Effects of Oral Administration of Purified Micronized Flavonoid Fraction on Increased Microvascular Permeability Induced by Various Agents and on Ischemia/Reperfusion in the Hamster Cheek Pouch

Abstract

The effects of a clinically used purified micronized flavonoid fraction (S 5682) containing 90% diosmin and 10% hesperidin on increased microvascular permeability induced by histamine, bradykinin, and leukotriene B₄ (LTB₄) were investigated by intravital microscopy in the hamster cheek pouch preparation. The authors also investigated the effects of S 5682 on macromolecular permeability increase and leukocyte adhesion during ischemia-reperfusion by using the same preparation. S 5682, suspended in 10% lactose solution, or vehicle (10% lactose) was administered orally to male hamsters for ten days at 20 mg/kg/day (10 mg/kg twice a day). Fluorescein isothiocyanate (FITC)-labeled dextran (mol wt 150,000) was given intravenously, thirty minutes after completion of the cheek pouch preparation. The leukocytes were stained by continuous IV infusion of acridine orange (0.5 mg/kg/minute). Histamine (2 μM), bradykinin (1 μM), and LTB ₄ (0.01 μM), applied topically for five minutes, increased the number of fluorescent vascular leakage sites in postcapillary venules. A temporary ischemia with total circula tory arrest of the cheek pouch was obtained by clamping the neck of the everted pouch. The maximum number of leaky sites (per cm² in the prepared area) that occurred either at five minutes after the beginning of each topical application or ten minutes after the onset of reperfusion was quantified in ultraviolet light microscopy. The results from 60 animals divided into 10 groups of 6 animals each are presented as means ±SEM. In comparison with vehicle, S 5682 significantly inhibited the macro molecular permeability-increasing effect of histamine (343.5 ±22.3 versus 207.5 ±32.0 leaks/cm²; P<0.01), bradykinin (345.2 ± 19.0 versus 206.2 ±21.6 leaks/cm²; P<0.01), and LTB₄ (353.3 ±27.5 versus 242.7 ±33.6 leaks/cm²; P<0.05). At reperfusion, after thirty minutes of ischemia, S 5682 significantly decreased the observed macromolecular permeability (103.6 ±15.4 versus 42.6 ±9.3 leaks/cm² ; P<0.01). Flavonoid-treated animals also displayed a statistically significant lower number of adhering leukocytes to the venular endothelium (83.5 ±9.5 versus 48.4 ± 12.3 per 6 mm²; P<0.05). These results demonstrate that oral administration of S 5682 for ten days at 20 mg/kg body weight/day had a protective effect against leakage of macromolecules after application of permeability-increasing substances and during ischemia-reperfusion in the cheek pouch microvasculature. Since firm leukocyte attachment to the endothelial wall and subsequent emigration of leukocytes into the interstitium is a mechanism for tissue damage during inflammation, attenuation of this phenomenon during conditions of ischemia-reperfusion can in part explain previous observations that this purified micronized flavonoid fraction decreases edema formation. The present data illustrating the inhibitory effect of a clinically relevant dose of S 5682 on the inflammatory processes induced in this in vivo model of microcirculation may serve as a rational basis to explain its clinical efficacy.