Inhibitory effects of Mannich bases of heterocyclic chalcones on NO production by activated RAW 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils

Some chalcones exert potent anti-inflammatory activities. Mannich bases of heterocyclic chalcones inhibited nitric oxide (NO) production in lipopolysaccharide and interferon-γ stimulated RAW 264.7 macrophages. Also Formyl-Met-Leu-Phe and cytochalasin B induced superoxide anion generation (O2·-) and elastase release in human neutrophils. Mannich bases of heterocyclic chalcone analogs exhibited potent inhibitory effects on NO production with IC(50) values ranges between 10.5 and 0.018 μM, O2·- generation (IC(50) 39.87-0.68 μM) and elastase release (IC(50) 39.74-0.95 μM). Compound 29 (IC(50) 0.055 μM) and 34 (IC(50) 0.018 μM) were showed excellent inhibition on NO production. On the other hand, compounds 2 and 8 showed potent inhibition on O2·- generation and elastase release. Therefore, these four compounds may be new leads for development of anti-inflammatory activities. The structure-activity relationships are also discussed.     

The synthesis and effect of fluorinated chalcone derivatives on nitric oxide production

Dimethoxy- and trimethoxychalcone derivatives, with various patterns of fluorination, were synthesized and evaluated for their influence on nitric oxide production. Some of them, chalcones 1, 5, 7, 10, 11 and 17, inhibited NO production with an IC(50) in the submicromolar range; 17 is especially noteworthy because of its potency (IC(50) 30nM). These effects were not the consequence of a direct inhibitory action on enzyme activity but the inhibition of enzyme expression.     

Inhibitory effects of sesquiterpenes from bay leaf on nitric oxide production in lipopolysaccharide-activated macrophages: structure requirement and role of heat shock protein induction

The methanolic extract from the leaves of Laurus nobilis (bay leaf, laurel) was found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, fourteen known sesquiterpenes were isolated from the active fraction and were examined for ability to inhibit the NO production. Seven sesquiterpene lactones (costunolide, dehydrocostus lactone, eremanthine, zaluzanin C, magnolialide, santamarine and spirafolide) potently inhibited LPS-induced NO production (IC50 = 1.2 approximately 3.8 microM). Other sesquiterpene constituents also showed the inhibitory activity (IC50 > or = 21 microM), but their inhibitory activities were less than those of sesquiterpene lactones. Alpha-methylene-gamma-butyrolactone also showed inhibitory activity (IC50 = 9.6 microM), while mokko lactone and watsonol A etc., reductants of the alpha-methylene-gamma-butyrolactone moiety by NaBH4 or DIBAL, and a 2-mercaptoethanol adduct of dehydrocostus lactone showed little activity (IC50 > or = 18 microM). These results indicated that the alpha-methylene-gamma-butyrolactone moiety is important for the activity. Furthermore, costunolide and dehydrocostus lactone inhibited inducible nitric oxide synthase (iNOS) induction in accordance with induction of heat shock protein 72 (HSP 72). These results suggested that, as one of their mechanisms of action, sesquiterpene lactones induce HSP 72 thereby preventing nuclear factor-kappaB activation followed by iNOS induction.     

Photocontrol of nitric oxide production in cell culture using a caged isoform selective inhibitor

