Overexpression and RNAi-mediated Knockdown of Two 3ß-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis.

3ß-hydroxy-Δ5-steroid dehydrogenases (3ßHSDs) are supposed to be involved in 5ß-cardenolide biosynthesis. Here, a novel 3ßHSD (Dl3ßHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3ßHSD1 and Dl3ßHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3ßHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3ßHSD1, Dl3ßHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3ßHSDs was realised by Agrobacterium-mediated transfer of Dl3ßHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3ßHSD1 and 35S:Dl3ßHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3ßHSD1 lines than in the controls. In the 35S:Dl3ßHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3ßHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3ß-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3ßHSD1 knockdown. These results can be taken as the first direct proof that Dl3ßHSD1 is indeed involved in 5ß-cardenolide biosynthesis.

Effects of Lipophilicity and Structural Features on the Antiherpes Activity of Digitalis Cardenolides and Derivatives.

There is growing interest in exploring Digitalis cardenolides as potential antiviral agents. Hence, we herein investigated the influence of structural features and lipophilicity on the antiherpes activity of 65 natural and semisynthetic cardenolides assayed in vitro against HSV-1. The presence of an α,ß-unsaturated lactone ring at C-17, a ß-hydroxy group at C-14 and C-3ß-OR substituents were considered essential requirements for this biological activity. Glycosides were more active than their genins, especially monoglycosides containing a rhamnose residue. The activity enhanced in derivatives bearing an aldehyde group at C-19 instead of a methyl group, whereas inserting a C-5ß-OH improved the antiherpes effect significantly. The cardenolides lipophilicity was accessed by measuring experimentally their log P values (n-octanol-water partition coefficient) and disclosed a range of lipophilicity (log P 0.75±0.25) associated with the optimal antiherpes activity. In silico studies were carried out and resulted in the establishment of two predictive models potentially useful to identify and/or optimize novel antiherpes cardenolides. The effectiveness of the models was confirmed by retrospective analysis of the studied compounds. This is the first SAR study addressing the antiherpes activity of cardenolides. The developed computational models were able to predict the active cardenolides and their log P values.

Elucidation of the mechanism of anti-herpes action of two novel semisynthetic cardenolide derivatives.

Human herpesviruses are among the most prevalent pathogens worldwide and have become an important public health issue. Recurrent infections and the emergence of resistant viral strains reinforce the need of searching new drugs to treat herpes virus infections. Cardiac glycosides are used clinically to treat cardiovascular disturbances, such as congestive heart failure and atrial arrhythmias. In recent years, they have sparked new interest in their potential anti-herpes action. It has been previously reported by our research group that two new semisynthetic cardenolides, namely C10 (3ß-[(N-(2-hydroxyethyl)aminoacetyl]amino-3-deoxydigitoxigenin) and C11 (3ß-(hydroxyacetyl)amino-3-deoxydigitoxigenin), exhibited potential anti-HSV-1 and anti-HSV-2 with selectivity index values > 1,000, comparable with those of acyclovir. This work reports the mechanism investigation of anti-herpes action of these derivatives. The results demonstrated that C10 and C11 interfere with the intermediate and final steps of HSV replication, but not with the early stages, since they completely abolished the expression of the UL42 (ß) and gD (γ) proteins and partially reduced that of ICP27 (α). Additionally, they were not virucidal and had no prophylactic effects. Both compounds inhibited HSV replication at nanomolar concentrations, but cardenolide C10 was more active than C11 and can be considered as an anti-herpes drug candidate including against acyclovir-resistant HSV-1 strains.

The Foxgloves (Digitalis) Revisited.

This review provides a renewed look at the genus Digitalis. Emphasis will be put on those issues that attracted the most attention or even went through paradigmatic changes since the turn of the millennium. PubMed and Google Scholar were used ("Digitalis" and "Foxglove" were the key words) to identify research from 2000 till 2017 containing data relevant enough to be presented here. Intriguing new results emerged from studies related to the phylogeny and taxonomy of the genus as well as to the biosynthesis and potential medicinal uses of the key active compounds, the cardiac glycosides. Several Eastern and Western Foxgloves were studied with respect to their propagation in vitro. In this context, molecular biology tools were applied and phytochemical analyses were conducted. Structure elucidation and analytical methods, which have experienced less exciting progress, will not be considered here in great detail.

Production of the Cytotoxic Cardenolide Glucoevatromonoside by Semisynthesis and Biotransformation of Evatromonoside by a Digitalis lanata Cell Culture.

