Planta Med 2013; 79 - PK23
DOI: 10.1055/s-0033-1352283

Qualitative and quantitative analysis of tumor-promoting and skin irritating diterpene esters in homeopathic mother tinctures by HPLC and MS

J Kummer 1, MF Melzig 1
  • 1Institute of Pharmacy – Pharmaceutical Biology, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany

Some plants from the family Euphorbiaceae were used in the past as traditional remedies. With ingenol melbutate there is now a highly active therapeutic for the topical field therapy of actinic keratosis [1]. The active ingredients are diterpene esters, which are known to be tumor-promoting and skin irritating since Hecker et al. discovered TPA (12-O-tetradecanoylphorbol-13-acetate) in the oil of Croton tiglium L. seeds [2].

There are two basic chemical structures such as the alcohols phorbol and ingenol. These two alcohols form a great variety of different esters with different activities. Available preparations of these esters are homeopathic mother tinctures. For standardization and the monographic description in pharmacopoeias it is necessary to establish an easy method to quantify these esters from herbal preparations, e.g. homeopathic mother tinctures.

To avoid the difficult analysis of the different esters and to quantify all the toxic esters it's necessary to hydrolyse them and to determine the resulting alcohols by HPLC. The alkaline hydrolysis is not suitable for all structures because of a bad recovery [3].

We will discuss the different approaches for gentle methods to hydrolyze the esters. The enzymatic hydrolysis with different enzymes such as esterase from Bacillus subtilis and the S9 fraction from rat liver ended almost in complete hydrolysis.

For the identification of the different diterpene esters their peaks where collected after HPLC separation and then analysed by mass spectrometry.

References:

[1] Lebwohl, M. et al., N Engl J Med 2012, 366: 1010 – 1019.

[2] Hecker, E., Schmidt, R., Prog Chem Org Nat Prod 1974 31: 377 – 467.

[3] Gläser, S., Untersuchungen zu einem möglichen Gesundheits- und Krebsrisiko durch pflanzliche Arzneimittel sowie industriell genutze Rohstoffe aus Euphorbiaceen – Quantitative Bestimmung von irritierenden und tumorpromovierenden Diterpenestern und Evaluierung durch biochemische und biologische Tests (Thesis, Universität Heidelberg) 1991