Progress 10/27/99 to 10/26/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Citrus contains a broad range of natural chemical substances. Some of these chemicals are altered by natural events to generate chemical compounds that reduce citrus quality and consumer acceptance. Delayed bitterness in citrus is a slow process that involves the generation of bitter compounds (limonoids) in citrus tissues that have undergone mechanical disruption (i.e., freezing). The bitterness problem is being addressed in this research through the development of citrus species that have been genetically altered to more efficiently convert the bitter limonoids to non-bitter limonoid glucoside derivatives thereby reducing the pool of bitter limonoid precursors responsible for delayed bitterness. Since large amounts of the non-bitter limonoids appear naturally in mature citrus fruit and in citrus
processing by-products, the research further examines the potential role of these compounds in human health leading to their potential utilization in functional foods to realize economic value from citrus processing by-products. The problem of delayed bitterness has a significant impact on the quality and value of citrus. Delayed bitterness in citrus initiated by climatic factors can reduce the consumer acceptability of fresh citrus. In 1990 and 1998, the California navel orange industry lost $260 million and $700 million, respectively, due to delayed bitterness caused by freezing. Many other citrus varieties require significant post-harvest processing to eliminate the limonoids responsible for delayed bitterness in order to make juice from these varieties acceptable to consumers. In either case, the citrus grower or citrus processor incurs losses or increased costs which reduce the value of a citrus crop. The genetic alteration of citrus varieties to achieve naturally debittered
citrus would reduce or eliminate the impact of climatic factors on citrus crop value and increase the scope of varieties available for juice production. Limonoids occur in large amounts in citrus. It is estimated that 15,000 tons of limonoids are produced annually as by-products of worldwide citrus juice production. Citrus limonoids in juice processing by- products are currently utilized solely in cattle feed. Research evidence has established that limonoids commonly found in high yield in juice processing by-products possess significant anti-tumor activity in mammals. If these limonoids can be demonstrated to impart important health and nutrition properties to humans, citrus processors could obtain higher value from the reclamation of the limonoids from processing by-products to be used as nutritional supplements than as cattle feed. These activities fall within National Program 306, Quality and Utilization of Agricultural Products and contributes to NP 107, Human Nutrition.
Specifically they address Problem Area 2a, New Product Technology of NP 306's Component 2. New Processes, New Uses, and Value- Added Foods and Biobased Products and Performance Goal 3.1.3., Nutritious Plant and Animal Products: Develop more nutritious plant and animal products for human consumption of NP 107's Component 6, Health Promoting Properties of Plant and Animal Foods. 2. List the milestones (indicators of progress) from your Project Plan. In FY 2000, we will provide analytical procedures necessary to assess initial attempts to insert the gene responsible for non-bitter limonoid precursor to non-bitter limonoid glucoside conversion in to a Citrus species. Isolation of amounts of individual limonoids and limonoid glucosides in quantities and purities compatible with biological screens will be initiated. New analytical methodology will be applied to characterize sources of biologically active limonoids in Citrus and citrus processing by-products and to develop definitive
chemotaxonomic information about limonoids relative to citrus breeding. Further information about the biological activity of limonoids as insect anti- feedants will be accumulated. A cooperative human nutrition study to determine the bioavailability of limonoids in humans will be initiated. In FY 2001, further efforts toward the creation of the non-bitter transgenic Citrus species will proceed. Biological screening of pure limonoids will continue and structure/activity relationships will be examined. The chemotaxonomic study will be concluded. The human bioavailability study will also conclude. Refinement of separation procedures of important limonoids from citrus processing products will proceed. Industrial scale isolation methodology for accumulation of important bioactive limonoids will be initiated. In FY 2002, research efforts to create the non-bitter transgenic Citrus species will continue. Based upon the determination of bioavailability of limonoids to human, application
for funding for a human clinical trial of limonoids as anti-cancer agents will be made. Industrial scale isolation procedures for purified biologically active limonoids will be attempted. In FY 2003, the final phases of development of a non-bitter transgenic Citrus species should be underway. Human clinical trials will be initiated to determine the chemopreventative properties of specific citrus limonoids. Synthetic or biosynthetic manipulation of limonoids to increase anti-cancer activity will be initiated. In FY 2004, evaluation of the viability of the transgenic Citrus sp. will be determined and if the transgenic species is commercially adaptable, commercialization plans will be initiated. Based upon prior test results, specific limonoids will be examined as potential cancer therapy agents. Genetic manipulation of selected Citrus sp. will be considered as a means to generate highly bioactive limonoids. 3a List the milestones that were scheduled to be addressed in FY 2005. For
each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. In FY 2004, evaluation of the viability of the transgenic Citrus sp. will be determined and if the transgenic species is commercially adaptable, commercialization plans will be initiated. Based upon prior test results, specific limonoids will be examined as potential cancer therapy agents. Genetic manipulation of selected Citrus sp. will be considered as a means to generate highly bioactive limonoids. Milestone Substantially Met 2. In collaboration with researchers in Japan, transgenic citrus tissues that express limonoid glucosyl transferase, the enzyme responsible for the conversion of limonoid A-ring lactones into glucosides, have been generated from callus tissue derived from Washington Navel. In 2004, efforts to regenerate transgenic plants capable of propagation have continued in order to generate plants suitable for commercial evaluation. Milestone Substantially Met 3. Prior in
vivo and in vitro bioassay results have established various limonoids from citrus and a mixture of limonoid glucosides to possess anti-tumor and cholesterol lowering properties. Our establishment of the bioavailability of limonin in humans fed limonin glucoside in 2003 provided the rationale for the development of a study to examine the anti- cholesterolemic properties of a pure limonoid glucoside in humans. In FY2004 a proposed collaborative human feeding study utilizing the resources of USDA/ARS/WRRC and USDA/ARS/WHNRC was drafted and submitted to potential study funding entities in the U.S. and Australia. The study was selected for funding by a major commercial beverage company with support from an Australian company. The study is underway. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Milestones for the
FY2005-FY2007 period are defined in a new 5-year Project Plan for a new project 5325-41430-009-00D, entitled "Improving Citrus Nutritional Properties and Quality to Benefit Human Health and Enhance Citrus Utilization". 4a What was the single most significant accomplishment this past year? Initiation of a collaborative study of anti-cholesterolemic properties of citrus limonoids in humans. Prior in vivo and in vitro bioassay results established various limonoids from citrus and a mixture of limonoid glucosides to possess anti-tumor and cholesterol lowering properties. These observations coupled with research conducted by the Processed Foods Research Unit, Albany, CA, and the Western Human Nutrition Research Center, Davis, CA, on establishment of the bioavailability of limonin in humans fed limonin glucoside in 2003 provided the rationale for a study of the anti-tumor or anti-cholesterolemic properties of a pure limonoid in humans. A study of the anti-cholesterolemic properties of a
