Received date: August 20, 2013; Accepted date: October 16, 2013; Published date: October 21, 2013
Citation: Nayak BN, Rempel CB, Pascoe D, Gary Fulcher RG (2013) 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. J Nutr Food Sci 3:232. doi: 10.4172/2155-9600.1000232
Copyright: © 2013 Nayak BN, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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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.
TNFα; RAW 264.7; ELISA; Oat β-glucan; Barley β glucan; Saskatoon berries; Curcumin
TNF α is a key pleiotropic pro-inflammatory cytokine involved in the pathophysiology of many human diseases due to its role in immunity, infection, cell proliferation and differentiation [1-3]. Quantification of TNF α is routinely carried out in cell culture supernatants by in vitro enzyme-linked immunosorbent spectrophotometric assays. Thus assays have served as a primary tool for in vitro evaluation of cytokine-mediated anti-inflammatory and immunomodulatory properties of biomolecules . Saskatoon berries (Amelanchieralnifolianutt) (Family: Rosaceae) are deeply pigmented berries that grow on deciduous shrubs in Canadian Prairie Provinces, and Western and North Central United States. They are a rich source of bioactive compounds with physiological benefits [5-8]. Lavola et al. have reported quercetin, hydroxyl cinnamic acids, protocatechuic acid, quercetin-3-glucoside, quercetin-3-galactosided, (-) epicatechin and chlorogenic acid in the leaves . Flavanone, flavonol glycosides, catechins and hydroxybenzoic acids were found in stem components.Eriodictyl-7-glucoside and proanthocyanidin were found both in leaves and stems. Berries, on the other hand, were low in proanthocyanidins.
Oat (Avenosativa) β-G glucans are linear, high molecular weight polysaccharides consisting primarily of (1-4)-beta-D glucans with occasional insertions of single (1-3) links . Recent evidence suggests that cereal β-glucans possess a number of health benefits including enhancing immune function [11-14], reducing blood cholesterol [15,16], maintaining blood glucose homeostasis [17-19], genoprotective antitumor  and protecting effects against pathogenic infection . Shen et al. have suggested that oat beta glucans have a greater impact on health than barley beta glucan, although there is considerable variation in molecular weight and linkage ration from cultivar to cultivar in each species . The cellular effects on mammalian systems of β-glucans from various sources are under intense scrutiny. β-glucans bind to Dectin-1, Complement Receptor (CR3) and Toll-like receptors TLR-2/6 and activate TNFα production in immune cells including macrophages, neutrophils, monocytes, natural killer cells and dendritic cells and modulate innate and adaptive response . Dectin-1 is predominantly expressed on the surface of monocytes/macrophages and neutrophil lineage  and mediates the biological effects of β-glucan . Curcumin (1,7-bis (4-hydroxy 3-methoxyphenyl)-1,6-heptadiene-3,5-Dione). (Diferuloylmethane) is a well-studied pleiotropic molecule which exerts its biological activities through a variety of mechanisms. Involvement of NFκB is one of the major mechanisms by which curcumin regulate inflammation and immune response [25-27]. LPS is a complex lipoglycanmolecule andis known to stimulate TNFα production in macrophages by specifically binding to Toll-Like-Receptor 4 (TLR 4) complex [28-31].
The present study aims at evaluating the critical parameters of the immunoassay by stringently applying the standardized method to examine the relative-and concentration-specific effects of two distinctly different, but common, types of plant-derived bioactive compounds that occur in commercial abundance on the Canadian (and northern US) prairies: polyphenol-enriched saskatoon berries (SKB), and mixed linkage polysaccharide, oat β-glucan. Both products are the subjects of current research related to existing or potential health claims in several countries.
All chemicals were obtained from Sigma-Aldridge (St. Louis, MO, USA). Cell culture medium, Fetal Bovine Serum (FBS), and penicillin/ streptomycin antibiotics were obtained from Life Technologies Inc. (Burlington, Ontario, Canada). Cell culture flasks, plates and other plastic wares were obtained from UWR International, Mississauga, Ontario, Canada. LPS was obtained from Sigma and Invivogen (San Diego, CA, USA). Mouse TNFα kits were obtained from e-Bioscience (San Diego, CA, USA) and Abcam, Inc. (Cambridge, MA, USA). Cell proliferation kit and MTT regent were obtained from ATCC and Sigma, respectively.
