Isolation and Characterization of a Suspected Phytoalexin from Wilted Red Maple Leaves

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.

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.

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

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

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

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

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 one of a number of co-oxidants present in the leaves. A second and distinct fraction also caused methemoglobin formation. Because of this, it was hypothesized that another unidentified compound was working with gallic acid to cause increased oxidative damage. GC-MS was used for initial screening, followed by an ultraviolet-visible (UV-Vis) quantification of gallic acid and gallotannin through the use of rhodamine. The structures of the identified compounds from this study are shown in Figure 1. The differences between wilted and fresh leaves were not explored in this paper, and the description of methods did not mention the time between collection and rapid drying, making a transition period of wilting possible.

Research has also been performed on a number of gallic acid glycosides as inhibitors of feeding to forest tent caterpillar. Abou-Zaid et al. (2001) used extracts of red maple leaves in a

study similar to that of Schwarze et al. This research group used extracts to deter feeding of

aspen leaves, the preferred diet of the caterpillar. Fresh leaves were extracted with ethanol at room temperature and then purified by column chromatography and high performance liquid

chromatography (HPLC), followed by nuclear magnetic resonance spectroscopy (NMR) and

mass spectral analysis for identification. As with the previous study, the time between removing

the leaves and extraction was not recorded, making wilting a possibility between removal of

leaves and extraction. The main compound responsible for the feeding deterrence was ethyl m-

digallate (Figure 2). This compound, as well as a few others, was documented in fresh leaves.

This research provided a background for the Boyer group in identifying their potential

compounds as gallic acid derivatives, as this class of compounds is well documented in red

maple.

R1 O

R1 HO R1 OH R 1 R1

HO O R1 O R1 O OH O Gallic acid O R1 O

R 1 R1 O O

OH OH O R1 O R1

R O O 1 O R1

2,3-dihydro-3,5-dihydroxy-6-methoxy-4H-pyran-4-one Gallotanin (R1=OH)

Figure 1: Constituents of red maple foliage identified by Boyer et al. (2002)

O HO HO

Figure 2: Ethyl m-digallate

Previous research at Ashland University has focused on differences between wilted and fresh red maple leaves, since toxicity to horses is associated with wilting. Elizabeth A. Miller

(Miller and Weidenhamer, unpublished results) analyzed fractions of red maple extracts before and after wilting. After extraction with methanol and ethyl acetate, she found a compound present in the wilted fraction that was not present in the fresh leaves. In certain TLC systems, this compound fluoresced blue. Miller tested the compound against known phytochemicals from red maple, only to determine that the compound was undocumented. She was unsuccessful, however, in isolating the compound. Continued research by Janna Pearson was generally unfruitful, but showed that selection of ideal mobile phases for TLC is of utmost importance. She found that the use of TLC mobile phases containing ethyl acetate and ammonia resulted in the synthesis of acetamide on the TLC plate, which contaminated the isolated products and made spectral characterization impossible (Pearson, Bergosh, and Weidenhamer, unpublished results).

G6PD Deficiency

A human red blood cell mutation has been discovered that mimics the symptoms associated with exposure to the red maple toxicity. The human deficiency is known as glucose-6- phosphate dehydrogenase deficiency, or G6PD deficiency. This mutation was discovered when a

5 group of persons developed hemolytic anemia in response to anti-malarial drugs (Beutler, 1994).

The mutation is the most common of human mutations, and affects approximately 400 million people worldwide. G6PD deficiency prevents oxidized hemoglobin from being reduced; if exposed to an oxidizing agent in diet or drug form, Heinz body formation occurs in 1-2 days, followed by dark urine and hemolytic anemia. The symptoms mimic that of horses after exposure to red maple leaves. Since the human deficiency has been studied extensively, it may provide insight into the types of compounds that cause hemolytic anemia in horses.

