BIOACTIVE CONSTITUENTS AND SPECIALTY FOOD FIBERS AS VALUE-ADDED PRODUCTS FROM CITRUS PROCESSING WASTE

Objective 1. Major objectives of this project were aimed at developing new products from citrus bioactive constituents and specialty food fibers derived from citrus processing waste streams. The first objective involved the study of the metabolism and pharmacokinetics of selected citrus peel flavonoids in an effort to understand the chemical factors influencing bioavailability and efficacy of these compounds. To this end, metabolites of citrus peel compounds, including the main flavanone glycosides eriocitrin and hesperidin, and the diverse polymethoxylated flavones, termed the polymothoxylated flavones, were purified and identified from samples obtained from pharmacokinetic investigations in rats and in humans. Early studies focused on the detection and identification of the main metabolites of nobiletin and tangeretin in rats. In addition to the identification of these compounds, the pharmacokinetic time courses of these compounds and of their metabolites were characterized. These studies showed the occurrence of major differences in the metabolism and pharmacokinetics of the polymothoxylated flavones and the flavanone glycosides. Yet, upon further chemical breakdown of these compounds, strong similarities in the chemical species present in the animal tissues were discovered. In a different human clinical trial conducted by a collaborator at Sao Paulo State University, 24 volunteers ingested two different doses of citrus flavonoids and extracts of plasma and urine samples were prepared for analyses by high performance liquid chromatography mass spectrometry. The metabolites in these tissue extracts were subsequently quantitatively and qualitatively analyzed. Flavonoid metabolites in humans exclusively occurred as sulfate and/or glucuronic acid conjugates. Negligible amounts of ring fission products occurred, thus eliminating this class of compounds as major contributors to beneficial biological actions in humans. The rates of appearance of the main metabolites in human plasma samples were measured and studied as a function of different orange juice extraction techniques (fresh squeezed versus commercially processed juice) . The two juices had notably different profiles of soluble and precipitate compounds, yet the results showed no difference in bioavailability of the orange juice compounds in the two styles of juices. The results of these studies provided evidence of additional metabolites originating from the minor-occurring polymethoxylated flavones and hydroxycinnamates in orange juice (also potentially healthful compounds). This is the first description of polymethoxylated flavone metabolites from orange juice measured in human studies. This is notably different from observations made with similar citrus peel flavonoids fed to rats. Isolation of metabolites of citrus peel polymethoxylated flavones allowed the quantification of this class of flavonoids in the human samples. These preliminary pharmacokinetic data will be immediately applicable to efforts to identify and validate beneficial biological effects of these citrus compounds in emerging new commercial products. In a separate study, the metabolites of eriocitrin, the major flavonoid in lemons, were analyzed in rat urine and blood samples in a pharmacokinetics feeding trial. Biological activities of these compounds were studied in several in vivo inflammation trials in mice fed a special high-fat diet and dramatic protective anti-inflammatory effects occurred. The induction of two powerful proinflammatory signaling proteins, IL-6 and MCP-4 was blocked. Recent work involved large scale purifications of three different polymothoxylated flavones for additional rat feeding trials with the aim to characterize the wide range of metabolites, which have thus far been detected in 4 main classes. Fifteen compounds were isolated and will be evaluated for their chemical structures and subsequently used in in vitro studies. Since these are metabolites already occurring in animals, the small-animal trials can be eliminated. Progress was also made in understanding the chemical mechanisms of grapefruit-drug interactions caused by grapefruit juice phytochemicals. A new fungal metabolite of an important grapefruit furanocoumarin was detected and structurally characterized, and was found to lack CYP3A4 inhibitory activity. CYP3A4 inhibitory activity of most grapefruit furanocoumarins is responsible for the grapefruit/drug interactions that occur in humans consuming both grapefruit and certain prescription medications. It was hypothesized that the ability to modify these grapefruit compounds by fungi may lead to new technologies to eliminate these deleterious drug interactions in humans. The sequestration of grapefruit furanocoumarins by foods was investigated by characterizing the binding between these compounds and foods with contrasting protein, fat, and carbohydrate compositions. Individual grapefruit furanocoumarins exhibited contrasting