Attenuation of methylglyoxal-induced glycation and cellular dysfunction in wound healing by Centella cordifolia.

Current pre-clinical evidences of Centella focus on its pharmacological effects on normal wound healing but there are limited studies on the bioactivity of Centella in cellular dysfunction associated with diabetic wounds. Hence we planned to examine the potential of Centella cordifolia in inhibiting methylglyoxal (MGO)-induced extracellular matrix (ECM) glycation and promoting the related cellular functions. A Cell-ECM adhesion assay examined the ECM glycation induced by MGO. Different cell types that contribute to the healing process (fibroblasts, keratinocytes and endothelial cells) were evaluated for their ability to adhere to the glycated ECM. Methanolic extract of Centella species was prepared and partitioned to yield different solvent fractions which were further analysed by high performance liquid chromatography equipped with photodiode array detector (HPLC-PDA) method. Based on the antioxidant [2,2-diphenyl-1-picrylhydrazyl (DPPH) assay] screening, anti-glycation activity and total phenolic content (TPC) of the different Centella species and fractions, the ethyl acetate fraction of C. cordifolia was selected for further investigating its ability to inhibit MGO-induced ECM glycation and promote cellular distribution and adhesion. Out of the three Centella species (C. asiatica, C. cordifolia and C. erecta), the methanolic extract of C. cordifolia showed maximum inhibition of Advanced glycation end products (AGE) fluorescence (20.20 ± 4.69 %, 25.00 ± 3.58 % and 16.18 ± 1.40 %, respectively). Its ethyl acetate fraction was enriched with phenolic compounds (3.91 ± 0.12 mg CAE/µg fraction) and showed strong antioxidant (59.95 ± 7.18 µM TE/µg fraction) and antiglycation activities. Improvement of cells spreading and adhesion of endothelial cells, fibroblasts and keratinocytes was observed for ethyl acetate treated MGO-glycated extracellular matrix. Significant reduction in attachment capacity of EA.hy926 cells seeded on MGO-glycated fibronectin (41.2%) and attachment reduction of NIH3t3 and HaCaT cells seeded on MGO-glycated collagen (33.7% and 24.1%, respectively) were observed. Our findings demonstrate that ethyl acetate fraction of C. cordifolia was effective in attenuating MGO-induced glycation and cellular dysfunction in the in-vitro wound healing models suggesting that C. cordifolia could be a potential candidate for diabetic wound healing. It could be subjected for further isolation of new phytoconstituents having potential diabetic wound healing properties.

The kainate receptor antagonist UBP310 but not single deletion of GluK1, GluK2, or GluK3 subunits, inhibits MPTP-induced degeneration in the mouse midbrain.

The excitatory neurotransmitter glutamate is essential in basal ganglia motor circuits and has long been thought to contribute to cell death and degeneration in Parkinson's disease (PD). While previous research has shown a significant role of NMDA and AMPA receptors in both excitotoxicity and PD, the third class of ionotropic glutamate receptors, kainate receptors, have been less well studied. Given the expression of kainate receptor subunits GluK1-GluK3 in key PD-related brain regions, it has been suggested that GluK1-GluK3 may contribute to excitotoxic cell loss. Therefore the neuroprotective potential of the kainate receptor antagonist UBP310 in animal models of PD was investigated in this study. Stereological quantification revealed administration of UBP310 significantly increased survival of dopaminergic and total neuron populations in the substantia nigra pars compacta in the acute MPTP mouse model of PD. In contrast, UBP310 was unable to rescue MPTP-induced loss of dopamine levels or dopamine transporter expression in the striatum. Furthermore, deletion of GluK1, GluK2 or GluK3 had no effect on MPTP or UBP310-mediated effects across all measures. Interestingly, UBP310 did not attenuate cell loss in the midbrain induced by intrastriatal 6-OHDA toxicity. These results indicate UBP310 provides neuroprotection in the midbrain against MPTP neurotoxicity that is not dependent on specific kainate receptor subunits.

