Progress 10/01/11 to 07/31/14

Outputs
Target Audience: chokeberry growers and horticultural scientists interested in polyphenol content of chokeberry cultivars scientists and clinicians concerned with the lipid-modulating, antioxidant, and anti-inflammatory mechanisms of polyphenol-rich foods food scientists utilizing chokeberry extracts as polyphenol-rich ingredients Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Sarah Kranz was trained in the analysis of antioxidant activity. Bethany McAvoy was trained to accomplish analysis of antioxidant activity; Dr. Francisco Sylvester provided training to Dr. Bolling and Derek Martin, a graduate student, for analysis of anti-inflammatory activity. Rod Taheri, a graduate student, was trained to analyze chokeberry polyphenols. Liyang Xie, a graduate student, was trained in analysis of chokeberry metabolites by Dr. Bollng. Dr. Kim, a postdoctoral fellow, and Casey Wegner, a graduate student were trained by Dr. Lee in the analysis of Caco-2 cells. How have the results been disseminated to communities of interest? In Spring 2014, Sarah Kranz presented a public seminar at the University of Connecticut Undergraduate Research Symposium on antioxidant activity of chokeberry polyphenols. Also in Spring 2014, Dr. Bolling presented an oral presentation at the Experimental Biology Conference about the anti-inflammatory activity of chokeberry polyphenols. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact: Consumption of chokeberries and other berries are thought to reduce cardiovascular disease risk, but the way this occurs is not well known. Understanding the processes by which berries and other foods reduce cardiovascular disease risk and reduce chronic inflammation will allow for more effective dietary recommendations to reduce disease risk. Furthermore, chokeberries are native to the US and being produced commercially, but are underutilized as a nutraceutical crop. The broad goal of this work is to better understand how chokeberry antioxidant and anti-inflammatory activity can reduce cardiovascular disease risk. During this reporting period, we generated and disseminated scientific knowledge as follows: 1) we identified in vitro interactions between chokeberry polyphenols and sugars and organic acids that inhibit antioxidant capacity; 2) we determined the in vitro anti-inflammatory activity of underutilized chokeberries, and 3) synthesized the in vitro and in vivo anti-inflammatory activities of chokeberries in mice immunocytes. These data have been disseminated by scholarly publication and conference presentations. This work has improved the understanding of how consumption of chokeberries may inhibit inflammation, and important contributor to cardiovascular disease risk. Specific accomplishments Aim 1: Characterize black chokeberry polyphenols. Major activities completed/experiments conducted. We characterized the in vitro antioxidant capacity of aronia polyphenols in combination with common juice components; to help determine their differential contributions to in vitro antioxidant capacity. Data collected. We assessed the DPPH and FRAP antioxidant capacity of aronia polyphenols in combination with sucrose, glucose, fructose, sorbitol, and citric acids, important components of aronia juice. Summary statistics and discussion of results. In our previous report, we found a considerable variability in chokeberry polyphenols during the harvest window, which could not be attributed directly to polyphenols. Sorbitol, but not fructose, glucose, or sucrose inhibited DPPH antioxidant activity. Citric acid also inhibited DPPH activity. Thus, certain sugars and organic acids may inhibit in vitro antioxidant activity. These are not expected to inhibit in vivo antioxidant capacity as these occur through different mechanisms. Key outcomes or other accomplishments realized. We are preparing a manuscript based on these data, which is expected to increase knowledge of chokeberry polyphenols. Furthermore, key data were presented at a local conference. Aim 2: Determine the effects of chokeberry treatment on lipoprotein concentrations, hepatic expression of genes related to cholesterol synthesis and transport, and oxidative stress in a mouse model of atherosclerosis. Major activities completed/experiments conducted. Our work in the mouse model of atherosclerosis was completed in the previous reporting period. Data collected. NA Summary statistics and discussion of results. NA Key outcomes or other accomplishments realized. The work in the mouse model led to the development and funding of an ongoing human intervention study. We are in the process of conducting an ongoing study to determine if supplemental aronia polyphenols can reduce cardiovascular disease risk in former smokers (NCT01541826). Aim 3: Characterize the components of black CB responsible for prevention of regulation of cholesterol synthesis and transport and oxidative stress. Major activities completed/experiments conducted. We used data from experiments conducted in the previous reporting period and published a scientific manuscript about the anti-inflammatory activity of chokeberry polyphenols in primary mouse