A number of drugs and food items have been known to cause symptoms in G6PD deficient humans, and therefore provide models of compounds for the red maple toxin. Humans with G6PD deficiency are sensitive to sulfur and nitrogen containing compounds, such as 8- aminoquinoline, phenazopyridine, and sulfacetamide (Figure 3). These compounds bind to iron, oxidizing it, and preventing it from binding oxygen. Fava beans cause Favism, the clinical term for hemolytic anemia as a result of fava bean consumption. Fava beans have nitrogen containing alkaloids, most notably vicine, convicine, and their corresponding aglycones, divicine and isouramil (Burbano et al., 1995). These compounds have been determined to be the cause of symptoms in G6PD deficient humans. Other foods have been documented as causes of hemolytic anemia in affected individuals, but it is worth noting that gallic acid and the gallic acid derivatives, which are significant components of black and green teas, have not been found to cause symptoms in humans (Anesini et al., 2008). Horses have been found to be more susceptible to accumulation of methemoglobin, as well as depletion of glutathione, which is needed to reduce hemoglobin (Robin and Harley, 1967). In effect, horses will experience symptoms of hemolytic anemia after exposure to nitrogenous compounds that oxidize hemoglobin, much in the same way that humans with G6PD deficiency do. Thus, it seems

unlikely that gallic acid derivatives would be responsible for the hemolytic anemia associated

with consumption of wilted red maple leaves.

N O H N N S

N O O

NH2 H2N N NH2 H2N

8-aminoquinoline phenazopyridine sulfacetamide

Figure 3: Compounds known to cause hemolytic anemia in G6PD deficient humans

Phytoalexin Overview

Plants produce a multitude of biologically active compounds that play a variety of roles

in defending the plant against pathogens and herbivores. These compounds can have unintended

side effects, such as having activity against human illness and cancers. The compound produced

by red maple upon wilting is suspected to be a phytoalexin, a compound produced to ward off

microbial attack, analogous to an animal immune system or defense mechanism. Phytoalexins are synthesized in response to environmental stressors, wounding, or microbial invasion

(Gottstein and Gross, 1992). They can be produced anywhere in the plant, and have been documented in roots, bark, and leaves.

Leaves are an area of particular interest, because phytoalexin production is regulated by stimuli in leaves, where synthesis in bark and root may be constant. Previous research has shown that wounding can initiate synthesis pathways for phytoalexins in leaves. Genes can be turned on for production of these compounds within minutes after exposure (Creelman et al., 1992).

Defensive compounds can be produced either before or after infection. Pre-infection compounds usually reside in the bark, roots, or corks of a plant. These are used to prevent infection from occurring in the plant. Produced constantly, these are found in the plant continually (Gottstein and Gross, 1992). Post-infection metabolites are produced after invasion has occurred, or as invasion begins. The plant’s response can be specific to the invading pathogen, or generalized.

Three major post-infection signaling pathways have been studied in plants. The first pathway uses jasmonic acid/ethylene as a signaling molecule. This pathway is a specific immunity, so it only responds to exposure of particular fungal or bacterial epitopes (Po-Wen et al., 2012). Increased levels of jasmonic acid and ethylene lead to activation of plant defensin 1

and 2 (PDF1,2) genes, which respond to specific bacteria (necrotrophic, or host-killing bacteria).

The second specific immunity pathway uses salicylic acid as a signaling molecule. This pathway

activated pathogensis related 1 (PR1) genes, which are also a target towards specific bacteria.

Lastly, plants have a general form of immunity known as pattern-triggered immunity. Much like

the inflammation response in animals, this pathway causes an increase in the overall defense of

the plant (Po-wen et al., 2012). This pathway causes cell walls to thicken, a defense mechanism

referred to as callose formation. Stomata also close in response to pattern-triggered immunity

activation.

The determination of the red maple defense pathway is crucial to understanding the

production of the toxins that cause such potent affects in horses. Jasmonic acid, ethylene, or

salicylic acid presence would indicate specific invasion of the plant tissue, while absence of

these cell signaling molecules would support an argument for pattern-triggered immunity

signaling. In the case of patterned triggered immunity, the maple leaves would produce the

phytoalexin as a general defensive response.

The synthesis of the phytoalexins may occur to prevent tissue invasion, no matter which

pathway is used. Genes turned on in an attempt to slow or stop microbial invasion must be fast-

acting. Small, toxic compounds allow for rapid production at a reduced cost for the plant.