Differentiation of Three Centella Species in Australia as Inferred from Morphological Characteristics, ISSR Molecular Fingerprinting and Phytochemical Composition.

Centella asiatica is one of the popular herbs used for inflammatory and neural conditions. Its differentiation from similar species is currently lacking. The aims of this study were to differentiate the three closely related Centella species using methods based on morphological characters, genetic biodiversity, phytochemical compositions and antioxidant activities. According to the morphological characteristics, the collected samples were identified as three species: C. asiatica, Centella cordifolia and Centella erecta and clustered into three groups based on their morphometric variability. Dendogram constructed on the basis of the intersimple sequence repeats (ISSR) analyses were consistent with the morphological grouping. Centella cordifolia had the highest triterpene glycosides, phenolics and antioxidant capacity, followed by C. asiatica, then C. erecta, therefore, was genetically and chemically closer to C. asiatica, while C. erecta was distinctively different from them. The results confirm the occurrence of the closely related three species of Centella in Australia, and the differentiation among them can be achieved via the combination of morphometric, molecular and phytochemical methods. This first comparative botanical study on Centella species provides a foundation for further systematic study and medicinal development of Centella.

Optimisation of Pueraria isoflavonoids by response surface methodology using ultrasonic-assisted extraction.

Puerariae Lobatae Radix (PLR) exerts cyto-protective effect against oxidative stress due to its high isoflavonoid content. In this study, the ultrasonic-assisted extraction condition for the maximum recovery of isoflavonoids with high cyto-protective effect was optimised by response surface methodology (RSM). A second-order polynomial fitted the experimental data (R2: 0.9736; p-value <0.0001). The optimal extraction parameters were determined as: extraction time 16.02min, ethanol concentration 41.41% and liquid-to-solid ratio 44.35mL/g. Practical experiments with extraction time 16.00min, ethanol concentration 41.00% and liquid-to-solid ratio 44.00mL/g were carried out in triplicate. This subsequently yielded a cell viability of 82.90±0.78% against hydrogen peroxide-induced oxidative stress on EA.hy926, and was comparable to the predicted of 85.60%. Five chemical constituents in the extract were identified to exert cyto-protective effect. Taken together, this method successfully integrated RSM and the partial least squares regression method to optimise the PLR extract with highest cyto-protective activity.

The Differential Binding of Antipsychotic Drugs to the ABC Transporter P-Glycoprotein Predicts Cannabinoid-Antipsychotic Drug Interactions.

Cannabis use increases rates of psychotic relapse and treatment failure in schizophrenia patients. Clinical studies suggest that cannabis use reduces the efficacy of antipsychotic drugs, but there has been no direct demonstration of this in a controlled study. The present study demonstrates that exposure to the principal phytocannabinoid, Δ9-tetrahydrocannabinol (THC), reverses the neurobehavioral effects of the antipsychotic drug risperidone in mice. THC exposure did not influence D2 and 5-HT2A receptor binding, the major targets of antipsychotic action, but it lowered the brain concentrations of risperidone and its active metabolite, 9-hydroxy risperidone. As risperidone and its active metabolite are excellent substrates of the ABC transporter P-glycoprotein (P-gp), we hypothesized that THC might increase P-gp expression at the blood-brain barrier (BBB) and thus enhance efflux of risperidone and its metabolite from brain tissue. We confirmed that the brain disposition of risperidone and 9-hydroxy risperidone is strongly influenced by P-gp, as P-gp knockout mice displayed greater brain concentrations of these drugs than wild-type mice. Furthermore, we demonstrated that THC exposure increased P-gp expression in various brain regions important to risperidone's antipsychotic action. We then showed that THC exposure did not influence the neurobehavioral effects of clozapine. Clozapine shares a very similar antipsychotic mode of action to risperidone, but unlike risperidone is not a P-gp substrate. Our results imply that clozapine or non-P-gp substrate antipsychotic drugs may be better first-line treatments for schizophrenia patients with a history of cannabis use.