Preparation of Saskatoon Berry Extract
Five grams of SKB Puree powder, Lot code PJ 3401-NSK BPP- 141011-T 13 (Food Development Centre, Portage La Prairie, Manitoba, Canada) were mixed in 50 mL of 80%ethanol made in deionized distilled water on a Max Q 4000 Shaker for 30 minutes at 25ºC. The mixture was centrifuged at 250X g for 25 minutes. The precipitate was dissolved in 50 mM Tris HCL buffer, pH 8.0, vortexed several times, and then left on a shaker. The solutions were transferred to Thermo Scientific Nalgene Oak Ridge centrifuge tube with sealing cap and was centrifuge at 1000 Xggat 4ºC for 15minutes, using Sorvall Legend RT (Mandel) (Fisher Scientific) bench top centrifuge. The supernatant was filtered with Whatman filter # 4, then, filtered again using 0.2 μM syringe filters. The filtrate was transferred to 1.5 ml Eppendorf tubes (1 ml per tube) and freeze dried in a temperature controlled UV S 400 Universal vacuum system (Speed Vac) for several hours. The contents were weighed and dissolved in advanced MEM medium, filtered through 0.2 μM filters. Final solutions were made at 0, 10, 25, 50, 100, 500, and 1000 μg/ml in complete medium (Life Technology) containing antibiotics.
Preparation of Curcumin Solution
Curcumin is a naturally occurring anti-inflammatory compound that is commonly present in turmeric, a major component in some diets. Curcumin (IUPAC Name: (1E,6E)-1,7-bis (4-hydroxy-3- methoxyphenyl)-1,6-heptadiene-3,5-dione) was obtained from Tocris Bioscience/Rand D Systems Co. It was dissolved in ethanol to make final stock solution 5 mM (1.8419 μg/ml) with rapid stirring, sonication, and gentle warming at 45-60ºC.The stock solutions were stored in tightly sealed vials at -80°C and used within 1 month. The working solutions were made at 1 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 100 μM in cell culture medium with antibiotics.
A mixed linkage oat β-glucan (>97% purity; MW ~150 kD) was obtained from Ceapro, Inc., Edmonton, CA. Oat β-glucan (OBG) stock solutions (2mg/ml) samples were dissolved in sterile DMEM (Sigma cat. # D5648, St. Louis, MO) medium at 65C for ~25 min using vigorous vortexing. Final solutions were made at 0.5, 1, 10, 25, 50, 100, 500 and 1000 μg/ml in cell culture medium. In order to produce a low molecular weight glucan comparison, enzyme-treated (i.e., hydrolyzed) OBG was produced by incubating dissolved cereal glucan samples at 40°C for 1 h with glucan hydrolase (Lichenase; 18 μ/mg; #E-LICHN, Megazyme Int. Ireland Limited). The reaction was stopped in a boiling water-bath (10 min).
HPLC and Mass Spectrometric Analysis of SKB Extracts
The saskatoon berry extracts were analyzed by HPLC using Nova Pack-C18 analytical column (3.9×150 mm, 5 um) UV at 250 nm. LC/MS was carried out using Agilent 1200 series HPLC (single wave length) coupled with 6100 B series single quadruple LC/MS System for LC/MS analysis. Solvent: 5 mM ammonium acetate in water (A) and acetonitrile (B), flow rate 1ml/min, ionization mode: ESI (positive) with linear gradient. The MS analysis was carried out using standard method for carotenoids analysis and Zorbax Eclipse XDB-C18, analytical (4.6×150 mm, 5 μM) (Table 2).
|Extract Concentration (µg/ml)||Deviations from the Control Absorbance Values (at 570 nm).|
|SKB 10||+0.4556 **|
Table 1: Effects of Different Concentrations of SKB, Curcumin & OBG on Cell Proliferation (The mean absorbance values expressed as deviations from the mean control absorbance valuesmeasured at 570 nm (MTT Assay).
|Compounds||Ionic Molecule/Molecular Weight|
|Cinnamic acid||148 M|
|Hydroxycinnamic acid||164 M|
|Coumaric acid||164 M|
|Caffeic acid||180 M|
|Ferulic acid||194 M|
|Sinapic acid||224 M|
|Chlorogenic acid||354 M+|
Table 2: Mass Spectral Analysis of Bioactive Compounds in Saskatoon Berry Extracts.