Over the past decade, multiphoton microscopy has progressed from a photonic novelty to a technique whose application is currently experiencing exponential growth in the biological sciences. A novel application of this technology with significant therapeutic potential is the control of drug activity by multiphoton photolysis of caged therapeutics. As an initial case study, the potent isoform selective inhibitor N-(3-(aminomethyl)benzyl) acetamidine (1400W) of inducible nitric oxide synthase (iNOS) has been conjugated to a caging molecule 6-bromo-7-hydroxy-4-hydroxyquinoline-2-ylmethyl acetyl ester (Bhc). Here we present the first report of a bulk therapeutic effect, inhibition of nitric oxide production, in mammalian cell culture by multiphoton photolysis of a caged drug, Bhc-1400W. Mouse macrophage RAW 264.7 cells induced with bacterial lipopolysaccharides to express iNOS were used to assess the therapeutic value of the conjugated inhibitor. Both 1400W and Bhc-1400W are stable in metabolically active cells and an optimal time interval for the photorelease of the inhibitor was determined. The ratios of the IC(50) values of Bhc-1400W over 1400W calculated in the presence of iNOS enzyme and in RAW 264.7 cell culture are 19 and 100, respectively, indicating that a broad therapeutic range exists in cell culture. Multiphoton uncaging protocols and therapeutic doses of inhibitors were not cytotoxic. Photocontrol of LPS induced nitric oxide production was achieved in mammalian cell culture using a single laser focal volume. This technology has the potential to control active drug concentrations in vivo, a lack of which is one of the main problems currently associated with systemic drug administration.     

Inducible nitric oxide synthase inhibitors of Chinese herbs. Part 2: naturally occurring furanocoumarins

Inducible nitric oxide synthase (iNOS)-dependent production of nitric oxide (NO) plays an important role in inflammation. The effects of various naturally occurring furanocoumarins on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells were evaluated in vitro. The results showed that angelicin, pimpinellin, sphondin, byakangelicol, oxypeucedanin, oxypeucedanin hydrate, xanthotoxin, and cnidilin are potential NO production inhibitors, and their IC50 values for inhibition of nitrite production were 19.5, 15.6, 9.8, 16.9, 16.8, 15.8, 16.6, and 17.7 microg/mL, respectively. Distinct structure-activity relationships were also revealed for the NO production inhibitory activities of these furanocoumarins. Activities of the angelicin type such as pimpinellin and sphondin were more potent than those of the psoralen type. Presence of a methoxy at the C6 position in the angelicin type seemed to be essential to augment the activity. Western blot analysis demonstrated that only sphondin dose-dependently inhibited the expression of the iNOS protein at 2.5-20 microg/mL. However, iNOS enzyme activity was stimulated with LPS for 12 h and sphondin was administered (20 microg/mL) for 24 h, which did not reasonably inhibit iNOS enzyme activity. L-NAME (100 microM), a known specific inhibitor of iNOS, was employed as a positive control with the same protocol and showed more than 50% inhibition activity. The results demonstrate that the NO production inhibitory activity of sphondin is due to the effect of iNOS expression, but not by direct inhibition of iNOS enzyme activity. Thus, sphondin may act as a potent inhibitor of NO production under tissue-damaging inflammatory conditions.     

Cycloamphilectenes,a new type of potent marine diterpenes: inhibition of nitric oxide production in murine macrophages

The inhibitory effect of a series of 6 cycloamphilectenes, novel marine diterpenes based on amphilectene skeletons and isolated from the Vanuatu sponge Axinella sp., on NO, PGE(2) and TNFalpha production in murine peritoneal macrophages was studied. These compounds reduced potently nitric oxide production in a concentration-dependent manner with IC(50) values in the submicromolar range (0.1-4.3 microM). Studies on intact cells and Western blot analysis showed that the more potent cycloamphilectenes reduced the expression of inducible nitric oxide synthase without affecting cyclo-oxygenase-2 expression. Among them cycloamphilectene 2, the unique compound bearing an exocyclic methylene group, was able to reduce NO production without affecting TNFalpha release. Cycloamphilectene 2, which is an inhibitor of the nuclear factor-kB pathway, exhibited topical anti-inflammatory activity.     

Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.

Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo.     