pure limonoid glucoside in humans was chosen to obtain validation of the in vivo and in vitro bioassay data. A commercial beverage manufacturer provided funding for the cholesterol study, collaborative between WRRC/ARS and WHNRC/ARS and subject feeding is expected to get underway in late 2005. The study should provide validating information about the importance of limonoids to the health of humans and thereby reinforce the consumption of citrus as an asset to human health. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Research within the scope of this project has established the chemical and biochemical factors associated with limonoid induced delayed bitterness in citrus. Fifty three (53) limonoids and limonoid glucosides have been isolated and characterized by WRRC/ARS scientist from Citrus and Citrus related genera. Extramural trust funded research conducted by WRRC scientists is currently examining the chemistry
of the limonoids responsible for delayed bitterness to develop new ways to chemically measure the impact of near freeze and freeze conditions on navel orange quality. The biochemical information achieved in this research project provides the basis for an ongoing collaborative genetic engineering research project with Japanese scientists to produce a delayed bitterness free transgenic citrus which would have significant value to both citrus growers and processors. Many of the limonoids isolated from citrus by ARS scientists have been supplied to collaborators for evaluation of their biological activity and have led to the discovery that several citrus limonoids are potent anti-tumor agents. This information has led to the development of isolation methods to obtain purified limonoids as potential value-added products from citrus processing by-products. A collaborative project between WRRC and Western Human Nutrition Research Center, Agricultural Research Service scientists has shown
that citrus limonoids commonly present in orange juice are bioavailable to humans following consumption of juice. The absolute correlation of activity of these compounds to the prevention of cancer in humans would have significant impact in human health and nutrition and provide a strong endorsement of citrus as an important component in human diets and would supply incentive to the citrus industry to isolate these compounds as high value citrus processing by-products. An Australian company is currently isolating a mixture of limonoid glucosides from citrus seed for marketing as a nutraceuticals. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The transfer of biochemical information to the sphere of genetic engineering has been accomplished in this
project. The WRRC/ARS and Japanese collaboration to develop a non-bitter transgenic citrus is ongoing and the level of success of this collaboration is an important component in an aggressive ARS/University of California Davis/University of California Riverside molecular biological collaboration, endorsed and funded by citrus commodity groups, to produce transgenic citrus species beneficial to the citrus growers. Technology for the transformation of Washington navel orange obtained by a WRRC/ARS scientist in a three-month visit in the lab of Japanese collaborators has made this technology available to USDA and US scientists. This technology transfer can be expected to lead to significant advance in the development of a transgenic citrus species over next five to seven years. The process for the isolation and purification of limonoids from citrus by-products has been established through this research. A patent for the isolation of limonoid glucosides from citrus by-products exists
and inquiries about the methodology incorporated in the patent continue. Licensing of the patent would be important in the commercialization of limonoid glucosides obtained from citrus processing by-products. Collaboration research involving WRRC/ARS and WHNRC/ARS has established the bioavailability of limonoids in humans. This research has led to an externally funded research program to examine the potential cholesterol- lowering effects of citrus limonoids in humans. Technology transfer from these types of studies can be expected to have significant influence on the role of limonoids in human health and nutrition and in the degree of interest in the reclamation of limonoids from citrus processing by- products over the next two to three years. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). A.P. Breksa III, G.D. Manners, Phytonutrient Recovery
and Utilization from Citrus, United Kingdom Foods Mission to WRRC, 7/9/04, Albany, CA. Log #167287. A.P. Breksa III, G.D. Manners, Phytonutrient Recovery and Utilization from Citrus, USDA-China MOST Workshop, 7/19-7/21/04, Monterey Park, CA. Log #167288.
Impacts
(N/A)
Publications
- Breksa III, A.P., Manners, G.D. 2004. Determination of limonin d-ring lactone hydrolase activity by solid phase extraction with indirect fluorescence detection. Journal of Agricultural and Food Chemistry. Vol 52, No. 12, p. 3772-3775.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Citrus contains a broad range of natural chemical substances. Some of these chemicals are altered by natural events to generate chemical compounds that reduce citrus quality and consumer acceptance. Delayed bitterness in citrus is a slow process that involves the generation of bitter compounds (limonoids) in citrus tissues that have undergone mechanical disruption (i.e. freezing). The bitterness problem is being addressed in this research through the development of citrus species that have been genetically altered to more efficiently convert the bitter limonoids to non-bitter limonoid glucoside derivatives thereby reducing the pool of bitter limonoid precursors responsible for delayed bitterness. Since large amounts of the non-bitter limonoids appear naturally in mature citrus fruit and in citrus
processing by-products, the research further examines the potential role of these compounds in human health leading to their potential utilization in functional foods to realize economic value from citrus processing by-products. The problem of delayed bitterness has a significant impact on the quality and value of citrus. Delayed bitterness in citrus initiated by climatic factors can reduce the consumer acceptability of fresh citrus. In 1990 and 1998, the California navel orange industry lost $260 million and $700 million, respectively, due to delayed bitterness caused by freezing. Many other citrus varieties require significant post harvest processing to eliminate the limonoids responsible for delayed bitterness in order to make juice from these varieties acceptable to consumers. In either case the citrus grower or citrus processor incurs losses or increased costs which reduce the value of a citrus crop. The genetic alteration of citrus varieties to achieve naturally debittered
citrus would reduce or eliminate the impact of climatic factors on citrus crop value and increase the scope of varieties available for juice production. Limonoids occur in large amounts in citrus. It is estimated that 15,000 tons of limonoids are produced annually as by-products of worldwide citrus juice production. Citrus limonoids in juice processing by- products are currently utilized solely in cattle feed. Research evidence has established that limonoids commonly found in high yield in juice processing by-products possess significant anti-tumor activity in mammals. If these limonoids can be demonstrated to impart important health and nutrition properties to humans, citrus processors could obtain higher value from the reclamation of the limonoids from processing by-products to be used as nutritional supplements rather than as cattle feed. These activities fall within National Programs 306, Quality and Utilization of Agricultural Products (70%), and NP 107, Human Nutrition (30%).
