Grindelic Acid Research Articles

The crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase reveals active site features controlling catalytic specificity

Diterpenoid natural products serve critical functions in plant development and ecological adaptation and many diterpenoids have economic value as bioproducts. The family of class II diterpene synthases catalyzes the committed reactions in diterpenoid biosynthesis, converting a common geranylgeranyl diphosphate precursor into different bicyclic prenyl diphosphate scaffolds. Enzymatic rearrangement and modification of these precursors generates the diversity of bioactive diterpenoids. We report the crystal structure of Grindelia robusta 7,13-copalyl diphosphate synthase, GrTPS2, at 2.1 Å of resolution. GrTPS2 catalyzes the committed reaction in the biosynthesis of grindelic acid, which represents the signature metabolite in species of gumweed (Grindelia spp., Asteraceae). Grindelic acid has been explored as a potential source for drug leads and biofuel production. The GrTPS2 crystal structure adopts the conserved three-domain fold of class II diterpene synthases featuring a functional active site in the γβ-domain and a vestigial ɑ-domain. Substrate docking into the active site of the GrTPS2 apo protein structure predicted catalytic amino acids. Biochemical characterization of protein variants identified residues with impact on enzyme activity and catalytic specificity. Specifically, mutagenesis of Y457 provided mechanistic insight into the position-specific deprotonation of the intermediary carbocation to form the characteristic 7,13 double bond of 7,13-copalyl diphosphate.

Flavonoids from Ericameria nauseosa inhibiting PI3K/AKT pathway in human melanoma cells

The PI3K/AKT and MAPK/ERK pathways are frequently mutated in metastatic melanoma. In a screen of over 2500 plant extracts, the dichloromethane extract of Ericameria nauseosa significantly inhibited oncogenic activity of AKT in MM121224 human melanoma cells. This extract was analyzed by analytical HPLC, and the column effluent was fractionated and tested for activity to generate the so-called HPLC-based activity profile. Compounds eluting within active time-windows of the chromatogram were subsequently isolated in a larger scale to afford 11 flavones (1-11), four flavanones (12-15), two diterpenes (16, 17), and a seco-caryophyllene (18). All isolated compounds were tested for activity, whereby only flavonoids were found active. Of these, flavones were shown to be more active than the flavanones. The most potent flavone was compound 9, that was displaying an IC50 of 14.7 ± 1.4 µM on AKT activity in MM121224 cells. The terpenoids (16-18) were found to be inactive in the assay. Both diterpenes, a grindelic acid derivative (16) and an ent-neo-clerodane (17) were identified as new natural products. Their absolute configuration was established by ECD. Compound 17 is the first description of a clerodane type diterpene in the genus Ericameria.

In Vitro and In Silico Cytotoxic and Antibacterial Activities of a Diterpene from Cousinia alata Schrenk

A biologically guided isolation of secondary metabolites from Cousinia alata Schrenk wild plant growing in Akmola region, Kazakhstan, led to the isolation of the bioactive diterpene grindelic acid (1). Six flavonoids were also isolated and identified as retusine (2), pachipodol (3), jaranol (4), penduletin (5), casticin (6), and 5, 7, 3′-trihydroxy-3, 4′-dimethoxyflavone (7). Penduletin (5) showed moderate cytotoxic activity assay. Grindelic acid exhibited promising cytotoxic activity against the Artemia salina nauplii and antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Salmonella enteritidis. The presence of the essential pharmacophoric features of histone deacetylase (HDAC) inhibitors in the structure of grindelic acid encouraged us to run a molecular docking study against the HDAC enzyme to understand its mechanism of action on a molecular level. Grindelic acid showed a binding mode of interaction similar to that of the cocrystallized ligand and exhibited good binding affinity against HDAC with the binding free energy of −18.70 kcal/mol. The structures of isolated compounds were determined by MS, 1D, and 2D NMR spectroscopy methods. Compounds (1–7) were isolated for the first time from Cousinia genus.

Grindelia squarrosa Extract and Grindelic Acid Modulate Pro-inflammatory Functions of Respiratory Epithelium and Human Macrophages.

