Analysis of urinary andrographolides and antioxidant status after oral administration of Andrographis paniculata leaf extract in rats

A simple high-performance liquid chromatography (HPLC) method was developed to determine the content of andrographolide (AP) and 14-deoxy-11,12-dideoxyandrographolide (DIAP) in a pooled urine of rat obtained within 24 h after an oral dose of Andrographis paniculata leaf extract at 1 g/kg body weight. Cumulative urinary excretion of AP and DIAP in 24 h after oral administration of the extract was 0.88% and 1.61% of oral dose administered, respectively. The extract showed significant reduction (p < 0.05) of MDA levels and elevation of total antioxidant status in rat urine samples collected in 24 after oral administration.     

Antioxidant and antilisterial effect of seed essential oil and organic extracts from Zizyphus jujuba

Hydrodistilled volatile oil from the seeds of Zizyphus jujuba was analyzed by GC–MS. Twenty three compounds representing 91.59% of the total oil was identified. The oil and organic extracts revealed a great potential of antilisterial effect against all five strains of Listeria monocytogenes ATCC 19111, 19116, 19118, 19166 and 15313. Also the oil had strong detrimental effect on the viable count of the tested bacteria. The samples were also subjected to screening for the antioxidant activity by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals scavenging activities assay. In the first case, the IC₅₀ value of the Z. jujuba essential oil was determined to be 5.21 ± 0.01 μg/ml. Among the extracts, the strongest activity was exhibited by the methanol extract with an IC₅₀ value of 20.44 ± 0.18 μg/ml. In the superoxide radicals scavenging activities assay, methanol extract was superior to all other extracts (IC₅₀ = 18.60 ± 0.3 μg/ml). Furthermore, the amount of total phenolic compounds was determined. The results indicate that the essential oil and extracts of Z. jujuba could serve as natural antimicrobial and antioxidant agents for the food industry.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana 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 36.93–68.76% 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 93.46 and 26.75 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 S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 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 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Magnolia liliflora Desr.

This study was carried out to assess the antioxidant and antidermatophytic activities of the essential oil and extracts of Magnolia liliflora Desr. 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 values = 10.11 and 16.17 μg/ml, respectively) as compared to butylatedhydreoxyanisole (BHA), (IC₅₀ value = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (96.13 mg/g of dry wt) as compared to the other extracts. Further, the oil (1000 μg/disc) and extracts (1500 μg/disc) revealed 42.36–63.12% and 19.07–54.14% antidermatophytic effect, respectively along with their respective MIC values ranging from 62.5 to 500 and 250 to 2000 μg/ml against the members of Trichophyton and Microsporum spp. Also the oil had strong detrimental effect on spore germination of tested fungal pathogens as well as concentration and time dependent kinetic inhibition of Microsporum canis KCTC 6348. The results of this study justify a potential role of M. liliflora to serve as a natural antioxidant and antidermatophytic agent.     

Antioxidant activities, metal contents, total phenolics and flavonoids of seven Morchella species

Seven Morchella species were analyzed for their antioxidant activities in different test systems namely β-carotene/linoleic acid, DPPH, reducing power, chelating effect and scavenging effect (%) on the stable ABTS⁺, in addition to their heavy metals, total phenolic and flavonoid contents. In β-carotene/linoleic acid system, the most active mushrooms were M. esculenta var. umbrina and M. angusticeps. In the case of DPPH, methanol extract of M. conica showed high antioxidant activity. The reducing power of the methanol extracts of mushrooms increased with concentration. Chelating capacity of the extracts was also increased with the concentration. On the other hand, in 40 μg ml⁻¹ concentration, methanol extract of M. conica, exhibited the highest radical scavenging activity (78.66 ± 2.07%) when reacted with the ABTS⁺ radical. Amounts of seven elements (Cu, Mn, Co, Zn, Fe, Ca, and Mg) and five heavy metals (Ni, Pb, Cd, Cr, and Al) were also determined in all species. M. conica was found to have the highest phenolic content among the samples. Flavonoid content of M. rotunda was also found superior (0.59 ± 0.01 μg QEs/mg extract).     

