Pmstaglandins Leukotrienes and Essential 0 Longman GroupUK Ltd 1991

Fatty Acids (1991) 42. 73-81

Acetyl Eugenol, a Component of Oil of Cloves (Syzygium aromuticum Z,.) Inhibits Aggregation and Alters Arachidonic Acid Metabolism in Human Blood Platelets K. C. Srivastava* and N. Malhotra’ *Department of Environmental Medicine, ISH, tInstitute of Chemistry, Odense University, J. B. Winslarws Vej 19, DK-5000 Odense C, Denmark (Reprint requests to KCS) ABSTRACT.

In continuation of our studies with the oil of cloves - a common kitchen spice and a crude drug for home medicine - we have isolated yet another active component identified as acetyl eugenol (AE); the earlier reported active component being eugenol. The isolated material (IM) was found to be a potent platelet inhibitor; IM abolished arachidonate (AA)-induced aggregation at ca. 12 PM, a concentration needed to abolish the second phase of adrenaline-induced aggregation. Chemically synthesized acetyl eugenol showed similar effects on AA- and adrenaline-induced aggregation. A dose-dependent inhibition of collagen-induced aggregation was also observed. AE did not inhibit either calcium ionophore A23187- or thrombin-induced aggregation. Studies on aggregation and ATP release were done using whole blood (WB). AA-induced aggregation in WB was abolished at 3 pg/ml (14.6 PM) which persisted even after doubling the concentration of AA. ATP release was inhibited. Inhibition of aggregation appeared to be mediated by a combination of two effects: reduced formation of thromboxane and increased generation of 1Zlipoxygenase product (12HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxBz formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids.

INTRODUCTION It is commonly assumed that consumption of spices is related to taste. But there may be more to spice consumption than mere taste. Lately many spices have made a cross-cultural penetration which has generated some curiosity about their biological effects and mechanisms of action. Onion and garlic, and especially the latter, serve to illustrate this point. Substantial anecdotal evidence supports the invaluable role that garlic has played in the therapy of numerous diseases since time immemorial. Yet scientific evidence confirming its role in medicine and prophylaxis has been provided from around the world only recently. Since our earlier observations with onion, garlic and ginger on their effects on blood platelet aggregation and inhibition of fatty acid oxygenases (l-5) we have screened some more spices for this particu-

Date received 14 April 1990 Date accepted 14 August 1990

lar biological action (6-8). Clove is frequently used in spice mixtures in the Indian subcontinent. Clove is used also as a crude drug for home medicine. Traditionally, clove has been reputed to be an aromatic stomachic. In the literature only a few pharmacological studies are mentioned - its active principles possess cholagogue effects to mention one (9). Earlier, we have reported that an ethereal extract (oil of cloves, OC) inhibited platelet aggregation, an effect which was largely mediated by reduced prostanoid formation (7). This was followed by isolation and characterization of an active component (Zmethoxy, 4-ally1 phenol, or eugenol) from the oil of cloves, and this component was found to be a strong inhibitor of platelet aggregation and of cyclooxygenase (CO). Furthermore, eugenol was found to preserve the platelet ultrastructure when stimulated by arachidonate in concentrations that produced irreversible platelet aggregation (10). The present study addresses itself to the effects of yet another component (acetyl eugenol, eugenol acetate, l-acetyl, 2-methoxy, 4-ally1 phenol) from clove on platelet aggregation and prostanoid metabolism.

74

Prostaglandins Leukotrienes and Essential Fatty Acids

MATERIALS

AND METHODS

Arachidonic acid (l-14C) (specific activity 58.3 mCi/ mmol) was purchased from the Radiochemical Centre, Amersham, England. Calcium ionophore A23187 was obtained from Sigma Chemical Co., and arachidonic acid from Nu Chek Prep. Inc. Elysian, MN, USA. Collagen [l mg of native collagen fibrils (type l)] was purchased from Chrono-Log Corporation, and adrenaline was obtained in ampules (1 mg/ml) produced locally. Thromboxane Bz was kindly provided free by ON0 Pharmaceutical Co Ltd, Osaka (Japan). Thin-layer chromatography (TLC) plates precoated with silica gel 60 F254r obtained from E. Merck, Darmstadt, West Germany, were used in autoradiography experiments. For other separation experiments, TLC silica gel G plates (0.25 mm) were prepared in our laboratory. All solvents were of analytical reagent grade. Fractionation of oil of cloves by thin layer-chromatography Cloves (pungent aromatic dried flower buds of tropical myrtle) (18 g) were powdered and extracted in 150 ml ether overnight at 4°C. The ethereal extract was filtered and the filtrate treated with anhydrous sodium sulphate. The extract was filtered again and the solvent was removed completely by distillation and bubbling nitrogen at 45°C. The oily extract was ensured to be free of ether, and then dissolved in ethanol for use in various experiments. A portion of the ethanolic solution was subjected to TLC using solvent benzene-acetone (8: 1, v/v) on a silica gel G plate prepared in our laboratory (9). TLC bands shown after a quick exposure to iodine vapour were marked. Several clearly separated bands were shown. The bands were marked and plates allowed to stay in a fume-cupboard until no stains were observable. The marked bands were scraped off and extracted in chloroform-methanol (2: 1, v/v). The band which corresponded to Rf 0.86 (this Rf value was determined using a TLC plate precoated with silica gel 60, F254) was scraped off and pooled from several plates. More material was collected by preparative TLC. A corresponding number of TLC plates, prepared in our laboratory, were developed in solvent benzeneacetone (8: 1, v/v) without the extract of cloves. Silica gel corresponding to Rf 0.86 was scraped off from the plates and extracted. This was dissolved in ethanol and the solution served as the vehicle in controls. Chemical synthesis of acetyl eugenol The amount of acetyl eugenol in OC is low compared to eugenol which constitutes more than 80% of OC. When it was confirmed by TLC behaviour

