In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice

Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.     

Pyrogenic effects of cytokines (IL-1β, IL- 6, TNF-α) and their mode of action on thermoregulatory centers and functions

The objective of this study was to compare the thermoregulatory responses of rabbits to fever-inducing doses of various cytokines (IL-1β, IL-6, TNF-α) injected peripherally (i.v.), or centrally (i.h.).TNF-α (1 μg kg⁻¹), when applied i.v. at thermoneutral conditions, induced a long lasting increase in hypothalamic temperature, which reached maximal values within 50 min and then persisted for at least 3 h. This monophasic fever-like response was due to extensive and long lasting attenuation of panting and due to transient vasoconstriction. Metabolic rate tended to increase during the early phase of the fever, only. On the other hand, IL-6 when applied i.v. in the same dose (1 μg kg⁻¹) and IL-1β, in a much lower dose (60 ng kg⁻¹), induced a short lasting monophasic hyperthermia due to transient attenuation of panting and due to transient vasoconstriction. No significant increase in metabolic rate was observed. The immediate attenuation of panting and induction of vasoconstriction rather resembled reflex (shock) responses than specific thermoregulatory reactions. Thirty minutes after i.v. administration of TNF-α, temperature thresholds for induction of cold thermogenesis, panting and vasomotion were shifted to higher body temperatures and remained elevated for at least 3 h. In case of IL-1β the increased temperature thresholds were observed 30 min after i.v. administration, only. I.v. applied IL-6 did not influence the thresholds neither 30 or 180 min after injection. Thus, peripheral IL-6 rather induced hyperthermia than fever.When injected i.h. all cytokines studied induced a long lasting increase in body temperature similar to that after i.v. injection of LPS. Temperature thresholds for induction of all thermoregulatory outputs were increased and remained increased for at least 3 h. No changes in hypothalamic thermosensitivity were observed. Data indicate a nonspecific effect of central cytokines on body temperature control.IL-1β appeared to be the most potent fever inducer. Nanogram doses of IL-1β injected i.v. induced a similar febrile response as microgram doses of other cytokines. On the other hand, TNF-α induced a longer lasting fever than other cytokines.     

The roles of aerobic exercise training and suppression IL-6 gene expression by RNA interference in the development of insulin resistance

ObjectiveTo demonstrate the hypothesis that aerobic exercise training inhibits the development of insulin resistance through IL-6 and probe into the possible molecular mechanism about it.MethodsRats were raised with high-fat diets for 8 weeks to develop insulin resistance, and glucose infusion rates (GIRs) were determined by hyperinsulinemic–euglycemic clamping to confirm the development of insulin resistance. Aerobic exercise training (the speed and duration time in the first week were respectively 16 m/min and 50 min, and speed increased 1 m/min and duration time increased 5 min every week following it) and/or IL-6shRNA plasmid injection (rats received IL-6shRNA injection via the tail vein every two weeks) were adopted during the development of insulin resistance. The serum IL-6, leptin, adiponectin, fasting blood glucose, fasting serum insulin, GIR, IL-6 gene expression levels, p-p38 in various tissues and p-STAT3/t-STAT3 ratio in the liver were measured.ResultsRats fed with high-fat diets for 8 weeks were developed insulin resistance and the IL-6mRNA levels of IL-6shRNA injection groups in various tissues were significantly lower than those of control group (P < 0.05), respectively. The development of insulin resistance in exercise rats significantly decreased, however, compared with that, the GIR of exercise rats injected by IL-6shRNA was lower (P < 0.05). The IL-6mRNA levels were highest in the fat tissue and lowest in the skeletal muscles in all the rats. The serum adiponectin levels decreased (P < 0.05) following the development of insulin resistance, and it increased (P < 0.05) when the rats were intervened by aerobic exercise training for 8 weeks at the same time. However, there were not significant differences when serum leptin concentrations were compared (P > 0.05). The p-p38 significantly increased in the rats fed with high-fat diets, however, p-p38 of the exercise high-fat diets rats in the liver and fat tissues significantly decreased than that (P < 0.05). The changes of p-p38 in exercise rats injected by IL-6shRNA were irregular. The activation of STAT3 in the liver significantly increased (P < 0.05) following the development of insulin resistance, and it decreased (P < 0.05) when the rats were intervened by aerobic exercise training for 8 weeks at the same time, and the gene silencing of IL-6 did not have effects on the activation of STAT3 in the liver (P > 0.05).ConclusionsIn conclusion, aerobic exercise training prevented the development of insulin resistance through IL-6 to a certain degree. The gene expression and secretion of IL-6 could inhibit the development of insulin resistance. The mechanism of the effects were possibly related with elevating the levels of serum adiponectin, and/or inhibiting the activation of STAT3 in the liver and p38MAPK in the skeletal muscles, liver and fat tissues.     

