Screening Botanical Extracts for Quinoid Metabolites Benjamin M. Johnson, Judy L. Bolton, and Richard B. van Breemen* Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612-7231 Received June 25, 2001 Botanical dietary supplements represent a significant share of the growing market for alternative medicine in the USA, where current regulations do not require assessment of their safety. To help ensure the safety of such products, an in vitro assay using pulsed ultrafiltration and LC-MS-MS has been developed to screen botanical extracts for the formation of electrophilic and potentially toxic quinoid species upon bioactivation by hepatic cytochromes P450. Rat liver microsomes were trapped in a flow-through chamber by an ultrafiltration membrane, and samples containing botanical extracts, GSH and NADP(H), were flow-injected into the chamber. Botanical compounds that were metabolized to reactive intermediates formed stable GSH adducts mimicking a common in vivo detoxification pathway. If present in the ultrafiltrate, GSH conjugates were detected using LC-MS-MS with precursor ion scanning followed by additional characterization using product ion scanning and comparison to standard compounds. As expected, no GSH adducts of reactive metabolites were found in extracts of Trifolium pratense L. (red clover), which are under investigation as botanical dietary supplements for the management of menopause. However, extracts of Sassafras albidum (Nutt.) Nees (sassafras), Symphytum officinale L. (comfrey), and Rosmarinus officinalis L. (rosemary), all of which are known to contain compounds that are either carcinogenic or toxic to mammals, produced GSH adducts during this screening assay. Several compounds that formed GSH conjugates including novel metabolites of rosmarinic acid were identified using database searching and additional LC-MS-MS studies. This assay should be useful as a preliminary toxicity screen during the development of botanical dietary supplements. A positive test suggests that additional toxicological studies are warranted before human consumption of a botanical product. Introduction Cytochromes P450 constitute an important family of metabolic enzymes that carry out a variety of Phase I reactions with broad substrate specificity. Xenobiotic substrates for these enzymes are usually converted to metabolites that are more hydrophilic than their precur- sors, allowing them to distribute preferentially in the blood and be excreted in the urine. However, some xenobiotics including botanical compounds might be activated by P450 to short-lived electrophilic intermedi- ates capable of alkylating cellular biomolecules or par- ticipating in redox cycling reactions and causing cellular damage. For example, a botanical electrophile might contain a para-alkoxyl- or ortho-hydroxy-substituted phenol that could be metabolized to a quinone methide or ortho-quinone, respectively. An important human defense against such reactive intermediates is the nu- cleophilic tripeptide GSH, which binds covalently to electrophiles to form stable hydrophilic conjugates. En- zymatic and nonenzymatic conjugation between electro- philic metabolites and GSH is common in the liver, where the concentration of glutathione is approximately 10 mM (1). However, liver cells are often slow to respond to depletions of the GSH supply, and a large quantity of electrophilic material can overwhelm defense mecha- nisms and cause damage (2). Consumer demand for dietary supplements is growing in the United States of America, where the market for botanical dietary supplements (herbal medicines) in particular now exceeds $5 billion/year (3). Herbal supple- ments are often perceived to be safer than drugs because of their “natural” origin, long-standing ethnomedical use, and over-the-counter availability. However, crude plant extracts typically consist of a complex mixture of com- pounds and might contain high concentrations of specific compounds recovered from a plant during processing. Given the molecular diversity of botanicals, it is possible that toxic compounds might be extracted and concen- trated during the preparation of botanical dietary supple- ments. Another cause for concern is the possibility that human metabolism might activate some of these com- pounds to form reactive and potentially toxic products. Few botanical dietary supplements have been screened for toxicity because safety testing is not required in the United States and animal and human safety studies are time-consuming and expensive. Pulsed ultrafiltration-mass spectrometry is a drug discovery technique invented in our laboratory for the affinity selection and mass spectrometric identification of small molecules that interact with a macromolecular receptor (4). We demonstrated the use of this technology in the early stages of drug development by rapidly screening for metabolites and metabolic stability (5) and by screening drugs for metabolic activation to electro- philic intermediates (6). Here we report the use of pulsed * To whom correspondence should be addressed. Phone: (312) 996-9353. Fax: (312) 996-7107. E-mail: breemen@uic.edu. 1546 Chem. Res. Toxicol. 2001, 14, 1546-1551 10.1021/tx010106n CCC: $20.00 © 2001 American Chemical Society Published on Web 10/18/2001