499

Anti-Inflammatory Activity of Gallic Acid B. H. Kroes13, A. I I van den Berg1, H. C. Quarles van Ufford', H. van Dijk2, andR. P. Labadie' Research Centre for Natural Products and Phytopharmaceuticals, Faculty of Pharmacy, University of Utrecht, Sorbonnelaan 16, 3508 TB Utrecht, The Netherlands 2 Section of Experimental Immunology, Laboratory of Microbiology, Medical Faculty, University of Utrecht, The Netherlands Address for correspondence

Abstract Gallic acid was found to possess antiinflammatory activity towards zymosan-induced acute food pad swelling in mice. In vitro studies on the mode of action of gallic acid revealed that this compound interferes with the functioning of polymorphonuclear leuko-

This paper deals with the anti-inflammatory activity of gallic acid and its possible mode of action. Gallic acid was tested in vivo in a zymosan-induced foot pad inflammation model. In vitro studies were focussed on PMN functions such as the generation and transformation of O and other reactive oxygen species (ROS). Structural requirements for the expression of anti-inflammatory activity by gallic acid analogues will be presented.

cytes (PMNs). Scavenging of superoxide anions, inhibition of myeloperoxidase release and activity as well as a possible interference with the assembly of active NADPH-oxidase may account for the inhibition of inflammatory process by gallic acid. Structure-activity relationship analysis showed that the o-dihydroxy group of gallic acid is important for the inhibitory activity in vitro. Key words

Gallic acid, anti-inflammatory activity, granulocytes, immunomodulation, chemiluminescence.

Materials and Methods Buffers and reagents Complement assays: Five times-concentrated veronal saline buffer, pH 7.35 (VSB-5x), prepared according to Mayer (8), served as a stock solution for the preparation of VSB', containing 0.5mM Mg2 and 0.15mM Ca2, and for EGTA-VB, con-

taining 2.5mM Mg2, and 8mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA, Aldrich).

Chemiluminescence assay: Experiments were performed in Hank's balanced salt solution buffered at pH 7.35 with NaHCO, (HBSS, Gibco, Paisly, Scotland). Before use, 0.1 % (w/v) of gelatin was added to the buffer to avoid cell aggregation (HBSS-gel).

Introduction For more than a decade an ethnopharmacognostic approach is used in our laboratory to search for immunomodulators of plant origin (1). Besides the iso-

lation of various active novel compounds (2, 3), this approach has also led to the discovery of new interesting properties of known plant constituents (4— 6).

Azadirachta indica bark is the main ingredient in the preparation of the ayurvedic drug Nimba

arishta which is applied in Sri Lanka to skin diseases (rashes) and as blood-purifier. By activity-guided frac-

tionation of an aqueous extract of Azadirachta bark one of the compounds we ended up with was gallic acid which was found to inhibit luminol-dependent chemilumines-. cence generated by zymosan-stimulated human polymorphonuclear leukocytes (PMNs) (5). Consequently, the inhibi-

tion of chemiluminescence by Nimba arishta was found to correlate with the gallic acid content of the preparation (7). Inhibition in the chemiluminescence assay may be indicative of anti-inflammatory activity in vivo.

Zymosan-induced inflammation Effects of gallic acid on zymosan-induced inflammation were studied in groups of five male BALB/c mice of 10—12 weeks. Inflammation was induced by injecting 300 pg of zymosan (Sigma Chemicals Co., St. Louis MO, USA), suspended in 25 p1 of sterilized saline in the left hind foot pad. Gallic acid (Carl

Roth OHG, Karlsruhe FRG) dissolved in 500 p1 of saline was administered intra-peritoneally (i.p.) immediately before and 3 hours after zymosan injection. Control mice were injected with saline. Foot pad swelling was measured 6 hours after induction. The thickness of the foot pad of individual mice before zymosan injection served as control.

Erythrocytes Sheep and rabbit blood in citrate-buffered glucose (Alsever's solution) served as sources of sheep erythrocytes (ShE) and rabbit erythrocytes (RaE), respectively. To elute possibly

adsorbed serum proteins, the erythrocytes were washed three

times with 0.16M sodium iodide before use. ShE were resuspended in VSB2 (4 x 108 cells/mi), and sensitized by incahation

with an equal volume of 1: 2000 diluted rabbit anti-ShE serum (hemolytic amboceptor) at room temperature for 10mm (the sensitized cells are referred to as ShEA). Subsequently the mixture

was centrifuged and ShEA were resuspended in VSB' (4 x io cells/mI). RaE were resuspended in EGTA-VB (3 x 108 cells/mi).

