Conclusion - Effects of Perilla frutescens on cell proliferation and gene expression 86

The potential of PF extracts in terms of cell proliferation inhibition has been reported. Their effects were immediate and correlated with the observed reducing potential. The results were verified by the determination of PF cytotoxicity and by the measurement of a panel of cell cycle key-regulator genes. In this regard, the absence of LDH release and the non-regulated expression of caspase 9 confirmed the suppression of cell proliferation not to be due to cell toxicity or apoptosis-inducing effects of PF. This is furthermore supported by the

87 downregulation of cell cycle regulator genes c-jun, cyclin C1 and cyclin B1, the latter showing an immediate decrease at time point 0h. 67LR, which is mainly discussed for its connection to cancerogenesis, was also detected to be downregulated at time points 0, 6 and 24h.

Moreover, control measurements were conducted, which included the detection of H₂O₂ generation, which was found to be a se t at the applied lo PF ua tit of . g . The assessment of PF extract stability in cell culture medium revealed PF compounds to be partly unstable in cell culture medium, which was reflected by a decrease of their abundance over time. However, effects of VS PF water, 50% EtOH and water extract on cell proliferation and the expression of cyclin D1 and 67LR were found to be immediate, i.e. at a time point at which non-degraded compounds would still be present in the medium. Furthermore PF concentration employed for the stability study was higher due to the implementation of several sample preparation procedures and thus dilution steps. For this reason a distinct generation of H2O2 as well as hydroxyl radicals, which would impair the compound stabilities, can be assumed. Although an effect of PF degradation products on the experimental outcome in the latter stages of experiments cannot be ruled out, the observed immediate impact of PF on cell proliferation and gene expression at time point 0 h is due to the presence of the extract rather than oxidation products.

88

5. Conclusion

This study is comprised of two main experimental parts, which consequently resulted in the establishment and employment of various methods in order to explore effects of PF extracts on enzymatic activity as well as on cell physiology. The impact of PF extract on the activity of three health-related enzymes was comparatively investigated by photometric as well as MS detection. Special emphasis was placed on the enzymatic assay adaption to MS detection as well as on the conduction of suitable control measurements to verify the results. In this regard MS revealed its great potential for the observation of multiple reaction intermediates as well as for enzymatic products non-detectable with classical photometric methods. For the finding of individual enzyme-regulatory compounds from complex mixtures an online coupled continuous flow mixing setup, which connects the advantages of MS detection, the implementation of a chromatographic separation and the ability to capture enzymatic activity in one single run, was employed. Eventually an inhibition of XOD and iAP was observed in the presence of chromatographically separated PF extracts. Results were verified by means of comprehensive control experiments, which included the injection of known inhibitors and alternative substrates.

Although the employed system required an extensive process of method establishment, online coupled continuous flow mixing system may prospectively obviate the need to conduct elaborate and time-consuming extract fractionation procedures, which are commonly performed in order to isolate and identify promising compounds from complex mixtures.

Apart from the observed regulation of enzymatic activity, further studies were conducted in order to identify the regulatory potential of PF extracts on the cell physiology of a porcine jejunal epithelial cell line. It was found that cell growth and gene expression of a panel of cell cycle and cancer related genes were downregulated by PF extracts, with a good correlation to the applied extracts respective reducing potentials. Based on the possible occurrence of a variety of artificial effects, which have often been neglected in past studies, comprehensive control experiments were conducted to verify the results. This included the determination of PF extract toxicity, which was measured by means of LDH release from the cells. Moreover H2O2 generation in cell culture medium and the stability of individual PF extract compounds

89 was assessed. In this regard H2O2 generation and PF toxicity could be excluded to be responsible for the detected inhibition of cell proliferation and regulation of gene expression. In contrast, PF compound stability was impaired in cell culture medium, which assumingly results in the generation of unknown degradation products. Nevertheless the detected immediate effect of PF extracts on cell physiology supports the assumption of regulatory potential of PF. Future investigations may however focus on the investigation of individual PF compounds and their effects on health and disease. Since most available studies investigate well-known PF components apigenin, luteolin or rosmarinic acid, whose beneficial effects are already established, the impact of e.g. glucuronidated and glucosylated flavonoids on physiological processes has yet to be determined. Moreover a comprehensive investigation of individual compounds promising with regard to enzyme-regulatory activity is required in vitro as well as in vivo. In order to assess the probability of them reaching their specific enzymatic target, their bioavailability in vivo has to be examined. This is of particular importance since a variety of flavonoids have been found to be metabolized and conjugated upon intestinal resorption.

Embedded in the context of detailed method establishment procedures, the here presented results thus reveal promising properties of PF. Various aspects of MS and photometric detection for the observation of enzymatic activities as well as PF effects on cell physiology have been discussed to outline advantages, but also various drawbacks of the employed methods.

90