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.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 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 OHHOHH 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 OHOH 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 OHH HHHH H H --103.2 ± 2.1** 6.7 ±0.7 -5 4 .2 ± 6.5** N.D. etin OHHH OHHHOH OH 100.0 ± 5.2** 2.3 ±0.3 101.7 ± 14.2** 3.3 ±0.2 empferol OHOHH OHHHH OH 74.4 ± 3.7** 4.5 ± 1.2 75.8 ± 8.9** 2.9 ±0.8 ercetin OH OHHOH H H OH OH 90.4± 0.9** 2.4 ±0.1 88.5 ± 2.7** 2.1 ± 0 .2 >rin OH OH H OHHOHH OH 36.3 ± 7.0** N.D. 0.7 ± 3.3 N.D. ercetagetin-7-O-Gl OH OH OH OG1 H HOHOH 72.4 ± 2.7** 6.9 ± 1.2 74.7 ± 6.2** 4.9 ± 1.0 nbuletin OH OH H OCH, OG1 H OH OCH-, 13.8± 2.8* N.D. 41.4 ± 15.0** N.D. tin ORu OHHOH HHOH OH 35.1 ±11.9* N.D. 12.3 ± 11.2 N.D. ixerutin ORu OH H OHEHHOHE OHE 36.9 ± 4.0 N.D. 38.3± 2.1 N.D. ssypin OH OH H OH OG1 HOH OH 15.5 ± 1.8 N.D. 0.5 ±10.3 N.D. lifolin OH OH H OH OGlr H OH OH 62.4 ± 4.0** 7.1 ± 1.0 9.3 ± 6.2 N.D.

ß-Glucuronidase Lysozyme ivanones R5 R7 R3 R4 %I IC50( x 10“5m) % I IC50( x 10~5 m) ringenin OH OH H OH 23.6 ±4.8** N.D. 8.0 ±2.0 N.D. ringin OH ORu H OH —1.9 ±1 .6 N.D. -5.5 ±7 .5 N.D.

Leucocyanidol

ß-Glucuronidase Lysozyme r IC50(x 10'5 m) IC50 (X 10-5 M) ivanols r 3 R4 r 5 r 7 3 R4 % I % 1

)-Catechin SOH H, H OH OHOHOH -7.5 ± 1.4 N.D. -4.4 ± 3.7 N.D. )-Epicatechin /?OH H, H OH OH OH OH -4.2 ± 2.5 N.D. 10.2 ±10.9 N.D. ucocyanidol 14.2 ± 1.2* N.D. 25.6 ± 2.3 N.D. 238 M. Tordera et al. • Flavonoids and Selected Rat Neutrophil Functions

ß-Glucuronidase Lysozyme Biflavones %I 1C50 ( x 10'5m) %I IC^ (x 10

Amentoflavone 131.6±2.4** 0.57±0.0 86.0±3.5** 2.5±0.3

Gl = Glucose, Ru = Rutinose, Glr = Glucuronic acid, HE = Hydroxyethyl. * P < 0.05, ** P < 0.01. The enzyme release in control tubes was 28.9 ± 1.7 and 67.5 ± 1.9 (mean ± SE, n = 30) % of the total enzyme content in cells, for ß-glucuronidase and lysozyme, respectively.

to blank samples (without flavonoid) were 7.0 x 10~5 m and 3.0 * 10“5 m, respectively. The flavonols fisetin, kaempferol and querceta- getin-7-O-glucoside, the flavones chrysin, apigenin and luteolin, as well as amentoflavone and to a very reduced extent, naringenin, significantly in­ hibited arachidonic acid release (Fig. 2). The refer­ ence compound, quercetin needed a concentration higher than those able to inhibit purified PLA2 ac­ tivity of different origins (Fawzy et al., 1988). We also found a significant correlation between inhi­ bition of ß-glucuronidase secretion and of arachi­

Fig. 1. Regression lines of amentoflavone (log. (4.M con­ donic acid release (r = 0.73, P < 0.001), as well as centrations) for inhibition of ß-glucuronidase (squares) between inhibition of lysozyme secretion and of and lysozyme (triangles) release. All points were statisti­ arachidonic acid release (r = 0.82, P< 0.001), for cally significant at least at P < 0.05, except for the two lower concentrations of amentoflavone on lysozyme the series of flavonoids studied. release.

pounds the potency exhibited for inhibition of the two enzymic markers was similar. 3-Hydroxyflavone was the only compound which exhibited a biphasic effect, since it inhibited ß-glucuronidase release without influencing lyso­ zyme release, but at higher concentrations it stim­ ulated degranulation in a significant manner (103.2 ±2.1 and 54.2 ±6.5% increase at 10~4 m, for ß-glucuronidase and lysozyme release, re­ spectively, P < 0.01). In another set of experiments carried out in the absence of any stimulus, 3-hy- Q QG K droxyflavone dose-dependently increased ß-gluc­ Fig. 2. Effect of flavonoids tested on arachidonic acid re­ uronidase and lysozyme release, with 282.8 ± 1.5 lease. C = chrysin, A = apigenin, L = luteolin, F = fise­ tin, Q = Quercetin, QG = quercetagetin-7-O-glucoside, and 139.5 ± 12.4% increase respectively (P< 0.01), K = kaempferol, N = naringenin and AF = amentofla­ at the concentration of 10“4 m. Concentrations vone. All results were significant at P < 0.01, except for causing approximately 50% increase with respect naringenin (P < 0.05). M. Tordera et al. • Flavonoids and Selected Rat Neutrophil Functions 239

