Screening and Isolation of Potential Anti-Inflammatory Compounds from Saxifraga atrata via Affinity Ultrafiltration-HPLC and Multi-Target Molecular Docking Analyses.

In this study, a 100 g sample of Saxifraga atrata was processed to separate 1.3 g of 11-O-(4'-O-methylgalloyl)-bergenin (Fr1) after 1 cycle of MCI GEL® CHP20P medium pressure liquid chromatography using methanol/water. Subsequently, COX-2 affinity ultrafiltration coupled with reversed-phase liquid chromatography was successfully used to screen for potential COX-2 ligands in this target fraction (Fr1). After 20 reversed-phase liquid chromatography runs, 74.1 mg of >99% pure 11-O-(4'-O-methylgalloyl)-bergenin (Fr11) was obtained. In addition, the anti-inflammatory activity of 11-O-(4'-O-methylgalloyl)-bergenin was further validated through molecular docking analyses which suggested it was capable of binding strongly to ALOX15, iNOS, ERBB2, SELE, and NF-κB. As such, the AA metabolism, MAPK, and NF-κB signaling pathways were hypothesized to be the main pathways through which 11-O-(4'-O-methylgalloyl)-bergenin regulates inflammatory responses, potentially functioning by reducing pro-inflammatory cytokine production, blocking pro-inflammatory factor binding to cognate receptors and inhibiting the expression of key proteins. In summary, affinity ultrafiltration-HPLC coupling technology can rapidly screen for multi-target bioactive components and when combined with molecular docking analyses, this approach can further elucidate the pharmacological mechanisms of action for these compounds, providing valuable information to guide the further development of new multi-target drugs derived from natural products.

Biological evaluation of a natural steroid ester, Stigmasta-5(6), 22(23)-dien-3-beta-yl acetate isolated from the Himalayan herb Astilbe rivularis as potential antitumor agent.

BACKGROUND: Cancer remains a major world health issue due to its high morbidity and mortality rate. Plant based natural products (NPs) have played vital role in discovery of valuable anti-cancer drugs. Darjeeling Himalayan region has a rich diversity of therapeutic plants that can be utilized for development of novel drugs. AIM: We previously reported cytotoxic potential of rhizome extract of A.rivularis, a Darjeeling himalayan herb. Present study reports isolation and characterization of a phytosteroid from the plant rhizome in a bioassay-guided approach and evaluation of its anti-tumorigenic potential. RESULTS: The phytosteroid was characterized as stigmasta-5(6), 22(23)-dien-3-beta-yl acetate (A11) by various spectrometric techniques (IR, NMR, MS etc.). The catalytic inhibition and structural alteration of human dihydrofolate reductase (hDHFR) by A11 was evaluated using methotrexate (MTX), a DHFR inhibitor anticancer drug as a reference. A11 inhibited hDHFR activity with IC50 values of 1.20 µM A11 caused concentration dependent quenching of tryptophan fluorescence of hDHFR suggesting its effect on alteration of enzyme structure. Molecular docking of A11 on crystal structure of hDHFR revealed significant interaction with free energy of binding and Ki values of -10.86 kcal/mol and 11 nM, respectively. Subsequent in vitro studies at cellular level showed a relatively greater cytotoxic effect of A11 against human kidney (ACHN, IC50 60 µM) and liver (HepG2, IC5070 µM) cancer cells than their respective normal cells (HEK-293, IC50 350 µM and WRL-68, IC50 520 µM). Scanning electron microscopy of A11 treated cells revealed the morphological feature of apoptosis, like cell rounding and surface detachment, membrane blebbing, loss of cilia and increased number of pores of decreased sizes. A11 mediated apoptosis of cancer cells was found to be correlated with induction of intracellular of reactive oxygen species (ROS) level and fragmentation of genomic DNA.

Effect of UV radiation and altitude characteristics on the functional traits and leaf optical properties in Saxifraga hostii at the alpine and montane sites in the Slovenian Alps.

