Identification of Anthocyanin Composition of Berries and Their Antioxidant and Anti-inflammatory Properties

IDENTIFICATION OF ANTHOCYANIN COMPOSITION OF BERRIES AND THEIR ANTIOXIDANT AND ANTI-INFLAMMATORY PROPERTIES

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

COMPLETE

Funding Source

HATCH

Reporting Frequency

Annual

Accession No.

0229964

Grant No.

(N/A)

Cumulative Award Amt.

(N/A)

Proposal No.

(N/A)

Multistate No.

(N/A)

Project Start Date

Oct 1, 2012

Project End Date

Sep 30, 2017

Grant Year

(N/A)

Program Code

[(N/A)]- (N/A)

Recipient Organization
UNIV OF CONNECTICUT
438 WHITNEY RD EXTENSION UNIT 1133
STORRS,CT 06269

Performing Department
Nutritional Sciences

Non Technical Summary
Recently, increased attention has been given to the possible health benefits of anthocyanins in preventing chronic and degenerative diseases including cardiovascular disease (CVD), and cancer. These effects were mainly attributed to their antioxidant and anti-inflammatory effects. Although the multi-mechanistic actions of berry anthocyanins have been extensively studied in vitro, there are still important gaps in our knowledge concerning the influence of different anthocyanin compositions on biological and physiological functions in vivo. Thus, studies into the metabolism of berry anthocyanins at cellular and molecular levels are necessary and defining structural and compositional relationships with their biological and physiological functions in cellular and animal models are critical in the overall evaluation of the role that berry anthocyanins play in the prevention and treatment of chronic human diseases. Therefore, in the proposed study, we aim to 1) identify and characterize the anthocyanin composition of different berries (blueberry, blackberry, and black currant), 2) test the antioxidant capacity of berry extracts and define its relationship with anthocyanin composition, 3) define the impact of different anthocyanin compositions of berry extracts on their antioxidant and anti-inflammatory properties in a cellular model, and 4) evaluate the health benefits of berry anthocyanins in the prevention of obesity-associated inflammation in an animal model. In the animal study proposed, a diet-induced obese (DIO) mouse model has been specifically chosen since it better represents obese adults on a typical American diet. This is a five-year project and once this project is successfully completed, the study findings will contribute to expanding current knowledge upon which recommended dietary intake of anthocyanin-rich berries can be provided to ameliorate obesity-induced inflammation.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
701	1120	1010	10%
701	1129	1010	20%
702	1120	1010	15%
702	1129	1010	35%
724	1120	1010	10%
724	1129	1010	10%

Knowledge Area
724 - Healthy Lifestyle; 702 - Requirements and Function of Nutrients and Other Food Components; 701 - Nutrient Composition of Food;

Subject Of Investigation
1120 - Blueberry; 1129 - Berries and cane fruits, general/other;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

Goals / Objectives
An overwhelming body of research has now firmly established that the dietary intake of berries has a positive and profound impact on human health, performance, and disease. Although berries contain micro- and macronutrients including vitamins, minerals, and fiber, their various biological properties have been largely related to their high levels and wide variety of phenolic compounds, in particular anthocyanins. For research to progress in this area, it is critical to understand the distribution and actual chemical structures of anthocyanins in foods. Few studies have reported the composition of anthocyanins in berries. Furthermore, whether anthocyanins with different chemical structures may exert significantly different chemical or physiological properties is of interest. However, information on structures and concentrations is still incomplete mainly due to limitations of analytical instruments. Recently, with an advancement of new technology which has provided us additional and powerful ways to identify the minor and uncommon anthocyanins in foods, increased attention has been given to their possible health benefits in preventing chronic and degenerative diseases including CVD and cancer. These effects have been mainly attributed to their antioxidant and anti-inflammatory effects. Although the multi-mechanistic actions of berry anthocyanins have been extensively studied in vitro, there are still important gaps in our knowledge concerning the influence of different anthocyanin compositions on the biological and physiological functions in vivo. Thus, studies into the metabolism of berry anthocyanins at cellular and molecular levels are necessary and defining structural and compositional relationships with their biological and physiological functions in cellular and animal models are critical in the overall evaluation of the role that berry anthocyanins play in the prevention and treatment of chronic human diseases. Therefore, in the proposed study, we aim to identify and characterize anthocyanin composition of different berries (blueberry, blackberry, and black currant), test antioxidant capacity of berry extracts and define its relationship with anthocyanin composition, define the impact of different anthocyanin compositions of berry extracts on their antioxidant and anti-inflammatory properties in a cellular model, and evaluate the health benefits of berry anthocyanins in the prevention of obesity-associated inflammation by using an animal model. Once this project is successfully completed, the study findings will contribute to expanding on the recommended dietary intake of anthocyanin-rich berries that can ameliorate obesity-induced inflammation.