and mass spectrometry that the material in the band with Rf 0.86 was acetyl eugenol, the compound was chemically synthesized to give sufficient material for a detailed study. This was necessitated because it was not available commercially. A mixture of 4-ally, 2-methoxypehnol (eugenol) (3.38 g, 0.02 mol) and acetic anhydride (4.08 g, 0.04 mol) containing dry pyridine (10 ml) was refluxed on an oil bath for 4 h. After completion of the reaction (as judged by TLC), pyridine, acetic anhydride and acetic acid were distilled off under vacuum to give acetyl eugenol. Platelet aggregation Blood was collected from healthy volunteers who had not taken aspirin or any other drug known to affect platelet function for about 7-10 days prior to giving blood. The standard procedure of differential centrifugation was used for the preparation of plateletrich plasma (PRP) and platelet-poor plasma (PPP) which were used in the aggregation experiment. Aggregation was performed in PRP samples by the Born turbidimetric technique (11) using threshold concentrations of arachidonate, adrenaline, collagen, ADP and ionophore A23187. Aggregation in whole blood (WB) was performed also using an electronic aggregometer (lumiaggregometer model 500 VS Chronolog Corporation) (12) which operates on the principle that when platelets aggregate, a fall in conductance (increase in impedance) is observed. The effect of acetyl eugenol (separated from oil of cloves and synthesized) on platelet aggregation was examined by treating PRP samples with varying concentrations of this agent. Acetyl eugenol was added to the platelet sample (PRP) in the aggregometer while stirring at 37°C for 30 s, and then the sample was allowed to remain for 2 min 30 s min at room temperature (RT) before adding an agonist to produce aggregation. Eicosanoid formation from (l-‘4C)arachidonate in blood platelets Washed platelet suspensions were prepared in Ringer-citrate-dextrose (RCD) as described earlier (13). Platelet suspensions (200 ~1, 1 X 10’ platelets) were incubated with the vehicle (control) or AE at 37°C for 5 min followed by arachidonic acid (8.2 pM) for 15 min at 37°C. All incubations were made in duplicate. The lipids were extracted after acidification (pH 3.0) in ether (3 ml, twice). The pooled ether extract was washed with 1 m1 water and evaporated under nitrogen. The residue was dissolved in 200 ~1 chloroform-methanol (2: 1, v/v). The lipids, including AA metabolites were resolved by TLC. Twenty-five ~1 of the extract (200 ~1) were resolved by using TxB2 as a reference standard in

Effect of AE on Aggregation and AA Metabolism in Human Blood Platelets

solvent I (upper organic phase of ethyl acetateisooctane-acetic acid-water, 110:50:20: 100, v/v after 5 min mixing). Thromboxane B2 (Rf 0.40) was separated from other eicosanoids. lZlipoxygenasederived products (12-hydroxyeicosatetraenoic acid, 12HETE) and the hydroxy acid (12_hydroxyheptadecatrienoic acid, 1ZHHT) produced by the cyclooxygenase were resolved from TxBz, prostaglandins PGF*,, PGE2, PGD& phospholipids and AA by resolving another 25 ~1 of the same extract using solvent II (n-hexane-ether-acetic acid, 80: 20: 1, v/v). The hydroxy acids (lZHETE, 12-HHT) were located between the application point (Rf 0.00, prostaglandins, TxB2, phospholiopids) and AA (Rf 0.35). The counts for the lipoxygenase products were calculated by subtracting TxB2 counts from the total counts due to the lipoxygenase products and 12-HHT (6, 10). This was based on the assumption that TxB2 and 12-HHT are produced in equal amounts in platelets (14-20, see Discussion). Hereinafter, 12-HHT and 12-HETE are referred as HHT and HETE respectively. Autoradiography platelets

of eicosanoids

produced in

Twenty-five ~1 of the incubation extract (200 ~1) were resolved in solvent I, II or III (chloroformacetic acid, 90:3 v/v) on TLC platelets precoated with silica gel 60 Fls4 (0.25 mm, Merck). After a run of ca. 17 cm. solvent was allowed to evaporate from the plates to which were exposed X-ray film (X-ray autoradiography) for 7 days. Labelling of platelets with (14C)arachidonic acid: effect of acetyl eugenol on the incorporation of arachidonic acid and formation of eicosanoids