Typical phytoalexins are conjugated structures consisting of 1-4 rings. They generally contain

oxygen, carbon, and hydrogen, but may incorporate nitrogen or sulfur as well (Gottstein and

Gross, 1992). For this reason, isolation of small, aromatic compounds is the goal of this research, as data showing that the compound is small and aromatic may confirm its use as a plant defense

compound. Shown below (Figure 4) are some typical phytoalexin compounds that have been

previously documented in plants.

O O

H O O O

S O S

Pisatin (peas) Allicin (garlic)

Benzophenanthridine (poppy)

Figure 4: Documented phytoalexins in garlic, peas, and poppy plants

Research has shown that phytoalexins can be produced under wilting conditions. Plumbe and Willmer (1985) showed that pisatin, a phytoalexin from pea plants, is produced in severely water-stressed leaves. Their study also determined that pea plants do not produce phytoalexins in response to mild water stress; severe wilting and drying are needed in order for the pisatin to be made. This compound inhibited growth of fungi, which led to the conclusion that it is likely

synthesized to prevent fungal invasion while the plant is in a stressed state (Perrin and

Bottomley, 1961). This work relates to red maple because the compound is only present in wilted

leaves, so it is possible that red maple would make compounds only during wilting as well.

Phytoalexins may also contain sulfur and nitrogen groups, which are known to cause

hemolytic anemia humans with G6PD deficiency. Allicin is a phytoalexin isolated from garlic

that contains two sulfur atoms (Ankri and Mirelman, 1999). Benzophenanthridine is an alkaloid

phytoalexin that has been documented in California poppy. The basic backbone of

benzophenanthridine (Figure 4) can have substitutions that alter its functionality. Studies of this

alkaloid and its related compounds have shown that is produced in response to fungal spores

(Roos et al., 1998). When poppy seed was exposed to yeast, the production of

benzophenanthridine alkaloids could be measured in the plant. This supports the idea that red

maple could produce a phytoalexin in response to fungal invasion.

The objective of this research was to isolate and characterize the unknown fluorescent

compound previously isolated in wilted red maple leaves. After isolation, the biological activity

of this presumed phytoalexin should be explored. It is hypothesized that this compound is

produced in response to wilting or pathogen invasion, and may be responsible for the known

toxicity of red maple to horses.

Materials and Methods

Collections. Leaves from red maple trees were collected from branches and ground, and then stored in mesh bags to air dry in laboratory storage.

Extraction. Approximately 770g of leaves were used for extraction. Leaves were crushed and placed into three 2L Erlenmeyer flasks. Mixtures of leaves and hexane were allowed to sit for one day before decanting to remove chlorophylls and waxes. Ethyl acetate extractions were attempted to remove the compound of interest, but were determined to be unsuccessful. HPLC grade methanol was then added to the flasks, allowed to sit for one day, and then decanted and dried down (~300mL brown tar). An accurate mass of extract was difficult to determine, as the mixture easily boiled over during drying. A simple silica thin layer chromatography separation with tetrahydrofuran: isopropanol: ammonia (55:35:10, v:v) was used to view the blue fluorescent band with an Rf of approximately 0.35 under long UV light.

Initial Separation. A basic alumina column was used to provide initial separation of the compound. A gradient of ethyl acetate to methanol was used (0, 5, 10, 25, 50, 75% ethyl acetate).

A final wash of four columns volumes of 100% methanol was used. Location of the compound could be tracked under long UV light. The compound of interest was found to be located in the methanol wash, or on the column (depending on the amount of alumina used). If on the column, the alumina was extracted with hot methanol, filtered, and dried down. Multiple preparative separations were run in attempts to purify the unknown compound.

Preparative TLC. Two different preparative TLC separations were used. A 1000µm silica plate was used with the tetrahydrofuran: isopropanol: ammonium hydroxide (55:35:10) mobile phase for further purification. Approximately 80-90mg was spotted per plate. The band was visualized under long wavelength UV light, and the fluorescent spot was scraped and extracted.

A second 1000µm basic alumina plate was used for further purification with methanol: ethyl

acetate: water (90:5:5) as a mobile phase. The blue band was then scraped and extracted for further purification.