High intensity interval exercise decreases IL-8 and enhances the immunomodulatory cytokine interleukin-10 in lean and overweight–obese individuals

PurposeTo compare the effects of two interval exercises with different intensities on acute inflammatory response in lean and overweight–obese subjects.MethodsTen lean (BMI < 24.9 kg/m²) and 12 overweight–obese (BMI 25 to <34.9 kg/m²) males performed two conditions in randomly assigned: (1) high intensity interval exercise (HIIE) 10 × 60 s (85–90%P_Max)/75 s (50%P_Max); (2) moderate intensity interval exercise (MIIE) 10 × 60 s (70–75%P_Max)/60 s (50%P_Max), with blood collections at pre, immediately and 30 min post each exercise bouts to evaluate total and differential leukocyte counts, serum creatine kinase (CK), lactate dehydrogenase (LDH) and systemic levels of IL-1ra, IL-6, IL-8, IL-10, IL-17a and CCL2.ResultsIn lean group, HIIE induced a significant increase in total leukocytes and monocyte, while MIIE session did not change the number of leukocytes. Overweight–obese group presented similar increase in leukocytes, monocytes and lymphocytes in both HIIE and MIIE sessions. At baseline, overweight–obese group showed high levels of CK, IL-8, IL-6 and CCL2 and lower concentrations of IL-10 compared to lean group. The MIIE did not alter the cytokine concentrations in both groups, independently of the time analysis. The HIIE induced significant decrease in IL-8 levels 30 min post session in both the groups, and a progressive elevation in IL-10 levels immediately and 30 min post in lean and overweight–obese. Regarding IL-6, overweight–obese subjects presented progressive increase either immediately and 30 min after HIIE, while lean individuals presented significant increase only 30 min after exercise.ConclusionsThe acute inflammatory response to interval exercise is intensity-dependent. Although obesity influences the basal concentrations of several cytokines, only HIIE induced important alterations in IL-8 and IL-10 levels, which may have important implications in the control of chronic low-grade inflammation in obesity.     

Acute high-dose sodium selenite administration improves intestinal microcirculation without affecting cytokine release in experimental endotoxemia

We evaluated the effects of acute high-dose sodium selenite (SEL) administration on the intestinal microcirculation and the release of the cytokines TNF-α, IL-1β, IL-6 and IL-10 in experimental endotoxemia (induced by lipopolysaccharide-LPS). Three groups of animals (n=30) were studied: control group, endotoxemic group (15 mg kg^?1 i.v. LPS from E. coli) and SEL-treated LPS group (100 μg kg^?1 SEL i.v.). SEL treatment resulted in a significant reduced number of firmly adhering leukocytes in intestinal submucosal venules and reduced significantly the impairment of the intestinal functional capillary density. Despite of the improvement of microcirculatory parameters, we did not detect any changes in the pattern of cytokine release. In conclusion, administration of high-dose sodium SEL attenuates leukocyte adhesion and improves capillary perfusion within the intestinal microcirculation without affecting release of the cytokines TNF-α, IL-1β, IL-6 and IL-10 in experimental endotoxemia.     

