The limited number of studies on blueberry phytochemicals and cell culture models of intestinal inflammation, the diversity of cell lines used, and parameters measured speak to the need for more studies to determine how blueberries modulate gut function and health.Because both inflammation and oxidative stress are frequently associated with the development of chronic diseases , it is important to understand how dietary factors impact these outcomes. Here, we have reviewed studies reporting the effects of blueberry phytochemicals on cell culture models of inflammation and/or oxidative stress . Blueberry phenolic compounds and more broadly, phytochemicals, exert regulatory effects including a decrease in proinflammatory gene expression/production in part through the modulation of the NF-κB pathway. A modulation of the MAPK pathway by blueberry phytochemicals is less evident with contradictory observations reported but may also play a role. Blueberry phytochemicals decreased DNA damage in cells in vitro, via the reduction of ROS production, lipid peroxidation, and an increase in antioxidant enzyme activities. Despite many in vitro studies on blueberry extracts, no specific compounds have emerged as singly responsible for the regulatory effects on inflammation and oxidative stress. Virtually all studies have focused on blueberry phenolic extracts or fractions, with a large emphasis on anthocyanins. Health effects of dietary anthocyanins have been extensively reported and discussed , and berries provide an excellent vector for anthocyanin consumption. Blueberries have a complex anthocyanin profile and both major anthocyanidin derivatives, planting gutter malvidin and delphinidin, have demonstrated a reduction of inflammatory markers in different in vitro models of intestinal inflammation and endothelial dysfunction .

Although it is highly likely that anthocyanins largely contribute to the health benefits provided by blueberries, as supported by the number of studies focusing on those compounds, it is doubtful that they are entirely responsible for the bioactivities. Several in vitro studies compared different fractions of blueberry phytochemicals, with reports of similar or better effects by other phenolic fractions and/or whole blueberry extract compared with anthocyanins . These different studies highlight that mechanisms of action of individual blueberry compounds and fractions are context and/or model specific. More studies comparing the effect on individual compounds and well-defined combinations of molecules in different systems are needed to investigate the impact of a system’s environment or system-specific regulation on the bioactivity of blueberries. Although the amplitude of the effect of individual compounds appears to be widely specific to the model studied, the use of whole fractions of the fruits seems to alleviate inflammation and/or oxidative stress more consistently across models, despite not always demonstrating the strongest effects compared with specific blueberry fractions. As the health effects of polyphenols have been extensively described, more data on other phytochemicals should be gathered as they may also exert health benefits. Other notable phytochemicals in blueberries include ascorbic acid , polysaccharides , and volatile compounds and could contribute to inflammatory or oxidative responses of cells to stimuli. A blueberry volatile extract, high in monoterpenes , modulated the inflammatory response in LPS-induced RAW 264.7 cells through inhibition of the NF-κB pathway . Phenolic compounds, although carrying anti-inflammatory and antioxidant modulatory effects, may not be solely responsible for the health benefits of blueberries. Whether the phytochemicals act in synergy or target different molecular pathways remains to be elucidated.

