Inhibitory effect on soybean lipoxygenase and docking studies of some secondary metabolites,isolated from Origanum vulgare L. ssp. hirtum

In this study, five secondary metabolites (caffeic acid, rosmarinic acid, lithospermic acid B, 12-hydroxyjasmonic acid 12-O-β-glucoside and p-menth-3-ene-1,2-diol 1-O-β-glucopyranoside) isolated from the polar extracts of the plant Origanum vulgare L. ssp. hirtum, were tested in vitro for their ability to inhibit soybean lipoxygenase. Among the examined compounds, lithospermic acid B demonstrated the best inhibitory activity on soybean lipoxygenase with IC₅₀ = 0.1 mM. Docking studies have been undertaken as an attempt for better understanding the interactions of these compounds within the active site of soybean lipoxygenase. The predicted binding energy values correlated well with the observed biological data.     

大紫丹参的多酚类化合物

从云南丽江产大紫丹参（ Ｓａｌｖｉａ ｐｒｚｅｗａｌｓｋｉｉ Ｍａｘｉｍ ．） 的根部分离得到１１ 个多酚类化合物, 其中８ 个鉴定为已知的原儿茶醛, 原儿茶酸, 咖啡酸, Ｒ－ （ ＋ ） － β－ Ｄ－ （３ , ４ － 二羟基苯基） － 乳酸, 迷迭香酸, 迷迭香酸甲酯, 紫草酸和紫草酸Ｂ。另外３ 个为紫草酸Ｂ的甲酯化衍生物, 即紫草酸Ｂ二甲酯, ９″－ 紫草酸Ｂ 单甲酯和９－ 紫草酸Ｂ 甲单酯。它们的结构通过波谱方法得到鉴定。研究结果表明, 大紫丹参含有与正品丹参相似的酚类化合物。     

Conditions for the formation of the oxo derivatives of arachidonic acid from platelet 12-lipoxygenase and soybean 15-lipoxygenase

Three carbonyl compounds derived from arachidonic acid have recently been characterized in human platelets, namely, 12-ketoeicosatetraenoic acid and two isomeric 12-oxododecatrienoic acids. The conditions for the synthesis of these compounds and for the synthesis of analogous products from soybean lipoxygenase, i.e., 15-ketoeicosatetraenoic acid and 15-oxopentadecatetraenoic acids, were compared with regard to the role of oxygen and fatty acid availability, and heme catalysis. Using platelet homogenates or soybean lipoxygenase and arachidonic acid as a substrate, it was found that the establishment of anaerobic conditions during the incubation was mandatory only for the synthesis of 15-oxopentadecatetraenoic acids. Anaerobic conditions, however, greatly increased the formation of 15-ketoeicosatetraenoic acid and, to a lesser extent, of 12-oxododecatrienoic acids. On the other hand, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) was transformed into 12-ketoeicosatetraenoic acid and 12-oxododecatrienoic acids by platelet homogenates or soybean lipoxygenase. This transformation was increased when the incubation was performed in anaerobic conditions and in the presence of a fatty acid substrate of the enzyme. These data suggest that oxygen deprivation and excess fatty acid could play a stimulatory role in the synthesis of 12-oxo compounds by platelets. Finally, we have compared the heme-catalyzed generation of the 12-oxo and 15-oxo derivatives from their hydroperoxide precursors: whereas 12-oxododecatrienoic acids and 12-ketoeicosatetraenoic acid were formed in the proportion of 8.5: 1.5 from 12-HPETE incubated with hematin (150 nM), 15-ketoeicosatetraenoic acid was the only carbonyl compound generated from 15-HPETE in the same conditions, emphasizing the unique reactivity of the 12-HPETE.     

Evaluation of aldose reductase inhibition and docking studies of some secondary metabolites, isolated from Origanum vulgare L. ssp. hirtum

Five polar constituents of Origanum vulgare L. ssp. hirtum were investigated for their ability to inhibit aldose reductase (ALR2), the first enzyme of the polyol pathway implicated in the secondary complications of diabetes. The most active compound was found to be lithospermic acid B. Caffeic acid was inactive as it showed no inhibitory activity against the enzyme. The order of the inhibitory activity of the remaining compounds was: rosmarinic acid >12-hydroxyjasmonic acid 12-O-beta-glucopyranoside > p-menth-3-ene-1,2-diol 1-O-beta-glucopyranoside. Docking studies have been undertaken to gain insight into the binding mode of the investigated compounds at the active site of ALR2. The predicted hydrogen bonding and hydrophobic interactions may explain the observed inhibitory activity.     

