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ARS Home » Midwest Area » Columbia, Missouri » Cropping Systems and Water Quality Research » Research » Publications at this Location » Publication #304428
Title: A simple method for isolation and purification of DIBOA-Glc from Tripsacum dactyloides (L.)

Author
item WILLETT, CAMMY - University Of Missouri
item Lerch, Robert
item GOYNE, KEITH - University Of Missouri
item LEIGH, NATHAN - University Of Missouri
item LIN, CHUNG - University Of Missouri
item ROBERTS, CRAIG - University Of Missouri

Submitted to: Natural Product Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/10/2014
Publication Date: 9/12/2014
Citation: Willett, C.D., Lerch, R.N., Goyne, K.G., Leigh, N.D., Lin, C.H., Roberts, C.A. 2014. A simple method for isolation and purification of DIBOA-Glc from Tripsacum dactyloides (L.). Natural Product Communications. 9(9):1283-1286.

Interpretive Summary: Long-term and widespread use of the corn herbicide atrazine has led to significant contamination of surface and ground water resources across the U. S. Corn Belt. New strategies are needed to remove atrazine residues from soil and prevent its off-site transport. Forage grasses, such as Eastern gamagrass (EG), have been identified as potential sources of chemicals that can enhance the breakdown of atrazine in soils. The use of plants to rid the environment of a contaminant is called phytoremediation and this strategy has been applied to other point and non-point source pollutants. Recently, we isolated an atrazine-degrading phytochemical, referred to as DBG, from EG that shows promise for enhancing atrazine degradation in soils. This, or related phytochemicals, could lead to the development of a commercial product that could be used alone or in concert with existing phytoremediation or agricultural management practices to further enhance atrazine degradation in soils and minimize contamination of water resources. Detailed chemical studies of compounds like DBG have been hindered by an inability to obtain sufficient quantities of purified material for use as authentic standards. Previous methods to synthesize or isolate atrazine-degrading phytochemicals involved highly specialized equipment and/or dangerous chemicals. The objective of this research was to develop a simple purification and isolation method for obtaining bulk quantities of highly purified DBG. The developed method was based on solvent extraction of EG roots followed by separation and isolation of DBG from the extract. The purity of the obtained material was >99% DBG. The newly developed method will facilitate future research to characterize the chemical behavior of DBG and determine its potential as an atrazine mitigation and remediation tool. Researchers interested in phytoremediation can apply this method for isolation and study of other phytochemicals that could lead to new strategies for remediating contamination of soil and water resources.

Technical Abstract: Naturally occurring benzoxazinone (Bx) compounds have been studied extensively over the last several decades and are highly reactive natural compounds. Recently, we identified 2-Beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) as the atrazine-degrading compound present in the roots of Eastern gamagrass (Tripsacum dactyloides L.). However, characterization of the DIBOA-Glc /atrazine degradation reaction has been limited due to obstacles in attaining sufficient quantities of purified DIBOA-Glc. The objective of the study was to develop a simple purification and isolation method for obtaining bulk quantities of highly purified DIBOA-Glc. T. dactyloides roots were extracted with 90 percent aqueous methanol, and the crude extract was fractionated using high-performance liquid chromatography (HPLC) equipped with a C8 semi-prep column and fraction collector. Ultra high-performance liquid chromatography tandem mass spectrometry with diode array detection (UHPLC-MS/MS-DAD) was used to confirm the identity of DIBOA-Glc in the collected fractions. Analysis by 13C and 1H nuclear magnetic resonance (NMR) and DAD indicated that 542 mg of DIBOA-Glc with a purity of > 99 percent was obtained. A solution containing 1000 uM DIBOA-Glc and 10 uM atrazine resulted in degradation of 48.5 plus or minus 1.2 percent (SD) of the atrazine in 16 hours. This simple approach offers several advantages over existing extraction and synthesis methods, which are more cumbersome, use hazardous chemicals, and yield only small quantities of purified compound. The newly developed method will facilitate future research to characterize the chemical behavior of DIBOA-Glc and determine its potential as an atrazine mitigation and remediation tool.