Streptomyces chiangmaiensis SSUT88A mediated green synthesis of silver nanoparticles: characterization and evaluation of antibacterial action against clinical drug-resistant strains.

This study involved the characterization of AgNPs synthesized from soil isolate Streptomyces sp. SSUT88A and their antimicrobial activities. The strain SSUT88A revealed 98.8% similarity of the 16s rRNA gene to Streptomyces chiangmaiensis TA4-1T. The AgNPs were synthesized by mixing either intracellular or extracellular cell-free supernatant of strain SSUT88A with AgNO3. The synthesized AgNPs from intracellular cell-free supernatant and extracellular cell-free supernatant were designated as IS-AgNPs and ES-AgNPs, respectively. The IS-AgNPs showed maximum absorbance of UV-vis spectra at 418 nm, while ES-AgNPs revealed maximum absorbance at 422 nm. The TEM observation of synthesized AgNPs revealed a spherical shape with an average diameter of 13.57 nm for IS-AgNPs and 30.47 nm for ES-AgNPs. The XRD and XANES spectrum profile of both synthesized AgNPs exhibited similar spectrum energy, which corresponded to AgNPs. The IS-AgNPs revealed antimicrobial activity against clinical isolate drug-resistant bacteria (Acinetobacter baumannii, Escherichia coli 8465, Klebsiella pneumoniae 1617, and Pseudomonas aeruginosa N90PS), while ES-AgNPs had no antimicrobial activity. When compared to commercial AgNPs, IS-AgNPs exhibited antibacterial efficacy against all clinical isolate bacteria including A. baumannii, one of the most threatening multi-drug resistant strains, while commercial AgNPs did not. Thus, IS-AgNPs has potential to be further developed as an antimicrobial agent against drug-resistant bacteria.

Fabrication of Piezoelectric Electrospun Termite Nest-like 3D Scaffolds for Tissue Engineering.

A high piezoelectric coefficient polymer and biomaterial for bone tissue engineering- poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP)-has been successfully fabricated into 3D scaffolds using the wet electrospinning method. Three-dimensional (3D) scaffolds have significant advantages for tissue engineering applications. Electrospinning is an advanced method and can fabricate 3D scaffolds. However, it has some limitations and is difficult to fabricate nanofibers into 3D shapes because of the low controllability of porosity and internal pore shape. The PVDF-HFP powders were dissolved in a mixture of acetone and dimethylformamide with a ratio of 1:1 at various concentrations of 10, 13, 15, 17, and 20 wt%. However, only the solutions at 15 and 17 wt% with optimized electrospinning parameters can be fabricated into biomimetic 3D shapes. The produced PVDF-HFP 3D scaffolds are in the cm size range and mimic the structure of the natural nests of termites of the genus Apicotermes. In addition, the 3D nanofiber-based structure can also generate more electrical signals than the conventional 2D ones, as the third dimension provides more compression. The cell interaction with the 3D nanofibers scaffold was investigated. The in vitro results demonstrated that the NIH 3T3 cells could attach and migrate in the 3D structures. While conventional electrospinning yields 2D (flat) structures, our bio-inspired electrospun termite nest-like 3D scaffolds are better suited for tissue engineering applications since they can potentially mimic native tissues as they have biomimetic structure, piezoelectric, and biological properties.

One-Pot Synthesis of Coumarin-Indomethacin Hybrids as COX-2 Targeting Probes for Cancer Imaging.

Facile synthesis of 6- or 7-substituted coumarin-indomathacin hybrids (Coum-IDM) has been developed for specific cyclooxygenase-2 (COX-2) binding along with their intrinsic fluorescent properties. A mild and rapid condensation/dehydrative cyclization of 2-hydroxy benzaldehyde with activated indomethacin was carried out in one step under ultrasound irradiation. Coum-IDM4 was found to be the best of this series as it presented significant binding to COX-2 and exhibited higher fluorescent intensity in cancer cells than in normal cells. Therefore, in the light of drug development tools, this new hybrid system could be a potential targeted probe for COX-2-overexpressed inflammation and cancer-cell tracking.

