Inhibitory effect of plant phenolics on fMLP-induced intracellular calcium rise and chemiluminescence of human polymorphonuclear leukocytes and their chemotactic activity in vitro

Figures & data

Figure 1. Typical changes of [Ca²⁺]_i in human PMNs after (a) direct stimulation with 5 μM ferulic acid; (b) after exposure to control solvent (DMSO in 0.9% saline, final DMSO concentration 1 mM); (c) cells preincubated for 30 min with DMSO in 0.9% saline and then stimulated with 0.5 μM fMLP; (d) cells preincubated for 30 min with 5 μM quercetin and then stimulated with 0.5 μM fMLP.

Table 1. Effect of polyphenols on fMLP-induced [Ca²⁺]_i response of human PMNs in vitro.

	fMLP-stimulated increase of [Ca^2+]i in human PMNs [nM]
Tested polyphenol (5 µM)	Preincubated with control solvent (Δ[Ca^2+]i C)	Preincubated with polyphenol (Δ[Ca^2+]i P)	Mean ratio of Δ[Ca^2+]i P/Δ[Ca^2+]i C
Quercetin	148.7 ± 54.5 (154.4)	100.5* ± 33.3 (96.3)	0.68 ± 0.14 (0.62)
Catechol	108.9 ± 25.0 (100.2)	97.5# ± 29.0 (89.1)	0.88 ± 0.10 (0.88)
3,4-Dihydroxycinnamic acid	108.9 ± 25.0 (100.2)	96.8 ± 27.4 (90.2)	0.89 ± 0.17 (0.96)
Phlorizin	148.7 ± 54.5 (154.4)	128.4 ± 37.1 (137.6)	0.90 ± 0.32 (0.83)
Chlorogenic acid	108.9 ± 25.0 (100.2)	97.0 ± 21.9 (98.0)	0.91 ± 0.21 (0.85)
Quinic acid	108.9 ± 25.0 (100.2)	108.1 ± 29.7 (99.8)	0.99 ± 0.13 (0.97)
Phloroglucinol	148.7 ± 54.5 (154.4)	142.1 ± 41.9 (144.1)	0.99 ± 0.33 (0.89)
Ferulic acid	108.9 ± 25.0 (100.2)	107.8 ± 19.5 (107.7)	1.00 ± 0.10 (0.98)
3,4-Dihydroxyhydrocinnamic acid	129.9 ± 30.4 (117.3)	133.6 ± 29.8 (139.8)	1.02 ± 0.13 (1.04)
3,4-Dihydroxybenzoic acid	148.7 ± 54.5 (154.4)	153.2 ± 50.0 (155.3)	1.04 ± 0.15 (1.04)
Catechin	129.9 ± 30.4 (117.3)	141.3 ± 24.4 (145.2)	1.09 ± 0.09 (1.10)
4-Hydroxybenzoic acid	129.9 ± 30.4 (117.3)	144.8 ± 32.3 (137.3)	1.12 ± 0.19 (1.04)
3,4-Dihydroxyphenylacetic acid	129.9 ± 30.4 (117.3)	149.6 ± 41.7 (156.5)	1.14 ± 0.19 (1.10)
Gallic acid	129.9 ± 30.4 (117.3)	155.4 ± 53.0 (155.8)	1.17 ± 0.28 (1.10)

Δ[Ca²⁺]_i P/Δ[Ca²⁺]_i C ratio reflects the compound’s ability to suppress increase of [Ca²⁺]_i after PMNs activation with fMLP, values less than 1 indicate the suppression. Results obtained from six series of experiments with cells from nine donors are expressed as a mean ± SD (median).

*p < 0.006 versus control, #p < 0.04 versus control.

