Progress 04/01/23 to 03/31/24
Outputs
Target Audience:The target audience for this project remains largely unchanged compared to the previous year, continuing to focus on key groups such as the citrus industry, scientists working in nutrition and natural products chemistry, and the general public. As orange peel is a major by-product of orange juice processing, our work to explore its potential use in disease prevention and human health improvement remains relevant and valuable to these groups. In the past, orange peel was primarily used as cattle feed, but our findings highlight its potential to benefit the citrus industry economically by offering new applications in food systems for health improvement. This year, however, our reach has expanded. The publication of our work, along with a news releaseand presentations, has allowed us to connect with a broader audience. Following the news release of our research, we received inquiries from the general public, reflecting growing interest in the health benefits of orange peel and its compounds. Additionally, the citrus industry has shown increased interest, hoping to find pathways to further support and extend this work. We continue to engage citrus growers, processors, packers, and food ingredient companies, explaining how orange peel can improve heart health and discussing the specific compounds responsible for these effects. Feedback from stakeholders has been positive, with growers eager to understand how these findings can be applied to increase economic returns and ingredient companies excited about developing new products using orange peel byproducts. We have shared our results at scientific meetings and industry extension meetings, including a presentation at the ACS meeting in 2023, and another one in August 2024 and a presentaion at the ICBC meeting in September, 2024. These efforts ensure that our findings continue to benefit and engage a wide audience across both scientific and industry sectors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This year, we continued to provide valuable training and professional development to our postdoctoral researchers and graduate students through this project. Both postdocs from Dr. Wang's and Dr. Yokoyama's labs received ongoing support to strengthen their writing and presentation skills. Each had the opportunity to present their research at scientific meetings, enhancing their professional experience.In Dr. Wang's lab, the postdoc collaborated with thepostdoc in Dr. Yokoyama's lab on a publication that garnered significant public attention, further highlighting their research impact. Both postdocs have successfully secured positions--one in academia and the other in industry--demonstrating the effectiveness of the training provided.Additionally, the graduate student in Dr. Niu's lab continues to receive training in data analytics and processing, building expertise in data science, which is essential for their future career development. How have the results been disseminated to communities of interest?The results of this project have been disseminated to communities of interest through multiple channels. We published a paper that includes the key findings reported in the accomplishments section, specifically detailing how orange peel extracts decrease TMA and TMAO levels by inhibiting enzyme activities and identifying the effective compounds responsible for these effects. This publication received significant public attention following a news release that explained the research in layman's terms, making the findings accessible to a broader audience.In addition to the publication, we presented these results at various scientific meetings, where we engaged with researchers and industry professionals, further disseminating our findings and fostering discussions on the potential applications of orange peel in promoting heart health. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, as our project concludes in March 2025, we will focus on wrapping up our remaining studies. All the objectives and commitments outlined in the original proposal have been completed, but we still have unpublished data, specifically related to the metabolism of the effective compounds in orange peel. We plan to compile these findings and publish them in a separate paper. ?Another key step will be disseminating our final results. I will be presenting our findings at various scientific meetings and events to ensure our work reaches a wide audience. Additionally, we will ensure that all results from our students' research are communicated effectively to stakeholders, including the citrus industry, scientists, and other relevant parties. This will help maximize the impact of our project and ensure that the findings are well-integrated into future research and industry applications.
Impacts
What was accomplished under these goals?
