Progress 10/27/04 to 10/26/09
Outputs
Progress Report Objectives (from AD-416) The primary goals of this research project are to establish the role of naturally occurring biologically active citrus phytochemicals, in particular citrus limonoids, in human health and nutrition, to enhance citrus co-product utilization and to utilize molecular level methods to improve citrus quality and optimize important biologically active phytochemicals in new citrus varieties. Approach (from AD-416) 1) Characterize naturally occurring phytochemicals, in particular limonoids, isolated from citrus fruit and juice, and assess their biological activity, determine their potential contribution to the improvement of human health and nutrition, and establish the nature and scope of their action in biological systems. 2) Develop and refine methodology to reclaim pure biologically active citrus limonoids important to human health and nutrition from citrus processing co- products. 3) Determine the origins and modulating chemical and biochemical factors for natural phytochemicals in citrus and develop molecular level strategies to modify and/or enhance the character or amount of these compounds to achieve improve citrus quality, increased consumer acceptance and optimized nutritional value of citrus. This project was replaced by 5325-41430-010-00D. Please see the report for the replacement projct 5325-41430-010-00D for progress report.
Impacts
(N/A)
Publications
|
Progress 10/01/08 to 09/30/09
Outputs
Progress Report Objectives (from AD-416) The primary goals of this research project are to establish the role of naturally occurring biologically active citrus phytochemicals, in particular citrus limonoids, in human health and nutrition, to enhance citrus co-product utilization and to utilize molecular level methods to improve citrus quality and optimize important biologically active phytochemicals in new citrus varieties. Approach (from AD-416) 1) Characterize naturally occurring phytochemicals, in particular limonoids, isolated from citrus fruit and juice, and assess their biological activity, determine their potential contribution to the improvement of human health and nutrition, and establish the nature and scope of their action in biological systems. 2) Develop and refine methodology to reclaim pure biologically active citrus limonoids important to human health and nutrition from citrus processing co- products. 3) Determine the origins and modulating chemical and biochemical factors for natural phytochemicals in citrus and develop molecular level strategies to modify and/or enhance the character or amount of these compounds to achieve improve citrus quality, increased consumer acceptance and optimized nutritional value of citrus. Replaces 5325-41430-008-00D (01/2005). Significant Activities that Support Special Target Populations The Metabolic Analysis of Mandarin Orange - For this project, we have continued our analysis of Satsuma mandarin orange juices obtained from 10 different orchards located in Placer County and expanded the scope of the study to include metabolite profiling and molecular analyses. Profiling experiments target carotenoids, limonoids and volatile juice components. Molecular analyses focused on the expression of enzymes in the synephrine biosynthetic pathway. No progress on the analyses yet. The CDFA-Funded Citrus Project - This multi-step project consists of four steps that include the collection of citrus samples, primary analysis of physicochemical properties, metabolite profiling by both GC- MS and LC-MS, and data analysis. During the past growing session we have collected commercial samples of Navel, Valencia and Mandarin orange varieties from multiple locations throughout California and have completed the primary chemical analysis of these samples. Additionally, we have established schedules to collect additional samples during the coming months and have secured samples from Florida and the Citrus Variety Collection. Trust - Efforts in support of this project have been directed at the continued recovery of limonin glucoside from available starting materials and the isolation and characterization of the degradation products resulting from the vitamin B2 catalyzed oxidation of limonin glucoside. Additionally, the evaluation and introduction of various pretreatment methods resulted in improved recovery efficiencies downstream and led to the recovery of enough limonin glucoside for testing on another four study participants. Technology Transfer Number of Other Technology Transfer: 10
Impacts
(N/A)
Publications
- Dragull, K.D., Breksa III, A.P., Cain, B.R. 2008. Synephrine Content of Juice from Satsuma Mandarins (Citrus unshiu Marcovitch). Journal of Agricultural and Food Chemistry. 56:8874-8878.
- Breksa III, A.P., Dragull, K.D. 2009. Development and Validation of a Decigram-Scale Method for the Separation of Limonin from Limonin Glucoside by C-18 Flash Chromatography. Food Chemistry. 113:1308-1311.
- Breksa III, A.P., Hidalgo, M., Lee-Yuen, M. 2009. Liquid Chromatography- Electrospray Ionization Mass Spectrometry Method for the Rapid Identification of Citrus Limonoid Glucosides in Citrus Juices and Extracts. Journal of Food Chemistry. 117(4)739-744.