Project Methods
Preparation of Berry Phenolic Extracts: Berry powders (blueberry, blackberry, and blackcurrant) will be provided by VDF FutureCeuticals, Inc (Momence, IL). The phenolics in berries will be extracted from 8 g of ground freeze-dried samples using 80% aqueous methanol with 0.1% HCl by homogenization method, and ultrasound-assisted method. Fractionation of Berry Anthocyanins: For the separation of anthocyanins during HPLC analysis, a simple fractionation of berry phenolic extracts will be performed using preconditioned C18 Sep-Pak cartridges to separate anthocyanins from nonanthocyanin phenolics. HPLC Analysis of Anthocyanins: HPLC analysis of berry anthocyanins will be performed using a reversed-phase HPLC system with a C18 reversed-phase Symmetry Analytical column. ABTS assay: The antioxidant capacity of berries will be measured based on ABTS assay modified by Kim et al. Real Time Quantitative PCR Analysis of mRNA Expression: RAW 264.7 cells will be incubated in 25 cm2 flasks for 24, 48, or 96 h with combinations of TNF-a (50 ng/ml) and berry extracts. Total RNA will extracted from these cultures using a Sigma genelute RNA isolation kit and reverse transcribed with M-MLV reverse transcriptase using random nonamer primers. Real time-PCR will be performed on a StepOne PCR system (Applied Biosytems, UK) using the DNA-binding dye SYBR green for detection of PCR products. Mice and Diets: Male DIO mice will be purchased from Jackson Laboratories at 13 wk of age and housed individually at the University of Connecticut (Uconn) animal facility. Ethical treatment of animals will be assured by UConn Institutional Animal Care and Use Committee. After several days of acclimation, mice will be assigned to 4 groups (n = 10 mice/group) that received 1 of 4 high-fat diets (HFD) for 12 wk (Research Diets). These will be a 60% high-fat diet (HFD), a modified HFD supplemented with 5% of blueberry freeze-dried powder (HFD + BB), 5% of blackberry freeze-dried powder (HFD + BK), and 5% of black currant freeze-dried powder (HFD + BC). After 12 weeks of feeding, all mice will be deprived of food overnight and killed under sodium pentobarbital anesthesia (40 mg/kg body weight). Blood will be collected from the abdominal cava vein into heparinised tubes. Plasma will be prepared by centrifugation at 12,000g for 2 min, and samples will be stored at −80C. Major organs, epididymal fat, and retroperitoneal fat tissues will be harvested, washed with ice-cold phosphate-buffered saline and weighed. Tissues will be immediately frozen in liquid nitrogen and stored at −80C until the time of analysis. Blood Collection and Analyses The measurement of a range of inflammatory and antioxidant markers, will be conducted, respectively. Serum clinical chemistry tests included the measurement of alanine aminotransferase, aspartate aminotransferase, glucose, insulin, and CRP will be analyzed by using immunoturbidimetric techniques. The antioxidant enzyme activities (catalase, SOD, GPx) will be measured using immunoabsorbent assay kits. Determination of Plasma Antioxidant Capacity The plasma antioxidant capacity will be determined using the ORAC assay.