Under our long-term goal of improving gut health through the use of orange peel to prevent cardiovascular disease and promote heart health, we made significant progress in understanding the mechanisms behind the reduction of TMA (trimethylamine) and TMAO (trimethylamine-N-oxide) levels. Our previous results demonstrated that both polar and non-polar extracts of orange peel effectively decreased TMA and TMAO levels in plasma and urine. However, 16S rRNA sequencing did not reveal significant changes in gut microbiota composition, suggesting an alternate mechanism responsible for the reduction in TMA and TMAO levels. 1. Enzyme Inhibition Model:To explore this potential mechanism, we set up a carnitine monooxygenase/reductase enzyme model to test if orange peel extracts inhibit enzyme activity, thereby reducing TMA levels. We applied carnitine alone, carnitine with orange peel polar extract, carnitine with orange peel non-polar extract, and the positive control berberine to the enzyme model. Our findings revealed that the orange peel polar extract significantly inhibited carnitine enzyme activity compared to the non-polar extract, leading to a notable reduction in TMA levels. 2. Metabolomics Profiling and Bioactive Component Identification:We applied untargeted metabolomics to profile the orange peel extracts and identified individual compounds with inhibition activity. Among the compounds tested, feruloylputrescine was identified as significantly inhibiting carnitine enzyme activity and decreasing TMA levels, even more effectively than the positive control berberine. 3. In Vivo Validation:To validate the activity of feruloylputrescine in vivo, we conducted a study in mice. We compared the effects of pure feruloylputrescine, orange peel polar extract, orange peel non-polar extract, and a control group treated with carnitine. The results confirmed that feruloylputrescine significantly decreased TMA and TMAO formation in the in vivo model, further solidifying its potential as a key bioactive compound in the prevention of atherosclerosis through gut microbiome modulation.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Lee, H., Koh, GY., Lee, H., Alves, P., Yokohama, W., Wang, Y. Discovery of a novel bioactive compound in orange peel polar fraction on the inhibition of thrimethylamine and trimethyl N-Oxide through metabolomics approaches and in vitro and in vivo assays: feruloylputrescine inhibits trimethylamine via suppressing cntA/B enzyme. J. Agric. Food Chem., 2024, 72, 14, 7870-7881
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Lee, H., Xin, L., An, J., Wang, Y. Identification of polymethoxyflavones (PMFS) from orange peel and their inhibitory effects on the formation of thrimethylamine (TMA) and thimethylamine-N-oxide (TMAO) using cntA/B and cutC/D enzymes and molecular docking. J. Agric. Food Chem. 2023, 71, 43, 16114-16124
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2023
Citation:
Wang, Y., Lee, H. The effects of polymethoxyflavones (PMFs) from orange peel on the inhibition of trimethylamine (TMA) and trimethylamine-N-oxide (TMAO) producing enzymes and TMA/TMAO production: in vitro study and docking analysis. 266th American Chemical Society National Meeting & Exposition, San Francisco, 2023
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Progress 04/01/22 to 03/31/23
Outputs
Target Audience:The target audience of this project in the past year was citrus industry, scientists working in theareas of nutrition and natural products chemistry as well as the general public. Orange peel is the major by-productgenerated during orange juice processing. In the past, the main use of orange peel is the cattle feed. If the orange peel can be used in the food system to prevent diseases and improve human health, it could largely benefit the entire citrus industry economically. Therefore, we communicated our findings with entities such as citrus growers, processers, packers and food ingredient company using citrus peel as ingredients. We explained to them the potential of using orange peel to improve heart health and indicated the potential compounds in the peel showing the health benefits as well as the mechanisms. We are preparing an extension article for the citrus industry and hope could be published by the end of the year. Meanwhile, we also presented our results at the scientific meetings to disseminate our findings such as what compounds in the peel would prevent the transformation from carnitine to TMAO.One presentation will be given at the upcoming ACS meeting in August 2023. The comments we received from the stakeholders so far were very positive. The growers would like to know more how the results could be used by the industry and increase economic returns. The ingredient companies are excited, and they would like to consider how to develop new products using the orange peel left from juice processing. Changes/Problems:Originally, we didn't plan to test more natural compoundsin the cntA/B and cutC/D models. However, the results from our planned studies were so interesting, so we hope we could test more before the project ends. In addition, the animal study at USDA-ARS were delayed previously due to the pandemic closure then lack of the facility staff. It has been gradually coming back and we hope we could complete all planned