Role of α-CGRP in the regulation of neurotoxic responses induced by kainic acid in mice

Kainic acid (KA) is an excitatory and neurotoxic substance. The role of α-calcitonin gene-related peptide (α-CGRP) in the regulation of KA-induced hippocampal neuronal cell death was investigated in the present study. The intracerebroventricular (i.c.v.) administration with KA (0.07 μg) increased hippocampal α-CGRP mRNA level in ICR mice. The α-CGRP mRNA level began to increase at 1 h, reached at maximal level at 6 and 12 h, and returned to the control level by 24 h after i.c.v. administration with KA. In addition, KA-induced hippocampal CA3 neuronal death in C57BL6 (wild type) group was more pronounced compared to KA-induced hippocampal CA3 pyramidal cell death in α-CGRP knock-out (KO) group. Furthermore, sumatriptan, a CGRP releasing inhibitor, significantly protected the pyramidal cell death in CA3 hippocampal region induced by KA administered i.c.v. in ICR mice. Our results suggest that α-CGRP may play an important role in the regulation of KA-induced pyramidal cell death in CA3 region of the hippocampus.     

Involvement of potassium channels in the antidepressant-like effect of venlafaxine in mice

AimsStudies have shown that the acute administration of venlafaxine elicits an antidepressant-like effect in the mouse forced swim test (FST) by a mechanism dependent on the l-arginine–nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway. Because it has been reported that NO activates different types of potassium (K⁺) channels in the brain, this study investigated the involvement of K⁺ channels in the antidepressant-like effect of venlafaxine in the mouse FST.Main methodsMale adult Swiss mice were pretreated with different K⁺ channel inhibitors or openers 15 min before venlafaxine administration. After 30 min, the open-field test (OFT) and FST were carried out.Key findingsIntracerebroventricular (i.c.v.) pretreatment of mice with subeffective doses of tetraethylammonium (TEA, a non-specific inhibitor of K⁺ channels, 25 pg/site), glibenclamide (an ATP-sensitive K⁺ channel inhibitor, 0.5 pg/site), charybdotoxin (a large- and intermediate-conductance calcium-activated K⁺ channel inhibitor, 25 pg/site) or apamin (a small-conductance calcium-activated K⁺ channel inhibitor, 10 pg/site) was able to potentiate the action of a subeffective dose of venlafaxine (2 mg/kg, i.p.). Moreover, the reduction in the immobility time elicited by an effective dose of venlafaxine (8 mg/kg, i.p.) in the FST was prevented by the pretreatment of mice with the K⁺ channel openers cromakalim (10 µg/site, i.c.v.) and minoxidil (10 µg/site, i.c.v.). The drugs used in this study did not produce any change in locomotor activity.SignificanceThe results demonstrate that the neuromodulatory effects of venlafaxine, via the inhibition of K⁺ channels, are possibly involved in its anti-immobility activity in the mouse FST.     

Microinjection of melanin concentrating hormone into the lateral preoptic area promotes non-REM sleep in the rat

The ventrolateral preoptic area (VLPO) has been recognized as one of the key structures responsible for the generation of non-REM (NREM) sleep. The melanin-concentrating hormone (MCH)-containing neurons, which are located in the lateral hypothalamus and incerto-hypothalamic area, project widely throughout the central nervous system and include projections to the VLPO. The MCH has been associated with the central regulation of feeding and energy homeostasis. In addition, recent findings strongly suggest that the MCHergic system promotes sleep. The aim of the present study was to determine if MCH generates sleep by regulating VLPO neuronal activity. To this purpose, we characterized the effect of unilateral and bilateral microinjections of MCH into the VLPO on sleep and wakefulness in the rat. Unilateral administration of MCH into the VLPO and adjacent dorsal preoptic area did not modify sleep. On the contrary, bilateral microinjections of MCH (100 ng) into these areas significantly increased light sleep (LS, 39.2 ± 4.8 vs. 21.6 ± 2.5 min, P < 0.05) and total NREM sleep (142.4 ± 23.2 vs. 86.5 ± 10.5 min, P < 0.05) compared to control (saline) microinjections. No effect was observed on REM sleep. We conclude that MCH administration into the VLPO and adjacent dorsal lateral preoptic area promotes the generation of NREM sleep.     