The effect of eugenol acetate was examined on i) the incorporation of (14C)AA in platelet phospholipids, and (ii) the release of AA from labelled platelets and subsequent formation of ( 14C)TxBz and (‘4C)OH-acids (HHT, HETE) on stimulation with ionophore A23187; stimulation of labelled platelets by thrombin and collagen was done mainly to examine AE’s effect on deacylation of phospholipids. In the first experiment, PRP was treated with AE prior to incubation with (14C)AA for 2 h at 37°C (3). In the second experiment, platelets (PRP) were first incubated with trace amounts of (14C)AA for 2 h at 37”C, separated from the plasma by centrifugation, washed with and resuspended in RCD. This was followed by incubation of labelled platelets with AE for 30 s in the aggregometer and allowed to remain at RT for ca. 5 min. Control platelets were treated with the vehicle. Control as well as AE-treated platelets were finally stimulated by adding A23187 (5 PM), thrombin (5 units/ml) or

75

collagen (10 pg/ml) and stirred for 30 s at 37°C in the aggregometer. Platelet samples were allowed to remain at RT for 10 min, transferred quantitatively to unsiliconized test tubes and finally extracted in chloroform-methanol (2: 1, v/v) overnight at 4°C. Details of preparation of the lipid extract, and its resolution into various eicosanoids (TxB2, HHT, HETE) from AA and phospholipids are described elsewhere (3, 6, 10). Statistical analysis

Statistical significance was evaluated by Student’s ttest for paired data.

RESULTS Chemical characterization band with Rf 0.86

of the component

in the

Thin-layer chromatography separations of various components contained in the ethereal extract of cloves (OC) were achieved as described earlier (9, 10). The material present in the band with Rf 0.86 was isolated and used in a part of this study. The material showed in its mass spectrum a base peak at m/z 206 (M+). When tentatively identified (based on TLC behaviour and mol wt) to be acetyl eugenol, it was chemically synthesized to obtain in sufficient amounts for NMR spectroscopy and biological study. The synthesized material in its NMR spectrum showed signals at 6 6.93 (d, lH, ArH), 6.75 (d, 2H, ArH). 5.87-6.03 (m, lH, -CH=), 5.06 (t, 2H, -CH& 3.8 (s, 3H, OCH& 3.36 (d, 2H, =CHZ) and 2.28 (s, 3H, CHs). Its mass spectrum showed a molecular ion peak at m/z 206 (M+). Other prominent peaks in the mass spectrum were present at m/z 207 (M+ + l), 164 (207-COCH3). 149 (164-CH3), 77 (C&Is) and 43 (COCH3). The spectral data are consistent with the structure l-acetyl, 2-methoxy, 4-ally1 phenol (eugenyl acetate, acetyl eugenol). Effect of acetyl eugenol on platelet aggregation

The effect of the isolated material (identified as acetyl eugenol) from the oil of cloves was examined on AA- and adrenaline-induced aggregation. The material was found to be a potent inhibitor of AAinduced aggregation; in the concentration range 2.5-3.75 pg/rnl it abolished almost completely the aggregation. The second phase of adrenalineinduced aggregation was abolished at 2.0-4.0 pg/ml (Fig. 1). A similar inhibition pattern was observed with synthesized AE in AA- and adrenaline-induced aggregation (Fig. 2). Collagen-induced aggregation was also inhibited by AE in a dose-dependent manner (Fig. 3). However, AE was not effective in

76

Prostaglandins Leukotrienes ADRENALINE ~lrn0ml

and Essential Fatty Acids ARACHIDONATE 1 lO3rnU~

Collagen IZpg/mll

Effect of acetyl eugenol on collagen-induced platelet aggregation. 1 Control tracing using collagen (1 &ml PRP); 2 Control tracing with 2 &ml collagen; 3-6 Treated with acetyl eugenol at 50 pg, 5 pg. 2.5 pg and 1.5 cLg/ml respectively. Fig. 3

Fig. 1 Effect of the isolated material (identified as acetyl eugenol) from OC on platelet aggregation. A. 1 and 4 Control tracings produced by adding adrenaline (final cont. 10 PM) to RPP treated with the vehicle; 2 Treated with the isolated material 2.0 &ml; 3 Treated with the isolated material 4.0 rg/ml. B. 1 Control tracing produced by adding sodium arachidonate (final cont. 0.5 mM) to PRP treated with the vehicle; 2 Control tracing produced by adding arachidonate (final cont. 0.3 mM) to PRP treated with the vehicle; 3 and 4 Treated with the isolated material respectively at 2.5 pg/ml and 3.75 pg/ml. Tracings 2-4 were produced with arachidonate at 0.3 mM. The numbers on the tracings indicate the sequence in which aggregation was performed. A and B were obtained with the same blood sample.

