New hybrid molecules combining benzothiophene or benzofuran with rhodanine as dual COX-1/2 and 5-LOX inhibitors: Synthesis,biological evaluation and docking study

New molecular hybrids combining benzothiophene or its bioisostere benzofuran with rhodanine were synthesized as potential dual COX-2/5-LOX inhibitors. The benzothiophene or benzofuran scaffold was linked at position -2 with rhodanine which was further linked to various anti-inflammatory pharmacophores so as to investigate the effect of such molecular variation on the anti-inflammatory activity. The target compounds were evaluated for their in vitro COX/LOX inhibitory activity. The results revealed that, compound 5h exhibited significant COX-2 inhibition higher than celecoxib. Furthermore, compounds 5a, 5f and 5i showed COX-2 inhibitory activity comparable to celecoxib. Compound 5h showed selectivity index SI = 5.1 which was near to that of celecoxib (SI = 6.7). Compound 5h displayed LOX inhibitory activity twice than that of meclofenamate sodium. Moreover, compounds 5a, 5e and 5f showed significant LOX inhibitory activity higher than that of meclofenamate sodium. Compound 5h was screened for its in vivo anti-inflammatory activity using formalin-induced paw edema and gastric ulcerogenic activity tests. The results revealed that, it showed in vivo decrease in formalin-induced paw edema volume higher than celecoxib. It also displayed gastrointestinal safety profile as celecoxib. The biological results were also consistent with the docking studies at the active sites of the target enzymes COX-2 and 5-LOX. Also, compound 5h showed physicochemical, ADMET, and drug-like properties within those considered adequate for a drug candidate.     

Dual inhibitory effect of Glycyrrhiza glabra (GutGard™) on COX and LOX products

Glycyrrhiza glabra and its phytoconstituents have been known to possess widespread pharmacological properties as an anti-inflammatory, anti-viral, antitumour and hepatoprotective drug. In this study, we examined the inhibitory potential of extract of G. glabra (GutGard™) root and its phytoconstituents (glabridin, glycyrrhizin, and isoliquiritigenin) on both cyclooxygenase (COX) and lipoxygenase (LOX) products in order to understand the mechanism of its anti-inflammatory action. Inhibitory effect of GutGard™ and its phytoconstituents on lipopolysaccharide (LPS) induced prostaglandin E₂ (PGE₂), calcimycin (A23187) induced thromboxane (TXB₂), and leukotriene (LTB₄) release was studied using murine macrophages (J774A.1) and human neutrophil (HL-60) cells. Results revealed that, G. glabra and glabridin significantly inhibited PGE₂, TXB₂ (COX) and LTB₄ (LOX), while, isoliquiritigenin exerted inhibitory effect only against COX products but failed to suppress LOX product. However, glycyrrhizin at the tested concentrations failed to exhibit inhibitory effect on both COX and LOX products. Here, we report for the first time that G. glabra (almost devoid of glycyrrhizin) exhibits anti-inflammatory property likely through the inhibition of PGE₂, TXB₂ and LTB₄ in mammalian cell assay system, which could be influenced in part by glabridin and isoliquiritigenin.     

n‐Octyl Gallate as Inhibitor of Pyruvate Carboxylation and Lactate Gluconeogenesis

The alkyl gallates are found in several natural and industrial products. In the latter products, these compounds are added mainly for preventing oxidation. In the present work, the potencies of methyl gallate, n‐propyl gallate, n‐pentyl gallate, and n‐octyl gallate as inhibitors of pyruvate carboxylation and lactate gluconeogenesis were evaluated. Experiments were done with isolated mitochondria and the isolated perfused rat liver. The potency of the gallic acid esters as inhibitors of pyruvate carboxylation in isolated mitochondria obeyed the following decreasing sequence: n‐octyl gallate > n‐pentyl gallate > n‐propyl gallate > methyl gallate. A similar sequence of decreasing potency for lactate gluconeogenesis inhibition in the perfused liver was found in terms of the portal venous concentration. Both actions correlate with the lipophilicity of the compounds. The effects are harmful at high concentrations. At appropriate concentrations, however, octyl gallate should act therapeutically because its inhibitory action on gluconeogenesis will contribute further to its proposed antihyperglycemic effects.     