|
Progress 10/01/07 to 09/30/08
Outputs
Progress Report Objectives (from AD-416) The primary goals of this research project are to establish the role of naturally occurring biologically active citrus phytochemicals, in particular citrus limonoids, in human health and nutrition, to enhance citrus co-product utilization and to utilize molecular level methods to improve citrus quality and optimize important biologically active phytochemicals in new citrus varieties. Approach (from AD-416) 1) Characterize naturally occurring phytochemicals, in particular limonoids, isolated from citrus fruit and juice, and assess their biological activity, determine their potential contribution to the improvement of human health and nutrition, and establish the nature and scope of their action in biological systems. 2) Develop and refine methodology to reclaim pure biologically active citrus limonoids important to human health and nutrition from citrus processing co- products. 3) Determine the origins and modulating chemical and biochemical factors for natural phytochemicals in citrus and develop molecular level strategies to modify and/or enhance the character or amount of these compounds to achieve improve citrus quality, increased consumer acceptance and optimized nutritional value of citrus. Replaces 5325-41430-008-00D (01/2005). Significant Activities that Support Special Target Populations Efforts in support of this project have been directed at the continued isolation of limonin glucoside from available starting materials and the evaluation of alternative starting materials. Concurrent with our isolation efforts, a 200-day study examining the stability of limonin glucoside in beverage matrices was completed. Results from this study provided evidence that limonoid glucoside is stable in common beverage matrices and resistant to degradation into limonin. Of the matrix components tested, only vitamin B2 was found to be detrimental. In addition, the human study portion of the project has commenced and six participants have already completed the study regime. In addition, we determined the synephrine concentrations found in the juices of Satsuma mandarin fruits obtained from 10 different orchards located in Placer County, CA, USA. The analyses for synephrine were complemented with physiochemical characterization of the juices. Synephrine concentrations found in Satsuma mandarins were on average higher than those reported for sweet and sour oranges, and in a range similar to other mandarins or tangerines. Results for some samples suggests that the consumption of one standard cup of juice could provide a dose of synephrine in the upper range of those available in oral weight loss supplements. The data also revealed an up to 2-fold difference among groves in the same county suggesting that factors including microclimate or localized agricultural practices may influence synephrine concentrations. This research supports NP306, Component II, New Processes, New Uses, and Value-Added Foods and Biobased Products, d) New and Improved Processes and Feedstocks.
Impacts
(N/A)
Publications
- Breksa III, A.P., Dragull, K.D., Wong, R.Y. 2008. Isolation and Identification of the First C-17 Limonin Epimer, Epilimonin. Journal of Agricultural and Food Chemistry. 56:5595-5598.
- Breksa III, A.P., Manners, G.D., Ibarra, Jr., P. 2008. Clarification of reconstituted frozen orange juice concentrate by continuous flow centrifugation for limonin glucoside solid phase extraction. Journal of the Science of Food and Agriculture. 88:2213-2218.
- Breksa III, A.P., Hidalgo, M.B., Wong, R.Y. 2008. Stability of Limonin Glucoside in Beverage Matrices. Journal of the Science of Food and Agriculture. 88:2194-2200.
|
Progress 10/01/06 to 09/30/07
Outputs
Progress Report Objectives (from AD-416) The primary goals of this research project are to establish the role of naturally occurring biologically active citrus phytochemicals, in particular citrus limonoids, in human health and nutrition, to enhance citrus co-product utilization and to utilize molecular level methods to improve citrus quality and optimize important biologically active phytochemicals in new citrus varieties. Approach (from AD-416) 1) Characterize naturally occurring phytochemicals, in particular limonoids, isolated from citrus fruit and juice, and assess their biological activity, determine their potential contribution to the improvement of human health and nutrition, and establish the nature and scope of their action in biological systems. 2) Develop and refine methodology to reclaim pure biologically active citrus limonoids important to human health and nutrition from citrus processing co- products. 3) Determine the origins and modulating chemical and biochemical factors for natural phytochemicals in citrus and develop molecular level strategies to modify and/or enhance the character or amount of these compounds to achieve improve citrus quality, increased consumer acceptance and optimized nutritional value of citrus. Replaces 5325-41430-008-00D (01/2005). Accomplishments Colorimetric Method for Estimating Limonoid Concentrations. Citrus limonoids is one of the last remaining families of compounds important to citrus quality, human health and possessing potential health-promoting properties, detection methods for which are lacking. Researchers in the Processed Foods Unit, Albany, CA, filled a critical need by developing and subsequently disseminating a simple, rapid and inexpensive method for estimating total limonoid concentrations in juices. This method will allow researchers and citrus producers alike to evaluate the quality and value of their citrus products. This research supports NP306, Component I, Quality Characterization, Preservation and Enhancement, d) Preservation and/or Enhancement of Quality and Marketability. Robust and Green Method for the Isolation of Limonin Glucoside. Methods that overcome the hindrances to commercialization of citrus due to limonin glucoside are critical to U.S. agriculture. Researchers in the Processed Foods Research Unit, Albany, CA, have developed a robust and green method for the isolation of limonin glucoside. The utility and robustness of the method was demonstrated on a pilot scale as it was used to obtain, from a variety of sources and without modification, over 350 grams of high purity material (greater than 98%). Methods designed to utilize readily scalable food grade materials and minimize the number of steps and waste are now available. This research supports NP306, Component II, New Processes, New Uses, and Value-Added Foods and Biobased Products, d) New