Carlos L. Céspedes

Tyrosinase Inhibitors from Galls of Rhus javanica Leaves and Their Effects on Insects

Abstract As a defense mechanism of the leaves of Rhus javanica (Anacardiaceae) against the aphid Melaphis chinensis (Aphididae) attack, tannic acid is rapidly accumulated and forms galls along the midrib of the leaves resulting in a unique natural medicine Gallae Rhois. Tannic acid was found to inhibit the oxidation of ʟ-3,4-dihydroxyphenylalanine (ʟ-DOPA) catalyzed by tyrosinase (EC 1.14.18.1) with an IC50 of 22 μᴍ. The aphid would detoxify the ingested toxic tannic acid to relatively nontoxic gallic acid, whereas the non-adapted pink bollworm Pectinophora gossypiella larvae are sensitive to the ingested tannic acid.

Acetylcholinesterase and Insect Growth Inhibitory Activities of Gutierrezia microcephala on Fall Army worm Spodoptera frugiperda J. E. Smith

From the aerial parts of Gutierrezia microcephala (Asteraceae), four oxyflavones were isolated, namely 5,7,2′-trihydroxy-3,6,8,4′,5′-pentamethoxyflavone (1); 5,7,4′-trihydroxy-3,6,8-trimethoxyflavone (2); 5,7,2′,4′-tetrahydroxy-3,6,8,5′-tetramethoxyflavone (3); 5,2′-dihydroxy- 3,6,7,8,4′,5′-hexamethoxyflavone (4), and an ent-clerodane, bacchabolivic acid (5). Compounds 1-5, the synthetic methyl ester (6 ), n-hexane and MeOH extracts were evaluated against the fall armyworm (Spodoptera frugiperda). Gedunin, a known insect growth regulator isolated from Cedrela spp. was used as a positive control. When tested for activity on neonate larvae into the no-choice artificial diet bioassay, flavone (1), clerodane (5), its methyl ester (6), MeOH and n-hexane extracts caused significant larval mortality with MC50 of 3.9, 10.7, 3.46. 7.95 and 7.5 ppm at 7 days, respectively, as well as growth reduction. They also increased the development time of surviving larvae and a significant delay in time to pupation and adult emergence. Acute toxicity against adults of S. frugiperda was also found, 5, 6 , gedunin and n-hexane extract had the most potent activity with LD50 value of 6.59, 15.05, 10.78, and 12.79 ppm, respectively. In addition, MeOH, n-hexane extracts, 5, 6 and gedunin caused acetylcholinesterase inhibition with 93.7,100,90.2,62.0 and 100% at 50.0 ppm, respectively; whereas 1-4 exhibited only moderate inhibitory activity. Compounds 1,5 and 6 showed inhibitory activities comparable with gedunin. These compounds could be responsible of the insect growth inhibitory activity of this plant.

Insect growth regulator and insecticidal activity of beta-dihydroagarofurans from Maytenus spp. (Celastraceae).

From the aerial parts of Maytenus disticha, we have isolated 9β-benzoyloxy-1α,2α,6β,8α,15- penta-acetoxy-dihydro-β-agarofuran (1) and from seeds of Maytenus boaria 9β-furoyloxy-1α, 6β, 8α-triacetoxy-dihydro-β-agarofuran (2). These compounds and their MeOH and hexane/ethyl acetate (1:1 v/v) extracts were evaluated for their effects on the fall armyworm (Spodoptera frugiperda). Toosendanin, a commercial insecticide derived from Melia azedarach was used as a positive control. When tested for activity using neonate larvae in a nochoice artificial diet bioassays, the agarofurans 1, 2 and toosendanin as well as the MeOH and hexane/EtOAc extracts caused significant growth inhibitory effects with GC50 of 7.55; 3.84; 1.75; 14.0 and 7.3 ppm at 7 days, respectively. Compounds 1 and 2 caused 100% larval mortality at 25 and 15 ppm, respectively. MeOH and hexane/EtOAc extracts caused 100% larval mortality at 25.0 ppm, respectively, they also increased the development time of surviving larvae and a significant delay for the time of pupation and adult emergence. These compounds showed comparable potency of activity with toosendanin. Acute toxicity against adults of S. frugiperda was also found, for hexane/EtOAc extract and 2 had the most potent activity with LD50 value of 4.7 and 1.9 ppm, respectively. MeOH extract, hexane/EtOAc extract, 1 and 2 caused acetylcholinesterase inhibition with 78.0, 89.2, 79.3 and 100% inhibition at 15.0 ppm, respectively. Therefore, the furoyloxy agarofuran may be responsible for the insecticidal activity of these plants

Tyrosinase inhibition kinetics of anisic acid.

