旋覆花提取物对朱砂叶螨的生物活性及酶活性的影响

本文研究了旋覆花石油醚提取物对朱砂叶螨的毒力作用及其对相关酶活性的影响。结果表明：旋覆花石油醚提取物的杀螨活性较高,浓度为2mg?mL-1 时,校正死亡率达到92.05％。通过对旋覆花石油醚提取物进一步萃取、柱层析分离、薄层层析,发现最终得到的38个流分中,杀螨效果较好的是流分7,其校正死亡率为85.53%。流分7经GC/MS鉴定为羽扇豆醇,其校正死亡率达到66.46%。进一步测定羽扇豆醇对朱砂叶螨谷胱甘肽-S-转移酶、Ca2 -ATPase、过氧化物歧化酶的活性以及总蛋白含量的影响,结果表明经羽扇豆醇处理后,螨体内过氧化物歧化酶被激活,而谷胱甘肽-S-转移酶和Ca2 -ATPase均受到不同程度的抑制,蛋白总量在这个过程中没有明显的变化。上述结果表明旋覆花提取物羽扇豆醇可以有效地杀死朱砂叶螨,这为旋覆花作为新型植物源农药提供一定的理论依据。     

Dietary Effects of Eicosapentaenoic and Docosahexaenoic Acid Esters on Lipid Metabolism and Immune Parameters in Sprague-Dawley Rats

Sprague-Dawley rats were fed eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) ethyl esters at the 2% level for 3 weeks to clarify their effects on immune functions. In the rats fed EPA or DHA, serum cholesterol, triglyceride, and phospholipid (PL) levels were significantly lower than those in the rats fed safflower oil. In PL fractions of serum, liver, lung, splenocytes, and peritoneal exudate cells (PEC), increases in linoleic and dihomo-γ-linolenic acid contents and a decrease in arachidonic acid (AA) content were observed in the rats fed EPA or DHA. In addition, the EPA content increased in the rats fed EPA and DHA. In the rats fed EPA or DHA, a decrease of LTB₄ productivity and an increase of LTB₅ productivity were observed in the PEC, in response to the treatment with 5 μM calcium ionophore A23187 for 20 min. The changes in leukotriene production were more marked in EPA-fed rats than in DHA-fed rats. These results suggest that dietary EPA affects lipid metabolism and leukotriene synthesis more strongly than DHA.     

A validated liquid chromatography-mass spectrometry method for the determination of leukotrienes B4 and B5 produced by stimulated human polymorphonuclear leukocytes

A validated high-performance liquid chromatography (HPLC)-mass spectrometry method has been developed for the simultaneous assay of leukotrienes (LTs) B4 and B5, derived from omega-6 arachidonic acid and omega-3 polyunsaturated fatty acids (PUFA), respectively, produced by human polymorphonuclear leukocytes (PMNLs) stimulated with calcium ionophore A23187. The HPLC separation of PMNL ether extracts was performed on a reversed-phase column using a gradient elution program of 15 mM ammonium acetate and MeOH. Detection was performed by electrospray ionization-single quadripole mass spectrometry using single ion reaction monitoring in the negative mode at m/z 333.3 [M-H](-) and m/z 335.2 for prostaglandin B2/LTB5 and LTB4, respectively. The calibration curves for LTB4 and LTB5 were linear over the ranges 165-990 and 0.825-13.2 ng/ml, respectively. The lower limit of quantification for LTB5 was 0.66 ng/ml. The mean absolute recoveries for LTB4 and LTB5 were 81+/-4.8% and 82+/-5.9%, respectively. The method is precise with mean interday CVs for LTB4 and LTB5 within 7.1-10.7, and 3.8-9.4%, respectively, and accurate (range of interday deviations for LTB4 and LTB5 were -7.8 to 1, and -5 to 9% , respectively). The method has been validated and is being applied to the simultaneous quantification of the leukotrienes B4 and B5 in stimulated PMNLs in a clinical protocol studying the influence of a diet enriched in omega-3 PUFA on various surrogate markers of inflammation in young cystic fibrosis patients.     

