Influence of Anti-Inflammatory Flavonoids on Degranulation and Arachidonic Acid Release in Rat Neutrophils Marí a Tordera, Marí a Luisa Ferrándiz and María José Alcaraz Departamento de Farmacologia de la Universidad de Valencia, Facultad de Farmacia, Avda. Vicent Estelles s/n, 46100 Burjassot, Valencia, Spain Z. Naturforsch. 49c, 235-240 (1994); received December 30, 1993 Flavonoids, Anti-Inflammatory Flavonoids, Arachidonic Acid, Lysosomal Enzymes, Rat N eutrophil We assessed the effects of 24 flavonoid derivatives, reported as anti-inflammatory, on lyso­ somal enzyme secretion and arachidonic acid release in rat neutrophils. Amentoflavone, quer- cetagetin-7-O-glucoside, apigenin, fisetin, kaempferol, luteolin and quercetin were the most potent inhibitors of ß-glucuronidase and lysozyme release. The first compound was also able to inhibit basal release. These flavonoids besides chrysin and to a reduced extent, naringenin, significantly inhibited arachidonic acid release from membranes. A correlation between de­ granulation and arachidonic acid release was found for this series of compounds. Structure- activity relationships and implications for the anti-inflammatory effects of these flavonoids were discussed. Introduction of flavonoids on reactive oxygen species genera­ Besides a wide range of biological activities tion in neutrophils (Limasset et al., 1993). (Havsteen, 1983) some members of the flavonoid Secretion is a crucial event induced by leukocyte class display anti-inflammatory effects in animals activation and resulting in the release of degrada- (Alcaraz and Jimenez, 1988; Ferrändiz and Alca­ tive enzymes which play an important role in tissue raz, 1991). The mechanism of action is unclear but damage during inflammation. There are several it may involve the inhibition of eicosanoids prod­ possible links between arachidonic acid release uction by intact cells with different selectivity to­ and secretion. Thus, products derived from PLA2 wards cyclo-oxygenase or lipoxygenase pathways activity could be required for the fusion of lyso- depending on their structural features (Moroney somes and plasma membranes, or arachidonic acid et al., 1988). Other actions possibly account for the released would induce degranulation and other molecular mechanisms leading to control the in­ neutrophil responses (Smolen and Weissmann, flammatory process. As suggested for many non­ 1993). steroidal anti-inflammatory agents (Smolen and The objective of this study was to obtain evi­ dence as to whether a series of flavonoids endowed Weissmann, 1993; Kaplan et al., 1984; Neal et al., 1987; Smolen et al., 1991; Inoue et al., 1991; Zim- with anti-inflammatory properties, modify select­ merli et al., 1991) the suppression of leukocyte ed neutrophil responses which contribute to the in­ functions may provide a basis for the anti-inflam­ flammatory process. Thus, we have investigated matory effects of flavonoids. In this respect, recent the capacity of a series of flavonoids to inhibit de­ attention has focused upon the inhibitory effects granulation in rat neutrophils and we have includ­ ed as reference some compounds, like quercetin, previously reported as inhibitors of leukocyte functions in other species (Bennett et al., 1981; Middleton Jr. et al., 1987; Showell et al., 1981; Abbreviations: BSA, bovine serum albumin; DM SO, Blackburn et al., 1987; Berton et al., 1980). In dimethyl sulfoxide; F M L P, N-formyl-L-methionyl- order to understand better the modification of L-leucyl-L-phenylalanine; PBS, phosphate buffer saline; eicosanoid generation by flavonoids in relevant in­ PLA 2, phospholipase A 2; PLC, phospholipase C; PLD, phospholipase D. flammatory cells, we have also assessed in the Reprint requests to Prof. Maria Jose Alcaraz. present study the influence of these flavonoids on the release of arachidonic acid from leukocyte Verlag der Zeitschrift für Naturforschung, D-72072 Tübingen membranes, a process mainly dependent on PLA2 0939-5075/94/0300-0235 $03.00/0 activity. 236 M. Tordera et al. ■ Flavonoids and Selected Rat Neutrophil Functions Materials and Methods Arachidonic acid release (Croft et al., 1987) Drugs Cells were suspended in Hank's solution at a Sideritoflavone was isolated from Sideritis leu- concentration of 107 ml and incubated with arachi­ cantha and hypolaetin-8-O-glucoside from Sideri­ donic acid (0.1 |iCi/ml) for 60 min at 37 °C. Leu­ tis mugronensis, following known procedures (Vil- kocytes were then washed in PBS three times and lar et al., 1985; Jimenez et al., 1986). Oroxindin resuspended in H ank’s solution + 0.1 % BSA. Test from Oroxylum indicum; quercetagetin-7-O-gluco- compounds were preincubated for 15 min at 37 °C side from Tagetes erecta, gossypin