An in vivo microdialysis measurement of harpagoside in rat blood and bile for predicting hepatobiliary excretion and its interaction with cyclosporin A and verapamil

Harpagoside, a major bioactive iridoid glucoside in genus Scrophularia, has been widely used in clinical practice for the treatment of pain in the joints and lower back for its neuroprotective and anti-inflammation activities. To investigate the pharmacokinetics and hepatobiliary excretion, an in vivo microdialysis method coupled with high performance liquid chromatography was developed to monitor the concentration of harpagoside in blood and bile. The harpagoside bile-to-blood distribution ratio (AUC_bile/AUC_blood) up to 986.28 ± 78.46 significantly decreased to 6.41 ± 0.56 or 221.20 ± 18.92 after co-administration of cyclosporin A or verapamil. The results indicated that harpagoside went through concentrative elimination from the bile which was probably regulated by P-glucoprotein, providing possible clinical trials of co-administration of transporter inhibitors to decrease drug efflux, thus to enhance the curative effects.     

A New Species of Rhaponticoides (Asteraceae) from Southern Italy

A new diploid (2n?=?30) species, Rhaponticoides calabrica, is described from Calabria and Basilicata (southern Italy). This species differs from the closely related R. centaurium – an endemic of Apulia and northern Basilicata – by different flower colour (white in the former, purplish in the latter), width of membranous margin of phyllaries (1.8?±?0.4 mm vs 0.8?±?0.3 mm) and pappus length (6.8?±?0.9 mm vs 9.1?±?1.0 mm). The two species are completely allopatric and seem to have also different chromosome numbers.     

Harpagoside: from Kalahari Desert to pharmacy shelf

Harpagoside is an iridoid glycoside that was first isolated from Harpagophytum procumbens (devil’s claw, Pedaliaceae), a medicinal plant in which it is the major constituent of the iridoid pool. Both the pure compound and devil’s claw extracts have potent anti-rheumatic, anti-inflammatory and analgesic effects. According to the European Pharmacopoeia commercial devil’s claw products should contain at least 1.2% harpagoside. However, the compound has also been isolated from several other plant species and in vitro plant culture systems. Recent advances in knowledge of harpagoside distribution, biosynthesis/accumulation and pharmacology are summarized in this review. We also discuss the possible synergism and/or antagonism between major constituents in harpagoside-containing phytopharmaceutical products. Finally, future perspectives for its potential application are highlighted.     

Metabolic differentiations and classification of Verbascum species by NMR-based metabolomics

The genus Verbascum L. (mulleins) comprises of about 360 species of flowering plants in the Scrophulariaceae family. Mulleins have been used in the traditional folk medicine for centuries, for treatment of a wide range of human ailments, inter alia bronchitis, tuberculosis, asthma, and different inflammations. Despite all applications the knowledge of the metabolites, accumulated in different mullein species, is still limited and based mainly on determination of the major compounds. Here we report the application of ¹H NMR metabolic fingerprinting in combination with principal component analyses (PCA) in five different Verbascum species. Based on the obtained results mulleins were divided in two groups: group A (Verbascum phlomoides and Verbascum densiflorum) and group B (Verbascum xanthophoeniceum, Verbascum nigrum and Verbascum phoeniceum). Further it was found that the plants in group B accumulate higher amounts of bioactive iridoid and phenylethanoid glycosides. V. xanthophoeniceum and V. nigrum accumulate higher amounts of the pharmaceutically-important harpagoside (∼0.5% on dry weight basis) and verbascoside, forsythoside B and leucosceptoside B (in total 5.6–5.8% on dry weight basis), which underlines the possibility for their application in pharmaceutical industry. To the best of our knowledge this is the first report on the analyses of Verbascum sp. leaf metabolome.     