The cardioprotective effect of different doses of vasopressin (AVP) against ischemia–reperfusion injuries in the anesthetized rat heart

The aim of the present study was to investigate the protective effect of various doses of exogenous vasopressin (AVP) against ischemia–reperfusion injury in anesthetized rat heart. Anesthetized rats were randomly divided into seven groups (n = 4–13) and all of them subjected to prolonged 30 min regional ischemia and 120 min reperfusion. Group I served as saline control with ischemia, in treatment groups II, III, IV and V, respectively different doses of AVP (0.015, 0.03, 0.06 and 1.2 μg/rat) were infused within 10 min prior to ischemia, in group VI, an AVP-selective V1 receptor antagonist (SR49059, 1 mg/kg, i.v.) was administrated prior to effective dose of AVP injection and in group VII, SR49059 (1 mg/kg, i.v.) was only administrated prior to ischemia. Various doses of AVP significantly prevented the decrease in heart rate (HR) at the end of reperfusion compared to their baseline and decreased infarct size, biochemical parameters [LDH (lactate dehydrogenase), CK-MB (creatine kinase-MB) and MDA (malondialdehyde) plasma levels], severity and incidence of ventricular arrhythmia, episodes and duration of ventricular tachycardia (VT) as compared to control group. Blockade of V1 receptors by SR49059 attenuated the cardioprotective effect of AVP on ventricular arrhythmias and biochemical parameters, but partially returned infarct size to control. AVP 0.03 μg/rat was known as effective dose. Our results showed that AVP owns a cardioprotective effect probably via V1 receptors on cardiac myocyte against ischemia/reperfusion injury in rat heart in vivo.     

Importance of IL-10 and IL-6 during chronic hepatitis c genotype-1 treatment and their relation with IL28B

This paper investigates serum levels of interleukin 10 (IL-10) and interleukin 6 (IL-6) in patients with chronic hepatitis C genotype 1 (CHC-GT1), the relation of each with clinical and virological characteristics, how they affect the response to combined therapy and their relation with the IL28B polymorphisms rs12979860. Serum level expression and the polymorphism of IL-10, IL-6 and IL28B were determined in 138 CHC-GT1 patients, treated with pegylated interferon/ribavirin (pegIFN-α/RBV) for 48 weeks, in the following samples: baseline, week-12 (during treatment) and week-72 (post-treatment). 77 patients (56%) presented Sustained Virological Response (SVR) and 61 (44%) were non-SVR. Multivariate logistic regression showed that age ? 40 years (aOR = 3.7, 95%CI = 1.5–8.9, P = 0.004), low activity of gamma glutamyl transferase (GGT) (aOR = 0.9, 95%CI = 0.98–0.99, P = 0.028), CC genotype of IL28B polymorphim (aOR = 2.7, 95%CI = 1.0–7.2, P = 0.044) and low IL-6 (aOR = 0.5, 95%CI = 0.3–1.0, P = 0.038) were predictor factors of virological response. In all patients, following treatment, IL-6 decreased at week-12 (P = 0.004) from baseline and had returned to basal values at week-72. Serum IL-10 concentration was significantly decreased at week-72 only in SVR patients (P ? 0.001). When patients were stratified by IL28B polymorphisms rs12979860 CC vs non-CC patients, a statistically significant decrease in IL-10 at week-72 in both groups was observed (P = 0.003 and P ? 0.001, respectively). None of the polymorphisms of IL-10 or IL-6 studied were associated with SVR.ConclusionsCC genotype of IL28B and low IL-6 serum concentration are factors associated independently with SVR. Moreover, decreased IL-10 at week-72 is associated with SVR in both CC and non-CC patients, and both factors are important to determine the effectiveness of treatment.     

