Plant Glycosides Research Articles

Activity levels of six glycoside hydrolases in apple fruit callus cultures depend on the type and concentration of carbohydrates supplied and the presence of plant growth regulators

Sucrose presence and concentration modulated in different ways and to different extents the activity of six plant glycoside hydrolases (PGHs) extracted from apple callus cultures, both in the water soluble fraction (WS-F) and in the NaCl-released fraction (NaCl-F). β-d-Glucosidase activity increased because of sucrose starvation and the addition of sucrose decreased both WS-F and NaCl-F β-d-glucosidase from calli grown in a Murashige and Skoog’s basal medium with (MSH) or without (MS0) plant growth regulators (PGRs). WS-F and NaCl-F α-l-arabinofuranosidase, NaCl-F β-d-galactosidase and NaCl-F β-d-xylosidase activity reached a maximum when 0.045 M sucrose was added to the MS0 medium with an ensuing decline at higher sucrose concentrations. α-d-Galactosidase and α-d-xylosidase activity reached a maximum when 0.045 M sucrose was supplied and did not decline significantly in 0.09 M sucrose-supplied calli. When the effects of PGR presence or absence were analysed, NaCl-F β-d-glucosidase, α-d-galactosidase, β-d-galactosidase, α-d-xylosidase and β-d-xylosidase activities were found to be higher in MS0 than in MSH. To assess whether sugar effects were sucrose-specific, other sugars (glucose, fructose, galactose, maltose, lactose, raffinose, sorbitol and mannitol) were tested, with or without PGR supplementation. In general, sugar alcohols (mannitol, sorbitol) and some monosaccharides (fructose and glucose in particular) were better inducers of NaCl-F α-l-arabinofuranosidase, β-d-galactosidase and β-d-xylosidase activity than disaccharides (sucrose, maltose, and lactose) or the trisaccharide raffinose. This trend was not widespread to all PGHs assessed since sucrose-supplemented calli displayed higher NaCl-F α-d-galactosidase than those supplemented with glucose, galactose, sorbitol or mannitol. These results show that sugars supplied to callus tissue cultures as a carbon source can also modulate PGH activity. Modulation is different for each PGH, sugar-specific and, at least in the case of sucrose, concentration-dependent. Results also suggest the existence of regulatory interactions between PGRs and sugars as part of an intricate sensing and signalling network. Combination of PGR, sugar type and concentration should be taken into account to maximize each PGH activity for further enzyme studies.

Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes

Plant family 32 glycoside hydrolase enzymes include hydrolases (cell wall invertases, fructan exohydrolases, vacuolar invertases) and fructosyltransferases. These enzymes are very similar at the molecular and structural levels but are functionally different. Understanding the basis of the functional diversity in this family is a challenging task. By combining structural and site-directed mutagenesis data, Asp239 in AtcwINV1 was identified as an amino acid critical for binding and stabilizing sucrose. Plant fructan exohydrolases lack such an Asp239 equivalent. Substitution of Asp239 led to the loss of invertase activity, while its introduction in fructan exohydrolases increased invertase activity. Some fructan exohydrolases are inhibited by sucrose. The difference between the inhibitor (fructan exohydrolase) and the substrate (invertase) binding configurations of sucrose can be explained by the different orientation of Trp82. Furthermore, the evolutionary hydrolase/transferase transition could be mimicked and the difference between S-type fructosyltransferases (sucrose as donor) and F-type fructosyltransferases (fructan as donor) could be unravelled.

A Versatile Transport Network for Sequestering and Excreting Plant Glycosides in Leaf Beetles Provides an Evolutionary Flexible Defense Strategy

The larval defenses of chrysomeline leaf beetles comprise components that are either synthesized de novo or sequestered from their food plants. Both biosynthetic modes are based on glucosides that serve as substrates and forms of transport. The defensive glands import the compounds through highly selective glucoside transporters from a circulating pool in the hemolymph. Here we address the selectivity of the different transport systems with larvae of Chrysomela populi, an obligate sequestering species, and with larvae of Phaedon cochleariae, producing monoterpene [corrected] iridoids. Both species possess an interconnected network of transport systems for uptake and excretion. The glucosides are imported by the gut membrane with low selectivity. Their excretion by the Malpighian tubules is similarly unselective, but the uptake of the glucosides from the hemolymph into the defensive system is specific. Only the genuine glucoside precursors made de novo or sequestered from the plant are imported. The successful combination of the precursor-adapted pathways of excretion and defense has probably allowed many leaf beetle species to adaptively radiate onto, and coevolve with plants that offer appropriate glucoside precursors.