Discussion donic acid release than on lysosomal enzyme secre­ tion; nevertheless, the structural features related to In our experiments lysozyme release levels were their inhibitory activity are similar for both neu­ higher than ß-glucuronidase release levels as trophil responses. reported by some authors in other systems (Ward The precise mechanism of flavonoid inhibition et al., 1984; Cockcroft and Stutchfield, 1989). of leukocyte functions is unclear. Recently, it has This was probably due to a higher sensitivity of been suggested a possible interaction of the less hy­ specific granules against different stimuli. This fact drophilic flavonoids with the lipid environment of could explain that some flavonoids have been the membrane FMLP receptor or with the recep­ more effective on azurophilic granules secretion. tor itself, while compounds with at least four hy­ Quercetin has been included in our study as a re­ droxyl groups would act at a later stage of the acti­ ference compound since its inhibitory effects on vation process and thus they would influence leu­ lysosomal enzyme release have been demonstrated kocyte responses in a non-specific way (Limasset in a number of systems such as rabbit neutrophils et al., 1993). However, interaction of polyhydroxy­ (Bennett et al., 1981; Showell et al., 1981) and hu­ lated flavonoids with membranes has been report­ man neutrophils (Blackburn et al., 1987) stimulat­ ed (Ratty et al., 1988) and it has been related to the ed by FMLP or concanavalin A (Berton et al., protective effects of these compounds on different 1980). Our results are in accordance with those cell types (Perrissoud and Testa, 1986). It is inter­ previously reported for quercetin, apigenin and esting to note that the inhibition of PLA2 activity luteolin on ß-glucuronidase release in human neu­ by quercetin has been explained in part by its abili­ trophils (Middleton et al., 1987), although we have ty to interact with phospholipidic substrates used a different cell system. We have also obtained (Fawzy et al., 1988). In addition, the behaviour of a better potency for flavone and a similar effect for amentoflavone which decreases basal secretion, 3-hydroxyflavone, with respect to results reported suggests a non-specific stabilizing action on cell in rabbit neutrophils (Bennett et al., 1981). membranes by this flavonoid, while 3-hydroxyfla­ Hypolaetin-8-O-glucoside failed to significantly vone could have the opposite effect, partially affect lysosomal enzyme secretion. This is not sur­ masked in the presence of stimulus due to some prising since many glycosides show a low level of likely interference with the activation pathway. activity on in vitro systems. Nevertheless, in an ex­ We have demonstrated that this series of anti­ perimental model of inflammation in rats, we pre­ inflammatory flavonoids can affect neutrophil viously demonstrated that this flavonoid inhibited function through mechanisms not attributable to ß-glucuronidase release (Villar et al., 1987). their effects on either the cyclo-oxygenase or The flavonoids able to inhibit lysosomal enzyme lipoxygenase pathway. Flavonoids have not exhib­ release share some structural features. They are ited a high potency on these in vitro tests, neverthe­ polyhydroxylated aglycones of the flavone or fla- less high doses of flavonoids are usually needed to vonol types and the presence of a free hydroxyl exert inhibitory effects on in vivo models of inflam­ group at C4' increases the inhibitory activity mation (Villar et al., 1987; Alcaraz and Jimenez, (chrysin-apigenin). Other important features are 1988; Ferrändiz and Alcaraz, 1991) where they the keto group at 4 position and the C 2-C 3 dou­ may achieve a range of concentrations enough to ble bond. In contrast, the glycosylation (quercetin- interact with leukocyte functions. Thus, with the rutin, naringenin-naringin) or the introduction of limitations derived from an in vitro study, it is like­ a free hydroxyl at C2' (kaempferol-morin) seems ly that quercetagetin-7-O-glucoside, apigenin, fise- to be detrimental. In summary, the most active tin, kaempferol, luteolin, quercetin and especially, compounds were C2-C3 unsaturated, hydroxy- amentoflavone may, at least in part, affect the in­ lated at C7 and C4' and with some additional hy­ flammatory response through their action on neu­ droxyl groups (at C5, C3 or C3'), characteristics trophils. included in those found for inhibition of other leu­ A cknowledgemen ts kocyte functions such as reactive oxygen species production by human neutrophils stimulated by This work was supported by grant PM 90-0 126, FMLP (Limasset et al., 1993). from C.I.C.Y.T., Spanish Ministerio de Education Flavonoids have been less effective on arachi- y Ciencia. 240 M. Tordera et al. ■ Flavonoids and Selected Rat Neutrophil Functions

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