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INVESTIGATOR: Sang, S.; Hanner, T.

PERFORMING INSTITUTION:
NORTH CAROLINA A&T STATE UNIV
1601 EAST MARKET STREET
GREENSBORO, NORTH CAROLINA 27411

WHEAT BRAN FOR COLON CANCER PREVENTION: A TARGETED METABOLOMIC APPROACH

NON-TECHNICAL SUMMARY: Colorectal cancer (CRC) is a leading cause of cancer deaths in the United States. Many studies have found that CRC is preventable and dietary factors play an important role on the development of CRC. Epidemiological studies have suggested that dietary fibers, especially fiber from cereal sources, protects against colon cancer. Human and animal studies indicated that not all cereal fibers are equally efficacious. Wheat bran is the only cereal bran that shows consistent protection against colon cancer in laboratory animal models, however, the active components in wheat bran are still unknown. The objectives of this project are to identify the active components in wheat bran for colon cancer prevention and study the exposure markers of wheat bran for future dietary assessment of whole grain wheat and wheat bran consumption using a targeted metabolomic approach. The development of this proposal is to build North Carolina A&T State University's research capacity in the area of bioactive food components for cancer prevention. New research activities created in this project will strengthen North Carolina A&T State University's research capacity to study the beneficial health effects of functional foods and their bioactive components and to work with food industries to develop value-added functional foods to prevent chronic diseases, such as cancer.

OBJECTIVES: The objectives of this project are to identify the active components in wheat bran (WB) for colon cancer prevention and study the exposure markers of WB for future dietary assessment of whole grain wheat and WB consumption using a targeted metabolomic approach. Upon the completion of this project, the measurable outcomes will be 1) the elucidation of the structures of the major phytochemicals of WB and their metabolites and 2) the development of the LC/MS methods to analyze the chemical and metabolic profiles of WB; 3) the determination of the inhibitory effects of WB and its fraction F7 on the formation of aberrant crypt foci (ACF) in rats; and 4) the identification of the major cancer preventive components of WB and of the exposure markers of WB consumption.

APPROACH: In Aim 1, we will measure the chemical profiles of phytochemicals in wheat bran (WB) and their metabolic profiles in rat plasma, urine, feces and colon tissues using a targeted metabolomic approach. We will modify our established HPLC-APCI/MS assay for the measurement of the major phytochemicals in WB. The major unknown components will be purified using column chromatography and be identified using 1D and 2D NMR. Colon tissues, plasma, urine, and feces will be collected from rats after two-week treatment with WB. The chemical profile of WB and the metabolic profile of WB in colon tissues, plasma, urine, and feces will be studied using data-dependent MS/MS analysis. In Aim 2, we will evaluate the efficacy of wheat bran and the fraction enriched with 5-alkylresorcinols (ARs) against aberrant crypt foci (ACF) formation in the AOM-treated rat model and identify the exposure markers of wheat bran. Blood, urine, and fecal samples will be collected every two weeks during the experiment and at the end of the experiment. Colon tissues will also be collected at the end of the experiment. The inhibitory action observed will be correlated with the levels of the major phytochemicals in WB and their related metabolites. We will place special emphases on ARs and their metabolites.