Progress 10/01/12 to 09/30/17

Outputs
Target Audience:The ultimate goal of this project is to elevate clinical or experimental evidence of the health benefits of berries to the public health arena and contribute to expanding the recommended dietary intake of anthocyanin-rich berries by reducing the risk of aging-associated chronic diseases. Under this goal, a special lecture on the protective effects of black currant on aging-associated bone loss was given to the undergraduate senior and junior students in the NUSC 4260 Dietary Supplements and Functional Foods class (2017 fall semester). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?While conducting these animal and cell studies as well as chemical compositional analyses for the project, our graduate students who participated in this project gained the knowledge and learned the science relevant to the study and also acquired technical skills needed to conduct these studies. How have the results been disseminated to communities of interest?Findings were disseminated to scientific communities via journal articles, professional society conferences, and invited seminars. Dring the 5 year project period,we published6 papers and1 book chapter, and gave7 presentations at conferences and one invited seminar as well astwo classroomlecturesto the communities of interest. Publications: Sang Gil Lee, Taoran Wang, Terrence M. Vance, Patrice Hubert, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Validation of analytical methods for plasma total antioxidant capacity by comparing with urinary 8-isoprostane level. J Microbiol Biotech. 27(2):388-394, 2017 Feb. doi: 10.4014/jmb.1604.04053. Sang Gil Lee, Terrence Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Evaluation of pH Differential and HPLC Methods Expressed as Cyanidin-3-glucoside Equivalent for Measuring the Total Anthocyanin Contents of Berries. J Food Measurement and Characterization. Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. J Medicinal Food 19(4):390-7, 2016. Sang Gil Lee, Terrence M. Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, and Ock K. Chun. Contribution of Anthocyanin Composition to Total Antioxidant Capacity of Berries. Plant Food and Human Nutrition. 70(4): 427-432, 2015. 10.1007/s11130-015-0514-5 Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Jiyoung Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Relationship between oxidative stress and bone mass in obesity and effects of berry supplementation on bone turnover: an exploratory study. J Med Food 18(4): 476-82, 2015 Apr Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism. J Nutr Biochem 25(4): 404-11, 2014 Apr. Book Chapter: Sang Gil Lee, Terrence M. Vance, Ock K. Chun. Chapter 9. Antioxidative and anti-inflammatory effects and health implications of berry polyphenols: evidence from compositional and structural background to cell, animal, and human studies. Bagchi D, Swaroop A, Bagchi M (eds.), Nutraceuticals and Functional Foods in Human Health and Disease Prevention (2nd Edition), CRC Press/Taylor & Francis, Boca Raton. pp. 155-165, 2015. Presentations: Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. 2014 Experimental Biology Meeting, San Diego, CA, April 26-30, 2014 Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Antioxidant and anti-inflammatory effects of three berries having different anthocyanidin compositions. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral) Bohkyung Kim, Sang Gil Lee, Chai Siah Ku, Youngki Park, Yue Yang, Tho X Pham, Casey Wegner, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Comparison of hypolipidemic effects of three berries in diet-induced obese C57BL/6J mice. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral) Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Jiyoung Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral) Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Jiyoung Lee, Sung I. Koo, Ock K. Chun. Anthocyanin compositions and contents influence the antioxidant capacities of berries. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Sang Gil Lee, Young-ki Park, Jiyoung Lee, Sung I. Koo, Ock K. Chun. Berry anthocyanin fractions inhibit LPS-induced expression of inflammatory mediators in RAW 264.7 macrophages. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Comparison of analytical methods for anthocyanin quantification in berries: HPLC and pH differential methods. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Seminar: Invited Talk at the Seminar of the Department of Pathobiology and Veterinary Science, UConn: Protective Effect of Anthocyanin-Rich Blackcurrant Extract on Ovariectomy-Induced Bone Loss on April 2, 2015. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplishment during the 5th year (2016-2017): Under these major goals, during the Year 5 (final period) of the project, as part of method development and validation under this project, we evaluated the accuracy of pH differential (PD) method and HPLC method expressed as cyanidin-3-glucoside equivalent (CGE) for measuring total nthocyanin (TA) contents of five berries. In addition, we validated four conventional analytical methods for measuring plasma TAC including ABTS assay, DPPH assay, FRAP assay, and ORAC assay by comparing with urinary 8-isoprostane concentration. Accomplishment of the project for 5 years (2012-2017): During the five year project period, we investigated the beneficial role of berry anthocyanins in the prevention and treatment of chronic human diseases by defining structural and compositional relationships of berry anthocyanins with their biological and physiological functions in cellular and animal models. From the in vitro cell studies, we found that berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism. These findings led us to animal studies with two disease models, diet-induced obesity and estrogen-deficiency-induced osteoporosis and we reported the relationship between oxidative stress and bone mass in obesity and effects of berry supplementation on bone turnover and also documented that anthocyanin-rich blackcurrant extract attenuates ovariectomy-induced bone loss in mice. In addition, we conducted compositional analyses of berry anthocyanins for the evaluation of two different measurement methods of total anthocyanin contents in berries, pH Differential and HPLC methods, for the accuracy and efficacy and also investigated the contribution of anthocyanin composition to total antioxidant capacity of berries. Altogether, these study findings may contribute to the scientific basis for expanding on the recommended dietary intake of anthocyanin-rich berries that can ameliorate chronic disease risk induced by inflammation and oxidative stress.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Sang Gil Lee, Terrence Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Evaluation of pH Differential and HPLC Methods Expressed as Cyanidin-3-glucoside Equivalent for Measuring the Total Anthocyanin Contents of Berries. J Food Measurement and Characterization. 10(3): 562568, 2016. DOI: 10.1007/s11694-016-9337-9
Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Sang Gil Lee, Taoran Wang, Terrence M. Vance, Patrice Hubert, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Validation of analytical methods for plasma total antioxidant capacity by comparing with urinary 8-isoprostane level. J Microbiol Biotech. 27(2):388-394, 2017. doi: 10.4014/jmb.1604.04053.
Type: Book Chapters Status: Published Year Published: 2015 Citation: Sang Gil Lee, Terrence M. Vance, Ock K. Chun. Chapter 9. Antioxidative and anti-inflammatory effects and health implications of berry polyphenols: evidence from compositional and structural background to cell, animal, and human studies. Bagchi D, Swaroop A, Bagchi M (eds.), Nutraceuticals and Functional Foods in Human Health and Disease Prevention (2nd Edition), CRC Press/Taylor & Francis, Boca Raton. pp. 155-165, 2015. *The publisher didn't allow us to add a section to acknowledge funding sources of the study in this book chapter.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The target audience for this reporting period includes researchers in nutrition and health science areas, and the food and nutraceutical industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?While conducting these animal and cell studies for the project, the graduate students who participated in this project gained knowledge and learned the science relevant to the study and also acquired technical skills needed to conduct these studies. How have the results been disseminated to communities of interest?Findings were disseminated to scientific communities via journal articles, professional society conferences, and invited seminars. To date, we published4 papers, gave 6 presentations at conferences and presented the study findings at an invited seminar. . What do you plan to do during the next reporting period to accomplish the goals?As part of method development and validation under this project, we plan to evaluate the accuracy of pH differential (PD) method and HPLC method expressed as cyanidin-3-glucoside equivalent (CGE) for measuring total anthocyanin (TA) contents of five berries. In addition, we will validate four conventional analytical methods for measuring plasma TAC including ABTS assay, DPPH assay, FRAP assay, and ORAC assay by comparing with urinary 8-isoprostane concentration.

