Isolation and characterization of natural inhibitors of post-proline specific peptidases from the leaves of Cotinus coggygria Scop

Ethnopharmacological relevance: Cotinus coggygria has a number of applications in traditional medicine most of which are connected with its anti-inflammatory and anti-oxidant properties. Since inflammation and oxidative stress are recognized as triggering factors for cancer, anti-cancer activity has also been documented and the possible mechanisms of this activity are under investigation. Important components of C. coggygria extracts are shown to be hydrolysable gallotannins of which pentagalloyl-O-glucose has been studied in details. This com-pound inhibits various enzymes including prolyl oligopeptidase which is involved in tumorigenesis and tumour growth. According to our pilot studies, oligo-O-galloylglucoses with more than five galloyl residues are also presented in the herb of Bulgarian origin, but their activities have not been examined.Aim of the study: To establish an extraction method by which it is possible to concentrate high molecular hydrolysable gallotannins from dried leaves of Cotinus coggygria and to determine their inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein alpha.Materials and methods: Dried leaves of C. coggygria were extracted using different solvents in single-phase or biphasic systems under various extraction conditions. Main compounds of the extracts were identified by using high performance liquid chromatography and liquid chromatography - high resolution mass spectrometry. The extracts' inhibitory properties towards prolyl oligopeptidase and fibroblast activation protein alpha were studied on recombinant human enzymes by enzyme kinetic analyses using a fluorogenic substrate.Results: Ethyl acetate/water (pH 3.0) extraction of dried plant leaves proved to be the most efficient method for isolation of high molecular hydrolysable gallotannins which can be further concentrated by precipitation of dicyclohexylammonium salts in ethyl acetate. The main components of those extracts were oligo-O-galloyl glucoses with more than five gallic acid residues. They were shown to inhibit both enzymes studied but were about 30 times more effective inhibitors of prolyl oligopeptidase.Conclusions: C. coggygria from Bulgarian origin is shown to possess a substantial quantity of oligo-O-galloyl glucoses with more than five gallic acid residues which has not been described thus far in the same herb from other sources. An extraction method useable for concentrating those compounds is established. They are found to inhibit prolyl oligopeptidase with a very good selectivity to fibroblast activation protein alpha. The previously described antitumor activity of this plant may be at least in part due to the inhibition of the above enzymes which has been shown to participate in the genesis and development of various types of tumors.