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens

Comparative phytochemical analysis of wild and in vitro-derived greenhouse-grown tubers, in vitro shoots and callus-like basal tissues of Harpagophytum procumbens was done. Dried samples were ground to fine powders and their total iridoid (colorimetric method), phenolic [Folin-Ciocalteu (Folin C) method] and gallotannin (Rhodanine assay) contents as well as anti-inflammatory activity [cyclooxygenase assays (COX-1 and COX-2)] were determined. The tissue culture-derived tubers had the highest total iridoid content which was significantly higher than that of the tubers collected from the wild and other tissue cultured materials evaluated. This suggests that cultivated plants can be a viable alternative source of the active principle(s). The total phenolic and gallotannin contents of the wild tubers were significantly higher than the tissue culture-derived tubers and other in vitro-derived plant materials. The presence of phenolic compounds including gallotannins in the tissue cultured materials is of interest from a pharmacological point of view given the pharmacological role of phenolics. In general, extracts from wild tubers demonstrated better inhibitory activities in both COX-1 and COX-2 assays when compared to the tissue culture-derived tubers. All the petroleum ether (PE) and dichloromethane (DCM) extracts showed moderate (50-70%) to good (> 70%) inhibitory activities whereas the ethanol (EtOH) extracts showed poor or no inhibition in both assays. Based on previous reports indicating weak inhibition of COX-2 enzyme by harpagoside, the inhibitory activities of both COX enzymes exhibited by PE and DCM extracts in the current study could be due to the presence of other constituents in the extracts. This points towards the need to identify other active constituents and evaluate their role and mode of action in relation to harpagoside — the main active principle.     

Investigations on Genetically Modified Maize (Bt-Maize) in Pig Nutrition: Chemical Composition and Nutritional Evaluation

The objective of the present study was to determine the composition and the nutritional value of parental and transgenic maize seeds fed to pigs. The parental maize line was genetically modified to incorporate a gene from Bacillus thuringiensis (Bt) expressing a toxin against the European corn borer (Ostrinia nubilalis). Both (parental and transgenic) maize lines were analyzed for crude nutrients, starch, sugar, non-starch polysaccharides (NSP), amino acids, fatty acids, as well as for selected minerals. Furthermore, four complete diets were mixed and were analyzed for the same nutrients and some selected ingredients. The diets contained 70% maize to attain a high effect level. To evaluate the feeding value of one variety of genetically modified maize (transgenic) compared to the feeding value of the unmodified maize (parental) line, a balance study with twelve pigs was designed. Three collecting periods were used for each maize line each with six animals. The collected faeces were analyzed for crude nutrients. All measured parameters were virtually the same ( e.g. crude protein 11.59% vs. 11.06% in DM), especially the digestibility of crude protein (85.8 ± 2.3% vs. 86.1 ± 1.8%), the amount of nitrogen-free-extract (92.8 ± 0.6% vs. 93.2 ± 0.6%) and the metabolizable energy (15.7 ± 0.2% vs. 15.8 ± 0.2% MJ/kg DM) for both maize lines. Compared to the parental line, the chemical composition and digestibility of crude nutrients and energy content were not significantly affected by the genetic modification of maize. Therefore, from the view of a nutritional assessment, the genetically modified maize can be regarded as substantially equivalent to the parental maize line.     

Physcion,a tetra-substituted 9,10-anthraquinone,prevents homocysteine-induced endothelial dysfunction by activating Ca2+- and Akt-eNOS-NO signaling pathways