Specifically they address Problem Area 2a, New Product Technology of NP 306's Component 2. New Processes, New Uses, and Value-Added Foods and Biobased Products and Performance Goal 3.1.3., Nutritious Plant and Animal Products: Develop more nutritious plant and animal products for human consumption of NP 107's Component 6, Health Promoting Properties of Plant and Animal Foods. 2. List the milestones (indicators of progress) from your Project Plan. In FY 2000, we will provide analytical procedures necessary to assess initial attempts to insert the gene responsible for non-bitter limonoid precursor to non-bitter limonoid glucoside conversion into a Citrus species. Isolation of amounts of individual limonoids and limonoid glucosides in quantities and purities compatible with biological screens will be initiated. New analytical methodology will be applied to characterize sources of biologically active limonoids in Citrus and citrus processing by-products and to develop definitive
chemotaxonomic information about limonoids relative to citrus breeding. Further information about the biological activity of limonoids as insect anti- feedants will be accumulated. A cooperative human nutrition study to determine the bioavailability of limonoids in humans will be initiated. In FY 2001, further efforts toward the creation of the non-bitter transgenic Citrus species will proceed. Biological screening of pure limonoids will continue and structure/activity relationships will be examined. The chemotaxonomic study will be concluded. The human bioavailability study will also conclude. Refinement of separation procedures of important limonoids from citrus processing products will proceed. Industrial scale isolation methodology for accumulation of important bioactive limonoids will be initiated. In FY 2002, research efforts to create the non-bitter transgenic Citrus species will continue. Based upon the determination of bioavailability of limonoids to human, application
for funding for a human clinical trial of limonoids as anti-cancer agents will be made. Industrial scale isolation procedures for purified biologically active limonoids will be attempted. In FY 2003, the final phases of development of a non-bitter transgenic Citrus species should be underway. Human clinical trials will be initiated to determine the chemopreventative properties of specific citrus limonoids. Synthetic or biosynthetic manipulation of limonoids to increase anti-cancer activity will be initiated. In FY 2004, evaluation of the viability of the transgenic Citrus sp. will be determined and if the transgenic species is commercially adaptable, commercialization plans will be initiated. Based upon prior test results, specific limonoids will be examined as potential cancer therapy agents. Genetic manipulation of selected Citrus sp. will be considered as a means to generate highly bioactive limonoids. 3. Milestones: In FY 2004, evaluation of the viability of the transgenic
Citrus sp. will be determined and if the transgenic species is commercially adaptable, commercialization plans will be initiated. Based upon prior test results, specific limonoids will be examined as potential cancer therapy agents. Genetic manipulation of selected Citrus sp. will be considered as a means to generate highly bioactive limonoids. In collaboration with researchers in Japan, transgenic citrus tissues that express limonoid glucosyl transferase, the enzyme responsible for the conversion of limonoid A-ring lactones into glucosides, have been generated from callus tissue derived from Washington Navel. In 2004, efforts to regenerate transgenic plants capable of propagation have continued in order to generate plants suitable for commercial evaluation. Prior in vivo and in vitro bioassay results have established various limonoids from citrus and a mixture of limonoid glucosides to possess anti-tumor and cholesterol lowering properties. Our establishment of the bioavailability
of limonin in humans fed limonin glucoside in 2003 provided the rationale for the development of a study to examine the anti- cholesterolemic properties of a pure limonoid glucoside in humans. In FY2004 a proposed collaborative human feeding study utilizing the resources of USDA/ARS/WRRC and USDA/ARS/WHNRC was drafted and submitted to potential study funding entities in the U.S. and Australia. A CRADA is being established to support this study. Milestones for the FY2005-FY2007 period are defined in a new 5-year Project Plan for a new project entitled "Improving Citrus Nutritional Properties and Quality to Benefit Human Health and Enhance Citrus Utilization". 4. What were the most significant accomplishments this past year? A. Various limonoids from citrus and a mixture of limonoid glucosides possess anti-tumor and cholesterol lowering properties. These observations coupled with the research conducted in the Processed Food Research Unit, Albany, CA, on the bioavailability of
limonin in humans fed limonin glucoside in 2003 provided the rationale for a study of the anti-tumor or anti-cholesterolemic properties of a pure limonoid in humans. Since an anti-tumor study would be excessive in cost and clinically complicated, a study of the anti-cholesterolemic properties of a pure limonoid glucoside in humans was chosen to obtain validation of the in vivo and in vitro bioassay data. A proposal for a collaborative study of the effect of human consumption of a pure citrus limonoid on levels of cholesterol by research personnel at USDA/ARS/WRRC and USDA/ARS/WHNRC was drafted and selected for CRADA funding by a commercial partner. The study, which will get underway in FY2005, should provide validating information about the importance of limonoids to the health of humans and thereby reinforce the consumption of citrus as an asset to human health. B. Increasing interest in the biological activity of citrus limonoids in relation to human health has increased efforts
to isolate and identify new limonoids from citrus sources. Scientists in the Processed Food Research Unit, Albany, CA, developed a new LC/MS method that correlates mass spectral fragmentation patterns of limonoids separated by chromatographic techniques with specific structural characteristics. The method allows the rapid identification of known limonoids in citrus extracts and provides basic information important to the characterization of unknown limonoids in those extracts. The technique will allow researchers attempting to isolate new limonoids from citrus sources to select new unknown limonoids and avoid isolating known limonoids. A manuscript describing the method has been accepted for publication. Freeze damage or physical damage to citrus fruit, including juicing, initiates the formation of the bitter compounds and is a major problem for both fresh fruit and juice producers worldwide. Scientists in the Processed Foods Research Unit, Albany, CA, developed a method for the
evaluation of LDLH activity associated with delayed bitterness in citrus. Compared to other reported procedures, this method is more sensitive, does not require radioactive materials, and is more efficient. The method is available for use by the scientific community. A method was developed for the isolation of two key metabolites (limonoate and nomilionate a-ring lactone) in the limonoid biosynthetic pathway critical to citrus quality. Scientists in the Processed Foods Research Unit, Albany, CA, isolated and characterized the two compounds from young Chandler pummelo seedlings. Their isolation will facilitate the development of methods to improve citrus quality and identify new Citrus cultivars important to human nutrition. Information will be made available to user groups and the scientific community. C. Significant Activities that Support Special Target Populations: None. D. Progress Report: None. 5. Describe the major accomplishments over the life of the project, including
their predicted or actual impact. Research within the scope of this project has established the chemical and biochemical factors associated with limonoid-induced delayed bitterness in citrus. Fifty-three (53) limonoids and limonoid glucosides have been isolated and characterized by WRRC/ARS scientists from Citrus and Citrus related genera. Extramural trust-funded research is currently examining the chemistry of the limonoids responsible for delayed bitterness to develop new ways to chemically measure the impact of near- freeze and freeze conditions on navel orange quality. The biochemical information achieved in this research project provides the basis for an ongoing collaborative genetic engineering research project with Japanese scientists to produce a delayed bitterness-free transgenic citrus which would have significant value to both citrus growers and processors. Many of the limonoids isolated from citrus by ARS scientists have been supplied to collaborators for evaluation of
their biological activity and have led to the discovery that several citrus limonoids are potent anti- tumor agents. This information has led to the development of isolation methods to obtain purified limonoids as potential value-added products from citrus processing by-products. An extramural trust-funded collaborative project between WRRC and Western Human Nutrition Research Center, ARS scientists has shown that citrus limonoids commonly present in orange juice are bioavailable to humans following consumption of juice. The absolute correlation of activity of these compounds to the prevention of cancer in humans would have significant impact in human health and nutrition and provide a strong endorsement of citrus as an important component in human diets and would supply incentive to the citrus industry to isolate these compounds as high value citrus processing by-products. An Australian company has recently developed a method, based upon a USDA/ARS patented process, to isolate
limonoid glucosides from citrus molasses to market as functional food additives and nutraceuticals. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The transfer of biochemical information to the sphere of genetic engineering has been accomplished in this project. The WRRC/ARS and Japanese collaboration to develop a non-bitter transgenic citrus is ongoing and the level of success of this collaboration is an important component in an aggressive ARS/University of California Davis/University of California Riverside molecular biological collaboration, endorsed and funded by citrus commodity groups, to produce transgenic citrus species beneficial to the citrus growers. Technology for the transformation of the Washington navel orange obtained by a
WRRC/ARS scientist in a three- month visit in the lab of Japanese collaborators has made this technology available to USDA and U.S. scientists. This technology transfer can be expected to lead to significant advancements in the development of a transgenic citrus species over the next five to seven years. The process for the isolation and purification of limonoids from citrus by-products has been established through this research. An ARS patent for the isolation of limonoid glucosides from citrus by-products was written and an Australian company is applying the methodology and considering obtaining an exclusive license. This technology transfer should lead to the first commercialization of limonoid glucosides obtained from citrus processing by-products. Collaborative research involving WRRC/ARS and WHNRC/ARS has established the bioavailability of limonoids in humans. This research has led to an externally funded research program with a commercial partner to examine the potential
cholesterol-lowering effects of citrus limonoids in humans that will get underway in the Fall of 2004. Technology transfer from these studies will have significant influence on the role of limonoids in human health and nutrition and in the degree of interest in the reclamation of limonoids from citrus processing by-products over the next two to three years. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Presentations: Manners, G.D. Health Promoting Properties of Citrus Limonoids, CIFAR Conference XIX, U.C. Davis Medical School, May 25, 2004. Breksa III, A.P. Current Status of Citrus Genomics Efforts. Washington Free Tree Commission. February 10, 2004. Albany, CA. Breksa III, A.P., Manners, G. D., Zukas, A. A. Improving Citrus Nutritional Properties and Quality to Benefit Human Health and Enhance Citrus Utilization. CRB Biotechnology Workshop. April 29, 2004. Oakland, CA. Breksa III, A.P.,
Manners, G. D. Enhanced and Enriched Citrus By- Products. UK Food Mission. July 9, 2004. Albany, CA. Breksa III, A.P., Manners, G. D., Zukas, A. A. Phytonutrient Recovery and Utilization from Citrus. USDA-MOST, China Workshop. July 21, 2004. Monterey Park, CA.
