Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

Antioxidant activity and total phenolic content of ethanolic extract of Caesalpinia bonducella seeds

The aim of this study was to assess the in vitro potential of ethanolic extract of Caesalpinia bonducella seeds as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 38.93–74.77% as compared to ascorbic acid (64.26–82.58%). The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 74.73 and 26.68 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of C. bonducella was achieved using Folin–Ciocalteau reagent containing 62.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 109.85, 102.65 and 89.84 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 70.79, 65.98 and 36.68 μg/ml respectively. The results obtained in this study clearly indicate that C. bonducella has a significant potential to use as a natural antioxidant agent.     

Inhibition of inflammatory mediators by polyphenolic plant extracts in human intestinal Caco-2 cells

The mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) are involved in transduction cascades that play a key role in inflammatory response. We tested the ability of preselected natural polyphenolic extracts (grape seed, cocoa, sugar cane, oak, mangosteen and pomegranate) to modulate intestinal inflammation using human intestinal Caco-2 cells treated for 4 h with these extracts and then stimulated by cytokines for 24 or 48 h. The effect of polyphenolic extracts, at 50 μmol of gallic acid equivalent/l, was investigated on inflammation-related cellular events: (i) NF-κB activity (cells transfected with a NF-κB-luciferase construct), (ii) activation of Erk1/2 and JNK (western blotting), (iii) secretion of interleukin 8 (IL-8) (ELISA), (iv) secretion of prostaglandin (PG) E₂ (ELISA), (v) production of NO (Griess method). Results show that: (i) sugar cane, oak and pomegranate extracts inhibited NF-κB activity (from 1.6 to 1.9-fold) (P < 0.001); (ii) pomegranate slightly inhibited Erk1/2 activation (1.3-fold) (P = 0.008); (iii) oak and pomegranate decreased NO synthesis by 1.5-fold (P < 0.001) and that of IL-8 by 10.3 and 6.7-fold respectively; (iv) pomegranate and cocoa decreased PGE₂ synthesis by 4.6 (P < 0.0001) and 2.2-fold (P = 0.001), respectively. We suggest that pomegranate extract could be particularly promising in dietary prevention of intestinal inflammation.     

Evaluation of in vitro cytotoxicity of 6-benzylaminopurine carboplatin derivatives against human cancer cell lines and primary human hepatocytes

A series of seven platinum(II) cyclobutane-1,1-dicarboxylato (cbdc) complexes {[Pt(cbdc)(L_n)₂], 1-7}, derived from carboplatin by a substitution of two NH₃ molecules for two 2,6,9-trisubstituted 6-benzylaminopurine-based N-donor ligands (L_n), was studied by the MTT assay for their in vitro cytotoxic activity against seven human cancer cell lines, i.e. lung carcinoma (A549), cervix epithelioid carcinoma (HeLa), osteosarcoma (HOS), malignant melanoma (G361), breast adenocarcinoma (MCF7), ovarian carcinoma (A2780) and its cisplatin-resistant analogue (A2780cis), and against two primary cultures of human hepatocytes (LH31 and LH32). The prepared complexes were cytotoxic against several cancer cells, in some cases even more than cisplatin. The best results were achieved for complexes 1 (IC₅₀ = 17.4 ± 2.0 μM) and 2 (IC₅₀ = 14.8 ± 2.1 μΜ) against HOS cells, 1 (IC₅₀ = 15.1 ± 6.8 μM), 2 (IC₅₀ = 13.6 ± 5.2 μM) and 6 (IC₅₀ = 19.0 ± 6.6 μM) against MCF7, 6 (IC₅₀ = 6.4 ± 0.1 μM) against A2780, and 1-6 (IC₅₀ = 15.6 ± 4.0, 12.9 ± 3.7, 15.8 ± 3.8, 16.6 ± 5.5, 22.1 ± 2.5, and 5.6 ± 1.7 μM, respectively) against A2780cis. Viability of human hepatocytes was not declined by the tested complexes up to the concentration of 50 μM (for 1, 3-7) and 20 μM (for 2; caused by lower solubility of this complex).     

