Controlled non-invasive transdermal iontophoretic delivery of zolmitriptan hydrochloride in vitro and in vivo

The objective was to investigate the transdermal delivery kinetics of zolmitriptan from an iontophoretic patch system in Yorkshire swine in vivo. Preliminary in vitro experiments showed that cumulative drug transport during a 6-h current application (0.25 mA cm⁻²) was independent of patch load (263.7 ± 92.7, 357.2 ± 85.9, 374.9 ± 74.3 and 335.9 ± 27.7 μg cm⁻² for 7.5, 15, 45 and 90 mg patch loads, respectively; ANOVA, p < 0.05); the steady-state flux was ∼92 μg cm⁻² h⁻¹. The in vivo studies used multistep current profiles to demonstrate (i) rapid drug uptake and (ii) the effect of superposing a bolus input on basal drug levels. In both studies, zolmitriptan was detected in the blood after 2.5 min; drug levels were 7.1  1.7 and 10.4 ± 3.5 ng ml⁻¹ at t = 30min in Studies 1 and 2, respectively. In Study 2, increasing current intensity from 0.2 to 1.4 mA (0.05-0.35 mA cm⁻²) at t = 180 min caused zolmitriptan levels to rise from 9.38 ± 0.93 ng ml⁻¹ at t = 180 min to 13.57 ± 1.85 ng ml⁻¹ at t = 190 min; a ∼50% increase in 10 min. Extrapolation of these results to humans suggests the feasibility of delivering therapeutic amounts of zolmitriptan at faster rates than those from existing dosage forms.     

Feed contaminated with Fusarium toxins alter lymphocyte proliferation and apoptosis in primiparous sows during the perinatal period

Two groups of pregnant primiparous sows (day 89 ± 2 of gestation) were 54 days (±1 day) fed either with an experimental diet (5.08 mg kg⁻¹ deoxynivalenol – DON, 0.09 mg kg⁻¹ zearalenone and 21.61 mg kg⁻¹ fusaric acid) or control diet (0.25 mg kg⁻¹ DON). The consummation of feed was significantly higher in the control group. Lymphocyte stimulation in culture from peripheral blood lymphocytes (PBL) was measured by BrdU incorporation test using two different concentrations of mitogen PHA 10 and 20 μg ml⁻¹, two different concentrations of DON (5 and 1 μg ml⁻¹) and a combination of both, PHA and DON (PHA 10 + DON 5, PHA 10 + DON 1 and PHA 10 + DON 0.1 μg ml⁻¹). The lymphocyte proliferation, except for PHA 10 μg ml⁻¹, was significantly higher in the experimental group. Further on, using the photometric enzyme immunoassay, the apoptosis was studied in non-stimulated 72 h lymphocyte culture or stimulated with 1 μg ml⁻¹ of DON. The significantly higher apoptosis was in non-stimulated lymphocyte cultures from the experimental group (P = 0.036). The results suggest that such feed may affect the peripheral lymphocyte population in the direction of their proliferation response and programmed cell death.     

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 10⁵Alexandrium minutum dinoflagellate cells g⁻¹ (containing 12 ± 3.0 μg STX-equivalent 100 g⁻¹, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g⁻¹ in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g⁻¹ tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g⁻¹ after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.     

Chemical composition,antifungal and antiaflatoxigenic activities of Ocimum sanctum L. essential oil and its safety assessment as plant based antimicrobial

The study deals with the efficacy of Ocimum sanctum essential oil (EO) and its major component, eugenol against the fungi causing biodeterioration of food stuffs during storage. O. sanctum EO and eugenol were found efficacious in checking growth of Aspergillus flavus NKDHV8; and, their minimum inhibitory concentrations (MICs) were recorded as 0.3 and 0.2 μl ml⁻¹, respectively. The O. sanctum EO and eugenol also inhibited the aflatoxin B₁ production completely at 0.2 and 0.1 μl ml⁻¹, respectively. Both of these were found superior over some prevalent synthetic antifungals and exhibited broad fungitoxic spectrum against 12 commonly occurring fungi. The LD₅₀ value of O. sanctum EO on mice was found to be 4571.43 μl kg⁻¹ suggesting its non-mammalian toxic nature. The findings of present study reveals the possible exploitation of O. sanctum EO and eugenol as plant based safe preservatives against fungal spoilage of food stuffs during storage.     

