Plants have been used by humans since ancient times due their antimicrobial and medicinal properties. Essential oils (EOs) are complex mixtures of secondary plant metabolites, including terpenoids, phenylpropanoids, and other aromatic compounds. Cinnamomun verum and Thyme vulgaris EOs and their organic extracts exert numerous biological activities because of their major compounds, particularly thymol, carvacrol, eugenol, and benzoic and cinnamic acid. The structural motifs presented by these phytochemicals are responsible for their biological activities. Modification or hybridization of these structures could lead to new bio-based compounds with improved efficacy or multiple modes of action. In this work, we aimed to develop reliable methods of obtaining six hybrid molecules from the major constituents of C. verum and T. vulgaris EOs. For the first time, we tested their efficacy in the inhibition of the mycelium growth and spore germination of two of the most important phytopathogenic fungi, Fusarium oxysporum and Colletotrichum gloeosporioides, and one opportunistic human pathogen, Aspergillus niger. The cytotoxic activity of the obtained hybrids was assessed using the brine shrimp lethality assay. In addition, we report for the first time a biocatalytic process for the obtention of these bioactive hybrid molecules. The results of this work enable the possibility of using hybrid molecules based on the major constituents of EOs as active ingredients in strategic industries such as agriculture, aquaculture, and pharmaceuticals.

The extensive use of synthetic pesticides has created considerable concern for both human health and the environment, which has prompted the search for safer alternatives, such as the resin of guayule (Parthenium argentatum). Thus, in the present study, we aimed to test the biopesticide activity of crude guayule resin and three derived fractions and compare them to reference products that act against four of the most economically significant plant pests: Tetranychus urticae, Bemisia tabaci, Myzus persicae and Frankliniella occidentalis. None of the guayule products caused plant damage. The crude guayule resin and the hexane and ethyl acetate fractions displayed moderate to high contact mortality against T. urticae and B. tabaci, as well as moderate to high antifeedant activity against T. urticae, B. tabaci and M. persicae. No significant activity was observed against F. occidentalis. A correlation analysis of the activity and fraction composition revealed that guayulins C and D, isoargentatins A and B, argentatins A, B and D and an unknown compound C6 were likely responsible for the contact mortality. By contrast, the antifeedant activity appeared to be caused by guayulins A and B against T. urticae and B. tabaci and by guayulins C and D and argentatin B against M. persicae. The feeding reduction in F. occidentalis was associated with an unknown compound C2 and argentatin C. Therefore, guayule appears to be a promising novel biopesticide.

Botanical and fungal biopesticides, including endophytes, are in high demand given the current restrictive legislations on the use of chemical pesticides. As part of an ongoing search for new biopesticides, a series of fungal endophytes have been isolated from selected medicinal plants including Lauraceae species. In the current study, an extract from the endophytic fungus Trichoderma sp. EFI 671, isolated from the stem parts of the medicinal plant Laurus sp., was screened for bioactivity against plant pathogens (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi) and plant parasites (Meloidogyne javanica), with positive results against M. persicae. The chemical study of the neutral fraction of the active hexane extract resulted in the isolation of a triglyceride mixture (m1), eburicol (2), β-sitostenone (3), ergosterol (4) and ergosterol peroxide (5). The free fatty acids present in the acid fraction of the extract and in m1 (oleic, linoleic, palmitic and stearic) showed strong dose-dependent antifeedant effects against M. persicae. Liquid (potato dextrose broth, PDB and Sabouraud Broth, SDB) and solid (corn, sorghum, pearl millet and rice) growth media were tested in order to optimize the yield and bioactivity of the fungal extracts. Pearl millet and corn gave the highest extract yields. All the extracts from these solid media had strong effects against M. persicae, with sorghum being the most active. Corn media increased the methyl linoleate content of the extract, pearl millet media increased the oleic acid and sorghum media increased the oleic and linoleic acids compared to rice. The antifeedant effects of these extracts correlated with their content in methyl linoleate and linoleic acid. The phytotoxic effects of these extracts against ryegrass, Lolium perenne, and lettuce, Lactuca sativa, varied with culture media, with sorghum being non- toxic.