splenocytes. We also determined the potential of chokeberry to induce hepatic antioxidant enzymes in mice. Data collected. We determined hepatic quinone reductase activity, glutathione peroxidase activity, glutathione transferase activity and total thiols from mice fed aronia or control diets. Summary statistics and discussion of results. Chokeberry polyphenols (from red, purple, and black cultivars) inhibited lipopolysaccharide-induced IL-6. ‘Viking’ black chokeberry extract induced IL-10 in splenocytes. Incubation of isolated polyphenols (anthocyanins, hydroxycinnamic acid, flavonols) did not explain the anti-inflammatory efficacy of extracts. Inflammation is a key event for inducing oxidative stress. Thus, more work is needed to determine how anti-inflammatory activity of chokeberry polyphenols relates to its prevention of oxidative stress. Chokeberry-rich diets also modulate baseline antioxidant function in liver of wild-type mice. Glutathione transferase activity of aronia fed mice was less than control (P < 0.05). However, quinone reductase activity was significantly increased in mice fed chokeberries (P < 0.05). Therefore, aronia differentially modulated antioxidant enzyme activities in mice. Further work is needed to confirm the importance of these changes to cardiovascular disease risk. Key outcomes or other accomplishments realized. A knowledge change was delivered by publication of a peer-reviewed manuscript of this work (Martin et al., 2014). Furthermore, a presentation was delivered on this work at a national scientific conference.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Martin DA, Taheri R, Brand MH, Draghi Ii A, Sylvester FA, Bolling BW. Anti-inflammatory activity of aronia berry extracts in murine splenocytes. Journal of Functional Foods. 2014;8:68-75.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bolling B, Brand M, Draghi A, Martin D. Differences between the ex vivo and in vivo anti-inflammatory activities of Aronia mitschurinii (black chokeberry) polyphenols in C57/BL6 mice (134.3). The FASEB Journal. 2014;28.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: chokeberry growers and horticultural scientists interested in polyphenol content of chokeberry cultivars scientists and clinicians concerned with the lipid-modulating, antioxidant, and anti-inflammatory mechanisms of polyphenol-rich foods food scientists utilizing chokeberry extracts as polyphenol-rich ingredients Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Training activities: Bethany McAvoy was trained to accomplish analysis of antioxidant activity; Dr. Francisco Sylvester provided training to Dr. Bolling and Derek Martin, a graduate student, for analysis of anti-inflammatory activity. Rod Taheri, a graduate student, was trained to analyze chokeberry polyphenols. Liyang Xie, a graduate student, was trained in analysis of chokeberry metabolites by Dr. Bollng. Dr. Kim, a postdoctoral fellow, and Casey Wegner, a graduate student were trained by Dr. Lee in the analysis of Caco-2 cells. Professional development: Dr. Bolling presented this work and discussed implications of this work at the following conferences and seminar series: University of Connecticut Center on Aging Seminar Series and the 7th International Symposium of Ayurveda and Health, Farmington CT; Rod Taheri presented a public seminar at the University of Connecticut Department of Nutritional Sciences on his work with chokeberry polyphenols. How have the results been disseminated to communities of interest? The results of this work have primarily been disseminated via publication of three peer-reviewed publications in scholarly journals. Additionally, two abstracts of conference proceedings were published and these papers were presented at the Experimental Biology 2013 conference. The works have been presented at other seminar series as described above, primarily reaching other scientists or interested individuals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Impact: Consumption of chokeberries and other berries are thought to reduce cardiovascular disease risk, but the way this occurs is not well known. Understanding the processes by which berries and other foods reduce cardiovascular disease risk will allow for more effective dietary recommendations to reduce disease risk. Furthermore, chokeberries are native to the US and being produced commercially, but are underutilized as a nutraceutical crop. The broad goal of this work is to better understand how chokeberry antioxidant and anti-inflammatory activity can reduce cardiovascular disease risk. During this reporting period, we generated and disseminated scientific knowledge as follows: 1) we characterized the polyphenol composition of commercial and underutilized chokeberry cultivars; 2) we characterized how polyphenol content of chokeberry changes through the harvest; 3) we described key antioxidant actions of chokeberry in a mouse model of cardiovascular disease; 4) we described the cholesterol-lowering actions of dietary chokeberry in a mouse model and cultured cells. These data have been disseminated by scholarly publication and conference presentations. This work has improved