Preparation of sterile stock solution of LPS (Sigma)
A stock solution of 5 μM LPS was prepared by dissolving 5 mg of LPS (Sigma-Aldridge) in 1 ml of sterile deionized distilled water. It was vortexed until complete solubilisation. Final concentrations at 0.5, 10, 100, 500,600, 700, 800, and 1000 ng/ml made with complete medium.
Preparation of LPS (Invivogen)
Aliquoted LPS-EB Ultrapure, 5×106 EU from E. coli 0111:B4-TLR4 ligand specific was obtained from Invivogen. 1 mg of lyophilized, gamma-irradiated powder (Sigma) was dissolved in 1 ml of complete medium, gently swirled until the powder dissolved. Reconstituted product was further diluted to desired concentrations with complete medium. Reconstituted aliquots were stored frozen at -20°C.
RAW264.7 cells represent a macrophage-like Abelson leukemia virus-transformed murine macrophage/monocyte cell line derived from BALB/C mice. The cell line was obtained from the American Type Culture Collection (ATCC). The cells were grown in Dulbecco’s modified Eagles media (DMEM) (Life Technology), supplemented with 10% heat inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100μg/ml streptomycin solution. The cells were grown in 25 cm2 tissue culture flasks (Fisher Scientific Co.) in a humidified incubator at 37ºC with 5% CO2. Once the cells were 80-85% confluent, they were detached using mechanical scrapers; gently re-suspended, then placed in a sterile 15 ml centrifuge tube and centrifuged at 1000 RPM for 3 minutes to separate the cells. The supernatant was removed and the cells were re-suspended in fresh complete medium. Cell counts were performed using a haemocytometer. The cells were then sub cultured into 96-well tissue culture plates, adding 5×104 cells per well and then incubated at 37°C in 5% CO2 for 24 hours. After cells were grown for 24 hrs in 96-well plate, the old medium was removed. (Note cells can be treated with extracts after cells are grown for 6-8 hrs.). Pre-warmed medium at 37°C containing various concentrations of extracts with and without LPS was added for 18 hrs. The condition medium was recovered, micro centrifuged to remove particulate materials. The supernatant was collected into sterile labeled Eppendorf tubes and were either used immediately for ELISA or stored at -80ºC for further analysis.
MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) Assay
Cell proliferation and viability were determined by MTT assay (ATCC) . This assay is based on the cleavage of the yellow tetrazolium salt, MTT, to form a soluble blue formazan product by mitochondrial enzymes, and the amount of formazan produced is directly proportional to the number of living, not dead cells, present during MTT exposure. The cells were plated at 2.5×104 cells/ml in 200 μl volume/well in 96- well Nunclon surface (Nunc) plates and incubated for 24 hr. Cells were then treated with new complete medium containing extracts at the appropriate concentrations, and allowed to grow for a further 24 hr. We carefully removed 50 μl of medium and added 20 μl of MTT solution (ATCC) to each well. After incubation for 4h at 37°C, 100 μl detergent solution (ATCC) was added to each well. The optical density was then measured at 570 nm. The average values for the blank was subtracted from the average values from triplicate readings of treated wells. Absorbance values in treated wells that are lower than the control cells indicate a reduction in the rate of cell proliferation. Conversely, a higher absorbance rate indicates an increase in cell proliferation.
Enzyme-linked Immunosorbent Spectrophotometric Assay
To assess the anti-inflammatory activity of the extracts, TNF-α was quantified using ELISA kits (e-Bioscience Inc., Cincinnati, OH, USA; Abcam Inc. Cambridge, MA, USA). The assays were performed according to manufacturer instructions, using BD Falcon 96-well ELISA plates and adhesive film (VWR Co.). The amount of TNFα in pg/ ml was calculated from the standard curve using recombinant mouse TNFα protein standard (e-Bioscience Inc. or Abcam Inc.). Each plate had its own standards, positive (with LPS) and negative (without LPS) controls. Standards in replicate were placed in columns 1 and 2, rows A and B in ascending concentrations. Stop solutions were added to each well prior to absorbance readings. After 30 minutes, absorbance was read with a Varian 50 MPB Cary Microplate Reader with dual beams at 450 nm.
