Carlos L. Céspedes, Juan R. Salazar, Armando Ariza-Castolo, Lydia Yamaguchi, José G. Ávila,
Pedro Aqueveque, Isao Kubo,
Basic Science Department, Faculty of Sciences, University of Bío Bío, Andres Bello Av, s/n, Chillán, P.O. Box 447, Ñuble 3780000, Chile.
The effects of persistent organic pollutants (POPs) on humans and biodiversity are multiple and varied. Nowadays environmentally-friendly pesticides are strongly preferred to POPs. It is noteworthy that the crop protection role of pesticides and other techniques, i.e. biopesticides, plant extracts, prevention methods, organic methods, evaluation of plant resistance to certain pests under an integrated pest management (IPM), could improve the risks and benefits which must be assessed on a sound scientific basis. For this directive it is crucial to bring about a significant reduction in the use of chemical pesticides, not least through the promotion of sustainable alternative solutions such as organic farming and IPM. Biopesticides are derived from natural materials such as animals, plants, bacteria, and certain minerals. Most of them are biodegradable in relatively short periods of time. On this regard, substances from Calceolaria species emerge as a strong alternative to the use of POPs. The American genus Calceolaria species are regarded both as a notorious weeds and popular ornamental garden plants. Some have medicinal applications. Other taxa of Calceolaria are toxic to insects and resistant to microbial attack. These properties are probably associated with the presence of terpenes, iridoids, flavonoids, naphthoquinones and phenylpropanoids previously demonstrated to have interesting biological activities. In this article a comprehensive evaluation of the potential utilization of Calceolaria species as a source of biopesticides is made. The chemical profile of selected members of the Chilean Calceolaria integrifolia sensu lato complex represents a significant addition to previous studies. New secondary metabolites were isolated, identified and tested for their antifeedant, insect growth regulation and insecticidal activities against Spodoptera frugiperda and Drosophila melanogaster. These species serve as a model of insect pests using conventional procedures. Additionally, bactericidal and fungicidal activity were determined. Dunnione mixed with gallic acid was the most active fungistatic and fungicidal combination encountered. Several compounds as isorhamnetin, combined with ferulic and gallic acid quickly reduced cell viability, but cell viability was recovered quickly and did not differ from that of the control. The effect of these mixtures on cultures of Aspergillus niger, Fusarium moniliforme, Fusarium sporotrichum, Rhizoctonia solani, and Trichophyton mentagrophytes, was sublethal. However, when fungistatic isorhamnetin and dunnione were combined with sublethal amounts of both ferulic and gallic acid, respectively, strong fungicidal activity against theses strains was observed. Thus, dunnione combined with gallic acid completely restricted the recovery of cell viability. This apparent synergistic effect was probably due to the blockade of the recovery process from induced-stress. The same series of phenolics (iridoids, flavonoids, naphthoquinones and phenylpropanoids) were also tested against the Gram-negative bacteria Escherichia coli, Enterobacter agglomerans, and Salmonella typhi, and agaisnt the Gram-positive bacteria Bacillus subtilis, Sarcinia lutea, and Staphyllococcus aureus and their effects compared with those that of kanamycin. Mixtures of isorhamnetin/dunnione/kaempferol/ferulic/gallic acid in various combinations were found to have the most potent bactericidal and fungicidal activity with MFC between 10 and 50 μg/ml. Quercetin was found to be the most potent fungistatic single compound with an MIC of 15 µg/ml. A time-kill curve study showed that quercetin was fungicidal against fungi assayed at any growth stage. This antifungal activity was slightly enhanced by combination with gallic acid. The primary antifungal action of the mixtures assayed likely comes from their ability to act as nonionic surfactants that disrupt the function of native membrane-associated proteins. Hence, the antifungal activity of isorhamnetin and other O-methyl flavonols appears to be mediated by biophysical processes. Maximum activity is obtained when the balance between hydrophilic and hydrophobic portions of the molecules of the mixtures becomes the most appropriate. Diterpenes, flavonoids, phenylpropanoids, iridoids and phenolic acids were identified by chromatographic procedures (HPLC-DAD), ESI-MS, and NMR hyphenated techniques.