Evidence for immunotoxic effects of crude Ginkgo biloba L. leaf extracts using the popliteal lymph node assay in the mouse

Allergic reactions due to contact with different parts of the ancient tree Ginkgo biloba L. have repeatedly been reported. Provocation tests in patients and animal experiments have identified alkylphenols such as ginkgolic acids as causative constituents. Leaf extracts from Ginkgo are widely used to treat peripheral or cerebral circulatory disorders and Alzheimer's disease. Since alkylphenols are also present in leaves, potential allergic and other immunological hazards of such preparations have to be carefully controlled. Thus, we have evaluated if the popliteal lymph node assay (PLNA) in the mouse may represent a suitable model for the detection of constituents with immunotoxic properties in a complex mixture of biologically active agents such as plant extracts. Subplantar injection (2 mg) of a crude aqueous-ethanolic extract from Ginkgo leaves caused a significant lymphoproliferative reaction (LPR) in the ipsilateral popliteal lymph node. PLNA-active compounds in this extract could be enriched in the lipophilic phase by liquid-liquid partition between heptane and water. Chemical analysis of the heptane extract revealed the presence of a high concentration of alkylphenols (approx. 30%) and further subfractionation indicated that the enlargement of the popliteal lymph node was mainly due to the content of ginkgolic acids. This presumption was corroborated by observing a similar LPR following injection of a purified mixture of ginkgolic or hydroginkgolic acids. Thus, our experiments confirm that Ginkgo leaf extracts may contain constituents with immunotoxic properties, underlining the need to apply adequate production procedures to guarantee the completest possible removal of these compounds. The PLNA appears to represent a simple test model for the detection, characterisation and control of ingredients with potential immunotoxic side effects in complex herbal drugs.