Analytical and preparative HPLC. An Agilent 1100 HPLC/DAD equipped with a Restek

Ultra C18 (150x4.6mm, 5µm) column was used to analyze the purity of the extract. A Supelco

Supelcosil SPLC-18-DB semi-preparative column (250x10mm, 5µm) was used for final

preparative purification. The compound of interest was monitored at 257nm, which was based on

the results of spectrofluorimetric analysis. The original solvent system was a methanol: water

gradient (1ml/min), but a second solvent system with acetonitrile, water, and formic acid

produced faster runs with similar separation, and was also used for HPLC-MS analysis. A mobile

phase gradient of acetonitrile: water: formic acid (2:98.9:0.1) to acetonitrile: water: formic acid

(97.9:2:0.1) (Table 1) was also used for prep HPLC and to assess the purity of the extracts. The illustration in Figure 5 summarizes the extraction and separation methods used in this research.

Solvent

Sand

(Kent, 2013) Extract

Alumina

Hexane,

Methanol Glass Wool

PREP

HPLC

HPLC, NMR Repeat UV-VIS, GC/MS

Figure 5: Extraction and isolation schematic

Instrumental Methods. UV absorbance and fluorescence spectral data was gathered on the compound to assist with characterization and following the compound throughout purification.

An RF-5301PC Shimadzu spectrofluorophotometer was used for fluorescence analysis. Proton

NMR was performed on a JOEL 400MHz NMR with CD3OD as solvent. High resolution liquid chromatography/mass spectroscopy work (LC/MS) was performed at Ohio State University with a Thermo Scientific Acclaim PAIL column (C18, 300µmx15cm, 3µm, 120 Å) at 2µl/min and an acetonitrile: water: formic acid (2:97.9:0.1→97.9:2:0.1) gradient solvent system, as seen in

Table 1. A Bruker maXis 4 QqUHR-TOF was used to determine accurate mass of the purified

extract with a lock mass of 622.0290. Optimal electrospray ionization conditions were used to

create positive ions with a voltage of 1.5KV, and 150˚C capillary temperature. The mass

spectrometer acquisition range (m/z) was 50-1000 at a rate of 2Hz.

Table 1: Mobile phase gradient, acetonitrile: water: formic acid (v: v: v) Time 2: 97.9: 0.1 97.9: 2: 0.1 0 98 2 35 2 98 38 2 98 38.1 98 2 50 98 2

Results

Preliminary separations determined a set of potential TLC solvents which allowed the

compound to be visualized. After an enriched fraction was obtained, it was characterized by spectrofluorophotometry. Figure 6 shows a flask under normal lighting (1) and long UV light

(2). The characteristic blue color was used to check for presence of the compound after various separations, as well as analytical TLC verification with a mobile phase of THF: isopropanol: ammonia (55:35:10, v:v).

(1) (2)

Figure 6: Enriched extract under white (1) and long-UV (2) light

Since the compound was fluorescent, the absorbance and emission wavelengths of an

enriched fraction were determined. The data after preparative TLC separation showed that the compound had a maximum absorbance at 257nm, while a shoulder of absorbance extended to

300nm. Maximum emission from the extract occurred at 333nm, which was the in the ultraviolet range. The tail end of the emission spectrum was in the violet/blue visible range, so the tailing was likely responsible for the blue color under long UV light (Figure 7).

Spectrofluorophotometer Data 300 Ultraviolet Light Violet/Blue 250 Emission 200

Excitation 150 Intensity 100

0 220 240 260 280 300 320 340 360 380 400 Wavelength (nm)

Figure 7: Spectrofluorophotometric Data

The absorbance spectrum obtained by spectrofluorophotometry was then used in HPLC analysis to assess the purity of the extract mixture after each separation technique (Figure 8).

The peaks were checked to see if they matched the compound’s maximum absorbance of 257nm, as well as the shoulder that extended to 300nm. In a mobile phase with methanol and water, the compound was found to elute at approximately 15.3 minutes (black arrow). In a separation with acetonitrile, water, and formic acid (solvent system B), the compound eluted earlier, at approximately 7.5 minutes (black arrow).

After Extraction A.

A b s o r b a n c e @ 2 5 7 n m

Retention time (minutes)

After Alumina Column B. A b s o r b a n c e @ 2 5 7 n m

Retention time (minutes)

Figure 8: Chromatograms showing enrichment of the target compound through successive

purifications.

