Essential Oil Composition of Grindelia squarrosa from Southern Idaho.

Grindelia squarrosa is an arid lands herb that has been used in Native American traditional medicine, is a potential source of pharmacologically active compounds, and has been explored as a source of biofuel. The purpose of this work was to examine the essential oil composition of G. squarrosa from southern Idaho. Gas chromatographic methods revealed the essential oil of G. squarrosa var. serrulata to be rich in monoterpenoids, α-pinene (21.9%), limonene (17.1%), terpinolene (10.6%), and borneol (6.5%). The essential oil composition of G. squarrosa from Idaho is similar to that previously reported from specimens collected from Montana and confirms the volatile phytochemistry of plants growing in North America. The major essential oil components were screened for antimicrobial activity against respiratory and dermal pathogens. (-)-ß-Pinene showed strong antibacterial activity against Streptococcus pneumoniae (MIC 39.1 µg/mL) and (-)-borneol showed strong activity against Staphylococcus aureus (MIC 78.1 µg/mL).

Neutrophil Immunomodulatory Activity of (-)-Borneol, a Major Component of Essential Oils Extracted from Grindelia squarrosa.

Grindelia squarrosa (Pursh) Dunal is used in traditional medicine for treating various diseases; however, little is known about the immunomodulatory activity of essential oils from this plant. Thus, we isolated essential oils from the flowers (GEOFl) and leaves (GEOLv) of G. squarrosa and evaluated the chemical composition and innate immunomodulatory activity of these essential oils. Compositional analysis of these essential oils revealed that the main components were α-pinene (24.7 and 23.2% in GEOFl and GEOLv, respectively), limonene (10.0 and 14.7%), borneol (23.4 and 16.6%), p-cymen-8-ol (6.1 and 5.8%), ß-pinene (4.0 and 3.8%), bornyl acetate (3.0 and 5.1%), trans-pinocarveol (4.2 and 3.7%), spathulenol (3.0 and 2.0%), myrtenol (2.5 and 1.7%), and terpinolene (1.7 and 2.0%). Enantiomer analysis showed that α-pinene, ß-pinene, and borneol were present primarily as (-)-enantiomers (100% enantiomeric excess (ee) for (-)-α-pinene and (-)-borneol in both GEOFl and GEOLv; 82 and 78% ee for (-)-ß-pinene in GEOFl and GEOLv), while limonene was present primarily as the (+)-enantiomer (94 and 96 ee in GEOFl and GEOLv). Grindelia essential oils activated human neutrophils, resulting in increased [Ca2+]i (EC50 = 22.3 µg/mL for GEOFl and 19.4 µg/mL for GEOLv). In addition, one of the major enantiomeric components, (-)-borneol, activated human neutrophil [Ca2+]i (EC50 = 28.7 ± 2.6), whereas (+)-borneol was inactive. Since these treatments activated neutrophils, we also evaluated if they were able to down-regulate neutrophil responses to subsequent agonist activation and found that treatment with Grindelia essential oils inhibited activation of these cells by the N-formyl peptide receptor 1 (FPR1) agonist fMLF and the FPR2 agonist WKYMVM. Likewise, (-)-borneol inhibited FPR-agonist-induced Ca2+ influx in neutrophils. Grindelia leaf and flower essential oils, as well as (-)-borneol, also inhibited fMLF-induced chemotaxis of human neutrophils (IC50 = 4.1 ± 0.8 µg/mL, 5.0 ± 1.6 µg/mL, and 5.8 ± 1.4 µM, respectively). Thus, we identified (-)-borneol as a novel modulator of human neutrophil function.

Inula helenium and Grindelia squarrosa as a source of compounds with anti-inflammatory activity in human neutrophils and cultured human respiratory epithelium.

ETHNOPHARMACOLOGICAL RELEVANCE: During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1ß and TNF-α) release and downregulation of adhesive molecules. AIM OF THE STUDY: During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. MATERIALS AND METHODS: The effect on IL-8, IL-1ß and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. RESULTS: We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of ß2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1ß release comparable with budesonide, used as a positive control. CONCLUSIONS: The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I. helenium and G. squarrosa for a treatment of inflammation-based diseases in respiratory tract.