Crotalic and emarginellic acids: two triterpenes from Crotalaria emarginella and anti-inflammatory and anti-hepatotoxic activity of crotalic acid

The aerial parts of Crotalaria emarginella Vatke (Leguminosae) has afforded two triterpenes, characterized as 3alpha-hydroxy-arbor-12-ene-28-carboxylic acid, designated as crotalic acid (1), and 2beta,3beta,21-trihydroxy-arbor-12-ene-29-carboxylic acid, designated as emarginellic acid (2). The structures of the isolated products were elucidated on the basis of spectral and chemical studies. On screening the biological activity, the crotalic acid (1) exhibited a significant anti-inflammatory activity (dose: 10mg/kg), which showed 53% inhibitory effect. Whereas, the standard oxyphenyl butazone (100mg/kg) exhibited 69% inhibition with respect to carrageenan (0.05ml, 1%) used to cause inflammation in rat paw method. In addition, it also showed anti-hepatotoxic activity by 13-30% with respect to standard silybon-70 (35-57%) against CCl(4) induced toxicity in Wistar rats.     

Crystal structure of the complex formed between a group I phospholipase A2 and a naturally occurring fatty acid at 2.7 A resolution

This is the first evidence of a naturally bound fatty acid to a group I Phospholipase A(2) (PLA(2)) and also to a PLA(2) with Asp 49. The fatty acid identified as n-tridecanoic acid is observed at the substrate recognition site of PLA(2) hydrophobic channel. The complex was isolated from the venom of Bungarus caeruleus (Common Indian Krait). The primary sequence of the PLA(2) was determined using the cDNA method. Three-dimensional structure has been solved by the molecular replacement method and refined using the CNS package to a final R factor of 19.8% for the data in the resolution range from 20.0 to 2.7 A. The final refined model is comprised of 912 protein atoms, one sodium ion, one molecule of n-tridecanoic acid, and 60 water molecules. The sodium ion is located in the calcium-binding loop with a sevenfold coordination. A characteristic extra electron density was observed in the hydrophobic channel of the enzyme, into which a molecule of n-tridecanoic acid was clearly fitted. The MALDI-TOF measurements of the crystals had earlier indicated an increase in the molecular mass of PLA(2) by 212 Da over the native PLA(2). A major part of the ligand fits well in the binding pocket and interacts directly with His 48 and Asp 49. Although the overall structure of PLA(2) in the present complex is similar to the native structure reported earlier, it differs significantly in the folding of its calcium-binding loop.     

Anti-inflammatory effects of a triterpenoid isolated from Wilbrandia ebracteata Cogn

Wilbrandia ebracteata (WE), a Brazilian medicinal plant used in folk medicine for the treatment of rheumatic diseases, displays anti-inflammatory properties and constitutes a rich source of cucurbitacins and cucurbitacin-related compounds. The current study investigated the potential anti-inflammatory properties of Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound isolated from roots of WE, in some in vivo and in vitro experimental models. Intraperitoneal treatment of mice with DHCB reduced both carrageenan-induced paw edema (0.3, 1 and 3 mg/kg caused inhibitions of 26, 44 and 56 % at 2 h after stimulation, respectively) and pleurisy (10 mg/kg inhibited leukocyte numbers and LTB(4) levels in the pleural fluid by 51 and 75% at 6 h after cavity challenge, respectively). In vitro, DHCB (up to 10 microg/mL) failed to modify LTB(4) production by human neutrophils or PGE(2) production by COS-7 cells transfected with COX-1, but PGE(2) production by COX-2 transfected COS-7 cells was markedly inhibited (by 72%). The levels of COX-1 or COX-2 proteins in IL-1alpha-stimulated NIH3T3 cells were unaffected by DHCB. The results corroborate the potential anti-inflammatory properties ascribed to W. ebracteata Cogn. in folk medicine and suggest that they might be attributed, at least in part, to the capacity of one of this plants main constituents, DHCB, to inhibit COX-2 activity (but not its expression) during inflammation.     