Liquid chromatography/electrospray tandem mass spectrometry method for the determination of cefuroxime in human plasma: Application to a pharmacokinetic study

A rapid, selective and sensitive high performance liquid chromatography–tandem mass spectrometry method (LC–MS/MS) was developed and validated for the determination and pharmacokinetic investigation of cefuroxime in human plasma. Cefuroxime and the internal standard (IS), cefoxitin, were extracted from plasma samples using solid phase extraction with Oasis HLB cartridges. Chromatographic separation was performed on a LiChrospher^® 60 RP Select B column (125 mm × 4 mm i.d., 5 μm particle size) using acetonitrile:5 ± 0.2 mM ammonium acetate solution:glacial acetic acid (70:30:0.020, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Detection of cefuroxime and cefoxitin was achieved by tandem mass spectrometry with an electrospray ionization (ESI) interface in negative ion mode. The calibration curves were linear over the range of 81.0–15976.2 ng/mL with the lower limit of quantitation validated at 81.0 ng/mL. The intra- and inter-day precisions were within 7.6%, while the accuracy was within ±6.3% of nominal values. No matrix effect was observed in this method. The validated LC–MS/MS method was successfully applied for the evaluation of pharmacokinetic and bioequivalence parameters of cefuroxime after an oral administration of 500 mg cefuroxime tablet to 36 healthy male volunteers.     

Determination of mitoxantrone in rat plasma by liquid chromatography–tandem mass spectrometry method: Application to a pharmacokinetic study

A rapid, sensitive and specific high performance liquid chromatography–tandem mass spectrometric (HPLC–MS/MS) method has been developed for quantification of mitoxantrone in rat plasma. The analyte and palmatine (internal standard) were extracted from plasma samples with diethyl ether–dichloromethane (3:2, v/v) and separated on a C₁₈ column. The chromatographic separation was achieved within 2.5 min using methanol–10 mM ammonium acetate containing 0.1% acetic acid as the mobile phase at a flow rate of 0.2 mL/min. The method was linear over the range of 0.5–500 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL. Finally, the method was successfully applied to a pharmacokinetic study of mitoxantrone in rats following intravenous administration.     

Models of hemorrhagic shock: Differences in the physiological and inflammatory response

IntroductionThe hemorrhagic shock (HS) model is commonly used to initiate a systemic post-traumatic inflammatory response. Numerous experimental protocols exist and it is unclear how differences in these models affect the immune response making it difficult to compare results between studies. The aim of this study was to compare the inflammatory response of different established protocols for volume-controlled shock in a murine model.MethodsMale C57/BL6 mice 6–10 weeks and weighing 20–25 g were subjected to volume-controlled or pressure-controlled hemorrhagic shock. In the volume-controlled group 300 μl, 500 μl, or 700 μl blood was collected over 15 min and mean arterial pressure was continuously monitored during the period of shock. In the pressure-controlled hemorrhagic shock group, blood volume was depleted with a goal mean arterial pressure of 35 mmHg for 90 min. Following hemorrhage, mice from all groups were resuscitated with the extracted blood and an equal volume of lactated ringer solution. Six hours from the initiation of hemorrhagic shock, serum IL-6, KC, MCP-1 and MPO activity within the lung and liver tissue were assessed.ResultsIn the volume-controlled group, the mice were able to compensate the initial blood loss within 30 min. Approximately 800 μl of blood volume was removed to achieve a MAP of 35 mmHg (p < 0.001). No difference in the pro-inflammatory cytokine (IL-6 and KC) profile was measured between the volume-controlled groups (300 μl, 500 μl, or 700 μl). The pressure-controlled group demonstrated significantly higher cytokine levels (IL-6 and KC) than all volume-controlled groups. Pulmonary MPO activity increased with the severity of the HS (p < 0.05). This relationship could not be observed in the liver.ConclusionVolume-controlled hemorrhagic shock performed following current literature recommendations may be insufficient to produce a profound post-traumatic inflammatory response. A decrease in the MAP following blood withdrawal (300 μl, 500 μl or 700 μl) was usually compensated within 30 min. Pressure-controlled hemorrhagic shock is a more reliable for induction of a systemic inflammatory response.     

