Abstract

The objective of this research was to describe the effect of Vaccinium floribundum (VF) Kunth, a wild ber-ry species of Ericaceae family growing in Ecuador’s Andean highlands, commonly known as mortiño, on primary culture of porcine aortic endothelial cells (pAEC). In particular way, due to the protective effects of several natural compound, the aim of the research was to phytochemical characterization of FVM and investigate the effect of VF microencapsulated (VFM) on angiogenesis and on anti-inflammatory proper-ties. The phytochemical characterization of the microencapsulated extract was carried out through HPLC-DAD (High-Performance Liquid Chromatography with Photodiode Array detection) and LC-MS (Liquid chromatography–mass spectrometry) assays and it’s in vitro antioxidant capacity was evaluated via spec-trophotometric assays. The in vitro antioxidant capacity was evaluated through DPPH and ORAC assays. The pAEC were then treated with different doses (1 ng/mL, 10 ng/mL, 100 ng/mL, 1μg/mL, 10μg/mL, 100μg/mL for 24h or cultured with 1 mg/mL of VFM for 5h in presence or absence of LPS (25μg/mL) The viability was evaluated by MTT test. After RNA extraction, PCR Real Time reactions (SYBR GREEN) were perfomed for the following interest genes: VEGF (Vascular Endothelial Growth Factor), the two VEGF re-ceptors FLT1 and FLK1 (fms related receptor tyrosine kinase 1 and kinase insert domain receptor respec-tively) and HO1 (heme oxygenase 1) to investigate the effect of FVM on angiogenesis. Moreover, the ability of pAECs to form capillary-like structures in presence of FVM (0.1 and 1mg/ml) was evaluated using a tube formation assay on a three-dimensional extracellular matrix. Gene expression of IL6 (interleukin 6), IL8 (interleukin 8) was performed to evaluate the effect on inflammatory response. The reference genes used on PCR Real Time were GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and β-ACT (β Actin). The phytochemical characterization of the microencapsulated extract was carried out through HPLC-DAD and HPLC-MS analysis to target specific compounds belonging to different phenolic classes. Fi-nally, in in vivo model (mice), in which edema has been caused into the right hind paw, we evaluate the ability of FVM oral administration to reduce the damage. The paw volume was measured with a ple-thysmometer to determine the difference between the right and left paws at 1, 2, 3, 4, 5, and 6 h after carrageenan injection. The results allowed to identify and quantify fifteen chemical compounds belonging to phenolic acids, fla-vonols, flavan-3-ols, dihydrochalcones, and anthocyanins. The in vitro antioxidant capacity was evaluated through DPPH and ORAC assays, and both tests confirmed an antioxidant capacity of the extract, ex-pressed as Trolox Equivalents/g dry weight (see data sets VFM_pAEC_LCMS_14012026.csv; VFM_pAEC_HPLC_14012026.csv; VFM_pAEC_DPPH_14012026.csv; VFM_pAEC_ORAC_14012026.csv). Our results showed that VFM at 1 mg/mL significantly increased cell viability compared to the standard condi-tion (VFM_pAEC_Viability_FVM_14012026.csv) while no significant differences were observed between control and other VFM doses. Regarding gene expression of VEGF, FLT1, FLK1 and HO1 our data showed only significantly increased of FLT1 in pAEC treated with FVM 0.1mg/ml after 5h. in relation to the control and VFM 1mg/ml. No significant differences were observed in gene expression at 24h. treated cells while the exposure to VFM (see data set VFM_pAEC_qPCR_14012026.csv). The pro-angiogenic potential of VFM investigated by an in vitro capillary-like tube formation assay showed statistically significant differ-ences between control and both treatment concentrations for all evaluated parameters (number of mas-ter junctions, total master segment length, total branching length, total segment length, number of meshes, and total mesh area) (see data set VFM_pAEC_IVCLTFA_14012026.csv) About the inflammatory model (treatment with LPS), our data showed that VFM (1mg/ml) was able to restore viability when LPS (25ug/ml) was added (see data set VFM_pAEC_Viability_FVMLPS_14012026.csv sheet 2). The qPCR data demonstrated that VFM 1mg/ml significantly reduced the level of expression of both cytokines (IL6 and IL8) in pAEC cultured with LPS (5h). In vivo results (see data set VFM_pAEC_PAW_EDEMA_14012026.csv) showed the efficacy of FVM to reduce edema.