A Versatile Transport Network for Sequestering and Excreting Plant Glycosides in Leaf Beetles Provides an Evolutionary Flexible Defense Strategy

A Versatile Transport Network for Sequestering and Excreting Plant Glycosides in Leaf Beetles Provides an Evolutionary Flexible Defense Strategy

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.

Functional and Structural Characterization of a Flavonoid Glucoside 1,6-Glucosyltransferase from Catharanthus roseus

Sugar-sugar glycosyltransferases play an important role in structural diversity of small molecule glycosides in higher plants. We isolated a cDNA clone encoding a sugar-sugar glucosyltransferase (CaUGT3) catalyzing 1,6-glucosylation of flavonol and flavone glucosides for the first time from Catharanthus roseus. CaUGT3 exhibited a unique glucosyl chain elongation activity forming not only gentiobioside but also gentiotrioside and gentiotetroside in a sequential manner. We investigated the functional properties of CaUGT3 using homology modeling and site-directed mutagenesis, and identified amino acids positioned in the acceptor-binding pocket as crucial for providing enough space to accommodate flavonoid glucosides instead of flavonoid aglycones. These results provide basic information for understanding and engineering the catalytic functions of sugar-sugar glycosyltransferases involved in biosynthesis of plant glycosides.

Analytical Procedures for Determination of Quercetin and its Glycosides in Plant Material

Quercetin and its glycosides are widely distributed in the plant kingdom and belong to the most abundant of the flavonoid molecules. Besides their important biological roles in plant pigmentation, these flavonols possess anti-cancer and anti-inflammatory properties, which are the consequence of their affinity for proteins and their anti-oxidant properties. The content of quercetin and its glycosides in different plants have been summarized in various papers, although quantitative data are not so reliable due to the wide diversity of extraction and determination procedures. The general analytical strategy involves the isolation from a sample matrix followed by separation, identification and measurement. The recovery step usually involves solvent extraction using a range of solvents. Separation is commonly achieved by high-performance liquid chromatography (HPLC) and capillary electrophoresis. This paper intends to present and discuss the analytical procedures for determination of quercetin—aglycone as well as its conjugates—in different plant materials by means of routine and more recently developed separation techniques. Selected applications are included to illustrate the scope and limitations of the various approaches.

Saponins as Tool for Improved Targeted Tumor Therapies

Saponins are plant glycosides that consist of a steroid, steroid alkaloid or triterpenoid aglycone and one or more sugar chains that are covalently linked by glycosidic binding to the aglycone. Glucose, galactose, glucuronic acid, xylose and rhamnose are commonly bound monosaccharides. Saponins are found in all organs of a variety of higher plants. Due to the great variability of their structures, diverse functions have been described for distinct saponins; including foaming and pore forming properties as well as selective removal of protozoa from the rumen. The most interesting properties are, however, favorable anti-tumorigenic effects. Several saponins inhibit tumor cell growth by cell cycle arrest and apoptosis with half maximal inhibitory concentrations of down to 0.2 microM. A drawback of saponins in tumor therapy is the non-targeted spreading throughout the whole body. Surprisingly, certain saponins were identified that drastically enhance the efficacy of targeted chimeric toxins bearing the ribosome-inactivating protein saporin as cell-killing moiety. It was demonstrated that this effect is substantially more pronounced on target cells than on non-target cells, thus not only preserving the target specificity of the chimeric toxin but also broadening the therapeutic window with simultaneous dose lowering. This review describes the role of saponins as drug in general, their use as single drug treatment in tumor therapy, their combination with conventional tumor treatment strategies and the synergistic effects with particular targeted tumor therapies that are based on recombinant proteins.

