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Jose L. Perez, G.K. Jayaprakasha, and Bhimanagouda S. Patil

Grapefruit has potential health-promoting properties due to the presence of multitude bioactive compounds. Ongoing cell culture and animal studies in our lab using limonoids and flavonoids have provided strong evidence of their protective properties for preventing chronic diseases. Studies related to D-glucarate, a natural, nontoxic bioactive compound found in grapefruit, has not been explored. One of the derivatives, such as D-glucaro-1,4-lactone, is reported to be a potent ß-glucuronidase inhibitor. With the inhibition of ß-glucuronidase enzyme, glucuronidation will be favored. Glucuronidation is a conjugation process through which potentially carcinogenic environmental toxins can be neutralized. In this context, quantification of glucarate using HPLC was developed. Samples from grapefruits were prepared by heating fruit extract with distilled water. Further, the extract was homogenized and centrifuged. The supernatant was treated with petroleum ether to remove non-polar substances. Then the extract was subject to ion exchange chromatography. Fractions were collected and analyzed by analytical HPLC for the quantification of D-glucarate content and its lactone. This project was supported by the USDA-CSREES grant for Designing Foods for Health through the Vegetable and Fruit Improvement Center.

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Xin Zhao, Edward Carey, James Nechols, Kim Williams, and Weiqun Wang

Implications of dietary phenolic compounds for human health and disease prevention have been indicated by a body of literature. A greenhouse pot study was performed to investigate the impacts of fertilizer source and preventive insecticide application on phenolic compound levels in pac choi [Brassica rapa (L.) cv. Mei Qing]. A two-way randomized complete-block design with five replications was used in this experiment. Fertilizer source consisted of two levels: conventional fertilizer (pre-plant application of Osmocote slow-release fertilizer), and organic fertilizer (pre-plant application of vermicompost and fertigation with compost tea and fish emulsion). Insecticide application consisted of three levels: organic (pyrethrin) vs. conventional (permethrin), and a plain water control. At harvest, total phenolics and individual phenolic compounds in pac choi leaves (blades) were analyzed by Folin assay and HPLC, respectively. Head weight of pac choi was significantly higher under conventional fertilizer treatment, while it was not affected by insecticides. Total phenolic content of pac choi was significantly increased by organic fertilizer treatment. HPLC results indicated that organic fertilizer treatment resulted in significantly higher levels of individual phenolic compounds, including chlorogenic acid and ferulic acid. In contrast, preventive insecticide application showed little effect on the phenolics in pac choi. Correlation analysis excluded the influence of plant size (head weight) on phenolic content in pac choi. Differential N-forms, rates of nutrient release, and/or variable nutrient content in organic and conventional fertilizer treatments may contribute to elevated phenolic content in organically fertilized pac choi.

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Shibu M. Poulose*, Jennifer S. Brodbelt, Leonard M. Pike, and Bhimanagouda S. Patil

Limonoids, chemically related triterpinoids predominantly found in citrus and neem relatives, are known to play a pivotal role in the prevention of different types of cancer and cardiovascular diseases. Since the concentrations of these compounds are low in the plant tissues, the isolation of pure compounds is the limiting factor for the individual activity studies in animal models. In this study, combinations of chromatographic techniques were used to isolate limonoid aglycones and limonoid glucosides from citrus byproducts such as seeds and molasses. The compounds were initially extracted with different polar solvents and the concentrated extracts were passed through a series of adsorbent resin (SP-70) and ion-exchange resins (WA-30, Dowex-50, Q-sepharose) to remove further impurities. The use of increasing ionic strength of NaCl from 0 to 800 mm to release the exchanged compounds from the ion exchange columns further separated the limonoids from flavonoids, which was confirmed through TLC, UV, and analytical HPLC methods. Individual compounds were further purified using flash chromatography and preparative HPLC methods and identified by using LC-MS analysis. Direct crystallization of limonin resulted in a 17% increase in the yield as compared to the previously reported methods. The results suggest that application of these purification methods are useful for the bulk purification of compounds in order to further investigate their biological activity.

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David Graper and Will Healy

Non flowering Alstroemeria `Regina' plants were divided into aerial components: stems and apical and basal leaves or underground components: rhizome, storage roots, stele and fibrous roots. Samples were collected from distal and proximal ends of the rhizome to allow comparisons between structures of different ages. Ethanol soluble sugars were extracted and measured using HPLC. Starch was degraded to glucose using amyloglucosidase and measured.

There were no age differences in the starch, total soluble sugar (TSUGAR) or total soluble carbohydrates (TCHO) in the rhizome or aerial portions of the plant. There was a preferential partitioning of starch, sucrose, TSUGAR and TCHO to underground plant parts. The storage roots were the primary sink for the stored carbohydrates. Stems contained large concentration of glucose while fructose was found in storage roots and old stems. Sucrose was found primarily in old steles and storage roots. Starch was partitioned almost exclusively into the storage roots with no difference due to age of the storage root. Up to 42% of the TCHO in the old storage roots was composed of a carbohydrate which co-chromatogramed with melezitose using HPLC.