the understanding of how consumption of chokeberries may reduce cardiovascular disease risk. Specific accomplishments Aim 1: Characterize black chokeberry polyphenols. Major activities completed/experiments conducted. We published our data characterizing chokeberry polyphenols reported in the previous reporting period (Taheri, et al., 2013). Furthermore, we completed an additional study of the polyphenol composition of chokeberry juice prepared from different times within the harvest window. Data collected. We analyzed the total phenol, antioxidant, polyphenol content of chokeberry juices prepared from different times within a seven-week harvest window. Summary statistics and discussion of results. Chokeberry juice anthocyanin content increased ~ 227% by week 5 of the edible harvest window. Chokeberry juice proanthocyanidin content increased by 67% at week 7, the last harvest date. In contrast, chokeberry juice hydroxycinnamic acids decreased 33% during harvest. Changes in chokeberry juice antioxidant activity were not attributed to a single polyphenol. These data indicates that there is a considerable variability in chokeberry polyphenols during the harvest window. If a particular polyphenol or combination of polyphenols or other dietary bioactives are found to be optimal for reducing oxidative stress and inflammation, certain weeks may have favorable bioactivity. Key outcomes or other accomplishments realized. We published these data (Taheri et al., 2013) and presented this work at a scientific meeting. Thus, a change in knowledge was realized for this objective. Aim 2: Determine the effects of chokeberry treatment on lipoprotein concentrations, hepatic expression of genes related to cholesterol synthesis and transport, and oxidative stress in a mouse model of atherosclerosis. Major activities completed/experiments conducted. We further explored the basis of cholesterol-lowering activity in Caco-2 cells; we published two manuscripts describing the work reported in the previous reporting period (Kim et al., 2013a; Kim et al., 2013b). Data collected. Caco-2 LDL uptake was evaluated after co-incubation with chokeberry extract. Sirtuin gene expression was determined in Caco-2 cells after co-incubation with chokeberry extract. Summary statistics and discussion of results. Caco-2 cells had increased LDL update when incubated with polyphenol-rich chokeberry extract. Furthermore, Sirtuin -1, -3, -4, and -5 mRNA expression was increased, but Sirtuin-2 mRNA expression was decreased. These data suggest an association of epigenetic changes and cholesterol transport upon exposure of intestinal cells to chokeberry polyphenols. Key outcomes or other accomplishments realized. A knowledge change was realized through our publications (Kim et al., 2013a; Kim et al., 2013b). Specifically, the ability of chokeberry appear to lower cholesterol appear to be governed by intestine, rather than hepatic changes in gene expression. Aim 3: Characterize the components of black CB responsible for prevention of regulation of cholesterol synthesis and transport and oxidative stress. Major activities completed/experiments conducted. We developed UHPLC-MS methods necessary to evaluate the in vivo bioavailability of chokeberry polyphenols and metabolites. Furthermore, we conducted experiments to determine the anti-inflammatory activity of chokeberry polyphenols using primary mouse splenocytes. Data collected. We determined recoveries, limits of detection, and limits quantification for the following metabolites from urine and plasma, assessed by UHPLC: 3, 4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, hippuric acid, ferulic acid, 3-(4-hdroxyphenyl) propionic acid, cyanidin-3-O-galactoside, cyandin-3-O-glucoside, cyanidin-3-O-arabinoside, and peonidin-3-O-galcatoside. From in vitro studies, we assessed cellular excretion of IL-10 and IL-6, key cytokines involved in inflammation after exposure to chokeberry extracts or their isolated polyphenols. Summary statistics and discussion of results. Acceptable recoveries, ranging from 60-110% were achieved for chokeberry polyphenols and their metabolites, except for 3-(4-hdroxyphenyl) propionic acid which had 0% recovery from plasma. Limits of quantification for these compounds ranged from ~5 pg/column for hippuric acid to ~500 pg/column for cyanidin-3-O-glucoside. Limits of detection were less than 200 pg/column for all compounds. Thus, these methods are appropriate for assessing bioavailability of chokeberry polyphenols. Chokeberry polyphenols (from red, purple, and black cultivars) inhibited lipopolysaccharide-induced IL-6. ‘Viking’ black chokeberry extract induced IL-10 in splenocytes. Incubation of isolated polyphenols (anthocyanins, hydroxycinnamic acid, flavonols) did not explain the anti-inflammatory efficacy of extracts. Inflammation is a key event for inducing oxidative stress. Thus, more work is needed to determine how anti-inflammatory activity of chokeberry polyphenols relates to its prevention of oxidative stress. Key outcomes or other accomplishments realized. A knowledge change was delivered by publication of an abstract and scientific presentation (Martin et al., 2013).

