Progress 01/01/04 to 12/31/04
1. Several citrus limonoids were isolated, characterized and purified as indicated in the proposal. Different citrus species and components (seeds, molasses and peel) were used to isolate several milligrams of limonin, limonin glucoside (LG), deacetyl nomilin acid glucoside, deacetylnomilin, iso-obacunoic acid glucoside, obacunone glucoside, nomilinic acid glucoside (NAG), nomilin, and obacunone. Structures of these compounds have been identified using 1H, 13C NMR spectra and mass spectral data. 2. Isolated and purified limonoids were obtained by chromatographic methods developed in the Patil group. The extracts were solubilized and diluted, then infused by electrospray ionization into a tandem mass spectrometer for analysis. The glucosides were analyzed as deprotonated species. The limonoid aglycones were analyzed as protonated species or sodium complexes. The fragmentation patterns of the limonoids confirmed their identities in the extracts. The major limonoids were
identified. Robust and reliable LCMS methods for direct analysis of limonoids in mixtures were developed. 3. Production of synthetic limonoids from limonin and nomilin is ongoing. 4. Analysis of aberrant crypt foci (ACF), proliferation and apoptosis has been completed. Immunoblotting procedures have been implemented that allow analysis of iNOS, COX-2 and GAPDH (loading control protein) from the same blot. Rats injected with the carcinogen and consuming the basal diet had more high multiplicity ACF, compared to all the experimental diets. The proportion of cells within a crypt undergoing proliferation was decreased by the experimental diets, compared to the basal diet. Only rats consuming the basal diet had a significant increase in the proliferative index as a result of carcinogen injection. The apoptotic index was elevated in rats injected with the carcinogen and consuming diets containing the grapefruit pulp powder, naringin or limonin. 5. More efficient in vitro cell culture model
to investigate the effect of citrus limonoids in murine hepatoma cell, Hepa 1c1c7 have been standardized. These studies are now being extended in intestine and stomach cell lines which seem to show greatest effect of limonoids in the in vivo models. In a similar experiment, the cells were harvested after the limonoid incubation and homogenized using a sonicator using hypotonic phosphate buffer. 6. Limonoid glucosides inhibited human CYP1B1 Ethoxy resorufin (EROD) activities to a greater extent than did the aglycones. Competitive mode of inhibition exhibited by LG on CYP19 with dibenzyl flurescein as substrate whereas NAG resulted in a non competitive mode of inhibition on the same substrate. Limonoid glucosides inhibited human CYP1B1 EROD activities to a greater extent than did the aglycones. 7. Coloring Our Plate with Fruits and Vegetables has been developed and pilot tested in Dallas County. Feedback from county Extension agents and clientele have been very positive. 8.
Phytochemicals course has been a great success and planned to expand to the Ohio State University in spring 2005.
A rapid method of purifying and isolation of citrus limonoids is developed which has less unit of operation as compared to previous method (patent application pending). Biological studies with specific citrus limonoids will provide the structural features associated with activity and provide, at the same time, critical information that can be used to determine the molecular mechanism of action. Furthermore, knowing the relative contribution of specific compounds on detoxifying enzymes will impact citrus industry in understanding their role in several types of cancer. Results in animal studies indicate that carcinogen induced colon cancer could be protected by citrus limonoids. In vitro study of phase I enzymes provide good evidence to the potential of citrus bioactive compounds for their anticancer properties. The unique course has received national and international attention and new land grant universities such as Ohio State University showed an interest in
participating in the course. An International symposium entitled Potential Health Benefits of Citrus was organized during American Chemical Society in Philadelphia on 08/23/2004 and global level task force was formation was discussed. The meeting was attended by several key citrus industry representatives from Florida, California, and Texas and scientists around the world. American Chemical Society has approved to publish a book on the above title with contributing authors of the symposium. Our symposium outcome was covered in several national and international media.
