Cytotoxicity of natural ginseng glycosides and semisynthetic analogues

The cytotoxicity of natural glycosides from Ginseng, semisynthetic analogues and related triterpenes of the dammarane series, isolated from the leaves of the Far-East species of the genus Betula was studied in order to elucidate structure-activity relationships. Some of the compounds studied were active against the human lung carcinoma GLC4 and adenocarcinoma COLO 320 cell lines. The natural glycosides displayed the lowest cytotoxicity. The triterpenes of the dammarane series used as starting aglycones for semisynthetic derivatives were moderately cytotoxic. The dammarane triterpenes possessing keto groups and their semisynthetic glucosides were the most active compounds tested. Cytotoxic effects of the dammarane glucosides were inversely proportional both to the number of sugars attached to the aglycones and to the number of hydroxy groups of the aglycones. The type of side chain and the configuration of the hydroxy group at C-3 in aglycones did not have a significant influence on the cytotoxicity.     

Mucuna pruriens: Improvement of the biotechnological production of the anti-Parkinson drug L-dopa by plant cell selection

Routinely grown cell suspension cultures ofMucuna pruriens L. (Fabaceae) were able to endogenously accumulate the anti-Parkinson drug L-dihydroxyphenylalanine (L-dopa) in the range between 0.2 and 2% on a dry weight (DW) basis. The green colour that developed in light-exposed cultures, appeared to be a suitable marker to select cells with an increased L-dopa biosynthesis and/or phenoloxidase activity. For this purpose, saccharose concentrations from 0 to 4% (w/v), and light intensities of 1,000 and 2,000 lux, were involved in the selection procedure. After 6 months, photomixotrophic callus cultures with a rapid growth and a high L-dopa content of 0.9% (DW) were obtained on 2% saccharose and under 1,000 lux. The cell suspensions, derived from these calli, accumulated up to 6% (DW) L-dopa, which was the highest stable content ever measured in cultures ofM. pruriens. An L-dopa yield of approximately 1.2 g/l was calculated after 6 days of growth. In contrast, compared with the standard-grown parent cell line, the phenoloxidase activity, and consequently the bioconversion capacity as measured after entrapment in calcium alginate, of these high-producing cultures was approximately threefold lower.This publication is dedicated to the memory of Prof. Dr. Th.M. Malingre, who died on 10 April 1993.     

Role of membrane lipid composition in the cytotoxicity of the sesquiterpene lactone eupatoriopicrin

The aim of the present study was to investigate a possible role of lipid peroxidation in the cytotoxicity of eupatoriopicrin, the principal sesquiterpene lactone from Eupatorum cannabinum L. Incorporation of arachidonic acid acyl chains in the phospholipids of cellular membranes of mouse fibroblast LM cells enhanced their sensitivity to the cytotoxic effect of eupatoriopicrin. In the clonogenic assay (2 h exposure) 90% cell kill was found with 14.4 μM eupatoriopicrin in normal cells and 11.7 μM in cells enriched with arachidonic acid (PUFA cells). After incubation with eupatoriopicrin a more rapid loss of membrane integrity and more lysis was found for PUFA cells in the trypan blue exclusion assay. Using gas chromatography, it was shown that the spectrum of fatty acid acyl chains in the phospholipids of normal cells remained unchanged after 2 h incubation with 20 μM eupatoripicrin, followed by 24 h post-incubation. In PUFA cells the polyunsaturated fatty acids were found to degrade possibly due to lipid peroxidation. Using the alkaline unwinding assay and clamped homogeneous electric field electrophoresis, more DNA damage was found in PUFA cells than in normal cells after treatment with eupatoriopicrin. However, because in normal cells, in which PUFA were nearly absent, DNA damage was found as well, it is concluded that lipid peroxidation plays at most a minor role in eupatoriopicrin-induced cell killing.     

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.     

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.