Nuria Chinchilla

Plant biocommunicators: their phytotoxicity, degradation studies and potential use as herbicide models

The development of new bioactive molecules with potential application in pharmacology and agriculture by using natural products as templates has been a widely used approach in the recent years. Focusing our attention in phytochemicals with potential application in agriculture, allelochemicals (natural plant toxins) have been deeply researched with the main purpose of finding phytotoxic substances to use as new herbicidal templates. The development of new analytical methodologies, improved bioassay techniques and a complete understanding of the allelopathic phenomenon will provide new tools for natural herbicide models development, in the context of the new approaches to integrated pest management. A deeper knowledge of the release and assimilation of allelochemicals, and the degradation phenomena associated to them will aid to discover new chemical structures with potential utility, in addition to a better understanding on the ecological interactions mediated by phytochemicals. The research on these topics, made with promising allelochemicals such as benzoxazinones and their degradation derivatives, the improvements on analytical design, the degradation mechanisms elucidation and the novel bioassay techniques recently developed, are revised herein.

Bioactive steroids from Oryza sativa L.

Rice is one of the most interesting crops in the world from both the social and the economic point of views. The monoculture practices along with the heavy use of herbicides are characteristic of modern agriculture and are inducing the appearance of tolerant and/or herbicide resistant weed biotypes. This is the case the worlds main weed of rice barnyardgrass (Echinochloa crus-galli). Alternative strategies for weed suppression consist of the use of chemicals from rice due to necessity of obtaining new herbicides with new modes of action that could prevent resistance phenomena. 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 into three parts: fresh plant, dried plant, and fresh plant from Pluviotron. The aerial part was separated from roots in all cases and extracted in water, in organic solvents as well as with the Pluviotron device. The activity of the 12 extracts obtained was evaluated using a generalist bioassay, wheat etiolated coleoptiles bioassay, and a phytotoxic bioassay on barnyardgrass as target species. 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 in Oryza sativa. The most phytotoxic compounds on E. crus-galli were ergosterol peroxide and 7-oxo-stigmasterol. In the case of ergosterol peroxide the activity was higher than the commercial herbicide Logran. This is the first report of potential allelopathic activity of steroids on weeds based on their phytotoxicity.

Evidence for an Allelopathic Interaction Between Rye and Wild Oats

Allelopathy is a biological phenomenon in which an organism produces one or more biochemicals that influence the growth, survival, and reproduction of other organisms. Allelopathy has been the subject of a great deal of research in chemical ecology since the 1930s. The characterization of the factors that influence this phenomenon has barely been explored, mainly due to the complexity of this area. The main aim of the research carried out to date has been to shed light on the importance of these interactions in agroecosystems, especially in relation to the interactions between crops and weeds. Herein we report the characterization of a complete allelochemical pathway involving benzoxazinones, which are known to participate in allelopathic plant defense interactions of several plants of high agronomic interest. The production of the defense chemicals by a donor plant (crop), the route and transformations of the chemicals released into the environment, and the uptake and phytotoxic effects on a target plant (weed) were all monitored. The results of this study, which is the first of its kind, allowed a complete dynamic characterization of the allelopathic phenomenon for benzoxazinones.

Combined strategy for phytotoxicity enhancement of benzoxazinones.

Fifteen new derivatives of D-DIBOA, including aromatic ring modifications and the addition of side chains in positions C-2 and N-4, were synthesized and their phytotoxicity, selectivity, and structure-activity relationships evaluated. The most active compounds among the derivatives at the C-2 position were 6-Cl-2-Et-D-DIBOA and 6-F-2-Et-D-DIBOA. Of the derivatives at N-4, the most active compounds were 6-Cl-4-Pr-D-DIBOA and 6-Cl-4-Val-D-DIBOA. These four compounds showed high levels of inhibition in root length at very low concentrations in all species. The most remarkable result is the 70% inhibition observed for the root length of cress at 100 nM caused by the latter two compounds. These results support our previous research and conclusions regarding the steric, electronic, and solubility requirements to achieve the maximum phytotoxic activity.