and Improved Processes and Feedstocks. Pilot Scale Removal of Limonin. After learning that the limonin glucoside containing beverages to be used for the human study were too bitter for consumption due to the presence of limonin (less than 1%), a compound in the same family as limonin glucoside, but structurally different, researchers in the Processed Foods Research Unit, Albany, CA, quickly developed a pilot scale chromatographic method for removing residual limonin. Purity of the material treated by this polishing method increased by 1.5% to 99.5% with only a minimal loss of the starting material. The two-step process uses only ethanol and water as the solvents and affords a readily-scalable to a production scale method that may be used by producers whose customer requirements include increased purity. This research supports NP306, Component II, New Processes, New Uses, and Value-Added Foods and Biobased Products, d) New and Improved Processes and Feedstocks. Production Scale Quality Control Methods Developed. Methods are needed that can accurately evaluate potential feed-stock and isolated material for their limonoid glucoside content and character. Researchers in the Processed Foods Research Unit, Albany, CA, have developed analytical methods and implemented these methods as quality control processes in support of their pilot scale isolation of limonin glucosides. Methods, based on HPLC-MS and NMR, have been successfully utilized. The availability of ARS-developed methods, in light of the considerable public interest in the contents and character of food products, especially processed foods and supplements, will directly assist commercial enterprises pursuing the commercial scale isolation of limonoid glucosides by alleviating cost associated with in-house development of quality control methods. This research supports NP306, Component I, Quality Characterization, Preservation and Enhancement, d) Preservation and/or Enhancement of Quality and Marketability. Technology Transfer Number of Active CRADAS and MTAS: 7 Number of Non-Peer Reviewed Presentations and Proceedings: 2
Impacts
(N/A)
Publications
- Breksa III, A.P., Ibarra Jr, F.G. 2007. Colorimetric Method for the Estimation of Total Limonoid Aglycones and Glucoside Contents in Citrus Juices. Journal of Agricultural and Food Chemistry. (Article) 55(13):5013- 5017.
- Breksa III, A.P., Manners, G.D., Jacobs, R.A., Hasegawa, S. 2006. Citrus Limonoid Bioavailability in Humans. In: ACS Symposium Series No. 936, Potential Health Benefits of Citrus; Patil, B.S.; Turner, N.D.; Miller, E. G.; Brodbelt, J.S. Washington, DC: American Chemical Society. p. 95-101.
|
Progress 10/01/05 to 09/30/06
Outputs
Progress Report 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? Why does it matter? Citrus is recognized as a dietary component important to human nutrition. However, information about the role of the wide variety of natural chemicals (phytochemicals) present in citrus in human health and nutrition is limited. Relating specific phytochemicals or mixtures of phytochemicals to the improvement of human health would reinforce the importance of citrus in the human diet, promote increased citrus consumption and generally contribute to the improvement of human wellness. The identification of specific citrus phytochemicals beneficial to human health in citrus juice processing waste streams will provide the citrus processing industry an opportunity to realize increased revenues through the reclamation of these biologically active phytochemicals from these waste streams. Increased
demand for citrus with health promoting phytochemicals will expand existing citrus markets and open new markets for citrus varieties containing specific healthful phytochemicals. Understanding the origins of these phytochemicals in citrus will allow the development of genetic and molecular biological methods to enhance the amounts of these chemical compounds in citrus and will provide access to the biological mechanisms associated with the maintenance of citrus quality. Successful manipulation of these processes will lead to an expanded selection of higher quality citrus with increased content of recognized healthful phytochemicals that are beneficial to consumers. This research project primarily focuses on a specific class of biologically active phytochemicals (limonoids) that occur in large amounts in citrus juice and citrus processing by-products. Limonoids occur in orange juice in amounts comparable to vitamin C and it is estimated that 15,000 tons of limonoids are available
annually in by- products of worldwide citrus juice production. Research evidence has established that limonoids possess significant anti-cancer activity in animal tests and these compounds have been shown to become available to act in humans after ingestion. This research project will specifically assess pure citrus limonoids as agents that can act to intercede in preventative actions in humans that result in improved health. The research will concurrently develop methods to reclaim health-promoting limonoids from citrus processing by-products. Information about the health-promoting properties of specific citrus limonoids will also provide the basis for the biological examination of a variety of citrus species to accumulate new information to guide genetic or molecular biological manipulation protocols directed to generating new or improved high quality limonoid enriched citrus varieties. The project's research activities fall within Problem Area 2a. New Product Technology of
Component 2. New Processes, New Uses, and Value- Added Foods of NP306. Customers for the results of this research include citrus processors, citrus growers, the scientific community and the general public. 2. List by year the currently approved milestones (indicators of research progress) FY05 - Inventory amounts and purity of currently held limonoids, purify existing isolated limonoids, establish synthetic procedures for production of compounds for structure/activity studies with currently held limonoids, identify citrus sources for extraction of new limonoids/secondary metabolites, begin collection of citrus materials, develop bioassay-guided screening protocols, identify and establish bioassay screening resource. Human cholesterol/inflammation study - Marshal resources, establish study protocol and submit for clearance, accumulate limonin glucoside materials and prepare for study. Animal metabolism/toxicity study Accumulate pure limonoids for study. FY05 - Samples from