Chrysophyllum cainito stem bark extract induces apoptosis in Human hepatocarcinoma HepG2 cells through ROS-mediated mitochondrial pathway.

Hepatocellular carcinoma is the most common type of primary liver cancer in humans. This study aimed to demonstrate anticancer properties of an aqueous extract from Chrysophyllum cainito stem bark (CE) and its underlying mechanisms. Our MTT assay results showed that CE significantly reduced human hepatocellular carcinoma (HepG2) cell viability with the IC50of 100 µg/mL, while human dermal primary fibroblast (HDFa) cells showed less susceptibility in every concentration tested. Determined by Annexin V staining, the proportion of apoptotic HepG2 cells increased in a dose-dependent fashion after 24 hour-exposure of CE. The results from Western blot analysis confirmed that CE reduced procaspase-3, suggesting apoptosis by activating caspase-3 cleavage. Using the DCFH-DA and DiOC6 fluorescent probes, it was found that CE significantly stimulated the generation of reactive oxygen species (ROS) and reduced mitochondrial membrane potential (Δψ m), respectively. According to cell cycle analysis, CE (100 µg/mL) profoundly increased the percentage of cells in the sub-G1 phase, indicating cell apoptosis. These data suggest that CE induces apoptosis and cell death in human hepatocellular carcinoma via generation of intracellular ROS and disruption of Δψm. This is the first demonstration of the anticancer activity with proposed underlying mechanism of CE in liver cancer cells.

Development of Lipid-Based Nanocarriers for Increasing Gastrointestinal Absorption of Lupinifolin.

Lupinifolin, a plant flavonoid, has been reported to possess various pharmacological effects. It most likely exerts low oral bioavailability because of poor water solubility. The objective of this study was to develop lipid nanocarriers as drug delivery systems to increase the gastrointestinal absorption of lupinifolin extracted from Albizia myriophylla. Three types of nanocarriers, lupinifolin-loaded solid lipid nanoparticles, lupinifolin-loaded nanostructured lipid carriers, and lupinifolin-loaded nanoemulsions, were prepared by an emulsification-sonication technique. All three types of nanocarriers loaded with lupinifolin, lupinifolin-loaded solid lipid nanoparticles, lupinifolin-loaded nanostructured lipid carriers, and lupinifolin-loaded nanoemulsions, were successfully synthesized. The lipid components chosen to formulate nanocarriers were tripalmitin and/or medium chain triglyceride. Physicochemical characterizations along with releasing profiles of lupinifolin-loaded lipid nanocarriers were compared. It was found that the best lipid nanocarrier for lupinifolin was lupinifolin-loaded nanostructured lipid carriers, which demonstrated the particle size of 151.5 ± 0.1 nm, monodispersity distribution with a polydispersity index of 0.24, negative surface charge at - 41.2 ± 0.7 mV, high encapsulation (99.3%), and high loading capacity (5.0%). The obtained lupinifolin-loaded nanostructured lipid carriers exhibited prolonged release in a simulated circulatory system but produced a low release in gastrointestinal conditions (3.7%). Intestinal permeability of the nanocarriers was further evaluated in everted intestinal sacs. The results from the ex vivo study indicated that lupinifolin-loaded nanostructured lipid carriers significantly increased the absorption of lupinifolin compared to the native form. In conclusion, lupinifolin-loaded lipid nanocarriers were successfully formulated as delivery systems to enhance its oral bioavailability. Further in vivo experiments are needed to validate the results from this study.

Small-Intestine-Specific Delivery of Antidiabetic Extracts from Withania coagulans Using Polysaccharide-Based Enteric-Coated Nanoparticles.