Fig. 4 Effect of acetyl eugenol on arachidonate-induced aggregation in whole blood (WB). Four hundred fifty ~1 WB were mixed with 450 ~1 saline and 100 ~1 luciferin-luciferase reagent. Aggregation was produced by adding arachidonate (0.5 mM). Total inhibition was obtained with 3 &ml acetyl eugenol, and inhibition was maintained even with double (1.0 mM) of the concentration of arachidonate which produced irreversible aggregation in control platelets (B). A and B were obtained from the same blood sample.

Effect of acetyl eugenol (synthetic) on platelet aggregation. A. 1 Control tracing with arachidonate (0.5 mM); 2 and 7 Control tracings with arachidonate (0.3 mM); 3-5 Tracings obtained with PRP samples treated with acetyl eugenol at 5 pg, 3 pg and 2 &ml respectively (usin arachidonate (0.3 mM). 6 Tracing with acetyl eugenol 1 p J ml. B. 1 Control tracing with adrenaline; 2 and 3 Treated with acetyl eugenol at 2.5 pg and 5.0 &ml respectively. C. 1 and 4 Control tracings with arachidonate (0.5 mM); 2 and 3 Tracings obtained with PRP samples treated with acetyl eugenol at 5.0 PcLgand 2.5 &ml respectively. The numbers on the tracings indicate the sequence in which aggregation was performed. Whereas A was obtained with one blood sample, B and C show tracings utilizing blood sample obtained from another donor different from that used in A. Fig. 2

inhibiting aggregation induced by ADP, and ionophore A23187 (tracings not shown); with ADP a high concentration of AE were required to show a small inhibition of aggregation. Aggregation induced by AA in whole blood (WB) was inhibited by AE (Fig. 4). Release of ATP was also inhibited (data not shown). Effect of acetyl eugenol on the metabolism of exogenous, arachidonate in platelets Washed platelets were treated with several concentrations (0.63-10 pg/ml) of AE and control platelets with the vehicle prior to incubation with (14C)AA. Acetyl eugenol reduced the formation of TxB2 with a concomitant increase in 1Zlipoxygenase products

Effect of AE on Aggregation and AA Metabolism in Human Blood Platelets

77

Table 1 Effect of acetyl eugenol on the conversion of (14C)arachidonic acid to thromboxane B, and 1ZHETE in platelets Counts (DPM) recovered as 12-HETE’ TxB, Control (n = 4) Acetyl eugenol (0.63 &ml) Acetyl eugenol (1.25 pg/ml) Control (n = 6) Acetyl eugenol (2.5 pg/ml) Acetyl eugenol (5.0 pg/ml) Acetyl eugenol (10.0 pg/ml)

4398 3188 2420 4425 1203 769 622

+ 1002 rfr 492 f 527 f 1464 + 546* + 394* + 321*

9487 12586 14430 8207 14230 16379 16392

rt + f + + rt +

2493 1862 2564 3687 2801* 1614* 954*

Counts (Mean f SD) are l/8 of the total amount produced by lo* platelets present in the incubation medium. ‘Counts were calculated by subtracting TxB, counts from the total counts due to 1ZHHT and 12-HETE (see Methods). * p < 0.01 (Student’s t-test for paired data). Fig. 6 Effect of acetyl eugenol on platelet metabolism of arachidonic acid as shown by autoradiography. (a) C Control; l-3 Treated with acetyl eugenol at 2.5 pg, 5.0 pg and 10.0 pg/ml respectively. (b) C and 1-3 as in A. (a) was obtained by performing TLC using solvent chloroformacetic acid (90:3, v/v) and (b) was obtained by performing TLC using solvent II. Whereas in separation (b) thromboxane (TxBJ was localized at the application point it moved a little bit (Rf 0.02) (33. 34) from the application point in separation (a).

Table 2 Effect of acetyl eugenol on calcium ionophore A23187-induced phospholipid deacylation and on the subsequent formation of thromboxane B, and 1ZHETE

Control Acetyl eugenol (5 &ml) (n=4) Control Acetyl eugenol (20 pg/ml) (n=4) Fig. 5 Effect of acetyl eugenol on platelet metabolism of arachidonic acid as shown by X-ray autoradiography. C Control (washed platelets treated with the vehicle only); 1-3 Treated with acetyl eugenol at 2.5 pg, 5.0 pg and 10.0 pg/ml respectively. TLC separation was achieved on ready-made plates using solvent 1.