PROGRESS: 2012/09 TO 2017/08
Target Audience: Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Professional development training activities were provided to one undergraduate student and one postdoc. How have the results been disseminated to communities of interest? Results have been disseminated to communities of interest through scientific presentations and peer-reviewed journal article and book chapter publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2012/09 TO 2017/08
What was accomplished under these goals? 1. We purifiedand identified the major components in wheat bran and estabolished the chemical profile of phytochemicals in wheat bran. 2. We study the metabolic profole of alkylresorcinols in mice and human and identified several novel metabolites of alkylresorcinols. 3. Using targeted metabolic approach, we established the metabolic profiles of all the major phytochemicals in wheat bran. 4. Using un-targeted metabolic approach, we studied the impact of whole grain wheat intake on the profile of endogenous metabolites, and identified the potential biomarkers for the effects of whole grain wheat intake on the prevention of cancer, diabetes, and cardiovascular diseases. 5. Identified the major active components for colon cancer prevention using human colon cancer cells and rodent model. Upon the completion of this project, we had the following achievements. Organized symposium: "The Chemistry Behind Health Effects of Whole Grains" 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016. Organizers: Sang, S. and Landberg, R. Guest Editor (Special Issue): Molecular Nutrition & Food Research: The Chemistry Behind Health Effects of Whole Grains; Editors: Sang, S.; Landberg, R.; 2017, 61(7). Publications (peer-reviewed articles): 1. Zhu, Y.; Sang, S. Phytochemicals in Whole Grain Wheat and Their Health-Promoting Effects. Mol Nutr Food Res. 2017, 61, 1600852. 2.Sang, S.; Landberg, R. The chemistry behind health effects of whole grains. Mol Nutr Food Res. 2017, 61, 1770074. 3.Zhu, Y.; Wang, P.; Sha, W.; Sang, S. Urinary Biomarkers of Whole Grain Wheat Intake Identified by Non-targeted and Targeted Metabolomics Approaches. Sci. Rep. 2016, 6, 36278. 4.Zhu, Y.; Soroka, D.N.; Sang, S. Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human Colon Cancer Cell Growth: Identification, Chemical Synthesis and Biological Evaluation. J. Agric. Food Chem. 2015, 63(8), 2264-2276. 5.Zhu, Y.; Shurlknight, K.L.; Chen, X.; Sang, S. Identification and pharmacokinetics of novel alkylresorcinol metabolites in human urine: new candidate biomarkers for whole grain wheat and rye intake. J Nutr. 2014, 144 (2), 114-122. 6.Zhu, Y.; Soroka, D.; Sang, S. Structure Elucidation and Chemical Profile of Sphingolipids in Wheat Bran and Their Cytotoxic Effects against Human Colon Cancer Cells. J. Agric. Food Chem. 2013, 61(4), 866-874. 7.Zhu, Y.; Soroka, D.; Sang, S. Synthesis and inhibitory activities against colon cancer cell growth and proteasome of alkylresorcinols. J. Agric. Food Chem. 2012, 60(35), 8624-31. Book Chapter: Sang, S. and Zhu, Y. "Bioactive phytochemicals in wheat bran for colon cancer prevention" in "Wheat and Rice in Disease Prevention and Health", 2014, Chapter 10, 121-129. (Editors: Ronald R. Watson, Victor R. Preedy, and Sherma Zibadi; Elsevier publishing). Scientific Presentations: 1.Sang, S. "Biomarkers of whole grain wheat intake identified by targeted and non-targeted metabolomic approaches" 2016 Annual Conference of Functional Foods, Nutraceuticals, Natural Health Products, and Dietary Supplements, Orlando, FL, October 9-13, 2016. (Oral presentation) 2.Sang, S. "Identification of bioactive components in wheat bran: An example of team science" 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016. (Oral presentation) 3.Sang, S. "Phytochemicals in wheat bran for colon cancer prevention" 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016. (Oral presentation) 4.Zhu, Y.; Sha, W.; Wang, P.; Sang, S. "Biomarkers of whole grain wheat intake identified by targeted and non-targeted metabonomic approaches" 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016. 5.Sang, S.; Zhu, Y.; Sha, W.; and Wang, P. "Nutrimetabolomics fingerprinting to identify biomarkers of whole grain wheat intake" Experimental Biology 2016 Annual Meeting, Boston, MA, April 2-6, 2016. (Oral presentation) 6.Zhu, Y.; Sang, S. "Metabolites of wheat phytochemicals as the exposure biomarkers of whole grain wheat intake" 250th ACS National Meeting, Boston, MA, August 16-20, 2015. (Oral presentation) 7.Sang, S. "Bioactivity / Bioavailability of Phytochemicals in Grain Bran - How Much Do We Know?" 2015 AACCI Milling and Baking Spring Technical Conference, Savannah, GA, April 15-17, 2015 (Invited speaker) 8.Sang, S. "The chemistry behind the biological effect of whole-grain wheat" 248th ACS National Meeting, San Francisco, CA, August 10-14, 2014. (Oral presentation) 9.Sang, S.; Zhu, Y. "Novel alkylresorcinol metabolites in human urine as potential exposure biomarkers for whole grain wheat and rye intake" Experimental Biology 2014 Annual Meeting, San Diego, CA, April 26-30, 2014. (Oral presentation) 10.Sang, S. "Novel Metabolites of alkylresorcinols as the exposure markers to reflect whole wheat consumption in human" 246th AACC International Annual Conference, Albuquerque, NM, September 29-October 02, 2013. (Poster) 11.Zhu, Y. and Sang, S. "Metabolites of alkylresorcinols as the exposure markers to reflect whole wheat consumption in human" 246th ACS National Meeting, Indianapolis, IN, September 08-12, 2013. (Oral presentation) 12.1. Sang, S.; Zhu, Y. "Wheat bran for colon cancer prevention: Phytochemicals vs. Fiber". Experimental Biology 2013 Annual Meeting, Boston, MA, April 20-24, 2013. (Poster) 13.Zhu, Y.; Shurlknight, K.; Sang, S. "Metabolites of Alkylresorcinols as the Exposure Markers to Reflect Whole Grain Wheat Consumption in Human" North Carolina Research Campus 2nd Annual Catalyst Symposium, Kannapolis, NC, April 15, 2013. (Poster) 14.Zhu, Y.; Soroka, D.; Shurlknight, K.; Sang, S. "Bioactive Phytochemicals in Wheat Bran for Colon Cancer Prevention" North Carolina Research Campus 2nd Annual Catalyst Symposium, Kannapolis, NC, April 15, 2013. (Poster) 15.Zhu, Y.; Soroka, D.; Sang, S. "Alk(en)ylresorcinols as the Major Active Components in Wheat Bran Oil for Colon Cancer Prevention" 244th ACS National Meeting, Philadelphia, PA, August 19-23, 2012. (Oral presentation).

