Saiboku-To, a herbal extract mixture, selectively inhibits 5-lipoxygenase activity in leukotriene synthesis in rat basophilic leukemia-1 cells (original) (raw)
Related papers
Lipids, 1993
Leukotrienes have been shown to play an important role as mediators in various disease processes, including asthma and inflammation; thus, their synthesis is tightly regulated. The major precursor of leukotrienes is arachidonic acid (20:4n4~). Fatty acids which are structurally similar to 2~.4n4~, such as eicosatrienoic acid (2(k.3n4~; dihomogammalinolenic acid) and eicosapentaenoic acid (20:5n-3; timnodonic acid) have been found to inhibit leukotriene biosynthesis. Because of the structural similarity of octadecatetraenoic acid (18:4n~3; stearidonic acid) with 20:4n4~, the present study was undertaken to determine whether stearidonic acid also exerts an inhibitory effect on the ~iipoxygenase pathway. Human leukocytes were incubated with 18:4n-3 (20 ~M or 10 ~M), 20:5n-3 (20 ~Vl) or 20:3n4~ (20 ~M) and subsequently stimulated with 1 ~ ionophore A23187 and 20:4n~ (20 pM or 10 ~M). The 5-1ipoxygenase products were then measured by high-performance liquid chromatography. Leukotriene synthesis was reduced by 50% with 20 ~M 18:4n-3 and by 35% with 10 pM 18:4n-3. Formation of 5S,12S~ii-hydroxy~icosatetraenoic acid and of ~hydroxy~icosatetraenoic acid was decreased by 25% with 20 ~M 18:4n-3 and by 3% with 10 ~M 18:4n-3. The inhibition observed with 20 ~M 18:4n~3 appeared to be of the same order as that observed with 20 ~M 20:5n-3; the inhibition observed with 18:4n-3 was shown to be dose dependent. The inhibition produced by 20 ~M 20:3n4~ was greater than that observed with either 20 ~M 18:4n~3 or with 20 ~M 20:5n-3. The results suggest that stearidonic acid, which is found, for example, in blackcurrant seed oil (which also contains the 2~.3n4} precursor), may play a role in suppressing inflammation.
Inflammation, 1984
The effects of nordihydroguairetic acid (NDGA), 3-amino-1-trifluoromethyl-)-phenyl-2-pyrazoline (BW755c), eicostatetraynoic acid (ETYA), phenidone, quercetin, and indomethacin (INDO) on the synthesis of 15-hydroxyeicosatatetraenoic acid (15-HETE) from soybean 15-lipoxygenase, leukotriene B4 (LTB4 from 5-lipoxygenase, and prostaglandin E2 (PGE2 from cyclooxygenase enzymes of rat neutrophils and mouse peritoneal macrophages were investigated. All of the drugs caused a dose-related inhibition of increased oxygen consumption by soybean 15-lipoxygenase in the presence of arachidonic acid and the rank order of potency was phenidone ≥ BW755c > ETYA > quercetin > NDGA > indomethacin. The reduction in oxygen consumption correlated with a reduction of 15-HETE formation as identified by high-performance liquid chromatography. Apart from indomethacin, these drugs were also effective against the rat neutrophil 5-lipoxygenase, although the rank order of potency did not correlate with that obtained with soybean 15-lipoxygenase. Furthermore, in both A23187-activated rat neutrophils and zymosan-activated mouse peritoneal macrophages the synthesis of prostaglandins was inhibited by all of these drugs. In the neutrophils, the rank order of potency was INDO > ETYA > BW755c > quercetin > NDGA > phenidone, whereas in mouse peritoneal macrophages, the order was INDO > ETYA > BW755c > NDGA > quercetin > phenidone. These results suggest that putative lipoxygenase inhibitors exhibit both qualitative and quantitative differences in their effects on both lipoxygenases and cyclooxygenases.