In vitro and in vivo antioxidant activity of Symplocos cochinchinensis S. Moore leaves containing phenolic compounds

Methanol extract of Symplocos cochinchinensis S. Moore leaves was evaluated for its in vitro and in vivo antioxidant activity. The total phenolic content of the extract was 230 mg of gallic acid equivalents/g extract. The extract showed very good scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC₅₀ 620.30 ± 0.14 μg/ml), hydroxyl (IC₅₀ 730.21 ± 1.05 μg/ml), nitric oxide (IC₅₀ 870.31 ± 0.19 μg/ml) radicals, as well as high reducing power. The extract also showed strong suppressive effect on lipid peroxidation. In in vivo study CCl₄ induced oxidative stress produced significant increase in SGOT, SGPT and LDH levels along with reduction in liver SOD, CAT, GSH and GPx levels. Pre-treatment of rats with the extract (250 and 500 mg/kg) for 7 days showed significant reduction in the levels of SGOT, SGPT and LDH compared to CCl₄ treated rats. SOD, CAT, GSH and GPx levels were increased significantly due to treatment with the extract. The activity of the extract was comparable to the standard drug, silymarin (25 mg/kg). The results suggest that the leaves of S. cochinchinensis are a source of natural antioxidants.     

A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues

The pharmacology of Australian box jellyfish, Chironex fleckeri, unpurified (crude) nematocyst venom extract (CVE) was investigated in rat isolated cardiac and vascular tissues and in anaesthetised rats.In small mesenteric arteries CVE (0.01-30 μg/ml) caused contractions (EC₅₀ 1.15 ± 0.19 μg/ml) that were unaffected by prazosin (0.1 μM), bosentan (10 μM), CGRP_8-37 (1 μM) or tetrodotoxin (1 μM). Box jellyfish antivenom (5-92.6 units/ml) caused rightward shifts of the CVE concentration-response curve with no change in the maximum. In the presence of l-NAME (100 μM) the sensitivity and maximum response to CVE were increased, whilst MgSO₄ (6 mM) decreased both parameters. CVE (1-10 μg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation.Left atrial responses to CVE (0.001-30 μg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3 μg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3 μg/ml. These responses were unaffected by 1 μM of propranolol, atropine or CGRP_8-37. Antivenom (54 and 73 units/ml) caused rightward shifts of the CVE concentration-response curve and prevented atrial standstill in left and right atria.The effects of CVE do not appear to involve autonomic nerves, post-synaptic α₁- or β₁-adrenoceptors, or muscarinic, endothelin or CGRP receptors, but may occur through direct effects on the cardiac and vascular muscle. Box jellyfish antivenom was effective in attenuating CVE-induced responses in isolated cardiac and vascular tissues.     

Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities

The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC₅₀ value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c_max at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c_max. The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K_i values were determined. All K_i values exceeded c_max by at least a factor of 10-fold. According to FDA regulations 1 > c_max/K_i > 0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.     

Modulation of the expression of the π class of glutathione S-transferase by Andrographis paniculata extracts and andrographolide

Andrographis paniculata (Ap) is a commonly used herb for traditional medicine in many Southeast Asian countries. In the present study, we investigated the effect of Ap on the expression of the pi class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 25 or 50 μg/mL of ethanol or ethyl acetate extracts of Ap (ApEE or ApEAE) or 10 or 20 μM andrographolide, which is the major active diterpene lactone of Ap, for 48 h. ApEE, ApEAE, and andrographolide dose-dependently induced GSTP protein and mRNA expression. In a GST activity assay, GST activity was significantly higher in cells treated with the maximum concentrations of ApEE, ApEAE, and andrographolide than in control cells (P < 0.05). The pTA-2713 luciferase reporter construct containing rat GSTP enhancer 1 (GPE1) was transiently transfected into Clone 9 liver cells. Cells treated with ApEE, ApEAE, and andrographolide showed a dose-dependent increase in luciferase activity. GPE1 deletion abolished the induction efficiency of Ap. Also, the induction of GSTP expression by Ap was inhibited by wortmannin, which is an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These results indicate that ApEE, ApEAE, and andrographolide induce GSTP expression. This induction is likely related to the PI3K/Akt pathway, and GPE1, an enhancer element in GSTP promoter, is essential for the induction.     