Aneugenic effects of benzoxazinones in cultured human cells.

Benzoxazinones (BAs) are natural products that are present in Gramineae and represent part of the plant defence system against pests. In recent years, sprouts of maize, wheat and rye have been used for the production of dietary supplements. We have investigated the potential genotoxic activities of a diverse range of synthetic derivatives of the most abundant natural BA, namely DIBOA (2,4-dihydroxy-1,4-benzoxazin-3-one), proposed for use as a potential herbicide. We have tested 18 synthetic BAs for potential effects in cultured HeLa cells. We found significantly higher micronucleus (MN) induction over the background level, with the solvent DMSO used as an internal control. Concentration-dependent effects were found between 1nM and 20nM for all the synthetic compounds studied. Immunostaining with an anticentromere antibody showed that >80% of MN induced gave a centromere-positive signal. Similarly, fluorescence in situ hybridization (FISH) analysis with alphoid centromere probes showed a positive hybridization signal, indicating that all compounds analyzed are aneugenic. Chemical modification of the N in the heterocyclic aromatic amine served us to suggest a relationship between the structure and the aneugenic effects of the compounds analyzed. Our findings indicate that benzoxazinoids could be potential genotoxins for human cells.

Modified benzoxazinones in the system Oryza sativa-Echinochloa crus-galli: an approach to the development of biorational herbicide models.

The utility of benzoxazinones and some of their synthetic derivatives in the search for new leads for herbicide model development has been explored. The work described focuses on obtaining derivatives that present selectivity in the system Oryza sativa- Echinochloa crus-galli. To achieve this goal the influence of lipophilicity in this system has been studied by preparing 14 ester derivatives at the N-4 position of D-DIBOA along with other compounds with different functionalization and chain lengths at position C-2. These compounds have been tested in the aforementioned system, and the dose-response profiles have been compared. The most active compound was 2-ethyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one, which presented higher selectivity than the specific herbicide Cotanil-35. These results confirm the potential of D-DIBOA as a lead herbicide for the control of Echinochloa spp. in rice crops.

Aromatic-ring-functionalised benzoxazinones in the system Oryza sativa–Echinochloa crus-galli as biorational herbicide models

BACKGROUND Barnyardgrass, Echinochloa crus-galli (L.) Beauv., is one of the most problematic weeds occurring in rice crops. Although efficient chemical control is provided by herbicides available on the market, resistant biotypes provoked by pressure selection have appeared in recent times. This emphasises the need for alternative treatments in which herbicidal compounds from a natural origin could be included. RESULTS A number of chemicals with a [2H]-1,4-benzoxazin-3(4H)-one (D-DIBOA) skeleton were tested on this weed, and also in rice, in order to achieve an optimal lead for herbicide composition development by taking into consideration phytotoxic effects and selectivity on the weed. 6-Cl-D-DIBOA causes the same effect as the commercial herbicide propanil at a concentration 15 times lower, while 6-F-D-DIBOA causes this inhibition at a dose 30 times lower. The phytotoxicities caused by 8-Cl-D-DIBOA (IC50 = 44 microM, R2 = 0.866) and 7,8-diF-D-DIBOA (IC50 = 52 microM, R2 = 0.9067) are also remarkable. 8-Cl-D-DIBOA was the compound that presented the highest selectivity on Echinochloa crus-galli. The structural requirements for optimal phytotoxicity and selectivity were elucidated by means of QSAR methodology, considering electronic and steric factors. One of the most important descriptors influencing the bioactivity was the dipole moment modulus. This was successfully correlated by employing a second-order polynomial model. CONCLUSION The in vitro phytotoxic profiles and selectivities shown for these chemicals make them truly promising candidates for higher-level studies. 6F- and 6Cl-D-DIBOA, for their high phytotoxicities, and 8-Cl-D-DIBOA, because of its high selectivity, were found to be the most interesting compounds from this point of view.

