Ginseng Affects Induction of Luminescence in Vibrio harveyi

1. I-080 Ginseng Affects Induction of Luminescence in Vibrio harveyi J.C. Makemson, B. Ramalingam, and K. Mathee Florida International University, Miami, FL ABSTRACT Experimental Methods: Growth and Luminescence Curves HEPES Minimal – First Experiment HEPES Minimal Containing 0.0001% YE and 1 μM Fe Water soluble molecules in ginseng affect quorum sensing of Vibrio harveyi depending upon the growth medium. In complex broth (peptone-yeast extract-glycerol 75% sea water) ginseng inhibits quorum induced synthesis of the luminescent system. But, in minimal medium (ammonium-glycerol- 75%sea water) ginseng stimulated induction of luminescence by over 3 orders of magnitude after a brief period of inhibition. Stimulation of the production of luminescence in minimal broth was not affected by the presence of arginine a known inducer of luminescence. HPLC purified ginsenosides Rb2 and Re are responsible for part of the stimulation (~1.5 orders of magnitude) in minimal medium; other ginsenosides (Rb1, Rc, Rd and Rg1 had no effect). The ginsenosides caused no inhibition of quorum sensing in complex broth but rather produced a mild stimulation of luminescence. Ginseng molecules have multiple effects on lux regulation. CHANGES 1. Ginsengosides had no effect. The first two experiments were repeated with another source of ginsengosides, they had no effect. Quorum sensing effects are not due to ginsengosides. 2.Ginseng extracts (water or methanol) provided growth stimulation in HEPES Minimal. This made us re-examine growth in HEPES Minimal: V. harveyi requires some growth factor in Yeast Extract that can be provided by 0.0001% Yeast Extract. 3. We re-examined ginseng extracts in Autoinducer Bioassay (AB) medium. 1. Peptone or AB Media: Inoculate a starter culture, overnight at 25oC with shaking (200 rpm). Experimental: Peptone or AB medium 2.5 ml + small vol (µl of extracts or chemicals) inoculate with 25 to 100 l of overnight culture. Incubate 25oC with shaking (200 rpm). Measure OD660 for Growth and Luminescence in a calibrated photometer from the same tubes. 2. Minimal Medium: Inoculate a starter culture, overnight at 25oC with shaking (200 rpm). Experimental: Inoculate a larger volume (50 ml) to a very low calculated OD, incubate 25oC with shaking (200 rpm) so that the next morning, at 9AM the OD will be 0.05 to 0.10. Split (2.5 aliquots) culture to tubes with µl amounts of extracts or chemicals and continue incubation. Measure OD660 for Growth, Luminescence in a calibrated photometer from the same tubes. Inhibition in Peptone vs AB Media Ginseng Extracts Inhibit Quorum Sensing in Peptone Media HEPES Minimal Supplemented GROWTH MEDIA Effect of Purified Ginsengoside Rb1 CONCLUSIONS 1. Ginseng contains quorum sensing inhibitory molecule(s). Ginseng contains nutrients that increase growth of V. harveyi in Minimal Media. Ginseng molecules do not inhibit the arginine effect. Purified Ginsengosides (Rb1, Rb2, Rc, Rd, Re, Rf, Rg1) neither stimulate or inhibit quorum sensing.