UV radiation affects the biochemical, physiological and morphological responses of plants. The effect is most pronounced at high altitude, such as alpine regions, and low latitude environments. The effect of UV radiation is impacted by different environmental conditions including temperature. We examined the response of the alpine plant Saxifraga hostii Tausch subsp. hostii growing at two altitudes (montane, 1100 m a.s.l. and alpine, 1500 m a.s.l.) in the Slovenian Alps. Selected ecophysiological, anatomical and pigment analyses along with measurements of the leaf optical properties were carried out during the growing season from July to September. Plants were grown under two different UV levels, near-ambient UV (UV) and reduced UV (UV-) radiation, and temperature conditions were monitored at both altitudes. Saxifraga hostii exhibited high photochemical efficiency of photosystem II and stomatal conductance under near-ambient UV radiation in August, which indicates that it is a well-acclimated plant. In September, photochemical efficiency was higher under reduced UV at the alpine altitude which together with a lower photosynthetic pigment content indicate delayed senescence for plants growing under reduced UV. Most leaf tissue thicknesses were not affected by UV radiation and altitude difference. There was a trend of increased stomatal density and reduced stomatal length on both leaf surfaces under near-ambient UV in August. However, there was no effect of UV attenuation or location at the alpine or montane site on the content of UV-B absorbing compounds, which implies the plant's tolerance of UV-B radiation. Saxifraga hostii leaves showed high absorption in the UV spectrum at higher altitudes, as shown by their optical properties. This study shows that Saxifraga hostii is well-acclimated to ambient UV radiation and to the environmental conditions at both altitudes. The effect of UV radiation is impacted by site conditions and this produces diverse plant responses, which contribute to the specific functional traits of Saxifraga hostii in the high-altitude environment.

Effects of genetic variation and environmental factors on bergenin in Rodgersia sambucifolia Hemsl.

ETHNOPHARMACOLOGICAL RELEVANCE: Bergenin is a well-known active compound that exhibits antioxidant, antiarrhythmic, hepatoprotective, and anti-inflammatory activities. However, the resource reserve of Rodgersia sambucifolia, one of the main raw materials for extracting bergenin, have sharply declined, and the bergenin content in different germplasms differs vastly, resulting in a serious shortage of the market supply of bergenin. AIM OF THE STUDY: To investigate the influence of genetic diversity and environmental factors on bergenin content in Rodgersia sambucifolia. MATERIALS AND METHODS: Fifty Rodgersia sambucifolia samples with a growth period of 2-3 years were collected from different areas across China and the bergenin content was determined via HPLC. Meanwhile the total genomic DNA was extracted and ISSR was performed. The bergenin content as measured using HPLC and the environmental data gathered from the meteorological stations and field work were combined and analyzed using correlation tests in XLSTAT 2018 to detect the key factors affecting bergenin content. The genetic UPGMA tree constructed based on genetic distances of the 50 samples and the chemical dendrogram constructed according to the distance between the bergenin content were compared to determine the correlation between genetic and chemical differentiation. RESULTS: Among the 50 individuals, bergenin content varied from 2.83 to 12.54%, with the highest content being 4.43-fold that of the lowest content. The survey of the 50 individuals produced a total of 193 amplified bands, 187 of which were polymorphic (96.89%). In the study, bergenin content was positively correlated with annual mean temperature (AMT) (r = 0.583, P < 0.0001) and 1-12 month monthly mean temperature (MMT) (P < 0.0001). A comparison of the genetic dendrogram with the AHC dendrogram found no corresponding relationship between them. Mantel correlation analyses also showed that there was no significant correlation between them (r = 0.144). CONCLUSIONS: There were large differences in bergenin content among different germplasms that were not correlated with the high genetic variation in Rodgersia sambucifolia but were significantly correlated with environmental factors, such as temperature. This study lays the foundation for subsequent superior germplasm selection and artificial breeding of Rodgersia sambucifolia to improve the bergenin content and meet market demands.