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M.D. Whiting, G. Paliyath, and D.P. Murr

Apple fruits (Malus domestica Borkh. cv. `Red Delicious') stored for 6 months at 2°C in air were analyzed for headspace volatiles by SPME-GC and for surface components by HPLC of hexane extracts. Analysis of headspace volatiles evolved from whole fruit showed five major volatiles that were identified previously as: acetic acid, hexyl ester; hexanoic acid, butyl ester; octanoic acid, propyl ester; hexanoic acid, hexyl ester; and the sesquiterpene, α-farnesene. No significant differences existed in these volatiles between scald-developing and non-scald developing apples. To explore potential differences in volatile evolution, fruit developing scald were cut (axial plane) into scalding and non-scalding halves for analysis. In all cases, volatile emission was much higher from the non-scalding side of the fruit, and the ratio of volatile levels from non-scalding to scalding averaged greater that 2. Various regions of tissue from the same fruit were extracted in hexane for estimation of levels of α-farnesene and its potential catabolites by HPLC. The levels and proportions of the components were nearly identical to those observed during headspace volatile analysis of half fruit. The results suggest that there are potential differences in α-farnesene metabolism an/or permeability of apple cuticle to volatiles between scald-developing and non-scald developing regions of apple fruit.

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Jose E. Villarreal, Leonardo Lombardini, and Luis Cisneros-Zevallos

Pecans nuts from `Kanza' and `Desirable' cultivars were irradiated with 0, 1.5, and 3.0 kGy using electron beam (E-beam) irradiation and stored under accelerated conditions (40 °C and 55% to 60% RH). Antioxidant capacity (AC), phenolic (TP) and condensed tannin (CT) content, HPLC phenolic profile, tocopherol content, peroxide value (PV), and fatty acid profile were evaluated in kernels after 0, 7, 21, 55, and 134 days of storage. Irradiation had no detrimental effects in AC and TP; however, variation was found throughout storage. Tocopherol content of 1.5 and 3.0 kGy kernels decreased after irradiation, but no further decrease was observed thereafter. Irradiated `Desirable' samples had greater PV than controls, while `Kanza' 1.5 kGy samples had increased PV only after 134 days of storage. No change in fatty acid composition was detected for any cultivar. Color modification induced by storage included a decrease in lightness and yellowness and an initial increase of redness followed by a decrease after 98 days of storage. No differences in phenolic profile were observed after irradiation. Compounds identified by HPLC in hydrolyzed extracts were gallic and ellagic acid, catechin, and epicatechin. In general, beside the decrease in tocopherol content, no detrimental effects were found in antioxidant composition caused by irradiation treatments. While a faster oxidation rate was seen in irradiated kernels for `Desirable' cultivar, no other quality attribute was affected by E-beam irradiation.

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Jane E. Lancaster, Julie Farrant, and Martin L. Shaw

1 Scientist; e-mail: . 2 Research assistant. Research funded by New Zealand Institute for Research Science and Technology. Statistical assistance of Fred Potter and Ruth Butler and HPLC analytical assistance of Kevin Sutton is

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C.R. Brown, C.G. Edwards, C.-P. Yang, and B.B. Dean

and technical support for the HPLC analysis of these materials. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to

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Susan E. Trusty and William B. Miller

Exudation of phloem sap into EDTA (ethylenediaminetetraacetic acid) solutions has been found to be a successful technique for qualitatively determining translocated assimilates in many plants. Mature Chysanthemum leaves were excised under a solution of 10 mM EDTA (pH 7.0). The petioles of these leaves were placed in EDTA, and leaf exudate was collected at intervals for 24 h. Soluble carbohydrates were determined with HPLC. While numerous sugars were present in the leaf, sucrose was the only sugar found in the EDTA solutions. The greatest rate of sucrose exudation occurred in the first two h after excision. Diurnal fluctuations of soluble sugars in Chrysanthemum leaves were also monitored in greenhouse-grown plants (late winter in Arizona). Sucrose exhibited a clear diurnal fluctuation, and nearly doubled in concentration (to appx. 25 mg/g DWT) in the afternoon relative to the low in the morning. Other leaf carbohydrates, including glucose, starch, and fructans showed diurnal variations as well.

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Jorge H. Siller-Cepeda, Leslie Fuchigami, and Tony H. H. Chen

Many seeds of woody plants require low temperature or other treatments to overcome dormancy. Changes in catalase activity and glutathione has been proposed to be associated with the breaking of dormancy. We examined the level of glutathione and catalase activity of cherry seeds (Prunus mahaleb cv. Lambert) exposed to several dormancy breaking agents. Seeds imbibed in water for 24 hrs. were either stratified at 4°C or at 25°C for up to 12 weeks, or exposed to other dormancy breaking agents. Germination test, glutathione and catalase activity were determined weekly and/or after treatment. Analysis of levels and state of glutathione were performed by high pressure liquid chromatography (HPLC), and catalase activity was assayed spectrophotometrically. Total glutathione in dry and imbibed seeds were similar, but, ratio between the reduced and oxidized form were different. Low temperature stratification for 12 weeks increased the reduced form of glutathione six-fold, while percent germination increased up to 94%.