Progress 01/01/15 to 12/31/18
Outputs
Target Audience:Target audiences include scientific colleagues, through conference talks and university seminars, undergraduate students through lectures in courses and mentoring in research projects and younger students through outreach efforts. The results were presented in the 253th American Chemical Society National meeting & Exposition, April 2-6, San Francisco, CA, USA (Oral Presentation). Vegetable and Fruit Improvement Center provide several presentations to undergraduate students and high school students. During this project, several high school students were able watch the research activities related to this project during their visit. A science podcast for children, called Brains On, will feature a presentation on the circadian clock of plants and potential impact on human health, as a result of an interview and recording with Dr. Braam. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral researcher and one graduate student have been responsible for the work done in the Braam lab, with support from an undergraduate student. The postdoc also gained teaching and mentoring experience through bringing plant biology experimental projects to an introductory undergraduate biology lab course. The graduate student participated in the annual plant biology conference through a poster presentation. These researchers collaborated closely with two senior personnel and one postdoc from the Dashwood lab, who were involved in using the Pirc colon cancer model for testing of entrained spinach extracts. Together these personnel gained valuable experience in carrying out in vivo tumor studies using the rat model, and tracking tumor suppression by endoscopy. A postdoctoral research associate and an undergraduate student were responsible for quantifying and identifying the phytochemicals in spinach. The undergraduate was involved in sample preparation for the analysis of carotenoids, flavonoids total phenolics, and antioxidant activity. The postdoctoral researcher developed extraction methods for various phytochemicals using different solvents and rapid analytical techniques. How have the results been disseminated to communities of interest?Work was presented by Patil at the 253th American Chemical Society National meeting & Exposition, San Francisco, CA, USA, as an oral presentation, and Patil gave a keynote presentation in Brazil Vegetable Congress and partial results of this project were highlighted. Braam disseminated new knowledge to the scientific community through publications, including reporting on how the circadian clock affects postharvest crop longevity, tissue integrity and phytochemical maintenance, in addition to seminar presentations at international conferences (Alberta Institute of Agrologists, "Health and Food: Is There a Future?" Banff, Alberta, Canada; Gordon Research Conference, "Chronobiology", Girona, Spain) and University seminars (Washington University, St. Louis, MO, University of Virginia, Texas A&M, College Station, TX). In addition, Dr. Braam has been involved in high school student outreach, speaking to local high school biology classes. Braam also contributed to an educational podcast, Brains On, and student-led poster presentations at the annual plant biology conference. The research of Braam and Dashwood was also highlighted as the cover article in a recent Texas Medical Center publication. A novel endoscopy procedure in Pirc rats has been published by F Eterm et al., in GI Endoscopy. Dr. Patil is involved in diverse outreach activities that include students, producers, and retailers. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
A long-term goal of the project was to improve the nutritional quality of plant-based diets, by increasing awareness and exploitation of daily variation of beneficial phytochemicals and metabolites in plants. In addition, the project sought to identify novel metabolites that impact, human health. The knowledge gained will be of potential importance to everyone, but in particularly at-risk populations and people with health disparities, where maximum health benefits from plant-diets are sorely needed. Our research aimed to determine whether the health benefits of crop plants may differ depending upon the time of day that the plant material is ingested and/or preserved. We know that plant physiology and biochemistry differs dependent upon time of day, through the control of the circadian clock. Previous results demonstrated, for example, that defense hormones and some phytochemicals vary in levels depending upon the time of day the plants are harvested. If human health bio-actives in plants also differ by time of day, then there may be health benefits from harvesting, storing, and/or ingesting plant materials at specific times of day. We sought to test whether health impacting bio-actives vary by time of day in typical plant crops, including spinach and broccoli. We use a caterpillar-based bioassay to verify differential phytochemical accumulation in plant materials and seek to test whether the differential accumulation is significant enough to affect mammalian health using a rat model. In addition, we sought to identify diverse phytochemicals that may be under time-of-day regulation. Because of the