Recent studies demonstrate that cardiac glycosides, known to inhibit Na+/K+-ATPase in humans, have increased susceptibility to cancer cells that can be used in tumor therapy. One of the most promising candidates identified so far is glucoevatromonoside, which can be isolated from the endangered species Digitalis mariana ssp. heywoodii. Due to its complex structure, glucoevatromonoside cannot be obtained economically by total chemical synthesis. Here we describe two methods for glucoevatromonoside production, both using evatromonoside obtained by chemical degradation of digitoxin as the precursor. 1) Catalyst-controlled, regioselective glycosylation of evatromonoside to glucoevatromonoside using 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide as the sugar donor and 2-aminoethyldiphenylborinate as the catalyst resulted in an overall 30 % yield. 2) Biotransformation of evatromonoside using Digitalis lanata plant cell suspension cultures was less efficient and resulted only in overall 18 % pure product. Structural proof of products has been provided by extensive NMR data. Glucoevatromonoside and its non-natural 1-3 linked isomer neo-glucoevatromonoside obtained by semisynthesis were evaluated against renal cell carcinoma and prostate cancer cell lines.

Three-Step Test System for the Identification of Novel GABAA Receptor Modulating Food Plants.

Potentiation of γ-amino butyric acid (GABA)-induced GABAA receptor (GABAAR) activation is a common pathway to achieve sedative, sleep-enhancing, anxiolytic, and antidepressant effects. Presently, a three-component test system was established for the identification of novel GABAAR modulating food plants. In the first step, potentiation of GABA-induced response of the GABAAR was analysed by two-electrode voltage clamp (TEVC) for activity on human α1ß2-GABAAR expressed in Xenopus laevis oocytes. Positively tested food plants were then subjected to quantification of GABA content by high-performance liquid chromatography with fluorescence detection (HPLC-FLD) to exclude test foods, which evoke a TEVC-response by endogenous GABA. In the third step, specificity of GABAA-modulating activity was assessed by TEVC analysis of Xenopus laevis oocytes expressing the homologous glycine receptor (GlyR). The three-component test was then applied to screen 10 aqueous extracts of food plants for their GABAAR activity. Thus, hop cones (Humulus lupulus) and Sideritis sipylea were identified as the most potent specific GABAAR modulators eliciting significant potentiation of the current by 182 ± 27 and 172 ± 19 %, respectively, at the lowest concentration of 0.5 µg/mL. The extracts can now be further evaluated by in vivo studies and by structural evaluation of the active components.

Antiherpes activity of glucoevatromonoside, a cardenolide isolated from a Brazilian cultivar of Digitalis lanata.

Cardiac glycosides, known ligands of the sodium pump, are widely used in the treatment of heart failure, such as digoxin and digitoxin. Besides this important activity, other biological activities, such as the antiviral activity, have been described for this group. HSV are responsible for many infections of oral, ocular and genital regions. Treatment with nucleoside analogs such as acyclovir is effective in most cases; however drug-resistance may arise due to prolonged treatment mainly in immunocompromised individuals. In this study, an antiherpes screening was performed with 65 cardenolide derivatives obtained from different sources, and one natural cardenolide, glucoevatromonoside, inhibited HSV-1 and HSV-2 replication at very low concentrations. This cardenolide showed viral inhibitory effects if added up to 12h p.i. and these effects appear to take place by the inhibition of viral proteins synthesis (ICP27, U(L)42, gB, gD), the blockage of virus release and the reduction of viral cell-to-cell spread. This compound also showed synergistic antiviral effects with acyclovir and anti-Na(+)K(+)ATPase activity, suggesting that cellular electrochemical gradient alterations might be involved in the mechanism of viral inhibition. These results suggest that cardenolides might be promising for future antiviral drug design.

Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH.

Plants of the genus Digitalis produce 5 beta-cardenolides that are used in the therapy of cardiac insufficiency in humans. 3 beta-Hydroxysteroid dehydrogenase (3 beta-HSD) and progesterone 5 beta-reductase (P5 betaR) are both supposed to be important enzymes in the biosynthesis of these natural products. Activity and gene expression were demonstrated for both enzymes in cardenolide-accumulating leaves of Digitalis lanata but also in cardenolide-free permanent cell suspension cultures initiated from D. lanata leaf tissue. Enzyme activities were determined and quantified by HPLC and GC-MS methods. Expression of the respective genes, namely AY585867.1 (P5betaR gene) and DQ466890.1 (3beta-HSD gene), was made evident by real-time polymerase chain reaction (qPCR) analysis. We demonstrate for the first time that the P5betaR gene, encoding an enzyme described as a key enzyme in cardenolide biosynthesis, is also expressed in cardenolide-free tissues of cardenolide-containing plants.

Immobilization of flavonol 3-O-beta-heterodisaccharidase on porous glass and production of rutinose from rutin.

Flavonol 3- O-beta-heterodisaccharidase (FHG 1) was isolated from the dried herb of Fagopyrum esculentum, immobilized on porous glass, and used for the release of rutinose from rutin. The stability of the enzyme in its free or immobilized form was observed continuously at two different temperatures (4 degrees C and 25 degrees C). T(1/2) values were determined to be about 48 h for the free enzyme and about 300 h for the immobilized enzyme. The rutinose released was isolated by fractionated ethanol precipitation.