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Received: December 16. 1991

500 PlontaMed. 58 (1992)

in a total volume of 550 pl (HBSS). After incubation, the test mixture was centrifugated at 1300rpm and 4°C for 15mm. Subsequently, 250 p1 of the supernate was mixed with 625 p1 of HBSS, 750 pl of

Human pooled serum (HPS) from five healthy volunteers was used as source of complement. Classical pathway

0.1M potassium phosphate buffer (pH 6.2), l25pl of 3,3'-dimeth-

(CP) and alternative pathway (AP) activities were determined by a modified version of the microassay described by Klerx et al. (9).

Belgium; 2.5 mg/mi), and 125 p1 of 0.05% H202. The mixture was

The test was performed in U-well microtitre plates (no. 651101; Greiner Labor-technik, Nurtingen, FRG). Logarithmic dilutions of gallic acid were prepared in VSB2 (CP) or EGTA-VB (AP). To each well containing 100 p1 of these dilutions, 25 p1 of 10_16 dilution of HPS in VSB2 (CP) or 25 p1 of a 1002 dilution of HPS in EGTA-VB

(AP) were added after which the microtitre plates were preincubated for 30mm at 37°C. Subsequently, 25 p1 of ShEA (4 x 108 cells/mi VSB2*) (CP) or RaE (uncoated) (3 x 108 cells/mi EGTA-VB) (AP) were added. The plates were incubated at 37°C for 60 mm (CP) or 30 mm (AP). After incubation, the plates were centrifuged at 3000 rpm for 5mm to precipitate intact cells and cell ghosts. To determine the degree of hemolysis, 5Opl of the supernatants were mixed with 200 p1 of water in 96 wells flat-bottom microtitre plates (no 655101; Gremner). The light absorbance at 405 nm was measured using an automatic Elisa reader (STL instruments, model SF

plus). Controls in this assay consisted of similarly treated supernatants of erythrocytes incubated with water (100% hemolysis), or VSB2, EGTA-VB, or heat-inactivated (56°C for 30 mm) serum

dilutions (0% hemolysis), or buffer supplemented with the appropriate dilution of HPS (0% inhibition).

Chemiluminescence assay Polymorphonuclear leukocytes (PMNs) were isolated from venous blood of healthy volunteers as described by Verbrugh et al. (10). Zymosan was opsonized by incubation with diluted HPS for 30mm at 37°C (serum-treated zymosan, STZ). In 2 ml

oxybenzidine dihydrochioride (from Janssen Chimica, Beerse incubated at room temperature for 15 mm, after which 125pl of 1 % sodium azide was added. MPO levels were quantified by measuring the absorb ance at 460 nm. Galllc acid was added to the incubation mixture containing PMNs to assess its effect on both MPO release and MPO activity. The effect of gallic acid on MPO activity only, was assessed by addition of the substance to the mixture con-

taining the supernate.

Results Anti-inflammatory activity of gallic acid in vivo

Graded amounts of gallic acid were tested

in mice for their activity towards zymosan-induced inflammation. As shown in Fig. 1, gallic acid was found to exhibit a dose-dependent anti-inflammatory activity in vivo. Since the foot pad swelling was measured 6 hours after in-

duction, the model represents an acute inflammatory response. This type of inflammation is mediated by complement and PMNs (13, 14). Therefore in vitro studies on mechanistic aspects of the anti-inflammatory activity of gallic acid initially were focussed on both the complement system and PMNs.