A chronic oral exposure of pigs with deoxynivalenol partially prevents the acute effects of lipopolysaccharides on hepatic histopathology and blood clinical chemistry

Lipopolysaccharides (LPS), a cell wall component of gram-negative bacteria, and deoxynivalenol (DON), a prevalent Fusarium-derived contaminant of cereal grains, are each reported to have detrimental effects on the liver. A potentiating toxic effect of the combined exposure was reported previously in a mouse model and hepatocytes in vitro, but not in swine as the most DON-susceptible species. Thus, pigs were fed either a control diet (CON) or a Fusarium contaminated diet (DON, 3.1 mg DON/kg diet) for 37 days. At day 37 control pigs were infused for 1 h either with physiological saline (CON_CON), 100 μg/kg BW DON (CON_DON), 7.5 μg/kg BW LPS (CON_LPS), or both toxins (CON_DON/LPS) and Fusarium-pigs with saline (DON_CON) or 7.5 μg/kg BW LPS (DON_LPS). Blood samples were taken before and after infusion (−30, +30, +60, +120, and +180 min) for clinical blood chemistry. Pigs were sacrificed at +195 min and liver histopathology was performed. LPS resulted in higher relative liver weight (p < 0.05), portal, periportal and acinar inflammation (p < 0.05), haemorrhage (p < 0.01) and pathological bilirubin levels (CON_CON 1.0 μmol/L vs. CON_LPS 5.4 μmol/L, CON_DON/LPS 8.3 μmol/L; p < 0.001). DON feeding alleviated effects of LPS infusion on histopathology and blood chemistry to control levels, whereas DON infusion alone had no impact.     

The pharmacology of Malo maxima jellyfish venom extract in isolated cardiovascular tissues: A probable cause of the Irukandji syndrome in Western Australia

The in vitro cardiac and vascular pharmacology of Malo maxima, a newly described jellyfish suspected of causing Irukandji syndrome in the Broome region of Western Australia, was investigated in rat tissues. In left atria, M. maxima crude venom extract (CVE; 1-100 μg/mL) caused concentration-dependent inotropic responses which were unaffected by atropine (1 μM), but significantly attenuated by tetrodotoxin (TTX; 0.1 μM), propranolol (1 μM), Mg²⁺ (6 mM) or calcitonin gene-related peptide antagonist (CGRP_8-37; 1 μM). CVE caused no change in right atrial rate until 100 μg/mL, which elicited bradycardia. This was unaffected by atropine, TTX, propranolol or CGRP_8-37. In the presence of Mg²⁺, CVE 30-100 μg/mL caused tachycardia. In small mesenteric arteries CVE caused concentration-dependent contractions (pEC₅₀ 1.03 ± 0.07 μg/mL) that were unaffected by prazosin (0.3 μM), ω-conotoxin GVIA (0.1 μM) or Mg²⁺ (6 mM). There was a 2-fold increase in sensitivity in the presence of CGRP_8-37 (3 μM). TTX (0.1 μM), box jellyfish Chironex fleckeri antivenom (92.6 U/mL) and benextramine (3 μM) decreased sensitivity by 2.6, 1.9 and 2.1-fold, respectively. CVE-induced maximum contractions were attenuated by C. fleckeri antivenom (−22%) or benextramine (−49%). M. maxima CVE appears to activate the sympathetic, but not parasympathetic, nervous system and to stimulate sensory nerve CGRP release in left atria and resistance arteries. These effects are consistent with the catecholamine excess thought to cause Irukandji syndrome, with additional actions of CGRP release.     

Antioxidant activities of leaf extract of Salvia miltiorrhiza Bunge and related phenolic constituents

Leaf of Salvia miltiorrhiza Bunge, the waste part during the root harvest, is rich in health-promoting phenolics and is a novel resource of natural antioxidants. The acetone and methanol extracts of leaves (AL and ML, respectively) of S. miltiorrhiza were evaluated by various in vitro antioxidant assays. The total phenolic contents of AL and ML were 39.0 ± 1.13 and 54.3 ± 1.1 mg gallic acid equivalents/g extract tested, respectively. EC₅₀ of ML was 7.0 ± 0.28 μg/mL in DPPH radical scavenging assay and 246.5 ± 10.35 μg/mL in superoxide radical quenching assay. It was also found that ML has prominent effects on the inhibition of linoleic acid oxidation (93.2%), which was equivalent to the positive control, butylated hydroxytoluene (BHT, p > 0.05), and was significantly higher than α-tocopherol (VE, p < 0.05). The reducing power of leaf extracts was as strong as roots (p > 0.05). HPLC and correlation analysis show that salvianolic acid B and rosmarinic acid constitute the most abundant phenolic compounds. They are the major contributors to antioxidant activities. The results suggested that S. miltiorrhiza leaves could be considered as a new potential source of natural phenolic antioxidants for food, pharmaceutical, cosmetics or nutraceutical industries.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Metasequoia glyptostroboides Miki ex Hu