animal studies before the project ends. What opportunities for training and professional development has the project provided?The new hired postdoc by Dr. Wang's group in the area of natural products chemistry started her work in the beginning of year 2022. She used this opportunity to learn compound isolation and identification as well as molecular docking. She is also trained to use machine learn models to predict bioactivity based on chemical structures. In addition, she learnt how to write scientific publications and how to give scientific presentations. The postdoc. hired by Dr. Yokoyama's group focused on the training of developing animal models and bioinformatics. She also has been trained for grant writing. We hope the training provided by this project could well prepared the trainee for the future academic jobs. In addition, Dr. Niu enrolled a graduate student who has been trained in the data science and data processing. How have the results been disseminated to communities of interest? We submitted one publication based on our cntA/B and cutC/D results. Based on all our current data, we are preparing another two publications.One focused on our animal study which provide a novel perspective on how orange peel inhibit TMAO and TMAO formation other than just altering the composition of gut microbiota. The one focus on the relationship of chemical structures and inhibition efficacy. These scientific publications will possess a large number of readers from academia and industry. We will organize our findings into an extension article which much more focuses on stakeholders. Additionally, some of the results have been and will be presented at the educational workshop and scientific conferences. What do you plan to do during the next reporting period to accomplish the goals?1. Complete the paper writing for the animal study which focus on the mechanisms of how orange peel inhibit TMAO and TMA formation. 2. Apply more natural compounds onto cntA/B and cutC/D models to have a data repositoryon structural-activity relationship particularly to complete the machine learning prediction models to predict inhibition efficacy of TMAO and TMA formation in the future just based on chemical structures. 3. Complete the testing such as TMAO, TMA, carnitine levels etc. in the biosamples from theApoe−/−mice study.
Impacts
What was accomplished under these goals?
As known, there is a lot of fibers in the orange peel which can alter the gut microbiota composition significantly then leading to the decrease of TMA and TMAO. However, in our study, we focused on more other polyphenol compounds and their function other than just changing the gut microbiota composition. In our study, we have five groups of mice: 1) treated with standard chow; 2) treated with standard chow plus carnitine; 3) treated with standard chow+ carnitine +orange peel polar fraction 4) treated with standard chow+ carnitine +orange peel non-polar fraction 5) treated with standard chow+ carnitine +antibiotics cocktail. As a result, we didn't observed a significant gut microbiota composition change between groups treated with polar or non-polar orange peel extracts and group just treated with carnitine. However, we did observe TMAO and TMA levels decreased within groups of treated with orange peel extracts indicating other mechanisms were presented. In fact, cnt A/M enzyme activity has been significantly inhibited by orange peel extracts indicating inhibiting enzyme activity is a potential way to decrease TMAO level. We completed the study of testing 12 PMFs to inhibit cntA/B, cutC/D and FMO3 activities. We used molecular docking to analyze the relationship between chemical structures and inhibition efficacy indicating the key structural characteristics corresponding to each individual enzyme. The work has been submitted to Journal of Agriculture and Food Chemistry and it is under review now. Apoe−/−mice studies were conducted. Four treatments were performed: 1) treated with standard chow; 2) treated with standard chow plus carnitine; 3) treated with standard chow+ carnitine +low orange peel extract 4) treated with standard chow+ carnitine +high orange peel extract. So far, the body weight, food and water intake were monitored and didn't see significant changes among treatments. The further analyses will be done in the following months.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Koh, G.Y., and Wang, Y. (2022). Implications of type 1 and type 2 taste receptors on obesity-induced inflammation. J. Food Bioact. 20:2-10
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Lee, H., Liu, X., An, J., Wang, Y. Identification of polymethoxyflavones (PMFs) from orange peel and their inhibitory effects on the formation of trimethylamine (TMA) and trimethylamine-N-oxide(TMAO) using cntA/B and cutC/D enzymes and molecular docking. Submitted to Journal of Agricultural and Food Chemistry in July 2023 and now it is under review.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2023
Citation:
Wang, Y., Lee, H., et al. In vitro study of polymethoxyflavones (PMFs) from orange peel for their potential to inhibit trimethylamine (TMA) and trimethylamine-N-oxide (TMAO)-producing enzymes and reduce TMA/TMAO production. Division of Agricultural and Food Chemistry, Sustainable Agriceuticals, ACS Fall 2023.