The TNFα in serum samples was analyzed using ELISA (BD opt EIA, #55-5268, BD Biosciences, San Diego, CA, USA
All experiments were done with at least three replicates and controls. The absorbance values and amounts of TNFα were reported as mean ± SD. IBM SPSS independent and paired sample-t-tests were used with one-way of analysis of variance p value<0.05 was considered significant.
The cell culture supernatants obtained from the unstimulated (without LPS) RAW264.7 cells yielded approximately 925 ng/ml of TNFα. Cells stimulated with 500 ng/ml LPS (Invivogen) for 18 hr produced 16,954 (an increase of 18 X) ng/ml of TNFα. Cells treated with 1000 ng/ ml (1 μg/ml) produced 20,496 ng/ml TNFα (an increase of 22 X).LPS 500 ng/ml was considered an acceptable concentration as it produced measurably adequate amounts of TNFα without causing cytotoxicity. Simultaneous treatment with SKB extracts and LPS for 18hat 0.34×104 cells/ml in 96-well plate produced greater TNFα inhibition. Table 1 presents the effects of different concentrations of SKB, curcumin and OBG on cell proliferation as measured by the MTT assay. The mean absorbance at 570 nm was expressed as deviations from the mean value of the controls.SKB extracts significantly promoted cell growth at all concentrations tested (p>0.05). Maximum cell growth was obtained at 1000 μg/ml and 2000 μg/ml. Curcumin significantly inhibited (p<0.05) cell growth between 5 μg/ml to 500 μg/ml, with maximum cell growth at 50 μg/ml. OBG significantly promoted (p<0.05) cell growth between 25-100 μg/ml and significantly inhibited (p<0.05) cell growth at 500, 1,000 and 10,000 μg/ml. Figure 1 demonstrates the relative effects of different concentrations of saskatoon berry extracts and oat beta glucan in comparison to curcumin on TNFα, expressed as the percent difference of TNFα (pg/ml) from positive controls (LPS treated). At 10, 25, 50, 100, 500 and 1000 μg/ml SKB significantly (p<0.05) inhibited TNFα. Maximum inhibition was observed between 50 to 100 μg/ml. Curcumin inhibited TNFα at all concentrations tested. The maximum TNF α occurred at 4 g/ml. OBG at 1, 10 and 50 μg/ml significantly up regulated TNF α. However, OBG at 100 μg/ml showed significant inhibition on TNFα expression. For comparison, Figure 2 shows the stimulatory effect of oat β-glucans (μg/ml) on TNFα (as shown by Pascoe ) in murine macrophages. The TNF concentration was expressed in fg/ml.
Figure 1: Relative and Concentration Specific Effects of Various Extracts (μg/ ml) on TNFα Expressed as Percent difference from controls treated with LPS (500 ng/ml). Extracts (μg/ml) and LPS (500 ng/ml) were incubated together for 16 hrs. With RAW 264.7 cells (cell density=0.34 ×10 4 cells/ml in 96-well plate.
Based on the analysis of mass spectral data, the following compounds were tentatively identified in SKB extract: cyanidin-3- glucoside, peonidine-3-glucoside, chlorogenic acid, cinnamic acid, hydroxyl cinnamic acid, caffeic acid, sinapic acid, ferulic acid coumaric acid, pelargonidin 3-glucoside, malvidin, epicatechin, β-sitosterol, luteolinidin, pelargonidin, kaempferol, dihydrokaekaempferol, aurantinidin, cyanidin-3 arabinoside, ascorbic acid, malvidin, and eriodictyol-7-glucoside (Table 1). Quercetin, biotin and ascorbic acid were not detected in our SKB sample extracts. Results of this study show that curcumin at 10 μM (5 μg/ml) completely abrogated the effects of TNFα evoked by LPS (500 ng/ml).We also found that curcumin inhibited the endogenous TNFα in unstimulated cells. However, in cells treated with curcumin (10 μM) for 24 hrs, followed by treatment with curcumin (10 μM) and LPS (500 ng/ml), TNFα was upregulated. We also tested turmeric extract prepared in aqueous alkaline pH 10 at similar concentrations and found less inhibition of TNFα (in comparison to curcumin). The turmeric alkaline extract was found to show reduction in activity, which we consider was probably due to degradation in storage.
