Isolation and Characterization of a Suspected Phytoalexin from Wilted Red Maple Leaves by Jared T. Baisden A capstone project submitted in partial fulfillment of graduating from the Academic Honors Program at Ashland University May 2013 Faculty Mentor: Dr. Jeffrey D. Weidenhamer, Trustees’ Professor of Chemistry Additional Reader: Dr. Robert Bergosh, Assistant Professor of Chemistry Abstract Wilted red maple leaves are toxic to horses, causing death by oxidation of hemoglobin and inducing anemia. Gallic acid derivatives have been identified as the main oxidants present in the leaves. However, our work has found that a previously unknown phytoalexin is produced by wilting red maple leaves. Phytoalexins are defensive compounds produced by plants in response to fungal attack. These compounds often have a range of biological activities. The unidentified compound from red maple, which fluoresces blue in certain TLC systems, is present only after wilting. The objective of this study is to identify and characterize this compound so that its toxicity can be determined. Wilted leaves were collected, dried, and extracted with methanol. Leaf extracts have been purified through repeated thin layer chromatography and column chromatography. After successful purification, the structure of the compound will be confirmed by NMR and mass spectral analysis. This research will provide insight regarding the mechanism of fungal defense in Acer rubrum and may also be relevant to the known toxicity of wilted red maple leaves to horses. i Acknowledgements I am very thankful to Dr. Jeffrey Weidenhamer; his invaluable guidance and relentless passion for this project has fueled my own love for lab work and this research. I would also like to thank Dr. Robert Bergosh for his help with my initial exposure to the project and his expertise with interpreting NMR data. Nan Kleinholz, at Ohio State University, greatly aided in the high resolution mass spectral work. Lastly, I would like to thank Ashland University for providing me the opportunity and funding to perform this research. Support for this work was funded by the College of Arts and Sciences at Ashland University, and the Chemistry, Geology, and Physics Department at Ashland University. This work was supported by a grant from the National Science Foundation (CHE/MRI-0922921) for the JEOL ECS-400 NMR spectrometer, and a National Science Foundation Award (1040302) for the Bruker maXis 4 QqUHR-TOF at Ohio State University. ii Table of Contents Page Abstract………………………………………………………………………………………….....i Acknowledgements…………………………………………………………………….………….ii List of Tables…………………………………………………………………………….……….iv List of Figures…………………………………………………………………………….….........v Introduction………………………………………………………………………………………..1 Materials and Methods…………………………………………………………………………...11 Results……………………………………………………………………………………………14 Discussion………………………………………………………………………………………..26 Further Research………………………………………………………………………………....26 References......................................................................................................................................28 Author’s Biography…..………………………………………………………………………….30 iii List of Tables Table Page 1: HPLC mobile phase gradient, acetonitrile: water: formic acid ………..…..………..……..…14 2: Major components of each peak in HPLC chromatogram....…………………………………24 iv List of Figures Figure Page 1: Constituents of red maple foliage identified by Boyer et al. (2002)...................................…...4 2: Constituent of red maple foliage identified by Abou-Zaid et al. (2001)............………………5 3: Compounds known to cause hemolytic anemia in G6PD deficient humans…………………...7 4: Documented phytoalexins in various plant……………………………………………………..9 5: Extraction and isolation schematic……………………………………………………………13 6: Enriched flourescent extract under white and long-UV light…………………………………14 7: Spectrofluorophotometric data of enriched extract.....………………………………………..15 8: Chromatograms showing enrichment of target compound through purification………….16-17 9: Spectrum of compound of interest………………...………………………………………….18 10: Aromatic region of pNMR…………………………………………………………………..19 11: Chromatograph of LC/MS of purified compound………………………………….………..20 12: High Resolution mass spectrum of suspected phytoalexin……………………….………….21 13: Example of a doubly charged (2+) ion………………………………………………………22 14: Example of a single charged (1+) ion………………………………………………………..23 15: Possible structures for identified compounds………………………………….