Impacts
What was accomplished under these goals? Under these major goals, during Year 4 of the project, we evaluated the effect of blackcurrant anthocyanins on bone mass in an estrogen deficiency mouse model. Fourteen-week-old C57BL/6J mice (n = 54) were ovariectomized or sham operated. The ovariectomized mice were divided into two groups, basal diet (OVX) or basal diet containing 1% anthocyanin-rich blackcurrant extract (OVX+BC), and sacrificed at 4, 8, and 12 weeks. Femoral bone mineral density (BMD) and trabecular bone volume by dual-energy X-ray absorptiometry and micro-computed tomography, respectively, and serum bone markers were measured. Ovariectomy significantly reduced BMD and trabecular bone volume at all time points (P < .05). Blackcurrant supplementation attenuated ovariectomy-induced bone loss measured by BMD and trabecular bone volume at 8 weeks (P = .055 and P = .057) and the effect was more pronounced at 12 weeks (P = .053 and P < .05). Ovariectomy and blackcurrant treatment did not alter serum biomarkers of bone formation and resorption. Bone marrow cells extracted from OVX mice significantly induced osteoclast-like (OCL) cell formation compared with cells from sham controls (P < .05). Blackcurrant treatment decreased the number of TRAP(+) OCL compared with OVX mice at 8 and 12 weeks (P < .05). Furthermore, blackcurrant supplementation reduced bone resorption activity when measured by resorption pit assay, compared with OVX group (P < .05). These results demonstrate that blackcurrant may be effective in mitigating osteoclast-induced postmenopausal bone loss. With the findings, we have published one paper to a peer-reviewed journal.