An investigation of the kinetic and anti-angiogenic properties of plant glycoside inhibitors of thymidine phosphorylase

We investigated the potential of symplocomoside (1) and symponoside (2), glycosides isolated from the bark of Symplocos racemosa to inhibit thymidine phosphorylase (TP) activity and associated angiogenesis. Compound 1 was a reversible, noncompetitive inhibitor of deoxythymidine binding to TP (IC50 = 65.45 ± 5.08 μM; K i = 62.83 ± 2.10 μM) and 2 was a reversible, uncompetitive inhibitor (IC50 = 94.17 ± 4.05 μM; K i = 101.95 ± 1.65 μM). Molecular modeling analysis indicated that both compounds bound at the active site of the enzyme but not solely to amino acid residues involved in catalysis. Both compounds were active in in vitro angiogenic assays inhibiting endothelial cell migration and invasion in Matrigel, but did not inhibit growth factor-induced proliferation and were not cytotoxic. Compound 1 may have potential as an anti-angiogenic and anti-tumor agent.

Anti-Oxidant, Anti-Inflammatory and Anti-Allergic Activities of Luteolin

Luteolin is a flavone which occurs in medicinal plants as well as in some vegetables and spices. It is a natural anti-oxidant with less pro-oxidant potential than the flavonol quercetin, the best studied flavonoid, but apparently with a better safety profile. It displays excellent radical scavenging and cytoprotective properties, especially when tested in complex biological systems where it can interact with other anti-oxidants like vitamins. Luteolin displays specific anti-inflammatory effects at micromolar concentrations which are only partly explained by its anti-oxidant capacities. The anti-inflammatory activity includes activation of anti-oxidative enzymes, suppression of the NFkappaB pathway and inhibition of pro-inflammatory substances. In vivo, luteolin reduced increased vascular permeability and was effective in animal models of inflammation after parenteral and oral application. Although luteolin is only a minor component in our nutrition (less than 1 mg/day) epidemiological studies indicate that it has the potential to protect from diseases associated with inflammatory processes such as cardiovascular disease. Luteolin often occurs in the form of glycosides in plants, but these are cleaved and the aglycones are conjugated and metabolized after nutritional uptake which has to be considered when evaluating in vitro studies. Some data for oral and topical bioavailability exist, but more quantitative research in this field is needed to evaluate the physiological and therapeutical potential of luteolin.

Use of digitoxin and digoxin as internal standards in HPLC analysis of triterpene saponin‐containing extracts

Saponins are widely distributed complex plant glycosides possessing a variety of structure-dependent bioactivities. Quantitation of individual saponins is difficult due to lack of available standards, mainly as a consequence of purification difficulties. Determination of total saponin content can be problematic, often relying on non-specific methods based on butanol solubility, haemolytic activity or formation of coloured derivatives. To develop a general quantitative method based on the use of the readily available cardenolides, digitoxin (1) and digoxin (2), as internal standards in an HPLC-PAD-based analysis. The cardenolides were run at a variety of concentrations to establish linearity and reproducibility of detector response and then evaluated as internal standards for quantitation of triterpene saponins in several plant-derived extracts by HPLC-PAD. Mixtures of saponins, largely freed from other extractables, were obtained by fractionation of total extracts on solid phase extraction columns (SPE) employing a water-methanol gradient and used for construction of calibration curves. Saponin identification and structural information was obtained via a single quadrupole mass detector using electrospray ionisation in negative ion mode (ESI(-)). Saponin contents in six samples from five species were determined and compared with literature results and a gravimetric method based on butanol-water partitioning. Results were generally consistent with literature reports and superior to gravimetric butanol-water partitioning. Digitoxin and digoxin are useful as internal standards in HPLC estimation of saponin content. Saponins from different species having similar structures and molecular weights afford similar calibration curves.

Immunochromatographic assay for the detection of pseudojujubogenin glycosides

Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.