…………….25 v Introduction Toxicity of red maple foliage to horses The red maple tree, Acer rubrum, is a widely distributed species of deciduous tree in eastern North America. This species derives its name from red-orange flowers, fruit, buds, and petioles. It is best known for its brilliant red leaf color in fall, making it popular for landscaping and shading. The characteristics of the V-shaped, three lobed leaves and smooth gray bark allow this tree to be easily identified. Wilted leaves from the red maple tree are toxic to horses. Ingestion of 700g (1.5lbs) can be toxic, while as little as 1,400g (3lbs) can be lethal for a full sized horse (Purdue, 2006). Ingestion of wilted, fallen, or damaged leaves can cause symptoms to develop in one to two days, while fresh leaves have not caused toxic effects. Symptoms of exposure are similar to those associated with low oxygen levels: lethargy, deep and heavy breathing, increased heart rate, coma, and death (Boyer et al., 2002). Dark urine and discoloration of the mucous membranes are unique symptoms that result from exposure to the toxin. Dark urine is a result of protein, red blood cells, and methemoglobin being secreted in the urine. The reported fatality rate is 50-75% for affected horses (Purdue, 2006). The clinical symptoms of this toxicity are consistent with the idea that the compound or compounds responsible are oxidizing agents (Boyer et al., 2002). Consumption of the wilted leaves causes oxidation of the blood, preventing hemoglobin from carrying oxygen. This causes the suffocation-like symptoms by creating Heinz bodies, which appear as dark clumps of methemoglobin (Tennant et al., 1981). The dark clumps cannot transport oxygen, as the ferrous (2+) iron has been oxidized to its ferric form (3+). The toxin (or a related compound) also increases cell membrane fragility, causing red blood cells to lyse (Schall and Lehman, 2011). The Heinz bodies, along with the damaged red blood cells, are 1 filtered out by the kidneys and secreted in the urine. In a study to determine the hemolysis of equine erythrocytes after exposure to various maple species, Schall and Lehman collected fresh leaves of red, silver, Norway, black, and sugar maples (as well as boxelder). Leaves were allowed to wilt and dry before storage (-20˚C). The results of the study showed that dried leaves of maples, including silver, sugar, and red maple (but not black or Norway maple) contained large amounts of oxidizing agents, which resulted in formation of methemoglobin and hemolysis after exposure to leaf extracts. Treatment for this toxin focuses on symptoms, as well as preventing further ingestion of leaves. To begin with, leaves are removed from the horse’s environment. Secondly, mineral oil or activated charcoal is administered to the stomach to prevent absorption of any leaves that remain. Blood transfusions are performed to replace damaged cells while intravenous fluids are given to increase renal function (Plumlee, 2003). Ascorbic acid has been successfully used to reduce methemoglobin to hemoglobin, reversing the oxidation process and allowing hemoglobin to carry oxygen. Ascorbic acid (30-50mg/kg) can be added to IV fluids twice daily to counteract exposure. The newest treatment option is Oxyglobin (purified bovine hemoglobin). Successful treatments of red maple poisoning occurred at Tufts University, where an Oxyglobin and blood transfusion mixture was intravenously administered to affected horses. The Oxyglobin carries oxygen in the blood until symptoms lessen or blood transfusions become available (Plumlee, 2003). Currently a treatment of low dose ascorbic acid, Oxyglobin, and blood transfusions appears to be the most effective way to ensure horse survival. Red maple has also been shown to inhibit feeding of other animals, including beaver. In a 1994 study, the palatability of red maple was assessed using a number of techniques (Muller- Schwarze et al., 1994). When compared with other trees in a feeding study, maple trees were 2 least preferred. In another experiment in this study, red maple extracts were applied to aspen logs, the most preferred diet of beaver. After the extracts were applied to the logs, they became unpalatable. The beaver avoided eating the treated logs, showing that a chemical compound (or a number of chemical compounds) found in the red maple deterred the beavers from feeding. Whether the palatability of these extracts is related to the toxicity of red maple to horses is unknown. Chemistry of wilted red maple Previous research has identified several potential oxidants in red maple. Boyer et al. (2002) used thin layer chromatography (TLC) solvents, bioassays, and gas chromatography-mass spectroscopy (GC-MS) to determine possible oxidizing agents found in red maple leaf extracts. Unlike Schall and Lehman, these researchers rapidly dried their leaves in a 60˚C oven, reducing the effect of wilting on chemical composition. They identified gallic acid as the main oxidizing agent, but also mentioned that 2,3-dihydro-3,5-dihydroxy-6-methoxy-4H-pyran-4-one may be