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Correlating Civil War folklore with a natural products discovery.

ABSTRACT

We collected twenty-six samples of Humic Substances (HS) from the headwaters of the Suwannee River in the Okeefenokee Swamp to its delta in the Gulf of Mexico. Mass Spectrometry (MS) was used to analyze the samples. In all the samples that were rich in naturally occurring organic matter we were able to identify chincodine, a molecule similar to quinine in structure and medicinal value. We were able to correlate this find with trade levels of the chincodine molecule in dogwood trees around Valdosta. In addition, this discovery of chincodine in dogwood trees correlates with Civil War medicinal folklore. Civil War folklore describes how people would boil parts of the dogwood tree for medicinal applications when quinine did not make it through the Union blockade.

Key words: humic, quinine, chincodine, Suwannee River

INTRODUCTION

Research with Humic Substances (HS) has been an ongoing endeavor for a variety of sciences and engineering disciplines over the past century. Subsequently HS's have been divided into three groups, fulvic acid, which is soluble at all pH's, Humic acid (HA) which precipitates out of aqueous solutions below a pH of 2.0 and humin, the insoluble, nonpolar organic component. HS's are generally attributed to the molecular constituents of plant and animal decay in nature but may undergo further structural changes due to exposure to UV light from the sun, microbial decay, various oxidizing agents such as oxygen, and other environmental changes such as metal binding, pH shifts, and changes in ionic strength.

HS's play an important role in environmental chemistry. For example, they can bind and transport metals through the environment, solubilize nonpolar compounds (i.e. herbicides, pesticides, and petroleum products), fertilize soil, buffer soil and water, impact dissolved oxygen levels in the aqueous phase, etc. (1-4). The international standard used for HA, as recognized by the International Humic Substances Society, is taken from the Suwannee River in Fargo, Georgia (5, 6, 7).

Past work in this lab with HS's has included using Multiangle Laser Light Scattering (MALLS) to measure the average size and molar mass of HA aggregations under one set of conditions (8) to a variety of thermodynamic type studies (9, 10, 11). It has been proposed and supported by experimental data that HS's can form micelles and these micelles subsequently solubilize DDT (12). Specifically, humics have a large, nonpolar component that aggregates in solution providing a region that is chemically and physically compatible for larger nonpolar organic species. Our research has shown HA aggregates have an average size of 0.45 um and a molar mass of [10.sup.9] [amu's.sup.8] What should be emphasized is that HS's are an aggregation of many molecules present over a wide range of concentrations, including aliphatic and aromatic structures (13, 14), multiply substituted carboxylates (14), amino acids and peptides (15, 16), sugars (17), cellulose and lignin fractions (18), and functional groups including thiols, amines and phenols (19). Techniques routinely used to characterize HS include CHNOS analysis (13, 20-22), C-NMR (23, 24), Pyrolysis GC-MS (25-27), UV/VIS (28-29), fluoresecne (30, 31), X-Ray analysis (32, 33), Size Exclusion Chromatography (34, 35), and FT-IR (36-38). A large number of binding studies involving different forms of HS's from different locations globally have also been conducted. These studies include various elemental binding (i.e. [Ca.sup.+2], [Cu.sup.+2], lanthanides, actinides, etc.) and the trapping of various organic compounds such as herbicides and pesticides (39-46). HS can bind, concentrate, and trap an organic species for extended periods of time. It follows that HS are a window to the molecular composition of the surrounding ecosystem. The term Natural Products (NP) describes molecules that come from living organisms that have some medicinal value. Some well known examples include the anti-malaria drug quinine, which was isolated from tree bark, and the cancer drug taxol, which was originall y extracted from the yew tree. The Suwannee River Valley, where samples were collected, encompasses communities mapped under Southeastern Coastal Plain Ecosystems and further subdivided as Upland Pine Forests, Upland Hardwood Forests, Alluvial Wetlands and Paludal Wetlands. Serenoa repens (saw palmetto) dominates the understory of many upland hardwood forests and mesic pine communities in upland forests of the Suwannee River drainage.