1.75'

flatbottom vials (Sterilin Ltd, Middlesex, UK), 50 p1 of a suspension of PMNs (1 x io cells/mi HBSS-gel), 100 p1 of a luminol solution (3OpM in HBSS-gel) or lucigenin (0.7mM), and a metal spin bar were added to 500 p1 of logarithmic dilutions of gallic acid (Roth) in HBSS-gel. Subsequently, the vials were placed in a Packard Picolite Model 6500 Luminometer (Packard United Technologies, Downers Grave IL, USA) to equilibrate at 37°C under gentle stirring. Chemiluminescence was induced by adding 50pi of STZ (0.6 mg/mI) or phorbol myristate acetate (PMA; 140 ng/ml), and monitored every 2 mm for 5 sec. The peak levels (PL), which were reached after 8 to 10 mm, were used to calculate the activity of the sample in comparison with a control (identical incubates without the test compound or mixture). Modulatory effects on the chemilumiriescence are expressed as PLsampie/PLcontroj x 100%. Tests were performed in duplicate.

Assay for O-scavenging activity Scavenging activity was determined spectro-

photometrically by monitoring the effect of gallic acid on the reduction of cytochrome C by O (11). O anions were produced in a cell free system containing hypoxanthmne (HX) and xanthine oxidase (XO). The mixtures in the sample cell consisted of 1mM HX (Sigma Chemicals Co., St. Louis MO, USA), 0.63 mg/mi of cytochrome C (Sigma, no. C 7752; from horse heart, type IV), exponential

dilutions of gallic acid (Roth, Karlsruhe, FRG), and 29 mU XO (Sigma, no. X 4500; from butter-milk, grade III) in 0.1 M phosphate buffer (pH 7.4). The reference cell contained identical mixtures supplemented with 0.6 mU of superoxide dismutase (SOD; Sigma).

All experiments were performed at 37°C. For quantification of scavenging activity, reduction rates of cytochrome C were measured at 550 nm.

Myeloperoxidase assay Myeloperoxidase (MPO) activity was assessed by the method described by Simons et al. (11). MPO was produced by 1.3 x io zymosan-stimulated PMNs incubated at 37°C for 15 mm

E E

1.50' 1.25

C w

1.00

p < 0.05

0.75' (V

0.

0.50 -I 0 0 U0.25

0

II 0

1

p < 0.01

10

20

Sample dose (mg/mouse) Fig. 1 Effects of gallic acid on acute foot pad swelling in mice induced by zymosan. Statistical evaluation was performed using a two-sided Student's t-test. Vertical bar-values represent mean T SD.

In vitro immunomodulatory activity In line with previous findings (5, 7) gallic acid was found to be a potent inhthitor of lummnol-depend-

ent chemiluminescence effected by zymosan-stimulated human PMNs (IC50 value [concentration of the sample giving 50% inhibition in the test system) = 5 pg/mi). The anticompiementary activities of gallic acid, however, were

found to be rather moderate (classical pathway of complement: IC50 = 340 pg/mi; alternative pathway of complement: IC50 = 756 pg/mi). These findings suggest that the

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Hemolytic assay for human complement activity

B. H. Kroes etal.

Planta Med. 58(1992) 501

Anti-Inflammatory Activity of Gallic Acid

inhibition of zymosan-induced inflammation by gallic acid is rather due to interference with PMN function(s). Con-

sequently, further immunomechanistic studies were re-

that gallic acid exhibited inhibitory effects on STZ- as well as PMA-induced chemiluminescence. This finding suggests that the effect of gallic acid on the chemiluminescence is

stricted to this part of the immune system.

not primarily due to interference with complement receptor triggering by STZ.

Cytotoxicity of gallic acid

graded amounts of gallic acid at 37°C for 15 mill. Subsequently, the cells were washed and chemiluminescencë was measured upon stimulation with zymosan as described above. As shown in Fig. 2, the chemiluminescence was not affected by preincubation of cells with gallic acid. This indicates that the inhibitory activity of gallic acid is not to be ascribed to cytotoxic effects versus PMNs.

Studies with myeloperoxidase-deflcient PMNs

and cell-free systems have shown that luminol-dependent

chemiluminescerice is dependent on myeloperoxidase (MPO) (15, 16). To find out whether interference of gallic acid with MPO release and MPO activity was involved in the inhibition of chemiluminescence, STZ-stimulated PMNs and cell-free supernatants were incubated with gallic acid as described. It was found that gallic acid decreased MPO activity when added to the incubation mixture of PMNs and

1400 -

STZ, or to the corresponding supernatant after MPO release (Fig. 4). The latter indicates that gallic acid inhibits MPO activity. Yet, since the inhibition was more pronounced when the compound was added to the incubation

1200 -

0 1000 800 600

mixture containing PMNs, it is most likely that gaffic acid, besides inhibiting MPO activity, also interferes with the release of MPO by STZ-stimulated PMNs.