Withania coagulans is an Indian medicinal herb, the natural extracts of which are purported to have health-benefiting properties. In this study, the extract was encapsulated in nature-derived polymers with the aim of enhancing its bioavailability. The aqueous extract obtained from the plant W. coagulans was found to elicit the glucose-lowering effect by means of promoting insulin secretion from pancreatic ß cells. The cells treated with the extract showed a nearly 2-fold increase in insulin secretion compared to untreated cells. A delivery system for the extract was developed based on electrosprayed chitosan nanoparticles coated with food-based starch. The enteric starch coating retarded (by 2.5 times) the release of the extract in the stomach. The bioactivity of the encapsulated extract was subsequently tested in vitro on mouse-derived pancreatic ß cells, whereby the delivery system was found to promote insulin secretion. Finally, the extract-encapsulated oral delivery system was tested on diabetic mice and was validated to decrease blood glucose levels by 60%. In summary, it could be inferred that food-grade enteric-coated polysaccharide-based particles increase the bioavailability of the extracted compounds from the plant W. coagulans.

Antidiabetic activity, glucose uptake stimulation and α-glucosidase inhibitory effect of Chrysophyllum cainito L. stem bark extract.

BACKGROUND: Chrysophyllum cainito L., a tropical fruit tree, has been used as an alternative medicine for the treatment of diabetic patients in many countries. However, there is very limited scientific rationale for this medical use. The present study aimed to evaluate the antidiabetic activity of the extract from C. cainito stem bark and the possible mechanisms underlying this activity. METHODS: Phytochemistry and in vitro antioxidant capacity of the extract were studied. Hypoglycemic activity of the extract was examined in normal and alloxan-induced diabetic mice. The effect of C. cainito extract on glucose absorption and glucose uptake were conducted using mouse isolated jejunum and abdominal muscle, respectively. Finally, an in vitro effect of C. cainito extract on α-glucosidase activity was evaluated. RESULTS: C. cainito extract possessed a strong antioxidant activity comparable to the ascorbic acid and butylated hydroxytoluene. The extract at 500 mg/kg significantly reduced the area under curve of blood glucose level in oral glucose tolerance test in normal mice. In alloxan-induced diabetic model, similar to glibenclamide, a single dose of the extract significantly decreased fasting blood glucose level from 387.17 ± 29.84 mg/dl to 125.67 ± 62.09 mg/dl after 6 h of administration. From the isolated jejunum experiment, the extract at any doses used did not inhibit glucose absorption. However, the extract at 50 µg/ml significantly increased the amount of glucose uptake by abdominal muscles in the presence of insulin (P < 0.05). Lastly, it was found that the extract produced stronger inhibition of α-glucosidase activity (IC50 = 1.20 ± 0.09 µg/ml) than acarbose (IC50 = 198.17 ± 4.74 µg/ml). CONCLUSION: Direct evidence of antidiabetic activity of C. cainito stem bark with possible modes of action, glucose uptake stimulation and α-glucosidase inhibitory effect, was reported for the first time herein. These data support the potential use of this plant for the treatment of diabetic patients.

Lupinifolin from Derris reticulata possesses bactericidal activity on Staphylococcus aureus by disrupting bacterial cell membrane.

In this study, lupinifolin, a prenylated flavonoid, was isolated from Derris reticulata stem, identified by NMR spectra and confirmed with mass spectrometry. Lupinifolin was freshly prepared by solubilizing in 0.1 N NaOH and immediately diluted in Müller-Hinton broth for antibacterial testing. The data showed that Gram-positive bacteria were more susceptible to lupinifolin than Gram-negative bacteria. Of four strains of Gram-positive bacteria tested, Staphylococcus aureus was the most susceptible. Using the two-fold microdilution method, it was found that lupinifolin possessed antimicrobial activity against S. aureus with minimum inhibitory concentration and minimum bactericidal concentration of 8 and 16 µg/ml, respectively, which is less potent than ampicillin. However, from the time-effect relationship, it was shown that lupinifolin had faster onset than ampicillin. The faster onset of lupinifolin was confirmed by scanning electron microscopy. To investigate the mechanism of action of lupinifolin, transmission electron microscopy (TEM) was performed to observe the ultrastructure of S. aureus. The TEM images showed that lupinifolin ruptured the bacterial cell membrane and cell wall. Due to its fast onset, it is suggested that the action of lupinifolin is likely to be the direct disruption of the cell membrane. This hypothesis was substantiated by the data from flow cytometry using DiOC2 as an indicator. The result showed that the red/green ratio which indicated bacterial membrane integrity was significantly decreased, similar to the known protonophore carbonyl cyanide 3-chlorophenylhydrazone. It is concluded that lupinifolin inhibits the growth of S. aureus by damaging the bacterial cytoplasmic membrane.