Positive effects of cyanogenic glycosides in food plants on larval development of the common blue butterfly

Cyanogenesis is a widespread chemical defence mechanism in plants against herbivory. However, some specialised herbivores overcome this protection by different behavioural or metabolic mechanisms. In the present study, we investigated the effect of presence or absence of cyanogenic glycosides in birdsfoot trefoil (Lotus corniculatus, Fabaceae) on oviposition behaviour, larval preference, larval development, adult weight and nectar preference of the common blue butterfly (Polyommatus icarus, Lycaenidae). For oviposition behaviour there was a female-specific reaction to cyanogenic glycoside content; i.e. some females preferred to oviposit on cyanogenic over acyanogenic plants, while other females behaved in the opposite way. Freshly hatched larvae did not discriminate between the two plant morphs. Since the two plant morphs differed not only in their content of cyanogenic glycoside, but also in N and water content, we expected these differences to affect larval growth. Contrary to our expectations, larvae feeding on cyanogenic plants showed a faster development and stronger weight gain than larvae feeding on acyanogenic plants. Furthermore, female genotype affected development time, larval and pupal weight of the common blue butterfly. However, most effects detected in the larval phase disappeared for adult weight, indicating compensatory feeding of larvae. Adult butterflies reared on the two cyanogenic glycoside plant morphs did not differ in their nectar preference. But a gender-specific effect was found, where females preferred amino acid-rich nectar while males did not discriminate between the two nectar mimics. The presented results indicate that larvae of the common blue butterfly can metabolise the surplus of N in cyanogenic plants for growth. Additionally, the female-specific behaviour to oviposit preferably on cyanogenic or acyanogenic plant morphs and the female-genotype-specific responses in life history traits indicate the genetic flexibility of this butterfly species and its potential for local adaptation.

Saponins in Tumor Therapy

Saponins are plant glycosides with favorable anti-tumorigenic properties. Several saponins inhibit tumor cell growth by cell cycle arrest and apoptosis with IC50 values of up to 0.2 microM. We discuss diverse groups of saponins (dioscins, saikosaponins, julibrosides, soy saponins, ginseng saponins and avicins) investigated in relation to tumor therapy and focus on cellular and systemic mechanisms of tumor cell growth inhibition both in vitro and in vivo. The review also describes saponins in combination with conventional tumor treatment strategies, which result in improved therapeutic success. Some combinations of saponins and anti-tumorigenic drugs induce synergistic effects with potentiated growth inhibition.

Physiological roles of plant glycoside hydrolases

The functions of plant glycoside hydrolases and transglycosidases have been studied using different biochemical and molecular genetic approaches. These enzymes are involved in the metabolism of various carbohydrates containing compounds present in the plant tissues. The structural and functional diversity of the carbohydrates implies a vast spectrum of enzymes involved in their metabolism. Complete genome sequence of Arabidopsis and rice has allowed the classification of glycoside hydrolases in different families based on amino acid sequence data. The genomes of these plants contain 29 families of glycoside hydrolases. This review summarizes the current research on plant glycoside hydrolases concerning their principal functional roles, which were attributed to different families. The majority of these plant glycoside hydrolases are involved in cell wall polysaccharide metabolism. Other functions include their participation in the biosynthesis and remodulation of glycans, mobilization of energy, defence, symbiosis, signalling, secondary plant metabolism and metabolism of glycolipids.

Crystal Structure of Glycoside Hydrolase Family 78 α- L-Rhamnosidase from Bacillus sp. GL1

α- L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 α- L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 Å resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3–956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are β-sandwich structures designated as domains N, D1, D2, and C, and an (α/α) 6-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase ( Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K i = 1.8 mM) was also determined and refined at 2.1 Å with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (α/α) 6-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of α- L-rhamnosidase and identification of its clan-L (α/α) 6-barrel as a catalytic domain.

