Oleuropein-rich leaf extract affects intestinal microbiota and free fatty acids in Apc-mutant and wt rats

Epidemiological and experimental studies point out to several risk factors for colorectal cancer (CRC) such as dietary habits, a pro-inflammatory status and dysbiosis in the intestine. Much attention has been paid to explore the beneficial properties of natural products in the treatment/prevention of CRC. It has been reported that Oleuropein, a secoiridoide polyphenol, present mainly in the leaves of olive tree (Olea Europeae L.), but also, in moderate quantities, in extra virgin olive oil, shows anti-cancer activity in different cell lines. We previously reported that an oleuropein-rich leaf extract (ORLE) has beneficial effect on Apc-mutated PIRC rats, an experimental model of CRC. Since it has been documented that Oleuropein is able to modify the composition of the intestinal microbiota in experimental models of metabolic diseases, we were also interested in investigate whether the beneficial effect of ORLE observed in PIRC rats may be linked to variation in the microbiome and in metabolites such as free fatty acids FFAs. While many studies are uniquely devoted to the determination of short-chain fatty acids (SCFAs) arising from gut microbiota metabolism, the determination of additional FFAs is also interesting. Accordingly, our recent data suggest that the level of medium-chain fatty acid (MCFAs) such as octanoic and decanoic acids may be associated to gastrointestinal diseases, including CRC. Therefore, it was developed an isotopic dilution gas-chromatography coupled mass spectrometry (ID/GC-MS) method for the targeted analysis of both linear and branched FFAs (SCFAs, MCFAs, and LCFAs) in fecal water samples as specific markers for both microbiota and host metabolic variations. - 69 - Experimental We studied the effect of treatment with ORLE in PIRC rats (F344/NTacApcam1137) mutated in the onco-suppressor gene Apc (Adenomatous polyposis coli) and developing spontaneous tumours in the colon, as well in F344 wt rats. PIRC and wt rats were randomly assigned to control diet (AIN-76) or to the same diet containing ORLE (2,7 g/kg of diet) for one week. Fecal samples were collected at the end of the treatment and analysed for microbiota composition; FFAs in fecal waters were analysed by ID/GC-MS method by using an Agilent GC–MS system composed with single quadrupole mass spectrometer, gaschromatograph and autosampler as described by Vitali and colleagues. Results We previously showed that ORLE was able to inhibit tumour and macrophage iNOS in the PIRC rats. Here we document that ORLE promotes apoptosis and decreases proliferation in colon tumours and normal mucosa of Apc-Mutant Rats. Regarding microbiota composition, PCoA (principal coordinate analysis) based on Bray–Curtis distances showed a significant effect of the treatment with ORLE in both PIRC and wt rats. In addition, Lefse analysis able to determine the taxonomic units that most likely explain differences between the groups, showed a significant increase in the abundance of the genera Sporobacter, Anaerotruncus and Oscillibacter in ORLE group compared to the CTR group. Regarding FFAs, while SCFA were similar among groups, we observed that MCFA were higher in PIRC rats compared with wt rats, with no effect of dietary treatment. Conclusions Our previous data indicate that ORLE decreases inflammation, promotes apoptosis and decrease proliferation in PIRC rat tumours. Our present data, although preliminary, indicate that in PIRC rats but also in wt animals, ORLE, is able to modulate intestinal microbiota, a result that could be linked to the beneficial effects observed in carcinogenesis. Regarding FFAs, while we did not observe variation due to ORLE treatment, the fact that MCFA are higher in PIRC rats than in wt animals, may be linked to the presence of intestinal tumours, as also observed in CRC patients.