PUBLICATIONS (not previously reported): 2012/09 TO 2017/08
1. Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu, Y.; Sang, S. Phytochemicals in Whole Grain Wheat and Their Health-Promoting Effects. Mol Nutr Food Res. 2017, 61, 1600852.
2. Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, Y.; Wang, P.; Sha, W.; Sang, S. Urinary Biomarkers of Whole Grain Wheat Intake Identified by Non-targeted and Targeted Metabolomics Approaches. Sci. Rep. 2016, 6, 36278.
3. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sang, S. ?Biomarkers of whole grain wheat intake identified by targeted and non-targeted metabolomic approaches? 2016 Annual Conference of Functional Foods, Nutraceuticals, Natural Health Products, and Dietary Supplements, Orlando, FL, October 9-13, 2016.
4. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Sang, S. ?Identification of bioactive components in wheat bran: An example of team science? 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016.

PROGRESS: 2015/09/01 TO 2016/08/31
Target Audience: The target audience of this research are members of the scientific community who have interests in the area of bioactive food components for cancer prevention; also the food industries who may be looking to develop value-added functional foods to prevent chronic diseases such as cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development training activities were providedfor oneundergraduate student and a postdoc. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through scientifc presentations. What do you plan to do during the next reporting period to accomplish the goals?1.Scientists will further study the efficacy and molecular mechanism of the cancer preventive phytochemcials in wheat bran. 2. Summarize all the data and submit a new manuscript on the efficacy and molecular mechanism of wheat bran phytochemicals. 3. Submit a review manuscript on the bioactive phytochemicals in whole grain wheat to Molecular Nutrition and Food Research.

IMPACT: 2015/09/01 TO 2016/08/31
What was accomplished under these goals? After finishing the chemical profile of wheat phytochemicals,researchers conducted both a targeted and a non-targeted metabolomic analysis of the wheat phytochemicals. The targeted metabolomic analysis provides the metabolic profile of wheat phytochemicals. The non-targeted metabolomic analysis provides the changes of endogenous metabolites affected by whole grain wheat intake. The combination of the targeted and non-targeted metabolomic analysis leadsreserachers to identify the exposure markers of whole grain wheat intake. Results from this study have been summarized ina manuscript that has been submitted for publishing which is undergoing minor revisions.

PUBLICATIONS: 2015/09/01 TO 2016/08/31
1. Type: Journal Articles Status: Under Review Year Published: 2016 Citation: 1. Zhu, Y.; Wang, P.; Sha, W.; Sang, S. Biomarkers of Whole Grain Wheat Intake Identified by Non-targeted and Targeted Metabonomic Approaches. Sci. Rep. 2016 (Under revision).
2. Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: 1. Sang, S.; Zhu, Y.; Sha, W.; and Wang, P. ?Nutrimetabolomics fingerprinting to identify biomarkers of whole grain wheat intake? Experimental Biology 2016 Annual Meeting, Boston, MA, April 2-6, 2016. 2. Sang, S. ?Phytochemicals in wheat bran for colon cancer prevention? 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016. 3. Zhu, Y.; Sha, W.; Wang, P.; Sang, S. ?Biomarkers of whole grain wheat intake identified by targeted and non-targeted metabonomic approaches? 252th ACS National Meeting, Philadelphia, PA, August 21-25, 2016.

PROGRESS: 2014/09/01 TO 2015/08/31
Target Audience:The target audience of this research would include the scientific community who have interests in the area of bioactive food components for cancer prevention; also the food industries who may be looking to develop value-added functional foods to prevent chronic diseases such as cancer. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Anundergraduateandpostdoc were trainedusing this project. How have the results been disseminated to communities of interest?Results have been disseminated to communities of interest through scientific presentations. What do you plan to do during the next reporting period to accomplish the goals?Researchers will complete the in vivo and in vitro efficacy study of the phytochemicals in wheat bran and also finalize the exposure markers study.