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Taheri, R, Connoly, B, Brand, M, Bolling, B. Underutilized chokeberry (Aronia melanocarpa, arbutifolia, prunifolia) are rich sources of anthocyanins, flavonoids, hydroxycinnamic acids, and proanthocyanidins. Journal of Agricultural and Food Chemistry, 2013;61:8581-8588.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, B, Park, Y, Bolling, B, Lee, J. Polyphenol-rich Aronia melanocarpa (chokeberry) extract regulates expression of cholesterol and lipid metabolism genes in Caco-2 cells. Journal of Nutritional Biochemistry, 2013;24:1564-1570.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Kim, B, Ku, CS, Pham, TX, Park, Y, Martin, DA, Xie, L, Taheri, R, Lee, J, Bolling, B. Aronia melanocarpa (chokeberry) polyphenol rich extract improves antioxidant function and reduces total plasma cholesterol in apolipoprotein E knockout mice. Nutrition Research, 2013;33:406-13.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Taheri R, Durocher S, Brand M, Bolling, B. Impact of harvest timing on the polyphenol content of black chokeberry (Aronia melanocarpa, cv Viking) juice. Experimental Biology, Boston, MA, April 20-23, 2013. FASEB J April 9, 2013 27:lb272.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Martin D, Taheri R, Brand M, Draghi II A, Sylvester F, Bolling B. Polyphenol-rich red and purple aronia berry extracts inhibit interleukin-6 from mouse splenocytes. Experimental Biology, Boston, MA, April 20-23, 2013. FASEB J (2013). 27:348.3

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: We developed a UHPLC-DAD-MS method to simultaneously quantitate chokeberry anthocyanins, phenolic acids, and flavonols. The sensitivity, inter-assay, and intra-assay variation of these compounds in relation to standards were determined to validate this method. We further implemented an HPLC-FLD method to quantitate chokeberry proanthocyanidins. Also, we determined the polyphenol content of chokeberry extracts using the Total Phenols assay and the DMAC method. Using these methods, we determined the polyphenol content of a commercial chokeberry powdered extract used in animal studies. We also determined the polyphenol content of extracts from 12 chokeberry accessions, including Viking, a commercial chokeberry cultivar, Aronia melanocarpa (black chokeberry), Aronia arbutifolia (red chokeberry), and well as Aronia prunifolia (purple chokeberry). Black chokeberry extract was utilized to test for its ability to reduce cholesterol, triglycerides, and oxidative stress in the apolipoprotein E knockout mouse model. Viking chokeberry extract was supplemented at 0.005% or 0.05% in a high-fat high-cholesterol diet and fed to apolipoprotein E (ApoE) knockout mice for 4 weeks (n=10/group). We further evaluated the ability of black chokeberry to modulate gene expression related to lipid metabolism in Caco-2 cells. We also determined the ability of chokeberry extracts to inhibit endotoxin-induced inflammation in mouse splenocytes. We have disseminated this work by a presentation at the University of Connecticut Cornucopia event, papers submitted to peer-reviewed scientific journals, and presentations at scientific conferences. PARTICIPANTS: Dr. Bradley Bolling coordinated all aspects of this work. Dr. Jiyoung Lee coordinated and was responsible for experiments performed in ApoE mice and Caco-2 cells. Dr. Bohkyung Kim, Dr. Youngki Park, and graduate students Chai Siah Ku, Tho Pham, Derek Martin, Liyang Xi, Rod Taheri, and Bethany McAvoy assisted and/or were trained to accomplish aspects of this project. Dr. Mark Brand provided chokeberries for analysis. Dr. Francisco Sylvester provided training to Dr. Bolling and Derek Martin. TARGET AUDIENCES: The target audiences for this work include chokeberry growers and horticultural scientists interested in polyphenol content of chokeberry cultivars; scientists and clinicians concerned with the lipid-modulating, antioxidant, and anti-inflammatory mechanisms of polyphenol-rich foods; food scientists utilizing chokeberry extracts as polyphenol-rich ingredients. