Tm Malingre

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 cultures.

PRODUCTION OF THE NEW ANTIMALARIAL DRUG ARTEMISININ IN SHOOT CULTURES OF ARTEMISIA-ANNUA L

From aseptically grown Artemisia annua plantlets, shoot cultures were initiated. Using different concentrations of auxine, cytokinine and sucrose, a suitable culture medium was developed, with respect to the growth of the shoots and their artemisinin accumulation. Nitrate concentration and conductivity appeared to be suitable growth parameters. The artemisinin content was measured gas chromatographically. The shoot cultures were maintained in the developed standard medium, consisting of a half concentration of MS-salts with vitamins, 0.2 mg l-1 BAP, 0.05 mg l-1 NAA and 1% sucrose. The growth of the shoots and the artemisinin content remained stable for a longer period. They showed considerable photosynthetic activity and generally contained ca. 0.08% artemisinin on a dry weight basis. The highest artemisinin content found was 0.16% in the above mentioned standard medium, but also on the same medium with 0.5% sucrose. Attempts were made to further improve the artemisinin production by varying the medium composition through addition of gibberellic acid or casein hydroly-state; by omitting plant growth regulators; by precursor feeding, i.e. mevalonic acid; by influencing the biosynthesis routing through inhibition of the sterol synthesis by miconazole, naftifine or terbinafine; by changing gene expression with 5-azacytidine or colchicine; and by elicitation, using cellulase, chitosan, glutathione or nigeran. Enhanced artemisinin production was found with 10 mg l-1 gibberellic acid, 0.5 g l-1 casein hydrolysate, 10 mg l-1 or 20 mg l-1 naftifine. Relative increases of 154%, 169%, 140% and 120% were found, respectively. Other additions caused the growth to cease and the artemisinin contents to drop.

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, β-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.

Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

SummaryCell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of β-cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with β-cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. β-Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble β-D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with β-cyclodextrin, but coniferin is not commercially available.

Epicuticular wax composition of some European Sedum species

Abstract Epicuticular waxes from 30 species of Sedum and 2 species of Sempervivoideae, i.e. Aeonium spathulatum and Sempervivum nevadense , have been analysed by GC and GC-MS. The Sedum taxa examined were S. acre , S. album , S . series Alpestria (13 species), S. anglicum , S. brevifolium , S. litoreum , S. lydium , S . series Macaronesia (four species), S. melanantherum and S . series Rupestria (five species) of S . sect. Sedum and S. meyeri-johannis of S . sect. Africana . The waxes consist of alkanes, alkanols, fatty acids, fatty acid methyl esters, aldehydes, wax esters and triterpenes. Some 14 triterpenes were detected in waxes of Sedum , the major triterpenes being β-amyrenyl acetate, germanicyl formate (not previously reported from a natural source), multiflorenyl acetate and taraxeryl acetate. Waxes of the pruinose and glaucous taxa of Sedum were found to have a high triterpene content. In waxes of Aeonium spathulatum and Sempervivum nevadense , no triterpenes could be detected. Variation in the alkane and triterpene profiles proved to be of significant systematic value. In general, the distribution of the triterpenes in Sedum agrees with the infrageneric classification based on hybridization patterns and related morphological characters.

The accumulation and isolation of coniferin from a high-producing cell suspension of Linum flavum L.

Cell suspensions of Linum flavum L. contained large amounts (2 g·l−1) of the glucoside coniferin which was accumulated endogenously up to 12.4% on a dryweight basis. Callus material contained 5.6%, while in leaves of in-vitro-grown plantlets, the origin of the callus and suspension cultures, no coniferin could be detected. Leaf, callus and suspension material were compared for metabolite accumulation and associated enzyme activities. High coniferin contents corresponded with low 5-methoxypodophyllotoxin levels. A reciprocal relationship between β-glucosidase (E.C. 3.2.1.21) activity and coniferin accumulation was found. No relationship between peroxidase (E.C. 1.11.1.7) activity and coniferin accumulation or 5-methoxypodophyllotoxin could be demonstrated. Finally, a rapid and effective isolation procedure for coniferin was developed.