BackgroundHomocysteine (Hcy) induced vascular endothelial dysfunction is known to be closely associated with oxidative stress and impaired NO system. 1,8-Dihydroxy-3-methoxy-6-methylanthracene-9,10-dione (physcion) has been known to has antioxidative and anti-inflammatory properties.PurposeThe purpose of the present study was to define the protective effect of physcion on Hcy-induced endothelial dysfunction and its mechanisms involved.Study Design and MethodsHyperhomocysteinemia (HHcy) rat model was induced by feeding 3% methionine. A rat thoracic aortic ring model was used to investigate the effects of physcion on Hcy-induced impairment of endothelium-dependent relaxation. Two doses, low (L, 30 mg/kg/day) and high (H, 50 mg/kg/day) of physcion were used in the present study. To construct Hcy-injured human umbilical vein endothelial cells (HUVECs) model, the cells treated with 3 mM Hcy. The effects of physcion on Hcy-induced HUVECs cytotoxicity and apoptosis were studied using MTT and flow cytometry. Confocal analysis was used to determine the levels of intracellular Ca²⁺. The levels of protein expression of the apoptosis-related markers Bcl-2, Bax, caspase-9/3, and Akt and endothelial nitric oxide synthase (eNOS) were evaluated by western blot.ResultsIn the HHcy rat model, plasma levels of Hcy and malondialdehyde (MDA) were elevated (20.45 ± 2.42 vs. 4.67 ± 1.94 μM, 9.42 ± 0.48 vs. 3.47 ± 0.59 nM, p < 0.001 for both), whereas superoxide dismutase (SOD) and nitric oxide (NO) levels were decreased (77.11 ± 4.78 vs. 115.02 ± 5.63 U/ml, 44.51 ± 4.45 vs. 64.18 ± 5.34 μM, p < 0.001 and p < 0.01, respectively). However, treatment with physcion significantly reversed these changes (11.82 ± 2.02 vs. 20.45 ± 2.42 μM, 5.97 ± 0.72 vs. 9.42 ± 0.48 nM, 108.75 ± 5.65 vs. 77.11 ± 4.78 U/ml, 58.14 ± 6.02 vs. 44.51 ± 4.45 μM, p < 0.01 for all). Physcion also prevented Hcy-induced impairment of endothelium-dependent relaxation in HHcy rats (1.56 ± 0.06 vs. 15.44 ± 2.53 nM EC₅₀ for ACh vasorelaxation, p < 0.05 vs. HHcy). In Hcy-injured HUVECs, physcion inhibited the impaired viability, apoptosis and reactive oxygen species. Hcy treatment significantly increased the protein phosphorylation levels of p38 (2.26 ± 0.20 vs. 1.00 ± 0.12, p <0.01), ERK (2.11 ± 0.21 vs. 1.00 ± 0.11, p <0.01) and JNK. Moreover, physcion reversed the Hcy-induced apoptosis related parameter changes such as decreased mitochondrial membrane potential (MMP) and Bcl-2/Bax protein ratio, and increased protein expression of caspase-9/3 in HUVECs. Furthermore, the downregulation of Ca²⁺, Akt, eNOS and NO caused by Hcy were recovered with physcion treatment in HUVECs.ConclusionPhyscion prevents Hcy-induced endothelial dysfunction by activating Ca²⁺- and Akt-eNOS-NO signaling pathways. This study provides the first evidence that physcion might be a candidate agent for the prevention of cardiovascular disease induced by Hcy.     

New phenolic cinnamic acid derivatives as selective COX-2 inhibitors. Design,synthesis, biological activity and structure-activity relationships

Selective inhibition of cyclooxygenase (COX)-2 enzyme is an important achievement when looking for potent anti-inflammatory agents, with fewer gastrointestinal side effects. In this work, a new series of cinnamic acid derivatives, namely hexylamides, have been designed, synthesized and evaluated in human blood for their inhibitory activity of COX-1 and COX-2 enzymes. From this, new structure-activity relationships were built, showing that phenolic hydroxyl groups are essential for both COX-1 and COX-2 inhibition. Furthermore, the presence of bulky hydrophobic di-tert-butyl groups in the phenyl ring strongly contributes for selective COX-2 inhibition. In addition, a correlation with the theoretical log P has been carried out, showing that lipophilicity is particularly important for COX-2 inhibition. Further, a plasma protein binding (PPB) prediction has been performed revealing that PPB seems to have no influence in the activity of the studied compounds. From the whole study, effective selective inhibitors of COX-2 were found, namely compound 9 (IC₅₀ = 3.0 ± 0.3 μM), 10 (IC₅₀ = 2.4 ± 0.6 μM) and 23 (IC₅₀ = 1.09 ± 0.09 μM). Those can be considered starting point hit compounds for further optimization as potential non-steroidal anti-inflammatory drugs.     