Publications

Type: Journal Articles Status: Published Year Published: 2016 Citation: Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. J Medicinal Food 19(4):390-7, 2016
Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Antioxidant and antinflammatory effects of three berries having different anthocyanidin compositions. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral)
Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Bohkyung Kim, Sang Gil Lee, Chai Siah Ku, Youngki Park, Yue Yang, Tho X Pham, Casey Wegner, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Comparison of hypolipidemic effects of three berries in diet-induced obese C57BL/6J mice. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral)
Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. (2012) Anthocyanin compositions and contents influence the antioxidant capacities of berries. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Sang Gil Lee, Young-ki Park, Jiyoung Lee, Sung I. Koo, Ock K. Chun. Berry anthocyanin fractions inhibit LPS-induced expression of inflammatory mediators in RAW 264.7 macrophages. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25.
Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Comparison of analytical methods for anthocyanin quantification in berries: HPLC and pH differential methods. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25.
Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Ock Chun (2015) Seminar presentation. Protective effect of anthocyanin-rich black currant extract on ovariectomy-induced bone loss. University of Connecticut, Department of Pathobiology and Veterinary Science, April, 2, 2015.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:The ultimate goal of this project is to elevate clinical or experimental evidence of the health benefits of berries to the public health arena and contribute to expanding the recommended dietary intake of anthocyanin-rich berries byreducing oxidative stress andinflammation. Under this goal, the evaluation ofthe contribution of anthocyanin composition to the total antioxidant capacity of berries having different anthocyanin compositioncan provide a scientific basis for the future studies planned. The potential target audience of this study includes researchers in nutrition and health science areas, the food and neutraceutical industry, public health policy makers, nutrition educators, any gender, especially overweight or obese, postmenopausal women, and anyone interested in increasing antioxidant consumption. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student who participated in this project gained knowledge, learned the science relevant to the study and acquired the technical skills needed to conduct the compositional analytical studies. How have the results been disseminated to communities of interest?Findings were disseminated to scientific communities via journal articles, professional society conferences, and seminars. To date, 3 papers were published, and study findings were presented at 6 conferences and 1 invited seminar. Publications: Sang Gil Lee, Terrence M. Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, and Ock K. Chun. Contribution of Anthocyanin Composition to Total Antioxidant Capacity of Berries. Plant Food and Human Nutrition. 70(4): 427-432, 2015. 10.1007/s11130-015-0514-5 Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Jiyoung Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Relationship between oxidative stress and bone mass in obesity and effects of berry supplementation on bone turnover: an exploratory study. J Med Food 18(4): 476-82, 2015 Apr Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism. J Nutr Biochem 25(4): 404-11, 2014 Apr. Presentations: Xi Zheng, Se Hwan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. 2014 Experimental Biology Meeting, San Diego, CA, April 26-30, 2014 Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Antioxidant and anti-inflammatory effects of three berries having different anthocyanidin compositions. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral) Bohkyung Kim, Sang Gil Lee, Chai Siah Ku, Youngki Park, Yue Yang, Tho X Pham, Casey Wegner, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Comparison of hypolipidemic effects of three berries in diet-induced obese C57BL/6J mice. 2013 Experimental Biology Meeting, Boston, MA, April 20-24, 2013 (Oral) Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Jiyoung Lee, Sung I. Koo, Ock K. Chun. Anthocyanin compositions and contents influence the antioxidant capacities of berries. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Sang Gil Lee, Young-ki Park, Jiyoung Lee, Sung I. Koo, Ock K. Chun. Berry anthocyanin fractions inhibit LPS-induced expression of inflammatory mediators in RAW 264.7 macrophages. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Sang Gil Lee, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Comparison of analytical methods for anthocyanin quantification in berries: HPLC and pH differential methods. 2012 Experimental Biology Meeting, San Diego, CA, April 20-25, 2012 Seminar: Invited Talk at the Seminar of the Department of Pathobiology and Veterinary Science, UConn: Protective Effect of Anthocyanin-Rich Blackcurrant Extract on Ovariectomy-Induced Bone Loss on April 2, 2015. What do you plan to do during the next reporting period to accomplish the goals?We plan to continue mechanistic studies to investigate how anthocyanin-rich black currant extract attenuates ovariectomy-induced bone loss in mice. By addinggene expression data, we willupdateand submit one manuscript to a peer-reviewed journal. In addition, to expand our research interest to different types ofantioxidants, we are currentlyworking on a review of previous articleson soy isoflavones and osteoporotic bone loss with an emphasison modulation of bone remodeling. Onceit is ready, we willsubmitit to a peer-reviewed journal for publication.