Delta 5-3beta-hydroxysteroid dehydrogenase (3 beta HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme.

During the biosynthesis of cardiac glycosides, Delta (5)-3beta-hydroxysteroid dehydrogenase (3 beta HSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3beta-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3 beta HSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3 beta HSD was isolated by RT-PCR from the same source. A SPH I /KPN I 3 beta HSD cDNA was cloned into the pQE30 vector and then transferred into E. COLI strain M15[pREP4]. 3 beta HSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3 beta HSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). pQ3 beta HSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3beta-hydroxypregnanes but not cholesterol were 3beta-oxidised by pQ3 beta HSD when NAD was used as the co-substrate. Testosterone (4-androsten-17beta-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3 beta HSD has also 17beta-dehydrogenase activity. pQ3 beta HSD was able to reduce 3-keto steroids to their corresponding 3beta-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3 beta HSD genes were isolated and sequenced from another 6 species of the genus DIGITALIS. Gene structure and the deduced 3 beta HSD proteins share a high degree of similarity.

Cardenolide genin pattern in Isoplexis plants and shoot cultures.

An HPLC method for the quantitative analysis of cardenolides was developed and applied. The procedure was optimised for analysing small samples (40 mg dw) of plant and tissue culture material. ISOPLEXIS CANARIENSIS plants obtained from seeds accumulated cardenolides to about 20 - 40 micromol g (-1) dw as calculated from the levels of cardenolide genins released after acidic hydrolysis of methanolic extracts. The relative contents of xysmalogenin, digitoxigenin, uzarigenin and canarigenin were 5 - 15 %, 0 - 5 %, 10 - 15 % and 70 - 90 %, respectively. Shoot cultures were initiated from seeds, established as permanent cultures and cultivated on agar-solidified or in liquid medium. Shoot cultures maintained on solid medium contained an average of about 6 micromol cardenolides g (-1) dw. A relatively high proportion of digitoxigenin was found in two-thirds of the shoot cultures examined. The cardenolide content of amphibian shoot cultures averaged to about 1 micromol g (-1) dw. Plants regenerated from shoot cultures and maintained under hydroponic conditions accumulated the same amount of cardenolides as plants collected in the field. No cardenolides could be detected in callus cultures.

Molecular cloning and expression of progesterone 5beta-reductase (5beta-POR) from Isoplexis canariensis.

A full-length cDNA clone that encodes progesterone 5beta-reductase (5beta-POR, EC 1.3.1.3) was isolated from ISOPLEXIS CANARIENSIS leaves. The reading frame of the IC5beta-POR gene is 1170 nucleotides corresponding to 389 amino acids. The SPHI /SALI IC5beta-POR fragment was cloned into the pQE vector system and then transformed into ESCHERICHIA COLI strain M15[pREP4]. The gene was functionally expressed and the recombinant enzyme was characterised. K(m) and V(max) were calculated to be 0.215 mM and 46.4 nkat/mg protein, respectively, using progesterone as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The 5beta-POR from I. CANARIENSIS shows a significant homology to the putative progesterone 5beta-reductases isolated from other plant species, such as DIGITALIS LANATA and ARABIDOPSIS THALIANA.

Isolation and characterisation of a cysteine protease (phytolacain G), from Phytolacca americana roots.

Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl- L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed "lectin-like" activities such as haemagglutination and mitogenic effects towards human lymphocytes.

Purification and characterization of a flavonol 3-O-beta-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench.

A flavonol-3-O-beta-heterodisaccharide glycosidase (FHG I) was isolated from dried aerial tissues of Fagopyrum esculentum Moench (Fagopyri herba). It has a specific enzyme activity of ca. 3.5 nkat mg(-1) protein in buffered extracts when rutin (quercetin-3-O-rutinoside) was used as substrate and an optimal enzyme activity was seen at around pH 4.8 and 30 degrees C. FHG I was purified about 156-fold to apparent homogeneity by hydrophobic interaction, anion exchange and size exclusion chromatographic steps. The apparent molecular mass of FHG I was 74.5+/-2 kDa as determined by SDS-PAGE and it is a monomeric glycoprotein with a carbohydrate content of 23%. The isoelectric point as determined by isoelectric focusing was 5.7 and the energy of activation was 32 kJ mol(-1). FHG I exhibits a high substrate specificity, preferring flavonol 3-O-glycosides comprising the disaccharide rutinose. The K(m) and V(max) values for the natural substrate rutin were calculated to be 0.561 microM and 745 nkat mg (-1) protein, respectively. Two oligopeptide fragments obtained after enzymatic digestion of FHG I were sequenced and showed similarities to sequences of beta-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3-O-beta-heterodisaccharide glycosidase (FHG II) could also be detected in buckwheat herb, having a molecular mass of 85.3+/-2 kDa and an isoelectric point between pH 6.0 and 6.5.