challenge in obtaining large enough quantities of store-bought, circadian-entrained, spinach, we successfully identified an alternative crop to add to our analyses. Circadian entrained broccoli greens were successfully grown in large quantity.We redesigned animal studies, using oral gavage, to reduce the amount of plant material needed. To verify circadian entrainment of the plants, we used gene-specific probes for circadian clock genes and showedcircadian entrainment of the broccoli tissues. Wesuccessfully verified that the basal circadian variation in phytochemicals in freeze-dried plant materials can be profound enough to causedifferential organismal effects. We found that loopers prefer to feed onplant material harvested at specific times of day, indicating that the basal crop composition differs in a time-of-day dependent manner. Given the periodicity of the time-dependent bio-effects of the broccoli greens when fed to loopers, we postulated that the same biomaterials could exhibit time-dependent efficacy in animals. We selected the polyposis in rat colon (Pirc) model of human colorectal cancer mouse as a mammalian model to test time-dependent health effects of the plant material. The Pirc mouse harbors a significant colon tumor burden due to amutation in the murine adenomatous polyposis coligene, closely modeling familial adenomatous polyposisin humans (PNAS 2007;104:4036). In an acute dosing protocol lasting 3 consecutive days, the diurnally entrained broccoli samples were administered orally to 10-month-old Pirc females (freeze-dried broccoli at 250 mg/rat; 1 g/kg body wt.). Using a novel murine colonoscopy-polypectomy procedure (Gastrointest Endosc 2016;83: 1272), colon polyps were resected 6 h after the last dose of entrained broccoli, and at 24 h during necropsy, and colon tissues were processed for immunoblotting. At the early 6 h time point, entrained broccoli inhibited the expression of oncogenic factors associated with colon cancer, including b-catenin, cell cycle and apoptosis regulator 2, and histone deacetylases1, 2 and 3. Loss of protein expression in colon polyps coincided with the time point at which loopers fed least effectively on the same broccoli samples. However, in colon polyps removed at 24 h, loss of expression of oncogenic factors was greatest for the 0- and 20-hr entrained broccoli samples. This may reflect the pharmacokinetics of different phytochemicals/ bioactives in the broccoli samples. Importantly, the ability to downregulate oncogenic factors in colon polyps at both time points (6 and 24 post- gavage) is encouraging, and supportive of likely cancer preventive outcomes with long term feeding. The proof of concept experiments were repeated with additional rats to verify the reproducibility of initial results. Related transcriptional targetswere corroborated via RT-qPCR , including c-Myc and MMP7 for b- catenin/CCAR2-dependent signaling, and p21 and p16 for HDAC inhibition/loss in colon tissues after treatment with entrained broccoli. As a complementary approach, plant samples collected from different time points were investigated for levels of flavonoids and carotenoids. Identification of compounds was achieved by liquid chromatography-high resolution mass spectrometry. Results demonstrated that two major flavonoids varied incontent by time of day. Carotenoids such as lutein, β-carotene, chlorophyll a and b also showedvariation. Spinach glucuronides, especially 5,3',4'-trihydroxy-3-methoxy-6:7-methylen- dioxyflavone-4'-β-D-glucuronide, showhighest in vitro antioxidant and promising anti-allergic activities. Spinach was extracted with different solvents, analyzed with ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry . UHPLC-HR-QTOF-MS facilitated the identification and quantification of five unknown flavonoid spinacetin derivatives. In addition, five more unidentified glucuronide derivatives were also identified from and 5,3',4'-trihydroxy-3-methoxy-6:7-methylen-dioxyflavone-4'-β-D-(2′-O-feurloyl-glucuronide) was found to be prominent derivative among those identified compounds. This is the first report on identification and quantification of new spinacetin and glucuronide flavonoids derivatives in spinach. We purified 5,3',4'-trihydroxy-3-methoxy-6:7-methylen-dioxyflavone-4'-β-D-glucuronide by semi-preparative HPLC and elucidated the structure by 1H and 13C nuclear magnetic resonance spectroscopy. To understand the accumulation of glucosinolates implicated in human health impact in post-harvest plants, a rapid method for the quantitation of desulfoglucosinolates without using column chromatography was also developed. The methodology successfully identifieddesulfoglucosinolates in green kohlrabi and green cabbage. The rapid method showed high recovery of certain GLS and reproducibility because of its minimal unit operations as compared with the chromatographic technique. The use of HR-ESI-QTOF-MS provides a more reliable approach to identify desulfoGLS. The developed methodology is simple and can be easily applied to investigate glucosinolates in other Brassicaceae species. Thisstrong team of plant physiologists, natural product chemists, plant biochemists, cancer cell biologists, and epigenetic experts has contributed to our understanding of how diurnal changes in plant-based diets may enhance exploitation of beneficial phytochemicals and plant metabolites. The novel phytochemicals and plant metabolites identified might impacthuman health outcomes. Future work is needed to develop best practices in plant growth, post-harvest treatment, and storage with a view to capitalizing on phytochemical circadian rhythms for human health benefits, including via epigenetic mechanisms.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
