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In traditional medicine, Ayurveda, several spices and herbs are held to possess medicinal properties. Earlier we have reported that extracts from several spices, including turmeric, inhibit platelet aggregation and modulate eicosanoid biosynthesis. Due to their eicosanoid-modulating property, it was suggested that the spices may serve to provide clues to drugs directed to arachidonic acid (AA) pathway enzymes as pharmacological targets. Curcumin, a major component of turmeric, inhibited platelet aggregation induced by arachidonate, adrenaline and collagen. This compound inhibited thromboxane B2 (TXB2) production from exogenous [14C] arachidonate in washed platelets with a concomitant increase in the formation of 12-lipoxygenase products. Moreover, curcumin inhibited the incorporation of [14C]AA into platelet phospholipids and inhibited the deacylation of AA-labelled phospholipids (liberation of free AA) on stimulation with calcium ionophore A23187. Curcumin's anti-inflammatory property may, in part, be explained by its effects on eicosanoid biosynthesis.

When garlic cloves are chopped or crushed several dialkyl thiosulfinates are rapidly formed by the action of the enzyme alliin lyase or alliinase (EC 4.4.1.4) on S(+)-alkyl-L-cysteine sulfoxides. Allicin (diallyl thiosulfinate or allyl 2-propene thiosulfinate) is the dominant thiosulfinate released. A variety of sulfur containing compounds are formed from allicin and other thiosulfinates depending on the way in which garlic is handled. One such compound identified recently is ajoene which has been reported to possess antithrombotic properties. We present here data on the antiplatelet properties of ajoene together with its effects on the metabolism of arachidonic acid (AA) in intact platelets. Thus, ajoene was found to inhibit platelet aggregation induced by AA, adrenaline, collagen, adenosine diphosphate (ADP) and calcium ionophore A23187; the nature of the inhibition was irreversible. In washed platelets stimulated by labelled arachidonate, ajoene inhibited the formation of thromboxane A2; 12-lipoxygenase product(s) were reduced at higher ajoene concentrations. This garlic-derived substance inhibited the incorporation of labelled AA into platelet phospholipids at higher concentration. In labelled platelets, on stimulation with either calcium ionophore A23187 or collagen, reduced amounts of thromboxane and 12-HETE (12-hydroxyeicosatetraenoic acid) were produced in ajoene-treated platelets compared to control platelets. This substance had no effect on the deacylation of platelet phospholipids. The results suggest that at least one of the mechanisms by which ajoene shows antiplatelet effects could be related to altered metabolism of AA.

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 microM, 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 micrograms/ml (14.6 microM) 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 12-lipoxygenase product (12-HPETE). These effects were observed by exposing washed platelets to (14C)AA or by stimulating AA-labelled platelets with ionophore A23187. Acetyl eugenol inhibited (14C)TxB2 formation in AA-labelled platelets on stimulation with thrombin. AE showed no effect on the incorporation of AA into platelet phospholipids.

Effects of labetalol on the arachidonic acid metabolism in washed human blood platelets, and in lung and aorta of the rat was studied. Effects of this drug on the aggregation induced by usual aggregation agents, and on the prostacyclin generating capacity of rat aorta as assessed by its antiaggregation effect were also studied. The following results were obtained. 1. Reduced generation of thromboxane and HHT in platelets was observed at 1 mM conc. of the drug. 2. This drug inhibited ADP-, collagen-, and epinephrine-induced aggregation in a dose dependent manner. 3. Arachidonate-induced aggregation was only slightly inhibited at 1 mM conc. of labetalol. 4. Aorta and lung synthesized more prostacyclin in its presence from exogenous labelled arachidonate. 5. Aorta synthesized more prostacyclin from its endogenous AA pool in the presence of labetalol as assessed by inhibition of platelet aggregation.