Impacts
What was accomplished under these goals? Under these major goals, during Year 3 of the project, weidentified and characterized the anthocyanin composition of different berries (blueberry, blackberry, and black currant) andtested the antioxidant capacity of berry extracts and defined its relationship with anthocyanin composition. Blackberry demonstrated the highest total antioxidant capacity (TAC), while it had the lowest total anthocyanin content among the three berries in both of the phenolic extract and anthocyanin fractions. On the other hand, black currant had the highest total anthocyanin content, but the lowest TAC. Cyanidin-3-O-glucoside (cya-3-glc) accounted for 94% of blackberry anthocyanins, and as one of the strongest antioxidants present in these three berries, it substantially contributed to the TAC of blackberry anthocyanin fraction (96.0%). Delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside in black currant had lower antioxidant capacities compared with delphinin-3-O-glucoside and cya-3-glc, resulting in its lowest TAC among berry anthocyanin fractions examined. Malvidin derivatives, major anthocyanins of blueberry, had considerably lower antioxidant capacity than other anthocyanidin derivatives, such as cyanidin or delphinidin, resulting in lower TAC of blueberry compared with blackberry. Our findings indicate that anthocyanin composition as well as the antioxidant capacity of individual anthocyanins contributes to the TAC of berries rich in distinct anthocyanins. Study findings were published in a peer-reviewed journal.

Publications

Type: Journal Articles Status: Published Year Published: 2015 Citation: Sang Gil Lee, Terrence M. Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, and Ock K. Chun. Contribution of Anthocyanin Composition to Total Antioxidant Capacity of Berries. Plant Food and Human Nutrition. 70(4): 427-432, 2015