(HETE). The effect was dose-related (Table 1). Such effects of AE were shown also by autoradiography (Figs 5 & 6). Effect of acetyl eugenol on ionophore A231874nduced deacylation of platelet phospholipids and subsequent conversion of the released (“C)AA into thromboxane Bz and lipoygenase-derived products Platelets prelabelled with (14C)AA when stimulated with the ionophore, released AA which was enzymatically converted into various oxygenation products of which TxB2 and HETE were deter-

IZHETE-”

Phospholipids

TxB,”

1125 f 141 1077 f 133

73 * 17 461 ?I 74 35 f 7 533 + 58

1513 f 257 229 1323 Y!Z

79 f 12 757 I!Z226 29 + 6 920 + 283

Washed platelets obtained from earlier treated PRP samples with (‘4C)arachidonic acid (0.21 PM) to allow incorporation into platelet phospholipids were stimulated with A23187 (5 PM); arachidonic acid oxygenation products were separated from phospholipids and AA by TLC and their radioactivities counted. Values (DPM, Mean + SD) are l/6 of the total amount produced by 0.4 X lo9 platelets (5 &ml AE-treated) and 0.5 x lo9 platelets (20 &ml AE-treated). ‘After subtracting the background counts.

mined. The effect of AE was examined on this reaction sequence. For this purpose two concentrations (5 pg and 20 pg/ml) of AE were used. This substance affected the deacylation reaction; it promoted this reaction by ca. 5% and 13% at 5 pg and 20 ps/rnl AE concentration. However, in its presence reduced TxB2 formation and an increased formation of HETE were observed. The latter effect could be accounted for by a combination of inhibition of CO-activity and increased substrate (AA) availability to the lipoxygenase pathway (Table 2).

78

Prostaglandins Leukotrienes

and Essential Fatty Acids

Effect of acetyl eugenol on thromhin- and collagen-induced deacylation of platelet phospholipids The effect of AE (10 pg/rnl) on the release of AA from platelet phospholipids induced by thrombin and collagen was examined. As there was no significant difference (ca. 5% inhibition of deacylation of phospholipids in platelets treated with 10 pg/ml AE on stimulation with thrombin occurred) between the radioactivities located in the phospholipid fraction in control and AE-treated platelets, it was concluded that AE was without effect on this reaction. TxBz formation was reduced in AE-treated platelets on stimulation with thrombin (5 U/ml) (data not shown). TxBz determination was not done in collagen-stimulated platelets.

Effect of acetyl eugenol on the incorporation of labelled AA into platelet phospholipids Incorporation of ( 14C)AA into platelet phospholipids was examined in the presence of AE at three concentrations - 10 pg, 50 pg and 100 &ml PRP. AE showed no effect (data not shown).

DISCUSSION In continuation of our studies with some frequently consumed spices on platelet aggregation and eicosanoid biosynthesis, we have shown recently that an ethereal extract of cloves (oil of cloves, OC) inhibited aggregation induced by platelet agonists, such as arachidonate, epinephrine and collagen; it was most effective against arachidonate-induced aggregation. Inhibition of aggregation was shown to be mediated by reduced formation of thromboxane in platelets; this was demonstrated both in intact (washed) and lysed platelet preparations. At low dose-levels of OC, an increased formation of lipoxygenase products was observed. This suggests a redirection of the substrate (AA) from the cyclooxygenase (CO) pathway to the lipoxygenase (LO) pathway. Formation of eicosanoids (TxB2, 12HETE) in AA-labelled platelets stimulated by ionophore A23187 showed that reduced formation of TxB2 was due to the effect of OC on the cyclooxygenase enzyme as no effect of OC on phospholipid degradation was observed in the experimental conditions used (7). A major component of the oil of cloves is eugenol (82% to 87%). The clove oil content in dry clove buds is ca. 15%. This would be equivalent to 120 to 130 mg of eugenol in 1 g of dry clove buds (21). We have isolated eugenol and examined its effects on platelet aggregation and eicosanoid biosynthesis. The isolated material was found to be anti-aggregatory,

abolishing AA-induced aggregation at 7.6 PM and the second phase of epinephrine-induced aggregation at 15.2 PM, both with PRP. Inhibition of aggregation was mediated apparently by reduced formation of thromboxane and an increased formation of lipoxygenase products (cf. oil of cloves). Increased formation of the lipoxygenase products was demonstrated in platelets challenged with labelled AA, and those prelabelled with AA and stimulated with A23187. As more counts were recovered in the AA fraction from the treated platelets, and as no effect was observed on the deacylation of platelet phospholipids, it was concluded that some enzyme(s) (the cyclooxygenase in particular) of the AA cascade was inhibited by the isolated material. These observations were confirmed by using pure eugenol (10). In this paper we report the results of a similar study with yet another active component which is present in the clove oil in a smaller amount, thus being a minor component in relation to eugenol. This component was as effective as eugenol in inhibiting platelet aggregation and thromboxane formation. It was consequently identified to be acetyl eugenol (eugenyl acetate) based on the TLC behaviour (9) and mass spectrometry. As the material in our hands was not sufficient for detailed study, we synthesized it chemically and established its structure by mass spectrometry and proton NMR spectroscopy. In the major part of the present study the synthesized material was used. Acetyl eugenol (AE) (isolated and synthesized) was found to be a potent inhibitor of platelet aggregation performed with PRP: at 2-5 pg/rnl it abolished/nearly abolished AA-induced aggregation, and at 1.5-2.5 ,ug/ml it abolished the second phase of adrenaline-induced aggregation. In whole blood AA-induced aggregation was abolished at 3 pg/rnl with a concomitant reduced release of ATP. Collagen-induced aggregation was inhibited at 1.25 to 5 &ml in a dose-dependent manner. However, with some blood samples higher doses of AE were needed to abolished collagen-induced aggregation. A23187-induced aggregation was not inhibited even at higher concentrations (50 pg/ml) of AE. Abolition of AA-induced aggregation of platelets in PRP and whole blood (plasma proteins by binding AE would reduce its effective concentration) by low concentrations of AE could not be explained by taking into account its effect on thromboxane formation alone. It may be speculated that other mechanism(s) besides reduced thromboxane formation - could be involved. One of the possibilities could be related to enhanced production of lipoxygenase products in the presence of AE. In fact, 1ZHPETE has been reported to inhibit platelet aggregation, and at higher concentrations it inhibits thromboxane formation in platelets (18).