Venom of Collett's snake (Pseudechis colletti) blocks the binding of α-bungarotoxin to acetylcholine receptors at chick but not human neuromuscular junctions: A histochemical study

Transverse cryosections, 6-8 μm thick, were cut from unfixed biventer cervicis muscles of chicks and quadriceps muscles of humans, mounted on glass slides and incubated for 1 h in either isotonic phosphate buffered saline, pH 7.3 (PBS), or crude venom of venom of Pseudechis colletti at concentrations between 2.1 and 210 μg ml⁻¹ in PBS. They were then exposed to a fluoresceine-conjugated α-bungarotoxin to label ACh receptor sites. Exposure to the crude venom of P. colletti prevented the labelling of acetylcholine (ACh) receptors in chick muscle in a dose-dependant manner; at a concentration of 2.1 μg ml⁻¹ labelling fell by 20% and at a concentration of 21 μg ml⁻¹ by more than 90%. In contrast, exposure to the venom at concentrations as high as 210 μg ml⁻¹ had no effect on receptor labelling in human skeletal muscle. The results suggest that ACh receptors in human skeletal muscle are relatively resistant to the postsynaptically active neurotoxins in the venom of P. colletti. The data explain the apparent anomaly that the venom blocks neuromuscular transmission in isolated nerve-muscle preparations of the chick whilst human subjects of envenoming bites by P. colletti exhibit no overt signs of neurotoxicity.     

Effects of Thai Musa species on prevention of UVB-induced skin damage in mice

The effects of oral administration of Musa sapientum and Musa suerier on prevention of UVB induced skin damages were investigated in male ICR mice. Animals were orally administered 50 mg/day ascorbic acid, or M. sapientum or M. suerier’s fruit pulps at dose of 0.5, 1 or 1.5 mg/g body weight/day for 12 weeks. Concurrently, the shaved backs of animals were irradiated with UVB for 12 weeks. The intensity of irradiation was progressively increased, from 54 mJ/cm² per exposure at week 1–126 mJ/cm² at week 11. A significant decrease (p < 0.05) in skin elasticity (from 0.82 ± 0.02 to 0.42 ± 0.09) and total glutathione (from (193.6 ± 18.7 to 152.7 ± 7.8 ng/mg protein) as compared with the control group (water-administered UVB-irradiated mice) was observed after 12 weeks of UVB exposure. When l-ascorbic acid (0.72 ± 0.01) or 1 mg/g body weight/day M. suerier (0.84 ± 0.06) were administered to UVB-irradiated mice, the reduction in skin elasticity was significantly inhibited (p < 0.05). Moreover, the significant increase (p < 0.05) in level of total glutathione was found in these groups (220.8 ± 13.3 ng/mg protein for l-ascorbic acid and 224.9 ± 20.1 ng/mg protein for M. suerier). These findings suggest the potential effect of daily consumption of M. suerier on prevention of skin damage from repeated UVB exposure.     

Growth and toxin production in the ciguatera-causing dinoflagellate Gambierdiscus polynesiensis (Dinophyceae) in culture

The growth and toxin production in a clonal strain of Gambierdiscus polynesiensis, TB-92, was examined in batch culture conditions. The mean growth rate at exponential phase was (0.13 ± 0.03) division day⁻¹. Regardless of the age of cultures, all mice injected with dichloromethanolic and methanolic extracts showed symptoms specific to ciguatoxin (CTX) and maitotoxin (MTX) bioactivity, respectively. The highest total toxicity assessed in TB-92 cultures was 10.4 × 10⁻⁴ mouse unit cell⁻¹. The toxin production pattern reveals an enhanced cellular toxin content with the age of the culture. CTX- and MTX-like compounds each accounted for approx. 50% of the total toxicity of TB-92 cultures, except in aged cells where CTXs were dominant. The high ciguatoxic activity of TB-92 was further confirmed in dichloromethanolic extracts by means of the receptor-binding assay. The highest CTX level monitored at late stationary phase was (11.9 ± 0.4) pg P-CTX-3C equiv cell⁻¹. Further HPLC and LC-MS analysis revealed the presence of five CTXs congeners in lipid-soluble extracts, i.e. CTX-3C, -3B, -4A, -4B and M-seco-CTX-3C, and of new CTX congeners. Toxin composition comparison between two G. polynesiensis strains suggests that the toxin profile is a stable characteristic in this species. G. polynesiensis clones also proved inherently more toxic than other Gambierdiscus species isolated from other geographical areas.     