1. Singh J., Jayaprakasha, G. K, and Patil, B. S (2018). An optimized solvent extraction and characterization of unidenti?ed ?avonoid glucuronide derivatives from spinach by UHPLC-HR-QTOF-MS. Talanta, 188, 763-771.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
2. Singh J., Jayaprakasha, G. K, and Patil, B. S (2017). Rapid and efficient desulfonation method for the analysis of glucosinolates by high-resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. J. Agricultural and Food Chemistry, 65 (50), 1110011108.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
3. Singh J., Jayaprakasha, G. K, and Patil, B. S (2017). Rapid ultra?high?performance liquid chromatography/quadrupole time?of?flight tandem mass spectrometry and selected reaction monitoring strategy for the identification and quantification of minor spinacetin derivatives in spinach. Rapid Communication of Mass spectrometry, 31, 18031812.
- Type:
Book Chapters
Status:
Published
Year Published:
2018
Citation:
1. Singh J., Jayaprakasha, G. K, and Patil, B. S. Extraction, identification, and potential health benefits of spinach flavonoids: a review. In Advances in Plant Phenolics: From Chemistry to Human Health, American Chemical Society, Oxford University Press, 2018, 1286, 1-437 (https://pubs.acs.org/isbn/9780841232969).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
1. Singh, J. G.K. Jayaprakasha and Bhimanagouda S. Patil (2017). Combination of green solvents using accelerated extraction of spinach and LC-HR-ESI-QTOF-MS led five new flavonoids and yielded higher antioxidants. 253th American Chemical Society National meeting & Exposition, April 2-6, San Francisco, CA,
|
Progress 01/01/17 to 12/31/17
Outputs
Target Audience:Target audiences include scientific colleagues, through conference talks and university seminars, undergraduate students through lectures in courses and mentoring in research projects and younger students through outreach efforts. The results were presented in the 253th American Chemical Society National meeting & Exposition, April 2-6, San Francisco, CA, USA (Oral Presentation). Vegetable and Fruit Improvement Center provide several presentations to undergraduate students and high school students. During this project, several high school students were able watch the research activities related to this project during their visit. A science podcast for children, called Brains On, will feature a presentation on the circadian clock of plants and potential impact on human health, as a result of a interview and recording with Dr. Braam. Changes/Problems:A relatively minor change was a switch to using Broccoli greens as a major source of experimental material because of the availability of large quantities, responsiveness to diurnal growth conditions, and tissue robustness required for the manipulations. In addition, we introduced the rat Pirc model for testing potential carcinogenesis effects because of the potential high value relevance of the results relative to our overall goal of determining whether time-of-day dependent changes in plant phytochemicals may affect human health. What opportunities for training and professional development has the project provided?One postdoctoral researcher and one graduate student have been responsible for the work done in the Braam lab, with support from an undergraduate student. These researchers collaborated closely with two senior personnel and one postdoc from the Dashwood lab. The graduate student in the Braam lab was able to work directly with the Dashwood team members, learning techniques for endoscopy in rat models of colon cancer, and evaluating target tissues for molecular endpoints pertinent to colorectal cancer etiology. In addition, one postdoctoral researcher worked in the Patil's lab along with undergraduate student. How have the results been disseminated to communities of interest?Work was presented by Patil at the 253th American Chemical Society National meeting & Exposition, April 2-6, San Francisco, CA, USA, as an oral presentation. In addition, Braam contributed to a educational podcast, Brains On. What do you plan to do during the next reporting period to accomplish the goals?We will follow up the initial rat Pirc model results with additional trials to verify a differential effect on carcinogenesis as a result of time-of-day harvested plant materials. These experiments have proven fruitful in terms of showing altered oncogenic targets in rat colon polyps after single acute dosing. However, they require follow-up studies of efficacy, i.e., suppression of colon tumor development, using a longer duration of treatment with entrained broccoli (possibly given in the diet). In addition, we will seek to identify the relevant phytochemicals whose levels correlate with differential health benefits.