Although the scope of this review is limited to blueberries, the anti-inflammatory and antioxidant effects and mechanisms are likely applicable to other commonly consumed berries. Berries are generally rich in polyphenols, particularly anthocyanins, flavonols, and proanthocyanidins, but the profile of each berry species, and even within varieties, harbors differences in terms of the individual compounds present and their respective concentration . Gasparrini et al. reviewed in detail the anti-inflammatory effects of several berries in cellular models using LPSinduced inflammation, and consistently report alleviation of inflammation by berry phytochemicals through inhibition of NF-κB and MAPK pathways. Other reviews also discuss and compare the anti-inflammatory properties of berries, in preclinical and human models . Moore et al. and Gu et al. have reported similar anti-inflammatory effects of berry volatiles compared with phenolic extracts for cranberries, blackberries, blueberries, red and black raspberries, and strawberries. Notably, the bioactivities of berry polyphenol extracts do not always explain the overall anti-inflammatory effects observed with whole berries , highlighting that potential health effects of berries as a group derived from highly diverse phytomolecules. After consumption, blueberries and their phytochemicals undergo metabolism through phase II enzymatic reactions in the enterocytes and hepatocytes or microbial metabolism in the gut . Evidence of the role of blueberry metabolites in the modulation of inflammation and/or oxidative stress has also been established . Metabolites of elderberry were tested in RAW 264.7 and dendritic cells, and p-coumaric, homovanillic, 4- hydroxybenzoic, ferulic, protocatechuic, caffeic, and vanillic acids [also reported to be blueberry metabolites ], exerted a dose-response inhibitory effect on NO . Studies regarding berry catabolites are less abundant than studies on berry parent phytochemicals but have gained interest in more recent literature. These studies of microbeand host-modified phytochemicals are extremely important to fully understand the potential anti-inflammatory effects of blueberry consumption. Although most of the evidence focuses on the effect of individual compounds, it is essential to consider the potency of these metabolites in profiles similar to what occurs physiologically.

To take the compound profile and physiologically available doses into account, Rutledge et al. treated LPS-induced rat microglial cells with serum from subjects having regularly consumed blueberry, strawberry, or a placebo powder blends over 90 d. The blueberry consumption decreased NO production, TNF-α secretion, iNOS expression, and moderately modulated COX-2 protein expression in the cells . This type of design allows the integration of a more realistic profile of parent compounds and metabolites from blueberry consumption, at physiological doses, within a cell-culturebased model. The current review summarizes the extensive amount of literature available on blueberry phytochemicals and inflammation using cell-based models. This choice comes with limitations, since it can be challenging to interpret results using specific concentrations of berry-derived molecules on cells when concentrations of these metabolites at the site of the target organs may not be established. There have been major differences in concentrations used to treat the cells, ranging anywhere from tens of μg/mL to mg/mL for total polyphenols and from tens of ng/mL to ≤1.2 mg/mL for anthocyanin fractions. Some of these concentrations are much higher than the blood concentrations that would be present in the body after consumption, as bio-availability of anthocyanins in the body is estimated to be lower than 2%, and peaking at 100 nmol/L after consumption of grape/blueberry juice . The relevance of the findings of cell-culture-based studies in complex human systems needs further investigation. These studies should comprise of well-controlled clinical trials, with the relevant choice of placebo controls and inclusion criteria depending on the specific blueberry phytochemical and physiological condition investigated. Future studies should also quantify the entire suite of berry-derived molecules and derivatives in key pools such as the blood, concurrently with physiologic indices of inflammation and oxidative stress.General anti-inflammatory and antioxidant outcomes are consistently reported for blueberry extracts or derivatives across many studies. However, gutter berries results observed in diverse cell culture studies from different investigators are challenging to interpret due to the differences in protocol, treatment, cell line, and analyzed markers. More studies investigating the effects of blueberry extracts on different systems and using comparable conditions would be valuable. Cellculture-based models are not suitable to draw definitive conclusions on the effects of blueberry compounds on complex physiological processes occurring in the human body. Limitations include the compartmentalization of the observations in space and time: the compounds are only available in the form they are distributed and to the type of cells tested, outside of any regulatory processes by surrounding local tissues or on the whole-body scale, and tested on a one-time, acute, and usually high-dose treatment. Thus, precautions should be taken when drawing conclusions from simplified models, especially when using pharmacological doses of compounds. Despite the limitations, cell-culturebased studies have yielded critical information regarding mechanisms of action of blueberry phytochemicals, and have provided consistent evidence that components of blueberries have anti-inflammation and antioxidant properties, which likely contribute to health and functional benefits attributed to blueberries.The gastrointestinal tract, especially the large intestine, houses the most abundant and complex microbiota in humans. Most of intestinal bacteria belong to the phylum Firmicutes and Bacteroidetes , which make up more than 90% of known phylogenetic categories and dominate the distal gut microbiota. Other lower abundance bacteria include Actinobacteria, Fusobacteria, Proteobacteria, and Verrucomicrobia.