400 200

0

Myeloperoxidase release

/ 0

0.3

100

/ / 1

3

10 30

80

Gallic acid (pg/mI) Fig. 2 Effects of preincubation of PMNs with gallic acid on the luminol-dependent chemiluminescence. Bars represent the mean In = 4) S.D.

Interference with receptor-triggering

To investigate whether gallic acid interfered with the interaction of serum-treated zymosan (STZ) with C3b(i) receptors on PMNs, the cells were stimulated with either STZ or phorbol myristyl acetate (PMA; final concentration lOng/mi). The data presented in Fig. 3 show 100

60

0 .0

40

C

20

0 0.3

3.0

30

Gallic acid (pg/mi) Fig. 4 Inhibition of MPO (myeloperoxidase) release and MPO activty by gallic acid. MPO activity was measured in supernatants of STZstimulated PMNs. Gallic acid was added either to the incubation mixture containing STZ and PMNs (0, overall effect on MPO release and MPO activity), or to the supernatant (•, effect on MPO activity).

80

C

60

0

—0— STZ + LumnoI

.0 =

—.— STZ + Lucigenin —— PMA + Luminol —.— PMA + LucigenEn

40

20

Interference with reactive oxygen species (ROS); scavenging activity

To investigate the interference of gaffic acid with the formation or detection of specific reactive oxygen

species, luminol was replaced by lucigenin being a

0 0.01

0.10

GaDic acid (mM) Fig. 3 Effects of gallic acid on STZ- and PMA-induced luminol- and lucigenin-dependent chemiluminescence generated by PMNs. Vertical bars represent S.D. In 4).

selective probe of O (17, 18). As shown in Fig. 3, the inhibitory effect on chemiluminescence induced by STZstimulated PMNs was not affected. However, for PMAstimulated PMNs it was found that replacement of luminol by lucigenin resulted in a substantial decrease in inhibitory activity. Scavenging of O by gallic acid in a cell-free system generating O (hypoxanthine/xanthine oxidase) was

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To determine whether the inhibitory effect of gallic acid on chemiluminescence generated by PMNs could be due to cytotoxicity, PMNs were incubated with

502 Planta Med. 58(1992)

B. H. Kroes et at

investigated by measuring the effect of gallic acid on the rate of cytochrome C reduction by these fibS. The results presented in Fig. 5 show that gallic acid inhibits the reduction rate of cytochrome C. This effect was not caused by

interference with the formation of O, since inhibitory activity was also observed with concentrations of gallic acid which did not affect xanthine oxidase activity (Fig. 5).

Discussion

100

C

80

This paper deals with the in vivo and in vitro anti-inflammatory activity of gallic acid. So far, several biological activities have been attributed to this

60

enhances in vitro phagocytic activities of PMNs. Antimutagenic (20, 21) and anticarcinogenic (22) activities of

compound. Wagner et al. (19) demonstrated that gallic acid

gallic acid, as well as inhibition of thiopurine methyltransferase (23), and blocking of anion channels (24) have also been described. Here we report in vivo anti-inflammatory activity in a model based on acute, zymosan-induced foot pad swelling (Fig. 1).

0

40 C

20

0 0.1

1.0

10.0

Gaflic acid (pg/mI) Fig. 5 Dose-dependent inhibition of luminol-dependent chemiluminescence (A), reduction rate of cytochrome C (U), and xanthine oxidase activity (•) by gallic acid. Oxidation of hypoxanthine by xanthine oxidase results in uric acid. Uric acid formation (absorbance af 290 nm) was used as measure for xanthine oxidase activity.