Phenylsulphonyl urenyl chalcone derivatives as dual inhibitors of cyclo-oxygenase-2 and 5-lipoxygenase

Two series of phenylsulphonyl urenyl chalcone derivatives (UCH) with various patterns of substitution were tested for their effects on nitric oxide (NO) and prostaglandin E2 (PGE2) overproduction in RAW 264.7 macrophages. None of the tested compounds reduced NO production more than 50% at 10 microM but most of them inhibited the generation of PGE2 with IC50 values under the micromolar range. Me-UCH 1, Me-UCH 5, Me-UCH 9, Cl-UCH 1, and Cl-UCH 9 were selected to evaluate their influence on human leukocyte functions and eicosanoids generation. These derivatives selectively inhibited cyclo-oxygenase-2 (COX-2) activity in human monocytes being Me-UCH 5 the most potent (IC50 0.06 microM). Selected compounds also reduced leukotriene B4 synthesis in human neutrophils by a direct inhibition of 5-lipoxygenase (5-LO) activity, with IC50 values from 0.5 to 0.8 microM. In addition, lysosomal enzyme secretion, such as elastase or myeloperoxidase as well as superoxide generation in human neutrophils were also reduced in a similar range. Our findings indicate that UCH derivatives exert a dual inhibitory effect on COX-2/5-LO activity. The profile and potency of these compounds may have relevance for the modulation of the inflammatory and nociceptive responses with reduction of undesirable side-effects associated with NSAIDs.     

Nitric oxide production by cultured aortic endothelial cells in response to thiol depletion and replenishment

The requirements and influence of thiols on the production of nitric oxide (NO) were examined in cultured porcine aortic endothelial cells. NO production was diminished when cells were pretreated with thiol-depleting agents (IC50: N-ethylmaleimide, 30 microM; 1-chloro-2,4-dinitrobenzene, 200 microM; diamide, 1.5 mM; diethyl maleate, 20 mM). The depletion of glutathione (45-99% loss at the various IC50 values) and protein thiols (3-25% loss at IC50) showed no consistent relationship to decreased NO production. The effects of the agents on NO production were not linked to altered sensitivity to the stimulant (calcium ionophore A23187; maximal effect at 10 microM), but roughly paralleled the appearance of cell damage (17-44% lactate dehydrogenase release at IC50). The decrease in NO production due to 1-chloro-2,4-dinitrobenzene was partially reversed by cysteine, dithioerythritol, and dihydrolipoate, whereas cystine partially reversed the decrease due to diamide or diethyl maleate. On the other hand, several thiols diminished NO production in control cells. Overall, alterations of NO production did not parallel the depletion or replenishment of either glutathione, protein thiol, or soluble thiol pools, and so the results argue against hypotheses that cellular thiols are either substrates or necessary cofactors in the pathway of NO synthesis in endothelial cells.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Grifolin derivatives from Albatrellus caeruleoporus, new inhibitors of nitric oxide production in RAW 264.7 cells

Two new farnesyl phenols named grifolinones A and B, together with known grifolin and neogrifolin, were isolated from methanolic extract of the inedible mushroom Albatrellus caeruleoporus. Their structures were characterized by a combination of 2D NMR, MS, IR, and UV spectra. Grifolinone B was composed of two grifolin molecules, which were connected by a C-C bond. Grifolinones A and B, grifolin, and neogrifolin exhibited inhibitory activity against nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) in RAW 264.7 cells with IC50values of 23.4, 22.9, 29.0, and 23.3 microM, respectively.     

Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

To clarify the structure-activity relationships of flavonoids for nitric oxide (NO) production inhibitory activity, we examined the inhibitory effects of 73 flavonoids on NO production in lipopolysaccharide-activated mouse peritoneal macrophages. Among those flavonoids, apigenin (IC(50)=7.7 microM), diosmetin (8.9 microM), and tetra-O-methylluteolin (2.4 microM), and hexa-O-methylmyricetin (7.4 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the activities of flavones were stronger than those of corresponding flavonols; (2) the glycoside moiety reduced the activity; (3) the activities of flavones were stronger than those of corresponding flavanones; (4) the flavones and flavonols having the 4'-hydroxyl group showed stronger activities than those lacking the hydroxyl group at the B ring and having the 3',4'-dihydroxyl group; (5) the flavonols having the 3',4'-dihydroxyl group (catechol type) showed stronger activities than those having the 3',4',5'-trihydroxyl group (pyrogallol type); (6) the 5-hydroxyl group tended to enhance the activity; (7) methylation of the 3-, 5-, or 4'-hydroxyl group enhanced the activity; (8) the activities of isoflavones were weaker than those of corresponding flavones; (9) methylation of the 3-hydroxyl group reduced the cytotoxicity. In addition, potent NO production inhibitors were found to inhibit induction of inducible nitric oxide synthase (iNOS) without iNOS enzymatic inhibitory activity.     