EXPERIMENTAL

Twenty-six samples were collected during the first week of August 2000 on the Suwannee River, Withlachochee River, Santa Fe River, Ichnetuckee River, and at the delta of the Suwannee River in the Gulf of Mexico. The Suwannee River, its tributaries, and springs had either low flow or no flow due to a four-year drought when those samples were collected. The following extraction procedure was used for the Suwannee River HS's. Approximately 10 [cm.sup.3] of each solid sample was measured, allowed to settle for 48 hours and centrifuged. This removed granular sized limestone and gypsum found throughout Suwannee River basin. Second, the remaining solid was treated with 0.05 M [HNO.sub.3] to dissolve remaining salts and extract the fulvic component of naturally occurring organic material. Third, the sample was washed with 0.05 M KOH to remove the remaining soluble HA component. The remaining solid sample was allowed to dry, soaked for 48 hours, and extracted with a 50/50 methanol-chloroform mixture. The yellow colore d solution was dried with magnesium sulfate. For a variety of studies ranging from identification of DDT to quantitating stearic acid, we analyzed this extract with three types of mass spectrometers (LC-MS, GC-MS, MALDIMS). The MALDI is a Reflex produced by Bruker Daltonics. The matrix used was a saturated solution of alpha-cyano-4-hydroxycinnamic acid (Sigma) in a 50:50 solution of acetonitrile: water and 0.1% trifluoroacetic acid. The GO-MS is a Hewlett-Packard 5970 MSD with a 5890 GC. The LC-MS is an Applied Biosystems (ABI) solvent delivery system connected to a PE Sciex APII plus mass spectrometer equipped with an electrospray source. The sample was purified using a Kromasil 0-18 column (1 mm x 250 mm with a 5 [micro]m particle size and 100 A pore size) from Keystone Scientific Inc. HPLC was accomplished using an Applied Biosystems (ABI) solvent delivery system. Solvent A was water with 1% triflouroacetic acid and solvent B was acetronitrile. Sample (10 p1.) was injected at 100 percent A. After 12 minute s, the percent B was ramped to 40% in 3 minutes. Then over a 32 minute time period the percent B ramped to 70 percent. The run was terminated after another 5 minutes when B reached 100%. The HPLC flow rate was 30 [micro]L per minute. The UV at 214 nm was measured using an ABI 759 A absorbance detector. After flowing through the detector, the effluent was split so that 22 [micro]L per minute went into the PE Sciex APII plus mass spectrometer equipped with an electrospray source the remaining 8 [micro]L of eluent went to waste. Commercially available quinine from Sigma-Aldrich was used for calibration of LC-MS.

RESULTS AND DISCUSSION

The Suwannee River is rich with humics and has a diverse range of plant and animal life as well as chemical conditions (pH, DO, C content, ORP, etc.) as it winds 300 kilometers from the Okefenokee Swamp (Georgia) to the Gulf of Mexico (Florida). In summary, the molecule chincodine was identified in the Suwannee River basin for the first time. It is a derivative of quinine (see Figure 1) and has the same medicinal value (anti-malarial agent). Both are found in the bark of the Chinchona tree found in Ecuador (Andes Mountains, South America) (39-41). Using our extraction and detection technique, we found quantities of the molecule trapped in HS throughout the Suwannee River basin. This is the first record we were able to find in the southeastern United States, but we were able to correlate the find with folklore dating back to the United States Civil War and the locally abundant dogwood tree (Cornus florida).