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We characterized the major polyphenols present in chokeberry extracts using validated analytical chromatography methods. The polyphenol content of chokeberries on a per gram dry weight basis ranged from 0.48 to 14.84 micrograms anthocyanins, 5.19-17.28 micrograms phenolic acids, 0.47-1.33 micrograms flavonols, and 1.1-3.7 mg proanthocyanidins by HPLC, and 4.25 to 18.6 mg catechin equivalents by the DMAC method, and 127 to 250 mg gallic acid equivalents by the Total Phenols assay. Black chokeberry varieties had the highest anthocyanin content, but lower mean phenolic acid content. Red, purple, and black varieties had subtle differences in phenolic acid, flavonoid, and proanthocyanidin content. The anthocyanin content was primarily cyanidins in all chokeberry genotypes, mainly as cyanidin 3-galactoside. Chokeberry proanthocyanidins were mainly polymers >10, with <1% amounts of monomers to nonamers. Viking chokeberry extract (0.05%) reduced total cholesterol by 12% in ApoE knockout mice compared to unsupplemented control group. Chokeberry did not alter mRNA expression of genes related to cholesterol and lipid metabolism. Both concentrations of chokeberry supplementation increased hepatic glutathione peroxidase activity in ApoE mice. The 0.05% chokeberry extract supplemented group also had increased plasma paraoxonase activity in ApoE mice. Therefore, chokeberry extract reduced total cholesterol and modulated antioxidant enzyme activity in ApoE mice. Viking extract at doses of 50 to 100 microgram/mL modulated Caco-2 gene expression related to cholesterol metabolism, including increasing low density lipoprotein receptor (LDLR) protein and mRNA expression. Further, chokeberry extract decreased 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) and sterol-regulatory-element-binding protein-2 (SREBP-2) mRNA and protein in Caco-2 cells. Further, this extract increased Caco-2 uptake of cholesterol relative to untreated cells. Extracts of black, purple, and red chokeberries inhibited LPS-induced interleukin-6 (IL-6) release from C57/BL6 mouse splenocytes from 32% to 75% of untreated cells. This anti-inflammatory potency was correlated most strongly to phenolic acid and flavonol content. Taken together, chokeberry contains anthocyanin and non-anthocyanin polyphenols. Black, red, and purple chokeberries have unique polyphenol profiles which are associated with in vivo anti-inflammatory potency. The lipid-modulating actions of chokeberry extract previously observed in clinical studies was also observed in ApoE knockout mice fed high-fat, high-cholesterol diets, at doses approximating the effective doses of chokeberry in clinical studies. Thus, the ApoE knockout mouse appears to be a relevant model to study the cholesterol-modulating actions of chokeberry extract. This data was leveraged to pursue a clinical study to study the ability of chokeberry extract to modulate lipids, inflammation, and monocyte gene expression in relation to polyphenol metabolism in former smokers funded by the Connecticut Department of Public Health (NCT01541826).

Publications

• Kim, B., Park, Y., Taheri, R., Kimball, K., Roto, A., Lee, J., and Bolling, B. (2012) Polyphenol-rich Aronia melanocarpa (chokeberry) extract regulates expression of cholesterol and lipid metabolism genes in Caco-2 cells. The FASEB Journal 26, 251.252.
• Kim, B., Ku, C. S., Pham, T., Park, Y., Martin, D., Xie, L., Taheri, R., Lee, J., and Bolling, B. (2012) Aronia melanocarpa (chokeberry) polyphenol rich extract reduces plasma cholesterol and improves antioxidant function in Apolipoprotien E knockout mice. The FASEB Journal 26, 1026.1029.