Biological Kerker Effect Boosts Light Collection Efficiency in Plants.

Being the polymorphs of calcium carbonate (CaCO3), vaterite and calcite have attracted a great deal of attention as promising biomaterials for drug delivery and tissue engineering applications. Furthermore, they are important biogenic minerals, enabling living organisms to reach specific functions. In nature, vaterite and calcite monocrystals typically form self-assembled polycrystal micro- and nanoparticles, also referred to as spherulites. Here, we demonstrate that alpine plants belonging to the Saxifraga genus can tailor light scattering channels and utilize multipole interference effect to improve light collection efficiency via producing CaCO3 polycrystal nanoparticles on the margins of their leaves. To provide a clear physical background behind this concept, we study optical properties of artificially synthesized vaterite nanospherulites and reveal the phenomenon of directional light scattering. Dark-field spectroscopy measurements are supported by a comprehensive numerical analysis, accounting for the complex microstructure of particles. We demonstrate the appearance of generalized Kerker condition, where several higher order multipoles interfere constructively in the forward direction, governing the interaction phenomenon. As a result, highly directive forward light scattering from vaterite nanospherulites is observed in the entire visible range. Furthermore, ex vivo studies of microstructure and optical properties of leaves for the alpine plants Saxifraga "Southside Seedling" and Saxifraga Paniculata Ria are performed and underline the importance of the Kerker effect for these living organisms. Our results pave the way for a bioinspired strategy of efficient light collection by self-assembled polycrystal CaCO3 nanoparticles via tailoring light propagation directly to the photosynthetic tissue with minimal losses to undesired scattering channels.

Extreme diversification of floral volatiles within and among species of Lithophragma (Saxifragaceae).

A major challenge in evolutionary biology is to understand how complex traits of multiple functions have diversified and codiversified across interacting lineages and geographic ranges. We evaluate intra- and interspecific variation in floral scent, which is a complex trait of documented importance for mutualistic and antagonistic interactions between plants, pollinators, and herbivores. We performed a large-scale, phylogenetically structured study of an entire plant genus (Lithophragma, Saxifragaceae), of which several species are coevolving with specialized pollinating floral parasites of the moth genus Greya (Prodoxidae). We sampled 94 Lithophragma populations distributed across all 12 recognized Lithophragma species and subspecies, and four populations of related saxifragaceous species. Our results reveal an unusually high diversity of floral volatiles among populations, species, and clades within the genus. Moreover, we found unexpectedly major changes at each of these levels in the biosynthetic pathways used by local populations in their floral scents. Finally, we detected significant, but variable, genus- and species-level patterns of ecological convergence in the floral scent signal, including an impact of the presence and absence of two pollinating Greya moth species. We propose that one potential key to understanding floral scent variation in this hypervariable genus is its geographically diverse interactions with the obligate specialized Greya moths and, in some species and sites, more generalized copollinators.

Divergent gene expression levels between diploid and autotetraploid Tolmiea relative to the total transcriptome, the cell, and biomass.

PREMISE OF THE STUDY: Studies of gene expression and polyploidy are typically restricted to characterizing differences in transcript concentration. Using diploid and autotetraploid Tolmiea, we present an integrated approach for cross-ploidy comparisons that account for differences in transcriptome size and cell density and make multiple comparisons of transcript abundance. METHODS: We use RNA spike-in standards in concert with cell size and density to identify and correct for differences in transcriptome size and compare levels of gene expression across multiple scales: per transcriptome, per cell, and per biomass. KEY RESULTS: In total, ~17% of all loci were identified as differentially expressed (DEGs) between the diploid and autopolyploid species. The per-transcriptome normalization, the method researchers typically use, captured the fewest DEGs (58% of total DEGs) and failed to detect any DEGs not found by the alternative normalizations. When transcript abundance was normalized per biomass and per cell, ~66% and ~82% of the total DEGs were recovered, respectively. The discrepancy between per-transcriptome and per-cell recovery of DEGs occurs because per-transcriptome normalizations are concentration-based and therefore blind to differences in transcriptome size. CONCLUSIONS: While each normalization enables valid comparisons at biologically relevant scales, a holistic comparison of multiple normalizations provides additional explanatory power not available from any single approach. Notably, autotetraploid loci tend to conserve diploid-like transcript abundance per biomass through increased gene expression per cell, and these loci are enriched for photosynthesis-related functions.