The effects of four beta-adrenoceptor blocking agents on platelet aggregation, formation of thromboxane from exogenous arachidonic acid (AA) in platelets, on AA incorporation in platelet phospholipids, and on platelet phospholipase activity were studied. Of the four drugs pindolol inhibited thromboxane formation in a dose-related (0.25-1.0 mM) manner apparently by exerting its influence at the cyclooxygenase (CO) level. This drug also inhibited aggregation induced by AA, collagen, epinephrine and ADP. Atenolol and metoprolol though not showing inhibition of AA-induced aggregation did inhibit collagen- and ADP-induced aggregation; metoprolol reversed ADP-induced aggregation, and abolished second phase of epinephrine-induced aggregation. Timolol did not inhibit aggregation induced by all the aggregating agents. Atenolol inhibited (by ca. 10%) TxB2 formation in platelets from exogenous as well as endogenous AA at rather high concentrations (1.0 mM). Metoprolol and timolol did not do so. The observations reported here can be best explained by taking into account the membrane stabilizing effects and lipophilic properties of the drugs.

Effects of various concentrations (12.5-500 uM) of linoleic acid and docosahexaenoic acid (C22:6 omega 3)(sod. salt) were examined on the platelet metabolism of labelled arachidonate (AA) under two different incubation conditions. In the first platelets were pretreated with either fatty acid prior to incubation with labelled AA; and in the second incubation platelets were incubated with a mixture containing a fatty acid (linoleic acid or DHA) and labelled AA. At all concentrations the two fatty acids reduced the formation of TxB2. At lower concentrations (up to 200 uM) the fatty acids inhibited platelet cyclooxygenase as shown by a reduced formation of prostaglandins (PGs) and TxB2. At higher concentrations (400 and 500 uM), however, the fatty acids behaved differently. Although TxB2 formation was reduced, there was observed an increased formation of PGs. In DHA pretreated platelets only PGE2 increased (to double control values). Platelets pretreated with linoleic acid produced increased amounts of all PGs (PGF2 alpha, PGE2, PGD2), and this effect was greatest for PGE2 which increased by 5-6 fold of control values. DHA showed a dose-dependent inhibition of platelet aggregation induced by arachidonate, epinephrine and collagen.

Vitamin E (alpha-tocopherol) and tocopherol acetate produced a slightly increased amount of thromboxane in treated compared to untreated platelets. In tocopherol acetate-treated platelets significantly more lipoxygenase products were produced. alpha-tocopherol induced an increased, but not significant, production of thromboxane B2 during blood clotting. alpha-tocopherol was not found to affect platelet phospholipase activity as determined by its effect on the release of labelled arachidonic acid from platelet phospholipids by challenging the platelets with calcium ionophore A23,187. alpha-tocopherol potentiated the incorporation of labelled arachidonate in the platelet phospholipids. Inspite of having no effect on the arachidonic acid cascade in platelets, alpha-tocopherol inhibited aggregation induced by several aggregating agents including A23,187. Inhibition of aggregation may be explained by the ability of alpha-tocopherol to inhibit intracellular mobilization of sequestered calcium from the dense tubular system to the cytoplasm.

An ethereal extract of omum (Trachyspermum ammi; Hindustani: ajwan)--a frequently consumed spice--was found to inhibit platelet aggregation induced by arachidonic acid (AA), epinephrine and collagen; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by omum could be explained by its effect on platelet thromboxane production as suggested by the following experimental observation. (i) Omum reduced TxB2 formation in intact platelet preparations from added arachidonate, and (ii) it reduced the formation of TxB2 from AA-labelled platelets after stimulation with Ca2+-ionophore A23187 by a direct action on cyclooxygenase as it did not affect the release of AA from labelled platelets. An increased formation of lipoxygenase-derived products from exogenous AA in omum-treated platelets was apparently due to redirection of AA from cyclooxygenase to the lipoxygenase pathway.

