or resistant herbicides weed biotypes (De Prado et al. 1997; Valverde 2000). This is the case of Echinochloa crus-galli, world's main weed of rice (Holm et al. 1977). It has been described a number of resistant biotypes in the United Stated, Brazil, Spain, Canada, France, Italy, China, and other countries. Resistant biotypes affect different herbicide groups (i.e. synthetic auxines, thiocarbamates or dinitroanilins) and different modes of action such as photosystem II or ACCase inhibitors (Maun et al. 1986; Gadamski at al. 2000; Hoagland et al. 1999; Lopez Martinez and Prado 1998). An alternative strategy for weed suppression could be the use of chemicals from rice. It could be render new herbicide models with new modes of action that could prevent resistance phenomena. Our goals are the isolation and characterization of those metabolites present in rice which may be involved in its allelopathic activity, and the evaluation of their bioactivities and their potentialities in the control of the weed Echinochloa crus-galli.
Fresh plants of Oryza sativa cv. Puntal were collected in Isla Menor (Seville) in June 2002 in the stage next to harvest and were provided by the company Dow AgroScience. The plant material was divided into three parts i.e.: fresh plant, dried plant, and a third part of fresh plant to be extracted using Pluviotron. In each one the aerial plant was separated from roots.
Fresh parts were extracted in water for 24 h at room temperature in the dark. Plant residues were dried at room temperature and re-extracted with CH2Cl2 (48 h) and later with methanol. Dried parts were extracted in DCM and later in methanol for 24 h at room. Two extracts in water of aerial part and roots of fresh plant were carried out for 24 h in a rain simulator device (Pluviotron).
The twelve extracts obtained were bioassayed on wheat etiolated coleoptiles and on E. crus-galli. Extracts from dried aerial part in DCM and from fresh roots in DCM were found to be the most active. They were chromatographed on silica gel using hexane-EtOAc mixtures of increasing polarity as eluent. Fractions eluted between hexane-EtOAc 7:3 and 5:5 yielded 15 compounds, eight of them being isolated from Oryza sativa for the first time (Figure 1).
The evaluation of their phytotoxicity was performed using on E. crus-galli as target using with an experimental design analogous to that described previously for STS species (Macias et al 2000). For E. crus-galli, seeds were purchased from Herbiseed Co. (Twyford, England). The number of seeds in each Petri dish was 25, and 5 mL of treatment, control or internal reference solution were added to each Petri dish. Four replicates were used (100 seeds).
After adding seeds and aqueous solutions, Petri dishes were sealed with Parafilm to ensure closed-system models. Seeds were further incubated at 25°C in a Memmert ICE 700 controlled environment growth chamber, in absence of light. The bioassay took 5 days. After growth, plants were frozen at -10°C for 24 h to avoid subsequent growth during the measurement process. This helped the handling of the plants and allowed a more accurate measurement of root and shoot lengths.
In order to carry out a study that guides to the isolation of the most active compounds from rice, different extracts were achieved and their activities evaluated. So, all the plant material was divided up three parts: fresh plant, dried plant and a third part of fresh plant to be extracted using Pluviotron.
Figure 1. Compounds isolated from active fractions of rice extracts.
The activity of these twelve extracts was evaluated with wheat etiolated coleoptiles bioassay and against E. crus-galli. The chromatographic study of the most bioactive extracts (DCM extracts of fresh roots and dried aerial parts) led to the isolation of fifteen compounds (Figure 1) that were identified by spectroscopic methods. Eight of these compounds (2, 4, 5, 6, 8, 10, 11, and 12) have been isolated from Oryza sativa for the first time.
The evaluation of the phytotoxicity of the chemicals identified was performed on E. crus-galli. The results showed that 7-oxo-stigmasterol (4) and ergosterol peroxide (7) were the most active compounds with -50% and -60% inhibition for 500 µM 4 and 7, respectively. Ergosterol peroxide (7) is even more active than the commercial herbicide Logran. This is specially evident at 50 µM, when ergosterol peroxide is still active (-50 %) while Logran is not (Figure 2).
Figure 2. Effects of tested compounds on the root length of Echinochloa crus-galli.
Some structure-activity relationships can be inferred from these results. Compounds 4 and 7 showed the highest values of phytotoxicity. The different functionalization pattern at B-ring between 3 and 4 and the rest of compunds suggests that higher oxidation levels enhances the phytotoxic activity on E. crus-galli.
This is also linked with the presence of a double bond in the side-chain. Thus, compound 4 is clearly more active than 2.
Endoperoxides can be transformed by peroxydases, very common in vegetable tissue. Moreover, endoperoxides could induce the appearance of free radicals.
These results, suggest that steroids may be involved in the allelopathic defence mechanism of rice, pointing out the importance of compounds 4 and 7.
This research has been supported by the Ministerio de Ciencia y Tecnología, Spain (MCYT; Project No. AGL2001-3556 (AGR).
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