Chemical composition and antimicrobial activity of the essential oils from flowers and leaves of Grindelia integrifolia DC.

Essential oils from flowers and leaves of Grindelia integrifolia DC. were investigated for the first time in terms of chemical composition and antimicrobial activity. The GC-FID/MS analysis allowed for the identification of 58 and 72 volatiles, comprising 92.4 and 90.1% of the oils, respectively. The major components of the flower oil were α-pinene (34.9%) and limonene (13.1%), while myrcene (16.9%), spathulenol (12.3%), ß-eudesmol (11.9%) and limonene (10.1%) dominated among the leaf volatiles. The antimicrobial activity, evaluated against 12 selected bacteria and fungus, was found moderate, with the strongest effect of both oils observed against C. albicans (MIC = MBC: 0.63 and 0.31 mg/mL for flower and leaf oil, respectively).

Parallel Pleistocene amphitropical disjunctions of a parasitic plant and its host.

PREMISE OF THE STUDY: Aphyllon is a clade of holoparasites that includes closely related North American and South American species parasitic on Grindelia. Both Aphyllon (Orobanchaceae) and Grindelia (Asteraceae) have amphitropical disjunctions between North America and South America; however, the timing of these patterns and the processes to explain them are unknown. METHODS: Chronograms for the Orobanchaceae and Grindelia and their relatives were constructed using fossil and secondary calibration points, one of which was based on the inferred timing of horizontal gene transfer from a papilionoid legume into the common ancestor of Orobanche and Phelipanche. Elevated rates of molecular evolution in the Orobanchaceae have hindered efforts to determine reliable divergence time estimates in the absence of a fossil record. However, using a horizontal gene transfer event as a secondary calibration overcomes this limitation. These chronograms were used to reconstruct the biogeography of Aphyllon, Grindelia, and relatives using a DEC+J model implemented in RevBayes. KEY RESULTS: Aphyllon had two amphitropical dispersals from North America to South America, while Grindelia had a single dispersal. The dispersal of the Aphyllon lineage that is parasitic on Grindelia (0.40 Ma) took place somewhat after Grindelia began to diversify in South America (0.93 Ma). Using a secondary calibration based on horizontal gene transfer, we infer more recent divergence dates of holoparasitic Orobancheae than previous studies. CONCLUSIONS: Parallel host-parasite amphitropical disjunctions in Grindelia and Aphyllon illustrate one means by which ecological specialization may result in nonindependent patterns of diversity in distantly related lineages. Although Grindelia and Aphyllon both dispersed to South America recently, Grindelia appears to have diversified more extensively following colonization. More broadly, recent Pleistocene glaciations probably have also contributed to patterns of diversity and biogeography of temperate northern hemisphere Orobancheae. We also demonstrate the utility of using horizontal gene transfer events from well-dated clades to calibrate parasite phylogenies in the absence of a fossil record.

Cativic acid-caffeic acid hybrid exerts cytotoxic effects and induces apoptotic death in human neuroblastoma cells.

The development of hybrids from natural products is a promising strategy for drug discovery. In cancer therapy, there is a need to discover novel agents that can induce apoptosis in cancer cells. To contribute to this field of interest, we investigated the effect of a synthetic hybrid from cativic acid and caffeic acid (5) on viability, proliferation, and apoptosis in human neuroblastoma cells (IMR-32). Three hybrids were prepared via Mitsunobu esterification from 17-hydroxycativic acid (1) and natural phenols. Cell viability was analyzed by MTT assay. SYTOX green and LDH leakage were used to determine the cytotoxic effect. Caspase-3 activity, cell cycle phases, and proliferation were analyzed in order to characterize the biological effects of hybrid 5. The mitogen-activated protein kinase (MAPK) status was evaluated for elucidating the potential mechanisms involved in hybrid 5 effect. Hybrid 5 reduced the viability of IMR-32 cells in a time- and concentration-dependent manner (IC50 = 18.0 ± 1.3 µM) as a result of its antiproliferative effect through changes in the cell cycle distribution and induction of apoptosis associated with activation of caspase-3. Exposure to 5 triggered ERK1/2 activation and nuclear translocation. Hybrid 5 also promoted an increase in nuclear localization of the transcription factor c-Jun. Inhibition of ERK1/2 and JNK potentiated 5-induced inhibition of IMR-32 viability. Hybrid 5 displays cell growth inhibition by promoting cell cycle arrest and apoptosis, through ERK1/2 and JNK participation.