Antioxidant, α-glucosidase inhibitory activity and sub-chronic toxicity of Derris reticulata extract: its antidiabetic potential.

BACKGROUND: Antidiabetic activity of Derris reticulata extract on alloxan-induced diabetic rats has been reported. The extract was found to lower blood glucose and inhibit intestinal glucose absorption. The aim of this study was to further investigate mechanisms underlying the antihyperglycemic activity of D. reticulata extract in vitro. METHODS: The aqueous extract was obtained from D. reticulata stem. Phytochemical screening, total phenolic, and flavanoid contents were examined. ABTS and DPPH scavenging assays, and FRAP method were used to determine in vitro antioxidant activities. Measurement of cell viability on alloxan-induced cellular damage was performed in the insulin-secreting RINm5F cells by MTT assay. The effects of the extract on α-glucosidase activity and insulin release were studied. In addition, sub-chronic toxicity test in rats was also conducted. RESULTS: The results revealed that the extract, which consisted of terpenoids, saponins, tannins and flavonoids, possessed moderate radical scavenging activities. Pre-treatment of RINm5F cells with the extract was also found to exert moderate, but significant, in vitro protection against alloxan, an oxidative stress producing agent. Unlike glibenclamide, the extract did not stimulate insulin secretion. However, the extract was found to inhibit α-glucosidase activity similar to acarbose. It was found that in sub-chronic toxicity studies D. reticulata extract did not cause mortality or produce any remarkable haematological, biochemical and histopathological adverse effects in rats. CONCLUSIONS: The data suggest that the possible mechanisms underlying antihyperglycemic activity of D. reticulata extract are cytoprotective effect on pancreatic cells, presumably by its antioxidant activity, and inhibition of α-glucosidase. Sub-chronic toxicity study also provides scientific evidence to corroborate the safety of this plant as an alternative antidiabetic agent.

Cytoprotective and anti-diabetic effects of Derris reticulata aqueous extract

The current study was aimed to investigate pancreatic protective and anti-diabetic activities of the aqueous extract of Derris reticulata stem. First, we evaluated a cytoprotective potential of D. reticulata extract on alloxan-induced damage in vitro. Treatment with D. reticulata extract at the doses of 250 and 500 ìg/ml significantly increased cell viability of the pancreatic beta-cell line RINm5F after exposure of alloxan. The anti-hyperglycemic activity of D. reticulata extract was further studied in alloxan-induced diabetic rats. A significant reduction in blood glucose level along with an increase in body weight was observed in diabetic rats treated with D. reticulata extract at 250 mg/kg body weight for 15 days. Serum aspartate transaminase and alanine transaminase levels were also significantly decreased compared to diabetic control rats. In accordance with in vitro cytoprotective effect, histopathological examination revealed that pancreatic islet cells of the extract-treated diabetic rat were less damage than those of the untreated diabetic group. In order to find another possible mechanism of action underling hypoglycemic activity, the effect on glucose absorption was examined using everted sac jejunum. The results showed that D. reticulata extract suppressed glucose absorption from small intestine. To corroborate safety use of D. reticulata extract, acute oral toxicity was also conducted in rats. Our results showed that none of the tested doses (250, 500, 1,000, and 2,000 mg/kg) induced signs of toxicity or mortality after administration of the extract. The results suggested that D. reticulata extract possess anti-diabetic activity, which resulting from its pancreatic cytoprotective effect and inhibition of intestinal glucose absorption

Cytoprotective and anti-diabetic effects of Derris reticulata aqueous extract.