Characteristics of arachidonic acid metabolism of human endothelial cells in culture

Human umbilical endothelial cells in culture retain differentiated morphological and functional characterization in primary culture and even in the early subcultures, after which they begin to degenerate. We have studied the morphological and biochemical characterization (ability to produce prostacyclin, prostaglandin E₂ and thromboxane A₂ in culture) of endothelial cells in the first seven subcultures. In addition the influence of serum and endothelial cell growth factor added to the culture medium have been evaluated. With 20% normal human serum, cell proliferation is faster than with the same concentration of human fetal or bovine fetal serum.After the 3rd passage, morphological and growth alterations become observable in the endothelial cells. However, prostacyclin, prostaglandin E₂ and thromboxane A₂ production showed no variations during the study.     

Inhibitory effect on NO production of phenolic compounds from Myristica fragrans

Three new phenolics: ((7S)-8'-(benzo[3',4']dioxol-1'-yl)-7-hydroxypropyl)benzene-2,4-diol (1), ((7S)-8'-(4'-hydroxy-3'-methoxyphenyl)-7-hydroxypropyl)benzene-2,4-diol (2) and ((8R,8'S)-7-(4-hydroxy-3-methoxyphenyl)-8'-methylbutan-8-yl)-3'-methoxybenzene-4',5'-diol (3), along with four known compounds (4-7) were isolated from the seeds of Myristica fragrans. Their chemical structures were established mainly by 1D and 2D NMR techniques and mass spectrometry. Their anti-inflammatory activity was evaluated against LPS-induced NO production in macrophage RAW264.7 cells.     

Reduction of vinyl groups in naturally occurring chlorophylls-a

3,8-Divinyl-chlorophyll(Chl)-a possessing a phytyl ester was hydrogenated in acetone by rhodium catalyst on alumina to afford 3-vinyl-8-ethyl-, 3-ethyl-8-vinyl- and 3,8-diethyl-Chls. The ratio of produced 3-ethyl-8-vinyl- over 3-vinyl-8-ethyl-Chls was determined to be 1.2, indicating that the reactivity of the 3-vinyl group was slightly higher than that of the 8-vinyl group. Catalytic hydrogenation of divinyl-protochlorophyll-a possessing a porphyrin ??-skeleton (C17C18) instead of the above chlorin moiety (C17H-C18H) gave an equal amount of mono-reduced regioisomers. The slight (or no) selectivity is different from that in the enzymatic reduction of divinyl-(proto)chlorophyllides-a lacking a phytyl ester in the biosynthetic pathway of Chl-a where the sole 8-vinyl group is transformed to the ethyl group.     

Enzymatic hydrolysis and extraction of arachidonic acid rich lipids from Mortierella alpina

A novel method for efficient arachidonic acid rich lipids extraction was investigated. Six different enzymes (papain, pectinase, snailase, neutrase, alcalase and cellulase) were used to extract lipids from Mortierella alpina. The effects of enzyme concentration, temperature and hydrolysis time on oil recovery were evaluated using factorial experimental design and polynomial regression for each enzyme. Hydrolysis time is found to be the most important parameter for all enzymes. The ratios of enzyme mixtures were also studied. It showed that the mixtures of pectinase and papain (5:3, v/v), pectinase and alcalase (5:1, v/v) were better combined effects on oil yields. The effects of hydrolysis time and temperature were then analyzed by response surface methodology, and oil recoveries were satisfactory (104.6% for pectinase and papain and 101.3% for pectinase and alcalase). In the whole process, the lipid composition was not affected by the enzyme treatments according to fatty acid profile.     