Effect of AE on Aggregation and AA Metabolism in Human Blood Platelets

This may gain more significance in the light of a recent report (22) that albumin - a constituent of the plasma proteins - acts as a ‘conduit’ to divert free AA from platelet CO to lipoxygenase pathway. Of late, the lipoxygenase pathway has come to be considered important, partly because the lipoxygenase activity is more slowly and less readily inactivated as compared to CO activity (24). The radioactivities present in the phospholipid fractions from platelets treated with AE were similar to those from the control (untreated) platelets after challenging with A23187 or thrombin. This suggests that reduced formation of eicosanoids (14C-Tx&) was due to AE’s direct effect on the cyclooxygenase (cf. effect on the metabolism of exogenous AA, Table I). There are reports in the literature where several research groups have employed thrombin as well as A23187 for activating platelets prelabelled with (3H)AA or (14C)AA to investigate the liberation of AA, its subsequent conversion to eicosanoids and metabolism of phospholipids. The ionophore- and thrombin-induced phospolipid deacylation occurs with an accompanied formation of HHT, 12-HETE and AA. In this study we have used mainly calcium ionophore A23187 as a stimulant for studying the formation of eicosanoids from labelled platelets. Thrombin and collagen were used for studying the effect of AE on the deacylation of phospholipids. Under the experimental conditions (cont. of A23187 and platelet activation time) used, consistent results were obtained. For the determination of lipoxygenase products (12-HETE) we have made an indirect approach. This was based on the assumption that TxB2 and HHT were produced in equal quantities by thromboxane synthase which utilizes PGH2 as the substrate. Thus, from a molecule of PGH2 half a molecule each of TxA2, HHT, and malondialdehyde (MDA) are produced (4). Several authors have shown that TxBz and HHT are produced in ca. 1: 1 ratio (14-20). In some of our earlier experiments we have observed more HHT than TxB2 as has been reported by others (23, 24). Any excess of HHT production (i.e., more than TxB2 on a molar basis) should originate from non-enzymatic degradation of PG-endoperoxides. Acetyl eugenol failed to inhibit A23187-induced aggregation even at high concentrations, although it drastically reduced the formation of CO products at lower concentrations. This effect may be similar to that shown by indomethacin which effectively inhibits the CO but fails to inhibit A23187- and thrombin-induced platelet aggregation. This is due to the fact that indomethacin-sensitive aggregation is of a ‘secondary’ (i.e., second wave) type which is characteristically different from the events related to ‘primary’ aggregation initiated by ionophore and thrombin. In thrombin- and ionophore-induced

79

phosphatidic acid (PA) is quickly reactions, generated, and this can be detected even 2 s after addition of the agonists. PA is generated from 1,2diacylglycerol (DAG) by diacylglycerol kinase by way of its phosphorylation (23). Thus PA production (mediated by phospholipase C) is related to early events of platelet aggregation which apparently is not affected by AE. In phosphotidylinositol (PI) turnover, phosphatidic- and lysophosphatidic acids are formed, and they are characterized as calcium ionophores. This may explain why an agent which inhibits CO (reduced TxA2 formation) would fail to inhibit aggregation not related to TxA2 formation. Further, one needs to consider the effect of 12HPETE on platelet secretion here. It has been shown to inhibit (a) aggregation and ATP secretion induced by AA; (b) production of both thromboxane Bz and malondialdehyde in response to AA; (c) aggregation and secretion induced by prostaglandin endoperoxide analogs, 9, 11-azo-PGH? and U46619; (d) both aggregation and secretion induced by collagen. These observations suggest a role of prostaglandin-endoperoxides and thromboxane in platelet aggregation and release reaction (18). However, 12-HPETE was effective also in inhibiting secretion in aspirin-treated platelets by thrombin. Thus, a direct effect of 12-HPETE (in higher concentrations) on the secretory mechanisms independent of prostaglandin endoperoxide-induced aggregation was shown (18). These effects of 12-HPETE on platelet function may support our observations with AE in whose presence more of the former (assayed as 1ZHETE) is produced. Increased formation of 12-HETE was observed with several spices (25, 26). As we did not have the reference standards HHT and HETE, we devised a TLC separation schedule which enabled separation of these two substances from other AA-metabolites and AA; the hydroxy acids (HHT, HETE) lying between the application point (phospholipids, TxB2, PGs) and AA (Rf 0.35). Diacylglycerol (1-stearoyl-2-arachidonylsn-glycerol) may be located in an area close to the hydroxy acids in this TLC separation as in our TLC system reference standard dipalmitin, a diacylglycerol, moved close to the hydroxy acids. The amount of remaining DAG (produced by the action of phospholipase C on PI by receptormediated mechanisms) would depend on the activities of two enzymes, diacylglycerol-lipase and ATP-kinase, both utilizing DAG as substrate and producing respectively AA and PA. When A23187 is used as a stimulant, most of the AA would come from phosphatidyl choline, and only a small amount would be derived from PI. This finds support from the observation that PA (DAG -+ PA) is produced in much lower quantity in A23187-stimulated plate-