The mouse macrophage RAW264.7 cell assay is an effective initial screening procedure in evaluating the growth and TNFα modulatory effects of plant-derived bioactive compounds on TNFα or other cytokines. The amount of TNFα in cell culture supernatant in control and treated samples was quantified by ELISA method. Cell density was found to be a critical factor in differentiating effects of treatments, particularly in the evaluation of curcumin and turmeric on TNFα than SKB or OBG. Our results demonstrate that SKB extracts have strong TNFα inhibitory effect in LPS stimulated-mouse macrophage RAW264.7 cells, similar to that of the curcumin effect. On the other hand, oat beta glucan at several lower concentrations stimulated the production of TNFα, but at higher concentration, OBG inhibited TNF α. β-glucans of both higher plant (such as oat and barley) and fungal/yeast origins are predominantly soluble, linear polymers of glucose with either linear 1-.4 (e.g. oats and barley), or linear 1-3 (yeast/fungi) linkages as the primary construct. Both exhibit strong immunomodulatory properties. β glucans bind to immune receptors including Complement Receptor (CR3), Toll-like Receptor (TLR 2/6), and Dectin 1 [16,24]. (Note-ultrapure LPS bind specifically to TLR4 and less purified LPS, with other membrane contaminants, may bind to TLR 4/TLR2/TLR 6). At higher concentration β-glucans inhibit TNFα, signifying the biphasic property of some β-glucans. The inhibitory effect of oat beta glucan at a higher concentration does not appear to be related to the cytotoxicity as suggested by the MTT assay. This property may be associated with the co-inhibitory effects of receptor complex on the macrophage/monocytes cells.
Recent literature suggests that β-glucans possess a number of health benefits including beneficial effects on wound healing due to their ability to promote fibroblast collagen biosynthesis and epithelisation [33,34]. It is suggested that after β glucans are internalized by macrophages and the products activate, Reactive Oxygen Species (ROS) in turn activate NFκB, resulting in the production of pro-inflammatory cytokines, including TNFα. The processed beta glucans may also prime other immune cells including T helper cells. Murphy et al. examined the effect of oat β-glucan on the pro-inflammatory cytokines (Il-1β, IL-6 and TNF α) in peritoneal and lung macrophages obtained from mice and cultured with varying concentration of oat β-glucan (10, 100 and 1000 μg/ml) for 24 hrs . The culture supernatants were analyzed for pro-inflammatory cytokines with ELISA. In most cases oat beta glucan resulted in a concentration dependent increase in IL-1 β, IL-6 and TNFα in lung and peritoneal macrophages. We have shown that at very low concentration (0.5 μg/ml) oat beta glucan stimulated cell mitosis. Similar mitogenic effects have been reported for short chain fatty acids on colonic crypt cells in animals and humans fed with resistant starch . Recent research shows that some oat β-glucan preparations may have a greater impact on health than barley β-glucan probably due to the characteristic viscosity and solubility properties of oat β-glucan . In a feeding study of dietary barley β-glucan (six grams purified barley β-glucans daily for six weeks), did not find any significant (p>0.05) changes in serum TNFα concentrations between β-glucan treated and control human subjects . This may have been due to the large age range of the subjects, but our experience is that the quality of reagents, including the specificity of monoclonal antibodies may determine the accuracy of the ELISA results. Furthermore, it is likely that the TNFα in serum may lose its activity on storage or may bind to other macromolecules resulting in misrecognition by TNFα R1 receptor . Reports also suggest that the biological effects of barley β-glucan depend on the specific molecular size and other biophysical properties including viscosity and solubility. Queenan et al. in a human study found OBG given in diets (6 gm/day) for 6 weeks significantly reduced total cholesterol and LDL cholesterol . Lehne et al. found no difference in cytokine and immunoglobulin concentrations in the blood of humans after 5 days treatment with three different concentration of yeast β-glucan (SBG) compared to baseline, although IgA concentrations were increased in saliva, only when using a high concentration of β –glucan . Hoffman et al.  studied concentration specific effects of fungal β glucan in rat alveolar macrophages and found concentration of β glucans less than 500 μg/ml stimulated TNFα and concentration greater than 500 μg/ml resulted in suppression of TNFα. Our result show that purified oat β-glucan at 100 μg/ml inhibited TNFα production while lower concentrations increased TNFα. Pascoe has found when RAW264.7 cells were pretreated with barley bran extract, and then treated with oat β-glucan, significantly less TNFα production occurred when compared to macrophages treated with barley bran extract (p>0.05) . In the same study, Pascoe also showed that TNFα production nearly doubled when barley bran extracts were combined with oat-β-glucan . This increase in TNFα production may have been due to the low pH of this specific bran extract. Extraction conditions often dictate the functionality of the compounds extracted.