Increase in Pharmacological Activity of Drugs in their Clathrates with Plant Glycosides

Increase in Pharmacological Activity of Drugs in their Clathrates with Plant Glycosides

Estrogenic activity of triterpene glycosides in yeast two-hybrid assay

Estrogenic potency of six triterpene glycosides, Holothurin A, Holotoxin A 1, Frondoside A, Cucumarioside A 2-2 and Cauloside C, that are natural products and semi-synthesized Ginsenoside-Rh 2, were examined with yeast two-hybrid system, including expressed genes of human estrogen receptor, hERα, the co-activator TIF2 and lacZ as a reporter gene. Only Ginsenoside-Rh 2 exhibited significant moderate estrogenic activity in the concentration range of 10 −7 to 10 −6 M. Its effect was approximately 30% of the activity of 17β-estradiol applied at half-effective concentration. This indicates Ginsenosides-Rh 2 is a weak phytoestrogen. The sea cucumber triterpene glycosides, Holothurin A, Holotoxin A 1, Cucumarioside A 2-2 and Frondoside A, and plant glycoside Cauloside C had no appreciable estrogenic activity. Data obtained by yeast two-hybrid assay reflect structure–activity relationship between tested compounds and 17β-estradiol. Only Ginsenoside-Rh 2 has some similarity in chemical structure with 17β-estradiol that might explain affinity of this glycoside to the hERα receptor.

Cloning of a Cholesterol-α-glucosyltransferase from Helicobacter pylori

O-Glycans of the human gastric mucosa show antimicrobial activity against the pathogenic bacterium Helicobacter pylori by inhibiting the bacterial cholesterol-alpha-glucosyltransferase (Kawakubo, M., Ito, Y., Okimura, Y., Kobayashi, M., Sakura, K., Kasama, S., Fukuda, M. N., Fukuda, M., Katsuyama, T., and Nakayama, J. (2004) Science 305, 1003-1006). This enzyme catalyzes the first step in the biosynthesis of four unusual glycolipids: cholesteryl-alpha-glucoside, cholesteryl-6'-O-acyl-alpha-glucoside, cholesteryl-6'-O-phosphatidyl-alpha-glucoside, and cholesteryl-6'-O-lysophosphatidyl-alpha-glucoside. Here we report the identification, cloning, and functional characterization of the cholesterol-alpha-glucosyltransferase from H. pylori. The hypothetical protein HP0421 from H. pylori belongs to the glycosyltransferase family 4 and shows similarities to some bacterial diacylglycerol-alpha-glucosyltransferases. Deletion of the HP0421 gene in H. pylori resulted in the loss of cholesteryl-alpha-glucoside and all of its three derivatives. Heterologous expression of HP0421 in the yeast Pichia pastoris led to the biosynthesis of ergosteryl-alpha-glucoside as demonstrated by purification of the lipid and subsequent structural analysis by nuclear magnetic resonance spectroscopy and mass spectrometry. In vitro enzyme assays were performed with cell-free homogenates obtained from cells of H. pylori or from transgenic Escherichia coli, which express HP0421. These assays revealed that the enzyme represents a membrane-bound, UDP-glucose-dependent cholesterol-alpha-glucosyltransferase.

Midgut glycosidases activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri

Parnassius apollo (Lepidoptera, Papilionidae) declines on numerous localities all over Europe. Its local subspecies frankenbergeri, inhabiting the Pieniny Mts (southern Poland) and successfully recovered from extinction, is monophagous in larval stage. In natural conditions, it completes development on the orpine Sedum telephium ssp. maximum. Since proper quality and quantity of necessary nutritional compounds of the food plant ensure developmental success, the digestive processes in the insect midgut should reflect adaptation to a specific food source. The paper presents, for the first time, the activity of detected glycolytic enzymes in midgut tissue and liquid gut contents of the L 4 and L 5 instars of P. apollo larvae. α-Amylase plays the main role in utilization of carbohydrates, contrary to cellulase activity. Saccharase seems to be the main disaccharidase, and high activity of β-glycosidase enables hydrolysis of the plant glycosides. Trehalase activity was unexpectedly low and comparable to those of cellobiase and lactase. α-Amylolytic and other glycolytic activities indicate that larvae utilize starch and other carbohydrate compounds as energy sources. Possible use of some plant allelochemicals as energy sources by Apollo larvae is discussed. To cite this article: M. Nakonieczny et al., C. R. Biologies 329 (2006).