LC-MS/MS method for determination of hederacolchiside E, a neuroactive saponin from Pulsatilla koreana extract in rat plasma for pharmacokinetic study

A simple, rapid, and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was applied to pharmacokinetic study of a neuroactive oleanolic-glycoside saponin, hederacolchiside E from SK-PC-B70M, a standardized extract of Pulsatilla koreana in rat. Rat plasma samples were pretreated by protein precipitation with acetonitrile, eluted from C₁₈ column, and analyzed using electrospray ionization (ESI)-MS/MS in negative ion mode. Digoxin was used as an internal standard. The standard curves were linear (r > 0.997) over the concentration ranges of 2–500 ng/mL. The intra- and inter-day precisions were measured to be below 9% and accuracy between 90 and 111% for all quality control samples at 2, 20, 100, and 500 ng/mL (n = 5). The lower limits of quantification (LLOQ) for hederacolchiside E was 2 ng/mL and the limit of detection (LOD) 0.5 ng/mL using 20 μL of plasma sample. Subsequently, hederacolchiside E was determined in rat plasma samples after oral administration of SK-PC-B70M. The mean maximum plasma concentrations of hederacolchiside E were 0.07, 0.13, and 0.36 μg/mL and the mean areas under the plasma concentration versus time curve 0.56, 1.27, and 6.46 μg h/mL at doses of 100, 200, and 400 mg/kg, respectively, which indicated non-linear pharmacokinetic pattern. In conclusion, this method was successfully applied to the pharmacokinetic study of hederacolchiside E after an oral administration of SK-PC-B70M to rats.     

Antihypertensive and vasorelaxant activities of Laelia autumnalis are mainly through calcium channel blockade

The aim of the present study was to evaluate the possible mechanism of the vasorelaxant action of methanol extract from Laelia autumnalis (MELa) in isolated rat aortic rings, and to establish its antihypertensive activity in vivo. MELa (0.15→50 µg/mL) induced relaxation in aortic rings pre-contracted with KCl (80 mM), showing an IC₅₀ value of 34.61 ± 1.41 µg/mL and E_max value of 85.0 ± 4.38% (in endothelium-intact rings) and an IC₅₀ value of 45.11 ± 4.17 µg/mL and E_max value of 80.0 ± 12.1% (in endothelium-denuded rings). Serotonin (5-HT, 1 × 10^− 4 M) provoked sustained contraction, which was markedly inhibited by MELa (0.15→50 µg/mL) in a concentration-dependent and endothelium-independent manner. Pretreatment with MELa (15, 46, 150, 300 and 1500 µg/mL) also inhibited contractile responses to norepinephrine (NE 1 × 10^− 11 M to 1 × 10^− 5.5 M). In endothelium-denuded rings, the vasorelaxant effect of MELa was reduced partially by ODQ (1 µM), but not by tetraethylammonium (5 µM), glibenclamide (10 µM), and 2-aminopyridine (100 µM). The extract also reduced NE-induced transient contraction in Ca²⁺-free solution, and inhibited contraction induced by increasing external calcium in Ca²⁺-free medium plus high KCl (80 mM). The antihypertensive effect of MELa was determined in spontaneously hypertensive rats (SHR). A single oral administration of the extract (100 mg/kg) exhibited a significant decrease in systolic and diastolic blood pressure and heart rate (p < 0.05) in SHR rats. Our results suggest that MELa induces relaxation in rat aortic rings through an endothelium-independent pathway, involving blockade of Ca²⁺ channels and a possible cGMP enhanced concentrations and also causes an antihypertensive effect.     