Lipoxygenase is an abundant protein in cucumber exudates

The presence of lipoxygenase (LOX) has been reported in many plant organs. High LOX activity (1–2 katal/mg protein) was detected in exudates from cut cucumber (Cucumis sativus L.) stems and petioles. Exudate LOX had a pH optimum of 5.0, an estimated molecular weight of 95 kDa and cross-reacted on sodium-dodecyl-sulfate gels with anti-LOX antibodies raised against soybean leaf LOX isoenzymes. Lipoxygenase activity was detected on native gels stained with o-dianisidine using linoleic acid as a substrate. Enzyme activity was similar with linoleic and linolenic acid and 2 times greater with arachidonic acid as substrate. At pH 6.8, LOX metabolized linoleic acid into 13- and 9-hydroperoxides at a ratio of 12. Linolenic acid was preferentially oxidized at carbon 13. Lipoxygenase activity was inhibited by n-propyl gallate (IC₅₀ 300 nM) and nordihydroguaiaretic acid (IC₅₀ 25 nM), but not by nonsteroidal anti-inflammatory drugs. LOX activity was enhanced 4.5-fold by 300 mM Ca²⁺. Spermine at 1 mM, and putrescine and spermidine at 2 mM completely inhibited LOX activity, but at low concentrations spermine (100 mM) and spermidine (100–500 mM) significantly stimulated LOX activity: 8- and 4.5-fold, respectively. Tissue printing of stem, petiole and hypocotyl sections with subsequent incubation with the antiserum raised against soybean leaf LOX revealed the presence of LOX in the internal and external phloem and in the sieve tubes.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - 9(S)-HpOD 9-(S)-hydroperoxy-(E,Z)10,12-octadecadienoic acid - 13(S)-HpOD 13-(S)-hydroperoxy(Z,E)-9,11-octadecadienoic acid - IC inhibition constant - IEF isoelectrofocusing - LOX lipoxygenase - NDGA nordihydroguaiaretic acid - PBS phosphate buffered saline - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate We would like to thank Ulla Jarlfors for exellent technical assistance with the histological analysis. The research reported in this paper was supported in part by grants to J.K. from the R.J. Reynolds Tobacco Company and Cooperative Agreement 43YK-5-0030 of the USDA-ARS. Journal paper 93-11-12 of the Kentucky Agricultural Experiment Station, Lexington.     

In vitro,fluorescence-quenching and computational studies on the interaction between lipoxygenase and 5-hydroxy-3′,4′,7-trimethoxyflavone from Lippia nodiflora L.

Abstract

Lippia nodiflora L. is extensively used in traditional medicine for several medicinal purposes, including their use in inflammatory disorders. In this study, the folk use of L. nodiflora was validated using the isolated natural compound, 5-hydroxy-3′,4′,7-trimethoxyflavone (HTMF) by in vitro, fluorescence spectroscopic and molecular modeling studies with lipoxygenase (LOX), because LOX plays an essential role in inflammatory responses. In this perspective, the methanol extract and HTMF are shown to demonstrate prominent inhibitory activity against soybean lipoxygenase, with an IC₅₀ value of 21.12 and 23.97?µg/ml, respectively. The data obtained from the spectroscopic method revealed that the quenching of intrinsic fluorescence of LOX is produced as a result of the complex formation of LOX–HTMF. The binding mode analysis of HTMF within the LOX enzyme suggested that hydrogen bond formation, hydrophobic interaction and π–π stacking could account for the binding of HTMF. Molecular dynamics results indicated the interaction of HTMF with LOX and the stability of ligand–enzyme complex was maintained throughout the simulation. The computational results are reliable with experimental facts and provided a good representation for understanding the binding mode of HTMF inside the active site of lipoxygenase enzyme.     

Inhibitory effects of brown algal phlorotannins on secretory phospholipase A2s, lipoxygenases and cyclooxygenases

The inhibitory effects of brown algal phlorotannins on secretory phospholipase A₂s (sPLA₂s), lipoxygenases (LOXs) and cyclooxygenases (COXs) were determined with an in vitro assay. Oligomers of phloroglucinol; eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol (a hexamer) and 8,8-bieckol (a hexamer) isolated from the brown alga Eisenia bicyclis had pronounced inhibitory effects on sPLA₂ from porcine pancreas and bee venom (IC₅₀ 100–200 M). The phlorotannins inhibited LOX activity more effectively than the well-known LOX inhibitors; resveratrol and epigallocatechin gallate. 8,8-Bieckol, the strongest LOX inhibitor in this study, inhibited soybean LOX and 5-LOX with IC₅₀ values of 38 and 24 M, respectively. Negligible or very weak effects of the phlorotannins on COX-1 and COX-2 were found, except for an inhibitory effect of dieckol on COX-1 (74.7%) and of eckol on COX-2 (43.2%) at 100 M.     