In the current study, an ethyl acetate extract from the endophytic fungus Trichoderma sp. EFI 671, isolated from the stem parts of the medicinal plant Laurus sp. was screened for bioactivity against plant pathogens (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi) and plant parasites (Meloidogyne javanica). The chemical study of this insect antifeedant extract resulted in the isolation of 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (1), eburicol (2), β-sitostenone (3), ergosterol (4) and ergosterol peroxide (5). The free fatty acids present in compound 1 (oleic, linoleic and palmitic) showed strong dose-dependent aphid antifeedant effects against M. persicae. Liquid (PDB, and SDB) and solid (corn, sorghum, pearl millet and rice) growth media were tested in order to optimize the yield and bioactivity of the fungal extracts. Pearl millet and corn gave the highest extract yields. All the extracts from these solid media had strong effects against M. persicae with sorghum being the most active. Corn increased the content in linolenic, pearl millet the oleic and stearic and sorghum oleic and linolenic acids compared to rice. Their antifeedant effects correlated with linoleic /oleic acids. The phytotoxic effects of these extracts against Lolium perenne and Lactuca sativa varied with culture media, with sorghum being the least toxic.

Increasing pesticide resistance in plant pathogens is major concern in agriculture production. Research on ecofriendly alternatives of chemical pesticides are more in demand in pesticide industry. In the current study, an ethyl acetate extract from the endophytic fungus Trichoderma sp. EFI 671, isolated from the stem parts of the medicinal plant Laurus sp., was screened for bioactivity against plant pathogens (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum and Botrytis cinerea), insect pests (Spodoptera littoralis, Myzus persicae, Rhopalosiphum padi) and plant parasites (Meloidogyne javanica). The bioactive components have been characterized following a bioassay-guided isolation against M. persicae. The chemical study of this bioactive extract resulted in the isolation of 1-oleoyl-2-linoleoyl-3-palmitoylglycerol (1), eburicol (2), (24R)-stigmast-4-ene-3-one or β-sitostenone (3), ergosterol (4) and ergosterol peroxide (5). The free fatty acids present in compound 1 (oleic, linoleic and palmitic) showed strong dose-dependent aphid antifeedant effects against M. persicae. Liquid (PDB, and SDB) and solid (corn, sorghum, pearl millet and rice) growth media were tested in order to optimize the yield and bioactivity of the fungal extracts. Pearl millet and corn gave the highest extract yields. All the extracts from these solid media had strong effects against M. persicae with sorghum being the most active. Corn increased the content in linolenic, pearl millet the oleic and stearic and sorghum oleic and linolenic acids compared to rice. Their antifeedant effects correlated with linoleic /oleic acids. The phytotoxic effects of these extracts against Lolium perenne and Lactuca sativa varied with culture media, with sorghum being the least toxic.

The nematicidal activity of hydrolate by-products from the semi industrial vapor-pressure essential oil extraction of selected aromatic plant species (commercial: Lavandula × intermedia Emeric ex Loisel. var. super, Thymus vulgaris L., T. zygis Loefl ex L. and experimentally pre-domesticated: L. luisieri (Rozeira) Rivas-Martínez) was investigated against the root-knot nematode Meloidogyne javanica by in vitro and in vivo bioassays. Liquid-liquid extraction of hydrolates yielded the corresponding aqueous and organic fractions which were biological and chemically studied. Hydrolates from L. × intermedia var. super, L. luisieri , T. vulgaris , and T. zygis showed strong in vitro nematicidal effects against M. javanica (J2 mortality and suppression of egg hatching). In the case of the Thymus species, the active components were found in the organic fraction, characterized by thymol as major component. Conversely, the nematicidal activity of L. × intermedia var. super and L. luisieri remained in the corresponding aqueous fractions. In vivo tests on tomato seedlings at sublethal doses of the hydrolates/organic fractions induced a significant reduction of nematode infectivity. In pot experiments, all hydrolates tested on tomato plants significantly affect the infection frequency and reproduction rate of the nematode population. This study demonstrates that L. × intermedia var. super, L. luisieri , T. vulgaris , and T. zygis hydrolates could be an exploitable source of potential waste protection products on root-knot nematodes.

Several extracts from pre-domesticated Lavandula luisieri (Rozeira) Riv-Mart. (Lamiaceae) (essential oil, EO; hexane, Hx and the organic fraction of the residual hydrolate, WRO) showed larvicidal effects against the hard tick Hyalomma lusitanicum, being WRO the most active. Their content in the necrodane-type monoterpenes (2,2,3,4-tetramethyl-5-oxocyclopent-3-en-1-yl)-methyl acetate (2), 5-hydroxymethyl-2,3,4,4-tetramethylcyclopent-2-en-1-one (1) and 2-(hydroxymethyl)-3,4,4-trimethyl-5-methylenecyclopent-2-en-1-one (5) correlated with these effects. Among the isolated compounds, 3,3,4,5-tetramethyl-2H-pyran-2,6(3H)-dione (7) and (2,2,3,4-tetramethyl-5-oxocyclopent-3-en-1-yl)-methyl acetate (2) were the most potent ones, with activity levels within the range of the positive control thymol. These compounds represent a new class of ixodicidal agents.

