Identification of a highly promiscuous glucosyltransferase from Penstemon barbatus for natural product glycodiversification.

Glycosylation reactions mediated by UDP-glycosyltransferases (UGTs) are common post-modifications involved in plant secondary metabolism and significantly improve the solubility and bioactivity of aglycones. Penstemon barbatus is rich in phenylethanoid glycosides (PhGs), such as echinacoside and verbascoside. In this study, a promiscuous glycosyltransferase UGT84A95 was identified from P. barbatus. In vitro enzyme assays showed that UGT84A95 catalyzed the glucosylation of the phenol hydroxyl group of PhGs efficiently as well as other structurally diverse phenolic glycosides, including flavonoids, terpenoids, stilbene glycosides, coumarins, and simple polyphenols. By using UGT84A95, 12 glycosylated products were prepared and structurally identified by NMR spectroscopy, among which 7 are new compounds. These findings suggest that UGT84A95 could be a potential biocatalyst to synthesize multi-glycosylated glycosides.

The Perennial Penstemon: Variation in Defensive Chemistry Across Years, Populations, and Tissues.

Plants produce a variety of secondary metabolites that function as a defense against their natural enemies. Production of these secondary metabolites is genetically controlled, but is also phenotypically plastic and varies in response to both biotic and abiotic factors. Therefore, plant species may vary widely in their chemical defenses and such variation can be evident at temporal, spatial and tissue levels. Focusing on the chemical defenses of a native Colorado wildflower, Penstemon virgatus, we assessed the variation in iridoid glycoside (IG) content across two non-consecutive growing seasons, six natural populations and three tissue types: leaves, stems and flowers. Our results indicate that P. virgatus plants contain high concentrations of IGs (mean = 23.36% dry weight of leaves) and that IGs were differentially allocated among tissue types. Leaves contained the highest concentration of IGs, which varied quantitatively between sampling years, among plant populations, and plant parts. We also quantified leaf herbivore damage at all six populations but we found very little herbivore damage. Our study indicates that the IG concentrations of P. virgatus plants are both spatially and temporally variable. Furthermore, the high concentrations of secondary metabolites combined with the low levels of damage suggest that these plants are well defended against generalist herbivores.

In vitro COX-1 and COX-2 enzyme inhibitory activities of iridoids from Penstemon barbatus, Castilleja tenuiflora, Cresentia alata and Vitex mollis.

A group of sixteen iridoids isolated from plants used as anti-inflammatory remedies in Mexican folk medicine were evaluated for their potential to inhibit cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) enzymes. From these assays, loganic acid (10) was identified as the most promising compound with both COX-1 (36.0 ± 0.6%) and COX-2 (80.8 ± 4.0%) inhibition at 10 µM. Compound 10 shows a better inhibition against the COX-2 enzyme. Other iridoids tested in the present study showed weak or no inhibition against these enzymes. Furthermore, herein are presented key interactions of iridoid 10 with COX-1 and COX-2 enzymes through molecular docking studies. These studies suggest that 10 exhibits anti-inflammatory activity due to COX inhibition.

Spatiotemporal Floral Scent Variation of Penstemon digitalis.

Variability in floral volatile emissions can occur temporally through floral development, during diel cycles, as well as spatially within a flower. These spatiotemporal patterns are hypothesized to provide additional information to floral visitors, but they are rarely measured, and their attendant hypotheses are even more rarely tested. In Penstemon digitalis, a plant whose floral scent has been shown to be under strong phenotypic selection for seed fitness, we investigated spatiotemporal variation in floral scent by using dynamic headspace collection, respectively solid-phase microextraction, and analyzed the volatile samples by combined gas chromatography-mass spectrometry. Total volatile emission was greatest during flowering and peak pollinator activity hours, suggesting its importance in mediating ecological interactions. We also detected tissue and reward-specific compounds, consistent with the hypothesis that complexity in floral scent composition reflects several ecological functions. In particular, we found tissue-specific scents for the stigma, stamens, and staminode (a modified sterile stamen common to all Penstemons). Our findings emphasize the dynamic nature of floral scents and highlight a need for greater understanding of ecological and physiological mechanisms driving spatiotemporal patterns in scent production.

Phenotypic selection to increase floral scent emission, but not flower size or colour in bee-pollinated Penstemon digitalis.