Activin A Inhibits MPTP and LPS-Induced Increases in Inflammatory Cell Populations and Loss of Dopamine Neurons in the Mouse Midbrain In Vivo.

Parkinson's disease is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta region and a subsequent loss of dopamine within the striatum. A promising avenue of research has been the administration of growth factors to promote the survival of remaining midbrain neurons, although the mechanism by which they provide neuroprotection is not understood. Activin A, a member of the transforming growth factor ß superfamily, has been shown to be a potent anti-inflammatory following acute brain injury and has been demonstrated to play a role in the neuroprotection of midbrain neurons against MPP+-induced degeneration in vitro. We hypothesized that activin A may offer similar anti-inflammatory and neuroprotective effects in in vivo mouse models of Parkinson's disease. We found that activin A significantly attenuated the inflammatory response induced by both MPTP and intranigral administration of lipopolysaccharide in C57BL/6 mice. We found that administration of activin A promoted survival of dopaminergic and total neuron populations in the pars compacta region both 8 days and 8 weeks after MPTP-induced degeneration. Surprisingly, no corresponding protection of striatal dopamine levels was found. Furthermore, activin A failed to protect against loss of striatal dopamine transporter expression in the striatum, suggesting the neuroprotective action of activin A may be localized to the substantia nigra. Together, these results provide the first evidence that activin A exerts potent neuroprotection and anti-inflammatory effects in the MPTP and lipopolysaccharide mouse models of Parkinson's disease.

Behavioral and cognitive data in mice with different tryptophan-metabolizing enzymes knocked out.

This article demonstrates behavioral changes in mice in response to free adaptation and drinking session adaptation modules implemented in their social home environment, the IntelliCage. These data complement the study "Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function" (Too LK, Li KM, Suarna C, Maghzal GJ, Stocker R, McGregor IS, et al., 2016) [1]. Prior to programmed drinking sessions, all mice were exposed to a home cage adaptation module during which there was no time limit on water access - the free adaptation module. The exploratory behaviors are here expressed as percentages of visits with nosepokes and of visits with licks. The measurements by percentage of exploratory activity showed minimal genotype effects. The number of nosepokes or licks per corner visit also was compared between WT and gene knockout (GKO) IDO1 mice, WT and GKO IDO2 mice and WT and GKO TDO2 mice and demonstrated unremarkable behavioral changes during the free adaptation module. Analysis of drinking session adaptation behavior showed no genotype effect between WT and GKO of IDO1, IDO2 or TDO2 background. Notwithstanding the absence of genotype differences, each IDO1, IDO2 or TDO2 animal group displayed a specific pattern of adaptation to the drinking session modules. Furthermore, IDO1 GKO mice showed a more rapid recovery of lick frequency to the baseline level compared to the WT equivalents in a simple patrolling task during the first complete testing cycle (R1). TDO2 GKO mice on the other hand did not differ from their WT equivalents in terms of lick frequency over the three test days of complex patrolling and discrimination reversal tasks. Lastly, IDO2 GKO mice reduced their visits to the permanently non-rewarding reference corners by the same degree as did the WT mice.

Deletion of TDO2, IDO-1 and IDO-2 differentially affects mouse behavior and cognitive function.