The greatest opponent the Union and Confederate armies faced during the American Civil War was not battlefield casualties but disease. Second only to the losses posted by dysentery, post-war statistical analysis compiled by the Federals revealed there were 1,315,955 cases of "Intermittent and remittent fevers (malaria)" resulting in 10,063 deaths in the Northern army (43). Though exact figures for the Confederacy are not available, it seems that southern soldiers and civilians also suffered greatly from malaria because northern blockades on southern ports sharply curtailed the importation of quinine, the nineteenth century's most effective malarial antiperiodic (44). By 1864, humanitarian-minded northerners called for an end to blockaded medicines only to be met by a political chorus of partisan nay sayers and newspaper editorialists that wrote poetic diatribes (as found above) in northern newspapers. The south, and in particular the Deep South where malaria was an epidemic, needed options. Out of desperation and with limited success, homespun remedies such as tea made from dogwood bark were literally found in the south's own backyard. The chemical qualities of dogwood bark found in South Georgia have been little studied to this point, its curative powers largely the stuff of folklore and its ministrations often handled by local herbalists.

Obviously, malaria epidemics were not a calamity unique to the American Civil War, however, the period does provide a more recent and fairly well documented account of the steps taken when the options were few when battling malaria. Quinine, the preferred treatment, was first introduced to Europe in the 1640s by way of Spanish colonialists settled in the Andes Mountains. Quinine is derived from quinquina bark of the chinchona tree and first showed to be an effective yet unpredictable malarial cure. There are over seventy species of chinchona trees and only a small percentage of these proved useful in abating the debilitating and sometimes fatal cycles of fever and recovery that afflict those bitten by the disease carrying Anopheles mosquito. The search for a reliable and marketable way of chemically analyzing and globally exporting the powdered quinquina bark was left to those with the greatest means to do so. Imperialist nations such as Spain (particularly Jesuit priests who first monopolized the trade), Hol land, and Britain at times held sway in the global trade of the drug, putting a great deal of effort into transplanting chinchona trees and attempting to decipher the potency of certain barks relative to others. The feat of determining the potency of the different species of bark was not fully accomplished until about 1900. To countries with lesser means (both ideas applied to the Confederacy), quinine shortages meant, among other things, boiling dogwood bark and placing more faith in folkloric conveyance than chemistry (42-47). After detecting chinconide in all samples collected that had significant amounts of HS, we conducted a separate study of dogwood trees in the Valdosta area. Specifically, we collected root, leaf, bark and stem samples from six trees in various settings such as on the banks of the Withlachochee River, residential areas, old growth forests, and near slash pine tree stands re-planted for harvest every 16-22 years. We found the same molecular fingerprint (see Figure 2) for chincondine in these trees, but the results were not consistent. We were unable to correlate the fingerprint with a specific part of the tree or a specific location and this will be left to a separate study. The relatively low quantities we detected coupled with lack of consistency between different trees would account for dogwood trees not being actively pursued when compared to other sources of quinine.

CONCLUSION

We identified the natural product chincondine in humic samples collected along the Suwannee River basin for the first time. This collection was part of a large systematic study of the river basin and involved a number of studies including ICP-AES analysis for trace metals, CHN analysis of humics, ET-IR analysis of humics and an exploratory component seeking out natural products. We then tested a number of dogwood trees in the Valdosta area and found low levels of the molecule in some, but not all, of the samples. From this work within the Suwannee river valley (48), we were than able to correlate the natural products discovery with Civil War medicinal folklore.

[FIGURE 2 OMITTED]

ACKNOWLEDGEMENTS

We would like to thank the Valdosta State University faculty development fund for purchasing various supplies used in this work. We would also like to thank the Georgia TIP (3) (Traditional Industry Program in Pulp and Paper) for also purchasing various supplies and equipment used in this work. Valdosta State University is thanked for purchasing FT-IR used in this work. Dr. Richard Carter of the VSU biology is thanked for discussions concerning local flora. Dr. David Williams of the VSU history department is thanked for some historical references. Dr. Durham Bell (Tifton, GA) is also thanked for providing a key reference related to the quinine work. We'd like to thank the University of Georgia (Athens, GA) for the LC-MS and MALDI-MS.

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Thomas Manning *

* Corresponding author, tmanning@valdosta.edu
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Author:Manning, Thomas; Umberger, Tice; Strickland, Stacy; Lovingood, Derek; Borchelt, Ruth; Manning, James
Publication:Georgia Journal of Science
Geographic Code:0GULF
Date:Jun 22, 2003
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