- Turner, N.D., J. Vanamala, T. Leonardi, B.S. Patil, M.E. Murphy, N. Wang, L. M. Pike, R.S. Chapkin and J.R. Lupton. 2004. Grapefruit and its isolated limonoids and flavonoids as colon cancer chemoprotectants in rats. 228th American Chemical Society, National meeting, Philadelphia, PA, 08/23/2004 (Invited).
- Miller, E.G., R.P. Gibbins, S.E. Taylor, J.E. McIntosh, and B.S. Patil, 2004. Long term screening study on the potential toxicity of limonoids, 228th American Chemical Society, National meeting, Philadelphia, PA, 08/23/2004. (Invited).
- Jayaprakasha, G.K., J. S. Brodbelt, N.G. Bhat, and B.S. Patil. 2004. Rapid methods for the separation of bioactive compounds from citrus. 228th American Chemical Society, National meeting, Philadelphia, PA, 08/23/2004 (Invited).
- Ahmad, H., J. Li, M. Polson, K. Mackie, W. Quiroga and B.S. Patil. 2004. Citrus limonoids and flavonoids, Enhancement of phase II enzymes and its potential in chemoprevention. 228th American Chemical Society, National meeting, Philadelphia, PA, 08/23/2004 (Invited).
- Quiroga, W., Trini Garza, Monica Cantu, Jose Diaz-Miron, Natalia Maani, Bhimanagouda Patil and Hassan Ahmad. 2004. Induction of Phase-II Drug Metabolizing Enzymes in Mouse Tissues by Citrus Flavonoids. American Association of Biochemistry and Molecular Biology, 06/12/2004.
- Peacock, J.J., E.G. Miller, S.E. Taylor, J.M. Wright and B.S. Patil. 2004. Inhibition of oral carcinogensis by citrus flavonoids. Submitted for the IADR/ADDR/CADR Dental Conference, 82nd General session- Honolulu, 03/10/2004.
- Gibbins, R.P., E.G. Miller, S.E. Taylor, J.E. McIntosh and B.S. Patil. 2004. Long term screening study on the potential toxicity of limonoids. Submitted for the IADR/ADDR/CADR Dental Conference, 82nd General session- Honolulu, 03/10/2004.
- Yu, J., L.Wang, R.W. Walzem, R.W. E.G. Miller, L.M.. Pike, B.S. Patil. 2004. Citrus Functional Components and Antioxidant Activity. Fifth International Conference and Exhibition on Nutraceuticals and Functional Foods, San Francisco, CA, 11/10/2004 (Invited).
- Vanamala, J., L.Reddivari, L.M. Pike, and B.S. Patil, 2004. Variation in Bioactive Flavonoid Content of Commercial Brands of Orange Juices. American Society for Horticultural Sciences, 07/20/2004, Austin, TX.
- Yu, J., R.T. Toledo, R.K. Singh, L.M.Pike, B.S. Patil. 2004. Supercritical fluid extraction of limonoids from grapefruit seed. American Society for Horticultural Sciences-July 20-22, 2004, Austin, TX.
- Poulose, S., J. S. Brodbelt. L.M. Pike, B. S. Patil, 2004. Chromatographic Techniques to Purify Individual Limonoids from Seeds and Molasses of Citrus Fruits. American Society for Horticultural Sciences, 07/20/2004, Austin, TX.
- Jayaprakash, G.K., Patil, B.S. and N.G.Bhat. 2004. A Process for the Isolation of Antioxidant Limonoid Glucosides from Citrus Seeds (Patent application submitted).
- Patil, B.S., Jun Yu, R.K. Singh, R. Toledo, and L.M.Pike. 2004. Citrus bioactive limonoids and flavonoids extraction by supercritical fluid. 228th American Chemical Society, National meeting, Philadelphia, PA, 08/23/2004 (Invited).