The current study was aimed to investigate pancreatic protective and anti-diabetic activities of the aqueous extract of Derris reticulata stem. First, we evaluated a cytoprotective potential of D. reticulata extract on alloxan-induced damage in vitro. Treatment with D. reticulata extract at the doses of 250 and 500 µg/ml significantly increased cell viability of the pancreatic ß-cell line RINm5F after exposure of alloxan. The anti-hyperglycemic activity of D. reticulata extract was further studied in alloxan-induced diabetic rats. A significant reduction in blood glucose level along with an increase in body weight was observed in diabetic rats treated with D. reticulata extract at 250 mg/kg body weight for 15 days. Serum aspartate transaminase and alanine transaminase levels were also significantly decreased compared to diabetic control rats. In accordance with in vitro cytoprotective effect, histopathological examination revealed that pancreatic islet cells of the extract-treated diabetic rat were less damage than those of the untreated diabetic group. In order to find another possible mechanism of action underling hypoglycemic activity, the effect on glucose absorption was examined using everted sac jejunum. The results showed that D. reticulata extract suppressed glucose absorption from small intestine. To corroborate safety use of D. reticulata extract, acute oral toxicity was also conducted in rats. Our results showed that none of the tested doses (250, 500, 1,000, and 2,000 mg/kg) induced signs of toxicity or mortality after administration of the extract. The results suggested that D. reticulata extract possess anti-diabetic activity, which resulting from its pancreatic cytoprotective effect and inhibition of intestinal glucose absorption.

Antibacterial activity of Aquilaria crassna leaf extract against Staphylococcus epidermidis by disruption of cell wall.

BACKGROUND: Aquilaria crassna Pierre ex Lecomte has been traditionally used in Thailand for treatment of infectious diseases such as diarrhoea and skin diseases for a long time. The main objectives of this study were to examine antibacterial activity of the Aquilaria crassna leaf extract against Staphylococcus epidermidis and its underlying mechanism. The antioxidant activity and acute toxicity were studied as well. METHODS: Antioxidant activities were examined by FRAP, ABTS and DPPH scavenging methods. Antibacterial activity was conducted using disc diffusion assay and the minimum inhibitory concentration (MIC) was determined by dilution method. The minimum bactericidal concentration (MBC) was reported as the lowest concentration producing no growth of microbes in the subcultures. Morphological changes of the microbe were observed by scanning electron microscopy, while an inhibitory effect on biofilm formation was evaluated by phase contrast microscopic analysis. Bacterial cell wall integrity was assessed by transmission electron microscopy. Acute toxicity was conducted in accordance with the OECD for Testing of Chemicals (2001) guidelines. RESULTS: The extract exhibited considerable antioxidant activity. Staphylococcus epidermidis was susceptible to the extract with the MIC and MBC of 6 and 12 mg/ml, respectively. The extract caused swelling and distortion of bacterial cells and inhibited bacterial biofilm formation. Rupture of bacterial cell wall occurred after treated with the extract for 24 h. Acute toxicity test in mice showed no sign of toxicity or death at the doses of 2,000 and 15,000 mg/kg body weight. CONCLUSION: The aqueous extract of Aquilaria crassna leaves possesses an in vitro antibacterial activity against Staphylococcus epidermidis, with no sign of acute oral toxicity in mice, probably by interfering with bacterial cell wall synthesis and inhibiting biofilm formation.

Participation of citral in the bronchodilatory effect of ginger oil and possible mechanism of action.