Formation of free radical metabolites in the reaction between soybean lipoxygenase and its inhibitors. An ESR study

Recent studies showed that soybean lipoxygenase inhibitors like phenidone and nordihydroguaiaretic acid (NDGA) reduce the catalytically active ferric lipoxygenase to its inactive ferrous form. Addition of 13(S)-hydroperoxy-cis-9,trans-11-octadecadienoic acid (13-HPOD) regenerated the active ferric form. In this paper, it is shown that in such a system the inhibitors are oxidized to free-radical metabolites. Incubation of soybean lipoxygenase and linoleic acid with p-aminophenol, catechol, hydroquinone, NDGA, or phenidone resulted in the formation of the one-electron oxidation products of these compounds. Free-radical formation depended upon the presence of the lipoxygenase and 13-HPOD. The free radicals were detected by ESR spectroscopy, and their structure was confirmed by analysis of the spectra, using a computer correlation technique. These data support the proposed mechanism for the inhibition of lipoxygenase by phenolic antioxidants.     

Regeneration of plantlets from cotyledon and embryonal axis explants of Vigna mungo L. Hepper

Hyssopus officinalis transformed roots were induced by infection with Agrobacterium rhizogenes. The transformed roots grew well in hormone-free Woody Plant liquid medium producing high levels of phenolic compounds such as rosmarinic acid (maximum: 8.03% of dry weight) and lithospermic acid B (maximum: 3.89% of dry weight). This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of isoquinoline alkaloids on soybean lipoxygenase activity in vitro

Novel isoquinoline alkaloids were evaluated for their effect on the kinetics of a soybean lipoxygenase type I using linoleic acid as substrate. Some of these alkaloids were found to increase the initial reaction velocity, this property seems related to phenolic groups present in the molecule. The effect of these compounds on the soybean lipoxygenase activity was compared to that of others products which are known to affect this reaction. A reaction mechanism is then proposed : it appeared, in this reaction a correlative structure-activity of phenolic compounds were tested.     

Effect of isoquinoline alkaloids on soybean lipoxygenase activity in vitro

Novel isoquinoline alkaloids were evaluated for their effect on the kinetics of a soybean lipoxygenase type I using linoleic acid as substrate. Some of these alkaloids were found to increase the initial reaction velocity, this property seems related to phenolic groups present in the molecule. The effect of these compounds on the soybean lipoxygenase activity was compared to that of others products which are known to affect this reaction. A reaction mechanism is then proposed: it appeared, in this reaction a correlative structure-activity of phenolic compounds we tested.     

Self-inactivation by 13-hydroperoxylinoleic acid and lipohydroperoxidase activity of the reticulocyte lipoxygenase

1. The self-inactivation of lipoxygenase from rabbit reticulocytes with linoleic acid at 37 degrees C is caused by the product 13-hydroperoxylinoleic acid. This inactivation is promoted by either oxygen or linoleic acid. 2. Lipohydroperoxidase activity was demonstrated with 13-hydroperoxylinoleic acid plus linoleic acid as hydrogen donor under anaerobic conditions at 2 degrees C. The products were 13-hydroxylinoleic acid, oxodienes and compounds of non-diene structure similar to those produced by soybean lipoxygenase-1. 3. 13-Hydroperoxylinoleic acid also changed the absorbance and fluorescence properties of reticulocyte lipoxygenase. The results indicate that one equivalent of 13-hydroperoxylinoleic acid converts the enzyme from the ferrous state into the ferric state as described for soybean lipoxygenase-1. The spectral changes were reversed by sodium borohydride at 2 degrees C, but not at 37 degrees C; it is assumed that the ferric form of reticulocyte lipoxygenase suffers inactivation.     

Phenolic compounds with antioxidant activity from Anthemis tinctoria L. (Asteraceae)

From the aerial parts of Anthemis tinctoria L. subsp. tinctoria var. pallida DC. (Asteraceae), one new cyclitol glucoside, conduritol F-1-O-(6'-O-E-p-caffeoyl)-beta-D-glucopyranoside (1), has been isolated together with four flavonoids, nicotiflorin (2), isoquercitrin (3), rutin (4) and patulitrin (5). The structures of the isolated compounds were established by means of NMR, MS, and UV spectral analyses. Methanolic extract and pure isolated compounds were examined for their free radical, scavenging activity, using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free stable radical, and for their inhibitory activity toward soybean lipoxygenase, using linoleic acid as substrate. Compounds 1 and 5 showed a strong scavenging effect in the DPPH radical assay. In addition 5 also exhibited high inhibitory activity on soybean lipoxygenase.     