Impacts
What was accomplished under these goals?
Impact Our research is targeted at determining whether the health benefits of crop plants may differ depending upon the time of day the plant material is ingested and/or preserved. We know that plant physiology and biochemistry differs dependent upon time of day, through the control of the circadian clock. Previous results demonstrated, for example, that major defense hormones and phytochemical vary in levels depending upon the time of day the plants are harvested. If human health bio-actives in plants also differ by time of day, then there may be health benefits from harvesting, storing, and/or ingesting plant materials at specific times of day. We seek to test whether health impacting bioactives vary by time of day in typical plant crops, including spinach and broccoli. We use a caterpillar-based bioassay to verify differential phytochemical accumulation in plant materials and seek to test whether the differential accumulation is significant enough to affect mammalian health using a rat cancer model. In addition, we seek to identify diverse phytochemicals that may be under time-of-day regulation. Because of the challenge in obtaining large enough quantities of store-bought, circadian-entrained, spinach designed to test whether there are time-dependent differences in impact on carcinogenesis rates in cancer-model rats, we have successfully identified an alternative crop to add to our analyses. Circadian entrained broccoli greens have now been successfully grown in large quantity in the lab. In addition, we have re-designed planned animal studies, using gavage, to greatly reduce the amount of plant material needed. To verify circadian entrainment of the plant materials, we have generated gene-specific probes for known circadian clock genes in broccoli; with these probes, clear circadian entrainment of the plant tissues has been verified. In addition, before beginning the expensive animal studies, we successfully verified that the basal circadian variation in phytochemicals in freeze-dried plant materials can be profound enough to demonstrate differential organismal effects. We prepared freeze-dried samples of broccoli greens, harvested at 4 different time points of the diurnal cycle, prepared cabbage looper (T. ni) food, and assessed looper growth. We found that looper growth varied depending upon the diurnal phase of the plant material included in their food supply, indicating that the basal crop composition differs in a time-of-day dependent manner. These data provided proof-of-concept for pilot studies in the polyposis in rat colon (Pirc) model, see below. Given the periodicity of the time-dependent bio-effects of the broccoli greens when fed to loopers, we postulated that the same biomaterials may exhibit time-dependent efficacy in vivo. We selected the Pirc mouse as a mammalian model to test time-dependent health effects of the plant material. The Pirc mouse harbors a significant colon tumor burden due to an inherited mutation in the murine adenomatous polyposis coli (Apc) gene, closely modeling familial adenomatous polyposis (FAP) in humans (Amos-Landgraf et al., PNAS 2007;104:4036-41). In an acute dosing protocol lasting 3 consecutive days, the diurnally entrained broccoli samples were administered orally to 10-month-old Pirc females (freeze-dried broccoli was gavaged at 250 mg/animal; 1 g/kg body wt.). Using a murine colonoscopy-polypectomy procedure (Ertem et al., Gastrointest Endosc 2016;83: 1272-6), colon polyps were resected 6 h after the last dose of entrained broccoli, and at 24 h during necropsy, and colon tissues were processed for immunoblotting. At the early 6 h time point, entrained broccoli inhibited the expression of oncogenic factors associated with colon cancer, including b-catenin, cell cycle and apoptosis regulator 2 (CCAR2), and histone deacetylases (HDACs) 1, 2 and 3. The loss of protein expression in colon polyps coincided with the timepoint at which loopers fed least effectively on the same broccoli samples. However, in colon polyps removed at 24 h, loss of expression of oncogenic factors was greatest for the 0- and 20-hr entrained broccoli samples. This might reflect the pharmacokinetics of different phytochemicals/ bioactives in the entrained broccoli samples. Importantly, the ability to downregulate oncogenic factors in colon polyps at both