A After Preparative TLC (x2) b C. s o r b a n c e @ 2 5 7 n m

Retention time (minutes)

After Preparative HPLC A D. b s Solvent System B o r X- solvent b a n c

eAfter Alumina Column @ 2 5 7 n m

Retention time (minutes)

Figure 8 (continued): Chromatograms showing enrichment of the target compound through

successive purifications.

As stated before, the elution of the fluorescent compound was verified by a diode array

detector. The absorbance spectrum of the compound matched that determined by the spectrofluorophotometer (Figure 9). HPLC data showed that the initial mixture extracted from the leaves, which contained a large number of compounds (Figure 8A), had been reduced down to a fairly pure extract that matched the spectrum of the compound of interest (Figure 8D). The

final purification was sent to the Ohio State University for exact mass determination, even

though the final purification contained a small contaminant which eluted just before the compound of interest. Although the column used to assess purity was not able to give baseline separation of the contaminant, the column used for the high resolution-mass spectroscopy work

was able to, so an accurate mass for the contaminant was determined as well.

Solvent

A b s Max Absorbance o r b a n c Shoulder e

Wavelength (nm)

Figure 9: Spectrum of compound of interest (eluting at 7.5 minutes)

Before the sample was sent for high resolution analysis, NMR data was gathered on the

compound after preparative TLC separation (x2). Proton and carbon NMR were performed on

milligram quantities of sample. Carbon NMR did not give any noticeable signals, probably due

to the small amount of sample. Proton NMR (Figure 10) was performed to determine if aromatic

hydrogen was present in the compound. The fluorescence suggests that the compound has

conjugated unsaturation (multiple double bonds). The pNMR gave a large mass of peaks with

low chemical shifts (1-5ppm) that were impossible to separate or discern. However, the pNMR

did give a large peak with a chemical shift of 8.5ppm, which suggests the presence of aromatic

nitrogen or an electron withdrawn aromatic ring. Small peaks for aromatic and vinylic hydrogens

were noticeable around 7.7, 7.6, 6.9, 6.6, and 5.8 ppm, but were still not distinct after a large

number of scans.

pNMR of Purified Compound

S i g n a l

Chemical Shift (ppm)

Figure 10: Aromatic region of pNMR

The NMR data suggests that the sample is either: a massive compound with a large

number of alkyl hydrogens and some aromaticity, or contaminated with another compound (or

compounds) that interfered with the signal of the desired compound. The small amount of

sample and number of low chemical shift peaks suggested that another method would be needed

for accurate characterization. High resolution LC-MS was used because it would allow for a very

19 small amount of sample, and it would give an accurate mass of the compounds in the sample, allowing molecular formulas to be determined from the mass spectral data even in a mixture.

The chromatogram from the HPLC-MS analysis showed that a mixture of at least four compounds was present after purification (Figure 11). The small peak (15.7 min) before the larger peak (16.3 min) suggested that the compound of interest eluted in the middle of the run; the order and size of the two peaks mimicked the HPLC data from previous runs. Baseline separation of the two compounds was achieved. It was possible that the 16.9 minute peak was a degradation product of the compound after exposure to mobile or stationary phases, since it did not appear before. The last peak could be a possible contaminant, because it would be less polar, while the expected compound was water soluble. Exact masses were determined for each of the peaks and then analyzed against the background. Since a single mass spectrometer was used and not a tandem mass spectrometer/mass spectrometer, no structural information could be gathered about the compound of interest by the fragmentation of individual ions.

Figure 11: Chromatograph of LC/MS of purified compound

Data from the high resolution mass spectrometer was both unique and unexpected. The spectrum (Figure 12) showed a number of peaks ranging from 131 to 883 mass per charge (m/z).

(Note: peak at 89.5069 is due to the mobile phase). The mass to charge limit on this machine was

1000 m/z, so it is possible that larger ions were also present in the sample. The 883 mass was much higher than expected, but provided insight into the unique NMR problems associated with this compound. Because NMR signals are proportional to the molar concentration of the sample, a large molecule will require a larger amount of material to achieve an adequate signal. This provides an explanation for the weak and complicated signals previously observed in the proton

NMR spectrum. The individual peaks in the mass spectrum were then analyzed to determine if they were singly or doubly charged ions.

