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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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C.B. Ely, R.E. Frans, T.L. Lavy, R.E. Talbert, and J.D. Mattice

Two-year-old highbush blueberry bushes (Vaccinium corymbosum L. `Collins') were treated in Mar. 1985 with diuron or simazine at 2.2 or 4.5 kg a.i./ha. No residues were detected by reverse-phase high-performance liquid chromatography-ultraviolet absorbance detection (HPLC-UV) from treated berries that were harvested in June. Methiocarb was applied in May 1986 at 0.84 and 3 kg·ha-1 over the top of 3-year-old `Collins' when the berries began to ripen. Reverse-phase HPLC-UV of berries treated with methiocarb at 3 kg·ha-1 had combined residues of methiocarb and its sulfone and sulfoxide metabolites of 13.1 ppm from unrinsed and 7 ppm from rinsed berries harvested on the day of treatment; 4.9 ppm from unrinsed and 4 ppm from rinsed berries harvested 4 days after treatment; and 2.4 ppm from unrinsed and 2.5 ppm from rinsed berries harvested 8 days after treatment. Unrinsed berries treated with methiocarb at 0.84 kg·ha-1 had 5.7 ppm residue on the day of treatment and 1 ppm 8 days later. Residues from berries treated with methiocarb at 0.84 or 3 kg·ha-1 were below the legal tolerance level of 5 ppm after the required 7-day waiting period. Chemical names used: n'-(3,4-dichlorophenyl)-N,N -dimethylurea (diuron); 6-chloro- N,N' -diethyl-1,3,5-triazine-2,4-diamine (simazine); 3,5-dimethyl-4-(methylthio)phenol methylcarbamate (methiocarb).

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Vicky W. Lee, H.P. Vasantha Rupasinghe*, and Chung-Ja Jackson

Apples are excellent sources of dietary phenolics, in particular flavonoids and chlorogenic acid, which are potent antioxidants that may play important roles in the prevention of chronic diseases. This study investigated the major phenolics profiles of apple fruit in relation to (1) the distribution among 8 Ontario-grown cultivars, (2) the different fruit parts, and (3) the effect of processing of fresh-cuts. In addition, total antioxidant capacity (TAC) and total phenols content (TPC) were measured in apples by spectrophotometric assays. Flavonoids and chlorogenic acid were quantified using HPLC/PDA. Vitamin C was quantified using HPLC/Fluorescence. TAC, TPC and flavonoids levels were the highest in Honey Crisp and Delicious, moderate in Idared, Spartan, Granny Smith, and Cortland, and the lowest in Crispin and Empire. Apple peel contained 2 to 10-fold higher TAC, TPC and total of 10 major phenolics than that of core and flesh indicating peeling of apples during processing could reduced significantly the nutritional quality of fresh-cut apples. Dihydrochalcone (phloridzin) and chlorogenic acid levels were 2 to 21-fold higher in apple core than skin and flesh. TAC levels and vitamin C contents could be increased up to 3-fold and 14 to 20-fold, respectively by the post-cut dipping treatment with an ascorbic acid-based antioxidant formula. The phenolic profiles of sliced apples were stable up to 21 days at 4°C.

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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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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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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%.

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Naoki Yamauchi, Xiao-Ming Xia, and Fumio Hashinaga

Effects of flavonoid pigments on chlorophyll (Chl) degradation by Chl peroxidase in the flavedo of Wase satsuma mandarin (Citrus unshiu Marc. var. praecox Tanaka) fruits were studied. Chl was degraded when hydrogen peroxide was added in a reaction mixture containing Chl and a phosphate buffer extract from the flavedo. Chlorophyllide, which was formed by the action of chlorophyllase in the extract, was also degraded. The flavonoid contents decreased with the Chl degradation in the reaction mixture. Analysis of the flavonoid with HPLC showed that hesperidin and narirutin were contained in the flavedo as a major flavonoid, and that the former decreased significantly and the latter showed almost no change with the Chl degradation in the reaction mixture. In the ethylene-treated fruits, the hesperidin content in the flavedo also decreased with the degreening of stored fruits, suggesting that the flavonoid oxidation by Chl peroxidase could be involved in the Chl degradation.

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Isa Bertline and Carol J. Lovatt

Tryptophan is known to be a precursor of IAA in plants. The amount of IAA available for the development of avocado fruit might be a limiting factor for its growth. It is well known that IAA is not transported into developing fruit along its strictly basipetal transport route. Therefore, IAA present in fruit must be synthesized in situ. We investigated the possibility that tryptophan or its metabolites are transported from leaf to fruit.

An HPLC method was developed to quantitatively isolate and measure tryptophan and all well known intermediates in the synthesis of IAA. Avocado leaves were fed L-[side chain-3-14C] tryptophan and its transport and metabolism to IAA within the leaf and within the fruit were monitored over time. Significant movement of tryptophan or a metabolite from leaf to fruit occurs in 24 h.