The other potentially bioactive material which we tested included saskatoon berry extract. The SKB extracts show strong inhibition of TNFα production, and thus appear to contain potent anti-inflammatory agents. Our preliminary chromatographic and mass spectrometry analyses suggest that saskatoon berries contain a number of bioactive molecules ranging in molecular weights. Lavola et al. analyzed the bioactive polyphenols in leaves, stems, and berries of saskatoon berries and reported cyanidin-based anthocyanins, quercetin derived flavonol glycosides, hydroxycinnamic acid, protocatechuic acids in berries, flavanone and flavonol glycosides, catechins and hydroxyl benzoic acid in stems and quercetin-and kaempferol-derived glycosides and hydroxycinnamic acid, catechins, quercetin-3-galactosides, (-) epicatechin, chlorogenic acid in leaves . Hosseinian and Beta reported delphinidin 3-glucoside, malvidin-3-glucoside and malvidin- 3-galactoside in SKB grown in western Canada . Our sample did not show quercetin compared to the sample analyzed by Lavola et al. this may be due to the eco-geographic variations among berries grown in western Canada prairies as compared to the berries grown in Finland, or to differences in berry genetics . Hakkinen et al. have reported the presence of quercetin in edible berries including cranberries grown in Finland, but their study did not include SKB . A study with LPS-stimulated J774.1 cells showed that flavonoids such as luteolin, apigenin, kaempferol, quercetin, myricetin, naringenin, catechin, phloretin, butein, pelargonidin and cyanidins are potent inhibitors of TNFα with IC50 values ranging from 3 to 37 μM [3,41]. Xagorari et al., reported that luteolin, genistein, luteolin-7-glucoside, and quercetin inhibit LPS induced TNFα and IL-6, whereas, eridictyol and hesperetin only inhibited TNFα [3,42].
Results of the present study show that OBG at 1-50 μg/ml promoted TNF α production, but at 100 μg/ml OBG inhibited TNFα. These concentration-dependent effects may also be due to the pharmacokinetics and pharmacodynamics properties of the bioactive molecules and their interactive effects. Differences in TNF alpha secretion between the current OBG samples and those reported by Pascoe  may be due to the molecular weight, aggregation of β− glucan fragments, or the differences in interaction of these polymers with macrophage receptors. The ability of SKB to counteract toxicity produced by LPS is of clinical significance. Many Gram negative bacteria produce highly inflammatory LPS products, in conditions such as sepsis. Application of SKB may alleviate the severity of these conditions. We also used curcumin as a standard: curcumin is known as a potent anti-inflammatory agent acting through multiple pathways including the NFkB pathway. Curcumin attenuated LPS-induced and TNFα-mediated inflammation at 10-40 μM. 10 μM was an effective concentration that completely attenuated LPS elicited TNF α. A report by Chan  suggests that curcumin at 5 μM inhibits LPS-induced production of TNFα and IL-1 β in a human monocytic macrophage cell line. In our study at 1 and 5 μM curcumin did not effectively inhibit TNFα but effectively inhibited TNFα between 10-40 μM concentrations. Curcumin treated at a concentration of 40 μM stimulated G2/M arrest and apoptosis in malignant glioma cells. Curcumin significantly inhibited the increase of both IL-1β and TNFα a in a chronic model of inflammation in rats . Some human clinical trials claim the usefulness of curcumin in cancer therapy . Published reports so that curcumin 10 μM inhibits epidermal growth factor receptor kinase activity up to 90% in a concentration-and time-dependent manner and also inhibits epidermal growth factor induce tyrosine phosphorylation of EGF-receptor. Curcumin at 30 μM induces apoptosis in immortalized NIH 3T3 and malignant cancer cell