Figure 12: High Resolution mass spectrum of suspected phytoalexin

The charges of the ions in the spectrum were determined by close examination of isotopes. Upon further investigation, the isotopic peaks associated with the 883.2967 ion displayed a gap of 0.5 atomic mass units (AMU). For this peak (Figure 13), isotopic increases of one AMU only gave a 0.5 AMU increase in the corresponding ions; this meant that the ion

responsible for the peak at 883.2967 was a doubly charged ion (2+), making the actual mass

twice as large (approximately 1766). This information was incredibly useful when investigating this data as a whole.

The charges of all the ions in the spectrum were determined using this same type of analysis. Some of the larger masses had double charges, while a number of single charged peaks were also found in the sample. Some of these peaks had molecular weights that would be expected for small molecules (Figure 14). Each of the peaks was compared to the baseline in order to determine which peaks represented large amounts of sample. The peaks, mass to charge ratios, and monoisotopic masses can be found in Table 2.

Figure 13: Example of a doubly charged (2+) ion

Figure 14: Example of a single charged (1+) ion

The data collected contained a large amount of information. Even so, all of this data could be interpreted to show that only four compounds made up the sample. The peak before the compound of interest eluted at 15.7 minutes and was found to contain a single (+1) ion with a mass of 344.2282. The large peak, which contained the compound of interest, had some singly charged ions, including 131, 163, and 443. The other ions were determined to have larger masses, totaling 439, 740, 1082, 1424, and 1766 when accounting for charge. These masses were much larger than expected, but they do provide important clues to the structure of the molecule.

The differences between the large peaks (1766, 1424, 1082, and 740) were found to be uniform.

A gap of ~342.131 AMU suggests that a polyglycoside or other natural polymer could be present in the sample. The uniform gap in mass (342 AMU) is equal to the mass of the peak eluting at

15.7 minutes. This meant that parts of the polymer could have broken off and run separate from the large peak. The similar retention time would also be expected, since the larger compound would contain multiple small subunits of similar polarity. The small difference in mass (2 AMU) could potentially explained by the difference in creating a 1+ ion versus a 2+ ion. This would

mean that the 219 peak could be the same compound responsible for the 443 peak, as it may

have lost mass in order to create the second positive charge. The peak eluting at 16.9 minutes

was found to contain a compound with a molecular weight of 443.0643. This mass matched a

large ion in the 16.3 minute peak. The matching masses suggest that a portion of the molecule

with a mass of ~443.064 could have broken off and eluted later on the column, or that this smaller compound is also a component of the leaf extract. In combination, this data proposed the idea that two large components are responsible for the majority of the peaks, while two smaller molecular ions were also in the sample.

Table 2: Major components of each peak

Elution Time Major Peaks (m/z) Actual Mass (Amu) 15.7 344.2282(1+) 344.2282 16.3 131.0495(1+), 163.0758(1+), 219.5463(2+), 131.0495, 163.0758, 439.0926, 370.0993(2+), 443.0648(1+), 541.1652(2+), 740.1986, 443.0648, 1082.3304, 712.2311(2+), 883.2967(2+) 1424.4622, 1766.5934 16.9 443.0642(1+) 443.0642 24.1 292.2628(1+) 292.2628

The exact masses of each molecule were then used to determine a molecular formula.

Accurate masses from high resolution mass spectrometers are able to determine exact formulas from compounds, where lower resolution mass spectrometers are not. For example, if a mixture of benzamide (C7H8N2), acetophenone (C8H8O), and ethyl toluene (C9H12) was analyzed on a

low resolution mass spectrometer, the masses would all read the same, approximately 120.1

AMU. If the same mixture was run on a high resolution mass spectrometer, it would determine three distinct masses: 120.068 (benzamide), 120.058 (acetophenone), and 120.096 (ethyl toluene). The increase in resolution to three or four decimal places will allow a mass to be matched exactly with a molecular formula. For this reason, high resolution equipment was used

for sample analysis. The smaller compounds in the 16.3 elution peak represented possible small

molecules. Monoisotopic masses were calculated using an online exact mass calculator

(www.chemcalc.org; Patiny and Borel, 2013). Using this calculator, only a single molecular

formula of C9H7O (6.5 levels of unsaturation) was given for the molecular weight of 131.0495.