Impacts
(N/A)
Publications
- Breksa III, A.P., Manners, G.D. 2004. Determination of limonin d-ring lactone hydrolase activity by solid phase extraction with indirect fluorescence detection. Journal of Agricultural and Food Chemistry. Vol 52, No. 12, p. 3772-3775.
|
Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Citrus contains a broad range of natural chemical substances. Some of these chemicals are altered by natural events to generate chemical compounds that reduce citrus quality and consumer acceptance. Delayed bitterness in citrus is a slow process that involves the generation of bitter compounds (limonoids) in citrus tissues that have undergone mechanical disruption (i.e., freezing). The bitterness problem is being addressed in this research through the development of a citrus species that has been genetically altered to more efficiently convert the bitter limonoids to non-bitter limonoid glucoside derivatives thereby reducing the pool of bitter limonoid precursors responsible for delayed bitterness. Since large amounts of the non-bitter limonoids appear naturally in mature citrus fruit and in citrus processing by-products, the research further examines the potential role of these
compounds in human health leading to their potential utilization in functional foods to realize economic value from citrus processing by-products. 2. How serious is the problem? Why does it matter? The problem of delayed bitterness has a significant impact on the quality and value of citrus. Delayed bitterness in citrus initiated by climatic factors can reduce the consumer acceptability of fresh citrus. In 1990 and 1998, the California navel orange industry lost $260 million and $700 million, respectively, due to delayed bitterness caused by freezing. Many other citrus varieties require significant post-harvest processing to eliminate the limonoids responsible for delayed bitterness in order to make juice from these varieties acceptable to consumers. In either case, the citrus grower or citrus processor incurs losses or increased costs that reduce the value of a citrus crop. The genetic alteration of citrus varieties to achieve naturally debittered citrus would reduce or
eliminate the impact of climatic factors on citrus crop value and increase the scope of varieties available for juice production. Limonoids occur in large amounts in citrus. It is estimated that 15,000 tons of limonoids are produced annually as by-products of worldwide citrus juice production. Citrus limonoids in juice processing by- products are currently utilized solely in cattle feed. Research evidence has established that limonoids commonly found in high yield in juice processing by-products possess significant anti-tumor activity in mammals. If these limonoids can be demonstrated to impart important health and nutrition properties to humans, citrus processors could obtain higher value from the reclamation of the limonoids from processing by-products to be used as nutritional supplements than as cattle feed. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? This research is assigned to National Program 306 70%,
Quality and Utilization of Agricultural Products; and National Program 107 30%, Human Nutrition. This assignment includes fundamental research to clarify the roles of citrus composition on end use performance and to effect genetic modifications to improve end product quality and increase the utilization of citrus limonoid components to increase the value of citrus products and improve human health. 4. What were the most significant accomplishments this past year? A. The necessity to eliminate delayed bitterness in citrus has fostered a genetic engineering approach to produce a citrus species capable of higher efficiency in the natural production of tasteless glucosides of bitter limonoids from the precursor responsible for delayed bitterness in citrus. During a three-month interactive training visit with collaborators at the Citrus Research Station in Okitsu, Japan Research, personnel from the citrus program at WRRC/ARS obtained technology and skills necessary to genetically
transform Washington navel orange to a citrus species without delayed bitterness. The genetic transformation skills obtained by Western Regional Research Center (WRRC)/Agricultural Research Service (ARS) research personnel have been applied to expedite the generation of a genetically engineered non-bitter Washington navel orange. Success in the development of a genetically engineered non- bitter Washington navel orange would have major impact on increased consumption of citrus and reduction of citrus grower losses associated with delayed bitterness. B. Citrus limonoids have been shown to be potential cancer preventive agents in animal test screens and human cancer cell screens and the effectiveness of these compounds as potential preventative agents to cancer in humans is dependent upon their physiological availability following ingestion of citrus fruits, citrus juices or citrus products. A trust funded collaborative human feeding study conducted by research scientist at WRRC/ARS
and Western Human Nutrition Research Center (WHNRC) /ARS has established the presence of citrus limonoid metabolites in the blood plasma of humans fed a pure limonoid compound occurring in high yield in citrus juices. The very recent publication of the results of this study provides clear evidence, for the first time, that limonoid glucosides are metabolized by humans. This evidence strongly suggest that the limonoid glucoside commonly found in citrus fruits and juices can become bioavailable in humans to act as preventatives to cancer. Simplified, efficient, sensitive analytical methods are necessary to measure the amount of bitter limonoids present in citrus subjected to mechanical damage or freezing relative to citrus quality and for the quantification of very low amount of limonoids and limonoid metabolites in human biological fluids collected in human studies relating limonoids to human health and nutrition. WRRC scientists have examined the application of liquid
chromatography coupled to mass spectrometry as the means to achieve the necessary analysis. A new analytical method utilizing liquid chromatography-mass spectrometry has been developed and reported by WRRC scientists that is applicable to the quantification of limonoids in freeze impacted citrus and extracts of blood plasma from human subjects. The new method offers speedier analysis of "total bitter limonoids" in citrus derived samples and the sensitivity necessary for the detection of low levels of limonoid metabolites in human biological fluids. C. Significant Activities that Support Special Target Populations. None. D. Milestone: Significant progress has been made toward the commercial utilization of citrus limonoids from citrus processing by-products. USDA/ARS/WRRC research has established that citrus limonoids may be important to human health and nutrition and has developed patented methodology for the reclamation of these compounds from citrus processing by-products. An
Australian company is currently purifying limonoid glucoside from a citrus processing by-product and is manufacturing a purified limonoid glucoside extract utilizing methodology based upon the USDA/ARS/WRRC patented process. An agreement in principal between the Australian company (raw material supplier) and a Japanese food company (manufacturer) utilizing technical information from USDA/ARS/WRRC should lead to the marketing of a limonoid glucoside fortified foods and juice products for the Japanese market by the end of 2003. Marketing of the foods or drinks containing the limonoid glucosides will be the first commercial utilization of these compounds and will validate the potential importance of the compounds for human health and nutrition. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Research within the scope of this project has established the chemical and biochemical factors associated with limonoids-induced