A New Depigmenting-Antifungal Methylated Grindelane from Grindelia chiloensis.

The new methylated grindelane diterpenoid, 7ß-hydroxy-8(17)-dehydrogrindelic acid (1b), together with the known 7α-hydroxy-8(17)-dehydrogrindelic acid (2a), 6-oxogrindelic acid (3a), 4ß-hydroxy-6-oxo-19-norgrindelic (4a), 19-hydroxygrindelic acid (5a), 18-hydroxygrindelic acid (6a), 4α-carboxygrindelic acid (7a), 17-hydroxygrindelic acid (8a), 6α-hydroxygrindelic acid (9a), 8,17-bisnor-8-oxagrindelic acid (10a), 7α,8α-epoxygrindelic acid (11a), and strictanonic acid (12a) as methyl esters were obtained from an Argentine collection of Grindelia chiloensis (Cornel.) Cabrera. Their structures and relative configurations were established on the basis of spectroscopic analysis. CHCl3 extract from the aerial parts and their pure compounds were evaluated for their antifungal and depigmenting effects. Methyl ester derivative of 10a (10b) exhibited a remarkable mycelial growth inhibition against Botritis cinerea with an IC50 of 13.5 µg ml-1 . While the new grindelane 1b exerted a clear color reduction of the yellow-orange pigment developed by Fusarium oxysporum against UV-induced damage.

Bet-hedging against larval herbivory and seed bank mortality in the evolution of heterocarpy.

PREMISE OF THE STUDY: Bet-hedging strategies maximize long-term geometric fitness at the cost of reduced arithmetic fitness by offsetting different mortality risks. Heterocarpic systems accomplish bet-hedging through the production of two or more fruit types that vary in dormancy and dispersal ability. It is unknown whether heterocarpy also offsets predispersal mortality risks. To address this question, we investigated whether heterocarpy in Grindelia ciliata (Asteraceae) also offsets mortality risks posed by a seed predator Schinia mortua (Noctuidae) to increase plant fitness. METHODS: We conducted two manipulative experiments to quantify critical life history components of this plant-insect interaction. We measured predispersal achene mortality from herbivory, postdispersal achene mortality in the seed bank, and seedling emergence. These measurements were then used in deterministic models to evaluate evolutionary consequences of predispersal seed mortality in G. ciliata. KEY RESULTS: Dormant achene types were less vulnerable to herbivory but more susceptible to mortality in the seed bank due to delayed seed emergence. Nondormant achene types experienced high predispersal mortality but low seed bank mortality due to rapid germination. Our herbivore-dependent model improved fit between observed and expected proportions of dormant and nondormant G. ciliata achenes and showed that heterocarpy could evolve in the absence of postgermination mortality. CONCLUSIONS: Our study provides empirical support of how predispersal herbivory can be equally important to postdispersal seed mortality risks in the evolution and maintenance of a heterocarpic reproductive system and expands understanding of how bet-hedging theory can be used to understand this unique reproductive strategy.

Exploring diterpene metabolism in non-model species: transcriptome-enabled discovery and functional characterization of labda-7,13E-dienyl diphosphate synthase from Grindelia robusta.

Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surfaces, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts.