commercial citrus processing co-product streams will collect, limonoid glucoside content of co-product streams will be obtained, promising co-product streams will be selected and appropriate materials collected, initial separation of limonoid glucosides based upon patented technology will be conducted, methodology for isolation of pure limonoid glucosides will be formulated. FY05 - Characterization and engineering of limonoid glucosyltransferase (LGTs)-Complete collaborative DNA and protein sequence analysis of LGT, clone promising enzymes and express as recombinants for additional characterization. Characterization of Limonin D-ring Lactone Hydrolase (LDLHs) - Complete the cloning and recombinant expression of LDLH. Metabolic Profiling - Develop methods for the metabolic profiling of limonoid aglycones and glucosides in citrus cultivars and in collaboration with Tracy Kahn (UC Riverside) initiate the evaluation of metabolic profiling as a tool for selecting new cultivars. FY06, FY07
- Extract plant materials and evaluate extracts through bioassay screen protocols, identify extracts of interest, isolate and characterize biologically active components in extracts, assess toxicity of biologically active compounds, initiate synthetic procedures for structure/activity studies, begin accumulation of biologically active materials or the development of synthetic methods to provide adequate amounts for animal testing. Human cholesterol/inflammation study - Recruit study subjects, begin study, collect samples, analyze samples, report results, develop follow-up studies. Animal metabolism/toxicity study - Provide limonoids, obtain animals, begin study, record weight data, sacrifice animals, analyze tissues and fluids, report results, develop follow-up studies. FY06, FY07 - Formulated methodology for isolation of pure limonoid glucosides will be applied and altered to maximize yields, methodology alteration to accommodate raw material character (i.e., molasses vs. seed
extracts) will be developed, methodology will be maximized for efficiency, pilot scale-up will be formulated, methodology adjustment or development will begin to accommodate accumulation of new biologically active compounds identified in objective 1. Patent(s) will be submitted for lab- proven technology. FY06, FY07 - Characterization and engineering of LGTs - Engineer LGT for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Characterization of LDLHs - In collaboration with Jose Chapparro (USDA) complete the DNA and protein sequence analysis of hydrolases derived from various citrus cultivars. Evaluate the activity of hyrdolases derived from different cultivars and clone and express as recombinants for further characterization those enzymes identified with promising properties will be for additional characterization. Metabolic Profiling - Continue the evaluation of metabolic profiling of limonoid
aglycones and glucosides over the growing season and draw a conclusion to the usefulness of the method. FY08, FY09 - Report structure/activity results, establish bioactivity criteria and develop protocols for animal model tests of biologically- active compounds, develop analytical methodology to detect metabolites, conduct animal tests, identify metabolites, characterize pharmacokinetics, evaluate potential for human application, formulate plans for human tests. Human cholesterol/inflammation study Begin planning for further studies. Animal metabolism/toxicity study Begin planning for further studies. FY08, FY09 - Formulation of pilot-scale scale-up, CRADA partner search, pilot scale-up, pilot scale process validation, patent licensing, industrial scale application. FY08, FY09 - Characterization and engineering of LGTs - Optimize enzyme activity and generate additional transgenic plants and seek to patent the engineered enzyme. Characterization of LDLHs - Engineer hydrolase for
improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Metabolic Profiling - Release a metabolic profiling of limonoids methods to be used by breeders and researchers. 4a List the single most significant research accomplishment during FY 2006. Cholesterol Lowering Potential of Limonoid Glucosides. Limonoid glucosides, found in citrus processing by-products, offer potential as cholesterol lowering agents to improve human health. Researchers in the Processed Foods Research Unit, Albany, CA are collaborating with the Western Regional Nutrition Research Center, Davis, CA to perform a human clinical feeding study funded by a trust agreement with an industrial partner. Large amounts of pure limonoid glucosides are being extracted using a patent pending method. The human study will begin by the end of fiscal year 2006 and will be the most comprehensive evaluation of limonoid glucosides ever. Validation of the
cholesterol lowering properties should provide an economic incentive for the citrus processing industry to utilize their by-products as sources of valuable health promoting compounds. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306. 4b List other significant research accomplishment(s), if any. Potential Antioxidant Activity of Limonoids. Biological activity of many naturally occurring compounds has been correlated with their ability to act as antioxidants. Recurring reports of citrus limonoid antioxidant activity are present in the literature. Researchers in the Processed Foods Research Unit, Albany, CA found unequivocal evidence that citrus limonoids do not act as antioxidants. This information will correct existing literature and support future development of more accurate analytical profiles for determination of the antioxidant activity of naturally occurring compounds. This research supports Component 2, New Processes, New Uses,
and Value-Added Foods of NP306. 5. Describe the major accomplishments to date and their predicted or actual impact. Biological Activity of Citrus Limonoid Glucosides. It has been shown that limonoid glucosides in citrus are active as anti-tumor agents, possess anti-infective properties and act as anti-feedants for insects. Researchers in the Processed Foods Research Unit, Albany, CA, in collaboration with researchers in the Western Regional Nutrition Research Center, Davis, CA discovered for the first time that citrus limonoids commonly present in orange juice are bioavailable to humans up to 24 hours after ingestion. This research supports citrus as an essential component in the human diet. Correlation of the biological activity of citrus limonoids to improving human health also provides incentives to the citrus industry to isolate these compounds from citrus processing by- products. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306.