The absolute configuration (AC) of the naturally occurring ocimenes (-)-(3S,5Z)-2,6-dimethyl-2,3-epoxyocta-5,7-diene (1) and (-)-(3S,5Z)-2,6-dimethylocta-5,7-dien-2,3-diol (2), isolated from the essential oils of domesticated specimens of Artemisia absinthium, followed by vibrational circular dichroism (VCD) studies of 1, as well as from the acetonide 3 and the monoacetate 4, both derived from 2, since secondary alcohols are not the best functional groups to be present during VCD studies in solution due to intermolecular associations. The AC follows from comparison of experimental and calculated VCD spectra that were obtained by Density Functional Theory computation at the B3LYP/DGDZVP level of theory. Careful nuclear magnetic resonance (NMR) measurements were compared with literature values, providing for the first time systematic 1 H and 13 C chemical shift data. Regarding homonuclear 1 H coupling constants, after performing a few irradiation experiments that showed the presence of several small long-range interactions, the complete set of coupling constants for 3, which is representative of the four studied molecules, was determined by iterations using the PERCH software. This procedure even allowed assigning the pro-R and pro-S methyl group signals of the two gem-dimethyl groups present in 3.

Several preparations were obtained from the aerial parts of predomesticated Lavandula luisieri, including the essential oil and ethanolic, hexane, and ethyl acetate extractives. Additionally, pilot plant vapor pressure extraction was carried out at a pressure range of 0.5-1.0 bar to give a vapor pressure oil and an aqueous residue. A chemical study of the hexane extract led to the isolation of six necrodane derivatives (1, 2, and 4-7), with four of these (1, 2, 5, and 7) being new, as well as camphor, a cadinane sesquiterpene (9), tormentic acid, and ursolic acid. The EtOAc and EtOH extracts contained a mixture of phenolic compounds with rosmarinic acid being the major component. Workup of the aqueous residue resulted in the isolation of the necrodane 3 and (1R*,2S*,4R*)-p-menth-5-ene-1,2,8-triol (8), both new natural compounds. The structures of the new compounds were established based on their spectroscopic data. The phytotoxic and nematicidal activities of these compounds were evaluated.

Trabajo presentado en Natural Products and Biocontrol Congress, celebrado en Perpignan (France) del 21 al 23 de septiembre de 2016.

The objective of this study was the characterization of nematicidal activity and valorization of hydrolate obtained as a byproduct of the semi industrial vapor-pressure essential oil extraction of a domesticated Artemisia absinthium population (Teruel, Spain), in comparison with a population (Sierra Nevada, Spain) undergoing the domestication process. Hydrolates from three crops of these two populations showed strong in vitro nematicidal effects against the root knot nematode Meloidogyne javanica and similar chemical profiles. The hydrolate of the A. absinthium population from Teruel strongly suppressed nematode egg hatching (>95%) after 5 days of incubation. In vivo tests on tomato seedlings showed a significant reduction in the of root penetration rate of M. javanica J2 treated with a hydrolate sublethal concentration (33%). In pot experiments, the hydrolate of A. absinthium tested on tomato plants significantly affected the nematode population, the infection frequency and the reproduction rate. The extraction of the organic fraction of the hydrolate gave a nematicidal extract and an inactive aqueous residue. The chemical profile of the active organic extract showed the presence of (5Z)-2,6-dimethylocta5,7-diene-2,3-diol, previously described in the hydrodistilled essential oil of this A. absinthium population, among other compounds. This study demonstrates that the organic fraction of the A. absinthium hydrolate is a potential root-nematode control agent. Therefore, the hydrolate byproduct of the semi industrial vapor-pressure essential oil extraction could be a source of new nematicidal products. (C) 2016 Elsevier Ltd. All rights reserved.

The objective of this study was the characterization and valorization of the essential oil from a domesticated Artemisia absinthium population (Teruel, Spain), and its comparison with another one (Sierra Nevada, Spain) undergoing the domestication process. These populations are being experimentally cultivated in the same field since 2008. We studied their biomass and essential oil production (Clevenger hydrodistillation, HD and semi-industrial vapor-pressure, VP). The domesticated population showed lower chemical variation and higher biomass and essential oil yields, allowing for the registration of a new plant variety. The observed variations in oil composition (HD, VP) between the two populations were mostly quantitative. The oils were characterized by the presence of cis-epoxyocimene, (−)-cis-chrysanthenol, chrysanthenyl acetate, linalool and trans-caryophyllene. The insect antifeedant (Leptinotarsa decemlineata, Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi) and antifungal (Fusarium spp. and Botrytis cinerea) effects of their oils were also tested. All VP extracts showed strong antifungal effects and the active antifungal compounds have been identified.