timepoints (6 and 24 post-gavage) is encouraging, and supportive of likely cancer preventive outcomes with longterm feeding. These proof of concept experiments will be repeated with additional rats to verify the reproducibility of our initial results. Related transcriptional targets (i.e., mRNA levels) will be studied with qRT-PCR, including c-Myc and MMP7 for b-catenin/CCAR2, and p21 and p16 for HDAC inhibition/loss in colon tissues. In addition, we have continued to investigate the identities of antioxidant, anti-inflammatory, antiobesity, antiproliferative and lipid lowering bioactives from spinach. Spinach glucuronides derivatives, especially 5,3',4'-trihydroxy-3-methoxy-6:7-methylen-dioxyflavone-4'-β-D-glucuronide showed highest in vitro antioxidant and promising antiallegic activities. Thus, we sought to identify the minor compounds present in spinach using various solvents and ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry. The UHPLC-HR-QTOF-MS facilitated the identification and quantification of five unknown spinacetin flavonoid derivatives with spinacetin?3?O?β?D?(2?ρ?coumaroyl glucopyranosyl?(1→6)?[β?D?apiofuranosyl?(1→2)]?β?Dglucopyranoside was the predominant derivative (14.04 ± 0.30 μg/g FW), followed by spinacetin?3?O?β?D?(2?coumaroyl glucopyranosyl)?(1→6)?β?D?glucopyranoside (4.44 ± 0.20 μg/g FW). Spinacetin?3?O?β?D?(glucopyranosyl)?(1→6)?[β?glucopranosyl?(1→2)]?β?D?glucopyranoside and spinacetin?3?O?β?D?(2?feruloyl glucopyranosyl)?(1→6)?[β?glucopranosyl?(1→2)]?β?Dglucopyran-oside were present at 0.64 ± 0.10 μg/g FW and 1.86 ± 0.30 μg/g FW, respectively. Quantification of identified flavonoids was achieved by spinach glucuronide derivatives that were isolated and purified by semi-preparative HPLC. The structure of the isolated compounds was confirmed by nuclear magnetic resonance spectra. This is the first report on identification and quantification of five new spinacetin flavonoids derivatives in spinach. Whether these bioactives vary quantitatively by time of day remains to be determined.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
1) Rapid ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry and selected reaction monitoring strategy for the identification and quantification of minor spinacetin derivatives in spinach. Rapid Communications in Mass Spectrometry, 31:18031812 (2017).
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
2) A rapid and efficient desulfonation method for the analysis of glucosinolates by high resolution liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Journal of Agricultural and Food Chemistry, DOI: 10.1021/acs.jafc.7b04662 (2017).
|
Progress 01/01/16 to 12/31/16
Outputs
Target Audience:Target audiences include scientific colleagues, through conference talks and university seminars, undergraduate students through lectures in courses and mentoring in research projects and high school students through outreach efforts. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A postdoctoral researcher has been largely responsible for the work done in the Braam lab, with support from an undergraduate student. One more postdoctoral researcher worked in the Patil's lab along with undergraduate student. Senior staff have been supported in the Dashwood lab. How have the results been disseminated to communities of interest?During the past year, Braam has given one research seminar (University of Virginia) where this research was highlighted. The research of Braam and Dashwood was also highlighted as the cover article in a recent Texas Medical Center publication. Patil has given akeynote presentation in Brazil Vegetable Congress and partial results of this project were highlighted. What do you plan to do during the next reporting period to accomplish the goals?During the next year, the Braam lab will conduct a higher resolution time course with both spinach and broccoli sprouts to identify the peak and troughs in bioactivity toward cabbage loopers. These peak time points will then be chosen for large scale preparative work to collect sufficient tissues for the animal carcinogenesis studies to be conducted in the Dashwood lab. Patil's lab will analyze the variation in the phytochemical levels like flavonoids, carotenoids, glucosinolates and vitamins in the spinach and broccoli sprouts, as materials are available. Antioxidant activities will be tested using in vitro assays.