The extract of ginger, the rhizomes of Zingiber officinale Roscoe (Zingiberaceae), has been reported to possess anti-hyperactivity and anti-inflammation on airway. The present study described brochodilatory activity of ginger oil and identified its active compound. Ginger oil was extracted by hydro-distillation. The compositions of ginger oil were analyzed by gas chromatography and mass spectrometer. Citral, eucalyptol and camphene were found to be the major components. Ginger oil and citral, but not camphene, suppressed rat tracheal contraction induced by carbachol (CCh). Consistent with previous report, eucalyptol showed a relaxing effect on rat airway. Since the content of eucalyptol in ginger oil was relatively low, the contribution of eucalyptol to the bronchodilatory effect of ginger oil was small. To elucidate the mechanisms responsible for the myorelaxing effect, propranolol (a ß-adrenergic receptor antagonist), indomethacin (a COX inhibitor) and L-NAME (a NOS inhibitor) were used to block the inhibitory effects of ginger oil and citral. It was found that propranolol, but not indomethacin and L-NAME, reversed bronchodilatory effects of both ginger oil and citral, suggesting that a possible mechanism involved ß-adrenergic receptor. This study provides the pharmacological basis supporting the therapeutic potential of Z. officinale rhizomes as a bronchodilator.

Comparison of the antioxidant and cytotoxic activities of Phyllanthus virgatus and Phyllanthus amarus extracts.

OBJECTIVE: To determine the antioxidant activity and cytotoxicity of Phyllanthus virgatus crude extract compared to Phyllanthus amarus. METHODS: Phenolic contents of the hydromethanolic extracts were measured using Folin-Ciocalteu reagent. Antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl hydrate free radical scavenging and antilipid peroxidation assays. Cytotoxicity on human hepatoma HepG2 cells was assessed by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assays. A stereomicroscope was used to observe and photograph the morphology of the cells. Oxygen consumption of the HepG2 cells was measured using a Clark oxygen electrode. RESULTS: The extract of P. virgatus, which contained more phenolic compounds than P. amarus extract, had higher cytotoxicity and showed higher free radical scavenging activity and more inhibition of peroxidation in a linoleic acid system. P. virgatus extract conspicuously increased the oxygen consumption of HepG2 cells, while P. amarus extract had little stimulatory effect. CONCLUSION: The hydromethanolic extract of P. virgatus had stronger antioxidant and cytotoxic action than P. amarus extract. The stimulation of HepG2 cell respiration by P. virgatus extract suggests the extract alters mitochondrial function; this action could play a role in the cytotoxicity of this plant.

The effects of wild ginger (Costus speciosus (Koen) Smith) rhizome extract and diosgenin on rat uterine contractions.

The aim of this study was to investigate the effects of wild ginger (Costus speciosus (Koen) Smith, Costaceae) rhizome extract on uterine contractility. We particularly examined the effects on spontaneous phasic contractions and the mechanisms whereby it exerts its effects. Wild ginger rhizomes were ethanolic extracted and their constituents analyzed. Isometric force was measured in strips of longitudinal myometrium and the effects of the extract studied. The extract (10 mg/100 mL) increased spontaneous contractions. The amplitude and frequency of the phasic contraction were significantly increased along with basal tension. Force produced in the presence of the extract was abolished by inhibition of l-type calcium channels or myosin light chain kinase (MLCK). The actions of the extract were not blocked by the estrogen receptor blocker, fulvestrant. Although significant amounts of diosgenin were present in the extract, we found that, depending upon its concentration, diosgenin had either no effect or was inhibitory on force. Interestingly, the extract induced significant amounts of force in the absence of extracellular calcium, which could be blocked by inhibition of the sarcoplasmic reticulum calcium-ATPase (SERCA), but not fulvestrant. We conclude that wild ginger rhizome extract stimulates phasic activity in rat uterus. Our data suggest that the uterotonic effect is due to nonestrogenic effects and not those of diosgenin. Wild ginger was able to increase contraction via calcium entry on l-type calcium channels and sarcoplasmic reticulum (SR) calcium release. We suggest that wild ginger rhizome extract may be a useful uterine stimulant.