Keto fatty acids not containing doubly allylic methylenes are lipoxygenase substrates

The soybean lipoxygenase I oxygenates the unusual substrate 12-keto-(9Z)-octadecenoic acid methyl ester as indicated by oxygen uptake and spectral changes of the incubation mixture. The main oxygenation products have been isolated by HPLC and identified as 9,12-diketo-(10E)-octadecenoic acid methyl ester and 12-keto-(10E)-dodecenoic acid methyl ester by UV and IR spectroscopy, cochromatography with an authentic standard, gas chromatography/mass spectroscopy, and 1H NMR. In the formation of both compounds the oxygenase and hydroperoxidase activities of the enzyme appear to be involved. These data and the earlier results on the oxygenation of furanoic fatty acids (Boyer et al., 1979) indicate that the lipoxygenase reaction is not restricted to substrates containing a 1,4-pentadiene structure.     

Soybean lipoxygenase-catalyzed formation of lipoxin A and lipoxin B isomers from arachidonic acid via 5,15-dihydroperoxyeicosatetraenoic acid

Soybean lipoxygenase converted arachidonic acid to a group of polar products (lambda max, 300-301 nm), which were increasingly formed during the continued incubation at 20 degrees C after the initial incubation (2 hrs, at 4 degrees C). These products were identified as lipoxin A and B isomers, based on the chromatographic and spectrometric analyses. In further chromatographic analyses, the lipoxin A and B isomers were separated into at least three isomers, respectively. The exposure of 5,15-dihydroperoxyeicosatetraenoic acid to the soybean lipoxygenase produced the identical product profile of chromatography, substantiating the intermediacy of 5,15-dihydroperoxyeicosatetraenoic acid in the soybean lipoxygenase-catalyzed formation of lipoxins. Based on these results, it is proposed that the conversion of arachidonic acid into lipoxins by soybean lipoxygenase may bear a mechanistic resemblance to the formation of lipoxins in the human leukocytes.     

New lipoxygenase-inhibiting constituents from Calligonum polygonoides

Calligonolides A (1) and B (2), two new butanolides, and a new steroidal ester, 3, have been isolated from the whole plant of Calligonum polygonoides, together with four known compounds, tetracosan-4-olide, beta-sitosterol and its glucoside, and ursolic acid. Their structures were elucidated by spectroscopic and mass-spectrometric studies. Compounds 1-3 showed moderate inhibitory potential against lipoxygenase from soybean.     

Appearance of new lipoxygenases in soybean cotyledons after germination and evidence for expression of a major new lipoxygenase gene

The appearance and subsequent disappearance of lipoxygenase activity at pH 6.8 in germinated cotyledons of soybean (Glycine max [L.]) was shown using a variant soybean cultivar (Kanto 101) that lacks the two lipoxygenase isozymes, L-2 and L-3, that are present in dry seeds of a normal soybean cultivar (Enrei). Three new lipoxygenases, designated lipoxygenase L-4, L-5, and L-6, were purified using anionic or cationic ion exchange chromatography. The major lipoxygenase in 5-day-old cotyledons of the variant soybean was lipoxygenase L-4. Lipoxygenases L-5 and L-6 preferentially produced 13(S)-hydroperoxy-9(Z), 11(E)-octadecadienoic acid (13S-HPOD) as a reaction product of linoleic acid, whereas lipoxygenase L-4 produced both 13S-HPOD and 9(S)-hydroperoxy-10(E), 12(Z)-octadecadienoic acid. All three isozymes have pH optima of 6.5, no activity at pH 9.0, and preferred linolenic acid to linoleic acid as a substrate. Partial amino acid sequencing of lipoxygenase L-4 showed that this isozyme shares amino acid sequence homology with lipoxygenases L-1, L-2, and L-3 but is not identical to any of them. This indicates that a new lipoxygenase, L-4, is expressed in cotyledons.     