80

Prostaglandins Leukotrienes

and Essential Fatty Acids

lets compared to those stimulated by thrombin (23). Thus, radioactivities present in the hydroxy acid fraction might contain components due to DAG. Clove and other spices studied by us are consumed regularly by a large section of the population in the Indian subcontinent and the Far East. They were found to contain components that interfered with the oxygenation of AA (25, 26). A direct conclusion of this observation could be that their consumption might help prevent diseases where elevated levels of eicosanoids have been reported. As an illustration we may quote our own preliminary observations on the beneficial effects of ginger consumption in muskuloskeletal disorders (29) in migraine headache (30) and in dysmenorrhoea (unpublished observations). Further, several spices are known to contain antioxidants. Such compounds might augment the effects of physiologically important dietary antioxidants (e.g., selenum and the vitamins A, C and E). A large-scale international epidemiological study has shown that there exists an inverse relationship between the ‘antioxidant’ status of the blood and the incidence of ischaemic heart disease (31). Thus, dietary intake of natural antioxidants might help body’s ,defence mechanisms against diseases mediated by peroxidation of lipids. Many antioxidants have lately been identified as anticarcinogens. Such materials from dietary plants may prove important in inhibiting carcinogeninduced tumorigenesis in humans (32). Acknowledgements We thank the personnel of the Blood Bank, Odense University Hospital for collection of blood samples. Ms. Ruth B. Alexandersen is thanked for skilful technical assistance. Ms. Kirsten Guldhoj of the X-ray Department, Odense University Hospital performed autoradiography. Mr. Jan Beyer and Ms. Winnie Most are thanked for providing assistance in some of our experiments. Secretarial assistance was provided by Ms. Inge Bogelund and Ms. Yrsa Kildeberg. This work was supported by a grant (12-9822 kg/mp) from the Danish Medical Research Council.

References 1. Srivastava K C. Effects of aqueous extracts of onion, garlic and ginger on platelet aggregation and metabolism of arachidonic acid in the blood vascular system: in vitro study. Prostaglandins Leukotrienes and Medicine 13: 227-235, 1984. 2. Srivastava K C. Aqueous extracts of onion, garlic and ginger inhibit platelet aggregation and alter arachidonic acid metabolism. Biomedica Biochimica Acta 43: S335S346, 1984. 3. Srivastava K C. Evidence for the mechanism by which garlic inhibits platelet aggregation. Prostaglandins Leukotrienes and Medicine 22: 313-321,1986. 4. Srivastava K C. Onion exerts antiaggregatory effects by altering arachidonic acid metabolism in platelets. Prostaglandins Leukotrienes and Medicine 24: 43-50,1986. 5. Srivastava K C. Isolation and effects of some ginger components on platelet aggregation and eicosanoid