New vernocuminosides from the stem barks of Vernonia cumingiana Benth

Seven new stigmastane-type steroidal glycosides, vernocuminosides A-G (1-7), have been isolated from the stem barks of Vernonia cumingiana Benth. The structural elucidation and stereochemistry determination were achieved by spectroscopic and chemical methods including 1D and 2D NMR ((1)H-(1)H COSY, HSQC, HMBC, and NOE) experiments, especially the employment of Snatzke's method expressed by the induced circular dichroism spectra. Anti-inflammatory activities and cytotoxicities of compounds 1-7 were evaluated.     

Characterization of enzymes involved in the interconversions of different forms of vitamin B6 in tobacco leaves

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP) and pyridoxine 5′-phosphate (PNP). PLP is a coenzyme required by more than 100 cellular enzymes. In spite of the importance of this vitamin, the understanding of VB₆ metabolic conversion in plants is limited. In this study, we developed a sensitive and reliable method to assay VB₆-metabolizing enzyme activities by monitoring their products visually using high-performance liquid chromatography. With this method, the reactions catalyzed by PL/PM/PN kinase, PMP/PNP oxidase, PM-pyruvate aminotransferase, PL reductase and PLP phosphatase were all nicely detected using crude protein extracts of tobacco leaves. Under optimal in vitro conditions, specific activities of those enzymes were 0.15 ± 0.03, 0.10 ± 0.03, 0.08 ± 0.02, 0.64 ± 0.13 and 23.08 ± 1.98 nmol product/min/mg protein, respectively. This is the first report on the conversion between PM and PL catalyzed by PM-pyruvate aminotransferase in plants. Furthermore, the PL reductase activity was found to be heat inducible. Our study sheds light on the VB₆ metabolism taking place in plants.     

Effects of bleaching on the pharmacological and toxicological activities elicited by the aqueous extracts prepared from two “fire corals” collected in the Mexican Caribbean

Hydrocorals of the genus Millepora are abundant skeleton-forming inhabitants of coral reefs around the world. These species are popularly known as “fire corals” since contact with them causes severe pain, skin eruptions and blisters as a result of the release of unidentified toxins. Millepora species associate with photosynthetic dinoflagellates of the genus Symbiodinium (“zooxanthellae”), and up to now the role of these symbionts in the toxic effects induced by the “fire corals” is unknown. In this study, we compared the hemolytic, vasoconstrictor, and phospholipase A₂ (PLA₂) activities of the crude aqueous extracts prepared from normal and bleached specimens of two hydrocorals collected in the Mexican Caribbean, Milleporaalcicornis and Millepora complanata. Electrophoretic analysis revealed some differences between the protein profiles of the extracts prepared from normal and bleached specimens. Bleaching decreased, but not abolished, the hemolytic effect induced by the hydrocorals extracts and the phospholipase A₂ activity of M. complanata extract. Furthermore, it did not modify the enzymatic activity of M. alcicornis extract and vasoconstriction elicited by both extracts. Our results suggest that the presence of the symbionts does not importantly influence the pharmacological and toxic effects induced by Millepora ssp. extracts, and indicate that cnidarians are the main source of the bioactive compounds.     

Structure-activity relationship of polyphenols on inhibition of chemical mediator release from rat peritoneal exudate cells

Summary The effect of phenolic compounds in foodstuffs on histamine and leukotriene B₄ (LTB₄) release from rat peritoneal exudate cells and their antioxidative activity were examined to assess their antiallergenic activities. Among them, triphenols such as pyrogallol and gallic acid inhibited histamine release from the cells, but diphenols did not. On the other hand, o- and p-diphenols such as catechol and hydroquinone with strong antioxidative activity inhibited LTB₄ release as strongly as pyrogallol, but an m-derivative resorcinol with weak antioxidative activity did not. Though carboxylated compounds and their noncarboxylated counterparts were antioxidative, the former exerted a much weaker inhibitory effect on the LTB₄ release than the latter. In flavonols, only myricetin with a triphenolic B ring strongly inhibited histamine release, but all flavonols strongly suppressed LTB₄ release irrespective of the number of OH groups in the B ring. Among flavonoids with an o-diphenolic B ring, flavonol and flavone with a C₄-carbonyl group strongly inhibited LTB₄ release, whereas the activity of anthocyan without C₄-carbonyl was much weaker than the above compounds. These results suggest that triphenolic structure is essential for the inhibition of histamine release. On the other hand, antioxidative activity and membrane permeability of phenolic compounds seemed to be essential for the inhibition of LTB₄ release. In addition, the C₄-carbonyl group seemed to be important for strongly inhibiting LTB₄ release.     