- Pikulski, M., M. Cho, J. Zhang, B. Davis, B. Girrennavar, N.G. Bhat, B. S. Patil and Jennifer S. Brodbelt. 2004. Analysis of Furanocoumarins, Limonoids, and Flavonoids in Citrus by Tandem Mass Spectrometry. American Society for Horticultural Sciences, 07/20/2004, Austin, TX.
- Miller, E.G. Peacock, J.J., S.E. Taylor, J.M. Wright and B.S. Patil. 2004. Reduction of Chemically Induced Tumorigenesis by Citrus Flavonoids. American Society for Horticultural Sciences, 07/20/2004, Austin, TX.
- Patil, B.S.; Jayaprakasha, G.K. and Mahesh Padanad. 2004. Isolation, characterization and role of functional components in fruits and vegetables, Presented at American Society for Horticultural Science, 07/20/2004.
- Quiroga, W., Garza, T., Cantu, M., Diaz-Miron, J., Maani, N., Patil, B., and Ahmad, H. 2004. Induction of Phase-II Drug Metabolizing Enzymes in Mouse Tissues by Citrus Flavonoids. Presented at the 2004 ASBMB Annual Meeting and 8th International Union of Biochemistry and Molecular Biology Conference to be held at Boston, 07/12/2004.
- Poulose, S., E.D. Harris, and B.S. Patil 2004. Pro-apototic and antioxidant properties of limonoid glucosides from citrus fruits. J. Nutrition (accepted).
- Raman, G.R., M. Cho, Minhee, J. Brodbelt, B.S. Patil. 2004. Isolation and Purification of Closely related Citrus Limonoid Glucosides by Flash Chromatography. Phytochemical Analysis (Accepted).
- Patil, B.S., J. Vanamala and G. Hallman. 2004. Irradiation and storage influence on functional components and quality of early and late season Rio Red grapefruit (Citrus paradisi Macf). Postharvest Biology and Technology.34:53-64.
- Raman, G., Jayaprakasha G.K. Minhee Cho, J. S. Brodbelt, and B. S. Patil 2004. Isolation of structurally similar citrus flavonoids by flash chromatography. Analytical Letters 37:3005-3016.
- Raman, G.R., Jayaprakasha, G.K., M. Cho, Minhee, J. Brodbelt, and B. S. Patil. 2004. Rapid Adsorptive Separation of Citrus Polymethoxylated Flavones in Non-aqueous Conditions. Separation and Purification Technology (Accepted).
- Vanamala, J., G. Cobb, N. Turner, J. Lupton, K. Yoo, L.M.Pike, and B.S. Patil. 2004. Bioactive compounds of grapefruit (Citrus paradisi cv Rio Red) respond differently to postharvest irradiation, storage and freeze drying. J. Ag. Food Chem. (Accepted).
Progress 01/01/03 to 12/31/03
1. Limonoid samples were sent to Dr. Brodbelt group for further confirmation by LC/MS. Several limonoid mixtures of along with limonin and irradiated grapefruit pulp were sent to Turner. Both aglycons and glucosides were isolated from molasses and seeds of citrus fruits and byproducts. Four aglycons and four glucosides were purified and sent to Mayer lab and in vitro study on several phase I enzymes were conducted. 2. Numerous limonoid-containing samples prepared in Patil lab have been analyzed by electrospray ionization mass spectrometry (ESIMS). Analytical feedback was provided to Patil group and assessing the purities of the samples to refine and optimize their extraction procedures. We have continued to develop a HPLC-ESIMS protocol for separation, quantification, and identification of limonoids. Characterization of limonoids by tandem mass spectrometry in order to create a database for the confirmation of limonoids in future applications is continued. 3.