Manufacture of Limonoid Glucosides from Citrus Processing By-Products. Scientists in the Processed Foods Research Unit, Albany, CA, developed an improved method for the isolation of pure limonoid glucosides and metal salts of limonoid glucosides (patent application submitted). This method utilizes selective preparative chromatographic methods to purify limonoid glucosides present in citrus processing by-products including citrus molasses, peel wash, pulp wash and seeds. This process is currently the only method for the manufacture of pure limonoid glucosides from citrus sources. These compounds have been shown to have significant anti-tumor activity in in vitro human cancer cell line tests and have been shown to be bioavailable when fed to humans. They have high potential as nutraceuticals and chemopreventatives for the improvement of human health and the patent pending method is available for utilization by the citrus industry. This research supports Component 2, New Processes,
New Uses, and Value-Added Foods of NP306. Method to Evaluate Citrus Juice Quality. A juice quality method for measuring the juices' potential of forming delayed-bitterness was developed by scientists in the Processed Foods Research Unit, Albany, CA. Freeze damage or physical damage to citrus fruit, including juicing, initiates the formation of the bitter dilactone limonoids from non-bitter monolactone precursors. This phenomenon is referred to as delayed bitterness and is a major problem for both fresh fruit and juice producers worldwide. We developed a rapid and sensitive method for the quantification of limonin and nomilin precursors to the development of bitterness. This method provides a valuable tool for citrus growers and juice producers to evaluate the susceptibility of a fruit or juice to delayed bitterness. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306. Extraction Methods for Analysis of Citrus Leaf Proteins. Human encroachment
on land traditionally used for citrus cultivation, environmental changes, emerging diseases and pests, in addition to changing consumer preferences have driven both researchers and commercial producers of citrus to search for methodologies to reduce the time and financial costs in generating new citrus varieties. A proteomics approach using two-dimensional electrophoresis analysis in combination with mass spectrometry has the potential to be a powerful tool in the selection and evaluation of new varieties. However, realization of the full potential of two-dimensional electrophoresis (2-DE) separation of citrus proteins is dependent on good sample preparation. Researchers in the Processed Foods Research Unit, Albany, CA, have developed general procedures for the extraction of Citrus leaf proteins for analysis by 2- DE. This research supports Component 2, New Processes, New Uses, and Value-Added Foods of NP306. 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 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 application for a second patent has been submitted. An Australian company is currently isolating a mixture of limonoid glucosides from a citrus processing by-product. This company has expressed interest in the isolation of pure limonoid glucosides from the same material and may be interested in licensing the newest patent. 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 an externally funded research program to examine the potential cholesterol- lowering effects of citrus limonoids in humans that will get underway in 2006. 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.
Impacts
(N/A)
Publications
- A.P. Breksa III, G.D. Manners, 2006. Evaluation of the Antioxidant Capacity of Limonin, Nomilin and Limonin Glucoside. Journal of Agricultural and Food Chemistry. V54:3827-3831.
- Manners, G.D., Breksa III, A.P. 2005. Manufacture of limonoid compounds. Patent Application, No. 11/289/240, June 1, 2006.