This study was undertaken to assess the antioxidant and antidermatophytic potential of the essential oil and extracts (hexane, chloroform, ethyl acetate and methanol) of Metasequoia glyptostroboides Miki ex Hu. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ = 9.1 and 14.24 μg/ml, respectively) as compared to butylatedhydroxyanisole (BHA), (IC₅₀ = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (93.26 mg/g of dry wt) as compared to the other extracts. Further, oil (1250 μg/disc) and extracts (1750 μg/disc) revealed 35.33–67.66 and 18.0–53.3% antidermatophytic effect, respectively, along with their respective MIC values (62.5–500 and 250–4000 μg/ml) against Trichophyton rubrum KCTC 6345, T. rubrum KCTC 6375, T. rubrum KCTC 6352, T. mentagrophytes KCTC 6085, T. mentagrophytes KCTC 6077, T. mentagrophytes KCTC 6316, Microsporum canis KCTC 6591, M. canis KCTC 6348 and M. canis KCTC 6349. The oil also had a strong detrimental effect on spore germination as well as concentration and time-dependent kinetic inhibition of M. canis KCTC 6591.     

Organic cation transporter 1 mediates the uptake of monocrotaline and plays an important role in its hepatotoxicity

Monocrotaline (MCT) is a kind of toxic retronecine-type pyrrolizidine alkaloids (PAs) from plants of Crotalaria, which can be bio-activated by cytochrome P450 (CYP) enzymes in liver and then induce hepatotoxicity. Since CYPs are localized in the endoplasmic reticulum, the influx of MCT to the liver is the key step for its hepatotoxicity. The objective of the present study was to investigate the role of organic cation transporter 1 (OCT1), a transporter mainly expressed in liver, in the uptake of MCT and in hepatotoxicity induced by MCT. The results revealed that MCT markedly inhibited the uptake of 1-methyl-4-phenylpyridinium (MPP⁺), an OCT1 substrate, in Madin–Darby canine kidney (MDCK) cells stably expressing human OCT1 (MDCK-hOCT1) with the IC₅₀ of 5.52 ± 0.56 μM. The uptake of MCT was significantly higher in MDCK-hOCT1 cells than in MDCK-mock cells, and MCT uptake in MDCK-hOCT1 cells followed Michaelis–Menten kinetics with the K_m and V_max values of 25.0 ± 6.7 μM and 266 ± 64 pmol/mg protein/min, respectively. Moreover, the OCT1 inhibitors, such as quinidine, d-tetrahydropalmatine (d-THP), obviously inhibited the uptake of MCT in MDCK-hOCT1 cells and isolated rat primary hepatocytes, and attenuated the viability reduction and LDH release of the primary cultured rat hepatocytes caused by MCT. In conclusion, OCT1 mediates the hepatic uptake of MCT and may play an important role in MCT induced-hepatotoxicity.     

A peripherally acting,selective T-type calcium channel blocker,ABT-639, effectively reduces nociceptive and neuropathic pain in rats