Effects of Phyllanthus urinaria extract on HepG2 cell viability and oxidative phosphorylation by isolated rat liver mitochondria.

ETHNOPHARMACOLOGICAL RELEVANCE: Phyllanthus urinaria is widely used as anti-inflammatory, anti-diarrheal and hepatoprotective medicines in Asian countries such as India, China and Thailand. In Thailand, Phyllanthus urinaria is traditionally used as an adjuvant or alternative medicine for cancer patients, including liver cancer. However, there is limited scientific evidence supporting its use in cancer particularly hepatocellular carcinoma. AIM OF THE STUDY: To investigate the cytotoxic effect of Phyllanthus urinaria extract on human hepatocellular carcinoma HepG2 cells and the effect on oxidative phosphorylation by isolated rat liver mitochondria. MATERIALS AND METHODS: HepG2 cells and isolated rat liver mitochondria were treated with the 50% methanolic extract of Phyllanthus urinaria. Cytotoxicity of the extract was assessed by trypan blue exclusion and MTT assay. Rates of oxygen consumption of isolated mitochondria were determined with a Clark oxygen electrode. RESULTS: It was found that the hydromethanolic extract induced cell death of HepG2 cells in a dose-dependent fashion. The IC(50) of Phyllanthus urinaria extract measured by trypan blue exclusion and MTT assay were 431+/-65 microg/ml and 445+/-62 microg/ml, respectively. Morphological changes of the cells were also observed. With isolated rat liver mitochondria, the extract slightly stimulated mitochondrial state 4 respiration but profoundly depressed state 3 respiration and respiratory control ratio. CONCLUSIONS: The extract impairs energy metabolism by acting as inhibitor of oxidative phosphorylation and weak mitochondrial uncoupler. These mitochondrial effects may play a role in the cytotoxic action of Phyllanthus urinaria extract on HepG2 cells. These results provide preliminary experimental evidence supporting the use of Phyllanthus urinaria against hepatocellular carcinoma and open the possibility of considering this plant an adjunctive medicine for the treatment of this deadly disease.

Proteomic analysis of phosphotyrosyl proteins in morphine-dependent rat brains.

Morphine has been used as a potent analgesic, having a high propensity to induce tolerance and physical dependence following their repeated administration. Although the mechanisms that underlie the development of dependence on morphine remain unclear, previous studies suggested that phosphorylations of diverse types of cellular proteins are crucial determinants of the neuroadaptive mechanisms associated with morphine dependence. Thus, understanding global phosphorylation events induced by chronic morphine administration is essential for understanding the complex signaling mechanisms of morphine dependence. This study characterized the alteration of tyrosine phosphorylation of frontal cortical proteins in morphine-dependent rat brains using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of morphine (26 nmol/microl/h) for 72 h via osmotic minipumps in rats. Phosphotyrosyl (p-Tyr) protein spots in brain frontal cortical regions were detected by two-dimensional electrophoresis (2-DE) and immunoblotting with anti-p-Tyr-specific antibodies. The protein spots showing significant changes in tyrosine phosphorylation were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Similar patterns of protein expression were detected by 2-DE gels in morphine-dependent and saline-treated control rat brains. However, phosphotyrosine 2-DE images of the frontal cortical proteins from saline-treated control and morphine-dependent rat brains were apparently different. The densities of most matched p-Tyr protein spots were increased in morphine-dependent rat brains compared with that of control samples. Additional p-Tyr protein spots were detected in 2-DE image of morphine-dependent rat brains. Fifty of p-Tyr protein spots, corresponding to 40 different proteins, were identified from 2-DE gels of morphine-dependent rat brains. The identified proteins include enzymes, cytoskeletal proteins, cell signaling molecules, and other proteins. In conclusion, the first available phosphotyrosine proteomic resources of morphine dependence were established using an animal model. The findings illustrate the potential of proteomics as an effective technique for studying phosphorylation events of morphine dependence in brains.