Adaptation of the Long-Lived Monocarpic Perennial Saxifraga longifolia to High Altitude.

Global change is exerting a major effect on plant communities, altering their potential capacity for adaptation. Here, we aimed at unveiling mechanisms of adaptation to high altitude in an endemic long-lived monocarpic, Saxifraga longifolia, by combining demographic and physiological approaches. Plants from three altitudes (570, 1100, and 2100 m above sea level [a.s.l.]) were investigated in terms of leaf water and pigment contents, and activation of stress defense mechanisms. The influence of plant size on physiological performance and mortality was also investigated. Levels of photoprotective molecules (α-tocopherol, carotenoids, and anthocyanins) increased in response to high altitude (1100 relative to 570 m a.s.l.), which was paralleled by reduced soil and leaf water contents and increased ABA levels. The more demanding effect of high altitude on photoprotection was, however, partly abolished at very high altitudes (2100 m a.s.l.) due to improved soil water contents, with the exception of α-tocopherol accumulation. α-Tocopherol levels increased progressively at increasing altitudes, which paralleled with reductions in lipid peroxidation, thus suggesting plants from the highest altitude effectively withstood high light stress. Furthermore, mortality of juveniles was highest at the intermediate population, suggesting that drought stress was the main environmental driver of mortality of juveniles in this rocky plant species. Population structure and vital rates in the high population evidenced lower recruitment and mortality in juveniles, activation of clonal growth, and absence of plant size-dependent mortality. We conclude that, despite S. longifolia has evolved complex mechanisms of adaptation to altitude at the cellular, whole-plant and population levels, drought events may drive increased mortality in the framework of global change.

Inhibitory effects of pentacyclic triterpenoids from Astilbe rivularis on TGFBIp-induced inflammatory responses in vitro and in vivo.

Transforming growth factor ß induced protein (TGFBIp) is an extracellular matrix protein which expression in several cell types is greatly increased by TGF-ß. TGFBIp is released by human umbilical vein endothelial cells (HUVECs), and functions as a mediator of experimental sepsis. Pentacyclic triterpenoids bearing a carboxyl group at C-27 position, 3ß,6α-dihydroxyolup-20(29)-ene (1), 3ß,6ß-dihydroxyolean-12-en-27-oic acid (2) and 3ß,24-dihydroxyolean-12-en-27-oic acid (3), are representative bioactive molecules in the genus Astilbe that possess cytotoxic, anti-inflammatory and wounds healing activities. Based on the biological effects of C-27 carboxylated pentacyclic triterpenoids, we investigated the anti-inflammatory effects of compounds 1-3 against TGFBIp-mediated vascular inflammatory responses. The anti-inflammatory activities of compounds 1-3 were determined by measuring permeability, leukocytes adhesion and migration, and activation of pro-inflammatory proteins in TGFBIp-activated human HUVECs and mice. We found that compounds 1-3 inhibited TGFBIp-induced barrier disruption, expression of cell adhesion molecules (CAMs) and adhesion/transendothelial migration of neutrophils to human endothelial cells. Each compound also suppressed TGFBIp-induced hyperpermeability and leukocyte migration in vivo. These results suggest that compounds 1-3 possess anti-inflammatory functions by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases.

Antioxidant activity and phenolic content of Bergenia crassifolia, B. x ornata and B. ciliata.