Tryptophan, an amino acid involved in routine energy metabolism, is a key modulator of sickness behaviors associated with inflammatory states and also plays roles in some psychiatric disorders. Tissue concentrations of tryptophan are regulated primarily by the enzymes indoleamine 2,3-dioxygenase 1 (IDO1), IDO2 and tryptophan 2,3-dioxygenase (TDO, encoded by TDO2). Altered IDO1 and TDO activities have been linked to the perturbed serotonergic neurotransmission that may underlie certain psychopathologies. Here we assessed mice genetically modified to be deficient in IDO1, IDO2 or TDO2 for their behavior and cognitive function using an automated home cage system, the IntelliCage™. A well-established behavioural and cognitive test battery was applied during two periods (Runs 1 and 2, "R1" and "R2") separated by one month. Various tryptophan-related neurochemicals also were measured in brain extracts. IDO1(-/-) mice displayed remarkable reductions of early diurnal exploration in the IntelliCage and this persisted in R2. In contrast, early diurnal hyperactivity was observed in IDO2(-/-) mice in both R1 and R2. TDO2(-/-) mice displayed increased diurnal and nocturnal exploration, but only in R2. Cognitive assessment suggested enhanced reference memory in IDO2(-/-) mice in a complex patrolling task, while TDO deficiency was associated with enhanced performance in complex patrolling and discrimination reversal tasks. Neurochemical measures showed attenuated brain serotonin levels in IDO1(-/-) mice and augmented tryptophan and serotonin levels in TDO2(-/-) animals, respectively. No neurochemical alterations were detected in IDO2(-/-) mice. Taken together, these findings reveal complex and dissimilar patterns of behavioral and cognitive changes induced by knockout of three different tryptophan-metabolizing enzymes.

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

Contrasting regional Fos expression in adolescent and young adult rats following acute administration of the antidepressant paroxetine.

Adolescents and adults may respond differently to antidepressants, with poorer efficacy and greater probability of adverse effects in adolescents. The mechanisms underlying this differential response are largely unknown, but likely relate to an interaction between the neural effects of antidepressants and brain development. We used Fos immunohistochemistry to examine regional differences in adolescent (postnatal day (PND) 28) and young adult (PND 56) male, Wistar rats given a single injection of the selective serotonin reuptake inhibitor paroxetine (10mg/kg). Paroxetine induced widespread Fos expression in both adolescent and young adult rats. Commonly affected areas include the bed nucleus of the stria terminalis (dorsolateral), medial preoptic area, paraventricular hypothalamic and thalamic nuclei and central nucleus of the amygdala. Fos expression was generally lower in adolescents with significantly greater Fos expression observed in young adults in the prelimbic cortex, supraoptic nucleus, basolateral amygdala, lateral parabrachial and Kölliker-Fuse nuclei. However, a small subset of regions showed greater adolescent Fos expression including the nucleus accumbens shell, lateral habenula and dorsal raphe. Paroxetine increased plasma corticosterone concentrations in young adults, but not adolescents. Plasma paroxetine levels were not significantly different between the age groups. These results indicate a different c-Fos signature of acute paroxetine in adolescent rats, with greater activation in key mesolimbic and serotonergic regions, but a more subdued cortical, brainstem and hypothalamic response. This suggests that the atypical response of adolescents to paroxetine may be related to a blunted neuroendocrine response, combined with insufficient top-down regulation of limbic regions involved in reward and impulsivity.

Curcumin Reduces Tumour Necrosis Factor-Enhanced Annexin V-Positive Microparticle Release in Human Vascular Endothelial Cells.

PURPOSE: Circulating microparticles have been highlighted as biomarkers of cardiovascular disease state and progression. The aim of this study was to evaluate the effects of curcumin on microparticle release from endothelial cells undergoing TNF-induced cell activation and apoptosis. METHODS: This study evaluated the effects of curcumin on microparticle release, cytotoxicity, apoptosis, cell adhesion molecule expression and monocyte adhesion in EAhy926 human endothelial cells. RESULTS: The results showed that the numbers of microparticles were increased by tumour necrosis factor (TNF) or the combination of TNF and cycloheximide (CHX). Curcumin attenuated microparticle release caused by TNF or TNF plus CHX treatments. The pretreatment by curcumin not only negated the accelerated cell death and apoptosis caused by TNF and CHX, but also diminished TNF-induced cell activation, as assessed by reduced surface expression of intercellular adhesion molecule 1, and adhesion of monocytes to endothelial monolayers. CONCLUSION: Curcumin reduced microparticle release from endothelial cells undergoing cell activation and apoptosis, which supports its protective role in TNF-associated endothelial dysfunction, and highlights its potential use as a nutraceutical agent for vascular inflammatory diseases. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

Seasonal Variation of Triterpenes and Phenolic Compounds in Australian Centella asiatica (L.) Urb.

