Anti-inflammatory properties of a dried fermentate in vitro and in vivo

Abstract

The aim of this study was to document anti-inflammatory properties of a dried fermentate derived from Saccharomyces cerevisiae (EpiCor(®)), hereafter referred to as dried fermentate in vitro using cell-based bioassays, and in vivo using a skin irritation model in healthy humans. In vitro testing involved parallel assessment of primary human polymorphonuclear (PMN) cell formation of reactive oxygen species (ROS) and migration toward the inflammatory mediator Leukotriene B4. In vivo evaluation used a single-blind placebo-controlled design, where dermal histamine-induced inflammation was used as a model for the complex intercellular signals involved in the initiation, escalation, and resolution of the inflammatory response. Microvascular blood perfusion was evaluated using noninvasive laser Doppler probes applied to the inner forearms of 12 healthy human subjects, where parallel sites were treated with either dried fermentate or saline (placebo). Subjective scores of dermal irritation were also collected. Treatment of PMN cells in vitro resulted in reduced ROS formation and migratory activity toward Leukotriene B4. Clinical results demonstrated significantly reduced microvascular inflammatory responses to histamine-induced skin inflammation, and significantly reduced subjective scores of irritation at the inflamed sites treated with dried fermentate compared with the sites treated with placebo (P<.05). Treatment of inflammatory cells in vitro with dried fermentate resulted in reduced inflammatory responses. This was confirmed in vivo, suggesting that the dried fermentate facilitates the resolution of inflammatory responses. The effects using a topical skin model suggest that similar events may happen when the dried fermentate is introduced across mucosal membranes after consumption.

Keywords: blood perfusion; histamine; laser Doppler; leukotriene B4; migration; polymorphonuclear; reactive oxygen species.

FIG. 1.

Anti-inflammatory effects were seen when polymorphonuclear cells were pretreated with the dried fermentate in vitro before the cells were tested in two different inflammation models. (A) Formation of reactive oxygen species (ROS) by polymorphonuclear cells in vitro. (B) Migration in response to the pro-inflammatory chemotactic mediator Leukotriene B4 (LTB4). Black bars show the inflammatory reactions by cells that were exposed to inflammatory stimuli without pretreatment with the dried fermentate (untreated: UT), and hatched bars show data from cells treated with the dried fermentate at the indicated doses in vitro before being assayed for the inflammatory responses. Each data point is based on triplicate measures (A) or quadruplicate measures (B). Levels of statistical significance shown by asterisks (*P<.05, **P<.01, and ***P<.001).

FIG. 2.

Diagram showing the principles and data generated from laser Doppler evaluation of microvascular blood perfusion. (A) Noninvasive application of a laser Doppler probe on the skin surface sends laser light into the skin. Data on reflected laser light of the same wavelength were not collected by the probe, however, laser light of a different wavelength, as a result of a Doppler shift, is collected and quantified as a measure of relative microvascular blood perfusion and red blood cell velocity. The Doppler shift only happens when laser light hits moving red blood cells. (B) Representative example of the laser Doppler data collected during a test run for this study: Baseline blood perfusion is evaluated for a minimum of 5 min, after which the probe is gently removed from its positioning on the skin. Ten microliters of a dilute solution of histamine (0.4 mg/mL), is applied to the site, and a small prick made using a Hollister–Stier lancet. After 1 min, a cotton tip is used to remove the histamine by gentle blotting without rubbing the skin site. Then, 10 microliters of either the dried fermentate (dose 0.1 g/mL) or saline is applied, the Doppler probe is re-applied to the site, and data were collected to measure the magnitude of the microvascular effects of the inflammatory insult.

FIG. 3.

Laser Doppler perfusion on two skin sites on the forearm. Both sites were treated with histamine and one site subsequently treated with the dried fermentate, while the other site was treated with saline. The treatments were blinded to the subject. (A) Blood perfusion on each site from a representative study participant. The number of minutes it took for the histamine-induced increase in blood perfusion to plateau and start to decline was identified as the Time to max (Tmax) for each site (saline versus dried fermentate). (B) T(max) and the slope for the subsequent reduction in blood perfusion was averaged for all study participants. The average and standard deviation are shown for T(max) and slope. The reduced T(max) and steeper slope for the sites treated with the dried fermentate were significantly lower than the T(max) for the sites treated with saline (*P<.05).

FIG. 4.

Diagram showing the initiation and resolution of the inflammatory response. (A) Circulating polymorphonuclear cells are shown as they adhere to the vessel wall, extravasate, produce pro-inflammatory compounds, produce anti-inflammatory compounds, and finally undergo apoptosis. (B) The normal wave of production of pro- versus anti-inflammatory compounds is shown to the left. To the right is shown an inflammatory response with inappropriate resolution, and prolonged production of pro-inflammatory compounds.