Fragrance is a putatively important character in the evolution of flowering plants, but natural selection on scent is rarely studied and thus poorly understood. We characterized floral scent composition and emission in a common garden of Penstemon digitalis from three nearby source populations. We measured phenotypic selection on scent as well as floral traits more frequently examined, such as floral phenology, display size, corolla pigment, and inflorescence height. Scent differed among populations in a common garden, underscoring the potential for scent to be shaped by differential selection pressures. Phenotypic selection on flower number and display size was strong. However, selection favoured scent rather than flower size or colour, suggesting that smelling stronger benefits reproductive success in P. digitalis. Linalool was a direct target of selection and its high frequency in floral-scent bouquets suggests that further studies of both pollinator- and antagonist-mediated selection on this compound would further our understanding of scent evolution. Our results indicate that chemical dimensions of floral display are just as likely as other components to experience selective pressure in a nonspecialized flowering herb. Therefore, studies that integrate visual and chemical floral traits should better reflect the true nature of floral evolutionary ecology.

Anti-inflammatory activity of Penstemon gentianoides and Penstemon campanulatus.

CONTEXT: Penstemon gentianoides (Kunth) Poir. and Penstemon campanulatus (Cav.) Willd. (Plantaginaceae) are important medicinal plants in Mexico used by indigenous people for their anti-inflammatory effects and to also reduce rheumatic pains. OBJECTIVE: In addition to radical scavenging activity, the anti-inflammatory activity of the extracts, fractions and compounds of these plants were investigated and reported here for the first time. MATERIALS AND METHODS: The anti-inflammatory activities of MeOH, CH(2)Cl(2), and ethyl acetate extracts and iridoid, flavonoids, and phenylpropanoids from Penstemon gentianoides and P. campanulatus were studied in the TPA-induced mouse ear edema model. In addition, antioxidant activity against DPPH, crocin and ß-carotene were investigated. RESULTS: All extracts were tested and a selection of known compounds significantly (p <0.05) inhibited mouse ear edema. The results showed that CH(2)Cl(2) extracts of roots and stems from P. gentianoides and ethyl acetate extracts of leaves from P. gentianoides and P. campanulatus, as well as luteolin, diosmetin, penstemide and verbascoside produced the most positive results. Of all substances tested, the CH(2)Cl(2) extract of P. gentianoides roots was the most powerful inhibitor (ED(50)=0.07 mg/ear), with activity comparable to that of indomethacin. These extracts, compounds purified, as well as known compounds, inhibited oxidation of ß-carotene and crocin. DISCUSSION AND CONCLUSION: These findings showed that the iridoid monoterpenes, flavonoids and phenylpropanoids present in these plants species may all contribute to the observed anti-inflammatory activity. Additionally, the observed antioxidant activity is correlated with the anti-inflammatory activity of these plants and the phytochemicals derived from them.

Antibiofilm phenylethanoid glycosides from Penstemon centranthifolius.

Bioassay-guided fractionation of the antibacterial ethyl acetate-ethanol (50 : 50) extract obtained from the aerial parts of Penstemon centranthifolius led to the isolation of six phenylethanoid glycosides (1-6) and eleven iridoid glycosides (7-17). Their structures were determined on the basis of spectroscopic analysis and comparison with the literature. Among them, two phenylethanoid glycosides, 4'''-O-acetylverbascoside (1) and verbascoside (2), were found to show significant inhibition of the formation of bacterial biofilms by Escherichia coli UTI89. Compound 1 showed 77% biofilm inhibition at 2.5 microg/mL, and compound 2 showed 60% inhibition at 5 microg/mL.

Pensteminoside, an unusual catalpol-type iridoid from Penstemon gentianoides HBK (Plantaginaceae).

From the MeOH and ethyl acetate extracts of aerial parts of Penstemon gentianoides HBK (Plantaginaceae) an unusual iridoid of the catalpol-type, was isolated and characterized as pensteminoside: (8-O-trans-cinnamoyl, 6-hydroxy, 1-[beta-D-glucopyranoside-6'-O-((4''-hydroxy)-cinnamoyl)]-catalpol) was isolated, along with the known iridoids: plantarelanoside and globularisicin, the flavone: luteolin and diosmetin, as well the phenylpropanoids, verbascoside and martynoside. Their structures were established by 1D and 2D NMR spectroscopic analyses.

[Analysis of phenolic acids in cultured cells and in Penstemon Serrulatus Menz]. / Zawartosc fenolokwasów w kulturach tkankowych i roslinach gruntowych Penstemon serrulatus Menz.

Qualitative and quantitative content of aromatic acids in callus tissues, cells in suspension cultures and intact plants of Penstemon serrulatus was determined by paper and gas chromatography. It has been observed that these acids occur in the investigated material mainly in bounded form. The largest quantities of phenolic acids were produced by callus tissues grown on Schenk and Hildebrandt's medium. The total content of aromatic acids found in the cells from suspension cultures was similar to the content of these compounds in intact plants, but twice lower than in callus tissue grown on the same medium.

Two new iridoids from selected Penstemon species--antimicrobial activity.