INTRODUCTION: Specific triterpenes, phenolic acids and flavonoids in Centella asiatica have been found to be bioactive. Harvesting the plant when these putative bioactive compounds are at their highest concentrations would provide consistency in their chemical profile, thus ensuring the quality and efficacy of derived medicinal products. OBJECTIVE: The aim of the study was to determine the impact of harvesting time on the contents of major triterpenoid and phenolic compounds in C. asiatica. METHODOLOGY: Australian C. asiatica was collected from a designated area in different months. The principal triterpenes (asiaticoside, madecassoside, asiatic acid and madecassic acid), flavonoid compounds (rutin, quercetin and kaempferol) and chlorogenic acid were quantitatively determined by HPLC-DAD analysis. RESULTS: Triterpenoid, kaempferol and chlorogenic acid content showed significant variation (p < 0.05) in different collecting months. The total content of the four triterpenes reached its highest levels in January and February (83.15 ± 0.16 mg/g and 78.41 ± 0.16 mg/g, respectively), the summer season of the southern hemisphere, and their lowest values in winter (June) and spring (October) seasons (35.65 ± 0.20 and 35.50 ± 0.55 mg/g, respectively). Similarly, the contents of chlorogenic acid and kaempferol were the highest in December and January (1.62 ± 0.01 and 0.33 ± 0.01 mg/g, respectively), and the lowest in June (0.06 ± 0.01 and 0.09 ± 0.01 mg/g, respectively). CONCLUSION: The results indicate that harvesting C. asiatica in summer returns the highest yield of the target triterpenoids, kaempferol and chlorogenic acid.

Activin A protects midbrain neurons in the 6-hydroxydopamine mouse model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by a significant loss of dopaminergic neurons within the substantia nigra pars compacta (SNpc) and a subsequent loss of dopamine (DA) within the striatum. Despite advances in the development of pharmacological therapies that are effective at alleviating the symptoms of PD, the search for therapeutic treatments that halt or slow the underlying nigral degeneration remains a particular challenge. Activin A, a member of the transforming growth factor ß superfamily, has been shown to play a role in the neuroprotection of midbrain neurons against 6-hydroxydopamine (6-OHDA) in vitro, suggesting that activin A may offer similar neuroprotective effects in in vivo models of PD. Using robust stereological methods, we found that intrastriatal injections of 6-OHDA results in a significant loss of both TH positive and NeuN positive populations in the SNpc at 1, 2, and 3 weeks post-lesioning in drug naïve mice. Exogenous application of activin A for 7 days, beginning the day prior to 6-OHDA administration, resulted in a significant survival of both dopaminergic and total neuron numbers in the SNpc against 6-OHDA-induced toxicity. However, we found no corresponding protection of striatal DA or dopamine transporter (DAT) expression levels in animals receiving activin A compared to vehicle controls. These results provide the first evidence that activin A exerts potent neuroprotection in a mouse model of PD, however this neuroprotection may be localized to the midbrain.

Comparing morphological, chemical and anti-diabetic characteristics of Puerariae Lobatae Radix and Puerariae Thomsonii Radix.