Diet is one of the important factors contributing to the gut microbial composition that ultimately affects human health. Obesity and associated metabolic diseases, including type 2 diabetes, are intimately linked to diet . A number of recent in vitro, in vivo, and human studies showed that polyphenols or polyphenol-rich dietary sources, particularly tea, wine, cocoa, fruits, and fruit juices, influence the relative abundance of different bacterial groups within the gut microbiota byreducing the numbers of potential pathogens and certain gramnegative Bacteroides spp. and enhance beneficial bifidobacteria and lactobacilli . Spices are derived from bark, fruit, seeds, or leaves of plants and often contain spice-specific phytochemicals. Spices have been used not only for seasoning of foods but also for medicinal purposes, and have a number of demonstrated disease preventive functions such as antimicrobial, antiinflammatory, antimutagenic activities, and are known to reduce the risk of cancer, heart disease, and diabetes . They are best known for their strong antioxidant properties that exceed most foods. It was reported that of the 50 food products highest in antioxidant concentrations among 1113 U.S. food samples, 13 were spices. Among them, oregano, ginger, cinnamon, and turmeric ranked #2, 3, 4, and 5, respectively . Previous research from our group reported that consumption of hamburger meat with spice mix added prior to cooking resulted in a reduction in the concentration of malondialdehyde, a lipid peroxidation marker, in the meat and in plasma and urine of healthy volunteers, and improved postprandial endothelial dysfunction in men with Type 2 diabetes . Subsequent study reported that commercial spices in dry or fresh form exhibited significant antioxidant capacity that correlated with total phenolic content butnot with the concentration of chemical biomarker . There is limited amount of information regarding the activity of culinary spice extracts against clinical isolated intestinal bacteria, and a limited number of bacterial strains have been assessed for their susceptibility or antimicrobial activity against spices. Gunes and colleagues reported that minimum inhibitory concentration of curcumin against 7 standard bacterial strains is in the range of 129 to 293 µg/mL . Cinnamaldehyde, a bio-active component of cinnamon, was shown to exhibit more potent in vitro antibacterial properties against 5 common foodborne pathogenic bacteria with MIC being 125 to 500 µg/mL as compared to crude cinnamon stick extract , but cinnamaldehyde did not modulate the population of selected Lactobacillus and Bifidobacterium counts in mouse cecal content . Supplementation of rosemary extract was reported to increase Bacteroides/Prevotella groups and reduce the Lactobacillus/Leuconostoc/Pediococcus group in the caecum of both obese and lean rats . Based on potential health benefits demonstrated from our group, this study investigated major chemical constituents, antioxidant activity, and in vitro effect of 7 spice extracts on the growth of 33 beneficial Bifidobacterium spp. and Lactobacillus spp., and established their antimicrobial activity against 88 intestinal, pathogenic, and toxigenic bacterial strains.Plants are some of the greatest chemists on our planet. They offer a vast, barely tapped repository of potentially bio-active compounds, with current estimates predicting over 200,000 unique specialized metabolites across the plant kingdom . Many of these metabolites act as therapeutic phytochemicals and essential nutrients in humans, making plants an invaluable source of bio-active compounds. However, barriers, such as the lack of access to healthy foods, limit the availability of these essential nutrients for human consumption . Plants also produce a wealth of therapeutic phytochemicals, both pharmaceuticals and nutraceuticals , that are difficult to chemically synthesize, leaving consumption of medicinal plants or plant extracts as the sole source of these important chemicals . Additionally, many important phytochemicals are expressed in plants that are difficult to cultivate or produce insignificant amounts of the desired phytochemical .