Actividad antituberculosa in vitro de los extractos etanólicos y agliconas de flavonoides de las especies: Grindelia boliviana Rusby (ch'iri ch'iri) y Chenopodium incisum Poir (Arq'a paiqo) sobre cepas de Micobacterium tuberculosis H37Rv / In vitro antituberculous activity of the ethanolic extracts and flavonoid aglycones of the species: Bolivian grindelia Rusby (ch'iri ch'iri) and Chenopodium incisum Poir (Arq'a paiqo) on strains of Mycobacterium tuberculosis H37Rv

El presente trabajo de investigación fue realizado con el propósito de determinar la actividad antituberculosa in vitre de los extractos etanólicos y agliconas de flavonoides de las especies Grindelia boliviana Rusby (Ch'iri ch'iri) y Chenopodium incisum Poir. (Arq'a paiqo) frente a la cepa de Micobacterium tuberculosis H37Rv. La investigación inicia con la recolección de las plantas, secado y molienda de las partes aéreas (hojas, tallos, flores y semillas) para luego ser extraída con solución de etanol al 70% por baño ultrasónico. Los extractos obtenidos se filtran y se depositan en un balón para ser concentrados, en el rotavapor a 40°C individualmente, posteriormente se realizaron las pruebas de solubilidad, identificación cualitativa de los metabolitos secundarios y porcentaje de extracción. Para la extracción de agliconas de flavonoides se tomó material vegetal seco de cada especie, realizándose la extracción con alcohol al 30% en baño ultrasónico, luego se filtró y centrifugó obteniéndose la fase acuosa de interés, los extractos así obtenidos fueron sometidos a una hidrólisis ácida con HCI 6N a la temperatura de 100°C a ebullición. El precipitado obtenido fue purificado en una columna de C-18, obteniéndose las agliconas de flavonoides los cuales fueron !dentificados por cromatografía líquida de alta resolución. Utilizando el método de proporciones de CANETTI, RIST Y GROSSET 1963, se procedió a comprobar la actividad antituberculosa in vitre de los extractos etanólicos y las agliconas de flavonoides, los cuales fueron incorporados al medio Lowenstein Jensen (MU) partiendo desde una concentración mínima hasta una concentración máxima. Luego se realizó el sembrado de las diluciones 10-3 mg/mL, 10-5 mg/mL y 10-6 mg/mL de Micobacterium tuberculosis H37Rv, a los tubos con los medios de Lowenstein Jensen de los extractos etanólicos, como de las agliconas de flavonoides. Finalmente se lleva a la incubadora a 37°c y se espera los 42 días para hacer las lecturas respectivas. Los resultados mostraron: Para los extractos etanólicos. El extracto etanólico de Grindelia boliviana Rusby (Ch'iri ch'iri) presentó una buena actividad antituberculosa a partir de la concentración de 33 mg/mL que sería la CMI, respecto al Chenopodium incisum Poir (Arq'a paiqo) presentó una mejor actividad antimicobacteriana con una CMI de 29 mg/mL, lo cual le confiere una significativa actividad antituberculosa. Para las agliconas de flavonoides. Las agliconas de flavonoides de Grindelia boliviana Rusby (Ch'iri ch'iri) presentó una CMI de 2.8 mg/MI, dicha actividad podría deberse a la presencia de las agliconas de flavonoides que no fueron identificadas. Respecto a las agliconas de Chenopodium incisum Poir (Arq'a paiqo) también presentó una CMI en 2.8 mg/mL. En el análisis por HPLC se logró identificar la aglicona de flavonoide (kaempferol) por comparación con su respectivo estándar, probablemente este metabolito secundario sería el responsable de la actividad antituberculosa. A partir de los resultados presentados se realizó la prueba estadística de ANOVA y POS ANOVA (TUKEY) teniendo como variable independiente las diferentes concentraciones de los extractos y como variable dependiente la actividad antituberculosa cuya significancia es 0.00, el cual indica que existe una diferencia significativa entre las distintas concentraciones