ETHNOPHARMACOLOGICAL RELEVANCE: Puerariae Lobatae Radix (PLR) and Puerariae Thomsonii Radix (PTR) are traditional Chinese medicines used for the treatment of diabetes and cardiovascular diseases. These two herbs are used interchangeably in clinical practice, even though they possess significantly different chemical profiles. In the case of Pueraria species, the misidentification is related to the multiple Chinese common names in clinical practice and variable pharmaceutical Latin names in different versions of the Pharmacopoeia of the People's Republic of China. In addition, there is lack of evidence demonstrating how the differences in the chemical profile would impact on the pharmacological activity of the two herbs. Therefore, the aim of this study was to compare the microscopic, phytochemical profiles and anti-diabetic activity of PLR and PTR so that the two species can be differentiated. MATERIALS AND METHODS: The microscopic characteristics of the PLR and PTR were observed and measured by an optical microscope. The major compounds were quantified by ultra-performance liquid chromatography (UPLC) and total flavonoid content (TFC) colorimetric assay. The free radical scavenging capacity was assessed by 2,2-diphenyl-2-picrylhydrazyl (DPPH) antioxidant assays. Anti-diabetic activity was determined by the inhibition of porcine pancreatic α-amylase and rat intestinal α-glucosidase activities. RESULTS: Microscopic results illustrated that the size of xylem vessels (PLR: 0.1390 ± 0.0184 mm; PTR: 0.0471 ± 0.0109 mm), number of fibre per bundle (PLR: 32.6800 ± 2.8780; PTR: 16.5900 ± 0.9982) and the size of fibre (PLR: 0.0075 ± 0.0003 mm(2); PTR: 0.0025 ± 0.0002 mm(2)) in PLR were significantly greater than that in PTR (p<0.01). PLR possessed a significantly lower total starch content (PLR: 0.5288 ± 1.2559 mg starch/g DM; PTR: 76.7954 ± 2.9905 mg starch/g DM) and total dietary fibre content (PLR: 4.2886 ± 0.3466 g/100g DM; PTR: 12.4148 ± 0.4541 g/100g DM) as compared to PTR. Isoflavonoids including puerarin, daidzin, genistin and daidzein were the major chemical constituents in both species. However, the average content of puerarin in PLR was found to be eleven times greater than that in PTR. Furthermore, the TFC, DPPH free radical scavenging capacity, anti-α-amylase and anti-α-glucosidase in the PLR extracts were 4.42, 4.91, 3.10 and 4.22 times greater than in the PTR extracts. CONCLUSIONS: This study provides a comprehensive investigation on the two medicinal valuable Pueraria species and allows differences to be ascertained. This information can be used to update monographs which will help practitioners and dispensers differentiate the herbs. Further study on the interchangeable use of PLR and PTR in clinical practice is urgently warranted.

Differentiating Puerariae Lobatae Radix and Puerariae Thomsonii Radix using HPTLC coupled with multivariate classification analyses.

Puerariae Lobatae Radix (PLR), the root of Pueraria lobata, is a traditional Chinese medicine for treating diabetes and cardiovascular diseases. Puerariae Thomsonii Radix (PTR), the root of Pueraria thomsonii, is a closely related species to PLR and has been used as a PLR substitute in clinical practice. The aim of this study was to compare the classification accuracy of high performance thin-layer chromatography (HPTLC) with that of ultra-performance liquid chromatography (UPLC) in differentiating PLR from PTR. The Matlab functions were used to facilitate the digitalisation and pre-processing of the HPTLC plates. Seven multivariate classification methods were evaluated for the two chromatographic methods. The results demonstrated that the HPTLC classification models were comparable to the UPLC classification models. In particular, k-nearest neighbours, partial least square-discriminant analysis, principal component analysis-discriminant analysis and support vector machine-discriminant analysis showed the highest rate of correct species classification, whilst the lowest classification rate was obtained from soft independent modelling of class analogy. In conclusion, HPTLC combined with multivariate analysis is a promising technique for the quality control and differentiation of PLR and PTR.

Gallic acid protects against endothelial injury by restoring the depletion of DNA methyltransferase 1 and inhibiting proteasome activities.

