- Kobayashi, Hideka
- Kentucky State University
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- Objective 1. Conduct chromatographic and spectrometeric study on chemical constituents of American persimmon and spicebush teas.
Objective 2. Investigate and report the phenolic content and antioxidant capacity of these teas, in comparison to green tea.
Objective 3. Investigate apoptosis induction and cell cycle regulation as well as Cyclooxygenase and Quinone Reductase activities by American persimmon and spicebush teas.
High school and undergraduate students will be mentored by research partners to experience hands-on research projects and be trained in using scientific methods. Special attention will be paid to the recruitment of minority and female students who will gain experience from working in different laboratories, and hopefully emerge from this initiative to pursue careers in food and agricultural sciences through networking.
The success of this project should possibly encourage persimmon growers and producers to participate in utilization of American persimmon and spicebush. Active participation by researchers, growers and possibly pharmaceutical companies will further lead to growth of the medicinal plant industry in Kentucky and development of a medicinal plant program at Kentucky State University.
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- Non-Technical Summary:
The state of Kentucky is facing serious health-related issues. Partly because of high smoking rate, Kentucky has the highest cancer death rate anywhere in our nation. One approach to combat cancer is through a healthy diet, and incorporating consumption of tea into diet has been suggested. Tea [Camellia sinensis (L.) Kuntze] has been consumed all over the world, and anticancer and chemopreventitive properties of the green tea are well documented. Despite the popularity of tea, domestic production is currently very much limited to South Carolina and Hawaii. In addition, the possible pesticide contamination and high concentrations of mineral elements such as aluminum, chromium, copper, and fluoride in imported teas can pose a serious health risk. Thus, it is an imperative pursuit to explore utilizing leaves of common and native plant species such as American persimmon (Diospyros virginiana L.) and spicebush (Lindera benzoin L.) for tea. The objectives of this projects are to: 1) identify mineral and phytochemical constituents of American persimmon and spicebush teas with chromatographic and spectrometric methods; 2) demonstrate antioxidant capacities of these two teas by performing assays on phenolic content and antioxidant capacities; and to 3) examine anticancer properties of both teas by subjecting cancerous cell lines to both teas and analyzing by flow cytometry and enzymatic assays. The expected changes in knowledge due to this project include determination on mineral and phenolic contents, antioxidant capacity and health promoting properties of spicebush and American persimmon teas. The possible changes in actions from this project include promotion of these teas for consumption, production of potentially safer and less expensive alternative products of the common tea, developing new products based on leaves of these species, and also cultivation of these native species for tea production. The possible changes in condition include an income diversification for American persimmon growers, an improvement on health of rural residents in Kentucky, rediscovering and raising awareness on native plant species in their backyard for alternative uses.
Objective 1. Leaves from ten each spicebush will be collected from multiple locations from Ohio and Kentucky. Leaves of four American persimmon cultivars will be collected from the University of Kentucky Arboretum, and from collaborators' farms. The aqueous extracts of teas will be prepared as described previously. The measurements of tea mineral content will be carried out using an atomic absorption spectrometer, and an electrothermal atomizer. Thin layer chromatograpgy separations will be made on silica gel 60 F-254 glass plates (0.25 mm) and visualized by spraying with 5% phosphomolybdic acid in EtOH and heating. High performance liquid chromatography analyses will be performed with an ESA CoulArray Multi-Channel ECD system. The concentrated tea extracts from different sources will also be analyzed by gas chromatography. Objective 2. The amount of soluble phenolic content will be quantified by Folin-Ciocalteu assay. The absorbance reading at 90 min. will be used to determine the total PC, expressed in mg of gallic acid equivalent per 100 g of dry weight. Antioxidant capacity of teas will be quantified by a modified ferric reducing antioxidant power assay. For conversion of antioxidant power in expressed in uM of Trolox equivalent per g of fresh weight, the absorbance readings at 60 min. will be used. Antioxidant capacity of RA-CD complexes will be measured by the CUPRAC method. The Trolox equivalent antioxidant capacities will be calculated by dividing the molar absorptivity of free and complexed polyphenol to the molar absorptivity of Trolox. Free radical scavenging activity will be evaluated using a benchtop electron spin resonance spectrometer. 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity will be measured using the method. Objective 3. COX-1 and COX-2 assays will be done by measuring prostaglandin E2 production from arachidonic acid using an enzyme-linked immunosorbent assay method. Induction of QR activity will be calculated from the ratio of specific enzyme activities of sample-treated cells in comparison with a solvent control, and CD values (g/ml, the concentration of sample needed to double QR induction) will be generated based on the average of two replicates. In addition, cytotoxicity of the tested crude extracts will be determined by comparing the response of the cells with that of the control. IC20, IC50 and IC90 values will be calculated by analysis of the percent inhibition of each tea extract or standard at ten concentrations. Two flow cytometric analyses will be performed. Cells after treatments will be analyzed for cell cycle and condensed chromatin apoptosis assay. HepG2 cells will be used for this assay, and cells analysis will be conducted on a flow Finally, Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay will be conducted following the instructions for a commercial in situ apoptosis detection kit supplied. Results from this project will be discussed and peer eveluated at regular meetings, and will be also presented at scientific conferences and published in journals and extension publication and other materials.
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- Nat'l. Inst. of Food and Agriculture
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