Eighteen secondary metabolites, belonging to three different chemical groups, were isolated from the methanolic extracts of the aerial parts of selected penstemon plants [Penstemon fruticosus (Pursh) Greene var. fruticosus, Penstemon palmeri Gray and Penstemon venustus Doug. ex Lindl.], and their structures were elucidated on the basis of spectral evidence. Six iridoid glucosides (1-6), three phenylpropanoid glucosides (13-15) and two acetophenone derivatives (16,17), obtained from P. fruticosus, five iridoids (2, 7-10), one phenylpropanoid glucoside (15) and two acetophenones (16, 18), isolated from P. palmeri while three iridoids (2, 11, 12) and three phenylpropanoids (13-15) were identified in P. venustus. Two of the iridoid glucosides (4, 5) from P. fruticosus are new natural products named accordingly as cis- and trans- forms of 10-O-p-methoxycinnamoylaucubin. All isolated compounds, as well as crude methanolic extracts, were evaluated for their antimicrobial activities against six Gram-positive and Gram-negative bacteria and three human pathogenic fungi.

Separation, purification and quantification of verbascoside from Penstemon barbatus (Cav.) Roth.

Echinacoside and verbascoside possess a spectrum of significant beneficial bioactivities. A new method of preparative high-speed counter-current chromatography for rapid separation and purification of verbascoside from the leaves of Penstemon barbatus (Cav.) Roth was introduced. The solvent system employed here was n-butanol-water (1:1, v/v). The structure was confirmed by IR, (1)H NMR and (13)C NMR. According to the quantification results, echinacoside and verbascoside were found to be as much as 5.25 ± 0.13 mg/g and 11.21 ± 0.16 mg/g, respectively, in the leaves of P. barbatus. This indicates that P. barbatus might be an ideal alternative resource for large scale preparation of echinacoside and verbascoside.

Chemical analysis of Penstemon campanulatus (Cav.) Willd.--antimicrobial activities.

A new iridoid glucoside named 10-isovaleroyl-dihydropenstemide (1), along with nine known compounds was isolated from the aerial part of Penstemon campanulatus (Cav.) Willd. The known compounds include three iridoid glucosides, one phenylpropanoid glucoside, one monoterpene glucoside, one monoterpene lactone and three flavonoids. Their structures were elucidated on the basis of spectral evidence. Additionally four phenolic acids as well as a fatty acid were determined through GC-MS analysis. All isolated compounds and the crude extracts were assayed for their antimicrobial activities against six Gram positive and negative bacteria, as well as against three human pathogenic fungi.

Separation and purification of echinacoside from Penstemon barbatus (Can.) Roth by recycling high-speed counter-current chromatography.

Echinacoside is an important bioactive compound extracted from Cistanche tubulosa which was endangered by overexploitation. It is imperative to find an alternative source. Echinacoside was isolated from Penstemon barbatus (Can.) Roth for the first time. The peak contents of echinacoside are 9.09±0.32mg/g and 7.25±0.36mg/g respectively in the leaves and roots annually. The methanolic extracts from 20g of dried powder of the roots of P. barbatus were pre-purified by AB-8 resin and the fraction containing echinacoside was further purified by conventional high-speed counter-current chromatography (HSCCC) and recycling HSCCC with the solvent system n-butanol-water (1:1, v/v). Totally 42.0mg echinacoside with a purity of 96.3% was recovered. The recovery rate of echinacoside by recycling HSCCC reached 91.0%. The structure of our echinacoside confirmed by IR, (1)H NMR and (13)C NMR is identical to the standard sample. This indicates that P. barbatus might be ideal source for preparation of large scale of echinacoside.

Anti-inflammatory evaluation and antioxidant potential of Senna crotalarioides and Penstemon roseus / Evaluación anti-inflamatoria y potencial antioxidante de Senna crotalarioides y Penstemon roseus

Chloroform, methanol and aqueous extracts of Penstemon roseus and Senna crotalarioides were investigated for their anti-inflammatory and antioxidant properties. The aqueous extract had no activity on TPA-induced ear edema, but the chloroform and methanol extracts of both plants caused a significant inhibition of the edema. The chloroform extracts showed activity on carrageenan-induced paw edema and mouse ear edema induced by multiple topical applications of TPA, and only P. roseus (dose of 100 mg/kg) exhibited anti-arthritic activity. The methanol extracts of P. roseus and S. crotalarioides had the most activity in the DPPH and reducing power tests. The LD50 values in mice of both chloroform extracts were considerate as slight acute toxic and higher than the doses used in this study.

Las propiedades antiinflamatorias y antioxidantes de los extractos de cloroformo, metanol y agua de Penstemon roseus y Senna crotalarioides fueron investigadas. El extracto acuoso no tuvo actividad sobre el edema auricular inducido con TPA, pero el extracto de cloroformo y etanol de ambas plantas inhibieron significativamente el edema. El extracto de cloroformo mostró actividad sobre el edema plantar inducido con carragenina y el edema auricular en ratón inducido por múltiples aplicaciones de TPA, y solo P. roseus y S. crotalarioides tuvieron la mejor actividad en las pruebas de DPPH y poder reductor. Los valores de la DL50 en ratón de ambos extractos de cloroformo son considerados con ligera toxicidad aguda y mayores que las dosis empleadas en el estudio.