Methyl gallate and its 3-glucoside promote rooting in bean cuttings

Methyl gallate stimulated adventitious root formation in cuttings of bean (Phaseolus vulgaris L.). This polyphenol was quickly metabolized into 3-glucosyl methyl gallate to such an extent that 4 h after application no methyl gallate was detected. The isolated glucoside when supplied exogenously at 0.5 mM also enhanced rooting; the effect was 2-fold greater than that of methyl gallate. The glucoside persisted in the cuttings for 72 h after treatment. Because methyl gallate is rapidly transformed to a stable glucoside, we suggest that the root stimulation effect could be ascribed to its glucoside.     

Kukoamine A analogs with lipoxygenase inhibitory activity

Kukoamine A (KukA) is a spermine (SPM) conjugate with dihydrocaffeic acid (DHCA), with interesting biological activities. The four possible regioisomers of KukA, as well as a series of KukA analogs incorporating changes in either the SPM or the DHCA structural units, were evaluated for their antioxidant activity and their inhibitory activity on soybean lipoxygenase (LOX) and lipid peroxidation. The reducing properties of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and found to be in the range 5–97.5%. KukA significantly inhibits LOX with IC₅₀ 9.5?μM. All tested analogs inhibited lipid peroxidation in the range of 11–100%. The most potent compounds KukA and its analog 3, in which the DHCA units had been replaced by O,O9-dimethylcaffeic acid units, were studied for their anti-inflammatory activity in vivo on rat paw edema induced by carrageenan and found to be of comparable activity to indomethacin. The results of the biological tests are discussed in terms of structural characteristics.     

Pharmacological evaluation and docking studies of α,β-unsaturated carbonyl based synthetic compounds as inhibitors of secretory phospholipase A2, cyclooxygenases,lipoxygenase and proinflammatory cytokines

Arachidonic acid and its metabolites have generated high level of interest among researchers due to their vital role in inflammation. The inhibition of enzymes involved in arachidonic acid metabolism has been considered as synergistic anti-inflammatory effect. A series of novel α,β-unsaturated carbonyl based compounds were synthesized and evaluated for their inhibitory activity on secretory phospholipase A₂ (sPLA₂), cyclooxygenases (COX), soybean lipoxygenase (LOX) in addition to proinflammatory cytokines comprising IL-6 and TNF-α. Six α,β-unsaturated carbonyl based compounds (2, 3, 4, 12, 13 and 14) exhibited strong inhibition of sPLA₂ activity, with IC₅₀ values in the range of 2.19–8.76 μM. Nine compounds 1–4 and 10–14 displayed inhibition of COX-1 with IC₅₀ values ranging from 0.37 to 1.77 μM (lower than that of reference compound), whereas compounds 2, 10, 13 and 14 strongly inhibited the COX-2. The compounds 10–14 exhibited strong inhibitory activity against LOX enzyme. All compounds were evaluated for the inhibitory activities against LPS-induced TNF-α and IL-6 release in the macrophages. On the basis of screening results, five active compounds 3, 4, 12, 13 and 14 were found strong inhibitors of TNF-α and IL-6 release in a dose-dependent manner. Molecular docking experiments were performed to clarify the molecular aspects of the observed COX and LOX inhibitory activities of the investigated compounds. Present findings increases the possibility that these α,β-unsaturated carbonyl based compounds might serve as beneficial starting point for the design and development of improved anti-inflammatory agents.     

Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity

Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down‐regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9‐hydroperoxyoctadecadienoic acid (9‐HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild‐type seeds by measuring the ultraviolet absorption of 9‐HPOD at 234 nm and by high‐performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS‐91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production.     