Abstract Essential oils (EOs) obtained from two crops and populations of thujone-free cultivated Artemisia absinthium were tested against two nematode models, the mammalian parasite Trichinella spiralis, and the plant parasitic root knot nematode Meloidogyne javanica. The EOs were characterized by the presence of (Z)-epoxyocimene and chrysanthenol as major components and showed time and population dependent quantitative and qualitative variations in composition. The EOs showed a strong ex vivo activity against the L1 larvae of the nematode Trichinella spiralis with a reduction of infectivity between 72 and 100% at a dose range of 0.5–1 mg/ml in absence of cytotoxicity against mammalian cells. Moreover, the in vivo activity of the EO against T. spiralis showed a 66% reduction of intestinal adults. However, these oils were not effective against M. javanica.

Artemisia absinthium is an aromatic and medicinal plant of ethnopharmacological interest and it has been widely studied. The use of A. absinthium based on the collection of wild populations can result in variable compositions of the extracts and essential oils (EOs). The aim of this paper is the identification of the active components of the vapour pressure (VP) EO from a selected and cultivated A. absinthium Spanish population (T2-11) against two parasitic protozoa with different metabolic pathways: Trypanosoma cruzi and Trichomonas vaginalis. VP showed activity on both parasites at the highest concentrations. The chromatographic fractionation of the VP T2-11 resulted in nine fractions (VLC1-9). The chemical composition of the fractions and the antiparasitic effects of fractions and their main compounds suggest that the activity of the VP is related with the presence of trans-caryophyllene and di-hydrochamazulene (main components of fractions VLC1 and VLC2 respectively). Additionally, the cytotoxicity of VP and fractions has been tested on several tumour and no tumour human cell lines. Fractions VLC1 and VLC2 were not cytotoxic against the nontumoural cell line HS5, suggesting selective antiparasitic activity for these two fractions. The VP and fractions inhibited the growth of human tumour cell lines in a dose-dependent manner.

Trabajo presentado en el European Congress on Natural Products and Biocontrol celebrado en Perpignan (Francia) del 24 al 26 de septiembre de 2014.

The objective of this study was the preparation of supercritical (SCE), conventional hydrodistilled (HDE) and organic solvent (OSE) extracts from two Iberian Lavandula luisieri populations (A and B) to study their chemical composition and their insect antifeedant properties against Spodoptera littoralis and Myzus persicae. HDE and SCE extracts had different chemical compositions and insect antifeedant effects, extracts from population A being more effective. Compounds 5-hydroxymethyl-2,3,4,4-tetramethylcyclopent-2-en-1-one (6) and (2,2,3,4-tetramethyl-5-oxocyclopent-3-en-1-yl)-methyl acetate (7)were the major components of the SCE fractions of both populations. The supercritical technique (SFE) improved the extraction of 6, 7, oxo-cadinol (8), hexadecanoic acid and several not identified compounds with respect to HDE. SCE extracts also exhibited increased antifeedant effects against S. littoralis and the aphid M. persicae (A-SCE). The biological effects of the active extracts cannot be accounted for those of their individual major components, suggesting additive or synergistic effects.

Trabajo presentado en la XXXIV Reunion Bienal de la Real Sociedad Espanola de Quimica, celebrada en Santander del 15 al 18 de septiembre de 2013.

The objective of this study was the valorization of the essential oils from Spanish Artemisia absinthium domesticated plants from Teruel and Sierra Nevada (Spain). These populations were experimentally cultivated in the field and under controlled conditions. The insect antifeedant properties of their essential oils collected yearly from two locations were tested against Spodoptera littoralis, Myzus persicae and Rhopalosiphum padi. Additionally we studied their phytotoxic, antifungal and antiparasitic effects. The oils from cultivated A. absinthium were characterized by the presence of cis-epoxyocimene, chrysanthenol, and chrysanthenyl acetate. The variations observed in oil composition were mostly quantitative but also qualitative. (Z)-2,6-Dimethyl-5,7-octadien-2,3-diol has been isolated and identified by NMR. Among the oil samples, these rich in cis-epoxyocimene and sesquiterpenes were the most active ones against S. littoralis. (Z)-2,6-Dimethyl-5,7-octadien-2,3-diol showed moderate activity against S. littoralis. The strongest antifeedant effects were found for commercial A. absinthium oil samples rich in thujones and sabinyl acetate. F. oxysporum and F. solani were affected by oils from cultivated A. absinthium and commercial oil samples. Oils from cultivated A. absinthium showed antiparasitic effects against Leishmania infantum and Trypanosoma cruzi with better results than the commercial samples.