affinities to foods, where the lipophilic bergamottin and several structurally related dimers bound to foods more tightly than the more polar 6',7'-dihydroxybergamottin. From the investigation of different classes of macromolecules in foods, water- soluble proteins were found to be the major constituents responsible for furanocoumarin sequestration. Studies using bovine serum albumin as a model protein demonstrated the dissociation of grapefruit furanocoumarins from the insoluble juice cloud particles and the subsequent formation of water-soluble bovine serum albumin-furanocoumarin complexes. Fluorescence binding assays further demonstrated the binding of bergamottin and 6',7'- dihydroxybergamottin to bovine serum albumin. These results demonstrate that proteins can be sequestration agents of these important dietary furanocoumarins. Objective 2. Progress was also made in two separate projects to detect biomarker compounds for citrus canker and Huanglongbing diseases. Key differences were detected in the profiles of compounds of healthy versus Huanglongbing-affected leaves and juice. Of particular importance was the discovery of the elevated levels of the bitter-tasting limonoids in Huanglongbing-symptomatic fruit. This marker of Huanglongbing-off-flavor has been widely adopted by the industry. The limonoids are first biosynthesized in the phloem, then transported to other plant tissues, and it is in the phloem that the Huanglongbing bacterial pathogen occurs. It was hypothesized that certain compounds in the phloem are essential to the growth of the Huanglongbing-causing bacterium. Extensive high performance liquid chromatograpy studies were done to identify and catalog these compounds including the energy requiring nucleotides, nucleosides, amino acids, and the secondary natural products, flavonoids, and hydroxycinnamates. High levels of the bitter-tasting limonin and nomilin were detected in the phloem tissues. Other work evaluated natural compounds for their antimicrobial activity against the citrus canker-causing bacterium Xanthomonas citri and problematic decay pathogens. Our investigations showed that the chemical make-up of canker lesions changes as the canker ages, and that there are increased levels of fluorescent coumarins in the lesions. Three of the four main compounds formed in response to the infection of citrus tissues by the pathogenic bacterium Xanthomonas citri were identified and quantified. Three additional compounds structurally related to these �phytoalexins� were identified. Other disease-induced compounds, including compounds newly observed in citrus, were detected and partially isolated. Objective 3. Progress was also made in enhancing the recovery of citrus peel bioactive compounds from steam-explosion technology. Steam explosion of either whole fruit or peel greatly improves pectin extraction, as well as the extraction of many other fruit compounds. New finding on the contents of water washes of steam-exploded fruit and peel, point to high recoveries of the fruit�s water soluble hydroxycinnamates, limonoid glucosides, and almost a third of the total polymothoxylated flavones. These clarified water washes could provide major portions of the world demand for many of these compounds based on mass balance studies of these wash fractions and total crop production. An additional application of this method is the product recovery from the fruit affected by the Huanglongbing disease. Due to this disease, large percentages of the total crop are lost to fruit drop, undersized fruit, or poor quality fruit. The recovery of the marketable compounds from the steam-explosion process represents a new opportunity for valuable co- product recovery from this �lost� portion of the citrus crop. Objective 4. The nanostructure of the pectin molecule is important for development of pectin products for food, medical or industrial use. Research into the polysaccharide structures of citrus byproducts used pectin methylesterase from commercial papaya enzyme extract to demethylate model pectin molecules. The resulting modifications to the pectin nanostructure were characterized, and indicate that reaction conditions (i.e., pH and enzyme/substrate ratios) affect the introduced nanostructural motifs. Accomplishments 01 Inhibition of inflammation occurring with high-fat diet in mice. Citrus compounds, termed flavonoids, have been shown to inhibit inflammation in certain animal assays and the study was conducted to test if these compounds could inhibit the oxidative stress and systemic inflammation induced by high-fat diet in mice. ARS researchers at Ft. Pierce, Florida and at Sao Paulo State University showed that the examined citrus flavonoids had protective effects against the inflammation and oxidative stress caused by the high-fat diet in mice and, therefore, prevented metabolic alterations associated with the development of cardiovascular diseases. This study supports the use of citrus as a health-promoting food, and increases the demand and value for these citrus byproduct flavonoids.