delayed bitterness in citrus. Fifty-three (53) limonoids and limonoid glucosides have been isolated and characterized by WRRC/ARS scientist from Citrus and Citrus related genera. Extramural trust-funded research conducted by WRRC scientists is currently examining the chemistry of the limonoids responsible for delayed bitterness to develop new ways to chemically measure the impact of near freeze and freeze conditions on navel orange quality. The biochemical information achieved in this research project provides the basis for an ongoing collaborative genetic engineering research project with Japanese scientists to produce a delayed bitterness- free transgenic citrus which would have significant value to both citrus growers and processors. Many of the limonoids isolated from citrus by ARS scientists have been supplied to collaborators for evaluation of their biological activity and have led to the discovery that several citrus limonoids are potent anti-tumor agents. This information
has led to the development of isolation methods to obtain purified limonoids as potential value-added products from citrus processing by-products. An extramural trust funded collaborative project between WRRC and Western Human Nutrition Research Center, Agricultural Research Service scientists has shown that citrus limonoids commonly present in orange juice are bioavailable to humans following consumption of juice. The absolute correlation of activity of these compounds to the prevention of cancer in humans would have significant impact in human health and nutrition and provide a strong endorsement of citrus as an important component in human diets and would supply incentive to the citrus industry to isolate these compounds as high value citrus processing by-products. An Australian company has recently developed a method, based upon a USDA/ARS patented process, to isolate limonoid glucosides from citrus molasses to market as functional food additives and nutraceuticals. 6. What do
you expect to accomplish, year by year, over the next 3 years? In FY 2004, collaboration with Japanese collaborators in efforts toward the creation of the non-bitter transgenic Citrus species will continue. The commercialization of limonoid glucosides as nutraceuticals or in functional foods should be underway. Methods for the efficient isolation of individual limonoid glucosides in quantities and purity compatible with human metabolic study needs will be continued. A collaborative study to examine the potential cholesterol lowering of properties of limonoids should be conducted. New chemical methods for the detection of delayed bitterness in field navel oranges will be completed. Cloning and characterization of enzymes related to the formation of bitterness in citrus initiated in FY 2003 will continue. In FY 2005, the final phases of development of a non-bitter transgenic Citrus species should be underway. Development and utilization of proteomic methods for citrus to
characterize attributes associated with quality and nutritive value should be underway. Preliminary limonoid metabolism studies in humans will be initiated. Application for funding for an expanded human clinical trial of limonoids as cancer chemopreventative agents will be made. In FY 2006, the development of a transgenic Citrus species should be nearing completion. Functional verification of proteins identified through proteomic methods using genetically transformed plants will be underway. Full limonoid metabolism studies in humans should be underway and cancer chemopreventative studies utilizing limonoids should be planned. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The transfer of biochemical information to the sphere of genetic
engineering has been accomplished in this project. The WRRC/ARS and Japanese collaboration to develop a non-bitter transgenic citrus is ongoing and the level of success of this collaboration is an important component in an aggressive ARS/University of California Davis/University of California Riverside molecular biological collaboration, endorsed and funded by citrus commodity groups, to produce transgenic citrus species beneficial to the citrus growers. Technology for the transformation of Washington navel orange obtained by a WRRC/ARS scientist in a three-month visit in the lab of Japanese collaborators has made this technology available to USDA and US scientists. This technology transfer can be expected to lead to significant advances in the development of a transgenic citrus species over next five to seven years. The process for the isolation and purification of limonoids from citrus by-products has been established through this research. A patent for the isolation of limonoid
glucosides from citrus by-products exists and an Australian company is applying the methodology and is considering obtaining an exclusive license. This technology transfer should lead to the first commercialization of limonoid glucosides obtained from citrus processing by-products. Collaboration research involving WRRC/ARS and WHNRC/ARS has established the bioavailability of limonoids in humans. This research has led to consideration of an externally funded research program to examine the potential cholesterol lowering effects of citrus limonoids in humans. Technology transfer from these types of studies can be expect to have significant influence on the role of limonoids in human health and nutrition and in the degree of interest in the reclamation of limonoids from citrus processing by-products over the next two to three years. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does
not replace your peer-reviewed publications listed below). Manners, G.D., Jacob, R.A., Breksa III, A.P., Schoch, T.K. Hasegawa, S. Bioavailability of Citrus Limonoids in Humans. American Society of Pharmacognosy. 44th Annual Meeting, July 12-17, 2003. Chapel Hill, NC. Log No. 151161. Manners, G.D. The feasibility of colorimetric analysis for the determination of freeze-impacted citrus quality. California Citrus Research Board. March 12, 2003. Riverside, CA. Breksa III, A.P., Manners, G.D. Current and Future Citrus Research Efforts at the USDA-ARS Western Regional Research Center. 4th Annual Fruit Biotechnology Research Conference, December 4-5, 2002. Tsukuba, Japan. Breksa III, A.P. Transformation Studies in Japan. California Citrus Research Board. March 12, 2003. Riverside, CA. Breksa III, A.P. Prevention of Limonin Bitterness. California Citrus Research Board. March 12, 2003. Riverside, CA.
Impacts
(N/A)
Publications
- Manners, G.D., Jacob, R.A., Breksa III, A.P., Schoch, T.K., Hasegawa, S. Bioavailability of citrus limonoids in humans. Journal of Agricultural and Food Chemistry. 2003. v. 51. p. 4156-4161.
- Manners, G.D., Breksa III, A.P., Schoch, T.K., Hidalgo, M.B. Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionization and electrospray ionization liquid chromatography - mass spectrometry. Journal of Agricultural and Food Chemistry. 2003. v. 51. p. 3709-3714.
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Progress 10/01/01 to 09/30/02
Outputs
(N/A)
Impacts
(N/A)
Publications
- Panter, K.E., Manners, G.D., Steglemeier, Lee, S., Gardner, D.R., Ralphs, M.H., Pfister, J.A., James, L.F. Larkspur poisonin: Toxicology and alkaloid structure-activity relationships. Biochemical Systematics and Ecology. 2002. v. 30. p. 113-128.
- Pfister, J.A., Ralphs, M.H., Gardner, D.R., Stegelmeier, B.L., Manners, G. D., Panter, K.E., Lee, S. Management of three toxic Delphinium species based on alkaloid concentration. Biochemical Systematics and Ecology. 2002, v. 30. p. 129-138.