Seven new vernocuminosides from the stem bark of Vernonia cumingiana Benth

Seven new stigmastane-type steroidal glycosides, vernocuminosides H-N, have been isolated from the stem bark of Vernonia cumingiana Benth. The structures of these compounds were determined by NMR spectrometric methods including HSQC, HMBC, ¹H-¹H COSY, and NOE, as well as circular dichroism experiments. Anti-inflammatory activities of vernocuminosides I and K-N were determined.     

Structures and mechanisms of enzymes in the leukotriene cascade

Leukotrienes are a family of proinflammatory lipid mediators of the innate immune response and are important signaling molecules in inflammatory and allergic conditions. The leukotrienes are formed from arachidonic acid, which is released from membranes by cPLA₂, and further converted by 5-lipoxygenase to form the labile epoxide leukotriene (LT) A₄. This intermediate is converted by either of the two enzymes, LTA₄ hydrolase or LTC₄ synthase, to form LTB₄ or LTC₄, respectively. In order for 5-lipoxygenase to work efficiently in cells, five-lipoxygenase-activating protein needs to be present. LTB₄ is one of the most powerful chemotactic agents whereas LTC₄ induces smooth muscle contractions, for example in the airways causing bronchoconstriction in asthmatic patients. The leukotrienes and the five enzymes/proteins involved in their formation have been subject to intense studies including drug design programs. Compounds blocking the formation or action of leukotrienes are potentially beneficial in treatment of several acute and chronic inflammatory diseases of the cardiovascular and respiratory systems. In order to succeed with drug development studies, knowledge of the molecular characteristics of the targets is indispensable. This chapter reviews the biochemistry, catalytic, and structural properties of the enzymes in the leukotriene cascade.     

Clearance and metabolism of arachidonic acid by C6 glioma cells and astrocytes

Effects of increased levels of arachidonic acid (AA) were analyzed in vitro by employment of C6 glioma cells and astrocytes from primary culture. The cells were suspended in a physiological medium added with arachidonic acid (AA) in a concentration range from 0.01 to 0.5 mM. The concentration profiles of the fatty acid and AA-metabolited were subsequently followed for 90 min. AA was measured by gas chromatography, whereas the AA-metabolites PGF₂ and LTB₄ by radioimmunoassay (RIA). Following administration of AA at 0.05 or 0.1 mM the medium was completely cleared from the fatty acid within 10 to 15 min. However, when 0.5 mM were added, AA concentrations of 0.36±0.055 mM were found at 20 min, while 0.275±0.045 mM at 90 min. Addition of AA (0.1 mM) to cell-free medium was also associated with a steady decline of its concentration, although the decrease was markedly delayed as compared to the clearance in the presence of glial cells. AA was subjected to dose-dependent metabolisation in the cell suspension as demonstrated by the production of PGF₂ and LTB₄. Following addition of 0.01 or 0.5 mM, concentrations of PGF₂ increased to a 1.9- or 4.9-fold level within 10 min, whereas those of LTB₄ rose to a 1.3- or 33.7-fold level. This was attenuated or completely blocked, respectively, by the cyclo- and lipoxygenase inhibitor BW 755C. Formation of both metabolites from AA was also observed when studying astrocytes from primary culture. The current findings demonstrate an impressive efficacy of C6 glioma cells and astrocytes to clear arachidonic acid from the suspension medium and to convert the lipid compound into prostaglandins and leukotrienes. Uptake and metabolisation of AA by the glial elements may play an important role in vivo, for example in cerebral ischemia.     

