In vitro evaluation of the cytotoxic potential of alkylphenols from Ginkgo biloba L.
Extracts from the leaves of Ginkgo biloba L. belong to the most widely used phytopharmaceuticals. In crude Ginkgo extracts, ginkgolic acids (GA) and related alkylphenols (e.g. cardanols and cardols) have been recognized as hazardous compounds with suspected cytotoxic, allergenic, mutagenic and carcinogenic properties. To further assess the cytotoxic potential of GA, their effect on the human keratinocyte cell line HaCaT and the rhesus monkey kidney tubular epithelial cell line LLC-MK(2) was investigated. The action of a defined mixture of GA on cell growth, viability and integrity was evaluated by the neutral red uptake assay as well as the release of lactate dehydrogenase (LDH) and acid phosphatase (ACP). Cell morphology was examined by electron microscopy. For comparison, the effect of the standardized Ginkgo extract EGb 761, which contains less than 5 ppm GA, was also investigated. Following incubation of cells with EGb 761, neutral red uptake was half-maximally inhibited at concentrations of 900 mg/l (HaCaT) and 1480 mg/ml (LLC-MK(2)). The corresponding IC(50)-values for the mixture of GA ranged between 22 mg/l (HaCaT) and 4.6 mg/l (LLC-MK(2)), respectively. In parallel to the inhibition of neutral red uptake, a concentration dependent release of LDH was observed when cells were incubated in the presence of GA (1-100 mg/l). In contrast, even at a concentration of 1800 mg/l EGb 761 did not cause release of LDH above controls. Since GA interacted with the assay for ACP, no index of lysosomal damage could be established by this method. Incubation of HaCaT cells with GA for 18 h increased the proportion of apoptotic cells from about 6% (control) to nearly 80% at concentrations of >or=30 mg/l. Electron microscopic analysis of HaCaT cells revealed a drug induced formation of myelinosomes possibly due to the inhibition of lysosomal enzymes, while morphological evaluation of LLC-MK(2) cells indicated that the cytotoxic activity of GA in these cells is primarily mediated by transformation of mitochondria, which is probably induced by uncoupling of oxidative phosphorylation.
Institute of Experimental and Clinical Pharmacology and Toxicology, Medical University of Lübeck, Ratzeburger Alle 160, Lubeck, Germany.
Helke
H
Hecker
Reiner
R
Johannisson
Egon
E
Koch
Claus Peter
CP
Siegers
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D020441:Ginkgo biloba / Q000737:chemistry*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects; D007684:Kidney Tubules / Q000187:drug effects / Q000648:ultrastructure; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008253:Macaca mulatta; D010085:Oxidative Phosphorylation / Q000187:drug effects; D010936:Plant Extracts / Q000494:pharmacology; D012459:Salicylates / Q000633:toxicity*; D000096983:Ginkgo Extract
D007770:L-Lactate Dehydrogenase; D010936:Plant Extracts; D012459:Salicylates; C063170:Ginkgo biloba extract; D000096983:Ginkgo Extract; C112485:ginkgolic acid