Research Areas

Bioactive Coumponds 2011-

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Researcher: Ricardo Elesbão Alves

Anti-inflammatory properties of extracts from native tropical fruit species as source of ingredients or raw-material for elaboration of new products

Research Team: Dr. Ricardo Elesbão Alves and Dr. Luis Cisneros-Zevallos.

Objectives: To achieve the main objective we intend to pursue the following specific objectives: (i) to characterize the bioactive compound profiles from different extracts of acerola leaf and fruits; (ii) to study the bioactivity of extracts and fractions against chronic inflammation; and (iii) to screen extracts from different varieties for enhanced bioactivities.

Approach: Activity A - Chemical characterization of extracts and fractions - Selected fruits will be tested initially by thin layer chromatography (TLC) to screen for different category of chemical compounds present. Samples of fruits and leaves will be studied and selected category of compounds will be further studied by HPLC-Mass spectrometry. Once compounds have been identified, we will proceed in fractionation studies of extracts to obtain fractions of different compounds to be tested in the biological studies; Activity B – Bioactivity of extracts and fractions - Selected extracts and fractions with high bioactive compound content will be used in this part of the project. They will be tested in fat cells, macrophages, human umbilical vein endothelial cells (HUVEC) and skin cells (SKU-1064 human skin fibroblast cells) in order to test the in vitro anti-inflammatory effects of the extracts. Cells will be treated with Lipopolysaccharide (LPS) or TNF- for adipocytes, macropahges and HUVEC cells and ultraviolet light (UVA, UVB) for skin cells to trigger the inflammatory response. Biomarkers for inflammation and antioxidant potential and underlying mechanisms will be studied by determining messenger-RNA expression of inflammation biomarkers including pro-inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-8, V-CAM, MCP1 using real time PCR. Inflammatory signaling through JNK and NF-κB pathways will also be determined using Western blot. To analyze adipocyte function, the production of adipokines such as adiponectin, leptin, resistin and angiotensin will be measured in the adipocytes using commercial assay kits. To determine adipocyte insulin sensitivity, the amount and phosphorilation state of Akt (an insulin signaling molecule) will be quantified using Western blot. In addition, we will measure expression of matrix metalloproteinase in human skin fibroblasts (Lee et al., 2008) and COX-2 and iNOS expression (Kim et al., 2008). The generation of reactive oxygen species (ROS) will be determined as described by Meng et al., 2008. The selected extracts and fractions will be chemically characterized using HPLC-DAD-MSn and appropriate fractionation procedures (Ozmianski et al., 1988); Activity C – Screening of extracts for enhanced bioactivity - A range of 5-10 fruit extracts from different fruits will be screened for anti-inflammatory properties. An ORAC antioxidant assay will be performed and the measurements of specific biomarker(s) for inflammation selected from Activity B (e.g., NFκ-B), Interleukin-1, Interleukin-6, and/or tumor necrosis factor alpha (TNF-α) will be determined. Selected species to be used in cosmetics or dietary supplements will be chemically characterized and the selected extracts will be standardized using HPLC-DAD-MSn.

Results: We aim at obtaining the bioactive compound profiles of acerola leave and fruits. The first preliminary results confirm the presence of phenolic compounds in fruits. Common carotenoids in leaves were recognized and preliminary identified as violaxanthin and neoxanthin, common carotenoids in the photosystem in plant. The TLC analysis of column chromatography fractions did not reveal any terpenoids in amount enough to be detected by the spraying reagents. Further analysis on phenolics compounds in fruit and leaves and on screening biological activity of leaves extracts will be necessary.