Inactivation of IL-6 and soluble IL-6 receptor by neutrophil derived serine proteases in cystic fibrosis

The ability of IL-6 to signal via both membrane bound and soluble receptors is thought to explain the capacity of this cytokine to act in both the initiation and resolution of acute inflammatory responses. In cystic fibrosis (CF), poorly resolved neutrophillic inflammation of the lungs is a primary cause of morbidity and mortality. Expression of IL-6 has been reported to be low in CF lung secretions, despite ongoing inflammation, but the status of soluble IL-6 receptor (sIL-6R) in these patients is unknown. We hypothesised that sIL-6R may be an important potentiator of IL-6 activity in CF associated lung disease. IL-6, sIL-6R and sgp130 (a natural antagonist of responses mediated by the sIL-6R) were analysed by ELISA and Western blot in bronchoalveolar lavage fluid (BALF) from 28 paediatric CF patients and nine non-CF controls. Total cell counts in CF were four fold higher compared to controls (median: 1.4 × 10⁶ cells/ml v. 0.35 × 10⁶ cells/ml in controls) (p < 0.001) and the infiltrate was dominated by neutrophils which were elevated by 89 fold (0.62 × 10⁶ cells/ml v. 0.007 × 10⁶ cells/ml in controls) (p < 0.001). Other markers of inflammation such as IL-8 and MCP-1 were elevated 17.5 and 3.8 fold respectively (IL-8; median: 1122 pg/ml v. 64 pg/ml in controls, p < 0.01 and MCP-1; median: 692 pg/ml v. 182 pg/ml in controls, p < 0.05). IL-6, although present in 23/32 CF BALF specimens compared to 1/9 controls (p < 0.01), was weakly expressed (median: 50 pg/ml). Expression of sIL-6R and sgp130 in CF was no different to control patients. We tested whether weak expression of all three molecules was due to degradation by CF BALF. Degradative activity was observed in association with BALF elastase activity and could be specifically blocked by serine protease inhibitors. Degradation of sIL-6R by purified serine proteases (elastase, cathepsin G and proteinase 3) was also observed leading to a loss of trans-signalling activity. Interestingly, sIL-6R was protected from proteolysis by interaction with IL-6. Our data identify and define a novel protease mediated deficiency of IL-6 signalling in the CF lung.     

Optimisation of preparative HPLC separation of four isomeric kaempferol diglycosides from Prunus spinosa L. by application of the response surface methodology

A fast and efficient preparative HPLC-PDA method was developed for the separation and isolation of four rare isomeric kaempferol diglycosides from leaves of Prunus spinosa L. The separation procedure of the enriched diglycoside fraction of the 70% (v/v) aqueous methanolic leaf extract was first optimised on analytical XBridge C18 column (100 mm × 4.6 mm i.d., 5 μm) and central composite design combined with response surface methodology was utilized to establish the optimal separation conditions. The developed method was directly transferred to preparative XBridge Prep C18 column (100 mm × 19 mm i.d., 5 μm) and the final separation was accomplished by isocratic elution with 0.5% acetic acid-methanol-tetrahydrofuran (75.2:16.6:8.2, v/v/v) as the mobile phase, at a flow rate of 13.6 mL/min, in less than 12 min for a single run. Under these conditions, four flavonoid diglycosides: kaempferol 3-O-α-l-arabinofuranoside-7-O-α-l-rhamnopyranoside, kaempferol 3,7-di-O-α-l-rhamnopyranoside (kaempferitrin), and reported for the first time for P. spinosa kaempferol 3-O-β-d-xylopyranoside-7-O-α-l-rhamnopyranoside (lepidoside) and kaempferol 3-O-α-l-arabinopyranoside-7-O-α-l-rhamnopyranoside, were isolated in high separation yield (84.8–94.5%) and purity (92.45–99.79%). Their structures were confirmed by extensive 1D and 2D NMR studies. Additionally, the UHPLC-PDA-ESI–MS³ qualitative profiling led to the identification of twenty-one phenolic compounds and confirmed that the isolates were the major components of the leaf material.     

