CYCLODEXTRINS AS A USEFUL TOOL FOR BIOCONVERSIONS IN PLANT-CELL BIOTECHNOLOGY

The application of cyclodextrins as precursor solubilizers in biotechnological processes, in which plant cells are involved, is new. In this paper the possibilities for cyclodextrin facilitated bioconversions by freely suspended and/or immobilized plant cells or plant enzymes are demonstrated. After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased from almost insoluble to 660 mu M. In addition, by complexation with beta-cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed to cell cultures of Podophyllum hexandrum in order to enhance the accumulation of podophyllotoxin. Finally, the glucosylation of podophyllotoxin by cell cultures derived from Linum flavum was investigated. Four cyclodextrins: beta-cyclodextrin, gamma-cyclodextrin, hydroxypropyl-beta-cyclodextrin and dimethyl-beta-cyclodextrin were used to improve the solubility of podophyllotoxin. Dimethyl-beta-cyclodextrin met our needs the best and the solubility of podophyllotoxin could be enhanced from 0.15 to 1.92 mM. Podophyllotoxin-beta-D-glucoside was formed at a rate of 0.51 mmol l(-1) suspension per day by the L. flavum cells growing in the presence of 1.35 mM podophyllotoxin, complexed with dimethyl-beta-cyclodextrin

ON THE IMPROVEMENT OF THE PODOPHYLLOTOXIN PRODUCTION BY PHENYLPROPANOID PRECURSOR FEEDING TO CELL-CULTURES OF PODOPHYLLUM-HEXANDRUM ROYLE

In order to improve the production of the cytotoxic lignan podophyllotoxin, seven precursors from the phenylpropanoid-routing and one related compound were fed to cell suspension cultures derived from the rhizomes of Podophyllum hexandrum Royle. These cell cultures were able to convert only coniferin into podophyllotoxin, maximally a 12.8 fold increase in content was found. Permeabilization using isopropanol, in combination with coniferin as a substrate, did not result in an extra increase in podophyllotoxin accumulation. Concentrations of isopropanol exceeding 0.5% (v/v) were found to be rather toxic for suspension growth cells of P. hexandrum. When coniferin was fed in presence of such isopropanol concentrations, beta-glucosidase activity was still present, resulting in the formation of the aglucon coniferyl alcohol. In addition, podophyllotoxin was released into the medium under these permeabilization conditions. Entrapment of P. hexandrum cells in calcium-alginate as such or in combination with the feeding of biosynthetic precursors, did not improve the podophyllotoxin production. Cell-free medium from suspension cultures at later growth stages incubated with coniferin, resulted in the synthesis of the lignan pinoresinol

THE ACCUMULATION OF PODOPHYLLOTOXIN-BETA-D-GLUCOSIDE BY CELL-SUSPENSION CULTURES DERIVED FROM THE CONIFER CALLITRIS-DRUMMONDII

Podophyllotoxin was produced by cell cultures derived from needles of Callitris drummondii. The needles of this conifer contained 1.56% podophyllotoxin on a dry weight basis, 32% being present in the β-glucosidic form. Trace amounts of desoxypodophyllotoxin and matairesinol were also detected. In dark-grown cell cultures, ca. 0.02 % podophyllotoxin was accumulated, 85-90 % in the β-D-glucosidic form. Moreover, traces of the lignans matairesinol, hinokinin and asarinin were detected. Illumination stimulated the endogenous production of podophyllotoxin-β-D-glucoside; contents of up to 0.11 % could be measured

DETECTION AND IDENTIFICATION OF PODOPHYLLOTOXIN PRODUCED BY CELL-CULTURES DERIVED FROM PODOPHYLLUM HEXANDRUM ROYLE

The phenylpropanoid derived lignan podophyllotoxin, occurring inPodophyllum species, is used as a starting compound for the chemical synthesis of the antitumour agents etoposide (VP-16-213) and teniposide (VM-26). At present, the availability of this lignan becomes increasingly limited. As an alternative source, cell cultures originating fromPodophyllum hexandrum Royle were initiated. Analysis of the cell extracts using different HPLC systems as well as TLC, indicated the presence of podophyllotoxin. After prepurification of the extracts by means of ITLC, the identity was confirmed by mass spectrometric analysis. Dark-grown cultures accumulated considerable higher amounts of podophyllotoxin in comparison with the light-grown culture

The large-scale isolation of deoxypodophyllotoxin from rhizomes of Anthriscus sylvestris followed by its bioconversion into 5-methoxypodophyllotoxin beta-D-glucoside by cell cultures of Linum flavum

Dried rhizomes of Anthriscus sylvestris contained 0.39% deoxypodophyllotoxin (1). An isolation procedure with an extraction efficiency of 51.2% was developed for this commercially unavailable lignan. The isolated 1 was subsequently complexed with dimethyl-P-cyclodextrin and fed to cell suspension cultures of Linum flavum by which it was bioconverted into 5-methoxypodophyllotoxin-beta-D-glucoside (2). After 7 days the cells contained 4.41% of this product on a dry-weight basis. An isolation procedure for compound 2 was developed, with an extraction efficiency of 83.4%

Structure-cytotoxicity relationships of a series of natural and semi-synthetic simple coumarins as assessed in two human tumour cell lines

The cytotoxicity of 22 natural and semi-synthetic simple coumarins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values > 100 mu M, following a continuous (96h) incubation, most coumarins exhibited only low cytotoxicity. Several compounds, however, displayed significant potencies. As far as the structure - cytotoxicity relationship is concerned, it is conspicuous that all the potentially active natural compounds possess at least two phenolic groups in either the 6,7- or 6,8-positions. In addition, the 5-formyl-6-hydroxy substituted semi-synthetic analogue was found to be potent, reflecting the importance of at least two polar functions for high cytotoxicity