Production of synthetic limonoids from limonin and nomilin is under progress. Most of the work on synthetic limonoids is being done by student and some of the assays are being run at the University of Mississippi. 4. Sprague Dawley rats were used to evaluate the effect of azoxymethane and diet (basal, grapefruit pulp powder, irradiated grapefruit pulp powder, naringin, and limonin) on the development of the preneoplastic lesions of colon cancer. Body weights were determined and food intake was measured. At the end of the study, samples collected to determine ACF number and multiplicity, rates of proliferation and apoptosis, as well as iNOS and COX-2 expression. Animals injected with the carcinogen and consuming the basal diet showed an increase in the number of high multiplicity ACF, compared to all the experimental diets. We may learn the mechanisms whereby diets containing citrus or citrus isolates protect against the formation of colon cancer preneoplastic lesions. 5. Glutathione
S-Transferases (GST) are efficient detoxification enzymes whose role in the metabolism of carcinogens or their metabolites is well established. Our results show that the limonoid aglycons, nomilin and aglycon mixture significantly induce GST activity in mouse liver and Intestine. 6. Phytochemicals were tested for the inhibitory effects on O-deethylase activity, O-demethylase activity and dibenzylflurescein reduction by CYP3A4 and CYP19. The total mixtures of both limonoids and flavonoids induced partial to high inhibitory effects at 10uM concentrations. Both limonoids and flavonoids inhibited metabolite formation by human CYP450s. Limonin has shown to reverse the inhibitory effect of other compounds when supplied with other compounds which induced independent inhibition. 7. Objectives were revised and revision of Phyte Phyte Phyte is ongoing. Development of prototype for consumer-based fact sheets is being planned. 8. The unique course is taught in spring 2003. The course is expanded
to Michigan State University and Purdue University including nine locations at Texas with a total enrollment of 64. Students can also access streaming video, threaded discussion.
Analytical methods for both qualitative and quantitative analysis of the limonoids in grapefruit are being developed. This research with synthetic and naturally-occurring limonoids will provide the structural features associated with activity and provide, at the same time, critical information that can be used to determine the molecular mechanism of action. Furthermore, knowing the relative contribution of specific compounds to the protection against colon cancer will impact citrus industry. In vitro study of phase I enzymes provide a good evidence to the potential of citrus bioactive compounds for their anticancer properties. The unique course has received national and international attention and students get major benefit and we hope the fruit and vegetable consumption will increase in the near future.
- Tian, Q., Li, D., Barbacci, D., Schwartz, S., and Patil, B.S. 2003. Electron ionization of mass spectrometry of citrus limonoids. Rapid Communication in Mass Spectrometry 17:2517-2522.
- Patil, B.S., R. Daniel Lineberger, K.M. Dooley, and J. Vanamala, 2003. Phytochemicals in Fruits and Vegetables: Use of Interactive Videoconferencing and World Wide Web for Teaching a Multidisciplinary Course. J. Food Science Education. 2:61-68.
- Vanamala, J., T. Leonardi, M. E. Murphy, S. S. Taddeo, B. S. Patil, L. M. Pike , R. S. Chapkin, J. R. Lupton3, and N. D. Turner. 2003. Natural and Irradiated Grapefruit Pulp and their Functional Compounds Suppress Aberrant Crypt Foci and Colonocyte Proliferation. Experimental Biology Annual Meeting, Washington, D.C.-April 2004.
- Leonardi, T., J. Vanamala, S.S. Taddeo, M.E. Murphy, B.S. Patil, N. Wang, R.S. Chapkin, J.R. Lupton, and N.D. Turner 2003. Apigenin and naringenin suppress high multiplicity aberrant crypt foci formation and cell proliferation in rat colon. Experimental Biology, Annual Meeting, Washington, D.C.-April 2004.
- Yu, J., L. Wang , R. L. Walzem, E. G. Miller, L.M. Pike, B.S. Patil. 2003. Antioxidant Activity of Citrus Limonoids, Flavonoids and Coumarins. International Citrus Congress to be held in Agadir, Morocco- February 15-20, 2004.
- Raman, G. S. Poulose, Ananthkrishnan, B. Patil. 2003. Utilization of Citrus By-products to Improve Human Health. 225th American Chemical Society, National meeting, New Orleans, LA, March 23-27, 2003.