Studies on the antioxidant activity of essential oil and different solvent extracts of Vitex agnus castus L. fruits from Turkey

This study is designed to examine the chemical composition and antioxidant activity of the essential oil and different solvent extracts of Vitex agnus castus. GC and GC–MS analysis was resulted in the detection of 27 components, representing 94.5% of the oil. Major components of the oil were 1,8-cineole (24.98%), sabinene (13.45%), α-pinene (10.60%), α-terpinyl acetate (6.66%), and (Z)-β-farnesene (5.40%). Antioxidant activities of the samples were determined by three different test systems, DPPH, β-carotene/linoleic acid and reducing power assays. In all systems, water extract exhibited excellent activity potential than those of other extracts (hexane, dichloromethane, ethyl acetate and methanol) and the oil. As expected, amount of total phenolics was very high in this extract (112.46 ± 1.22 μg GAEs/mg extract). Dichloromethane extract has been found to be rich in flavonoids. A positive correlation was observed between the antioxidant activity potential and total phenolic and flavonoid levels of the extracts.     

Hsian-tsao (Mesona procumbens Heml.) prevents against rat liver fibrosis induced by CCl4 via inhibition of hepatic stellate cells activation

In this study, the protective effect of extract of Hsian-tsao (Mesona procumbens) (EHT) against liver fibrogenesis in carbon tetrachloride (CCl₄)-injured rats was evaluated. The inhibitory effect of oleanolic acid (OA) and ursolic acid (UA), which are the active compounds in EHT, on the activation of hepatic stellate cells (HSC) was also determined. The results showed that EHT at a dosage of 1.2 g/kg of b.w. significantly reduced the liver injury induced by CCl₄ in rats. It also decreased the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the deposition of collagen in the liver. Oral administration of EHT reduced the levels of alpha-smooth muscle actin (α-SMA) and the activity of metalloproteinases (MMPs) in rats injured by treatment with CCl₄. In addition, we performed experiments with the rat hepatic stellate cell line HSC-T6 in which we induced the expression of MMP-2 and α-SMA with phorbol-12-myristate-13-acetate (PMA). Treating these cells with OA (20 μM) or UA (10 μM) caused a decrease in the levels of both proteins. Taken together, our data indicate that EHT can efficiently inhibit CCl₄-induced liver fibrosis in rats. EHT may therefore be a useful functional food for preventing liver fibrosis.     

A sensitive LC–MS/MS method to quantify loureirin B in rat plasma with application to preclinical pharmacokinetic studies

A novel method for the quantification of loureirin B in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC–MS/MS) was developed. Loureirin B and internal standard (buspirone) were extracted by liquid–liquid extraction and separated on a Agilent XDB C₁₈ column (50 mm × 4.6 mm, 5 μm). As mobile phase a binary mixture of methanol (containing 0.1% formic acid)–water (containing 0.1% formic acid) was delivered by a Shimadzu LC-20AD pump in gradient mode at a flow rate of 0.4 ml/min in a run time of 5.0 min. The detector was a Q-trap™ mass spectrometer with an electrospray ionization (ESI) interface operating in the multiple reaction monitoring (MRM) mode. The calibration curve of loureirin B in plasma showed good linearity over the concentration range of 0.08–100 ng/ml. The limit of detection and limit of quantification were 0.03 ng/ml and 0.08 ng/ml, respectively. Intra- and inter-day precisions (as relative standard deviation) in all samples were both within 15%. The validated method was successfully applied to a preliminary pharmacokinetic study of loureirin B in rats. After oral administration of 16 g/kg longxuejie to rats, the main pharmacokinetic parameters t_max, C_max, t_1/2, K_e and AUC_0–T were 0.8 h, 7.99 μg/l, 1.94 l h, 0.365/h, and 22.21 μg h/l, respectively.     

