Bioactive compounds in Aronia melanocarpa fruit and their potential anti-inflammatory and antiatherosclerotic effects

The aim of the project is the qualitative and quantitative analysis of compounds found in black chokeberry (Aronia melanocarpa) fruits, such as anthocyanins, procyanidins and hydroxycinnamic acids. Their content changes during maturation, depending on the time of harvest and the geographical location of the plantation. A novelty in our project is the use of NMR spectroscopy in combination with chemometric methods (PCA principal components analysis, PLS partial least squares method) to determine the composition of extracts and juices. The result will be a database of NMR parameters that will allow the analysis of the metabolite profile without the need to separate the mixture. The antioxidant properties of chokeberry extracts and juices will also be investigated and analyzed using chemometry to identify the main compounds responsible for these effects. A particularly important task will be to test whether chokeberry extracts or selected compounds have an anti-inflammatory effect on endothelial cells, and thus have potential anti-atherosclerotic properties. Additionally, the mechanisms underlying intracellular signaling will be investigated. The data obtained from the study of endothelial cells with the use of chemometry will allow to determine the compounds responsible for the biological activity of chokeberry extracts. Samples of Aronia melanocarpa fruit will be collected during three seasons from July to October on industrial, ecological crops in Poland. Most of the fruit will be freeze-dried, partially processed into juice. Extracts will be made from the lyophilized samples, the chemical composition of which will be determined by NMR spectroscopy (1H / 13C NMR in solution) using chemometric analysis. Known chemometric methods have been applied and developed in our Department. NMR spectra will be recorded on a Bruker 400MHz spectrometer. As the NMR spectra of the procyanidins in the mixture are difficult to interpret, support for chromatographic methods will be needed. Qualitative and quantitative analysis will be performed using an HPLC system with diode array detector (DAD-HPLC) according to published procedures. The purchase of HPLC-DAD equipment is planned from the project funds. All extracts will be tested for antioxidant properties: ORAC, FRAP as well as DPPH and lag-phase using EPR spectrometry. The EPR method is particularly suitable for measuring colored (e.g. dark red) as well as opaque samples. EPR spectra will be recorded on a 9.3 GHz MiniScope (Magnettech) EPR spectrometer. Studies on anti-inflammatory properties will be performed on human umbilical vein endothelial cells (HUVEC). Expression of adhesion molecules using ICAM-1 and VCAM-1 antibodies will be measured by flow cytometry. Secretion of the MCP-1 and IL-6 proteins will be performed with an ELISA kit. To determine the mRNA levels for ICAM, VCAM, MCP-1 and IL-6, quantitative real-time PCR analysis of the polymerase chain reaction will be performed on the LightCycler Kit. The generation of intracellular reactive oxygen species and the superoxide radical will also be studied. The phosphorylation of the p38 MAPK, ERK1 / 2, JNK and NF-kB p65 proteins will be assessed and the p65 protein level in the nuclear fraction measured using the Trans NF-kB p65 kit. Poland has the largest acreage of aronia cultivation in the world, and the consumption of this fruit may contribute to improving the health of EU citizens. Our research contributes to the documentation of EFSA health claims for chokeberry products, which will enable them to be promoted as a health-promoting product. The use of plant extracts as food, dietary supplements or herbal medicines requires them to be tested with analytical methods that allow complex mixtures to be tested. Such possibilities are provided by NMR spectroscopy, especially when metabolomics is combined with chemometric analysis. Project results will allow the standardization of chokeberry products, such as juices and extracts, while optimizing their antioxidant properties. Chokeberry fruits are characterized by the highest antioxidant activity among fruit (ORAC value> 160 μmol TE / kg), and may be valuable in the diet. Standardization is of paramount importance for in vivo testing. The divergent results of clinical trials are the result of the fact that the active compound profile varies according to harvest time, fruit origin and processing methods. Our research will allow us to better understand the bioactive aronia compounds and their antioxidant and anti-inflammatory effects. We expect that the preparations based on our results can be used in the prevention of atherosclerosis and cardiovascular diseases.