Activation of T-type Ca²⁺ channels contributes to nociceptive signaling by facilitating action potential bursting and modulation of membrane potentials during periods of neuronal hyperexcitability. The role of T-type Ca²⁺ channels in chronic pain is supported by gene knockdown studies showing that decreased Ca_v3.2 channel expression results in the loss of low voltage-activated (LVA) currents in dorsal root ganglion (DRG) neurons and attenuation of neuropathic pain in the chronic constriction injury (CCI) model. ABT-639 is a novel, peripherally acting, selective T-type Ca²⁺ channel blocker. ABT-639 blocks recombinant human T-type (Ca_v3.2) Ca²⁺ channels in a voltage-dependent fashion (IC₅₀ = 2 μM) and attenuates LVA currents in rat DRG neurons (IC₅₀ = 8 μM). ABT-639 was significantly less active at other Ca²⁺ channels (e.g. Ca_v1.2 and Ca_v2.2) (IC₅₀ > 30 μM). ABT-639 has high oral bioavailability (%F = 73), low protein binding (88.9%) and a low brain:plasma ratio (0.05:1) in rodents. Following oral administration ABT-639 produced dose-dependent antinociception in a rat model of knee joint pain (ED₅₀ = 2 mg/kg, p.o.). ABT-639 (10–100 mg/kg, p.o.) also increased tactile allodynia thresholds in multiple models of neuropathic pain (e.g. spinal nerve ligation, CCI, and vincristine-induced, and capsaicin secondary hypersensitivity). ABT-639 did not attenuate hyperalgesia in inflammatory pain models induced by complete Freund's adjuvant or carrageenan. At higher doses (e.g. 100 - 300 mg/kg) ABT-639 did not significantly alter hemodynamic or psychomotor function. The antinociceptive profile of ABT-639 provides novel insights into the role of peripheral T-type (Ca_v3.2) channels in chronic pain states.     

Pterostilbene from Vitis coignetiae protect H2O2-induced inhibition of gap junctional intercellular communication in rat liver cell line

In this study, we identified various stilbenoids derived from Vitis coignetiae and investigated the protective effect of the main component, pterostilbene, against the inhibition of gap junctional intercellular communication (GJIC) induced by H₂O₂ in WB-F344 rat liver epithelial cells. We analyzed seven kinds of stilbenoids, pterostilbene, astringin, piceid, vitisin, rhaponticin, resveratrol, and rhapontigenin, using DAD/UV HPLC. Total stilbenoid content was 127.37 ± 19.29 mg/100 g dry weight. Pretreatment with 0.5, 1.0, and 5.0 μM pterostilbene for 24 h was shown to recover GJIC blocked by 500 μM H₂O₂. Pretreatment with pterostilbene prevented the inhibition of GJIC via the down-regulation of connexin43 phosphorylation by the inactivation of ERK1/2 and p38 MAP kinase. Our results suggest that pterostilbene may be a functional chemopreventative agent and that dietary exposure of pterostilbene would be helpful for improving health.     

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A₂ and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA₂ (BmarPLA₂) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA₂ was isolated from the venom, and N-terminal amino acid sequencing of sPLA₂ showed high amino acid identity with other lysine K49 sPLA₂s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 μg/mL and MLC = 200 μg/mL. However, the BmarTV and BmarPLA₂ did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC₅₀ = 2.55 μg/mL and 2.86 μg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC₅₀ of 86.56 μg/mL for L. amazonensis and 79.02 μg/mL for L. chagasi. BmarPLA₂ did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.     

Antioxidant and hepatoprotective activities of phenolic rich fraction of Seabuckthorn (Hippophae rhamnoides L.) leaves

Present study was aimed to investigate antioxidant and hepatoprotective activities of phenolic rich fraction (PRF) of Seabuckthorn leaves on CCl₄ induced oxidative stress in Sprague Dawley rats. Total phenolic content was found to be 319.33 mg gallic acid equivalent (GAE)/g PRF and some of its phenolic constituents, such as gallic acid, myricetin, quercetin, kaempferol and isorhamnetin were found to be in the range of 1.935-196.89 mg/g of PRF as determined by reverse-phase high-performance liquid chromatography (RP-HPLC).Oral administration of PRF at dose of 25-75 mg/kg body weight significantly protected from CCl₄ induced elevation in aspartate aminotransferase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (GGT) and bilirubin in serum, elevation in hepatic lipid peroxidation, hydroperoxides, protein carbonyls, depletion of hepatic reduced glutathione (GSH) and decrease in the activities of hepatic antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-S-transferase (GST). The PRF also protected against histopathological changes produced by CCl₄ such as hepatocytic necrosis, fatty changes, vacuolation, etc. The data obtained in the present study suggests that PRF has potent antioxidant activity, prevent oxidative damage to major biomolecules and afford significant protection against CCl₄ induced oxidative damage in the liver.     