Synthesis and cholinesterase inhibition of cativic acid derivatives.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with memory impairment and cognitive deficit. Most of the drugs currently available for the treatment of AD are acetylcholinesterase (AChE) inhibitors. In a preliminary study, significant AChE inhibition was observed for the ethanolic extract of Grindelia ventanensis (IC50=0.79 mg/mL). This result prompted us to isolate the active constituent, a normal labdane diterpenoid identified as 17-hydroxycativic acid (1), through a bioassay guided fractionation. Taking into account that 1 showed moderate inhibition of AChE (IC50=21.1 µM), selectivity over butyrylcholinesterase (BChE) (IC50=171.1 µM) and that it was easily obtained from the plant extract in a very good yield (0.15% w/w), we decided to prepare semisynthetic derivatives of this natural diterpenoid through simple structural modifications. A set of twenty new cativic acid derivatives (3-6) was prepared from 1 through transformations on the carboxylic group at C-15, introducing a C2-C6 linker and a tertiary amine group. They were tested for their inhibitory activity against AChE and BChE and some structure-activity relationships were outlined. The most active derivative was compound 3c, with an IC50 value of 3.2 µM for AChE. Enzyme kinetic studies and docking modeling revealed that this inhibitor targeted both the catalytic active site and the peripheral anionic site of this enzyme. Furthermore, 3c showed significant inhibition of AChE activity in SH-SY5Y human neuroblastoma cells, and was non-cytotoxic.

Chemical composition of essential oils of Grindelia squarrosa and G. hirsutula.

The essential oils of Grindelia squarrosa (Pursh) Dunal and G. hirsutula Hook. & Am. cultivated in Romania were isolated by hydrodistillation. The essential oils were analyzed by a combination of GC-FID and GC-MS. The identification of the constituents was achieved from their retention indices and comparison of their MS data with computer library database and literature data. The fifty-six identified constituents accounted for 72.1-81.3% of the oils. The oils were found to contain a-pinene, beta-pinene, limonene, borneol, bornyl acetate and germacrene D as main constituents. The oils obtained from the two species showed small differences in chemical composition. However, menthol, menthone and pulegone were detected only in the essential oil of G. hirsutula.

Genetic and ecotypic differentiation in a Californian plant polyploid complex (Grindelia, Asteraceae).

Studies of ecotypic differentiation in the California Floristic Province have contributed greatly to plant evolutionary biology since the pioneering work of Clausen, Keck, and Hiesey. The extent of gene flow and genetic differentiation across interfertile ecotypes that span major habitats in the California Floristic Province is understudied, however, and is important for understanding the prospects for local adaptation to evolve or persist in the face of potential gene flow across populations in different ecological settings. We used microsatellite data to examine local differentiation in one of these lineages, the Pacific Coast polyploid complex of the plant genus Grindelia (Asteraceae). We examined 439 individuals in 10 different populations. The plants grouped broadly into a coastal and an inland set of populations. The coastal group contained plants from salt marshes and coastal bluffs, as well as a population growing in a serpentine grassland close to the coast, while the inland group contained grassland plants. No evidence for hybridization was found at the single location where adjacent populations of the two groups were sampled. In addition to differentiation along ecotypic lines, there was also a strong signal of local differentiation, with the plants grouping strongly by population. The strength of local differentiation is consistent with the extensive morphological variation observed across populations and the history of taxonomic confusion in the group. The Pacific Clade of Grindelia and other young Californian plant groups warrant additional analysis of evolutionary divergence along the steep coast-to-inland climatic gradient, which has been associated with local adaptation and ecotype formation since the classic studies of Clausen, Keck, and Hiesey.

Inhibition of NO production by Grindelia argentina and isolation of three new cytotoxic saponins.

