Immune Modifiers from Selected Prairie Crops: Concentration Effects of Mixed Linkage (1→3, 1→4) β-Glucans and Saskatoon Berry Extracts on TNFα Expression and Cell Growth in RAW264.7 Cells

Abstract

Many plants and herbal extracts have been shown to possess anti-inflammatory, antioxidant and anticancer properties. Identification, quantitation and characterization of active ingredients and evaluation of concentrations effects are important for understanding their potential therapeutic applications. Cell-based assays using reporter/ indicator cells such as macrophages are commonly used as a screening procedure to evaluate their anti-inflammatory properties. We examined the relative and concentration-dependent effects of a common cereal polysaccharide, mixed linkage (1-3, 1-4) oat β-D-glucan, and of polyphenol-enriched saskatoon berry extracts (in comparison with curcumin) on TNFα (tumor necrosis factor alpha) and cell growth in mouse macrophage/monocyte RAW264.7 cells. The test materials included: polyphenol-enriched saskatoon berry (SKB) extract, mixed linkage polysaccharide, oat β-Dglucan (OBG). We used ultrapure E. coli lipopolysaccharide (LPS) and curcumin as TNFα stimulatory and inhibitory agents, respectively. TNFα was measured using TNFα mouse ELISA kit (ab100747, Abcam Inc.). Cell proliferation was determined by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. LPS 500 ng/ml was used to stimulate TNFα production in RAW264.7 cells. Results show that SKB extract inhibited TNFα at 50, 100, 500 and 1000 μg/ml, and at 500 and 1000 μg/ml it promoted significant (p<0.05) cell growth. OBG stimulated TNFα at 10, 25, and 50 μg/ml and at 100 μg/ml it inhibited TNF. At 1000 and 10,000 μg/mlOBG was cytotoxic to RAW264.7 cells. Maximum cell growth was observed at 50 μg/ml. Curcumin at 10 μM to 40 μM) (optimal concentration10 μM) attenuated significantly the LPS-induced inflammation. Curcumin at 1 μMand 5 μM had no significant effect on TNFα or cell growth. Curcumin inhibited cell growth at13.6 μM (5 μg/ml) to 13.6 mM (500 μg/ml) with maximum inhibition was observed at 136 mM (50 μg/ml) (p<0.05). Chromatographic analysis of SKB extracts demonstrated several major peaks with retention times ranging from 1.179 to 8.21 minutes. Mass spectral analysis of SKB extracts (Table 2) revealed the following compounds in SKB; chlorogenic acid, kaempferol, epicatechin, luteolinidin, cyanidin-3-arabinoside, cyanidin-3-glucoside, peonidine-3-glucoside, pelargonidin-3- glucoside, malvidin, epicatechin, beta-sitosterol, aurantinidin, anderiodictyol-7-glucoside.