Dietary fatty acids affect the synthesis and thus ultimate availability of prostaglandin precursors. During processing of edible fats a significant portion of the naturally occurring cis fatty acids is changed to the respective trans isomers. Trans fatty acids could have a wide variety of effects including their effect on prostaglandin synthesis. A comparative study on the effect of cis (oleic and linoleic) fatty acids and their corresponding trans isomers (elaidic and linoelaidic acids) on the in vitro prostaglandin biosynthesis in human blood platelets was made. Linoleic acid (0.5, 1.0 mM) showed the same effect on the various arachidonic acid (AA) metabolites as reported earlier by us, that is, increased generation of prostaglandins and thromboxanes and reduced amounts of HHT and HETE (26). Linoelaidic acid at these two concentrations had the same effect but to a lesser extent. Both linoleic and linoelaidic acids at 0.25 mM concentration reduced TxB2 formation. Oleic and elaidic acids (0.5, 1.0 mM) decreased the formation of TxB2 though not significantly. With oleic acid, the results in respect of other AA metabolites were similar to those reported by us earlier (26). MDA was reduced significantly in the presence of linoleic, linoelaidic and oleic acids; cis isomers being more effective. In the presence of linoleic and linoelaidic acids, prostaglandin endoperoxides were reduced significantly; there was no change in the presence of oleic and elaidic acids. Thus fatty acids seem to affect the platelet cyclo-oxygenase activity; this being more pronounced in the case of cis fatty acids compared to their trans isomers. Reduction in cyclo-oxygenase activity also depends upon the number of double bonds in the acids.

The effects of some saturated (lauric, palmitic and stearic) an unsaturated (linoleic, gamma-linolenic, alpha-linolenic and oleic) fatty acids at 0.1. 0.25 and 0.5 mM concentrations on the in vitro metabolization of (1-14 C) arachidonic acid by washed human blood platelets have been studied. Effects of these fatty acids were studied with intact as well as lysed platelet preparations. With intact platelet preparations it was found that (i) all unsaturated fatty acids enhanced the biosynthesis of TxB2, PGE2, PGD2 and PGF2 alpha, (ii) unsaturated fatty acids reduced the formation of HHT and HETE with the exception of oleic acid which showed very little effect, (iii) unsaturated fatty acids reduced the formation of MDA, whereas palmitic and stearic acids increased its formation and (iv) all unsaturated fatty acids reduced the synthesis of prostaglandin endoperoxides. These results support our previous observations where effects of fatty acids were examined at higher concentrations (10). At 0.1 mM FA concentration, inconsistent results were obtained. With lysed platelet preparations all cyclooxygenase products were reduced in presence of unsaturated fatty acids, whereas HETE formation was reduced only in presence of linoleic and gamma-linolenic acids. Electron micrographs of washed platelet suspensions were obtained with untreated platelet preparations and platelet preparations treated with 0.25 and 0.5 mM linoleic acid concentrations. The results are discussed in the light of a possible soap-like effect of FA salt on platelets.

The effects of some vasodilatory (dipyridamole, nifedipine, verapamil) and antihypertensive (hydralazine, propranolol) drugs on the metabolism of exogenous arachidonic acid (AA), and on the production of thromboxane from endogenous AA stores were examined. In a coupled system of platelets and rat aorta, dipyridamole (100 microM), nifedipine (300 microM) and hydralazine (250 microM) potentiated the formation of 6-keto-F1 alpha from exogenous AA. Hydralazine in this system reduced the formation of TxB2. At higher concentrations the drugs (dipyridamole 500 microM; hydralazine 1 mM, verapamil 2mM; propranolol 2mM) were effective in reducing the formation of TxB2. Moreover, the drugs at lower concentrations produced reduced amounts of TxB2 in clotting blood. A dose-dependent inhibition of platelet aggregation induced by ADP, epinephrine, collagen and arachidonate was observed with these drugs. The results demonstrate that some of the beneficial effects of the drugs in the cardiovascular system can be explained on the basis of their effects on platelet aggregation and on AA metabolism.

Effect of furosemide at various concentrations (0.05-4 mM) was examined on the in vitro biosynthesis of prostaglandins in human platelets. At lower furosemide concentrations (0.05 and 0.1 mM) no effect was observed. At 1 and 2 mM concentrations, PGE2 significantly (P less than 0.01) increased. At 3 and 4 mM concentrations PGE2 increased though not significantly probably because of the small number of samples (n=5). A decrease in PGD2 formation was noted at 1-4 mM furosemide concentrations, though significantly only at 3 mM conc. At 1 and 2 mM concentrations, TxB2 and HHT increased whereas at 3 and 4 mM concentrations these metabolites were decreased. These effects were, however, not significant. No effect was observed on endoperoxide generation at 1 and 2 mM conc. Furosemide at 1 and 2 mM concentrations inhibited ADP- and arachidonic acid induced platelet aggregation. An increased platelet formation of PGE2 in the presence of furosemide may point to the fact that this drug shows its effect mainly on the formation of PGE2 which has been found to exert a profound effect on renal blood flow and thus ameliorates some forms of hypertension.