biosynthesis. Prostaglandins Leukotrienes and Medicine 25: 187-198, 1986. 6. Srivastava K C. Extract of a spice - omum (Trachyspermum ammi) - shows antiaggregatory effects and alters arachidonic acid metabolism in human platelets. Prostaglandins Leukotrienes and Essential Fatty Acids 33: l-6, 1988. 7. Srivastava K C, Justesen U. Inhibition of platelet aggregation and reduced formation of thromboxane and lipoxygenase products in platelets by oil of cloves. Prostaglandins Leukotrienes and Medicine 29: ll-18,1987. 8. Srivastava K C. Extracts of two frequently consumed spices - cumin (Cuminum cyminum) and turmeric (Curcuma longa) - inhibit platelet aggregation and alter eicosanoid biosynthesis in human blood platelets. Prostaglandins Leukotrienes and Essential Fatty Acids 37: 57-64, 1989. 9. Yamahara J, Kobayashi M, Saiki Y, Sawada T, Fujimura H. Biologically active principles of crude drugs. Pharmacological evaluation of cholaaogue substances in clove-and its properties. Journal of Pharmacobio-Dvnamics 6: 281-286.1983. 10. Srivastava K C,-Malhotra N. Characterization and effects of a component isolated from a common spice clove (Caryophylli flos) on platelet aggregation and eicosanoid production. Thrombotic and Haemorrhagic Disorders (In press). 11 Born G V R. Quantitative investigations into the aggregation of blood platelets. Journal of Physiology, London 162: 67P, 1962. 12. Cardinal D C, Flower R J. The electronic aggregometer: a novel device for assessing platelet behaviour in blood. Journal of Pharmacolosical v Methods 3: 135-158, 1980 13. Schmidt K G, Rasmussen J W. Preparation of platelet suspensions from whole blood in buffer. Description of a method which gives a large platelet yield. Scandinavian Journal of Haematology 23: 88-96, 1979. 14. Diczfalsy U, Falardeau P, Hammarstrom S. Conversion of prostaglandin endoperoxides to C17-hydroxy acids catalyzed by platelet thromboxane synthase. FEBS Letters 84: 271-274, 1977. 15. Butler A M, Gerrard J M, Peller J, Stafford S F, Rao G H R, White J G. Vitamin E inhibits the release of calcium from a platelet membrane fraction in vitro. Prostaglandins and Medicine 2: 203-216,1979. 16. Hamberg M, Svensson J, Samuelsson B. Prostaglandin endoperoxides. A new concept concerning the mode of action and release of prostaglandins. Proceedings of the National Academy of Sciences (USA) 71: 3824-3828, 1974. 17. Samuelsson B. The role of prostaglandin endoperoxides and thromboxanes in human platelets. In: Silver M J, Smith J B, Kocsis J J (editors) Prostaglandins in Hematology. Spectrum Publications, New York, P. 1. 18. Aharony D, Smith J B, Silver M J. Regulation of arachidonate-induced platelet aggregation by lipoxygenase product,. 12-hvdrooeroxveicosatetraenoic acid. Biochimica et Biophysics Acta 78: 192-200, 1982. 19. Malle E, Gleispach H, Kostner G M, Leis H J. Isotope dilution gas chromatography-mass spectrometry for the study of eicosanoid metabolism in human blood platelets. Journal of Chromatography (Biomedical Applications) 488: 283-293.1989. 20. Malmsten C, Hamberg M, Svensson J, Samuelsson B. Physiological role of an endoperoxide in human platelets: hemostatic defect due to platelet eyclooxygenase deficiency. Proceedings of the National Academy of Sciences (USA) 72: 1446-1450,1975.

Effect of AE on Aggregation and AA Metabolism in Human Blood Platelets 21. Council Report. Evaluation of the health hazards of clove cigarettes. Journal of American Medical Association 260: 3641-3644,1988. 22. Broekman M J, Eiora A M, Marcus A J. Albumin redirects platelet eicosanoid metabolism toward 12(S)-hydroxyeicosatetraenoic acid. Journal of Lipid Research 30: 1925-1932,1989. 23. Lapetina E G, Chandrabose K A, Cuatrecasas P. Ionophore A23187- and thrombin-induced platelet aggregation: Independence from cyclooxygenase products. Proceedings of the National Academy of Sciences (USA) 75: 818-822, 1978. 24. Lapetina E G, Cuatrecasas P. Rapid inactivation of cyclooxygenase activity after stimulation of intact platelets_Proceedings-of the National Academy of Sciences (USA) 76: 121-125. 1979. 25. Rattan S‘I S. Science behind spices: Inhibition of platelet aggregation and prostaglandin synthesis. BioEssays 8: 161-162, 1988. 26. Srivastava K C, Mustafa T. Spices: antiplatelet activity and prostanoid metabolism. Prostaglandins Leukotrienes and Essential Fatty Acids - Reviews 38: 255-266,1989. 27. Naito S. Yamaeuchi N. Yokoo Y. Antioxidative activities of vegetables of Album species. Studies on natural antioxidants. Part II. Nippon Shokuhin Kogyo Gakkaishi 28: 291-296, 1981. 28. Naito S. Yamaguchi N. Yokoo Y. Fraction of

29. 30.

31.

32.

33.

34.

antioxidant(s) extracted from garlic. Studies on natural antioxidants. Part III. Nippon Shokuhin Kogyo Gakkaishi 28: 465-470, 1981. Srivastava K C, Mustafa T. Ginger (Zingiber officinale) and rheumatic disorders. Medical Hypotheses 29: 25-28, 1989. Mustafa T. Srivastava K C. Ginger (Zingiber officinale) in migraine headache. Journal of Ethnopharmacology (in press). Gey K F. Inverse correlation between the plasma level of antioxidant vitamins and the incidence of ischemic heart disease (IHD) in cross sectional epidemiology. Agents and Actions 22: 343-344, 1987. Wattenberg L W. Inhibition of neoplasia by minor dietary constituents. Cancer Research 43 (Suppl): 2448-2453,1983. Bailey J M. Bryant R W, Feinmark S J, Makheja A N. Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin-layer chromatography. Prostaglandins 13: 479-492.1977. Srivastava K C, Tiwari K P, Awasthi K K. A convinient TLC schedule for differential separation of prostaglandins, thromboxanes, and hydroxy fatty acids formed from ( l-‘4C)arachidonic acid in human blood platelets. Microchemical Journal 27: 246-253. 1982.

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