Steady-state pharmacokinetics and pharmacodynamics of piperacillin/tazobactam administered by prolonged infusion in hospitalised patients

The objective of this study was to evaluate the steady-state pharmacokinetics and pharmacodynamics of piperacillin/tazobactam, administered by prolonged infusion, in hospitalised patients requiring antimicrobial therapy. Thirteen patients received 4.5 g every 8 h (q8h), infused over 4 h, and pharmacokinetic parameters were determined by non-compartmental methods. Monte Carlo simulations (10 000 patients) were performed to calculate the cumulative fraction of response (CFR) for seven Gram-negative pathogens using minimum inhibitory concentration (MIC) data from the Meropenem Yearly Susceptibility Test Information Collection (2004–2007, USA) as well as the probability of target attainment (PTA) at MICs ranging from 1 μg/mL to 64 μg/mL. The pharmacodynamic target was free piperacillin concentration remaining above the MIC for 50% of the dosing interval. Mean ± standard deviation maximum and minimum serum concentrations, half-life, volume of distribution at steady-state and systemic clearance of piperacillin were 108.2 ± 31.7 μg/mL, 27.6 ± 26.3 μg/mL, 2.1 ± 1.2 h, 22.1 ± 4.0 L and 8.6 ± 3.0 L/h, respectively. The CFR was >90% for Escherichia coli, Serratia marcescens and Citrobacter spp., 88.6% for Enterobacter spp., 87% for Klebsiella pneumoniae, 85.5% for Pseudomonas aeruginosa and 52.8% for Acinetobacter spp. The PTA was 100%, 81.1% and 12.3% at MICs of ≤16 μg/mL, 32 μg/mL and 64 μg/mL, respectively. Piperacillin/tazobactam 4.5 g q8h infused over 4 h provides excellent target attainment for bacterial pathogens with MICs ≤ 16 μg/mL. However, the CFR was <90% for four of the seven Gram-negative pathogens evaluated.     

Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats

The objective of present investigation was to study the effect of verapamil on the pharmacokinetics of irinotecan in order to evaluate the role of P-glycoprotein (P-gp) in irinotecan disposition. An in vitro study using Caco-2 intestinal cell monolayer was first carried out to determine the effect of verapamil on the function of intestinal P-gp. Verapamil (25 mg/kg) was administered orally 2 h before irinotecan oral (80 mg/kg) or intravenous (20 mg/kg) dosing in female Wistar rats. Plasma and biliary samples were collected at specified time points from control and treated animals to determine irinotecan and its metabolite, SN-38 concentrations. Bi-directional transport and inhibition studies in Caco-2 cells indicated irinotecan to be a P-gp substrate and the function of intestinal P-gp was significantly inhibited in presence of verapamil. After oral irinotecan dosing, the mean area under the plasma concentration–time curve (AUC) was found to be 14.03 ± 2.18 μg h/ml which was increased significantly, i.e. 61.71 ± 15.0 μg h/ml when verapamil was co-administered (P < 0.05). Similarly, the mean maximum plasma concentration of irinotecan increased from 2.93 ± 0.37 μg/ml (without verapamil) to 10.75 ± 1.0 μg/ml (with verapamil) (P < 0.05). There was approximately 4–5-folds increase in apparent bioavailability. On the other hand, the intravenous irinotecan administration with verapamil resulted in small but statistically significant effect on AUC (10.76 ± 2.0 to 23.3 ± 3.8 μg h/ml; P < 0.05) and systemic clearance (1206.4 ± 159.7 to 713.5 ± 78.2 ml/(h kg)). In addition, SN-38 showed significant change in oral pharmacokinetic parameters and minor changes in intravenous pharmacokinetic profile. Biliary excretion curves of both irinotecan and SN-38 were lowered by verapamil. The mean percent of irinotecan excreted into bile over 5 h following intravenous and oral administration was found to be 8% and 1%, respectively, which was further reduced to half when treated with verapamil. These results are quite stimulating for further development of a clinically useful oral formulation of irinotecan based on P-gp inhibition.     