This study focused on a phytochemical analysis of Bergenia crassifolia (L.) Fritsch., B. ciliata (Haw.) Sternb., and B. x ornata Stein. and evaluation of their free radical scavenging properties. Arbutin and total tannin contents of the leaves of the Bergenia species were determined during different seasons. The present study also aimed at analyzing, for the first time, environmental influence on concentrations of phenolic metabolites in Bergenia leaves. The highest total tannin content was found in the leaves of B. crassifolia (24.9-48.7 mg x g(-1) DW) and B. x ornata (36.9 mg.g(-1) DW). The highest amount of arbutin was in the leaves of B. x ornata (35.8-51.0 mg.g(-1) DW) and B. crassifolia (24.6-41.7 mg x g(-1) DW). Autumn was better than spring for the collection of Bergenia leaves for the highest amount of arbutin (B. x ornata: 51.0 mg x g(-1) DW). Free radical scavenging potential, in DPPH and ABTS assays, of the water leaf extracts revealed that extracts of B. crassifolia and B. x ornata are the most active radical scavengers. Antioxidant activity correlated well with the content of total tannin, especially in the ABTS assay, which suggests an important role for these compounds in antioxidant activity. It was shown that phenolic concentrations in Bergenia leaves are affected by seasonal factors. A significant correlation was found between arbutin and tannin contents and the average humidity.

[Research on growth state and physiological changes of Tianquan Bergenia purpurascens under different shade conditions].

OBJECTIVE: To study the growth index and physiological index of Tianquan Bergenia purpurascens under different shade conditions (100%, 80%, 60%, 40%, 20%), and investigate its adaptation mechanism to shade environment so that it can provide theoretical basis for cultivation and production. METHOD: The growth index such as the length, the width and the number of new leaves, and leaf area, fresh weight, dry weight and specific leaf weight of the samples which were collected from the beginning of March to late May under different shade conditions were measured. The physiological index including the contents of photosynthetic pigments, MDA, soluble sugar, soluble protein, proline and the activity of SOD were measured all together. RESULT: The growth and morphological index under light intensity 100%-60% were higher than those under light intensity 40%-20%. And except specific leaf weight, the growth and morphological index were significantly higher under light intensity 60% than those under the other shade conditions; The contents of Chla, Chlb, Chl (a + b) and Car under light intensity 100%-60% were higher than those under light intensity 40%-20%, while Chla/b was higher under light intensity 40%-20%; The activity of SOD, soluble sugar, soluble protein and proline contents were higher under light intensity 100%-60%, while MDA content was higher under light intensity 40%-20%. Chl(a+b) content, soluble sugar content and activity of SOD were the highest under light intensity 60%, but MDA content was the lowest. CONCLUSION: Sixty percent irradiance treatment was the most favorable to the growth of Tianquan B. purpurascens; 100%-80% irradiance treatments were favorable to its growth; 40%-20% irradiance treatment was negative for its growth.

Inflorescences of alpine cushion plants freeze autonomously and may survive subzero temperatures by supercooling.

Freezing patterns in the high alpine cushion plants Saxifraga bryoides, Saxifraga caesia, Saxifraga moschata and Silene acaulis were studied by infrared thermography at three reproductive stages (bud, anthesis, fruit development). The single reproductive shoots of a cushion froze independently in all four species at every reproductive stage. Ice formation caused lethal damage to the respective inflorescence. After ice nucleation, which occurred mainly in the stalk or the base of the reproductive shoot, ice propagated throughout that entire shoot, but not into neighboring shoots. However, anatomical ice barriers within cushions were not detected. The naturally occurring temperature gradient within the cushion appeared to interrupt ice propagation thermally. Consequently, every reproductive shoot needed an autonomous ice nucleation event to initiate freezing. Ice nucleation was not only influenced by minimum temperatures but also by the duration of exposure. At moderate subzero exposure temperatures (-4.3 to -7.7 °C) the number of frozen inflorescences increased exponentially. Due to efficient supercooling, single reproductive shoots remained unfrozen down to -17.4 °C (cooling rate 6 K h⁻¹). Hence, the observed freezing pattern may be advantageous for frost survival of individual inflorescences and reproductive success of high alpine cushion plants, when during episodic summer frosts damage can be avoided by supercooling.

