Source: TEXAS A&M UNIVERSITY submitted to
EXPLORING BIOLOGICAL ACTIVITIES OF CITRUS LIMONOIDS:A MULTIDISIPLINARY APPROACH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0189875
Grant No.
2001-52102-11257
Project No.
TEX08869
Proposal No.
2001-04533
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Sep 15, 2001
Project End Date
Sep 30, 2006
Grant Year
2001
Project Director
Patil, B. S.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
HORTICULTURAL SCIENCE
Non Technical Summary
Limonoids are naturally-occurring compounds found in citrus fruit. This multidisciplinary, multi-state, multi-institute integrated (research, extension and education) project will focus on the isolation, purification, identification and evaluation of biological activities of limonoids in citrus. The reactivities of limonoids will be tested based on several enzymatic, tumor cell, and animal models in order to evaluate the impact of specific limonoids on oral and colon cancer and the ability of limonoids to detoxify certain carcinogens, cancer-causing compounds. The ongoing successful multidisciplinary and multi-institute unique course 'Phytochemicals in Fruits and Vegetables to Improve Human Health' will be expanded to sites outside the state of Texas, i.e., the University of Wisconsin in Madison and the Johns Hopkins University in Baltimore. Citrus limonoids have potential for prevention of certain chronic diseases such as cancer and cardiovascular diseases. Exploring biological activities of limonoids is very essential. Educating dieticians and graduate students about phytochemicals is needed to improve human consumption of fruits and vegetables.
Animal Health Component
(N/A)
Research Effort Categories
Basic
45%
Applied
35%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7020910100015%
7020920100015%
7020930100015%
7020999100015%
7030910101010%
7030920101010%
7030930101010%
7030999101010%
Goals / Objectives
1. Isolation and purification of limonoids from ten citrus species so that detailed characterization and quantitation of the limonoids can be undertaken. Citrus processing and extraction methods will be optimized for this critical aspect of the proposed project, and three different extraction methods will be compared: conventional liquid-liquid extraction methods, solid phase extraction, and solid phase microextraction. 2. Development of electrospray ionization (ESI)/quadrupole ion trap tandem mass spectrometry for the structural characterization of limonoids in citrus fruit. 3. Evaluation of the effect of eight limonoids on the development of carcinogen-induced oral carcinomas. This research will provide information on the relationship between structure and anticancer activity. 4. Determination of whether three limonoids (limonin, obacunone, and limonin 17-b-D-glucopyranoside (LG)): a) protect against formation of preneoplastic lesions of colon cancer (aberrant crypt foci; ACF) and reduce foci multiplicity, and b) protect against oxidative challenge- determine whether the limonoids act as anti-oxidants in the colon. Optimal dietary levels to achieve chemoprotection will be established. 5. Determine the induction of glutathione S-transferases (GSTs) by citrus limonoids. Induction of GSTs has been linked to enhanced detoxification of carcinogens or their metabolites, and therefore prevention of carcinogenesis. GST induction by citrus limonoids will be investigated in mice. Detailed structural and functional (activity against carcinogens) characterization of GSTs will be carried out to evaluate their potential in chemoprevention. 