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Progress 10/01/00 to 09/30/01
Outputs
CESSED BY-PRODUCT UTILIZATION 1. What major problem or issue is being resolved and how are you resolving it?
Citrus contains a broad range of natural chemical substances. Some of these chemicals are altered by natural events to chemical compounds that reduce citrus quality and consumer acceptance. Delayed bitterness in citrus is a slow process that involves the generation of bitter compounds (limonoid) in citrus tissues that have undergone mechanical disruption (i.e. freezing). The bitterness problem is being addressed in this research through the development of citrus species that have been genetically altered to efficiently convert the bitter limonoids to non-bitter glucoside derivatives thereby reducing the pool of bitter limonoid precursors responsible for delayed bitterness. Since large amounts of the non-bitter limonoids appear naturally in mature citrus fruit, the research further examines the potential role of these compounds in human health and their potential utilization in functional foods to realize economic value from citrus processing byproducts.
2. How serious is the problem? Why does it matter?
The problem of delayed bitterness has a significant impact on the quality and value of citrus. Delayed bitterness in citrus initiated by climatic factors can reduce consumer acceptability of fresh citrus. In 1990 and 1998, the California navel orange industry lost $260 million and $700 million, respectively, due to delayed bitterness caused by freezing. Many other citrus varieties require significant post-harvest processing to eliminate the limonoids responsible for delayed bitterness and to make juice from these varieties acceptable to consumers. In either case, the citrus grower or citrus processor incurs losses or increased costs which reduce the value of a citrus crop. The genetic alteration of citrus varieties to achieve naturally debittered citrus would reduce or eliminate the impact of climatic factors on citrus crop value and increase the scope of varieties available for juice production. Limonoids occur in large amounts in citrus. It is estimated that 15,000 tons of
limonoids are produced annually as by-products of worldwide citrus juice production These limonoids presently have little commercial value and only find utilization as cattle feed. Limonoids are a problem to the citrus processing industry since certain limonoids impart severe bitterness to citrus juice and must be removed for consumer acceptance of the juices. Citrus processors incur increased production costs to remove the limonoids from the juice which may or may not be offset through the sale of the limonoid residues for cattle feed. If the limonoids had a higher value as separate product by virtue of a positive nutritional value, the citrus processor could afford to further purify the limonoids and market them for other uses such as fortification.
3. How does it relate to the National Program(s) and National Component(s)?
This research is assigned to National Program 306, Quality and Utilization of Agricultural Products. This assignment includes fundamental research to clarify the roles of citrus composition on end use performance and to effect genetic modifications to achieve the best end product quality as well as defining new uses for the limonoid components and adding value to citrus products. The research program includes collaboration with nutritional scientists of the ARS Western Human Nutrition Research Center funded extramurally by The Florida Citrus commodity board, research funded extramurally by a California citrus commodity board.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment: The natural abundance of limonoids as limonoid glucosides in citrus juices and their observed anti-tumor activity in animal and human cell in vitro tests suggest a potentially important role for humans but the biological fate when ingested is unknown. In collaboration with ARS nutritional scientists at the Western Human Nutrition Center, a human feeding study involving the ingestion of pure limonin glucosides obtained from citrus by-products by volunteer subjects has been initiated and periodic post-dose blood samples have been collected. A sensitive analytical method has been developed for the detection of limonoids or limonoid metabolites in plasma of these blood samples. Detection of limonoids or limonoid metabolites in the plasma of the human volunteers will provide substantial evidence that limonoids are biologically available to humans for potential action as cancer chemopreventatives. B. Other Significant Accomplishments: Elimination
of delayed bitterness in citrus has been sought to maintain the value of fresh fruit and prevent serious crop losses. A jointly patented genetic engineering approach has been applied by collaborators in Japan to produce a new citrus species capable of great efficiency in the natural production of citrus free of the bitter limonoids. These collaborators have successfully inserted a gene responsible for transforming bitter limonoids to non-bitter limonoids into the callus tissue of a citrus species with a limonoid bitterness problem and have verified that the callus produces tasteless limonoid glucosides from a bitter limonoid precursor. The successful generation of this transgenic callus tissue is a major step in the generation of a citrus species free of limonoid bitterness and would benefit citrus growers where periodic freezes are frequent. C. Significant Accomplishments/Activities that Support Special Target Populations: None. D. Progress report: Development of improved analytical
methods for the limonoid glucosides is a key milestone not only to improve our understanding of the occurence and distribution of limonoids in citrus fruits and citrus processing streams but also to lead to the ability to analyze for these compounds in human fluids. This methodology will be utilized in the human bioavailability studies recently initiated and described in 5325-41430-07-02-T, Bioavailability of Cancer Prevention Citrus Limonoids in Humans. If the limonoids or significant metabolites can be confirmed as bioavailable this should provide strong rationale for continued and increased ingestion of citrus and citrus byproduct and there should be significant increase in the citrus market. This research is an interesting example of how scientists applying basic science to understand the biochemistry of a quality problem in citrus is leading not only to the solution of that problem but also to the creation of a potentially unique health-promoting product.
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
Research within the scope of this project has clearly established the chemical and biochemical factors involved in the development of delayed bitterness in citrus. This information is the foundation for collaborative genetic engineering research (Japan) which is making significant progress toward the production of a transgenic citrus free from delayed bitterness. A delayed-bitterness-free trangenic citrus would have significant value to citrus growers and processors. The chemical characterization of 53 limonoids present in citrus and its closely related genera was accomplished within the scope of this research project and its antecedents. Many of these limonoids have been supplied to collaborators for evaluation of biological activity. These collaborations include three examining anti-cancer properties and one examining insect antifeedant properties. A current collaboration is examining the bioavailability of these compounds to humans and their role in human health and nutrition. The
correlation between limonoid ingestion to the prevention of cancer in humans would have significant impact in human health and nutrition and provide a strong endorsement of citrus as an important component in human diets. The validation of these compounds as anticancer agents or important insect antifeedants would supply incentive to the citrus industry to proceed aggressively to isolate these compounds as high value citrus processing byproducts.
6. What do you expect to accomplish, year by year, over the next 3 years?
Year 1: In conjunction with Japanese collaborators, further efforts toward the creation of the non-bitter transgenic citrus species will proceed. A cooperative human nutrition study to determine the bioavailability of limonoids in humans will continue.Isolation of individual limonoids and limonoid glucosides in quantities and purities compatible with biological screening will be continue. New industrial contacts for the commercialization of limonoid glucosides as nutritional supplements will be initiated. Year 2: The transgenic citrus species should be generated and be available for initial evaluation. Biological screening of pure limonoids will continue and structure/activity relationships will be examined. Limonoid metabolites from the human bioavailability study should be identified. Year 3: The final phases of development of a non-bitter transgenic Citrus species should be underway. Based upon the determination of bioavailability of limonoids to humans, application for funding
for a human clinical trial of limonoids as anti-cancer agents will be made.
7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product?