Animal protein-free OptiXcell and shortened equilibration periods can replace egg yolk-based extender and slow cooling for rhinoceros semen cryopreservation

OptiXcell (OP) was tested as an animal protein-free alternative to an egg yolk-based extender for rhinoceros semen cryopreservation and shorter chilling/equilibration periods were evaluated. Semen was collected from three rhinoceros species: black (Diceros bicornis; n = 2), white (Ceratotherium simum; n = 2), and greater one-horned (GOH; Rhinoceros unicornis; n = 3). Controls were diluted with equine extender (EQ) or OP and equilibrated for 1 h. Treatments were diluted with extender and cooled for 15 min (fast: FEQ; FOP) or not cooled (immediate: IEQ; IOP), prior to cryopreservation. Motility decreased post-thaw (EQ: 50.7 ± 5.2%; OP: 52.9 ± 3.4%) from fresh (82.9 ± 2.9%), was higher in OP than IOP (38.6 ± 4.9%; P ≤ 0.05) and decreased over time (P ≤ 0.05). Post-thaw acrosomal integrity was lower in EQ, FEQ, and IEQ (56.9 ± 0.7; 56.6 ± 4.5; 54.9 ± 2.9%) than OP, FOP, IOP (71.8 ± 4.7; 71.9 ± 3.8; 69.9 ± 4.5%) and fresh (72.6 ± 1.4%; P ≤ 0.05). Progression and viability were lower in EQ (2.8 ± 0.2; 61.9 ± 7.4%) and OP (3.1 ± 0.2; 53.4 ± 6.9%) than fresh (3.7 ± 0.2; 87.2 ± 1.3%), decreased over time (P ≤ 0.05) but not different among treatments (P > 0.05). Morphology did not differ between fresh (75.0 ± 4.9% normal) and any treatment group (70.0–77.8%) or over time (P > 0.05). OptiXcell is comparable to egg yolk-based EQ when used for rhinoceros semen cryopreservation. Furthermore, chilling/equilibration can be reduced with little impact on sperm characteristics.     

Synthesis, in vitro and in vivo biological evaluation,COX-1/2 inhibition and molecular docking study of indole-N-acylhydrazone derivatives

The objective of this work was to obtain and evaluate anti-inflammatory in vitro, in vivo and in silico potential of novel indole-N-acylhydrazone derivatives. In total, 10 new compounds (3a–j) were synthesized in satisfactory yields, through a condensation reaction in a single synthesis step. In the lymphoproliferation assay, using mice splenocytes, 3a and 3b showed inhibition of lymphocyte proliferation of 62.7% (±3.5) and 50.7% (±2), respectively, while dexamethasone presented an inhibition of 74.6% (±2.4). Moreover, compound 3b induced higher Th2 cytokines production in mice splenocytes cultures. The results for COX inhibition assays showed that compound 3b is a selective COX-2 inhibitor, but with less potency when compared to celecoxib, and compound 3a not presented selectivity towards COX-2. The molecular docking results suggest compounds 3a and 3b interact with the active site of COX-2 in similar conformations, but not with the active site of COX-1, and this may be the main reason to the COX-2 selectivity of compound 3b. In vivo carrageenan-induced paw edema assays were adopted for the confirmation of the anti-inflammatory activity. Compound 3b showed better results in suppressing edema at all tested concentrations and was able to induce an edema inhibition of 100% after 5?h of carrageenan injection at the 30?mg?kg^?1 dosage, corroborating with the COX inhibition and lymphoproliferation results. I addition to our experimental results, in silico analysis suggest that compounds 3a and 3b present a well-balanced profile between pharmacodynamics and pharmacokinetics. Thus, our preliminary results revealed the potentiality of a new COX-2 selective derivative in the modulation of the inflammatory process.     