line. Exposure of bovine aortic endothelial cells to curcumin (5-15 μM) resulted in both concentration and time dependent increase in hemeoxygenase activity . Motterlin et al. have reported that curcumin was cytotoxic to a wide variety of tumor cell lines through induction of cell apoptosis, the IC 50 ranged from 2 to 40 μg/ml depending on cell types . In transwell cell culture chamber assays, curcumin reduced the invasive capacity of lung adenocarcinoma CL 1-5 cells in a concentration range far below its level of cytotoxicity (20 μM) (7.3676 μg/ml) and this anti-invasive effect was concentration dependent. As an antioxidant agent curcumin has been reported to induce heme oxygenase-1 and protects endothelial cells against oxidative stress . Curcumin is a multifunctional molecule and exerts its biological activities by inhibiting NFκB, AP-1, TNF α and cyclin D1  and other pro-inflammatory enzymes and cytokines such as cyclooxygenases, IL-2, IL-1β and Il-6 . The anti-inflammatory effect of curcumin is reportedly due to its ability to inhibit NFκB signaling through the inhibition of nuclear translocation of NFκB p50 subunit . We examined the trend of TNF α production in LPS stimulated RAW cells over a period of 18 hrs. IL-6 was a low responder in RAW264.7 cells among all three cytokines tested. We found TNF α reached a peak at 6 hr. and maintains until 18 hr. In contrast, IL-6 and IL-1β showed peaks at 18 hr. Our previous work with ferulic acid, a metabolic product of curcumin showed that unlike curcumin, ferulic acid moderately inhibited IL-1 beta and had a variable effect on TNFα expression, without any appreciable effects on IL-6. Ferulic acid showed growth promoting effects on RAW264.7 cells. We have also found that human ovarian adenocarcinoma cell line (HeLa) treated with cranberry bark extract significantly promoted TNFα at 50 ug/ml but inhibited IL-6 (Nayak, B.N. unpublished data). In RAW264.7 cells cranberry bark extract inhibits IL-1 β and TNFα at 25 ug/ml. Ferulic acid did not appear to have much effect on IL-1β in RAW264.7 cells, but it inhibited IL-12.
TNFα is an important inflammatory cytokine involved in pathophysiology of many human diseases. It is up-regulated in rheumatoid arthritis, Crohn’s disease, and a number of other inflammation mediated diseases and sepsis. Production of TNFα by LPS evolves primarily through the activation of NFkB and p38 MAP kinase, as shown by Hoareau et al.  through their work with human adipocytes and with RAW264.7 cells. This study clearly demonstrates the significance of treatment concentration and possible role of extraction conditions such as pH and assay reagents, while evaluating the health and medicinal benefits of natural bioactive compounds. At lower concentrations it seems that these compounds exert antiinflammatory/ immunomodulatory effects and at higher concentrations they promote cell growth. Analysis of cell proliferation data supports the growth-promoting properties of Saskatoon berry extracts and oat β−glucans.
SKB and curcumin are strong inhibitors of TNFα and therefore may act as potent anti-inflammatory agents. On the other hand, oat β−glucans showed TNFα stimulation, and this may be a useful characteristic in wound healing. It is important to consider concentration related effects as some of these bioactive compounds show biphasic effects.
The study was supported by a grant through Manitoba Functional Food Opportunity Programs toRGF, CBR.The LC/MS analyses were kindly performed by Dr. A. Banskota, National Research Council, Halifax, Nova Scotia, Canada. Our special thanks also to Dr. SP Lall, Senior Research Officer, NRC, Halifax. Special thanks to Dr. Peter Jones, Richardson Centre for Functional Foods and Nutraceuticals, Dr. James Friel and Dr. Zakir Hossain, Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba for reviewing the manuscript.