A bioassay-guided phytochemical analysis of the ethanolic extract of Grindelia argentina Deble & Oliveira-Deble (Asteraceae) allowed the isolation of a known flavone, hispidulin, and three new oleanane-type saponins, 3-O-ß-D-xylopyranosyl-(1→3)-ß-D-glucopyranosyl-2ß,3ß,16α,23-tetrahydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (2), 3-O-ß-D-glucopyranosyl-2ß,3ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester, (3) and 3-O-ß-D-xylopyranosyl-(1→3)-ß-D-glucopyranosyl-2ß,3ß,23-trihydroxyolean-12-en-28-oic acid 28-O-ß-D-xylopyranosyl-(1→2)-ß-D-apiofuranosyl-(1→3)-ß-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl ester (4), named grindeliosides A-C, respectively. Their structures were determined by extensive 1D- and 2D-NMR experiments along with mass spectrometry and chemical evidence. The isolated compounds were evaluated for their inhibitory activities against LPS/IFN-γ-induced NO production in RAW 264.7 macrophages and for their cytotoxic activities against the human leukemic cell line CCRF-CEM and MRC-5 lung fibroblasts. Hispidulin markedly reduced LPS/IFN-γ-induced NO production (IC50 51.4 µM), while grindeliosides A-C were found to be cytotoxic, with grindelioside C being the most active against both CCRF-CEM (IC50 4.2±0.1 µM) and MRC-5 (IC50 4.5±0.1 µM) cell lines.

HPLC-DAD-ESI/MS(n) analysis of phenolic compounds for quality control of Grindelia robusta Nutt. and bioactivities.

The phenolic composition of herbal tea (HT) and hydromethanolic extract (HME) obtained from Grindelia robusta Nutt. was studied by HPLC-DAD-ESI/MS(n). Thirty six flavonoids and hydroxycinnamic acids were detected, from which thirty are described for the first time in this species. Quantification by HPLC-DAD showed that diosmetin-7-O-glucuronide-3'-O-pentoside+apigenin-7-O-glucuronide-4'-O-pentoside, apigenin-7-O-glucuronide+diosmetin-7-O-glucuronide and 3,5-dicaffeoylquinic acid+3,4-dicaffeoylquinic acid were the major compounds. Since the health-promoting effects of natural phenolic compounds against brain disorders is of increasing interest, HT and HME were also tested against oxygen and nitrogen reactive species and against enzymes related with Alzheimer's disease and depression. Extracts displayed strong in vitro scavenging activity and monoamine oxidase-A (MAO-A) inhibitory activity. The anti-MAO-A capacity was observed at non-toxic concentrations for SH-SY5Y human neuroblastoma cell line, reinforcing the benefits of G. robusta HT. However, no protection against hydrogen peroxide treatment was observed.

Phenolic acids in the flowers and leaves of Grindelia robusta Nutt. and Grindelia squarrosa Dun. (Asteraceae).

2D-TLC and RP-HPLC methods were applied to qualitatively determinate free phenolic acids and those liberated by acid and alkaline hydrolysis in the flowers and leaves of G. robusta and G. squarrosa. The presence of eleven phenolic acids, namely: caffeic, chlorogenic, p-coumaric, p-hydroxybenzoic, ferulic, gallic, protocatechuic, vanillic salicylic, p-hydroxyphenylacetic and ellagic acids was determined. Quantitative estimate of phenolic acids, expressed as caffeic acid, has been analyzed by the method described in the Polish Pharmacopoeia VIII. The content of phenolic acids in G. robusta reached 7.33 mg/g and 6.23 mg/g for flowers and leaves, respectively. The flowers and leaves of G. squarrosa were characterized by similar level of phenolic acids, namely 6.81 mg/g and 6.59 mg/g, respectively.

Active principles of Grindelia robusta exert antiinflammatory properties in a macrophage model.