[Selection of winter plant species for wetlands constructed as sewage treatment systems and evaluation of their wastewater purification potentials].

In order to establish an evaluation system for selection of winter wetland plants possessing high wastewater purification potentials in subtropics areas, designed sewage treatment experiments were carried out by introducing into the constructed wetlands 25 species of winter wetland plants. Cluster analysis was performed by including harmful environment-resistant enzyme and substrate enzyme activities into the commonly applied plant screening and assessment indexes system. The obtained results indicated that there were significant differences among the tested winter plants in their root length and vigor, leaf malonaldehyde (MDA), biomass, average nitrogen and phosphorus concentration and uptake, and urease and phosphoric acid enzyme activities in the root areas. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Oenanthe javanica, Brassicacapestris, Juncus effusu, Saxifragaceae, Iris pseudoacorus, Osmanthus fragrans and Iris ensata; those in the 2nd group possessing moderate purification potentials are Brassica oleracea var acephala, Calendula officinalis, Aucuba japonica, Ligustrum lucidu, Beta vulgaris, Rhododendron simsii and Ilex latifolia; and those in the 3rd group with low purification potentials are Brassica oleracea var acephala, Calistephus chinensis, Rosa chinensis, Antirrhinums, Liriope palatyphylla, Zephyranthes candida, Fatshedera lizei, Petunia hybrida, Ilex quihoui, Dianthus caryophyllus and Loropetalum chinensis.

Quality evaluation of golden saxifrage (Chrysosplenium alternifolium L.) through simultaneous determination of four bioactive flavonoids by high-performance liquid chromatography with PDA detection.

To control the quality of Chrysosplenium alternifolium L., a simple, fast and reliable method of high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA) was developed and validated for simultaneous quantitative determination of four bioactive polymethoxylated flavonoids, namely chrysosplenosides B and D, and chrysosplenols B and D. Separation of the four analytes was accomplished on a C18 Hypersil ODS column (5 microm, 125 mm x 4 mm, i.d.) with an acetonitrile 10-100% (v/v) elution gradient, recorded at 345 nm. The equilibration of the methanol extracts and standard solution to 30% (v/v) of water was found to be necessary when minimizing viscosity differences between injections and the mobile phase, and thereby when minimizing distortions of analyte peaks and maximizing the resolution of critical bands of chrysosplenosides B and D. The correlation coefficients of all the calibration curves showed excellent linearity (r=0.9999) over the wide test range. The relative standard deviation of the method was less than 3.53 and 4.41% for intra- and inter-day assays, and the average recoveries were between 95.3 and 103.5%. High sensitivity was demonstrated with detection limits between 0.012 and 0.029 microg/ml (0.24-0.58 ng). C. alternifolium was found to be a valuable source of the flavonoids with the total content ranging from 2.456 to 4.314% of dry weight, depending on harvest time and cultivation area. The total flavonoids were also determined using the pharmacopeial UV-spectrophotometric method and a notable underestimation was found in comparison to the developed HPLC method.

Photosynthetic characteristics and biomass distribution of the dominant vascular plant species in a high Arctic tundra ecosystem, Ny-Alesund, Svalbard: implications for their role in ecosystem carbon gain.