and hibifolin before addition of ionophore A 23187 (25 (ig/ml). from Hibiscus vitifolius, and tambuletin from After 10 min, tubes were placed on ice and centri­ Zhanthoxylum alatum were a gift from Prof. fuged as above. Radioactivity was determined in A. G. R. Nair (Pondicherry University, India). supernatants by liquid scintillation counting. Other flavonoids were commercially available: fla- Results were expressed as means ± SE for 3-6 vone, 3-hydroxyflavone and fisetin (Aldrich); tro- determinations. Compounds able to inhibit con­ xerutin (Almirall); quercetin (Merck); apigenin, lu- trol reactions at least by 50% were tested over a teolin and amentoflavone (Roth); leucocyanidol range of concentrations to determine their half- (Rovi); chrysin, rutin, morin, naringenin, naringin, maximal inhibitory concentration. Statistical (+)-catechin, (-)-epicatechin and kaempferol analysis was performed by using the Dunnett’s (Sigma). [14C]-arachidonic acid (100|iCi/ml) was t-test. purchased from DuPont. Ionophore A 23187, BSA, glycogen, cytochalasin B, FMLP and triton X-100 were purchased from Sigma. The rest of the Results chemicals were of analytical grade. In control incubations, the extent of lysozyme secretion was more than twice that of ß-glucuroni- dase either in the presence or in the absence of sti­ Isolation of leukocytes mulus (data not shown). A number of flavonoids Rat peritoneal leukocytes elicited by glycogen inhibited ß-glucuronidase and lysozyme release, (10 ml, 1%) were prepared by centrifugation and taken as markers of rat peritoneal leukocytes de­ hypotonic lysis of contaminating red cells (Moro- granulation (Table I). Amentoflavone, querceta- ney et al., 1988). The cell suspension contained getin-7-O-glucoside, apigenin, fisetin, kaempferol, 90% neutrophils and viability was greater than luteolin and quercetin yielded more than 60% of 95% assessed by Trypan blue exclusion. inhibition on both enzymes secretion. Besides, oroxindin, chrysin and hibifolin affected selective­ ly ß-glucuronidase release. The regression analysis of the curves obtained for these active compounds, Lysosomal enzyme secretion showing more than 60% of inhibition at 10"4 m , at Leukocytes were suspended at 6 x 106 cells/ml in a range of concentrations between 10“5 m and PBS and preincubated for 8 min at 37 °C with 10~4m (or 10“6m and 10-4m for amentoflavone), cytochalasin B (5 |ig/ml) followed by addition of allowed us the calculation of the inhibitory con­ test compound or vehicle (10|il DMSO). After centration 50% (IC50). Amentoflavone showed the 8 min, FMLP (10-6 m ) was added and incubation highest potency, especially for inhibition of ß-gluc- proceeded for 8 min. The cell suspensions were uronidase release, with an IC50 in the [IM range then placed on ice and centrifuged at 3000 r.p.m. (Fig. 1). At concentrations between 3 x 10-5 m and for 15 min at 4 °C. ß-Glucuronidase and lysozyme 1 0 -4 m amentoflavone gave percentages of inhibi­ levels in supernatants were determined by spectro- tion for ß-glucuronidase release higher than 100%, photometric procedures previously described indicating its ability to influence degranulation in (Gianetto and de Duve, 1955; Yuli et al., 1982) and basal conditions (in the absence of any stimulus). results were expressed as percentage of release with In this case, its IC50 was 4.5±0.1 x 10~5 m and respect to tubes treated with 10 |il 20% triton 6.2 ± 0.4 x 10'4 m, for ß-glucuronidase and lyso­ X-100. zyme basal release, respectively. For most com- M. Tordera et al. • Flavonoids and Selected Rat Neutrophil Functions 237 Table I. Structure of the flavonoids tested. Effect on leukocyte degranulation: percentage of inhibition at 10 4 m (% I) and inhibitory concentration 50% ( i c 50). 3' ß-Glucuronidase Lysozyme 5 m) % I IC50 (x 10~5 m) vones r 5 r 6 r 7 r 8 Ry R, % I IC50(xlO - vone H HHHH H 56.7 ±2.9** N.D. 32.6 ± 1.0** N.D. rysin OH H OH H H H 72.0± 4.8** 6.6 ±0.9 51.7 ± 3.3** N.D. igenin OH H OH H H OH 92.9 ±4.0** 2.6 ± 0.3 71.8 ± 3.3** 6.5 ±0.7 eolin OH H OH H OH OH 90.9 ±3.0** 3.5 ± 1.2 72.0 ± 1.3** 4.6 ±0.3 eritoflavone OH o c h 3 OCH, OCHj OH OH 32.7 ±2.1** N.D. 11.0 ±5.9 N.D. polaetin-8-O-Gl OH H OH OG1 OH OH 2.0 ± 2 .2 N.D. 12.2 ±2.4 N.D. )xindin OH H OGlr OCHj H H 70.8 ±3.1** 7.4 ± 1.1 44.3 ±2.0** N.D. ß-Glucuronidase Lysozyme r 2. r 3. % I IC jotx 10- 5 m) % 1 IC50 (X 10-5 M) vonols r 3 R-5 r 7 Rs r 4 fydroxyflavone OH H HHHH H H --103.2 ± 2.1** 6.7 ±0.7 -5 4 .2 ± 6.5** N.D. etin OH H H OH H H OH OH 100.0 ± 5.2** 2.3 ±0.3 101.7 ± 14.2** 3.3 ±0.2 empferol OH OH H OH H H H OH 74.4 ± 3.7** 4.5 ± 1.2 75.8 ± 8.9** 2.9 ±0.8 ercetin OH OH H OH H H OH OH 90.4± 0.9** 2.4 ±0.1 88.5 ± 2.7** 2.1 ± 0 .2 >rin OH OH H OH H OH H OH 36.3 ± 7.0** N.D.