Aim of the study: Both nasal and bronchial epithelial cells have evolved sophisticated mechanisms involved in cellular response to bacterial infection. Recognition of pathogens by TLR receptors activate the NF-κB transcription factor, and lead to production of wide spectrum of cytokines (TNF-α, IL-1β, IL-6 and IL-8). Released by epithelium proinflammatory cytokines intensify migration of macrophages to damaged tissues and modulate their pro-inflammatory functions. Based on traditional use of G. squarrosa aerial parts we hypothesized that successful treatment of cold-related diseases may arise from modulation of the pro-inflammatory functions of respiratory epithelium and human monocytes/macrophages. The biological activity of G. squarrosa extract and grindelic acid were compared with clarithromycin and budesonide used as positive controls. Methods: The expression of surface receptors (TLR-4, IL-10) and expression of adhesive molecules (ICAM-1, VCAM-1, E-selectin) was analyzed with flow cytometry. The macrophage attachment to the epithelial cells was assessed fluorimetrically. The p65 NF-κB concentration and cytokine production was measured spectrophotometrically using enzyme-linked immunosorbent assay. Antibacterial activity was examined by the standard disc-diffusion method and serial dilution method according to CLSI guidelines. Results: G. squarrosa extract and grindelic acid had no antimicrobial effect. However, we noticed significant modulation of pro-inflammatory functions of LPS-stimulated nasal and bronchial epithelium. G. squarrosa extract treatment resulted in decrease of TLR-4 expression and p65 NF-κB concentration and inhibition of cytokines synthesis (IL-8, TNF-α, IL-1β and IL-6) in both cellular models. Additionally, G. squarrosa extract slightly modulated ICAM-1 expression affecting on attachment of macrophages to epithelium. Only G. squarrosa extract was able to stimulate the anti-inflammatory functions of macrophages by inducing TGF-β release and IL-10 receptor surface expression. Grindelic acid, identified as a dominant compound in the plant extract, modulated pro-inflammatory functions of epithelium and macrophages slightly. Conclusion: The obtained results support traditional use of Grindelia squarrosa preparations for a treatment cold-associated diseases symptoms. In our opinion, the observed biological effect of extract may be a consequence of synergistic effect of all compounds present in the extract.

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

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-1β and TNF-α) release and downregulation of adhesive molecules. 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. The effect on IL-8, IL-1β 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. We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of β2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1β release comparable with budesonide, used as a positive control. 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.

Upgrading Biocrude of Grindelia Squarrosa to Jet Fuel Precursors by Aqueous Phase Hydrodeoxygenation

AbstractBio‐jet fuel is increasingly attracting attention in attempts to reduce the environmental impacts of aviation fuels. Feedstocks like camelina, jatropha and algae have been extensively studied, and exhibit fair performance in producing low‐density hydrocarbon biofuels, mainly from triglyceride lipids. Grindelia squarrosa is a promising feedstock since the primary diterpenoid, grindelic acid with the branched tricyclic structure, is the major component in its biocrude. Herein, a heterogeneous bi‐functional catalyst, palladium with the reductive properties supported on tungstate zirconia with acidic properties and good stability in hydrothermal media, was used for the conversion of Grindelia squarrosa biocrude to potential bio‐jet fuels. Different solid acid catalyst supports and a variety of reaction conditions, such as temperature, catalyst loading, hydrogen pressure, and reaction time, were systematically investigated. At the optimum conditions, the oxygen content deceased from 18.1 % in the Grindelia squarrosa biocrude feedstock to 5.6 % in the final product, and is ready for further industrial treatment. The upgrading method utilized in this work can produce jet fuel precursors for synthesizing high‐density cyclic hydrocarbons.

Production of High-Density Renewable Aviation Fuel from Arid Land Crop

Grindelia squarrosa biocrude is a promising feedstock for producing high-density cycloalkane fuels for high-performance aircraft due to the branched tricyclic nature of its primary diterpenoid component, grindelic acid. Herein we designed a novel biphasic tandem catalytic process (biTCP) for the conversion of Grindelia squarrosa biocrude into high-density aviation fuels with a superior carbon atom efficiency. The systematic experimental studies provided valuable insights into the effects of process conditions. The detailed speciation was carried out with the complementary chemical analysis techniques. Under the optimum conditions, a ∼90% carbon yield of aviation fuel components was produced from the Grindelia squarrosa biocrude. Multistep tandem reactions, including cyclic ether ring opening, dehydration, hydrogenation, and decarboxylation were carried out in the “one-pot” biTCP. The detailed catalytic reaction mechanism is discussed. A continuous process was designed integrating the extraction of biocrude and its conversion to dense jet fuels using biTCP. The techno-economic analysis has been performed, and in its current state, the proposed process is expected to deliver price-competitive high-density aviation fuel products.

Fast Pyrolysis of Opuntia ficus-indica (Prickly Pear) and Grindelia squarrosa (Gumweed)

Opuntia ficus-indica (prickly pear) and Grindelia squarrosa (gumweed) are two exceptionally drought tolerant plant species capable of growing in arid and semiarid environments. Additionally, they have unique cell wall structures. Prickly pear contains pectin and high levels of ash (16.1%) that is predominantly Ca and K. Gumweed has high levels of extractives that contain grindelic acid and monoterpenoids. The objective of this paper was to evaluate how these unique cell wall components alter the pyrolysis performance of prickly pear and gumweed. Using a tandem micropyrolyzer with GC-MS/FID/TCD, a detailed account of the product slate is given for products generated between 450 and 650 °C. Pyrolysis of prickly pear showed that the high levels of ash increase the amount of organics volatilized and shifted product pools, making it possible to generate up to 7.3% carbonyls vs 3.8% for Pinus taeda (loblolly pine) and 10.5% hydrocarbons vs 1.8% for pine depending on reaction conditions. Pyrolysis of gumweed sho...