Supressive effect of maslinic acid from pomace olive oil on oxidative stress and cytokine production in stimulated murine macrophages

The pentacyclic triterpene maslinic acid (MA) is a natural compound present in the non glyceride fraction of pomace olive oil, also called orujo olive oil. This compound has previously demonstrated antioxidant properties against lipid peroxidation in vitro, but its effects on reactive oxygen and nitrogen-derived species and pro-inflammatory cytokines generated by a cell system have not yet been investigated. In this study, we have tested the effect of MA upon oxidative stress and cytokine production using peritoneal murine macrophages. MA significantly inhibited the enhanced production of nitric oxide (NO) induced by lypopolysaccharide (LPS) when it was measured by the nitrite production with an inhibitory concentration 50% value (IC(50)) of 25.4 microM. This inhibiting effect seems to be consequence of an action at the level of the LPS-induction of the inducible nitric oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The secretion of the inflammatory cytokines interleukine-6 and TNF-a from LPS-stimulated murine macrophages was also significantly reduced (p < 0.05 and 0.01) by 50 and 100 microM of MA. In addition, reactive oxygen species were measured after stimulation with phorbol-12-myristate-13-acetate (PMA). Thus, pre-treatment with MA reduced the generation of hydrogen peroxide from stimulated macrophages in a dose-dependent manner (IC(50): 43.6 microM) as assayed by the oxidation of the peroxidase enzyme. However, no inhibitory effect on superoxide release, measured by the reduction of ferricytochrome c, was observed after the pretreatment with MA in the culture medium.These results suggest a potential biopharmaceutical use of this hydroxy-pentacyclic triterpene derivative, present in orujo olive oil, on preventing oxidative stress and pro-inflammatory cytokine generation.     

Effect of naturally occurring organosulfur compounds on nitric oxide production in lipopolysaccharide-activated macrophages

Excessive nitric oxide (NO) production is involved in cellular injury and possibly in the multistage process of carcinogenesis. In this study, we investigated the effect of organosulfur compounds (S-allyl cysteine, allyl sulfide, diallyl disulfide, allyl isothiocyanate, phenyl isothiocyanate, and benzyl isothiocyanate) that are found in allium or cruciferous vegetables on NO production in J774.1 macrophages activated with lipopolysaccharide (LPS). Diallyl disulfide, allyl, phenyl, and benzyl isothiocyanates inhibited NO production, as evaluated by nitrite formation at 25 microM. Allyl and benzyl isothiocyanates, the most active of the six organosulfur compounds, exhibited dose-dependent inhibition and had IC(50) values of 1.6 and 2.7 microM, respectively. Western blot analysis suggested that suppression of the induction of inducible NO synthase (iNOS) expression is responsible for the inhibition of NO production by allyl and benzyl isothiocyanates. In contrast, these isothiocyanates increased LPS-stimulated tumor necrosis factor alpha (TNF-alpha) release, suggesting their selective action on genes activated by LPS. Our results demonstrate that certain organosulfur compounds inhibit NO synthesis in LPS-activated macrophages, and the inhibitory effect may be a significant component of their anticarcinogenic activity.     