Optimized method for the determination of itopride in human plasma by high-performance liquid chromatography with fluorimetric detection

A high-performance liquid chromatographic method with fluorescence detection for the determination of itopride in human plasma is reported. The sample preparation was based on liquid–liquid extraction of itopride from plasma with t-butylmethylether and dichloromethane (70:30, v/v) mixture followed by a back extraction of the analyte to the phosphate buffer (pH 3.2). Liquid chromatography was performed on an octadecylsilica column (55 mm × 4 mm, 3 μm particles), the mobile phase consisted of acetonitrile–triethylamine–15 mM dihydrogenpotassium phosphate (14.5:0.5:85, v/v/v), pH of the mobile phase was adjusted to 4.8. The run time was 3 min. The fluorimetric detector was operated at 250/342 nm (excitation/emission wavelength). Naratriptan was used as the internal standard. The limit of quantitation was 9.5 ng/ml using 0.5 ml of plasma. The method precision and inaccuracy were less than 8%. The assay was applied to the analysis of samples from a bioequivalence study.     

Development and validation of a high throughput and robust LC–MS/MS with electrospray ionization method for simultaneous quantitation of diltiazem and its two metabolites in human plasma: Application to a bioequivalence study

A high throughput and specific method using ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) was developed for the simultaneous determination of diltiazem and its two metabolite (N-desmethyldiltiazem and O-desacetyldiltiazem) in human plasma. A one-step liquid–liquid extraction (LLE) with methyl-t-butyl ether (MTBE) involved for the extraction of diltiazem (DLTZ), metabolites (DMeD and DAcD) and internal standard. Analytes were chromatographed on a ACQUITY UPLC? BEH C₁₈ column (100 mm × 2.1 mm, i.d., 1.7 μm) with isocratic elution at a flow rate of 0.2 mL/min using 10 mM ammonium acetate buffer–acetonitrile (25:75, v/v). The Quattro Premier XE LC–MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Using 300 μL plasma, the method was validated over the concentration range 0.48–639.9 ng/mL for DLTZ and 0.24–320.1 for DMeD and 0.24–320.7 ng/mL for DAcD, with a lower limit of quantification of 0.48 ng/mL for DLTZ and 0.24 ng/mL for metabolites. The intra- and inter-day precision and accuracy were within 10.0%. The recovery was 77.4%, 76.0%, 74.5% and 74.1% for DLTZ, DMeD, DAcD and Ziprasidone, respectively. Total run time was 2.0 min only.     

Involvement of delta and mu opioid receptors in the acute and sensitized locomotor action of cocaine in mice

Analogs of deltorphins, such as cyclo(Nδ, Nδ-carbonyl-d-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) are functional agonists predominantly for the delta opioid receptors (DOR) in the guinea-pig ileum and mouse vas deferens bioassays. The purpose of this study was to examine an influence of these peptides (5, 10 or 20 nmol, i.c.v.) on the acute cocaine-induced (10 mg/kg, i.p.) locomotor activity and the expression of sensitization to cocaine locomotor effect. Sensitization to locomotor effect of cocaine was developed by five injections of cocaine at the dose of 10 mg/kg, i.p. every 3 days. Our results indicated that DK-4 and DEL-6 differently affected the acute and sensitized cocaine locomotion. Co-administration of DEL-6 with cocaine enhanced acute cocaine locomotion only at the dose of 10 nmol, with minimal effects at the doses 5 and 20 nmol, whereas co-administration of DK-4 with cocaine enhanced acute cocaine-induced locomotion in a dose-dependent manner. Similarly to the acute effects, DEL-6 only at the dose of 10 nmol but DK-4 dose-dependently enhanced the expression of cocaine sensitization. Pre-treatment with DOR antagonist – naltrindole (5 nmol, i.c.v.) and mu opioid receptor (MOR) antagonist, β-funaltrexamine abolished the ability of both peptides to potentiate the effects of cocaine. Our study suggests that MOR and DOR are involved in the interactions between cocaine and both deltorphins analogs. A distinct dose–response effects of these peptides on cocaine locomotion probably arise from differential functional activation (targeting) of the DOR and MOR by both deltorphins analogs.