Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey.

Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 µg ml(-1) thidiazuron (TDZ) and 0.25 µg ml(-1) indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 µg g(-1))>D. trojana (1385.75 µg g(-1))>D. cariensis (1038.65 µg g(-1))>D. davisiana (899.86 µg g(-1)) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides.

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.

Antimicrobial activity of endemic Digitalis lamarckii Ivan from Turkey.

Antimicrobial activity of the methanolic extracts of leaves and flowers of D. lamarckii Ivan, (Scophulariaceae), an endemic plant species of Turkey, was tested on ten bacterial and four yeast strains. Effective antibacterial activity was observed in four bacterial strains. Minimum inhibitory concentration (MIC) was calculated by use of liquid culture tests and in all the four effective bacterial strains, the MIC was found to be > or = 199.5 mg/ml. The minimum bactericidal concentration (MBC) of B. subtilis, S. aureus, and L. monocytogenes was calculated to be > or = 199.5 mg/ml, and MBC value for Shigella was calculated as > or = 399 mg/ml.

Use of commercial plant species in a hydroponic system to treat domestic wastewaters.

The objectives in this work were to investigate a conceptual layout for an inexpensive and simple system that would treat primary municipal wastewater to discharge standards. A commercial hydroponic system was adapted for this study and the wastewater was used to irrigate wooly digitalis (Digitalis lanata Ehrh.) and foxglove (Digitalis purpurea L.). These plants are medicinal and produce cardenolide compounds. Influent and effluent samples were collected once a month for six months and analyzed to determine the various parameters relating to water quality. The legal discharge levels for total suspended solids (SS), biochemical oxygen demand (BOD5), and chemical oxygen demand (COD) were reached for the two tested plants after 48 h of wastewater treatment; the removal was 82, 93, and 79%, respectively, for wooly digitalis and 92, 92, and 84%, respectively, for foxglove. Similar results were obtained during a 6-mo period although the sewage composition varied widely. The system tended to be unable to remove N and P to concentrations below regulated levels. Compared with the nutrient solution composition, the wastewater was more concentrated in Na+ and Cl- and less in N, K+, and Ca2+. These variations can lead to the decline of wooly digitalis plants. Foxglove developed a significant root system to increase mineral absorption wastewater being used as the unique nutritive source. After 10 wk all the wooly digitalis seedlings were dead. Despite this fact, however, the root system remained in place for a significant time (< 4 mo), thus continuing to filter wastewater and to be used as a bacterial support thus making it possible to have a security period to replace the dead plants.

Haploid plants regenerated from androgenic cell cultures of Digitalis lanata.

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.

12 beta-Hydroxylation of digitoxin by suspension-cultured Digitalis lanata cells: production of digoxin in 20-litre and 300-litre air-lift bioreactors.

A biotransformation process for the production of digoxin was developed using Digitalis lanata cell suspension cultures. Digitoxin was used as the substrate for biotransformation. Digoxin production was carried out in a variety of vessels, including 1-l exsiccators, 20-l glass reactors and a 300-l air-lift bioreactor. A culture volume of 200 l was established after 28 d and the cells were then cultured semi-continuously in a 300-l bioreactor employing the draw-fill cultivation method. Maximal digoxin production was achieved in an 8% glucose medium with a production optimum after 40-60 h of incubation in the presence of 0.65-0.8 mmol digitoxin per l. Levels of 0.52, 0.53 and 0.60 mmol digoxin per l suspension were achieved in 1-l, 20-l and 300-l vessels, respectively. About 80% of the digoxin produced was found in the bathing medium.

Cultivation and breeding of Digitalis lanata in the Netherlands.

After the second world war Marshall Aid funds were used to establish a cooperative organisation for growing, drying, and selling Digitalis lanata (and other medicinal, aromatic, and culinary herbs) in the Netherlands. The crop is sown in mid April and the fully mechanised harvest of the leaves takes place from September to late November. The leaves are dried for 10-12 hours at 50 degrees C maximum. The aim of breeding trials is to improve leaf production, erect leaf attitude, resistance to Septoria leaf spot and to bolting, and a higher dry matter and digoxin content.