The transfer of biochemical information to the genetic engineering community has been accomplished in this project. A patent for the gene has been approved by the ARS patent committee. The future development of a viable transgenic citrus (collaboratively with Japanese researchers and jointly patented) will include a transfer of genetically engineered plants to the citrus grower. This technology transfer can be expected to occur within the next five to seven years. The process for the isolation and purification of limonoids from citrus by-products has been established through this research. A patent for the isolation of limonoid glucosides from citrus by-products has been granted. A citrus industry commodity board funded research program is underway to develop an easy method to evaluate bitterness development in freeze impacted citrus in the field. Collaborative research has been initiated with the Western Human Nutrition Research Center to examine the bioavailability of limonoid
glucosides in humans. The technology transfer of limonoid glucoside anticancer research can be expected to occur within the next one or two years.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
None.
Impacts
(N/A)
Publications
- Schoch, T.K., Manners, G.D., Hasegawa, S. Analysis of limonoid glucosides from Citrus by electrospray ionization liquid chromatography-mass spectrometry. Journal of Agriculture and Food Chemistry. 2001. v.49. p. 1102-1108.
- Manners, G.D., Hasegawa, S. Citrus limonoids: Potential cancer chemopreventative agents. Proceedings of the International Society of Citriculture. 2001. p. 124-137.
- Manners, G.D., Schoch, T.K., Hasegawa, S. Citrus limonoids: Potential cancer chemopreventatives. American Society of Pharmacognosy. 2001. Abstract.P 157.
- Schoch, T.K., Manners, G.D., Hasegawa, S. Quantative analysis of limonoid glucosides from Citrus by LC/MS. American Chemical Society. 2000. Abstract.136.
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Progress 10/01/99 to 09/30/00
Outputs
CESSED BY-PRODUCT UTILIZATION 1. What major problem or issue is being resolved and how are you resolving it?
Citrus contains a broad range of natural chemical substances. Some of these chemicals are altered by natural events to chemical compounds that reduce citrus quality and consumer acceptance. Delayed bitterness in citrus is a slow process that involves the generation of bitter compounds (limonoid) in citrus tissues that have undergone mechanical disruption (i.e. freezing). The bitterness problem is being addressed in this research through the development of citrus species that have been genetically altered to efficiently convert the bitter limonoids to non-bitter glucoside derivatives thereby reducing the pool of bitter limonoid precursors responsible for delayed bitterness. Since large amounts of the non-bitter limonoids appear naturally in mature citrus fruit, the research further examines the potential role of these compounds in human health and their potential utilization in functional foods to realize economic value from citrus processing by-products.
2. How serious is the problem? Why does it matter?
The problem of delayed bitterness has a significant impact on the quality and value of citrus. Delayed bitterness in citrus initiated by climatic factors can reduce consumer acceptability of fresh citrus. In 1990 and 1998, the California navel orange industry lost $260 million and $700 million, respectively, due to delayed bitterness caused by freezing. Many other citrus varieties require significant post-harvest processing to eliminate the limonoids responsible for delayed bitterness and to make juice from these varieties acceptable to consumers. In either case, the citrus grower or citrus processor incurs losses or increased costs which reduce the value of a citrus crop. The genetic alteration of citrus varieties to achieve naturally debittered citrus would reduce or eliminate the impact of climatic factors on citrus crop value and increase the scope of varieties available for juice production. Limonoids occur in large amounts in citrus. It is estimated that 15,000 tons of
limonoids are produced annually as by-products of worldwide citrus juice production These limonoids presently have little commercial value and only find utilization as cattle feed. Limonoids are a problem to the citrus processing industry since certain limonoids impart severe bitterness to citrus juice and must be removed before the juice will be acceptable to consumers. Citrus processors incur increased production costs to remove the limonoids from the juice which may or may not be offset through the sale of the limonoid residues for cattle feed. If the limonoids had a higher value as separate product by virtue of a positive nutritional value, the citrus processor could afford to further purify the limonoids and market them for other uses such as fortification.
3. How does it relate to the National Program(s) and National Component(s)?
This research is assigned to National Program 306 (70%) New Uses, Quality, & Marketability of Plant & Animal Products. This assignment includes fundamental research to clarify the roles of citrus composition on end use performance and to effect genetic modifications to achieve the best end product quality as well as defining new uses for the limonoid components and adding value to citrus products. It also relates to NP107 Human Nutrition 30%.
4. What were the most significant accomplishments this past year?
A. Single Most Significant Accomplishment: Since earlier research has established that limonoids and limonoid glucosides possess anticancer activity, it was important to identify major sources of these compounds in citrus processing by-products to serve as sources for reclamation and purification. In collaboration with an industrial partner, specific processing stream by-product sources were examined and the most promising source was identified for limonoid glucoside reclamation. The by-product source was identified, the amounts and character of limonoids were determined, an improved method of purification of individual limonoid glucosides was developed and significant amounts of mixed limonoid glucosides (lesser amounts of individual limonoid glucosides) were obtained. The identification of this source of limonoids from a citrus processing by-product stream designates a source of biologically active limonoids which can be purified to marketable levels by the industrial partner
as a cancer chemopreventative and for other continuing biological assessments. B. Other Significant Accomplishments: The necessity to eliminate delayed bitterness in citrus has fostered a genetic engineering approach to produce a transgenic citrus species capable of higher efficiency in the natural production of tasteless limonoid glucosides from the limonoid precursor responsible for bitter limonoid formation. Japanese collaborators are developing a transgenic citrus species free from limonoid bitterness. These collaborators have successfully inserted the gene responsible for the conversion of the non-bitter limonoid precursor to non-bitter limonoid glucosides into the genome of a citrus species that has a limonoid bitterness problem. The successful insertion of this gene is a major step in the generation of a citrus species free of limonoid bitterness. C. Significant Accomplishments/Activities that Support Special Target Populations: D. Progress Report:
5. Describe the major accomplishments over the life of the project including their predicted or actual impact.
Research within the scope of this project has clearly established the chemical and biochemical factors involved in the development of delayed bitterness in citrus. This information has laid the foundation for collaborative genetic engineering research (Japan) to produce a transgenic citrus free from delayed bitterness. The development of the transgenic citrus would have significant value to citrus growers and processors. The chemical characterization of 53 limonoids present in citrus and its closely related genera was accomplished within the scope of this research project and its antecedents. Many of these limonoids have been supplied to collaborators for evaluation of biological activity. These collaborations include three examining anti-cancer properties and one examining insect antifeedant properties. The correlation of activity of these compounds to the prevention of cancer in humans would have significant impact in human health and nutrition and provide a strong
endorsement of citrus as an important component in human diets. A CRADA partner cooperating with a Japanese company, has decided to commercialize limonoid glucosides as anticancer agents. The validation of these compounds as anticancer agents or antifeedants would supply incentive to the citrus industry to proceed aggressively to isolate these compounds as high value citrus processing byproducts.