Trioxsalen derivatives with lipoxygenase inhibitory activity

Trioxsalen (TRX) is a 4,5′,8-trimethylated psoralen analog presenting interesting biological activities when irradiated with UVA light. A series of TRX derivatives, which where obtained by its chemical modification and incorporation of a variety of unsaturated functions at position 4′ of the psoralen ring-system, were evaluated for their antioxidant activity and their inhibitory activity on soybean lipoxygenase (LOX) and lipid peroxidation. The reducing properties of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assay and found to be very low, in the range 0–14%, with the exception of the hydroxamic acid 6 which showed almost identical activity to BHT. TRX derivative 3 significantly inhibited LOX, with IC₅₀ 9.4?μM. With the exception of TRX, all tested analogs inhibited lipid peroxidation in the range of 35–91%. The most potent compound, namely TRX derivative 3, was studied for its anti-inflammatory activity in vivo on rat paw edema induced by carrageenan, and was found to be of almost identical activity to indomethacin. The results of the biological tests are discussed in terms of structural characteristics.     

Induction of pumpkin glutathione S-transferases by different stresses and its possible mechanisms

Induction of pumpkin (Cucurbita maxima Duch.) glutathione S-transferases (GSTs) by different stresses and endogenous trans-2-hexenal content were determined in search of a common signal for GST induction. All of the stresses showed significant induction, As₂O₃ causing the highest induction followed by trans-2-hexenal. The trans-2-hexenal content was highest in trans-2-hexenal-treated seedlings and next-highest in methyl jasmonate-treated seedlings, whereas high temperature- and As₂O₃-treated seedlings had trans-2-hexenal contents lower than that of control seedlings. Induction of GST, lipoxygenase (LOX) and hydroperoxide lyase (HPL) was compared, since trans-2-hexenal and methyl jasmonate are the products of the LOX pathway. All four stresses showed weak LOX induction, high temperature causing the highest induction. However, only methyl jasmonate caused weak HPL induction. Both antioxidants or oxidants induced GST to different degrees. Glutathione contents of reduced glutathione (GSH) or oxidized glutathione (GSSG)-treated seedlings were significantly higher than the content of control seedlings, whereas those treated with other antioxidants or oxidants had contents similar to or less than control seedlings. The GSH:GSSG ratio was lowest in GSSG-treated seedlings and next-lowest in GSH-treated seedlings. The results of this study suggest that pumpkin GSTs are not induced through a common signalling pathway and that redox perturbation plays a role in pumpkin GST induction.     

Synthesis and biological properties of aryl methyl sulfones

A novel group of aryl methyl sulfones based on nonsteroidal anti-inflammatory compounds exhibiting a methyl sulfone instead of the acetic or propionic acid group was designed, synthesized and evaluated in vitro for inhibition against the human cyclooxygenase of COX-1 and COX-2 isoenzymes and in vivo for anti-inflammatory activity using the carrageenan induced rat paw edema model in rats. Also, in vitro chemosensitivity and in vivo analgesic and intestinal side effects were determined for defining the therapeutic and safety profile. Molecular modeling assisted the design of compounds and the interpretation of the experimental results. Biological assay results showed that methyl sulfone compounds 2 and 7 were the most potent COX inhibitors of this series and best than the corresponding carboxylic acids (methyl sulfone 2: IC₅₀ COX-1?=?0.04 and COX-2?=?0.10?μM, and naproxen: IC₅₀ COX-1?=?11.3 and COX-2?=?3.36?μM). Interestingly, the inhibitory activity of compound 2 represents a significant improvement compared to that of the parent carboxylic compound, naproxen. Further support to the results were gained by the docking studies which suggested the ability of compound 2 and 7 to bind into COX enzyme with low binding free energies.The improvement of the activity of some sulfones compared to the carboxylic analogues would be performed through a change of the binding mode or mechanism compared to the standard binding mode displayed by ibuprofen, as disclosed by molecular modeling studies. So, this study paves the way for further attention in investigating the participation of these new compounds in the pain inhibitory mechanisms. The most promising compounds 2 and 7 possess a therapeutical profile that enables their chemical scaffolds to be utilized for development of new NSAIDs.     