|
Progress 10/01/04 to 09/30/05
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 is recognized as a dietary component important to human nutrition. However, information about the role of the wide variety of natural chemicals (phytochemicals) present in citrus in human health and nutrition is limited. Relating specific phytochemicals or mixtures of phytochemicals to the improvement of human health would reinforce the importance of citrus in the human diet, promote increased citrus consumption and generally contribute to the improvement of human wellness. The identification of specific citrus phytochemicals beneficial to human health in citrus juice processing waste streams will provide the citrus processing industry an opportunity to realize increased revenues through the reclamation of these biologically active phytochemicals from these waste streams. Increased demand for
citrus with health promoting phytochemicals will expand existing citrus markets and open new markets for citrus varieties containing specific healthful phytochemicals. Understanding the origins of these phytochemicals in citrus will allow the development of genetic and molecular biological methods to enhance the amounts of these chemical compounds in citrus and will provide access to the biological mechanisms associated with the maintenance of citrus quality. Successful manipulation of these processes will lead to an expanded selection of higher quality citrus with increased content of recognized healthful phytochemicals that are beneficial to consumers. This research project primarily focuses on a specific class of biologically active phytochemicals (limonoids) that occur in large amounts in citrus juice and citrus processing by-products. Limonoids occur in orange juice in amounts comparable to vitamin C and it is estimated that 15,000 tons of limonoids are available annually in by-
products of worldwide citrus juice production. Research evidence has established that limonoids possess significant anti-cancer activity in animal tests and these compounds have been shown to become available to act in humans after ingestion. This research project will specifically assess pure citrus limonoids as agents that can act to intercede in preventative actions in humans that result in improved health. The research will concurrently develop methods to reclaim health-promoting limonoids from citrus processing by-products. Information about the health-promoting properties of specific citrus limonoids will also provide the basis for the biological examination of a variety of citrus species to accumulate new information to guide genetic or molecular biological manipulation protocols directed to generating new or improved high quality limonoid enriched citrus varieties. 2. List the milestones (indicators of progress) from your Project Plan. FY05 - Inventory amounts and purity of
currently held limonoids, purify existing isolated limonoids, establish synthetic procedures for production of compounds for structure/activity studies with currently held limonoids, identify citrus sources for extraction of new limonoids/secondary metabolites, begin collection of citrus materials, develop bioassay-guided screening protocols, identify and establish bioassay screening resource. Human cholesterol/inflammation study - Marshal resources, establish study protocol and submit for clearance, accumulate limonin glucoside materials and prepare for study. Animal metabolism/toxicity study Accumulate pure limonoids for study. FY05 Samples from commercial citrus processing co-product streams will collected, limonoid glucoside content of co-product streams will be obtained, promising co-product streams will be selected and appropriate materials collected, initial separation of limonoid glucosides based upon patented technology will be conducted, methodology for isolation of pure
limonoid glucosides will be formulated. FY05 Characterization and engineering of limonoid glucosyltransferase (LGTs)-Complete collaborative DNA and protein sequence analysis of LGT, clone promising enzymes and express as recombinants for additional characterization. Characterization of Limonin D-ring Lactone Hydrolase (LDLHs) - Complete the cloning and recombinant expression of LDLH. Metabolic Profiling - Develop methods for the metabolic profiling of limonoid aglycones and glucosides in citrus cultivars and in collaboration with Tracy Kahn (UC Riverside) initiate the evaluation of metabolic profiling as a tool for selecting new cultivars. FY06, FY07 Extract plant materials and evaluate extracts through bioassay screen protocols, identify extracts of interest, isolate and characterize biologically active components in extracts, assess toxicity of biologically active compounds, initiate synthetic procedures for structure/activity studies, begin accumulation of biologically active
materials or the development of synthetic methods to provide adequate amounts for animal testing. Human cholesterol/inflammation study - Recruit study subjects, begin study, collect samples, analyze samples, report results, develop follow-up studies. Animal metabolism/toxicity study Provide limonoids, obtain animals, begin study, record weight data, sacrifice animals, analyze tissues and fluids, report results, develop follow-up studies. FY06, FY07 Formulated methodology for isolation of pure limonoid glucosides will be applied and altered to maximize yields, methodology alteration to accommodate raw material character (i.e., molasses vs. seed extracts) will be developed, methodology will be maximized for efficiency, pilot scale-up will be formulated, methodology adjustment or development will begin to accommodate accumulation of new biologically active compounds identified in objective 1. Patent(s) will be submitted for lab- proven technology. FY06, FY07 Characterization and
engineering of LGTs - Engineer LGT for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Characterization of LDLHs - In collaboration with Jose Chapparro (USDA) complete the DNA and protein sequence analysis of hydrolases derived from various citrus cultivars. Evaluate the activity of hyrdolases derived from different cultivars and clone and express as recombinants for further characterization those enzymes identified with promising properties will be for additional characterization. Metabolic Profiling - Continue the evaluation of metabolic profiling of limonoid aglycones and glucosides over the growing season and draw a conclusion to the usefulness of the method. FY08, FY09 Report structure/activity results, establish bioactivity criteria and develop protocols for animal model tests of biologically- active compounds, develop analytical methodology to detect metabolites, conduct animal tests, identify