6. Elucidation of the effect of specific limonoids (i.e., limonin, nomilin, obacunone, and obacunone glucoside) on enzymatic activity of CPY1A1, CYP1A2 and CYP1B1. The mode of action of limonoids that causes the inhibition of reactions will be studied to confirm whether a mechanism-based inactivation of the cytochrome is operative. The selectivity of inhibition by the limonoids with respect to other isozymes will be determined. The fate of inhibitory compounds will be examined, as well as elucidation of the pathways of biotransformations. The establishment of structure-activity relationships (SAR) will be a key goal. 7. Development of educational materials about functional components of fruits and vegetables for specific target groups: all health educators across Texas and the United States including those in the Extension Service, dietitians, and educators of practitioners of dietetics, undergraduate and graduate students, fruit and vegetable producers, and consumers. Different delivery methods will be tested including web-based fact-sheets, animations, training sessions, classroom lectures, and face-to-face workshops. 8. Expansion of the ongoing unique and successful multidisciplinary and multi-institutional course 'Phytochemicals in Fruits and Vegetables to Improve Human Health' to sites outside the state of Texas, i.e., the University of Wisconsin in Madison and the Johns Hopkins University in Baltimore.
Project Methods
The proposed project will focus on characterizing and purifying various limonoids and their glucosides from ten citrus species including orange, grapefruit,lemon, lime, kumquat, and mandarin. The structures of the isolated compounds will be verified by tandem mass spectrometric methods. The limonoids will then be used for the various biological assays. Objective 1. Isolation and Purification. This aspect of the proposed work is critical for obtaining pure quantities of the limonoids that can be structurally characterized by mass spectrometry and used for biological activities in four labs. Objective 2. Structural Characterization by Tandem Mass Spectrometry.This aspect of the proposed work is aimed at the development of electrospray ionization quadrupole ion trap tandem mass spectrometry for the sensitive detection and characterization of limonoids in citrus fruit. Objective 3. Oral Carcinogenesis Assay. The test chemicals can either be incorporated into the diet or applied topically to the pouches. The principal advantage of topical applications is that only a small amount of the test chemical is needed for the assay. The model using topical applications has already been used to test twelve citrus limonoids for anticancer activity. Objective 4. Colon Cancer Assay. First, the ability of limonin, LG, and obacunone to protect against ACF will be examined in order to determine a) formation and multiplicity of ACF; b) in situ localization of proliferating cells; and c) in situ localization of apoptotic cells. The hypothesis is that ACF formation occurs through changes in proliferation and apoptosis in response to a carcinogen, and that limonoids may protect against ACF formation. To test this hypothesis we will determine ACF number and multiplicity, proliferation and apoptosis in the large intestine of rats challenged with a colon carcinogen. Objective 5. Induction of Glutathione S-Transferase. The effect of GST induction on GST-mediated detoxification systems in protection of cells against electrophilic carcinogens and xenobiotics will be studied. Mouse models will be selected to investigate induction of GST by the limonoids. Objective 6. Enzymatic Activities. Either microsomal preparations of human liver or those of three human lympho-blastoid cell lines will serve as the enzyme source of the cytochrome P450 isozymes CYP1A1, CYP1A2 and CYP1B1. Objective 7. The following training materials on functional foods are proposed. During the first year, we will develop animated graphic materials to show 1) atherosclerotic development and platelet aggregation leading to a heart attack, 2) functional foods that prevent atherosclerotic development and platelet aggregation, 3) phases of cancer development and 4) pictures of foods with icons that could be 'clicked on' to teach that functional foods protect against diseases. Objective 8. Multi-Institute Course. In the spring of 2003 and 2005, the unique course 'Phytochemicals in Fruits and Vegetables to Improve Human Health', will be offered at nine sites in Texas, the University of Wisconsin, and the Johns Hopkins University.

