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 compound 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. 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 α. 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 α were studied on recombinant human enzymes by enzyme kinetic analyses using a fluorogenic substrate. 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. 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 α. 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.

The present paper aims to assess the antitumor activity of ethyl acetate/water soluble fraction obtained from the extract of Tanacetum vulgare L. leaves and flowers (EATV). The chemical composition of EATV was determined by LC-HPMS. In vitro studies were performed on the HT-29 cell line (human colorectal carcinoma). The effect on the cell cycle and the pro-apoptotic activity were evaluated using flow cytometry analyses. Genotoxicity was analyzed by the comet assay. In vivo antitumor potential of EATV was assessed in Ehrlich’s tumor-bearing mice. Pathological, histological, and hematological analyses of the EATV-treated animals were performed, and the effect of the treatment on the lifespan was evaluated. The LC-HRMS analysis demonstrated a complex phytochemical profile of EATV comprising more than forty compounds, thirty-six of which were identified. The results showed that the antitumor activity of EATV towards HT-29 cells is due to a pronounced genotoxicity leading to cell cycle arrest and apoptosis of the cells. Pathological studies revealed more massive and frequently detected tumor necrosis, apoptosis, and fistulation in T. vulgare-treated mice compared to positive tumor-bearing controls. Furthermore, it was demonstrated that the intraperitoneal application of EATV prolonged the animal’s lifespan.

Epilobium parviflorum Schreb. is used in folk and modern medicine for the treatment of prostate diseases. It is also known to alleviate gastrointestinal ailments. The aim of the present study is to define the chemical composition of diverse extracts from the herb, to test their inhibitory properties toward post-proline-specific peptidases and to elucidate the mechanisms of their antitumor activity on colorectal carcinoma cells in vitro. The extractions were performed using mono- or biphasic systems of solvents. Their chemical compositions were defined by LC-HRMS. Inhibitory properties towards prolyloligopeptidase (POP) and fibroblast activation protein (FAP) were studied by kinetic assays on human recombinant enzymes. Antioxidant activity was measured by three methods. Genotoxicity to HT-29 colorectal carcinoma cells was analyzed with the comet assay. FACS analyses and flow cytometry were used to evaluate the extracts effect on the cell cycle and their pro-apoptotic properties on HT-29 cells. The extract derived using 80% ethanol was chosen for the next studies due to its efficient and selective inhibition of POP. It contains mainly oenotein B and myricetin-3-O-rhamnoside. Its antioxidant and moderate genotoxic activities can contribute to the antitumor effect on HT-29 cells. The extract has a small effect on the cell cycle but a pronounced pro-apoptotic action on those cells. In conclusion, the 80% ethanol extract of E. parviflorum concentrates the ellagitannin oenotein B, which is a selective inhibitor of POP. Antitumor activity of the extract towards HT-29 cells may be due to the inhibition of POP, the antioxidant, genotoxic and pro-apoptotic activities.