Black cumin seed, Nigella sativa L., and its oils have traditionally been used for the treatment of asthma and other inflammatory diseases. Thymoquinone (TQ) has been proposed to be one of the major active components of the drug. Since leukotrienes (LTs) are important mediators in asthma and inflammatory processes, the effects of TQ on leukotriene formation were studied in human blood cells. TQ provoked a significant concentration-dependent inhibition of both LTC4 and LTB4 formation from endogenous substrate in human granulocyte suspensions with IC50 values of 1.8 and 2.3 microM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC50 3 microM) as evidenced by suppressed conversion of exogenous arachidonic acid into 5-hydroxy eicosatetraenoic acid (5HETE) in sonicated polymorphonuclear cell suspensions. In addition, TQ induced a significant inhibition of LTC4 synthase activity, with an IC50 of 10 microM, as judged by suppressed transformation of exogenous LTA4 into LTC4. In contrast, the drug was without any inhibitory effect on LTA4 hydrolase activity. When exogenous LTA4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC4 synthase activity induced by TQ. The findings demonstrate that TQ potently inhibits the formation of leukotrienes in human blood cells. The inhibitory effect was dose- and time-dependent and was exerted on both 5-lipoxygenase and LTC4 synthase activity.

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Human platelets possess a specific membrane-bound leukotricnc (LT) C., synlhase, which catalyzes the conversion of LTA, to LTC,. Sti tnulation of the rcccptoi-s for thronibin, collagen or thromboxane A, provoked inhibition of this cnzyme, as judged by suppressed trmsformation of exogenous LTA, to LTC,.Similarly, direct activation of protein kinase (PK) C with riiltiomolur concentrations of 4/1-phorhol 12-myristate 13-acelate (PMA) inhibited the production of I,TC:,. Kinetic studies demonstrated that the i nhibition induced by thrombin and PMA was nori-compctitive. Elevation of inlracellular CAMP levels with carbacyclin did not affect basal LTC, formation, hut abolished the attenuation d' platelet LTC, synthasc activity induced by the thromboxane receptor agonisl U-46619. The unselective prolein kinasc inhibitor lstaurosporine prevented both receptor-mediated and PMA-induced suppression of LTC, formation. In contrast, two selective PKC inhibitors, Ro 31 4 2 2 0 and GF 100203X, reversed the inhibitory effect provoked by PMA, but failed to prevent thrombin-induced inhibition, Furthermore, the protein tyrosine phosphatase inhibitor, sodium orthovanadate, induced dose-dependent inhibition of LTC, production in platelet sonicates. In conclusion, receptor-mediated activation u f human platclets leads to decreased I,TC:., synthase activity via phosphoregulation. Although the present results demonstrate that platelet LIC, synthase can be regulated via PKC-dependent events, alternative mechanisms appears to be involved in the physiological regulation of this enzyme. The findings suggcst the possible importance of protein tyrosine phophorylations in this process.

Leukotriene A4 may be metabolized to 5(S),6(R)‐ and 5(S),6(S)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids by enzymatic or non‐enzymatic hydrolysis. Incubation of human platelet suspensions with these dihydroxy acids led to the formation of lipoxin A4 and 6(S)‐lipoxin A4 via lipoxygenation at C‐15. Furthermore, human platelets converted the two 5(R),6(S)‐ and 5(R),6(R)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids to tetraene‐containing trihydroxyeicosatetraenoic acids. In contrast, leukotrienes C4, D4 and E4 were not transformed to cysteinyl‐lipoxins, Time‐course studies of leukotriene A4 metabolism in human platelet suspensions indicated lipoxin formation via two pathways: (i) direct conversion of leukotriene A4, leading to formation of the lipoxin intermediate 15‐hydroxy‐leukotriene A4; and (ii) 15‐lipoxygenation of the 5(S),6(R)‐ and 5(S),6(S)‐dihydroxyeicosatetraenoic acids. The results demonstrate that lipoxygenation at C‐15 of 5,6‐dihydroxy‐7,9,11,14‐eicosatetraenoic acids may be an alternative novel pathway for platelet‐dependent lipoxin formation.

The results indicate that oral aspirin administration can lead to uncoupling of thromboxane A2-dependent negative feedback mechanisms, which may normally restrict the production of cysteinyl leukotrienes. This mechanism can be of potential interest in aspirin-induced asthma.