Isocostic Acid,a Promising Bioactive Agent from the Essential Oil of Inula viscosa (L.): Insights from Drug Likeness Properties,Molecular Docking and SAR Analysis

The chemical composition of the essential oil (LEO) and its volatile fractions (V₁–V₁₀) collected during the hydrodistillation process every 15 min from the fresh leaves of I. viscosa (L.), growing in Tunisia, were analyzed by GC‐FID and GC/MS. Eighty‐two compounds, representing 90.9–99.4 % of the total samples, were identified. The crude essential oil (LEO) and its fractions (V₁–V₁₀) were characterized by the presence of a high amount of oxygenated sesquiterpenes (82.7–95.8 %). Isocostic acid ( 1 ) was found to be the most abundant component (37.4–83.9 %) and was isolated from the same essential oil over silica gel column chromatography and identified by spectroscopic methods (¹H, ¹³C, DEPT 135 NMR and EI‐MS) and by comparison with literature data. Furthermore, the fresh leaves essential oil (LEO), its volatile fractions (V₁–V₁₀) as well as compound 1 were screened for their antibacterial, antityrosinase, anticholinesterase and anti‐5‐lipoxygenase activities. It was found that the isolated compound 1 exhibited an interesting antibacterial activity against Staphylococcus aureus ATCC 25923 (MIC=32 μg/mL) and Enterococcus faecalis ATCC 29212 (MIC=32 μg/mL) and the highest antityrosinase activity (IC₅₀=13.82±0.87 μg/mL). Compound 1 was also found to be able to strongly inhibit 5‐lipoxygenase with an IC₅₀ value of 59.21±0.85 μg/mL. The bioactivity and drug likeness scores of compound 1 were calculated using Molinspiration software and interpreted, and the structure‐activity relationship (SAR) was discussed with the help of molecular docking analysis.     

Time-dependent changes in the brain arachidonic acid cascade during cuprizone-induced demyelination and remyelination

Phospholipases A₂ (PLA₂) are the enzymatic keys for the activation of the arachidonic acid (AA) cascade and the subsequent synthesis of pro-inflammatory prostanoids (prostaglandins and tromboxanes). Prostanoids play critical roles in the initiation and modulation of inflammation and their levels have been reported increased in several neurological and neurodegenerative disorders, including multiple sclerosis (MS).Here, we aimed to determine whether brain expression PLA₂ enzymes and the terminal prostagland in levels are changed during cuprizone-induced demyelination and in the subsequent remyelination phase.Mice were given the neurotoxicant cuprizone through the diet for six weeks to induce brain demyelination. Then, cuprizone was withdrawn and mice were returned to a normal diet for 6 weeks to allow spontaneous remyelination.We found that after 4-6 weeks of cuprizone, sPLA₂(V) and cPLA₂, but not iPLA₂(VI), gene expression was upregulated in the cortex, concomitant with an increase in the expression of astrocyte and microglia markers. Cyclooxygenase (COX)-2 gene expression was consistently upregulated during all the demyelination period, whereas COX-1 sporadically increased only at week 5 of cuprizone exposure. However, we found that at the protein level only sPLA₂(V) and COX-1 were elevated during demyelination, with COX-1 selectively expressed by activated and infiltrated microglia/macrophages and astrocytes. Levels of PGE₂, PGD₂, PGI₂ and TXB₂ were also increased during demyelination. During remyelination, none of the PLA₂ isoforms was significantly changed, whereas COX-1 and -2 were sporadically upregulated only at the gene expression level. PGE₂, PGI₂ and PGD₂ levels returned to normal, whereas TXB₂ was still upregulated after 3 weeks of cuprizone withdrawal.Our study characterizes for the first time time-dependent changes in the AA metabolic pathway during cuprizone-induced demyelination and the subsequent remyelination and suggests that sPLA₂(V) is the major isoform contributing to AA release.