Investigation of cell physiology and phytoconstituents

Natural sources are widely and extensively investigated in order to find amelioration for a plethora of different health threats. Desired effects range from antioxidant, anti-allergic, anti-inflammatory, antimicrobial, antiviral, and anti-carcinogenic to the prevention and treatment of obesity and diabetes, to the improvement of wound healing processes, to anti-toxic effects e.g. in case of snake or scorpion bites and eventually to the alleviation of mental disorders and stress. Historically, plants have been the most important source for natural drugs, which led to the development of traditional medicinal treatments administered either by ingestion, topical application or subcutaneous injection. In former times, due to the lack of physiological and biochemical knowledge, therapies were necessarily approached by applying herbal medicines and observing the outcome, which then may have resulted in coincidental findings of effective plants. The development of diagnostic investigation and the increase of scientific understanding of matters connected to the body however led to more specific and targeted searches. In this regard, cell culture studies are widely used for the finding of natural extracts with proliferation-inhibiting properties, in order to identify new possibilities for the treatment of cancer. In this context, various extracts of botanical origin have been reported with inhibitory effects on cell growth and cell survival [101-104], including traditional Asian medicines like Masson pine pollen extracts [105], Ginkgo [106, 107], Ginger [108, 109], Perilla [110-112] or Ginseng [113-115] but also common herbs like Sage [116] and a variety of berries and fruits like blackberries, cherries, apples and plums

15 [117]. The effect of extracts can be detected by means of various methods. This includes the direct observation of tumor size and number of tumor incidents [113, 118], the assessment of cell cycle and apoptosis related gene expression [105, 110], the continuous long-term observation of cell growth [105], the determination of proliferating or apoptotic cells at specific time-points after treatment [116] or the detection of specific metabolic activities within the cells [101, 110-112] or in the medium, which are e.g. due to apoptotic events [119, 120].

1.2.1. Cell line IPEC-J2

The fate of secondary plant metabolites like polyphenols within the body is widely investigated due to their potential health-promoting effects with regard to the alleviation of a plethora of diseases ranging from cancer to cardiovascular diseases and inflammation-related disorders such as IBD or allergic reactions. Nevertheless the abundance of their intact forms in the plasma, i.e. their bioavailability, has been found to be low. Since most polyphenols are poorly absorbable in their glycosylated form, compounds demonstrated to enter the systemic circulation underwent preceding metabolic conversion by the gastrointestinal microbiota or by endogenous deglycosylating enzymes [121-123]. They are absorbed in the form of aglycones or microbial metabolites and can undergo further bioconversion to form e.g. glucuronic acids, glucuronide or sulfate conjugates, before exerting biological activity and being eventually excreted in the urine or transported back into the small intestine. The conversion of a particular compound is dependent on a variety of factors, including its chemical structure but also on the composition of the gastrointestinal tract microbiota. Although some compounds are likely to lose their ability to exert positive effects after the generation of conjugates or their metabolic conversion to smaller molecules like phenolic acids, some of them have been found to partly retain their biological activity, whereas others possessed even more beneficial potential [122, 124, 125].

In order to determine the fate of natural compounds as unambiguously as possible, in vivo animal or human intervention studies are considered the method of choice. It is however still not feasible to factor in all aspects of bioconversion due to the complexity of the system and individual microbial diversity. For these reasons initial screenings for the finding of active compounds or natural extracts are usually performed using well-established, fast and

16 more economical in vitro experiments. Although most studies rely on the utilization of cancer cell lines, non-transformed and non-tumorigenic cell lines may serve as in vitro model to mimic physiological conditions more precisely as it would be possible with transformed cell lines, which are likely to respond differently to external stimuli. One of only few non-transformed small intestinal cell lines is IPEC-J2, which is derived from the porcine jejunum.

IPEC-J2 cell line has been employed in a variety of different studies, including the detection of interactions between intestinal cells and microbial pathogens or probiotic microorganisms [126-128]. Moreover the cell line was employed for the assessment of mycotoxin [120, 129, 130] or essential oil component cytotoxicity [131] and for the detection of cell proliferation in the presence of plant tannin extracts [132]. The impact of infections by enteric pathogens, diet-induced cellular responses and the regulation of inflammatory parameters have been determined as well using IPEC-J2 cell line. The latter comprises the expression of inflammatory key genes like proinflammatory IL-6, IL-8 or TNF-α i the p ese e of atu al compounds such as apigenin by means of LPS-challenged IPEC-J2 cells [133-137]. IPEC-J2 cell line was also used for the determination of intracellular oxidative stress in the absence or presence of e.g. antioxidants like ascorbic acid [138-141]. Furthermore Kolodziejczak et al.

performed experiments with different cell lines including IPEC-J2 for the investigation of prion protein uptake to enterocytes by means of 67kDa laminin receptor (67LR), which is believed to take part in their internalization and thus in the development of neurodegenerative disorders like bovine spongiform encephalopathy (BSE) [142].

Although some differences between in vitro cell culture and in vivo experiments have been observed using IPEC-J2 cells in comparison to native epithelium [143, 144], they possess an overall similar morphology and functionality to in vivo intestinal epithelial cells, including their ability to form microvilli as well as tight junctions [133], which makes them an appropriate tool to address various physiological questions. The knowledge acquired by means of employing porcine gastrointestinal epithelial cells may also be considered transferable on the human gastrointestinal tract, due to a highly similar physiology and anatomy [145-147].

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