BACKGROUND: The aim of this study was to investigate the protective effects of gallic acid, a common phenolic compound naturally present in food and nutraceuticals, on endothelial cell death and the mechanisms involved. METHODS: Endothelial cell death was induced by the combination of homocysteine, adenosine and tumour necrosis factor (TNF) in human vascular endothelial cells (EAhy926 and HBEC-5i cells). The protective effects of gallic acid were evaluated against cytotoxicity, apoptosis and microparticle release. Underlying mechanisms were further investigated focusing on the involvement of DNA methyltransferase 1 (DNMT1) and proteasome activities. RESULTS: Our results showed that gallic acid dose-dependently arrested cytotoxicity in EAhy926 and HBEC-5i cells induced by the combination. Gallic acid showed anti-apoptotic effects and reduced the formation of microparticles. Notably, gallic acid reversed DNMT1 depletions at the protein level. The cytoprotective and anti-apoptotic effects of gallic acid were counteracted by the pre-treatment with DNMT1 inhibitor, 5-aza-2-deoxycytidine (5-aza-dC). Treatment with gallic acid led to the accumulation of ubiquitinated protein aggregates and the reduction in chymotrypsin-like proteasome activities indicating proteasome inhibition. CONCLUSION: Our results demonstrate for the first time that gallic acid is capable of protecting endothelial cells from injury induced by the combination of homocysteine, adenosine and TNF, at least in part, by restoring the depletion of DNMT1 and inhibiting proteasome activities.

Analysis of cannabis seizures in NSW, Australia: cannabis potency and cannabinoid profile.

Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ(9)-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales "Cannabis Cautioning" scheme. A further 26 "Known Provenance" samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The "Cannabis Cautioning" samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (<0.1%). "Known Provenance" samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed.

Differentiation of Pueraria lobata and Pueraria thomsonii using partial least square discriminant analysis (PLS-DA).

The aims of the study were to differentiate Pueraria lobata from its related species Pueraria thomsonii and to examine the raw herbal material used in manufacturing kudzu root granules using partial least square discriminant analysis (PLS-DA). Sixty-four raw materials of P. lobata and P. thomsonii and kudzu root-labelled granules were analysed by ultra performance liquid chromatography. To differentiate P. lobata from P. thomsonii, PLS-DA models using the variables selected from the entire chromatograms, genetic algorithm (GA), successive projection algorithm (SPA), puerarin alone and six selected peaks were employed. The models constructed by GA and SPA demonstrated superior classification ability and lower model's complexity as compared to the model based on the entire chromatographic matrix, whilst the model constructed by the six selected peaks was comparable to the entire chromatographic model. The model established by puerarin alone showed inferior classification ability. In addition, the PLS-DA models constructed by the entire chromatographic matrix, GA, SPA and the six selected peaks showed that four brands out of seventeen granules were mislabelled as P. lobata. In conclusion, PLS-DA is a promising procedure for differentiating Pueraria species and determining raw material used in commercial products.

High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine.

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

Vincristine chemotherapy trials and pharmacokinetics in tasmanian devils with tasmanian devil facial tumor disease.

Tasmanian Devil Facial Tumor Disease (DFTD) is a transmissible cancer threatening to cause the extinction of Tasmanian Devils in the wild. The aim of this study was to determine the susceptibility of the DFTD to vincristine. Escalating dosage rates of vincristine (0.05 to 0.136 mg/kg) were given to Tasmanian devils in the early stages of DFTD (n = 8). None of these dosage rates impacted the outcome of the disease. A dosage rate of 0.105 mg/kg, a rate significantly higher than that given in humans or domestic animals, was found to the highest dosage rate that could be administered safely. Signs of toxicity included anorexia, vomiting, diarrhea and neutropenia. Pharmacokinetic studies showed that, as with other species, there was a rapid drop in blood concentration following a rapid intravenous infusion with a high volume of distribution (1.96 L/kg) and a relatively long elimination half life (11 h). Plasma clearance (1.8 ml/min/kg) was slower in the Tasmanian devil than in humans, suggesting that pharmacodynamics and not pharmacokinetics explain the Tasmanian devil's ability to tolerate high dosage rates of vincristine. While providing base-line data for the use of vincristine in Tasmanian devils and possibly other marsupials with vincristine susceptible cancers, these findings strongly suggest that vincristine will not be effective in the treatment of DFTD.