Effects of some vasodilating (oxyfedrine, dipyridamole, verapamil and nifedipine) and antihypertensive (propranolol, hydralazine, prazosin and dihydralazine) drugs on the human blood platelet biosynthesis of prostaglandins from labelled arachidonic acid have been examined in vitro. Malondialdehyde (MDA) production in platelets and their aggregation in presence of these drugs were also studied. All the drugs inhibited the generation of thromboxane A2 which correlated well with the reduced platelet formation of MDA in presence of several of these drugs. In case of seven of the eight drugs, separation of HHT from HETE could be made. Values of these metabolites - especially those of HHT were found to be reduced, thus supporting the MDA and TxA2 data. With several of these drugs, ADP-induced platelet aggregation was inhibited either completely or partially. The results show that the well-known completely or partially. The results show that the well-known effects of these drugs might well be augmented by their effect on the platelet metabolism of arachidonic acid.

Effects of ascorbic acid at physiologically achieved concentrations were examined on the metabolism of exogenous dihomo-gamma -linolenic acid and arachidonic acid (AA) in washed human platelets, and of AA in rat aortic rings. In the presence of ascorbic acid an increased formation of PGF1 alpha, PGE1 and PGE2 was observed in platelets. Also this vitamin induced an increased production of prostacyclin (measured as its stable metabolite 6-keto-PGF1 alpha) from exogenously provided substrate in aortic rings. In addition, from endogenous stores of AA in aortic rings ascorbic acid induced an increased generation of prostacyclin as revealed by inhibition of ADP-induced platelet aggregation.

In vitro human platelet prostaglandin synthesis has been studied from added radioactive arachidonic acid (i) as function of substrate concentration, (ii) as function of platelet concentration and (iii) as function of pH. Platelets, as in platelet rich plasma when labelled with arachidonic acid, washed and treated with thrombin, released radioactivity mainly from phosphatidylcholine and phosphatidylinositol. The released radioactivity was mostly accounted for by the formation of the previously identified oxygenation products of arachidonic acid. Platelet utilization or arachidonic acid was also studied in presence of linoleic and dihomo-gamma-linolenic acids, the two essential fatty acids known for antithrombotic effect. At its high concentrations linoleic acid decreased platelet cyclo-oxygenase activity as seen by a decreased formation of endoperoxides from arachidonic acid. Dihomo-gamma-linolenic acid was found to be a mutually competitive substrate with arachidonic acid for the platelet prostaglandin synthetase thus causing reduced utilization of arachidonic acid as shown by measuring the various oxygenation products of arachidonic acid. These two acids were utilized differently by platelet prostaglandin synthetase.

In the traditional Indian system of medicine, Ayurveda, several spices and herbs are claimed to possess medicinal properties, such as being antithrombotic, antiatherosclerotic, hypolipidemic, anti-inflammatory etc. Earlier we have reported that extracts from several spices behave as antiaggregatory agents and inhibit eicosanoid synthesis. Similar studies with extracts prepared from cumin (Cuminum cyminum) and turmeric (Curcuma longa) were undertaken. Ethereal extract of both cumin and turmeric inhibited arachidonate-induced platelet aggregation. Extracts from these spices inhibited thromboxane B2 production from exogenous (14C) arachidonic acid (AA) in washed platelets; a simultaneous increase in the formation of lipoxygenase-derived products was observed. Less TxB2 was produced in blood samples treated with turmeric extract when they were allowed to clot. Turmeric extract inhibited incorporation of (14C)AA into platelet phospholipids and deacylation of AA-labelled phospholipids on stimulation with calcium ionophore A23187. Cumin extract was devoid of such effects. Extracts from the two spices reduced the formation of (14C)TxB2 from AA-labelled platelets when they were challenged with A23187. The anti-inflammatory property of turmeric may, in part, be explained by its effect on eicosanoid biosynthesis.