Grindelia squarrosa: A Potential Arid Lands Biofuel Plant

Gumweed (Grindelia squarrosa) has potential as a biofuel/biomaterial crop in arid lands. Two years of biomass production data at University of Nevada, Reno (UNR) field plots varied from 6700 kg/ha up to 14 900 kg/ha with an average of 9950 kg/ha. Gumweed was planted in 4 m × 4 m plots at spacings of 15, 20, 25, 30, and 37 cm with three replications each. Acetone extraction of the ground, dried biomass yielded about 12.5% extractable hydrocarbons, called biocrude or crude resin. Approximately 52% of the biocrude extract consists of the C20 diterpene acid grindelic acid, which is also approximately 6.5% of the dried plant biomass. Additional carboxylic acids bring the total carboxylic acid fraction to approximately 68% of the biocrude. Also found in the biocrude is approximately 4.7% monoterpenes, including pinene, limonene, germacrene, elemene, and camphene. Acid-catalyzed methylation of the biocrude followed by hexane extraction and removal of the hexane and remaining methanol produced an approximately 72...

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 invitro and invivo 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.

Derivatives of grindelic acid: From a non-active natural diterpene to synthetic antitumor derivatives

Using several reactions that include homologations and asymmetric epoxidations as well as Ugi and Huisgen couplings, we generated a small focused library of new derivatives from the labdane-type diterpene grindelic acid. These compounds were evaluated as cytotoxic agents against a panel of five human solid tumor cell lines (HBL-100, HeLa, SW1573, T-47D, and WiDr). The presence of the diamide functionalizations enhanced the cytotoxic effect. N-Benzyl-N-(1-(benzylamino)-2-methyl-1-oxopropan-2-yl)grindelicamide, proved to be the most active product in all cell lines tested, with values of 0.95 (±0.38) μM against HBL-100 cells.

Effects of elicitor and copper sulfate on grindelic acid production in submerged cultures of Grindelia pulchella

Grindelia pulchella callus and cell suspension cultures were established from seedling leaves. When several phytoregulator supplementations were assayed in solid Murashige and Skoog medium containing 3% (w/v) of sucrose (MS medium), combinations of indole-3-butyric acid (IBA) and N 6 -benzylaminopurine (BA) resulted the most appropriate conditions to generate fast growing friable calli with detectable levels of grindelic acid. Moreover, the same basal media supplemented with 20.0 µM IBA/4.4 µM BA was found to be optimal for cell growth in submerged cultures (µ max = 0.26 days -1 ) while the addition of 20.0 µM IBA/18.0 µM BA resulted in a relative higher metabolite production (4.55 mg/gDW) when the inocula was 5% (v/v). Furthermore, three different stress factors and combinations of them were used to elicit cell suspensions. These experiments demonstrated that the combination of CuSO 4 and dimethylsulfoxide (DMSO) increase the grindelic acid production to 2.63 mg/gDW in the elicited essay versus 0.756 mg/gDW in the control, at expense of cell growth. In contrast, the addition of jasmonic acid (JA) alone and combined with DMSO neither affected cell growth nor grindelic acid accumulation.

Effects of elicitor and copper sulfate on grindelic acid production in submerged cultures of Grindelia pulchella

Effects of elicitor and copper sulfate on grindelic acid production in submerged cultures of Grindelia pulchella

Stereo- and regioselective hydroxylation of grindelic acid derivatives by Aspergillus niger

Stereo- and regioselective hydroxylation of grindelane derivatives on position 3β was catalyzed by cultures of Aspergillus niger. Grindelic acid (1), methyl grindelate (2), 15-hydroxy-7(8)-en-9α,13(S)-oxide-ent-labdane (3) and 7α,8α-epoxymethylgrindelate (4) were bioconverted into the corresponding 3β-hydroxy derivatives as the only biotransformation products. 13(S),15-dihydroxy-8(9)-en-ent-labdane (5) remained unreacted under the same conditions. The conformational and electronic studies of the substrates are discussed.

Manoyl oxide alpha-arabinopyranoside and grindelic acid diterpenoids from Grindelia integrifolia.