Application of capillary-scale NMR for the structure determination of phytochemicals.

Employing a capillary-scale NMR probe enables the miniaturisation of structure determination and de-replication of purified natural products from plants using only 5-100 microg of material. Approximately 5 microg are required to perform one-dimensional proton and two-dimensional homonuclear (COSY and NOESY) NMR experiments; some 30 microg are needed to acquire HMQC- or HSQC-NMR spectra; ca. 75-100 microg are necessary to measure HMBC-NMR spectra; and around 200 microg of a compound are needed to perform 13C- and DEPT-NMR experiments. In order to illustrate the integration of the outputs from high-throughput natural product chemistry methods with the capabilities of the state-of-the-art CapNMR technology, the preparation of a natural product library from the extract of Penstemon centranthifolius, and the subsequent isolation, purification and structure determination of six known iridoid glycosides with 25-300 microg of material are presented.

Effects of extracts, flavonoids and iridoids from Penstemon gentianoides (Plantaginaceae) on inhibition of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) in LPS-Activated RAW 264.7 macrophage cells and their antioxidant activity / Efectos de extractos, flavonoides e iridoides de Penstemon gentianoides (Plantaginaceae) sobre la inhibición de oxido nitrico sintasa inducible (iNOS), ciclooxigenasa-2 (COX-2) celulas de macrofagos murinos RAW 264.7 activadas con LPS y sus actividades antioxidantes

Penstemon gentianoides (HBK) (Kunth) Poir (Plantaginaceae) is an evergreen shrub that grows the throughout high mountains from Guatemala, Mexico and Southern states of US. Its leaves and roots have been used therapeutically for inflammation-related conditions from Aztec times, but systematic studies of its anti-inflammatory activity are lacking and no specific active components have been identified. In this study, methanol, n-hexane, CH2Cl2, ethyl acetate and methanol/water (6:4) extracts, luteolin, diosmetin, verbascoside, martynoside, pensteminoside, globularisicin and plantarenaloside isolated from this plant were evaluated by determining their inhibitory effects on the production of proinflammatory mediators in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophage cells. Ethyl acetate extract, luteolin, and diosmetin exhibited potent anti-inflammatory and antioxidant activities. The results indicated that luteolin and diosmetin suppressed the LPS induced production of nitric oxide (NO), through the down-regulation of inducible nitric oxide synthases (iNOS) and cyclo-oxygenase 2 (COX-2) protein expressions and showed a potent antioxidant activity against DPPH, TBARS and DCFH. The inhibition of enzymes and NO production by selected extracts and compounds was dose-dependent with significant effects seen at concentration as low as 50 ìM. Thus, luteolin and diosmetin may provide a potential therapeutic approach for inflammation associated disorders.

Penstemon gentianoides (HBK) (Kunth) Poir (Plantaginaceae) es un arbusto perenne que crece a lo largo de las montañas altas de Guatemala, México y los estados del sur de los EE.UU.. Sus hojas y raíces se han utilizado terapéuticamente para afecciones relacionadas con inflamación desde la época de los aztecas, pero no existen estudios sistemáticos de su actividad anti-inflamatoria y ninguno de los metabolitos activos específicos han sido identificados. En este estudio, los extractos de metanol, n-hexano, CH2Cl2, acetato de etilo y metanol/agua (6:4), junto con, luteolina, diosmetina, verbascósido, martynoside, pensteminoside, globularisicin y plantarenaloside, aislados desde esta planta se evaluaron mediante la determinación de sus efectos inhibitorios sobre la producción de mediadores proinflamatorios en macrófagos murinos activados con lipopolisacárido (LPS)-RAW 264,7. El extracto de acetato de etilo, luteolina y diosmetina exhibieron una potente actividad anti-inflamatoria y antioxidante. Los resultados indican que luteolina y diosmetina suprimen la producción de óxido nítrico (NO), a través de la regulación de óxido nítrico sintasa-inducible (iNOS) y la ciclooxigenasa-2 (COX-2) ambas expresiones de proteínas. Ademas mostró una potente actividad antioxidante contra DPPH, TBARS y DCFH. La inhibición de las enzimas y la producción de NO por los extractos seleccionados y compuestos es dependiente de la dosis con efectos significativos visto en una concentración tan baja como 50 mM. Por lo tanto, luteolina y diosmetina puede proporcionar un enfoque terapéutico potencial para transtornos asociados a los procesos de inflamación.