Impacts
What was accomplished under these goals?
The long-term goals of the proposed project include improving the nutritional quality of plant-based diets by increasing awareness and exploitation of daily variation of beneficial phytochemicals and metabolites in plants and developing best practices in plant growth and post-harvest treatment and storage that capitalize on these rhythms for human health benefits. In addition, this work aims to identify novel phytochemicals and metabolites that have impact, either independently or in concert, on human health. Finally, this proposed project aims to provide fundamental knowledge about the potentially powerful impact of input stimuli on plant circadian clock function and output, which can influence the nutrient contents of the foods upon which we rely for nutrition and health. A challenge was encountered as we attempted to entrain a large enough quantity of store-bought, circadian-entrained, baby spinach for the animal studies designed to test whether there are time-dependent difference in carcinogenesis rates as a result of spinach-supplemented diets generated from differentially clock-phased spinach. Three strategies have been employed to overcome this challenge. One is to use field-grown spinach, which is naturally circadian entrained, but availability is seasonally restricted. Second is to use an alternative crop; we chose broccoli sprouts because broccoli contains glucosinolates and sprouts can be grown and circadian entrained in quantity in the lab. Our third strategy has been to re-design the animal studies in ways that greatly reduce the amount of plant material needed. To verify circadian entrainment of the plant materials, we have generated gene-specific probes for known circadian clock genes in both spinach and broccoli; with these probes, clear circadian entrainment of the plant tissues has been verified. Before beginning the expensive animal studies, we sought to test whether the basal circadian variation in phytochemicals can be profound enough to demonstrate differential effects. For this, we prepared samples of both spinach and broccoli, harvested at 4 different time points of the circadian cycle, prepared cabbage looper (T. ni) food, and assessed whether looper growth varied depending upon the circadian phase of the plant material included in their food supply. For both the spinach- and broccoli sprout-supplemented food, a significant difference in looper size was obtained, indicating that the basal crop composition differs in a time-of-day dependent manner. These data are proof of concept for the planned animal carcinogenesis studies. Field grown spinach samples collected at different time points were investigated for the levels of flavonoids and carotenoids by HPLC and compounds were confirmed by liquid chromatography-high resolution mass spectra. Results demonstrate that 5,3',4'-trihydroxy-3-methoxy-6:7-methylen-dioxy-flavone-4"- β-D- glucuronide was found to be a major flavonoid. At 7 AM, 552.24 µg/g was observed and reduce to 443.04 µg/g at 7AM than again increased to maximum level of 568.30 µg/g at 19 PM. Similarly another flavonoid 5,4'-dihydroxy-3,3'-dimethoxy-6:7-methylendioxy flavone-4'-β-D-glucuronide, had maximum value 148.26µg/g at 19 PM. Carotenoids such as lutein, chlorophyll a, chlorophyll b and β-carotene also show variation in levels. Results demonstrate that sample collected at 19 PM have higher levels of lutein (86.64 µg/g) and chlorophyll (612.25 µg/g) respectively. Similarly β-carotene showed the lowest value 38.07µg/g at 7AM and maximum value 50.7µg/g at 19 PM.