Biochemical Pharmacology, 1998
5-Lipoxygenase (EC 1.13.11.34) is the key enzyme in the regulation of leukotriene synthesis. Here, the effects of various substituted nitrobenzene compounds on 5-lipoxygenase activity and the formation of leukotriene B 4 (LTB 4) were studied in polymorphonuclear leukocytes (PMNL), B lymphocytes, and human whole blood. 1-Chloro-2,4,6-trinitrobenzene (TNCB) was found to inhibit calcium ionophore A23187-induced leukotriene synthesis in PMNL in a biphasic manner. Thus, 1.0 M TNCB caused 50% inhibition of LTB 4 formation, but only 16% inhibition was found at 10 times higher concentration. In contrast, this higher concentration of TNCB activated the synthesis of LTB 4 when PMNL were stimulated with arachidonic acid alone, demonstrating that TNCB can exert both stimulatory and inhibitory effects on leukotriene synthesis depending on the experimental conditions. The inhibitory effect of 1.0 M TNCB on ionophore A23187induced leukotriene synthesis could be circumvented by addition of exogenous arachidonic acid. At high concentrations of TNCB (25-100 M), the drug blocked ionophore A23187-induced leukotriene synthesis. TNCB also inhibited LTB 4 formation in B lymphocytes, as well as in human whole blood. The activity of recombinant 5-lipoxygenase was inhibited by TNCB, and reduced glutathione or -mercaptoethanol counteracted this inhibition. This suggests that TNCB might inhibit 5-lipoxygenase by alkylating thiol groups. TNCB possessed a high specificity for 5-lipoxygenase with only modest inhibitory effects on 12-lipoxygenase (EC 1.13.11.31), 15-lipoxygenase (EC 1.13.11.12), and phospholipase A 2 (EC 3.1.1.4) activities. Taken together, these results show that TNCB can both specifically inhibit and stimulate leukotriene formation and might be useful in further studies on the regulation of 5-lipoxygenase.
Journal of Ethnopharmacology, 1997
We examined the action of Shinpi-To (Formula divinita; T J-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC 4 and LTB 4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB 4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-1ipoxygenase (5-LO), LTC 4 synthase, and LTA 4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC 4 and LTB 4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC 4 and LTB 4 when LTAa-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca z + ([Ca 2+ ]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA 2 and 5-LO activities without affecting the mobilization of [ Ca2+ ]i.
The role of 5-lipoxygenase products in preclinical models of asthma
Journal of Allergy and Clinical Immunology, 1993
Background: The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. Methods: Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. Results: In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmollL. A 36fold selectivity for immunoreactive leukotriene C, versus immunoreactive prostaglandin D, inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C, release (maximum, 73%; bronchoalveolar lavage [BALI, 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced latephase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, 1-24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. Conclusions: In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.
British Journal of Pharmacology, 2012
BACKGROUND AND PURPOSE Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACH We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy. KEY RESULTS Tryptanthrin potently reduced LT-formation in human neutrophils (IC50 = 0.6 mM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca 2+ ]i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC50 = 10 mM) and reduced LTB4 levels in the rat pleurisy model after a single oral dose of 10 mg•kg-1. CONCLUSIONS AND IMPLICATIONS Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.
Inhibition of 5-lipoxygenase and Leukotriene C 4 Synthase in Human Blood Cells by Thymoquinone
Journal of Enzyme Inhibition and Medicinal Chemistry, 2004
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 LTC 4 and LTB 4 formation from endogenous substrate in human granulocyte suspensions with IC 50 values of 1.8 and 2.3 mM, respectively, at 15 min. Major inhibitory effect was on the 5-lipoxygenase activity (IC 50 3 mM) 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 LTC 4 synthase activity, with an IC 50 of 10 mM, as judged by suppressed transformation of exogenous LTA 4 into LTC 4 . In contrast, the drug was without any inhibitory effect on LTA 4 hydrolase activity. When exogenous LTA 4 was added to intact or sonicated platelet suspensions preincubated with TQ, a similar inhibition of LTC 4 synthase activity was observed as in human granulocyte suspensions. The unselective protein kinase inhibitor, staurosporine failed to prevent inhibition of LTC 4 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 LTC 4 synthase activity.
Advances in Applied Chemistry and Biochemistry, 2018
5-Lipoxygenase (5-LO) is the key enzyme in biosynthesis of inflammatory mediators known as leukotrienes which are responsible for asthma, allergic inflammation and innate immunity. This paper reports the evaluation of 30 species of Malaysian plant extracts potential in inhibiting 5-LO activity. Five plant extracts exhibited >80% inhibition against 5-LO activity which include Phyllanthus watsonii, Euphorbia hirta, Anacardium occidentale, Acalypha wilkensiana and Piper betle. Nine plant extracts exhibited moderate value of inhibition (79-40%), while sixteen others exhibited <40% inhibition. From the screening work via inhibition of 5-LO activity, P. watsonii showed the highest inhibition with value of 97.3%, followed by E. hirta with 90.9% inhibition. Both plant extracts were further fractioned using organic solvent which include Dichloromethane (DCM), Ethyl Acetate (EA) and Butanol (BuOH) for evaluation of their inhibiting effects on 5-LO activity. For P. watsonii, DCM fraction exhibited the highest value with 75% inhibition, while for E. hirta, EA fraction exhibited the highest value of 72.5% inhibition. Both active fractions were further subjected to isolation and purification work in order to identify the major compounds. Subsequently, four major compounds managed to be isolated and purified which further identified as quercetin-3-O-rhamnoside (E1), myricetin-3-O-rhamnoside (E2), 26-nor-D: A-friedoolean-14-en-3β-ol (PW1) and glochidonol (PW2) using spectroscopic technique and their data comparison with literature.