Synthesis and pharmacochemical evaluation of novel aryl-acetic acid inhibitors of lipoxygenase, antioxidants, and anti-inflammatory agents

Lipoxygenase catalyzes the first two steps of the transformation of arachidonic acid into leukotrienes which are implicated in host defense reactions. It is well known that many acids possess potent anti-inflammatory activity. Taking into account that compounds bearing a thienyl, naphthyl, pyrollyl, and 2,4-di-tert-butyl-phenol moieties possess anti-inflammatory activity which is related to their capacity to transfer electrons and to scavenge reactive oxygen species, we synthesized some new aryl-acetic acids and we explored their ability to inhibit soybean lipoxygenase, to present antioxidant and anti-inflammatory activities, and to interact with glutathione. The compounds have shown important antioxidant activity, medium anti-inflammatory activity, and very good inhibition of soybean lipoxygenase. Compound 3-(3,5-di-tert-butyl-2-hydroxy-phenyl)-2-phenyl-acrylic acid (1i) showed significant in vitro LO inhibition (IC(50) 65 microM). The results are discussed in terms of structural and physicochemical characteristics of the compounds. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity are experimentally determined by RPTLC method.     

New aspects of the inhibition of soybean lipoxygenase by alpha-tocopherol. Evidence for the existence of a specific complex

The formation of alpha-tocopherol--lipoxygenase complex was elucidated using immobilized affinity purified soybean lipoxygenase and [D-3H]alpha-tocopherol. The alpha-tocopherol--lipoxygenase complex did not dissociate on addition of linoleic acid. Iodoacetate modified immobilized lipoxygenase did not form the complex with alpha-tocopherol. Lipoxygenase attached to an aminoethyl linoleyl Sepharose column was eluted by alpha-tocopherol. DL-alpha-Tocopherol acetate at a concentration of 3 X 10(-3) M inhibited 80% of linoleate oxidation by soybean lipoxygenase. The lipoxygenase--alpha-tocopherol complex did not give the usual soybean lipoxygenase antigenic pattern in immunodiffusion. Digestion of the [3H]alpha-tocopherol--lipoxygenase complex with proteolytic enzymes showed that most of the radioactivity is incorporated into one peptide.     

The possible role of 9(S)-hydroperoxyoctadecatrienoic acid as a suicide substrate of soybean lipoxygenase

While incubation of soybean lipoxygenase with alpha-linolenic acid resulted in the gradual decrease of lipoxygenase activity, the incubation with linoleic acid had no change. The inactivation of soybean lipoxygenase during incubation with alpha-linolenic acid was markedly observed at pH 6.5, but not at pH 9.0. Among the lipoxygenation products of alpha-linolenic acid, only 9(S)-hydroperoxyoctadecatrienoic acid caused the inactivation of lipoxygenase. 9(S)-Hydroxyoctadecatrienoic acid, 13(S)-hydroperoxyoctadecatrienoic acid or 9,16-dihydroperoxy conjugated trienoic acid was without effect. Accordingly, it is suggested that the epoxide intermediate, one conversion product of 9(S)-hydroperoxyoctadecatrienoic acid, might be involved in the direct inactivation of lipoxygenase.     

Design, synthesis, and discovery of stilbene derivatives based on lithospermic acid B as potent protein tyrosine phosphatase 1B inhibitors

Dihydroxy stilbene derivatives were designed based on lithospermic acid B and were prepared from 4-(chloromethyl)benzoic acid. The inhibitory activities of the novel compounds against protein tyrosine phosphatase 1B (PTP1B) were evaluated. 3,4-Dihydroxy stilbene carbonyl compounds (7, 11b, 27b) inhibited PTP1B with IC50 values comparable to molybdate, while the conjugation-extended compound (15b) showed inhibition 3-fold better than preclinical RK682. The introduction of electron withdrawing groups or amides into the second phenyl ring, or extension of the conjugation into the stilbene molecule may increase stability of the generated radicals.     

Substituted furans as potent lipoxygenase inhibitors: Synthesis,in vitro and molecular docking studies

A number of 2-methyl-4-(2-oxo-2-phenyl-ethyl)-5-phenyl-furan-3-carboxylic acid alkyl ester derivatives (3a–j) were synthesized and evaluated for their in vitro inhibitory activity on soybean lipoxygenase enzyme. Among the screened compounds, 5-(4-bromo-phenyl)-4-[2-(4-bromo-phenyl)-2-oxo-ethyl]-2-methyl-furan-3-carboxylic acid methyl ester (3g) has been found to exhibit potent inhibitory activity with IC₅₀12.8 μM using nordihydroguaiaretic acid (NDGA) as standard. Molecular modeling was employed for better understanding of the binding between compounds and soybean lipoxygenase enzyme. The predicted binding energy values correlated well with the observed in vitro data.