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: The ultimate goal of this project is to elevate clinical or experimental evidence of the health benefits of berries to the public health arena and contribute to expanding the recommended dietary intake of anthocyanin-rich berries, which can ameliorate obesity-induced inflammation. Under this goal, the evaluation of themethods for measuring the total anthocyanin contents of berries can provide a scientific basis for the future studies planned.The potential target audienceof this study includes researchers in nutrition and health science areas, food and neutraceutical industry, public health policy makers, nutrition educators,overweight or obese population, postmenopausal women, and anyone interested in increasing antioxidant consumption. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? While conducting compositional analysis for the project, ourgraduate student who participated in this project gained the knowledge and learned the science relevant to the study and also acquired technical skills needed to conduct the analytical studies. How have the results been disseminated to communities of interest? Findings were disseminated to scientific communities via journal articles, professional society conferences, and invited seminars. What do you plan to do during the next reporting period to accomplish the goals? We plan to identify and characterize the anthocyanin composition of different berries (blueberry, blackberry, and black currant) and will test the antioxidant capacity of berry extracts and define its relationship with anthocyanin composition. In addition, we will continue to publish our findings in peer-reviewed journals and at international conferences.

Impacts
What was accomplished under these goals? Under these major goals, during Year 2 of the project, we evaluated the accuracy of the two methods for total anthocyanin (TA) analysis using Pearson correlation assay and Brand-Altman plot. The pH differential (PD) and HPLC methods expressed as cya-3-glc equivalents (CGE) were compared with the quantitative HPLC method (q-HPLC) using individual anthocyanin standards. In addition, the two methods using major anthocyanin equivalent (MAE) was also evaluated as an alternative unit. Blueberry, blackberry, black currant, raspberry, and cranberry were used for the study. Data showed that the values expressed as CGE considerably deviated from those obtained by q-HPLC. This discrepancy was due to the underestimated TA values when expressed as CGE in black currant and raspberry which contain disaccharides as major glycone. However, the TA values expressed as MAE were in better agreement with the actual values compared with CGE. Our findings indicate that the TA values expressed as MAE, rather than CGE, better approximate the actual TA contents. With the findings, we submittedone paper toa peer-reviewed journal, whichis under review: Sang Gil Lee, Terrence Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Evaluation of pH Differential and HPLC Methods for Measuring the Total Anthocyanin Contents of Berries. Food Chem (Submitted in 2014) Amongthe 3 papers which were under review or in preparation during the 1stproject reporting period, two werepublished in 2014and one paper is in preparation.In addition, we submitted one additional paper to a peer-reviewed journal and it is under review as detailed in the Products section.

Publications

Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Ji-young Lee. Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-kB independent of NRF2-mediated mechanism. J Nutr Biochem. 2014 Apr;25(4):404-11.
Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Ji-young Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Relationship Between Oxidative Stress and Bone Mass in Obesity and Effects of Berry Supplementation on Bone Remodeling in Obese Male Mice: An Exploratory Study. J Med Food. 2014 Sep 8. [Epub ahead of print]
Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Bohkyung Kim, Sang Gil Lee, Chai Siah Ku, Youngki Park, Yue Yang, Tho X Pham, Casey Wegner, Sung I. Koo, Ock K. Chun, Ji-young Lee. Blueberry, blackberry, and blackcurrant differentially affect plasma lipids and pro-inflammatory markers in diet-induced obesity mice. RSC Advances (Submitted in July 20, 2014)
Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Sang Gil Lee, Terrence Vance, Tae-Gyu Nam, Dae-Ok Kim, Sung I. Koo, Ock K. Chun. Evaluation of pH Differential and HPLC Methods for Measuring the Total Anthocyanin Contents of Berries. Food Chem (Submitted in May 9, 2014)