Abstract Anisic acid (p-methoxybenzoic acid) was characterized as a tyrosinase inhibitor from aniseed, a common food spice. It inhibited the oxidation of l-3,4-dihydroxyphenylalanine (ʟ- DOPA) catalyzed by tyrosinase with an IC50 of 0.60 mᴍ. The inhibition of tyrosinase by anisic acid is a reversible reaction with residual enzyme activity. This phenolic acid was found to be a classical noncompetitive inhibitor and the inhibition constant KI was obtained as 0.603 mᴍ. Anisic acid also inhibited the hydroxylation of ʟ-tyrosine catalyzed by tyrosinase. The lag phase caused by the monophenolase activity was lengthened and the steady-state activity of the enzyme was decreased by anisic acid.

Antifungal and Antibacterial Activities of Araucaria araucana (Mol.) K. Koch Heartwood Lignans

Abstract Five lignans (secoisolariciresinol, pinoresinol, eudesmin, lariciresinol, and lariciresinol-4- methyl ether) were isolated from an MeOH extract from Araucaria araucana (Mol.) K. Koch wood for the first time in this species and their structures determined with spectroscopic methods. The antimicrobial activities of these compounds were determined for the bacteria Citrobacter sp., Bacillus subtilis, Escherichia coli, Micrococcus luteus, Staphylococcus aureus, and Pseudomonas aeruginosa, and for the white rooting and staining fungi Mucor miehei, Paecilomyces variotii, Ceratocystis pilifera, Trametes versicolor, and Penicillium notatum, and in addition, the MeOH extract was evaluated against Aspergillus niger, Candida albicans, Fusarium moniliforme, F. sporotrichum and Trichophyton mentagrophytes. The most sensitive bacteria against pinoresinol were the Gram-positive. However, secoisolariciresinol exhibited a significant antifungal activity on fungi of white rooting and wood staining and this compound completely inhibited the mycelial growth of T. versicolor and C. pilifera at 300 and 400 μg per disc, respectively, whereas pinoresinol showed a moderate inhibitory activity. On the other hand, the MeOH extract had the highest activity against rooting and staining and pathogenic fungi as well as T. versicolor, Fusarium spp. and Trichophyton mentagrophytes, inhibiting completely the growth at 400 μg per disc

Biopesticides from plants: Calceolaria integrifolia s.l.

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 against the Gram-positive bacteria Bacillus subtilis, Sarcinia lutea, and Staphylococcus 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.

Insect growth regulatory activity of some extracts and compounds from Parthenium argentatum on fall armyworm Spodoptera frugiperda.

Abstract Argentatins, Insect Growth Regulators, Acetylcholinesterase The methanolic extract from aerial parts of Parthenium argentatum, afforded argentatin A and B. These compounds were evaluated for their effect on the fall armyworm (Spodoptera frugiperda). Toosendanin, a commercial insecticide derived from Melia azedarach was used as positive control. When tested for activity, using neonate larvae into the no-choice artificial diet bioassays, argentatin A, argentatin B and methanol extract caused significant growth inhibitory activity with GC50 of 17.8, 36.1 and 6.4 ppm at 7 days, respectively, and increased the development time of surviving larvae in a concentration-dependent manner with RGI values of 0.40, 0.60 and 0.26, at 25.0, 25.0 and 5.0 ppm. respectively. In addition, it was possible to observe in most of the treated groups a significant delay in the time of pupation, adult emergence and deformities. Acute toxicity against adults of S. frugiperda was also found, MeOH extract had the most potent activity with LD50 value of 3.10 ppm. In addition, MeOH extract and argentatin A caused acetylcholinesterase inhibition of 93.7% and 90.0%, at 5.0 and 50.0 ppm, respectively; whereas argentatin B had only slight inhibitory activity. Therefore, the MeOH extract was identified as insecticidal extract from P. argentatum with activity at concentrations above 15.0 ppm.

Tyrosinase inhibitors from Calceolaria integrifolia s.l.: Calceolaria talcana aerial parts.