Proteomic analysis of phosphotyrosyl proteins in the rat brain: effect of butorphanol dependence.

Butorphanol (17-cyclobutylmethyl-3,14-dihydroxymorphinan) tartrate (Stadol) is a mixed agonist-antagonist opioid analgesic agent that is about five to seven times as potent as morphine in analgesic effects. The chronic use of butorphanol produces physical dependence in humans and animals. Phosphorylation plays a very important role in developing butorphanol dependence; however, global phosphorylation events induced by chronic butorphanol administration have not been reported. The aim of this study is to determine the alteration of tyrosine phosphorylation of brain frontal cortical proteins in butorphanol-dependent rats using a proteomic approach. Dependence was produced by continuous intracerebroventricular (i.c.v.) infusion of butorphanol (26 nmol/microl/hr) for 72 hr via osmotic minipump in rats. Similar patterns of protein expression were detected by two-dimensional electrophoresis (2-DE) in brain frontal cortex of butorphanol-dependent and saline-treated control rats. All 65 phosphotyrosyl (p-Tyr) protein spots detected in pH 3-10 phosphotyrosine 2-DE of control rat brains were detected in butorphanol-dependent rat brains. The densities of most p-Tyr protein spots were increased in butorphanol-dependent rat brains compared to saline-treated control samples. Eighteen additional p-Tyr protein spots were detected in pH 3-10 2-DE images of butorphanol-dependent rat brains. Immobilized pH strips with three different narrow pH ranges were examined to improve the resolution of p-Tyr proteins in 2-DE gels. Fifty-three p-Tyr protein spots were identified as known proteins involved in cell cytoskeleton, cell metabolism, and cell signaling. This proteomic approach can provide useful information for understanding the complex mechanism of butorphanol dependence in vivo.

Nonspecific effects of the selective kappa-opioid receptor agonist U-50,488H on dopamine uptake and release in PC12 cells.

kappa-Opioid receptor agonists decrease the levels of extracellular dopamine in vivo and in vitro. However, the mechanism(s) underlying these actions are unclear. The objective of this study was to distinguish between an effect of the selective kappa-opioid receptor agonist U-50,488H ((trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide methanesulfonate) on secretion and reuptake of dopamine by PC12 cells. The data show that U-50,488H has both a modest effect to increase dopamine release and a more pronounced effect to inhibit dopamine uptake. Neither effect was sensitive to nor-binaltorphimine or naloxone, suggesting that they are not mediated through an opioid receptor.

Nonopioid receptor-mediated effects of U-50,488H on [Ca(2+)]i and extracellular dopamine in PC12 cells.

The present studies were carried out to determine the effects of a kappa-opioid receptor agonist on cytosolic Ca(2+) concentration, [Ca(2+)](i), and extracellular dopamine in undifferentiated PC12 cells. The kappa-opioid receptor agonist U-50,488H caused concentration-dependent increases in [Ca(2+)](i) and extracellular dopamine. Neither effect was blocked by the selective kappa-opioid receptor antagonist nor-binaltorphimine. Increases in extracellular dopamine content and [Ca(2+)](i) caused by U-50,488H were correlated positively in the presence of extracellular Ca(2+); however, reduction of extracellular Ca(2+) abolished the increase in [Ca(2+)](i), but not that in dopamine. The latter observation suggests that stimulation of exocytotic release is not the primary mechanism involved in the increase in extracellular dopamine caused by U-50,488H. Effects on dopamine synthesis or catabolism also seem unlikely because the enhancement of extracellular dopamine occurred rapidly, and the amount of a major metabolite of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), was not affected. In any event, neither the increase in [Ca(2+)](i) nor the increase in extracellular dopamine caused by U-50,488H is mediated by the kappa-opioid receptor.