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Theultimate goal of this project is to elevate clinical or experimental evidence of the health benefits ofberries to the public health arena andcontribute to expandingthe recommended dietary intake of anthocyanin-rich berries, which can ameliorate obesity-induced inflammation. Thus, thepotential target audience will include researchers in nutrition and health science areas, public health policy makers, nutrition educators, any gender,especially overweight or obese,postmenopausal women, and anyoneinterested in increasing antioxidant consumption. Changes/Problems: We modified the original project plan by adding an additionalspecific aim to examine the impact of berries on bone health as we hypothesized that berries' antioxidative and antiinflammatory properties might also attenuate bone loss mediated by oxidative stress and inflammation. This additional study will provide important information on how anthocyanin-rich berries improvebone metabolism and protect against bone loss mediated by oxidative stress and inflammation. The research findings will contribute to establishing effective dietary recommendations for the prevention of bone loss among people at high risk of bone loss such as older adults, menopausal women, smokers, and overweight or obese people. What opportunities for training and professional development has the project provided? While conducting a cell study andtwo animal studies for the project, our 4 PhD students who participated in this project gained the knowledge and learned the science relevant to each study and also acquired technical skills needed to conduct the studies. How have the results been disseminated to communities of interest? Our findings from this proposed research will facilitate the extension of new knowledge, the dispensing ofnutrition educational messages, and will applyto other diet-related chronic disease models in our future studies. What do you plan to do during the next reporting period to accomplish the goals? We planto identify and characterize the anthocyanin composition of different berries (blueberry, blackberry, and black currant) and willtest the antioxidant capacity of berry extracts and define its relationship with anthocyanin composition. In addition, we will continue animal studies for investigating the anti-inflammatory effectsof berriesin high fat diet-induced obese mice as well asbone protective effects of berries inOVX mice.

Impacts
What was accomplished under these goals? The ultimate goal of this project is to elevate clinical or experimental evidence of the health benefits of berries to the public health arena andcontribute to expandingthe recommended dietary intake of anthocyanin-rich berries, which can ameliorate obesity-induced inflammation. In addition, we modified the original project plan by adding a specific aim to examinethe impact of berries on bone health as we hypothesized that berries' antioxidative and antiinflammatory properties might also attenuate bone loss mediated by oxidative stress and inflammation. As the 1st year study of the project, we defined the impact of different anthocyanin compositions on the antioxidant and anti-inflammatory properties of berry extracts incellular models. We also conducted an animal study using diet-induced obese mice fed with different berries to investigate how anthocyanin-rich berries added in the diet improve bone mass in high fat diet-induced obese mice. In addition, we conducted a bone study using ovariectomized (OVX)female mice to examine whetherblack currant might attenuate OVX-induced bone loss. With the findings, we submitted two papers to peer-reviewed journals and one of them has been accepted and the other one is under review: Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Berry anthocyanin fractions repress pro-inflammatory gene expression and secretion by inhibiting nuclear translocation of NF-kB in RAW 264.7 macrophages. J Nutr Biochem (In press, Accepted in December 5, 2013) Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Jiyoung Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Blueberry and blackcurrant consumption increases bone mineral density and content in high fat diet-induced obese male mice. J Med Foods (Submitted in Dec 12, 2014) In addition, we also submitted one abstract to 2014 Experimental Biology Meeting as follows: Xi Zheng, Sewhan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Black currant extract attenuates ovariectomy-induced bone loss in mice (Submitted to 2014 Experimental Biology Meeting, Under Review)

Publications

Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Sang Gil Lee, Bohkyung Kim, Yue Yang, Youngki Park, Sung I. Koo, Ock K. Chun, Jiyoung Lee. Berry anthocyanin fractions repress pro-inflammatory gene expression and secretion by inhibiting nuclear translocation of NF-kB in RAW 264.7 macrophages. J Nutr Biochem (In press, Accepted in December 5, 2013)
Type: Journal Articles Status: Under Review Year Published: 2014 Citation: Sang Gil Lee, Terrence Vance, Do Yu Soung, Bohkyoung Kim, Jiyoung Lee, Sung I. Koo, M. Hicham Drissi, Ock K. Chun. Blueberry and blackcurrant consumption increases bone mineral density and content in high fat diet-induced obese male mice. J Med Foods (Under Review)
Type: Conference Papers and Presentations Status: Under Review Year Published: 2014 Citation: Xi Zheng, Sewhan Mun, Sang Gil Lee, Terrence Vance, Patrice Hubert, Sung I. Koo, Sun Kyeong Lee, Ock K. Chun. Black currant extract attenuates ovariectomy-induced bone loss in mice (Submitted to 2014 Experimental Biology Meeting, Under Review)