title: An experimental implementation of chemical subtraction.
abstract: A preparative analytical method was developed to selectively remove ("chemically subtract") a single compound from a complex mixture, such as a natural extract or fraction, in a single step. The proof of concept is demonstrated by the removal of pure benzoic acid (BA) from cranberry (Vaccinium macrocarpon Ait.) juice fractions that exhibit anti-adhesive effects versus uropathogenic Escherichia coli. Chemical subtraction of BA, representing a major constituent of the fractions, eliminates the potential in vitro interference of the bacteriostatic effect of BA on the E. coli anti-adherence action measured in bioassays. Upon BA removal, the anti-adherent activity of the fraction was fully retained, 36% inhibition of adherence in the parent fraction at 100mug/mL increased to 58% in the BA-free active fraction. The method employs countercurrent chromatography (CCC) and operates loss-free for both the subtracted and the retained portions as only liquid-liquid partitioning is involved. While the high purity (97.47% by quantitative (1)H NMR) of the subtracted BA confirms the selectivity of the method, one minor impurity was determined to be scopoletin by HR-ESI-MS and (q)HNMR and represents the first coumarin reported from cranberries. A general concept for the selective removal of phytoconstituents by CCC is presented, which has potential broad applicability in the biological evaluation of medicinal plant extracts and complex pharmaceutical preparations.
authors.affiliation: UIC/NIH Center for Botanical Dietary Supplements Research, Department of Medicinal Chemistry and Pharmacognosy & PCRPS, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Shao-Nong
authors.initials: SN
authors.lastname: Chen
authors.forename: Allison
authors.initials: A
authors.lastname: Turner
authors.forename: Birgit U
authors.initials: BU
authors.lastname: Jaki
authors.forename: Dejan
authors.initials: D
authors.lastname: Nikolic
authors.forename: Richard B
authors.initials: RB
authors.lastname: van Breemen
authors.forename: J Brent
authors.initials: JB
authors.lastname: Friesen
authors.forename: Guido F
authors.initials: GF
authors.lastname: Pauli
mesh_terms: D001422:Bacterial Adhesion / Q000187:drug effects*; D019817:Benzoic Acid / Q000302:isolation & purification* / Q000494:pharmacology; D001628:Beverages / Q000032:analysis*; D003377:Countercurrent Distribution / Q000379:methods*; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D014551:Urinary Tract; D029799:Vaccinium macrocarpon / Q000737:chemistry*
chemical_list: D019817:Benzoic Acid