Table 2. Effect of polyphenols on fMLP-induced luminol enhanced whole blood chemiluminescence in vitro (fMLP-LBCL).

	fMLP-induced whole blood chemiluminescence [RLU]
Tested polyphenol (5 µm)	Preincubated with control solvent	Preincubated with polyphenol	Percent inhibition (I%)
Catechol	12 161 ± 8442 (9431)	5656* ± 4282 (3884)	49 ± 5 (50)
Quercetin	12 745 ± 6715 (12 017)	6930* ± 4006 (6162)	42 ± 15 (44)
Gallic acid	10 920 ± 8957 (7373)	6413* ± 4663 (4259)	28 ± 18 (30)
3,4-Dihydroxyhydrocinnamic acid	10 920 ± 8957 (7373)	7581* ± 5936 (5275)	19 ± 14 (20)
Quinic acid	12 161 ± 8442 (9431)	9395* ± 7700 (6578)	17 ± 14 (14)
3,4-Dihydroxyphenylacetic acid	10 920 ± 8957 (7373)	7642* ± 6041 (4763)	16 ± 15 (13)
3,4-Dihydroxycinnamic acid	12 161 ± 8442 (9431)	9321* ± 7070 (5960)	15 ± 9 (15)
Chlorogenic acid	12 161 ± 8442 (9431)	9920 ± 8350 (6963)	14 ± 15 (17)
Catechin	10 920 ± 8957 (7373)	9047 ± 8380 (5476)	12 ± 15 (13)
Phloroglucinol	12 745 ± 6715 (12 017)	9829 ± 5279 (9516)	9 ± 26 (13)
3,4-Dihydroxybenzoic acid	12 745 ± 6715 (12 017)	10 455 ± 6200 (11 198)	9 ± 16 (6)
Phlorizin	12 745 ± 6715 (12 017)	10 759 ± 5132 (10 203)	6 ± 13 (4)
Ferulic acid	12 161 ± 8442 (9431)	10 736 ± 8905 (8472)	5 ± 14 (6)
4-Hydroxybenzoic acid	10 920 ± 8957 (7373)	10 472 ± 9349 (6368)	1 ± 17 (3)

Results expressed in arbitrary read light units (RLU) were obtained from eight series of experiments with blood from seven donors. Values are given as a mean ± SD (median).

*p < 0.05 versus control.

Table 3. Inhibitory effect of various concentrations of catechol, quercetin, and gallic acid on fMLP-induced luminol enhanced whole blood chemiluminescence in vitro.

	% inhibition of fMLP-induced whole blood chemiluminescence at given concentration (I%)
Tested polyphenol	0.5 µM	1 µM	2.5 µM	5 µM	7.5 µM	10 µM
Catechol	6 ± 9 (8)	13* ± 7 (13)	34* ± 9 (33)	49* ± 5 (50)	58* ± 7 (59)	67* ± 9 (68)
Quercetin	0 ± 12 (2)	11 ± 12 (12)	22* ± 10 (24)	42* ± 15 (44)	40* ± 24 (37)	53* ± 13 (58)
Gallic acid	1 ± 10 (0)	6 ± 10 (4)	10 ± 17 (11)	28* ± 18 (30)	39* ± 7 (41)	38* ± 14 (38)

Percent inhibition of chemiluminescent response (I%) by polyphenols, related to the control was calculated. Results obtained from eight series of experiments with blood from seven donors are expressed as a mean ± SD (median).

*Significant inhibition (p < 0.03 versus control).

Table 4. Chemoattractant properties of four plant phenolics for human PMNs: 4-dihydroxyhydrocinnamic acid, 3,4-dihydroxyphenylacetic acid, 4-hydroxybenzoic acid, and catechin.

	Tested polyphenol
Polyphenol concentration (µM)	3,4-Dihydroxy hydrocinnamic acid	3,4-Dihydroxy phenylacetic acid	4-Hydroxy benzoic acid	Catechin	Positive control fMLP (0.1 µM)	Negative control PBS
0.1	1.3 ± 0.8 (1.0)	1.2 ± 0.5 (1.2)	1.3 ± 0.6 (1.2)	1.2 ± 0.6 (1.1)	2.2*# ± 0.7 (2.1)	1.0 ± 0.0
1	1.2 ± 0.3 (1.1)	1.3* ± 0.2 (1.3)	1.3 ± 0.6 (1.2)	1.1 ± 0.4 (1.0)
5	1.9* ± 0.8 (1.6)	1.8* ± 0.7 (1.6)	1.6* ± 0.6 (1.6)	1.4* ± 0.2 (1.4)

Results expressed as a chemotactic index (C_i) were obtained from 12 series of experiments with cells from 44 donors shown as mean ± SD (median).

*p < 0.02 versus negative control (PBS with 1 mM DMSO), #p < 0.01 versus all tested polyphenols at all concentrations.