Plant extracts and/or secondary metabolites are receiving considerable attention as therapeutic agents for treating inflammatory diseases such as periodontitis, which affects the tooth supporting tissues. The aim of this study was to investigate the effect of a Grindelia robusta extract enriched in saponins and polyphenols on Aggregatibacter actinomycetemcomitans lipopolysaccharide (LPS)-induced inflammatory mediator (IL-6, TNF-a, RANTES, MCP-1, PGE(2) ) and matrix metalloproteinase (MMP-1, -3, -7, -8, -9, -13) secretion by macrophages. LPS induced a marked increase in the secretion of all inflammatory mediators and MMPs tested by macrophages, as determined by enzyme-linked immunosorbent assays. At non-cytotoxic concentrations, the G. robusta extract inhibited dose-dependently the secretion of IL-6, RANTES, MCP-1 and, to a lesser extent, PGE(2) and TNF-a. Such inhibition was also observed for MMP-1, -3, -7, -8, -9 and -13 secretion. This ability of G. robusta extract to reduce the LPS-induced secretion of inflammatory mediators and MMPs was associated with a reduction of nuclear factor-kappa B (NF-kB) p65 activation. The results suggest that G. robusta extract possesses an antiinflammatory therapeutic potential through its capacity to reduce the accumulation of inflammatory mediators and MMPs.

Allocation of biomass and photoassimilates in juvenile plants of six Patagonian species in response to five water supply regimes.

BACKGROUND AND AIMS: The growth-differentiation balance hypothesis (GDBH) states that there is a physiological trade-off between growth and secondary metabolism and predicts a parabolic effect of resource availability (such as water or nutrients) on secondary metabolite production. To test this hypothesis, the response of six Patagonian Monte species (Jarava speciosa, Grindelia chiloensis, Prosopis alpataco, Bougainvillea spinosa, Chuquiraga erinacea and Larrea divaricata) were investigated in terms of total biomass and resource allocation patterns in response to a water gradient. METHODS: One-month-old seedlings were subjected to five water supply regimes (expressed as percentage dry soil weight: 13 %, 11 %, 9 %, 7 % or 5 % - field water capacity being 15 %). After 150 d, plants were harvested, oven-dried and partitioned into root, stem and leaf. Allometric analysis was used to correct for size differences in dry matter partitioning. Determinations of total phenolics (TP), condensed tannins (CT), nitrogen (N) and total non-structural carbohydrates (TNC) concentrations were done on each fraction. Based on concentrations and biomass data, contents of TP and CT were estimated for whole plants, and graphical vector analysis was applied to interpret drought effect. KEY RESULTS: Four species (J. speciosa, G. chiloensis, P. alpataco and B. spinosa) showed a decrease in total biomass in the 5 % water supply regime. Differences in dry matter partitioning among treatments were mainly due to size variation. Concentrations of TP, CT, N and TNC varied little and the effect of drought on contents of TP and CT was not adequately predicted by the GDBH, except for G. chiloensis. CONCLUSIONS: Water stress affected growth-related processes (i.e. reduced total biomass) rather than defence-related secondary metabolism or allocation to different organs in juvenile plants. Therefore, the results suggest that application of the GDBH to plants experiencing drought-stress should be done with caution, at least for Patagonian Monte species.

Evaluation of inhibitory activities of plant extracts on production of LPS-stimulated pro-inflammatory mediators in J774 murine macrophages.

Whole plant methanolic extracts of 14 traditionally used medicinal herbs were evaluated for their anti-inflammatory activity. Extracts of Grindelia robusta, Salix nigra, Arnica montana, and Quassia amara showed up to 4.5-fold inhibition of nitric oxide (NO) production in the J774 murine macrophage cells challenged with LPS without cytotoxicity. These four selected extracts significantly reduced the protein levels of inducible NO synthase (iNOS) and the cyclooxygenase-2 (COX-2) as observed by Western blot analysis. Culture supernatants from cells treated with these extracts indicated 3-5-fold reduction of tumor necrosis factor-alpha (TNF-alpha). However, only G. robusta and Q. amara extracts significantly inhibited (by 50%) IL-1beta and IL-12 secretions. Furthermore, all these plant extracts were shown to prevent the LPS-mediated nuclear translocation of nuclear factor-kappaB (NF-kappaB). All the above observations indicate the anti-inflammatory potential of these plant extracts.