Stephen M. Boue

Screening and Identification of Glyceollins and Their Metabolites by Electrospray Ionization Tandem Mass Spectrometry with Precursor Ion Scanning

A method has been developed for screening glyceollins and their metabolites based on precursor ion scanning. Under higher-energy collision conditions with the employment of a triple quadrupole mass spectrometer in the negative ion mode, deprotonated glyceollin precursors yield a diagnostic radical product ion at m/z 148. We propose this resonance-stabilized radical anion, formed in violation of the even-electron rule, to be diagnostic of glyceollins and glyceollin metabolites. Liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) established that scanning for precursors of m/z 148 can identify glyceollins and their metabolites from plasma samples originating from rats dosed with glyceollins. Precursor peaks of interest were found at m/z 337, 353, 355, 417, and 433. The peak at m/z 337 corresponds to deprotonated glyceollins, whereas the others represent metabolites of glyceollins. Accurate mass measurement confirmed m/z 417 to be a sulfated metabolite of glyceollins. The peak at m/z 433 is also sulfated, but it contains an additional oxygen, as confirmed by accurate mass measurement. The latter metabolite differs from the former likely by the replacement of a hydrogen with a hydroxyl moiety. The peaks at m/z 353 and 355 are proposed to correspond to hydroxylated metabolites of glyceollins, wherein the latter additionally undergoes a double bond reduction.

Silver enhances the in vitro antifungal activity of the saponin, CAY-1

The fungicidal properties of purified CAY‐1, dissolved silver ion and ethylenediamine tetraacetic acid (EDTA) separately were studied in vitro as were the abilities of silver and EDTA to enhance CAY‐1 fungicidal properties. Non‐germinated and germinating conidia of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Fusarium verticillioides (Fusarium moniliforme), Fusarium oxysporum and Fusarium solani were incubated separately with CAY‐1 (0–24.8 μg ml−1), silver (0–111.1 μg ml−1), and EDTA (0–2400 μg ml−1). Controls consisted of non‐germinated or germinated conidia in test medium. To assess combined activity, compounds, based on the sub‐lethal doses of each as defined in the initial experiments, were combined and tested in bioassays. Controls for the mixed sets consisted of non‐germinated or germinated conidia only or with the sub‐lethal CAY‐1 test concentrations. The minimum inhibitory concentrations (MICs) for CAY‐1 and silver, both separate and combined, were determined. Viability assays showed CAY‐1 activity only against the germinating conidia of A. flavus, A. niger and F. solani. Silver was active against the germinating conidia of all fungi and the non‐germinated conidia of F. oxysporum and F. solani. Combined silver and CAY‐1 produced significant viability loss at concentrations not effective separately. EDTA was not fungicidal separately and did not enhance CAY‐1 fungicidal properties. MIC data showed that CAY‐1 plus silver had an additive effect. Results indicate that dissolved silver was fungicidal in vitro and enhanced the fungicidal properties of CAY‐1 at concentrations ineffective when tested separately.

Greenhouse and field evaluation of the natural saponin CAY-1 for control of several strawberry diseases.

The steroidal saponin from cayenne pepper, CAY-1, was tested as a potential fungicide in detached-leaf assays and field trials. Efficacy of CAY-1 against strawberry anthracnose was compared to the commercial fungicide azoxystrobin. Both fungicides prevented anthracnose leaf lesions when applied to the host 24 hours prior to inoculation with Colletotrichum fragariae, but neither prevented lesion development when applied 24 hours after inoculation. CAY-1 reduced the growth of several fungal pathogens in laboratory assays and prevented anthracnose development in detached-leaf assays, but it did not control foliar or fruit-rot diseases of strawberry in field trials.