Treasure from garden: chemical profiling,pharmacology and biotechnology of mulleins

The genus Verbascum (mulleins), belonging to the family Scrophulariaceae, comprises about 360 species of flowering plants. The leaves, flowers and whole aerial parts of Verbascum spp. have been widely used in traditional medicine for the treatment of respiratory and inflammatory disorders and also display powerful wound healing activity. Verbascum species are found to accumulate several groups of bioactive molecules, therefore they might be utilized as attractive sources of new (drug) leads. The present review attempts to provide an up-to-date comprehensive overview on phytochemical and pharmacological aspects of Verbacum spp. research along with some successful examples of growing (and transforming) mulleins in vitro.     

Seasonal variations in the harpagoside content of Scrophularia scorodonia L

Seasonal variations on the content of harpagoside in Scrophularia scorodonia L. (Scrophulariaceae) were investigated using plants collected monthly from January to December in 1995. During growth of this species the percentage of harpagoside was the highest during the maximum development of the plant, specially in July. Harpagoside levels differed among leaves, stems and flowers of S. scorodonia. Leaves were distinguished from other plant parts by higher levels of harpagoside. Drying at ambient temperature influenced the yield of harpagoside compared with the results of plant drying by microwave.     

<Emphasis Type="Italic">Myxobolus imparfinis</Emphasis> n. sp. (Myxozoa: Myxosporea), a new gill parasite of <Emphasis Type="Italic">Imparfinis mirini</Emphasis> Haseman (Siluriformes: Heptapteridae) in Brazil

A new species of myxozoan, Myxobolus imparfinis n. sp. is described based on material from the gills of Imparfinis mirini (Haseman) (Heptapteridae). Mature myxospores are round, measuring 7.1–8.4 (7.9 ± 0.3) μm in length, 4.5–6.2 (5.5 ± 0.5) μm in width and 3.1–4.2 (3.7 ± 0.3) μm in thickness. The polar capsules are of unequal size, the larger polar capsule measuring 3.4–4.5 (3.9 ± 0.3) μm in length and 1.4–2.0 (1.7 ± 0.1) μm in width and the smaller capsule measuring 3.1–3.8 (3.4 ± 0.2) μm in length and 1.2–1.8 (1.5 ± 0.2) μm in width. The polar filament presents 6–7 coils. Spores had a prevalence of infection of 75% (6/8). In histological analyses we detected the development site of spores in primary filaments, in afferent branchial artery, thus classifying the type of infection to the filamental type and vascular subtype. The phylogenetic analyses of a dataset including species Myxobolus Bütschli, 1882 and Henneguya Thélohan, 1892 from South America recovered M. imparfinis n. sp. as a sister species of Myxobolus flavus Carriero, Adriano, Silva, Ceccarelli & Maia, 2013. To our knowledge, this is the first record of a myxozoan species parasitising I. mirini.     

Effects of extender type on the quality of post-thaw giant panda (Ailuropoda melanoleuca) semen

Sperm cryopreservation is an essential approach for assisted reproduction and genetic resources conservation in captive giant pandas. Cryopreservation, however, leads to a significant decrease in sperm quality and, consequently, a low fertilization rate. Therefore, it is mandatory to disclose more suitable and efficient freezing strategies for sperm cryopreservation. In the present study, we compared for the first time the performance of two commercial freeze extender (INRA96 versus TEST) freezing methods on post-thawed semen quality. Semen cryopreserved with the INRA96 showed better total motility (73.00 ± 4.84% vs 57.56 ± 3.60%, P < 0.001), membrane integrity (60.92 ± 2.27% vs 40.53 ± 2.97%, P < 0.001) and acrosome integrity (90.39 ± 2.74% vs 84.26 ± 4.27%, P < 0.05) than stored with TEST. There was no significant difference in DNA integrity after thawing between the two extenders (95.69 ± 3.60% vs 94.26 ± 4.84%). In conclusion, the INRA96 method showed to be better for giant panda sperm cryopreservation and should therefore be recommended for use in order to increase success of artificial insemination.     