核桃青皮的化学成分研究——酚类化合物

核桃青皮是一种传统的中药材,含有大量的酚类化合物,具有镇痛、消炎、抑菌、抗肿瘤等功效。为了从核桃青皮中分离得到更多的酚类成分,以利于更好地阐明其作用机理,该研究采用大孔树脂Diaion HP-20SS、凝胶Sephadex LH-20,HPLC等方法对核桃青皮80%的乙醇提取物进行分离纯化。结果表明:共分离了10个单体化合物,它们的结构经质谱(MS)、一维核磁共振谱(~1H NMR和~(13)C NMR)、二维核磁共振谱(HSQC,HMBC)数据的分析及文献数据的比较确定为没食子酸(1),没食子酸甲酯(2),对羟基苯甲酸(3),3,4-二羟基苯甲酸甲酯(4),6-O-咖啡酸-D-葡萄糖(5),6-O-没食子酸-葡萄糖苷(6),4,8-二羟基-1-四氢萘醌(7),5,8-二羟基-4-甲氧基-1-四氢萘酮(8),5,8-二羟基-1-四氢萘酮(9),4-羟基-1-四氢萘酮(10)。其中,化合物5,化合物6为属内首次分离到。该研究结果为进一步深入研究核桃青皮的化学成分和药理作用提供了一定参考。     

Synthesis, in silico docking experiments of new 2-pyrrolidinone derivatives and study of their anti-inflammatory activity

A new class of 2-pyrrolidinone derivatives was designed, synthesized, and tested for their antioxidant and anti-inflammatory activities. The compounds were evaluated for their inhibitory activity against LOX. The most potent among them, 14d [IC(50) 0.08 (±0.005)mM], and 14e [IC(50) 0.0705 (±0.003)mM], were also tested in vivo. The compound 14d induced equipotent inhibition against rat paw edema, which is very close to the effect produced by the commonly used standard, namely indomethacin (47%). The LOX inhibitory activity of the compound 14e proceeds in parallel to the % inhibitory value of lipid peroxidation meaning that this LOX inhibitory activity is supported by the lipid peroxidation inhibition. The molecular features that govern their bioactivity were explored through in silico docking experiments. The results showed that acidic moieties must be placed in certain distance and orientation in the active site of LOX enzyme in order to productively exhibit inhibitory activity. In addition, the 2-pyrrolidinone template significantly contributes in the inhibitory properties of the new compounds.     

Biochemical properties of lipoxygenase from opium poppy chloroplasts

Lipoxygenase (LOX) from opium poppy (Papaver somniferum L.) chloroplasts was isolated and 126.1-fold purified to electrophoretic homogeneity by combination of ion-exchange chromatography on HA-Ultragel column and affinity chromatography on a linoleyl-aminopropyl agarose column. The relative molecular mass of the LOX determined by SDS-PAGE was 92 kDa. Kinetic properties of purified LOX were determined in spectrophotometric assay by using of linoleic acid (K_M = 1.78 mM and V_max = 11.4 μmol mg⁻¹ min⁻¹) and linolenic acid (K_M = 1.27 mM and V_max = 10.2 μmol mg⁻¹ min⁻¹). The optimum pH was 6.0 for both linoleic and linolenic acid dioxygenation catalyzed by LOX. HPLC analysis of the products revealed a dual positional specificity of linoleic acid dioxygenation at pH 6.0 with ratio of 9- and 13-hydroperoxide products being about 1:1. The activity of purified LOX was stimulated by Mg²⁺ and Ca²⁺.     

Expression of cucumber lipid-body lipoxygenase in transgenic tobacco: lipid-body lipoxygenase is correctly targeted to seed lipid bodies

 A particular isoform of lipoxygenase (LOX, EC 1.13.11.12) localized on lipid bodies has been shown by earlier investigations to play a role during seed germination in initiating the mobilization of triacylglycerols. On lipid bodies of germinating cucumber (Cucumis sativus L.) seedlings, the modification of linoleoyl moieties by this LOX precedes the hydrolysis of the ester bonds. We analyzed the expression and intracellular location of this particular LOX form in leaves and seeds of tobacco (Nicotiana tabacum L.) transformed with one construct coding for cucumber lipid-body LOX and one construct coding for cucumber LOX fused with a hemagglutinin epitope. In both tissues, the amount of lipid-body LOX was clearly detectable. Biochemical analysis revealed that in mature seeds the foreign LOX was targeted to lipid bodies, and the preferred location of the LOX on lipid bodies was verified by immunofluorescence microscopy. Cells of the endosperm and of the embryo exhibited fluorescence based on the immunodecoration of LOX protein whereas very weak fluorescent label was visible in seeds of untransformed control plants. Further cytochemical analysis of transformed plants showed that the LOX protein accumulated in the cytoplasm when green leaves lacking lipid bodies were analyzed. Increased LOX activity was shown in young leaves of transformed plants by an increase in the amounts of endogenous (2E)-hexenal and jasmonic acid. Received: 9 August 1999 / Accepted: 28 September 1999