Synthesis of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas and evaluation as modulators of the isoforms of nitric oxide synthase

Inhibition of the isoforms of nitric oxide synthase (NOS) has important applications in therapy of several diseases, including cancer. Using 1400 W [N-(3-aminomethylbenzyl)acetamidine], thiocitrulline and N(delta)-(4,5-dihydrothiazol-2-yl)ornithine as lead compounds, series of N-benzyl- and N-phenyl-2-amino-4,5-dihydrothiazoles and thioureas were designed as inhibitors of NOS. Ring-substituted benzyl and phenyl isothiocyanates were synthesised by condensation of the corresponding amines with thiophosgene and addition of ammonia gave the corresponding thioureas in high yields. The substituted 2-amino-4,5-dihydrothiazoles were approached by two routes. Treatment of simple benzylamines with 2-methylthio-4,5-dihydrothiazole at 180 degrees C afforded the corresponding 2-benzylamino-4,5-dihydrothiazoles. For less nucleophilic amines and those carrying more thermally labile substituents, the 4,5-dihydrothiazoles were approached by acid-catalysed cyclisation of N-(2-hydroxyethyl)thioureas. This cyclisation was shown to proceed by an S(N)2-like process. Modest inhibitory activity was shown by most of the thioureas and 4,5-dihydrothiazoles, with N-(3-aminomethylphenyl)thiourea (IC(50)=13 microM vs rat neuronal NOS and IC(50)=23 microM vs rat inducible NOS) and 2-(3-aminomethylphenylamino)-4,5-dihydrothiazole (IC(50)=13 microM vs rat neuronal NOS and IC(50)=19 microM vs human inducible NOS) being the most potent. Several thioureas and 4,5-dihydrothiazoles were found to stimulate the activity of human inducible NOS in a time-dependent manner.     

Inhibitors from the rhizomes of Alpinia officinarum on production of nitric oxide in lipopolysaccharide-activated macrophages and the structural requirements of diarylheptanoids for the activity

The 80% aqueous acetone extract from the rhizomes of Alpinia officinarum, a Chinese medicinal herb, were found to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. Through bioassay-guided separation, two diarylheptanoids [7-(4'-hydroxy-3'-methoxyphenyl-1-phenylhept-4-en-3-one and 3,5-dihydroxy-1,7-diphenylheptane] and a flavonol constituent (galangin) substantially inhibited LPS-induced NO production with IC50 values of 33-62 microM. To clarify structure-activity relationships of diarylheptanoids, related diarylheptanoids from Curcuma zedoaria were examined. Results indicate that the double bond or enone moiety at the 1-7 positions is important for the activity.     

In vitro anti-inflammatory activity of 3-O-methyl-flavones isolated from Siegesbeckia glabrescens

Four flavones, 3,4'-O-dimethylquercetin (1), 3,7-O-dimethylquercetin (2), 3-O-methylquercetin (3) and 3,7,4'-O-trimethylquercetin (4) were isolated as the inhibitors of nitric oxide production in activated microglia (IC(50) values: 11.1, 4.2, 3.8, and 25.1 microM, respectively). They suppressed the expression of protein and mRNA of inducible nitric oxide synthase. Furthermore, compounds 2 and 3 showed scavenging activity of peroxynitrite with SC(50) values of 1.75 and 0.77 microM, respectively.     

Inhibitors from rhubarb on lipopolysaccharide-induced nitric oxide production in macrophages: structural requirements of stilbenes for the activity.

By bioassay-guided separation, three stilbenes (rhapontigenin, piceatannol, and resveratrol), two stilbene glucoside gallates (rhaponticin 2"-O-gallate and rhaponticin 6"-O-gallate), and a naphthalene glucoside (torachrysone 8-O-beta-D-glucopyranoside) with inhibitory activity against nitric oxide (NO) production in lipopolysaccharide-activated macrophages were isolated (IC(50)=11--69 microM). The oxygen functions (-OH, -OCH(3)) of stilbenes at the benzene ring were essential for the activity. The glucoside moiety reduced the activity, while the alpha,beta-double bond had no effect. Furthermore, the active stilbenes (rhapontigenin, piceatannol, and resveratrol) did not inhibit inducible NO synthase activity, but they inhibited nuclear factor-kappa B activation following expression of inducible NO synthase.