Publications
|
Progress 01/01/15 to 12/31/15
Outputs
Target Audience:Target audiences include scientific colleagues, through conference talks and university seminars, undergraduate students through lectures in courses and mentoring in research projects and high school students through outreach efforts. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral researcher has been involved in the Braam lab. This researcher is new to plant biology, having been trained as a mouse developmental biologist. In addition to gaining hands on experience in plant biology, she has also gained teaching and mentoring experience through bringing plant biology experimental projects to an introductory undergraduate biology lab course. A postdoctoral research associate and an undergraduate student are quantifying and identifying the phytochemicals in spinach. The undergraduate is involved in sample preparation for the analysis of carotenoids, flavonoids total phenolics, and antioxidant activity. The psstdoctoral researcher involved is developing extraction methods for various phytochemicals using different solvents and rapid analytical techniques. Finally, one doctoral student has been involved in the Pirc colon cancer model for testing of spinach diets. She has gained valuable experience in carrying out in vivo tumor studies using this model, and tracking tumor suppression by endoscopy. How have the results been disseminated to communities of interest?The work has been disseminated to the scientific community through one publication reporting on how the circadian clock affects postharvest crop longevity, tissue integrity and phytochemical maintenance has been published, in addition to seminar presentations at international conferences (Alberta Institute of Agrologists, "Health and Food: Is There a Future?" Banff, Alberta, Canada; Gordon Research Conference, "Chronobiology", Girona, Spain) and University seminars (Washington University, St. Louis, MO, and Texas A&M, College Station, TX). In addition, Dr. Braam has been involved in high school student outreach, speaking to local high school biology classes. A novel endoscopy procedure in Pirc rats has been published by F Eterm et al., in GI Endoscopy. Dr. Patil is involved in diverse outreach activities that include students, producers, and retailers. What do you plan to do during the next reporting period to accomplish the goals?Our goals are to determine the optimal conditions to entrain postharvest spinach and cabbage leaves, both for phytochemical analyses and for carcinogenesis whole-animal assays and cell culture/enzyme assays. We will test the hypothesis that time-of-day phase of the vegetable tissue will have a significant differential effect on the health properties of the plant materials. Based on our observations from the spinach study (Fig 1), we will test different circadian entrained spinach (high vs low phytochemical accumulation) in the Pirc model and compare anti-tumor efficacy, level of Phase II detoxifying enzymes and HDAC inhibition in the colon tumor vs normal. In addition, we will identify the phytochemicals that demonstrate time-of-day differential accumulation.
Impacts
What was accomplished under these goals?
We characterized the benefits on both tissue longevity and phytochemical accumulation and maintenance by diurnal (light/dark) storage conditions on a variety of green leafy vegetables. This work was published in BMC Plant Biology. Ongoing efforts are underway to optimize circadian entrainment of postharvest spinach leaves using light dark cycles. Because the tissue degrades rapidly at room temperature, we are optimizing cooler temperatures with variant durations of light/dark cycles to entrain the clock. Concurrently, flavonoid and carotenoid accumulation and characterization is underway to begin to determine examples of phytochemicals that may be under the control of the circadian clock. Finally, we are developing a rodent colon cancer model system with which we plan to directly test whether there are time-dependent differences in carcinogenesis rates as a result of spinach-supplemented diets generated from differentially clock-phased spinach leaves. In vivo studies tested the anticancer activity of 10% spinach containing diet in the polyposis in rat colon (Pirc) colon cancer model. Male Pirc rats (n = 10) were administered controlAIN93 or AIN93-spinach diets, starting from one month of age, for a period of 6.5 months. The 10% spinach fed rats had a significant reduction in tumor multiplicity and overall tumor burden (56% reduction) as compared to rats fed standard AIN93 diet. Also, there was no adverse toxicity, body weight loss, or changes in food consumption. Dose-response parameters are being investigated to optimize the experimental protocol. In Pirc colon tumors, HDAC3 protein was strongly overexpressed, and was inhibited/turned over by single dose injections of dietary factors. This make the Pirc model excellent for testing tumor suppression by entrained spinach cultivars, with possible effects on HDAC3 protein expression or turnover. Finally, we are developing cell-based assays for detection of differential bioactive phytochemical content in clock-entrained time-of-day phase-dependent spinach including colon cancer cells and normal colonic epithelial cells.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Liu, J, Goodspeed, D, Sheng, Z, Baohua, L, Yang, Y, Kliebenstein, DJ, Braam, J. Keeping the rhythm: light/dark cycles during postharvest storage preserve the tissue integrity and nutritional value of leafy plants, BMC Plant Biology, 15:92 doi:10.1186/s12870-015-0474-9 (2015).
|