As a defense mechanism of the aerial parts of Calceolaria talcana (Calceolariaceae; formerly Scrophulariaceae) against herbivore offenses and insect pest attack, diterpenoids, triterpenoids, phenylethanoids, flavonoids, and iridoids are rapidly accumulated along the aerial parts, resulting in a unique natural biopesticide complex from this plant. In addition to verbascoside a series of known compounds were screened for their inhibitory activity against mushroom tyrosinase and protease enzymes. Ethyl acetate and n-hexane extracts, together with cyclopropyl-7,15-ent-pimaradiene (1), abietatriene (2), ursolic acid (3), α-lupeol (4), β-sitosterol (5), 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (6), α-dunnione (7), verbascoside (8), martynoside (9), and some known model compounds proved to be inhibitors of oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by tyrosinase (EC 1.14.18.1) with an IC50 between 10.0 and 200 ppm or μM, respectively, suggesting that phenolic moieties in the molecules assayed are important for the activity.

Inhibition of cholinesterase activity by extracts, fractions and compounds from Calceolaria talcana and C. integrifolia (Calceolariaceae: Scrophulariaceae)

Extracts, fractions and compounds from Calceolaria talcana and C. integrifolia exhibited strong inhibitory effects of the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using the in vitro Ellmańs method. The most active samples were from the ethyl acetate extract, which caused a mixed-type inhibition against AChE (69.8% and 79.5% at 100 and 200 μg/ml, respectively) and against BChE (98.5% and 99.8% at 100 and 200 μg/ml, respectively) and its major components verbascoside 8 (50.9 and 70.0% at 200 μg/ml, against AChE and BChE, respectively), martynoside 9, and fraction F-7 (which corresponds to a mixture of 8, 9, and other phenylethanoids and phenolics that remain unidentified) (80.2 and 85.3% at 100 and 200 μg/ml, against AChE, respectively and 99.1 and 99.7% at 100 and 200 μg/ml, against BChE, respectively) inhibited the acetylcholinesterase enzyme competitively. The most polar fraction F-5 from n-hexane extract (a mixture of naphthoquinones: 2-hydroxy-3-(1,1-dimethylallyl-1,4-naphthoquinone) 6, α-dunnione 7 and other polar compounds that remain unidentified) showed a mixed-type inhibition (71.5 and 72.1% against AChE and BChE at 200 μg/ml, respectively). Finally, the methanol-soluble residue presented a complex, mixed-type inhibition (39.9 and 67.9% against AChE and BChE at 200 μg/ml, respectively). The mixture F-3 with diterpenes was obtained from the n-hexane extract: (1,10-cyclopropyl-9-epi-ent-isopimarol) 1, 19-α-hydroxy-abietatriene 2, and F-4 a mixture of triterpenes α-lupeol 3, β-sitosterol 4, ursolic acid 5 together with a complex mixture of terpenes that did not show activity. In summary, extracts and natural compounds from C. talcana and C. integrifolia were isolated, identified and characterized as cholinesterase inhibitors.

Plant growth inhibitory activities by secondary metabolites isolated from Latin American flora

This chapter studies phytochemicals that are biodirected, to find botanical-origin biopesticides targeted against weeds. It describes the identification of some monoterpenes, diterpenes, sesquiterpene lactones, limonoids, triterpenes, coumarins, and flavonoids, including their chemical derivatives from plants belonging to the families Asteraceae, Celastraceae, Gomortegaceae, Lauraceae, Meliaceae, Monimiaceae, Poaceae, and Winteraceae. The findings show that some natural compounds or their derivatives possess plant growth regulatory (PGR) or herbicidal activities. Compounds with acetylated and α-β-unsaturated carbonyl derivatives showed mainly PGR activity. It seems that an α-methylene-γ-lactone and a group α,β-unsaturated carbonyl are important for structural activity requirements. Allelopathic activities were assayed with Triticum aestivum, T. vulgare, Trifolium pratense, T. alexandrinum, T. angustifolium, Lolium multiflorum, Lactuca sativa, Physalis ixocarpa, and Raphanus sativus as weed seeds models. All of them are weedy pests in wheat, oat, potatoes, corn, bean, and other important crops of Latin America. Very little is known about the inhibitory activity of these natural compounds and their derivatives. Natural compounds that we have isolated represent a valuable resource for the study toward noxious weedy species of the allelopathic activities of these plants and their control. Progress in biochemical and allelopathic characterization of this pathway is outlined.