IMPACT: 2014/09/01 TO 2015/08/31
What was accomplished under these goals? Researchershave elucidated the structures of the major steroids in wheat bran,established the chemical profile of steroids in wheat bran using LC/MS, and determined these compounds effects against the growth of human colon cancer cells, which has been published in Journal of Agricultural and Food Research (2015; 63(8):2264-76). We have further studied the efficacy and underlying molecular mechanisms of alkylresorcinols, the major active components in wheat bran,against colon cancer andare summarizing the data and preparing a manuscript. Further study is ongoing inthe exposure markers of wheat bran intake by looking at the metabolic profile of benzoxazinoids in wheat bran.

PUBLICATIONS: 2014/09/01 TO 2015/08/31
1. Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhu, Y.; Soroka, D.N.; Sang, S. Oxyphytosterols as Active Ingredients in Wheat Bran Suppress Human Colon Cancer Cell Growth: Identification, Chemical Synthesis and Biological Evaluation. J. Agric. Food Chem. 2015, 63(8), 2264-2276.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Stone, N.; Zhu, Y., Wang, P. and Sang, S. ?Alkylresorcinols: Purification from wheat bran and quantification in wholegrain wheat breads? 249th ACS National Meeting, Denver, CO, March 22-26, 2015. (Oral presentation)

PROGRESS: 2013/09/01 TO 2014/08/31
Target Audience: The target audience of this project includes graduate students, a postdoc and members of the general public. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Research staff are training one PhD graduate student using this grant. How have the results been disseminated to communities of interest? Results aredisseminated through presentations at scientific conferences. What do you plan to do during the next reporting period to accomplish the goals? Further study will be ongoingin vitro and in vivo efficacy and the metabolism of the active components identified from wheat bran.

IMPACT: 2013/09/01 TO 2014/08/31
What was accomplished under these goals? Scientists have identified all the major active components in wheat bran for colon cancer prevention.Researchers also studied the metabolism of alkylresorcinols and the potential to use alkylresorcinols and its major metabolites as the potential exposure markers to reflect whole grain wheat intake.

PUBLICATIONS: 2013/09/01 TO 2014/08/31
1. Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhu, Y.; Shurlknight, K.L.; Chen, X.; Sang, S. Identification and pharmacokinetics of novel alkylresorcinol metabolites in human urine: new candidate biomarkers for whole grain wheat and rye intake. J Nutr. 2014, 144 (2), 114-122.
2. Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Sang, S. ?The chemistry behind the biological effect of whole-grain wheat? 248th ACS National Meeting, San Francisco, CA, August 10-14, 2014. (Oral presentation) Sang, S.; Zhu, Y. ?Novel alkylresorcinol metabolites in human urine as potential exposure biomarkers for whole grain wheat and rye intake? Experimental Biology 2014 Annual Meeting, San Diego, CA, April 26-30, 2014. (Oral presentation)

PROGRESS: 2012/09/01 TO 2013/08/31
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Researchers presented results at different conferences including local, national and international conferences. What do you plan to do during the next reporting period to accomplish the goals? Researchers plan to further study the metabolic profile and the in vivo efficacy of phytochemicals in wheat bran. Researchers also plan to submit another manuscript on the chemical profile of wheat bran.

IMPACT: 2012/09/01 TO 2013/08/31
What was accomplished under these goals? Researchershave almost finished the chemical profile of the components in wheat bran. Using bioassay guilded purification, we purified and identified all the major components in wheat bran and identified several of them as the active components. Researchers are in the process of studying the in vivo efficacy and the metabolic profile of phytochemicals in wheat bran. Two manuscripts have been published. Results were presented at different scientific conferences.

PUBLICATIONS: 2012/09/01 TO 2013/08/31
1. Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhu, Y.; Soroka, D.; Sang, S. Structure Elucidation and Chemical Profile of Sphingolipids in Wheat Bran and Their Cytotoxic Effects against Human Colon Cancer Cells. J. Agric. Food Chem. 2013, 61(4), 866-874.
2. Type: Journal Articles Status: Published Year Published: 2012 Citation: Zhu, Y.; Soroka, D.; Sang, S. Synthesis and inhibitory activities against colon cancer cell growth and proteasome of alkylresorcinols. J. Agric. Food Chem. 2012, 60(35), 8624-31.