metabolites, characterize pharmacokinetics, evaluate potential for human application, formulate plans for human tests. Human cholesterol/inflammation study Begin planning for further studies. Animal metabolism/toxicity study Begin planning for further studies. FY08, FY09 Formulation of pilot-scale scale-up, CRADA partner search, pilot scale-up, pilot scale process validation, patent licensing, industrial scale application. FY08, FY09 Characterization and engineering of LGTs - Optimize enzyme activity and generate additional transgenic plants and seek to patent the engineered enzyme. Characterization of LDLHs - Engineer hyrdolase for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Metabolic Profiling - Release a metabolic profiling of limonoids methods to be used by breeders and researchers. 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. Inventory amounts and purity of currently held limonoids, purify existing isolated limonoids, establish synthetic procedures for production of compounds for structure/activity studies with currently held limonoids, identify citrus sources for extraction of new limonoids/secondary metabolites, begin collection of citrus materials, develop bioassay guided screening protocols, identify and establish bioassay screening resource. Human cholesterol/inflammation study - Marshal resources, establish study protocol and submit for clearance, accumulate limonin glucoside materials and prepare for study. Animal metabolism/toxicity study Accumulate pure limonoids for study. Milestone Substantially Met 2. Samples from commercial citrus processing co-product streams will collected, limonoid glucoside content of co-product streams will be obtained, promising co-product streams will be selected and appropriate materials
collected, initial separation of limonoid glucosides based upon patented technology will be conducted, methodology for isolation of pure limonoid glucosides will be formulated. Milestone Substantially Met 3. Characterization and engineering of LGTs -Complete collaborative DNA and protein sequence analysis of LGT, clone promising enzymes and express as recombinants for additional characterization. Characterization of LDLHs- Complete the cloning and recombinant expression of LDLH. Metabolic Profiling - Develop methods for the metabolic profiling of limonoid aglycones and glucosides in citrus cultivars and in collaboration with Tracy Kahn (UC Riverside) initiate the evaluation of metabolic profiling as a tool for selecting new cultivars. 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? FY06, FY07 Extract plant
materials and evaluate extracts through bioassay screen protocols, identify extracts of interest, isolate and characterize biologically active components in extracts, assess toxicity of biologically active compounds, initiate synthetic procedures for structure/activity studies, begin accumulation of biologically active materials or the development of synthetic methods to provide adequate amounts for animal testing. Human cholesterol/inflammation study - Recruit study subjects, begin study, collect samples, analyze samples, report results, develop follow-up studies. Animal metabolism/toxicity study Provide limonoids, obtain animals, begin study, record weight data, sacrifice animals, analyze tissues and fluids, report results, develop follow-up studies. FY06, FY07 Formulated methodology for isolation of pure limonoid glucosides will be applied and altered to maximize yields, methodology alteration to accommodate raw material character (i.e., molasses vs. seed extracts) will be
developed, methodology will be maximized for efficiency, pilot scale-up will be formulated, methodology adjustment or development will begin to accommodate accumulation of new biologically active compounds identified in objective 1. Patent(s) will be submitted for lab proven technology. FY06, FY07 Characterization and engineering of LGTs - Engineer LGT for improved activity, characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Characterization of LDLHs - In collaboration with Jose Chapparro (USDA) complete the DNA and protein sequence analysis of hydrolases derived from various citrus cultivars. Evaluate the activity of hyrdolases derived from different cultivars and clone and express as recombinants for further characterization those enzymes identified with promising properties will be for additional characterization. Metabolic Profiling - Continue the evaluation of metabolic profiling of limonoid aglycones and glucosides
over the growing season and draw a conclusion to the usefulness of the method. FY08, FY09 Report structure/activity results, establish bioactivity criteria and develop protocols for animal model tests of biologically active compounds, develop analytical methodology to detect metabolites, conduct animal tests, identify metabolites, characterize pharmacokinetics, evaluate potential for human application, formulate plans for human tests. Human cholesterol/inflammation study Begin planning for further studies. Animal metabolism/toxicity study Begin planning for further studies. FY08, FY09 Formulation of pilot-scale scale-up, CRADA partner search, pilot scale-up, pilot scale process validation, patent licensing, industrial scale application. FY08, FY09 Characterization and engineering of LGTs - Optimize enzyme activity and generate additional transgenic plants and seek to patent the engineered enzyme. Characterization of LDLHs - Engineer hyrdolase for improved activity,
characterize engineered enzymes and validate activity in transgenic cells or plants through a collaboration. Metabolic Profiling - Release a metabolic profiling of limonoid methods to be used by breeders and researchers. 4a What was the single most significant accomplishment this past year? The manufacture of pure limonoid glucosides from citrus processing by- products. Scientists in the Processed Foods Research Unit, Albany, CA, have developed an improved method for the isolation of pure limonoid glucosides and metal salts of limonoid glucosides (patent application submitted). This method utilizes selective preparative chromatographic methods to purify limonoid glucosides present in citrus processing by- products including citrus molasses, peel wash, pulp wash and seeds. The process utilizes only aqueous solutions (water, aqueous acid and base) and alcohol, and enables the isolation of the pure limonoid glucosides and limonoid glucosides and limonoid A-ring lactones as alkali