Aqueous extracts of onion, garlic and ginger inhibited platelet aggregation induced by several aggregation agents, including arachidonate (AA), in a dose-dependent manner. While onion and garlic extracts were found to be weak inhibitors of platelet thromboxane synthesis, ginger extract inhibited the platelet cyclooxygenase products and this effect correlated well with its inhibitory effects on the platelet aggregation induced by the above aggregation agents. These two effects were dose-dependent. Although the three aqueous extracts inhibited the biosynthesis of 6-keto-F1 alpha in rat aortic rings from labelled AA, they did not reduce prostacyclin production from endogenous AA pool in aortic rings. Aqueous ginger extract was extracted into three organic solvents in order of increasing polarity (n-hexane, chloroform, ethyl acetate). An analysis of the n-hexane extract revealed at least three clearly separated TLC bands containing materials that inhibited platelet thromboxane generation simultaneously increasing lipoxygenase products (HETE). The results indicate that if the same were happening in vivo, onion, garlic and ginger could be useful as natural antithrombotic materials.

Effects of various concentrations (12.5-50 microM) of eicosapentaenoic acid (EPA) on the metabolism of exogenous arachidonic acid (AA) in washed human platelets were examined under two different incubation conditions. First platelets were pretreated with EPA before exposing them to labelled AA. In the second incubation platelets were exposed to a mixture of EPA (12.5 and 25 microM) and labelled AA. At all concentrations EPA reduced thromboxane B2 (TxB2) formation, and at 25 and 50 microM this fatty acid reduced also prostaglandins (PGE2, PGF2 alpha). A reduced formation of PGs and TxB2 was confirmed by a decreased formation of PG-endoperoxides. While in EPA treated platelets less CO-(TxB2, HHT, PGs) and lipoxygenase (HETE) products were formed at 25 and 50 microM, more HETE seemed to be generated at lower EPA concentration (12.5 microM). Oleic acid reduced TxB2 formation at high concentration (100 microM). EPA showed a dose-dependent inhibition of platelet aggregation induced by arachidonate, epinephrine and collagen; it was most effective against AA-induced aggregation.

The effects of onion and ginger consumption on platelet thromboxane production were examined. Volunteers, all Danish women, consumed either 70 g raw onion or 5 g raw ginger daily for a period of 7 days. Each participant in each (onion or ginger) group served as her own control. TxB2 determination was made in serum obtained after blood clotting. The following are the results. TxB2 (pmol/ml serum): (i) onion group--before consumption 910 +/- 327, after consumption 1005 +/- 713 (Mean +/- SD, N = 5); (ii) ginger consumption 782 +/- 482, after consumption 498 +/- 164 (Mean +/- SD, N = 7).

The effect of some saturated and unsaturated fatty acids pre-incubated with human washed platelets was examined on the platelet utilization of (1-14C) arachidonic acid. The platelets were incubated with the radioactive arachidonic acid for periods of either 10 min or 30 sec. The following results were obtained. Conversion of arachidonic acid to thromboxane B2 was increased during 10 min in the presence of oleic, alpha-linolenic and lauric acids. Increased conversion of 14C-arachidonic acid to hydroxy fatty acids (HHT and HETE) was observed in the presence of stearic acid (10 min incubation) and lauric acid (30 sec incubation). Their synthesis, however, was decreased in presence of gamma-linolenic acid (10 min incubation). Endoperoxide generation from arachidonate was reduced by alpha-linolenic acid but increased by stearic acid. The conversion to PGE2 was not altered by these acids during 10 min incubation with arachidonic acid. However, in presence of gamma-linolenic, lauric and stearic acids conversion of arachidonic acid into PGE2 during 30 sec incubation was increased. Under these conditions, production of PGF2 alpha and PGD2 remained unchanged.

As a continuation of previous studies on the synthesis of prostaglandins in platelets of human venous blood, the present communication describes the synthesis of prostaglandins in experimental hypercholesterolemia induced in rabbits by dietary means. In such a condition, high serum level of cholesterol did not change the rate of prostaglandin synthesis.