6. What do you expect to accomplish, year by year, over the next 3 years?
FY 2001 In conjunction with Japanese collaborators, further efforts toward the creation of the non-bitter transgenic Citrus species will proceed. Industrial scale isolation for biologically active limonoid glucosides will be initiated. Isolation of individual limonoids and limonoid glucosides in quantities and purities compatible with biological screening will be initiated. A cooperative human nutrition study to determine the bioavailability of limonoids in humans will be initiated. FY 2002, the commercialization of limonoid glucosides as anticancer agents should be underway. Biological screening of pure limonoids will continue and structure/activity relationships will be examined. The human bioavailability study will be continues. FY 2003, the final phases of development of a non-bitter transgenic Citrus species should be underway. Based upon the determination of bioavailability of limonoids to humans, application for funding for a human clinical trial of limonoids as anti-cancer
agents will be made.
7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end user (industry, farmer, other scientists)? What are the constraints if known, to the adoption & durability of the technology product?
The transfer of biochemical information to the genetic engineering community has been accomplished in this project. A patent for the gene has been approved by the ARS patent committee. The future development of a viable transgenic citrus (collaboratively with Japanese researchers and jointly patented) will include a transfer of genetically engineered plants to the citrus grower. This technology transfer can be expected to occur within the next five to seven years. The process for the isolation and purification of limonoids from citrus by-products has been established through this research. A patent for the isolation of limonoid glucosides from citrus by-products has been granted. A CRADA has been initiated with a major industrial citrus processor to specifically examine the biological activity properties of these compounds and to develop better methods for their isolation and purification on an industrial scale. Collaborative research has been initiated with Western Human
Nutrition Research Center to examine the bioavailability of limonoid glucosides in human. The technology transfer of limonoid glucoside anticancer research can be expected to occur within the next one or two years.
8. List your most important publications in the popular press (no abstracts) and presentations to non-scientific organizations and articles written about your work (NOTE: this does not replace your peer-reviewed publications which are listed below)
Presentations: (A) Hasegawa, S. Citrus limonoids: Possible commercialization as anticancer agents. Toyotama International, Inc., Tokyo, Japan. May 18, 2000. (B) Hasegawa, S. Biological functions of citrus limonoids. Ross Products, Columbus, OH. June 22, 2000.
Impacts
(N/A)
Publications
- Murray,K.D., Hasegawa,S. Alford,A.R. Antifeedant activity of citrus limonoids against Colorado potato beetle: comparison of aglycones and glucosides. Journal of Chemical Ecology. 1999. v.92. p.331-334.
- Hasegawa,S., Lam,L.K.T. and Miller,E.G. Citrus limonoids: biochemistry and possible importance to human nutrition.Shahidi, F., Ho,C.T., editors. American Oil Chemists Society Press, Champaign, IL. Phytochemicals and Phytopharmaceuticals. 2000. p.79-94.
- Miller,E.G., Record,M.T., Binnie,W H. Hasegawa,S. Limonoid glucosides: systemic effects on oral carcinogenesis. Shahidi,F. and Ho,C.T., editors. American Oil Chemists Society Press, Champaign, IL.Phytochemicals and Phytopharmaceuticals. 2000. p.95-105.
- Kita,M. Hirata,Y., Moriguchi,T., Endo-Inagaki,T., Matsumoto,R., Hasegawa,S., Suhayda,C. Omura,M. Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase from Citrus. Federation of European Biochemical Societies Letters. 2000. v.469. p.173-178.
- Berhow,M.A, Hasegawa,S. Manners,G.D. editors. Citrus Limonoids. Functional Chemicals in Agriculture and Foods. American Chemical Society Symposium Series. 2000. v.(758). p.1-253.
- Hasegawa,S., Berhow,M.A. and Manners,G.D. Citrus limonoid research: an overview. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.1-8.
- Hasegawa,S. Biochemistry of limonoids in Citrus.Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.9-30.
- Hasegawa,S. Berhow,M. Analysis of limonoids by thin-layer chromatography. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.31-39.
- Manners,G., Hasegawa,S., Bennett,R.D., Wong,R. LC-MS and NMR techniques for the analysis and characterization of citrus limonoids. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.40-59.
- Miyake,M., Shimada,M., Osajima,Y., Inaba,N., Ayano,S., Ozaki,Y., Hasegawa, S. Extraction and recovery of limonoids with the supercritical carbon dioxide micro-bubble method. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). 96-106.
- Ozaki,Y., Miyake,M., Inaba,N., Ayano,S., Ifuku,Y., Hasegawa,S. Limonoid glucosides of Satsuma mandarin (Citrus unshiu Marcov.) and its processing products. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.107-119.
- Miller,E.G.,Taylor,S.E., Berry,C.W., Zimmerman, J.A,, Hasegawa,S. Citrus limonoids: Increasing importance as anticancer agents. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.132-144.
- Guthrie,N., Morley,K., Hasegawa,S., Manners,G. and Vandenberg,T. Inhibition of human breast cancer cells by citrus limonoids. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). 164-174.
- Kurowska,E., Banh,C., Hasegawa,S. and Manners,G. Regulation of Apo B production in HepG2 cells by citrus limonoids. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.175-184.
- Lam,L., Hasegawa,S. Bergstrom,C., Lam, S., Kenney, P. Limonin and nomilin inhibitory effects on chemical-induced tumorigenesis.Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.185-2000.
- Berhow,M., Hasegawa,S., Wong,K., Bennett,R.D. Limonoids and the chemotaxonomy of Citrus and the Rutaceae family. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p 212-229.
- Omura,M., Kita,M,. Endo-Inagaki,T., Moriguchi,T., Matsumoto,R., Suhayda,C., Hasegawa,S. Genetic evaluation and modification of the accumulation of limonoids in Citrus. Berhow,M.A., Hasegawa,S., Manners,G.D., editors. ACS Symposium Series, Washington, DC. Citrus Limonoids: Functional Chemicals in Agriculture and Foods. 2000. v.(758). p.230-237.
- Kita,M., Endo-Inagaki,T., Moriguchi,T., Shimada,T., Matsumoto,R., Hasegawa,S., Omura, M. Citrus limonoid glucosyltransferase: Isolation of cDNA and relationship between allelism and accumulation of limonoid glucosides. Nippon Agricultural Chemistry, Tokyo, 2000. Abstract #3H047a. p.367.
- Kita,M., Indo-Inagaki,T., Moriguchi,T., Shimada,T., Hirata,Y., Matsumoto,R., Hasegawa,S., Omura,M. Allelism and mapping on linkage group of limonoid UDP-gucosyltransferase gene of Citus. Japan Society of Horticultural Science, Kobe, Japan. 2000. Abstract #Po15, p.66.
- Kurowska1,E.M., Manthey,J.A., Hasegawa,S. Regulatory effects of tangeretin, a flavonoid from tangerines, and limonin, a limonoid from citrus, on Apo B metabolism in HEPG2 cells. Experimental Biology (American Physiological Society , American Society for Investigative Pathology, American Society for Nutritional Sciences, American Association of Anatomists). 2000. Abstract #231-2.
- Guthrie,N., Manthey,J.A., Hasegawa,S., Manners,G., Kurowska,E.M. In vitro studies on anti-cancer and cholesterol-lowering activities of citrus flavonoids and limonoids. Experimental Biology (American Physiological Society , American Society for Investigative Pathology, American Society for Nutritional Sciences, American Association of Anatomists). 2000. Abstract #408-2.
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