metal acid salts. This process is currently the only method for the manufacture of pure limonoid glucosides from citrus sources. These compounds have been shown to have significant anti-tumor activity in in vitro human cancer cell line tests and have been shown to be bioavailable when fed to humans. They have high potential as nutraceuticals and chemopreventatives for the improvement of human health. 4b List other significant accomplishments, if any. A method to evaluate citrus juice quality through measuring the juices potential of forming delayed-bitterness was developed by scientists in the Processed Foods Research Unit, Albany, CA. Freeze damage or physical damage to citrus fruit, including juicing, initiates the formation of the bitter dilactone limonoids from non-bitter monolactone precursors. This phenomenon is referred to as delayed bitterness and is a major problem for both fresh fruit and juice producers worldwide. The major limonoids associated with delayed bitterness
are limonin and nomilin. We developed a rapid and sensitive method for the quantification of limonin and nomilin precursors: limonoate A-ring lactone (LARL) and nomilinoate limonoate A-ring lactone (NARL). This method provides a valuable tool for citrus growers and juice producers to evaluate the susceptibility of a fruit or juice to delayed bitterness. Established extraction methods for analysis of Citrus leaf proteins by two-dimensional gel electrophoresis. Human encroachment on land traditionally used for citrus cultivation, environmental changes, emerging diseases and pests, in addition to changing consumer preferences have driven both researchers and commercial producers of citrus to search for methodologies to reduce the time and financial costs in generating new citrus varieties. A proteomics approach using two-dimensional electrophoresis analysis in combination with mass spectrometry has the potential to be a powerful tool in the selection and evaluation of new varieties.
However, realization of the full potential of two-dimensional electrophoresis (2-DE) separation of citrus proteins is dependent on good sample preparation. Researchers in the Processed Foods Research Unit, Albany, CA, have developed general procedures for the extraction of Citrus leaf proteins for analysis by 2-DE. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The biological assessment of citrus have shown several these compounds to be active as anti-tumor agents, to possess anti-infective properties and to be anti-feedants for insects. This information has led to the development of an earlier patented isolation method to obtain mixture of limonoid glucosides as potential value-added products from citrus processing by-products. The determination, by WRRC/ARS and WHNRC/ARS scientists, that citrus limonoids commonly present in orange juice are bioavailable to humans has provided increased rationale for methodology to
access pure limonoids from citrus processing by-products. WRRC/ARS scientists have developed this methodology within the scope of this research project (patent application submitted) and are currently obtaining pure limonoid glucoside from citrus processing by-products to utilize in a human feeding study to determine if these compounds can act to lower cholesterol in humans. Access to pure limonoid glucosides is necessary to confirm their role in human health. The absolute correlation of activity of these compounds to the lowering of cholesterol, the prevention of cancer or the control of infectious diseases in humans would have significant impact on human wellbeing and would strongly endorse citrus as an essential component in the human diet. Correlation of the biological activity of citrus limonoids to improving human health would also supply incentive to the citrus industry to isolate these compounds from citrus processing by-products. The methodology WRRC/ARS scientists have
developed for the isolation of these compounds will be available for utilization by the citrus industry when the industry determines market factors favorable to gain value-added revenue from limonoid glucoside reclamation. 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 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 application for a second patent has been submitted. An Australian company is currently isolating a mixture of limonoid glucosides from a citrus processing by-product. This company has expressed interest in the isolation of pure limonoid glucosides from the same material
and may be interested in licensing the newest patent. 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 an externally funded research program to examine the potential cholesterol- lowering effects of citrus limonoids in humans that will get underway in late 2005. 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). "Citrus Compound Ready to Help Your
Body!", Agricultural Research Magazine, February 2005. p. 16-17.
Impacts
(N/A)
Publications
- Zukas, A.A., Breksa III, A.P. 2005. Extraction Methods for Analysis of Citrus Leaf Proteins by Two-Dimensional Electrophoresis. Journal of Chromatography A. V. 1078:201-205.
- Manners, G.D., Breksa III, A.P. 2004. Identifying Citrus Limonoid Aglycones by HPLC-EI/MS and HPLC-APCI/MS Techniques. Phytochemical Analysis. V. 15:372-381.
- Breksa III, A.P., Zukas, A.A., Manners, G.D. 2005. Determination of Limonoate and Nomilinoate A-ring Lactones in Citrus Juices by Liquid Chromatography-Electrospray Ionization Mass Spectrometry. Journal of Chromatography A. V. 1064:187-191.
- Zukas, A.A., Breksa III, A.P., Manners, G.D. 2004. Isolation and Characterization of Limonoate and Nomilinoate A-Ring Lactones. Phytochemistry. V. 65:2705-2709.
- Zukas, A.A., Breksa III, A.P. 2004. Extraction methods for analysis of citrus leaf proteins by two-dimensional gel electrophoresis. ACS Symposium, Paper #74, October 2004, Sacramento, CA.
|
|