Pharmacological properties of ATP-sensitive purinergic receptors expressed in human G292 osteoblastic cells

We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca²⁺ level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC₅₀ of 0.5 ± 0.07 μM. While α,β-methylene-ATP (αβmeATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC₅₀ of 0.7 ± 0.2 μM. UTP and UDP also evoked responses with EC₅₀ of 2.0 ± 0.4 μM and 0.5 ± 0.1 μM respectively, but their responses were much smaller, resulting in an order of the response magnitude: ATP ~ ADP >> UTP ~ UDP. The responses evoked by ATP and ADP were blocked by pyridoxal-5'-phosphate-6-azophenyl-2,4,-disulfonate (PPADS) with IC₅₀ of 3.0 ± 0.05 μM and 5.0 ± 0.4 μM respectively, but not by suramin up to 30 μM. ATP-evoked responses were insensitive to inhibition by trinitrophenyl-ATP (TNP-ATP) and brilliant blue G. ADP-evoked responses were significantly inhibited by 2'-deoxy-N⁶-methyladenosine-3',5'-biphosphate (MRS2179) and 2-chloro-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279) with IC₅₀ of 48 ± 1.9 μM and 7.7 ± 0.9 μM respectively. Taken together, these results provide strong evidence for functional expression of ATP-sensitive P2Y receptors and particularly P2Y₁-like receptor in G292 cells.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the α-phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Systematic evaluation of the antioxidant potential of different parts of Foeniculum vulgare Mill. from Portugal

Fennel (Foeniculum vulgare Mill.) is a widespread perennial umbeliferous (Apiaceae) herb, traditionally used for medicinal purposes and human consumption. It is highly recommended for diabetes, bronchitis and chronic coughs, and for the treatment of kidney stones; some of those chronic diseases are related to the production of radical species involved in the oxidative stress. Therefore, the antioxidant potential of this herb might explain some of their empirical uses in folk medicine. This is the first time that a systematic study on different parts of fennel is performed, in order to understand differences in the antioxidant potential of shoots, leaves, steams, and inflorescences, particularly related to their composition in antioxidant compounds such as vitamins (ascorbic acid and tocopherols) and phenolics. The shoots seems to have the highest radical-scavenging activity and lipid peroxidation inhibition capacity (EC₅₀ values < 1.4 mg/ml), which is in agreement with the highest content in phenolics (65.85 ± 0.74 mg/g) and ascorbic acid (570.89 ± 0.01 μg/g) found in this part. The shoots also revealed high concentration of tocopherols (34.54 ± 1.28 μg/g) and were the only part with flavonoids.     

Hepatoprotective and antioxidant activity of methanol extract of <Emphasis Type="Italic">Ficus glomerata</Emphasis>

The present study was carried out to evaluate Ficus glomerata extracts for antioxidant and hepatoprotective properties. The methanol extract of the bark of F. glomerata showed potent in vitro antioxidant activity when compared to the root methanol extract. In the in vivo studies, the CCl₄ treated control rats showed a significant alteration in the levels of antioxidant and hepatoprotective parameters. The methanol extract of the bark when given orally along with CCl₄ at the doses of 250 and 500 mg/kg body weight showed a significant reversal of these biochemical changes towards the normal when compared to CCl₄-treated control rats in serum, liver and kidney. The results were comparable to those observed for standard sylimarin. Histological studies also confirmed the same. The results indicated the potent hepatoprotective and antioxidant nature of the bark extract.     