Enhanced cytostatic activity of the sesquiterpene lactone eupatoriopicrin by glutathione depletion

Eupatoriopicrin (EUP), a sesquiterpene lactone from Eupatorium cannabinum L., possesses cytostatic activity. This was demonstrated for FIO 26 cells in vitro with the aid of a clonogenic assay and in vivo by tumour growth delay in FIO 26 and Lewis lung tumour-bearing mice. In vitro the IC50 for 1 h exposure to EUP was 1.5 microgram ml-1 (4.1 nmol ml-1). This concentration depleted about 25% of its cellular GSH concentration. Pretreatment of FIO 26 cells with BSO, resulting in greater than 99%. GSH depletion, enhanced the cytotoxic effect of EUP. The dose-enhancement factor at the level of 10% cell survival was 2.3. Growth inhibition of the Lewis lung carcinoma and the FIO 26 fibrosarcoma, solidly growing in C57Bl mice, was found after i.v. injection of 20 or 40 mg kg-1 EUP, at a tumour volume of about 500 microliters. Pretreatment with BSO at a dose of 4 mmol kg-1 i.p., 6 h before EUP administration, resulted in a significantly stronger growth delay of both tumours compared with EUP only. At the time of EUP treatment, cellular GSH in the tumours was reduced by BSO treatment to about 60%. It is concluded that EUP possesses antitumour activity in vivo and that chemosensitisation of EUP may be accomplished by pretreatment with BSO, indicating that endogenous GSH protects against the cytostatic action of EUP.

Optimization of the biotransformation of l-tyrosine into l-dihydroxyphenylalanine (DOPA) by alginate-entrapped cells of Mucuna pruriens

The optimization of the biotransformation of l-tyrosine into l-dihydroxyphenylalanine (DOPA), and of formyl-tyrosine into formyl-DOPA by alginate-entrapped cells of Mucuna pruriens is reported. This optimization is discussed in terms of parameters that are relevant for the entrapped cell system (charge of the beads with cells, bead diameter, permeation of the cells) and some parameters that are relevant for the enzymatic transformation (pH, pO2, concentration of ascorbate). The optimization experiments resulted in the description of a biotransformation system which operates at a constant redox potential. With this transformation system a transformation efficiency of 70% could be obtained, at a biotransformation-rate of 435 μmol·h-1·g-1 (DW of cells) at a substrate concentration of 19 mM.

Flavones and flavonol glycosides from Eupatorium cannabinum L.

The 6-methoxyflavones hispidulin and eupafolin have been identified for the first time from the aerial parts ofEupatorium cannabinum L. The presence of the previously known flavonol glycosides astragalin, kaempferol-3-rutinoside, hyperoside, isoquercitrin and rutin could be confirmed. Hispidulin, eupafolin and rutin were screened for cytotoxicityin vitro.

Induction of DNA damage in Ehrlich ascites tumour cells by exposure to eupatoriopicrin

The sesquiterpene lactone eupatoriopicrin (EUP) from Eupatorium cannabinum L. has been shown to be cytotoxic in a glutathione (GSH)-dependent way. In order to assess possible DNA damage as a cause for cell death, the study reported was initiated. After 2 hr incubation of Ehrlich ascites tumour cells with EUP, the DNA damage, determined by the use of an alkaline DNA unwinding method, followed by hydroxylapatite column chromatography of degraded DNA, was observed at concentrations only slightly higher than those causing cell death in a clonogenic assay. The amount of EUP, requested to demonstrate DNA damage after a 24-hr post-incubation period lay within the concentration range that was effective in the clonogenic assay (1-10 micrograms/ml). Glutathione (GSH) depletion of the cells to about 99%, by use of buthionine sulphoximine (BSO), enhanced the extent of DNA damage. It is concluded that EUP-induced DNA damage may play a role in the observed cytotoxicity.