Structure activity relationship

Several structural congeners of gallic acid were screened for their ability to inhibit luminol-dependent chemiluminescence generated by PMNs upon stimulation with zymosan. It was found that replacement of the carboxyl (C0OH) group of gallic acid by COCH5 or C02C2H5

had no significant effect on the activity (Table 1). ComTable

1

Effects of gallic acid and structural congeners on luminol-dependent

Since the anticomplementary activity of galllc acid was found to be rather moderate in contrast to its marked inhibition in the chemiluminescence assay, further in vitro studies on mechanistic aspects of the antiinflammatory activity were focussed on the functioning of PMNs. As parameter for the activity of these phagocytes, the luminol-dependent chemiluminescence was chosen,

since this reflects their production of reactive oxygen species (lOS). 105 play a crucial role in inflammatory processes mediated by PMNs (25). For example, McCord and Wong (26) demonstrated that superoxide anions (O) play a major role in the formation of edema accompanying the phagocyte-mediated, acute inflammatory response. Furthermore, scavengers of 1105 llke a-tocopherol, catalase, and superoxide dismutase were found to block acute inflammation in several experimental models (27— 30). It was establlshed that inhibition of chemiluminescence by galllc acid cannot be ascribed to cytotoxic effects or interference with triggering of C3b(i)-receptors of PMNs by STZ. Consequently, gallic acid most likely interferes with the formation and/or detection of 105.

chemiluminescence generated by zymosan-stimulated polymorphonuclear

Since galllc acid is a well-known anti-

leukocytes.

oxidant, scavenging of 105 may (for some part) account for the observed activity. Scavenging activity of galllc acid for

H'

0 was assessed using a cell-free system, wherein it was experimentally confirmed that gallic acid is a scavenger of

H

0. This finding suggests that the inhibitory activity of

H4

gallic acid in the chemiluminescence assay is also mediated by scavenging of O. In line with this, it was found that replacing luminol by lucigenin, which largely monitors O5

1-9 R3

Compound No. Name

H1

1 Gallic acid

COOH OH

OH

OH

COOH COOH

OH OH

OH

COOH OH COCH, OCH3 OH

OH OH OH

R2

H4

H6

28

2 Protocatechuic acid

3 4 5 6 7 8 Vanillic acid

9 Veratric acid a Mean 8.0. (n = 4).

generation (16), did not affect the inhibitory activity of gallic acid in the chemiluminescence assay using STZ-

infl1M

CO2C2H502H5 OH

COOH OCH3 OCH3 OCH3 COOH OCH, OH COOH OCH, OCH3

2

30± 3

OH

523± 9 447 23

35± 5 33± 2

>1000 133± 9

> 1000

stimulated PMNs (Fig. 3).

Our results suggest that the in vivo antiinflammatory activity of gallic acid is largely based on scavenging of O anions. As described above, scavengers of 1105 have been shown to block phagocyte-mediated inflammatory responses. Anti-oxidants like a-tocopherol and

propyl gallate, which is a derivative of gallic acid, were found to inhibit experimental granuloma formation (27). In addition to the prevention of tissue damage, scavenging of

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parison of the activities of gallic acid and protocatechuic acid as well as compounds 5 and 6 revealed that the odihydroxy (catechol) group is important for the inhibitory effects. Absence or methylation of one of the o-hydroxy groups results in significantly reduced activities.

Planta Med. 58(1992) 503

Anti-Inflammatory Activity of Gallic Acid

Besides scavenging of O, gallic acid inhibited MPO activity and its release by STZ-stimulated

may contribute to the inhibitory activity of gallic acid towards inflammatory responses as well.

Acknowledgements The Netherlands Minister for Development

PMNs. As MPO is involved in the formation of more potent cytotoxic ROS (e.g. OCli, inhibition of MPO release and MPO activity by gallic acid is also likely to contribute to the anti-inflammatory activity of this compound.

Cooperation is kindly acknowledged for the financial support for this study.

Structure-activity relationship studies revealed that the o-dihydroxy group of gallic acid is important for its inhibitory activity. Protocatechuic acid showed not only identical activity in quantitative terms (Table 1), but also exhibited similarities with regard to the mode of action. That is, in analogy with gallic acid, the activity of this compound increased when STZ was replaced by PMA as stimulant in luminol-dependent chemiluminescence (4). Moreover, like gallic acid, protocatechuic acid exhibits more pronounced inhibitory effects on luminol-than on lucigenin-dependent chemiluminescence by PMA-stim-

Labadie, R. P.. Van der Nat, J. M.. Simons, J. M., Kroes, B. H.,

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8

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10

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B. H. Kroesetat

Anti-inflammatory activity of gallic acid.

Gallic acid was found to possess antiinflammatory activity towards zymosan-induced acute food pad swelling in mice. In vitro studies on the mode of ac...
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