A pharmacological examination of venom from the Papuan taipan: (Oxyuranus scutellatus canni)

The Papuan taipan (Oxyuranus scutellatus canni) is the third most venomous terrestrial snake in the world, however, little is know about the pharmacology of the venom. In the chick biventer cervicis muscle, venom (10 μg/ml) abolished nerve-mediated twitches (time to 90% inhibition (t₉₀) 44±5 min, n=9). This inhibition was unaffected by prior incubation of the venom with the phospholipase A inhibitor 4-bromophenacyl bromide (4-BPB; 0.72 mM) (t₉₀ 48±7 min, n=8). The mouse phrenic nerve diaphragm preparation displayed greater sensitivity to venom (10 μg/ml) (t₉₀ 25±1 min, n=6). In the chick biventer muscle, venom (10 μg/ml) significantly inhibited responses to acetylcholine (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. Venom (10 μg/ml) did not affect direct muscle stimulation. Venom (3–30 μg/ml) produced dose-dependant contractions of the guinea-pig ileum. Contractile responses were significantly inhibited by indomethacin (1 μM) or prior incubation of the venom with 4-BPB (0.72 mM) indicating involvement of a PLA component. In rat phenylephrine (0.3 μM) precontracted aortae, venom (3–100 μg/ml) produced endothelium-independent relaxation which was unaffected by prior incubation of venom (30 μg/ml) with 4-BPB (0.72 mM). In anaesthetised rats, 10 μg/kg (i.v.) venom produced rapid respiratory and cardiovascular collapse while 5 μg/kg (i.v.) venom produced only a small transient decrease in mean arterial blood pressure. Prior administration of 5 μg/kg (i.v.) venom enabled subsequent administration of 10 and 100 μg/kg (i.v.) venom without respiratory or cardiovascular collapse. Further work is required to identify specific toxins with the above pharmacological activity.     

Effects of Tityus serrulatus scorpion venom on lung mechanics and inflammation in mice

The present study evaluated the effects of an intramuscular injection of Tityus serrulatus venom (TsV) (0.67 μg/g) on lung mechanics and lung inflammation at 15, 30, 60 and 180 min after inoculation. TsV inoculation resulted in increased lung elastance when compared with the control group (p < 0.001); these values were significantly higher at 60 min than at 15 and 180 min (p < 0.05). Resistive pressure (ΔP₁) values decreased significantly at 30, 60 and 180 min after TsV injection (p < 0.001). TsV inoculation resulted in increased lung inflammation, characterised by an increased density of mononuclear cells at 15, 30, 60 and 180 min after TsV injection when compared with the control group (p < 0.001). TsV inoculation also resulted in an increased pulmonary density of polymorphonuclear cells at 15, 30 and 60 min following injection when compared to the control group (p < 0.001). In conclusion, T. serrulatus venom leads to acute lung injury, characterised by altered lung mechanics and increased pulmonary inflammation.     

Determination of ranolazine in human plasma by LC-MS/MS and its application in bioequivalence study

A simple, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantification of ranolazine in human plasma. The analytical method consists in the precipitation of plasma sample with methanol, followed by the determination of ranolazine by an LC–MS/MS. The analyte was separated on a Peerless Cyano column (33 mm × 4.6 mm, 3 μm) an isocratic mobile phase of methanol–water containing formic acid (1.0%, v/v) (65:35, v/v) at a flow rate of 1.0 ml/min. Protonated ions formed by a turbo ionspray in positive mode were used to detect analyte and internal standard (IS). The MS/MS detection was made by monitoring the fragmentation of m/z 428.20 → 279.50 for ranolazine and m/z 448.30 → 285.20 for internal standard on a triple quadrupole mass spectrometer. The method was validated over the concentration range of 5–2000 ng/ml for ranolazine in human plasma with correlation coefficient of 0.9937 (S.D.: ±0.00367, range: 0.9895–0.9963). The accuracy and precision values obtained from six different sets of quality control samples analyzed in separate occasions ranged from 94.53 to 117.86 and 0.14% to 4.56%, respectively. Mean extraction recovery was 82.36–94.25% for three quality control (QC) samples and 88.37% for IS. Plasma samples were stable for three freeze–thaw cycles, or 24 h ambient storage, or 1 and 3 months storage at −20 °C. Processed samples (ready for injection) were stable up to 72 h at autosampler (4 °C). The developed method was successfully applied for analyzing ranolazine in plasma samples for a bioequivalence study with 12 healthy volunteers.