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Progress 04/01/21 to 03/31/22
Outputs
Target Audience:The target audience of this project in the past year wascitrus industry andthe general public. The citrus industry is actually a big audience group containing various entities such as citrus growers, processers, packers and food ingredient company using citrus peel as ingredients.We have presented the results and findings from this work at the citrus extension workshop. We talked with these stakeholders. We explained to them the potential of using orange peel to improve heart health and indicated the potential compounds in the peel showing the health benefits. The comments we received from the stakeholders were very positive. The growers would like to know more if the similar compounds can be identified in the fruits. The processers are excited in addition to juice products, there might be a potential to use the juice processing by-products (orange peel).All entities expected to see more results from this study. Changes/Problems: The co-PI Dr. Wallace Yokoyama who mainly is in charge of the animal study locates in USDA-ARS California. In the past year, the entire ARS animal facility in California could not be fully open. We start to resume the animal study in May 2022. The animal study was hugely delayed.Therefore, it could take longer than we plannedto complete the entire study, and we may need another year extension. What opportunities for training and professional development has the project provided?After the first postdoc. left for an academic job, we hired another postdoc. in the beginning ofyear 2022 to continue this project. We use this position to train postdoc. in the areas of biochemistry and analytical chemistry as well as we train people to write scientific publications and presentation skills.We hope the training provided by this project could well prepared the trainee for the future academic jobs.In addition, the Co-PI enrolled a graduate student who has been trained in the data science and data processing. How have the results been disseminated to communities of interest? Based on the data we have already, we are preparing two publications.One focuses on the animal study and bacterial enzyme study, and the other one focuses on compound identification and the study of structure-activity. These scientific publications will possess a large number of readers from academia and industry. Additionally, some of the results have been presented at the educational workshop. We expect to give more presentations at the scientific meetings for disseminating information after our papers are published. What do you plan to do during the next reporting period to accomplish the goals? Start the study of usingApoe−/−miceto test the orange peel extract and effective compounds from the orange peel. Test the purified compounds from the bioactivity-guided fractionation of orange peel inC57BL/6mice as well as test these purified compounds using liver enzyme FMO3 which can transform TMA into TMAO.In this way, we could understand the mechanisms of inhibiting TMAO formation using these compounds from orange peel. Study the structure-activity relationship using molecular docking
Impacts
What was accomplished under these goals?
Here are the accomplishments we have acchived in the past year In addition to develop a carnitine enzyme (CntA/B) as we proposed in proposal, we developed a choline enzyme(Cut C/D) as well. Because during our study, we realizedthat some bioactive compounds may work differently on these two enzymes, even though they can both transform diet ingredients into TMA. We used these two enzymes to conduct bioactivity-guided compound isolation and identification in the orange peel extract. The inhibition efficacy of each individual compound has been tested and compared with positive control. The structure-activity are under studied. In total of 12 compounds have been identified from orange peel, which may inhibit the transformation from carnitine/choline to TMA.