Phytotoxic studies of naphthoquinone intermediates from the synthesis of the natural product Naphthotectone

Abstract1,4-Naphthoquinones exhibit strong activity as antimalarial, antibacterial, antifungal and anticancer agents. These kinds of compounds have also shown phytotoxic activity. The first example of an allelochemical to be described was naphthoquinone juglone (5-hydroxy-1,4-naphthoquinone). This fact led us to conduct a study of the phytotoxic activity and structure–activity relationships of 26 naphthazarin derivatives. The compounds belong to two main groups, 5,8-dioxygenated-1,4-naphthoquinones and 1,4,5,8-tetraoxygenated naphthalenes with different functionalizations and chains at positions 2, 4, 6 and 7. The results show that substitution with halogens and modification of the hydroxyl groups on the aromatic ring with methoxy or acetate groups seems to play an important role in enhancing the activity.

Soil biodegradation of a benzoxazinone analog proposed as a natural products-based herbicide

AimsBenzoxazinones with the 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one skeleton have been proposed as potentially successful models for the development of novel design leads. D-DIBOA has proven to be the most promising base structure in the search for novel herbicide models based on the benzoxazinone skeleton. The biodegradation dynamics of D-DIBOA in soil are therefore relevant and are the subject of this study.MethodsA previously optimized methodology for the assessment of biodegradation has been applied for the first time to a synthetic benzoxazinone.ResultsBiodegradability is a characteristic of natural benzoxazinones and a safety requirement for the development of herbicidal chemicals. The biodegradation phenomenon and its consequences for the development of new herbicide models are discussed. The half-life determined for D-DIBOA was much higher than those previously reported for the natural products DIBOA, DIMBOA and their benzoxazolinone derivatives.ConclusionsThis finding, together with its previously described potent phytotoxicity, suggests that D-DIBOA is a useful candidate for novel herbicide model development. The lactam D-HBOA, which is slightly less phytotoxic than its precursor, was discovered to be the first and principal metabolite resulting from D-DIBOA degradation.

Multifunctionalised benzoxazinones in the systems Oryza sativa–Echinochloa crus-galli and Triticum aestivum–Avena fatua as natural-product-based herbicide leads

BACKGROUND Fifteen novel derivatives of D-DIBOA, including aromatic ring modifications and the addition of side chains in positions C-2 and N-4, had previously been synthesised and their phytotoxicity on standard target species (STS) evaluated. This strategy combined steric, electronic, solubility and lipophilicity requirements to achieve the maximum phytotoxic activity. An evaluation of the bioactivity of these compounds on the systems Oryza sativa-Echinochloa crus-galli and Triticum aestivum-Avena fatua is reported here. RESULTS All compounds showed inhibition profiles on the two species Echinochloa crus-galli (L.) Beauv. and Avena fatua L. The most marked effects were caused by 6F-4Pr-D-DIBOA, 6F-4Val-D-DIBOA, 6Cl-4Pr-D-DIBOA and 6Cl-4Val-D-DIBOA. The IC(50) values for the systems Echinochloa crus-galli-Oryza sativa and Avena fatua-Triticum aestivum for all compounds were compared. The compound that showed the greatest selectivity for the system Echinochloa crus-galli-Oryza sativa was 8Cl-4Pr-D-DIBOA, which was 15 times more selective than the commercial herbicide propanil (Cotanil-35). With regard to the system Avena fatua-Triticum aestivum, the compounds that showed the highest selectivities were 8Cl-4Val-D-DIBOA and 6F-4Pr-D-DIBOA. The results obtained for 6F-4Pr-D-DIBOA are of great interest because of the high phytotoxicity to Avena fatua (IC(50) = 6 µM, r(2) = 0.9616). CONCLUSION The in vitro phytotoxicity profiles and selectivities shown by the compounds described here make them candidates for higher-level studies. 8Cl-4Pr-D-DIBOA for the system Echinochloa crus-galli-Oryza sativa and 6F-4Pr-D-DIBOA for Avena fatua-Triticum aestivum were the most interesting compounds.