The weight of 163.0758 gave a possible molecular formula of C10H11O2 (5.5 levels of

unsaturation). The unsaturation of these two compounds could result in conjugation and could be

used to explain the fluorescence of the sample. Possible structures of these two fragments can be

found in Figure 15. Both the 131 and 163 molecular weight compounds could be simple structures with a single ring. The monomer subunit of the sample, with a monoisotopic mass of

344.2282, gave a possible molecular formula of C13H28N8O3 (4 levels of unsaturation). The large peak at 443.0642 gave two potential molecular formulas, C24H13NO8 (19 levels of unsaturation)

and C23H7N8O3 (24.5 levels of unsaturation). The high unsaturation of the 443 peak could also be

used to explain the fluorescence of the sample. Data showing the presence of nitrogen in both of

these larger molecules could be used to explain the strong chemical shift in the NMR data. The

presence of nitrogen in the compound could explain the toxic effects noted in horses. Even

though the data generated for this sample is very powerful, more information (and sample) will

be needed to verify the exact molecular formulas of these compounds. The included structures

are simply speculation, but provide insight into possible structures of the small molecules.

O CH3

- O O

Figure 15: Possible structures for 131 and 163 molecular weight compounds

Discussion

The data gathered on the suspected phytoalexin is very unique, and may be unlike many

known phytoalexins. The fluorescent compound was found to be stable in solution (methanol) at

room temperature. The emergence of the small molecules with the larger molecular weight

elutions suggests that they may be covalently bonded into a large compound. Extraction after

preparative HPLC showed that the compound was water soluble, which was also unexpected.

The proton NMR, chromatography and fluorescent data, along with the extraction properties of the sample, reveal that this compound contains conjugated bonds and is very polar. The compound isolated seems to be a better candidate for the oxidative toxin than gallic acid derivatives previously identified by Boyer et al. for a number of reasons. Humans with G6PD

symptoms are not reported to experience sensitivity to such compounds as gallic acid. Also, the

larger molecular weight components of the sample analyzed in this work may contain nitrogen,

which is similar to compounds such as 8-aminoquinoline and phenzopyridine, which are known

to cause symptoms in G6PD deficient humans. Lastly, the extraction and separation techniques

used in this research are milder than those previously attempted. It is also likely that a large

compound such as the one discovered by this research would not be volatile enough for analysis

by GC/MS, the technique used for previous identification.

Further Research

A number of future experiments could be performed in order to learn more about the

suspected phytoalexin. Further preparative HPLC purification would allow for high field NMR

structural analysis. If sufficient sample cannot be obtained, then the use of an orbitrap or tandem

mass spectrometer/mass spectrometer could be used to determine more structural information about the compound with small quantities of sample.

Once the structure of the compound is confirmed, several additional studies could be

undertaken to answer important questions about this research. First, the role of the compound in

relation to the toxicity of red maple to horses could be determined. This could be assessed

through bioassays with equine erthyrocytes. Secondly, the concentrations of the compound could

be measured in the leaves at various points in leaf development and wilting. In this way, the

production of the compound could be understood more completely. The antifungal activities of

the compound could also be tested, which would verify the identification of it as a phytoalexin.

Other biological activities, such as anticancer and antibiotic properties could also be determined.

Lastly, if the compound is verified as a phytoalexin, the signaling pathway responsible for synthesis could be researched to understand the role of second messengers (salicylic acid, ethylene, jasmonic acid, or others) in triggering this plant defense.

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Author Biography

Jared Baisden was born in Wooster, Ohio. He grew up in Wooster and graduated from Wooster High School in 2009 with honors. At Ashland University, Jared is majoring in biology and biochemistry with a minor in chemistry. He is a National American Chemical Society Scholarship Recipient, the vice president of Ashland’s Beta Beta Beta Biological Honors Society, a Choose Ohio First Scholar, an Omicron Kappa Delta Leadership Honorary Member, a biology/chemistry/physics/calculus peer tutor, a member of Who’s Who Among Students in American Universities & Colleges, and a recipient of the outstanding senior award for Alpha Lambda Delta Honors Society.

Upon graduation, Jared plans to attend graduate school at University of North Carolina at Chapel Hill to pursue a research career in biochemistry or a related field.