Studies on terrestrial ecosystems in the high Arctic region have focused on the response of these ecosystems to global environmental change and their carbon sequestration capacity in relation to ecosystem function. We report here our study of the photosynthetic characteristics and biomass distribution of the dominant vascular plant species, Salix polaris, Dryas octopetala and Saxifraga oppositifolia, in the high Arctic tundra ecosystem at Ny-Alesund, Svalbard (78.5 degrees N, 11.5 degrees E). We also estimated net primary production (NPP) along both the successional gradient created by the proglacial chronosequence and the topographical gradient. The light-saturated photosynthesis rate (A (max)) differed among the species, with approximately 124.1 nmol CO(2) g(-1)leaf s(-1) for Sal. polaris, 57.8 for D. octopetala and 24.4 for Sax. oppositifolia, and was highly correlated with the leaf nitrogen (N) content for all three species. The photosynthetic N use efficiency was the highest in Sal. polaris and lowest in Sax. oppositifolia. Distributions of Sal. polaris and D. octopetala were restricted to the area where soil nutrient availability was high, while Sax. oppositifolia was able to establish at the front of a glacier, where nutrient availability is low, but tended to be dominated by other vascular plants in high nutrient areas. The NPP reflected the photosynthetic capacity and biomass distribution in that it increased with the successional status; the contribution of Sal. polaris reached as high as 12-fold that of Sax. oppositifolia.

Myriophyllum alterniflorum DC., biomonitor of metal pollution and water quality. Sorption/accumulation capacities and photosynthetic pigments composition changes after copper and cadmium exposure.

Watermilfoil genus Myriophyllum could be used in ecological surveys as in-situ biomonitors of metal pollution and water quality due to its ability to accumulate chemicals. The copper and cadmium sorption characteristics of Myriophyllum alterniflorum have been investigated. The Langmuir and Freundlich isotherms were used to model the metal sorption isotherms and the monolayer sorption capacities, as obtained by the Langmuir isotherm, were determined to be 13.9 mg/g and 11.1 mg/g for Cu2+ and Cd2+ respectively. Results have been compared with previous works on watermilfoils and are in accordance with those obtained on Myriophyllum spicatum. The sorption of the two metals was time-dependent and the kinetics fitted the pseudo-second-order equation well. The data were discussed in terms of ionic radii and HSAB concept. The phytotoxic effects assessed by classical (i.e. changes in biomass, node length) and photosynthetic pigments content endpoints have been investigated using chemometric techniques leading to an effect of cadmium onto photosynthetic pigments.

Ellagitannin biosynthesis: laccase-catalyzed dimerization of tellimagrandin II to cornusiin E in Tellima grandiflora.

An enzyme has been purified from leaves of the weed Tellima grandiflora (fringe cups, Saxifragaceae) that catalyzed the O2-dependent oxidation of the monomeric ellagitannin, tellimagrandin II, to a dimeric derivative, cornusiin E. The apparently homogeneous enzyme preparation had a Mr of ca. 160,000 (with four subunits of Mr 40,000), a pH-optimum and an isoelectric point at pH 5.2, and was most stable at pH 4.3. Inhibition studies revealed that this new enzyme, for which the systematic name 'tellimagrandin II: O2 oxidoreductase' is proposed, is a member of the laccase (EC 1.10.3.2) family of phenol oxidases. The properties of this enzyme differed from that of a related laccase that catalyzed the transition of 1,2,3,4,6-pentagalloylglucopyranose to tellimagrandin II, the preceding step in the biosynthetic route to cornusin E.

Biosynthesis of the dimeric ellagitannin, cornusiin E, in Tellima grandiflora.

First evidence for the in vitro synthesis of a dimeric ellagitannin has been obtained with cell-free extracts from the weed Tellima grandiflora (fringe cups, Saxifragaceae). Partially purified enzyme preparations from leaves of this plant catalyzed the oxidation of 1,2,3,4,6-pentagalloyl-beta-D-glucose to the monomeric ellagitannin, tellimagrandin II, followed by oxidative coupling of two units of this intermediate to yield a dimeric derivative. Chemical degradation, MALDI-TOF mass spectrometry, 1H and 13C nuclear magnetic resonance, and CD spectroscopy were employed to identify this enzyme reaction product as cornusiin E which is characterized by a (S)-valoneoyl bridge between glucose-positions 2, 4' and 6'. This result was supported by comparison with data obtained for cornusiin E that had been isolated from leaves of intact T. grandiflora plants. No indication for the earlier proposed existence of rugosin D (an isomer with a 1,4',6'-bound valoneoyl unit) in T. grandiflora has been obtained in this investigation.