Regulation of the fetal mouse ductus arteriosus is dependent on interaction of nitric oxide and COX enzymes in the ductal wall

Nitric oxide (NO) and cyclooxygenase (COX)-derived prostaglandins are critical regulators of the fetal ductus arteriosus. To examine the interaction of these pathways within the ductus wall, the ductus arteriosus of term and preterm fetal mice was evaluated by pressurized myography. The isolated preterm ductus was more sensitive to NOS inhibition than at term. Sequential NOS and COX inhibition caused 36% constriction of the preterm ductus regardless of drug order. In contrast, constriction of the term ductus was dependent on the sequence of inhibition; NOS inhibition prior to COX inhibition produced greater constriction than when inhibitors were given in reverse order (36 ± 6% versus 23 ± 5%). Selective COX-1 or COX-2 inhibition prior to N(G)-nitro-l-arginine methyl ester (l-NAME) induced the expected degree of constriction. However, NOS inhibition followed by selective COX-2 inhibition caused unexpected ductal dilation. These findings are consistent with NO-induced activation of COX in the ductus arteriosus wall and the production of a COX-2-derived constrictor prostanoid that contributes to the balance of vasoactive forces that maintain fetal ductus arteriosus tone.     

In-straw warming protocol improves survival of vitrified embryos and allows direct transfer in cattle

Vitrification is a superior method for cryopreservation of IVF embryos, but due to complicated warming protocols, it is not commonly used for commercial bovine embryos routine. To overcome the need of laboratory embryo preparation during warming, we developed an in-straw warming protocol compatible with most vitrification devices for embryo transfer without sucrose gradient steps and embryo evaluation. Surprisingly, one of the tested protocols improved embryo survival (95.0%* vs 83.1% expansion rate and 74.2%* vs 51.5% hatching rate) compared to conventional in-plate warming. Embryo quality was also increased, taken by the higher total cell numbers (160.7 ± 8.6* vs 99.0 ± 7.9) and lower apoptosis index (4.9 ± 0.6* vs 11.5 ± 2.4) 48 h after warming. Pregnancy rates were similar between vitrified-warmed embryos and fresh embryos (40% vs 43%). Based on our results, we suggest in-straw warming should always be used for vitrified embryos due to beneficial effects. Direct transfer can be safely performed using this protocol.     

Coronary aspirate TNFα reflects saphenous vein bypass graft restenosis risk in diabetic patients

Background

Patients with diabetes mellitus (DM) have an increased risk for periprocedural complications and adverse cardiac events after percutaneous coronary intervention. We addressed the potential for coronary microvascular obstruction and restenosis in patients with and without DM undergoing stenting for saphenous vein bypass graft (SVG) stenosis under protection with a distal occlusion/aspiration device.

Methods

SVG plaque volume and composition were analyzed using intravascular ultrasound before stent implantation. Percent diameter stenosis was determined from quantitative coronary angiography before, immediately after and 6?months after stent implantation. Coronary aspirate was retrieved during stent implantation and divided into particulate debris and plasma. Total calcium, several vasoconstrictors, and tumor necrosis factor (TNF)α in particulate debris and coronary aspirate plasma were determined.

Results

Patients with and without DM had similar plaque volume, but larger necrotic core and greater particulate debris release in patients with than without DM (20.3±2.7 vs. 12.7±2.6% and 143.9±19.3 vs. 75.1±10.4?mg, P<0.05). The TNFα concentration in particulate debris and coronary aspirate plasma was higher in patients with than without DM (15.9±6.6 vs. 5.1±2.4 pmol/mg and 2.2±0.7 vs. 1.1±0.2 pmol/L, P<0.05), whereas total calcium and vasoconstrictors were not different. Patients with DM had a greater percent diameter stenosis 6?months after stent implantation than those without DM (22.17±5.22 vs. 6.34±1.11%, P<0.05). The increase in TNFα immediately after stent implantation correlated with restenosis 6?months later (r=0.69, P<0.05).

Conclusion

In diabetics, particulate debris and coronary aspirate plasma contained more TNFα, which might reflect the activity of the underlying atherosclerotic process.