Progress 09/15/01 to 09/30/06

Outputs
This project (TEX08869) is transferred from Texas A&M University-Kingsville to Texas Agricultural Experiment Station, College Station due the transfer of Principal Investigator. Further reports of this project is being submitted in TEX09089.

Impacts
As the health promoting evidences of citrus have been increasing, our results provided proof-of the concept for the conventional wisdom of citrus limonoids benefit to prevent oral and colon cancer based on animal model studies. Using isolated limonoids, we have demonstrated that certain limonoids can inhibit the development of carcinogen-induced oral cancer. We also showed that limonoids can protect against early colon cancer lesions in a rat model of the disease. Together, these data suggest grapefruit and limonoids would likely confer the greatest protection against specific types of cancer. Educational activities such as coloring plate of fruits and vegetable will be used in different schools. This type of educational activities would increase fruits and vegetables consumption and expected to reduce health care costs. The unique phytochemical course which covers the benefits of fruits and vegetables and their role in disease prevention has impacted students, parents and society as whole. The importance of this research is increased by the fact citrus and juices contain high concentrations of these bioactive compounds.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
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.

Impacts
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.

Publications

  • 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

Outputs
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.

Impacts
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.

Publications

  • 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.


Progress 01/01/02 to 12/31/02

Outputs
Objective 1. Rapid isolation techniques such as crystallization to provide a method for producing the compounds in larger quantities in their pure form is being studied. Flash chromatography for rapid separation of glucosides and aglycones extracts have been conducted. We were able send 30 semi purified and several pure (90-95%) compounds to Dr. Brodbelt group for further confirmation by LC/MS. Pure limonin (97%) was sent to Dr. Miller. Objective 2. Seven limonoids have been characterized, including both glucosides and aglycones. The glucosides are best analyzed as deprotonated species. The fragmentation patterns show loss of water, carbon dioxide, acetic acid, glucose, and various cleavages of the rings. For HPLC-MS, a gradient elution at 0.4 ml/min flow rate with a mixture of water and acetonitrile (containing formic acid, pH 3.2) has been used with good linearity from 4 to 80 micromolar limonin glucoside. Objective 3. Our initial studies in this area have focused on the furan ring. Originally, it was assumed that this ring was one of the critical structural features needed for biological activity. Several results have now led us to question this assumption. Using a published procedure, we have been trying to produce two new compounds from limonin and nomilin. The reaction using ruthenium chloride and sodium periodate as the key ingredients is designed to replace a furan ring with a carboxyl group. In theory, this modification should leave us with an intact limonoid nucleus with a carboxyl group attached to the D ring. Pilot studies with limonin and nomilin have been run in our laboratory and in both cases the preliminary data suggest that the compounds were produced with yields of 55-80%. If additional tests confirm that we are in fact dealing with the expected products, then we will scale up the procedure and test these two new compounds for anticancer activity. Objective 4. Dried citrus pulp and isolated compounds will be provided to rats in order determine their ability to protect against colon cancer. The experimental design and planning for two experiments has been completed. The animal part of the research will be conducted starting in January for the first experiment and starting in March for the second experiment. Objective 5. Induction of GST has been linked to enhanced detoxification of carcinogens and thus prevention of carcinogenesis. Our results show that the limonoid aglycones, nomilin and aglycone mixture are effective inducers of GST activity. Objective 6. Planning for the animal experiment is under progress. Student has been working on isolation and purification of limonoids. Objective 7. Due to the sad demise of Dr. Sandi Simons, this objective has not progressed well. Alternative person has been identified to continue this objective. Objective 8. The unique course Phytochemicals in Fruits and Vegetable to Improve Human Health is scheduled to be taught in spring 2003. The course will be expanded to Purdue University and Michigan State University.

Impacts
Sensitive analytical methods for both qualitative and quantitative analysis of the limonoids in grapefruit are being developed, thus allowing assessment of the quantitites of limonoids in grapefruit, in addition to providing confirmation of the isolation and purity of specific limonoids prior to biological assays. This research with synthetic and naturally-occurring limonoids will allow us to pinpoint 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, compared to intact citrus pulp, will tell us which compounds are the most protective and which ones to focus on for further enhancement in fruit. In addition, our data suggest that the GST inducing ability of citrus limonoids may be linked to anticancer activities. The unique course has received national attention and students and general public will get major benefit and we hope the fruit and vegetable consumption will increase in the near future.

Publications

  • No publications reported this period


Progress 01/01/01 to 12/31/01

Outputs
The project money arrived to our sponsored office in November and account is established. In the mean time, the project director met with collaborators and discussed about the plan of action. Limonoid purification process was initiated and seeds were collected and extraction of limonoids by soxhlet is in progress. Students and post doc required for this project was announced in professional national meetings and in the web site. Initial contacts were made for the potential students and pdf were completed. The program director, Dr. Mel Mathias, meeting with collaborators in College Station is planned for January 8, 2002. Met with collaborator dealing with extension part of the proposal and discussed about developing a fact sheets of each phytochemicals. Intial contact has been made with the John Hopkins University and University of Wisconsin to expand the "Phytochemicals in Fruits and Vegetables to Improve Human Health" course to the respective universities.

Impacts
Potential improving human health by consumption of citrus. Education activities will be going beyond the geographical barriers.

Publications

  • No publications reported this period