Two new manoyl oxide-alpha-arabinopyranoside diterpenoids, 15-hydroxy-13-epi-manoyl oxide-14-O-alpha-L-arabinopyranoside (tarapacol-14-O-alpha-L-arabinopyranoside) (1) and 15-acetoxy-13-epi-manoyl oxide-14-O-alpha-L-arabinopyranoside (tarapacol-15-acetate-14-O-alpha-L-arabinopyranoside) (2), as well as a new grindelic acid derivative, 19-hydroxygrindelic acid (3), together with five known diterpenoids (tarapacol, tarapacanol A, grindelic acid, methyl grindeloate, 3beta-hydroxygrindelic acid, 4) were isolated from the aerial parts of Grindelia integrifolia. The structures of 1-3 were elucidated by spectral data analysis.

Phytotoxic Compounds from Xanthocephalum gymnospermoides var. eradiatum1

Investigation of the aerial parts of Xanthocephalum gymnospermoides var eradiatum led to the isolation of two new labdane-type of diterpenes, namely, 8 alpha,13S-epoxylabdane-14S,15-diol (1) and methyl grindelate (2). In addition, grindelic acid (3), 7 alpha, 8 alpha-epoxygrindelic acid (4), 7 alpha-hydroxy-8(17)dehydrogrindelic acid (5), 17-hydroxygrindelic acid (6) and 4,5-epoxy-beta-caryophyllene (7) were obtained. The isolated compounds were characterized by spectral means. The absolute configuration of compound 1 was established by chemical correlation with 8 alpha,13S-epoxy-15-nor-labdan-14-oic acid methyl ester of known absolute stereochemistry and by using the advanced Mosher's ester methodology. The results of the present investigation indicated that the known compound barbatol (8) could be an enantiomer of compound 1. Compounds 1-3 and 7 caused significant inhibition of the radicle growth of seedlings of Amaranthus hypochondriacus.

Grindelic Acid Production in Grindelia Pulchella Cell Suspension Cultures Elicited with CuSO4

4X. E. Hernandez, M.B. Kurina Sanz and O.S. GiordanoINTEQUI- Quimica Organica, Facultad de Quimica, Bioquimica y Farmacia, UNSL. Chacabuco yPedernera. (5700). San Luis, ArgentinaE-mail: xenia@unsl.edu.arIntroductionAbiotic elicitors affect both, biomass and secondary metabolite production in cell suspension cul-tures. In this work we have studied CuSO4 effect on the accumulation of grindelic acid in Grindeliapulchella cell suspension cultures.Material and MethodsCell suspension culturesMS media supplemented with indolbutiric acid and bencilamine purine was employed. Samples of20 ml were taken, after filtration biomass dry weight was evaluated.Grindelic acid production evaluationLiquid media was acidified to pH 5.00 with HCl 10% and submitted to liquid-liquid extraction pro-cedure with Et

Diterpenoid acids from Grindelia nana.

Two new norditerpenoid acids of the labdane-type (norgrindelic acids), 4,5-dehydro-6-oxo-18-norgrindelic acid (1) and 4beta-hydroxy-6-oxo-19-norgrindelic acid (2), as well as a new grindelic acid derivative, 18-hydroxy-6-oxogrindelic acid (3), were isolated from the aerial parts of Grindelia nana. In addition, the known compounds, 6-oxogrindelic acid, grindelic acid, methyl grindeloate, 7alpha,8alpha-epoxygrindelic acid, and 4alpha-carboxygrindelic acid were also isolated. The structures of the new compounds were characterized on the basis of spectroscopic analysis.

Production of 6-b-hydroxygrindelic acid from grindelic acid by Alternaria alternata

6-β-Hydroxygrindelic acid and 3-β-hydroxygrindelic acid were produced from grindelic acid by Alternaria alternata. The best yields for 6-β-hydroxygrindelic acid (25%) and for 3-β-hydroxygrindelic acid (55%) were obtained after 180 h.

Stereocontrolled Synthesis of ent-Grindelic Acid. A Useful Example of Diastereofacial Guidance in an Oxonium Ion-Initiated Pinacolic Ring Expansion

An enantioselective synthesis of (+)-grindelic acid is described, confirming that the dextrorotatory enantiomer is antipodal to the natural diterpenoid. The optically pure bicyclic ketone 5 representing the AB ring system is constructed from the levorotatory Wieland−Miescher ketone and must therefore possess the absolute configuration shown. Coupling of 5 with the 5-lithio derivative of optically active 2,3-dihydrofuran 3 derived from (R)-(−)-linalool was effected for the purpose of realizing acid-catalyzed rearrangement with generation of the appropriate spirocyclic framework. This key step is highly stereocontrolled, leading predominantly to 7. Once the advanced intermediate 15 is available in this fashion, its subsequent exposure to oxidation and dehydration steps led to the target molecule. The synthesis demonstrates unequivocally that natural (−)-grindelic acid is a true labdane diterpenoid.