Trial registration

URL: http://www.clinicaltrials.gov/ct2/results?term=NCT01430884; unique identifier: NCT01430884     

Bovine in vitro embryo production using media prepared with Milli-Q® Water or nanowater

The aim of this study was to compare the efficiency of in vitro embryo production (IVP) following the collection of bovine ovaries and 22-h in vitro maturation (IVM) of oocytes in media prepared with Milli-Q® Water (n = 509 oocytes) or nanowater (NW; n = 304 oocytes). The mean cleavage (63.8 ± 4.6 % vs. 63.6 ± 6.1 %, respectively; mean ± SEM) and blastocyst formation rate (16.3 ± 3.4 % vs. 16.7 ± 6.7 % of presumptive zygotes, respectively) did not vary (P > 0.05; Student t-test) between the two types of media diluents. NW is a safe substitute for Milli-Q® Water for IVM of bovine oocytes.     

In vivo anti-inflammatory activity and docking study of newly synthesized benzimidazole derivatives bearing oxadiazole and morpholine rings

In search of potential therapeutics for inflammatory disease, we report herein the synthesis, characterization and anti-inflammatory activities of a new series of 1-{(5-substituted-1,3,4-oxadiazol-2-yl)methyl}-2-(morpholinomethyl)-1H-benzimidazoles (5a-r). The anti-inflammatory activity of the compounds was evaluated using carrageenan induced rat paw edema test. Some compounds showed excellent anti-inflammatory activity in carrageenan induced rat paw edema test. 1-{(5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl)methyl}-2-(morpholinomethyl)-1H-benzimidazole (5g) showed maximum anti-inflammatory (74.17 ± 1.28% inhibition) with reduced ulcerogenic and lipid peroxidation profile and also showed significant COX-2 inhibition with IC₅₀ values of 8.00 μM. Compounds 5o and 5q were also found to exhibit good COX-2 inhibition with IC₅₀ values of 11.4 and 13.7 μM concentrations. Molecular docking study showed that morpholine and oxadiazole rings linked to the benzimidazole nucleus play an important role in binding with the COX-2.     

Recombinant expression and characterization of Lucilia cuprina CYP6G3: Activity and binding properties toward multiple pesticides

The Australian sheep blowfly, Lucilia cuprina, is a primary cause of sheep flystrike and a major agricultural pest. Cytochrome P450 enzymes have been implicated in the resistance of L. cuprina to several classes of insecticides. In particular, CYP6G3 is a L. cuprina homologue of Drosophila melanogaster CYP6G1, a P450 known to confer multi-pesticide resistance. To investigate the basis of resistance, a bicistronic Escherichia coli expression system was developed to co-express active L. cuprina CYP6G3 and house fly (Musca domestica) P450 reductase. Recombinant CYP6G3 showed activity towards the high-throughput screening substrates, 7-ethoxycoumarin and p-nitroanisole, but not towards p-nitrophenol, coumarin, 7-benzyloxyresorufin, or seven different luciferin derivatives (P450-Glo™ substrates). The addition of house fly cytochrome b₅ enhanced the k_cat for p-nitroanisole dealkylation approximately two fold (17.8 ± 0.5 vs 9.6 ± 0.2 min⁻¹) with little effect on K_M (13 ± 1 vs 10 ± 1 μM). Inhibition studies and difference spectroscopy revealed that the organochlorine compounds, DDT and endosulfan, and the organophosphate pesticides, malathion and chlorfenvinphos, bind to the active site of CYP6G3. All four pesticides showed type I binding spectra with spectral dissociation constants in the micromolar range suggesting that they may be substrates of CYP6G3. While no significant inhibition was seen with the organophosphate, diazinon, or the neonicotinoid, imidacloprid, diazinon showed weak binding in spectral assays, with a Kd value of 23 ± 3 μM CYP6G3 metabolised diazinon to the diazoxon and hydroxydiazinon metabolites and imidacloprid to the 5-hydroxy and olefin metabolites, consistent with a proposed role of CYP6G enzymes in metabolism of phosphorothioate and neonicotinoid insecticides in other species.