Simultaneous measurement of amiodarone and desethylamiodarone in human plasma and serum by stable isotope dilution liquid chromatography–tandem mass spectrometry assay

A stable isotope dilution liquid chromatography–electrospray ionization tandem mass spectrometry (LC–MS/MS) assay to measure amiodarone, the most frequently used agent for maintaining sinus rhythm in patients with atrial fibrillation, and its major metabolite desethylamiodarone in human plasma and serum was developed. Measurement of amiodarone and desethylamiodarone was performed during a 4.0-min run-time using amiodarone-D₄ and desethylamiodarone-D₄ as internal standards. Calibration curves covering 12 calibrators measured in four replicates each for the analysis of both amiodarone and desethylamiodarone were linear and reproducible in the range of 0.01–40.0 mg/L (r > 0.999). Limits of detection in plasma matrix were 2.7 μg/L for amiodarone and 1.9 μg/L for desethylamiodarone, and lower limits of quantification in plasma matrix were 7.5 μg/L for amiodarone and 2.5 μg/L for desethylamiodarone. Interassay imprecision and inaccuracy were <8% and <9% for both substances. Mean extraction yield was 99.6% (range 92.6–107.7%) for amiodarone and 90.2% (range 80.0–94.7%) for desethylamiodarone. Agreement was moderate for amiodarone (n = 162) and desethylamiodarone (n = 117), respectively, between the present method and a HPLC method with UV detection using a commercially available reagent set for the HPLC analysis of these drugs. The Passing–Bablok regression line was HPLC = 0.98 (LC–MS/MS) + 0.10 [mg/L]; r = 0.94 for amiodarone and HPLC = 1.05 (LC–MS/MS) + 0.02 [mg/L]; r = 0.90 for desethylamiodarone. This sensitive and interference-free LC–MS/MS assay permits rapid and accurate determination of amiodarone and desethylamiodarone in human plasma and other body fluids.     

Quantification of 4′-geranyloxyferulic acid,a new natural colon cancer chemopreventive agent,by HPLC-DAD in grapefruit skin extract

Oxyprenylated natural products (isopentenyloxy-, geranyloxy- and the less spread farnesyloxy-compounds and their biosynthetic derivatives) represent a family of secondary metabolites that have been consider for years merely as biosynthetic intermediates of the most abundant C-prenylated derivatives. Many of the isolated oxyprenylated natural products were shown to exert in vitro and in vivo remarkable anti-cancer and anti-inflammatory effects. 4′-Geranyloxyferulic acid [3-(4′-geranyloxy-3′-methoxyphenyl)-2-trans-propenoic] has been discovered as a valuable chemopreventive agent of several types of cancer. After development of a high yield and “eco-friendly” synthetic scheme of this secondary metabolite, starting from cheap and non-toxic reagents and substrates, we developed a new HPLC-DAD method for its quantification in grapefruit skin extract. A preliminary study on C18 column showed the separation between GOFA and boropinic acid (having the same core but with an isopentenyloxy side chain), used as internal standard. The tested column were thermostated at 28 ± 1 °C and the separation was achieved in gradient condition at a flow rate of 1 mL/min with a starting mobile phase of H₂O:methanol (40:60, v/v, 1% formic acid). The limit of detection (LOD, S/N = 3) was 0.5 μg/mL and the limit of quantification (LOQ, S/N = 10) was 1 μg/mL. Matrix-matched standard curves showed linearity up to 75 μg/mL. In the analytical range the precision (RSD%) values were ≤2% and the accuracy (bias%) between ±12%. This method was used to evaluate for the first time the presence of this analyte in natural extract of grapefruit. In conclusion, this method showed LOQ values able to selective quantification of this analyte in grapefruit skin extract.     

Clerodendron glandulosum.Coleb extract ameliorates high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis

This study evaluates the protective role of Clerodendron glandulosum.Coleb (CG) aqueous extract against high fat diet/fatty acid induced lipotoxicity in experimental models of non-alcoholic steatohepatitis (NASH). Supplementation of NASH mice with CG extract (1% and 3% in high fat diet for 16 weeks) prevented high fat diet induced elevation in liver enzymes, plasma and hepatic lipids, mitochondrial oxidative stress and compromised enzymatic and non-enzymatic antioxidant status and histopathological damage to hepatocytes. Furthermore, results from in vitro study indicates, addition of CG extract (20–200 μg/ml for 24 h) to HepG2 cells minimizes oleic acid induced lipid accumulation, higher lipid peroxidation, cytotoxicity and reduced cell viability. These in vivo and in vitro studies suggest that CG extract has the potential of preventing high fat/fatty acid induced NASH.