Publications
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Progress 04/01/20 to 03/31/21
Outputs
Target Audience: Target audience of this project is citrus industry and the general public. Florida is the largest citrus producer in the U.S., with nearly 95% of Florida oranges used for juice. The current juice recovery rate is approximately fifty percent with the remaining peel byproduct typically going to cattle feed. Expanding the use of orange peel to benefit human health is a solid way to promote sustainability of US agriculture. Plus, this could potentially provide greater economic returns to the citrusindustry. These scientific findings will be published in top journals such as Molecular Nutrition & Food Research, which possess a large number of readers from academia and industry. Additionally, these findings will be presented at scientific meetings for educating both industry and the public. In the past year, we have reached out citrus growers and people working in the citrus industry and provided information of how and why citrus peel could be used to adjust gut microbiota and improve heart health.An educational presentation was given at a citrus workshop.The feedback we received was very positive and many growers would like to contribute their fruits for the research study.? Changes/Problems:It was difficult for us to run animal studies during the pandemic. The co-PI Dr. Wallace Yokoyama who mainly is in charge of the animal study locates in USDA-ARS California. In the past year, the entire ARS animal facility in California could not be open. We still can't use the animal facility till maybe the end of April. Therefore, we conducted some animal studies at University of Florida where the PI Dr. Wang locates.Due to the issues above, the progress of both animal and analytical studies was delayed.In addition, the postdoc who we hired for conducting both the molecular biology and chemistry work has got a faculty job and we need to hire someone new to continue this work. Therefore, it could take a while to resume this project. What opportunities for training and professional development has the project provided?A postdoctoral associate was hired for this project.This project needs analytical and molecular biology techniques. It is a good chance for the postdoc. trainee to learn both. In addition, training for the presentation and writing skills were provided as well. This postdoc. submitted one abstract to a nutrition meeting but due to COVID-19, she didn't have a chance to present.However, she gave an educational presentation to the citrus industry including growers, processers and academic scientists working in citrus field. This postdoc. was hired in August, 2019, and via one and half year training, she successfully obtained a faculty position in a university in Texas.? How have the results been disseminated to communities of interest?These scientific findings will be published in top journals such as Molecular Nutrition & Food Research, which possess a large number of readers from academia and industry. Till now, we are still in the process of collectingdata and a publication can be expected in the near future.Additionally, some of the results have been presented at the educational workshop.We expect to give more presentations at the scientific meetings for disseminating information.? What do you plan to do during the next reporting period to accomplish the goals?We plan to repeat part of the short-term studies because we found some results were not consistent and need to be confirmed by more data. If the animal facility could fully open, we plan to run the long-term studies.
Impacts
What was accomplished under these goals?
A 6-week short term animal study with both genders has beencompleted to test how orange peel extracts ( polar and non-polar extracts) would affect gut microbiota composition and TMAO formation.Both TMA-lyase models (Cnt A/B and CUT C/D) were developed and purified. Many flavonoid compounds from orange peel were tested using this enzyme model.Polar and non-polar peel extracts were applied to this essay as well and will continue for the compounds isolation and purification.Levels of TMA, TMAO from both animal and enzyme models were tested using LC-MS/MS. Fecal samples has been extracted for the 16S rRNA. In addition, metabolites of orange peel in serum and urine were analyzed for additional information.
Publications
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Progress 04/01/19 to 03/31/20
Outputs
Target Audience:Target audience of this project is citrus industry and the general public.Florida is the largest citrus producer in the U.S., with nearly 95% of Florida oranges used for juice. The current juice recovery rate is approximately fifty percent with the remaining peel byproduct typically going to cattle feed. Expanding the use of orange peel to benefit human health is a solid way to promote sustainability of US agriculture. Plus, this could potentially provide greater economic returns to the citrus industry.These scientific findings will be published in top journals such as Molecular Nutrition & Food Research, which possess a large number of readers from academia and industry. Additionally, these findings will be presented at scientific meetings for educating both industry and the public. The previous year was the first year to conduct this porject. We haven't reached a solid result/conslusion to report to the industry and the general public. Changes/Problems:1.3% carnitine in the diet might be an issue for us to get a consistent result. Therefore we deducted it to 0.6%. What opportunities for training and professional development has the project provided?We hired a postdoctoral associate in August 2019. This projectneeds analytical and molecular biology techniques. It is a good chance for the postdoc. trainee to learn both. This postdoc. submitted one abstract to a nutrition meeting but due to COVID-19, she didn't have a chance to present. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We are conducting a longer-term animal study to understand effects of orange peel on improving gut health.In addition, we are using both metabolomics and bioactivity-guided isolation techniques to understand the effective compounds in orange peel.
Impacts
What was accomplished under these goals?
A short-term treatment study was completed to test how orange peel affect TMAO formaton via microbiota. Six-week-old male C57BL/6J mice was used to examine the capability of gut microbiota to generate TMA and TMAO following carnitine intake, as well as the inhibitory efficacy of orange peel compared with the positive control, resveratrol and antibiotic cocktail.
Publications
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