A probiotic feed additive containing spores of Bacillus subtilis and B. licheniformis does not prevent absorption and toxic effects of the Fusarium toxin deoxynivalenol in piglets

A 35-day piglet experiment starting from weaning (21 days of age, 7.7 ± 1.1 kg), was performed to examine the effects of feeding a control diet (CTR) and of a Fusarium toxin-contaminated diet (FUS) in the absence (−) or presence (+) of a probiotic additive (2.3 × 10⁶ colony-forming units per g diet of a one-to-one ratio of Bacillus subtilis and B. licheniformis) (deoxynivalenol [DON] concentrations of the CTR−, CTR+, FUS− and FUS+ diets were 0.21, 0.20, 2.75 and 2.45 mg/kg, respectively) on performance, blood concentration of DON and on liver function as evaluated by a breath test.     

Route of metabolization and detoxication of paralytic shellfish toxins in humans

Paralytic shellfish toxins (PST) are a collection of over 26 structurally related imidazoline guanidinium derivatives produced by marine dinoflagellates and freshwater cyanobacteria.Glucuronidation of drugs by UDP-glucuronosyltransferase (UGT) is the major phase II conjugation reaction in mammalian liver.In this study, using human liver microsomes, the in vitro paralytic shellfish toxins oxidation and sequential glucuronidation are achieved. Neosaxitoxin (neoSTX), Gonyautoxin 3/2 epimers (GTX3/GTX2) and Saxitoxin (STX) are used as starting enzymatic substrates. The enzymatic reaction final product metabolites are identified by using HPLC-FLD and HPLC/ESI-IT/MS. Four metabolites from GTX3/GTX2 epimers precursors, three of neoSTX and two of STX are clearly identified after incubating with UDPGA/NADPH and fresh liver microsomes. The glucuronic-Paralytic Shellfish Toxins were completely hydrolysed by treatment with β-glucuronidase. All toxin analogs were identified comparing their HPLC retention time with those of analytical standard reference samples and further confirmed by HPLC/ESI-IT/MS. Paralytic Shellfish Toxins (PST) were widely metabolized by human microsomes and less than 15% of the original PST, incubated as substrate, stayed behind at the end of the incubation.The apparent V_max and Km formation values for the respective glucuronides of neoSTX, GTX3/GTX2 epimers and STX were determined. The V_max formation values for Glucuronic-GTX3 and Glucuronic-GTX2 were lower than Glucuronic-neoSTX and Glucuronic-STX (6.8 ± 1.9 × 10⁻³; 8.3 ± 2.8 × 10⁻³ and 9.7 ± 2.8 × 10⁻³ pmol/min/mg protein respectively). Km of the glucuronidation reaction for GTX3/GTX2 epimers was less than that of glucuronidation of neoSTX and STX (20.2 ± 0.12; 27.06 ± 0.23 and 32.02 ± 0.64 μM respectively).In conclusion, these data show for the first time, direct evidence for the sequential oxidation and glucuronidation of PST in vitro, both being the initial detoxication reactions for the excretion of these toxins in humans. The PST oxidation and glucuronidation pathway showed here, is the hepatic conversion of its properly glucuronic-PST synthesized, and the sequential route of PST detoxication in human.     

Evaluation of the mutagenic effects of myosmine in human lymphocytes using the HPRT gene mutation assay

The minor tobacco alkaloid myosmine is implicated in DNA damage through pyridyloxobutylation similar to the tobacco-specific nitrosamines (TSNA). In contrast to TSNA, occurrence of myosmine is not restricted to tobacco. Myosmine is genotoxic to human cells in the comet assay. In this study, the mutagenic effect of myosmine was evaluated using the cloning hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutation assay. Four hour exposure of isolated peripheral blood lymphocytes from 14 subjects homozygous for the Leu84 wild-type of the O⁶-methylguanine-DNA-methyltransferase (MGMT) gene to 1 mM of myosmine increased mutant frequency from 0.73 ± 0.58 × 10⁻⁶ in control to 1.14 ± 0.89 × 10⁻⁶ lymphocytes (P < 0.05). These new data further confirm the mutagenic effects of myosmine.     

First report of anatoxin-a-producing cyanobacterium Aphanizomenon issatschenkoi in northeastern Germany

The neurotoxin anatoxin-a (ATX), has been detected in several northeast German lakes during the last two decades, but no ATX producers have been identified in German water bodies so far. In 2007 and 2008, we analyzed phytoplankton composition and ATX concentration in Lake Stolpsee (NE Germany) in order to identify ATX producers. Sixty-one Aphanizomenon spp. strains were isolated, morphologically and phylogenetically characterized, and tested for ATX production potential by liquid chromatography-tandem mass spectrometry (LC-MS/MS). New primers were specifically designed to identify a fragment of a polyketide synthase gene putatively involved in ATX synthesis and tested on all 61 Aphanizomenon spp. strains from L. Stolpsee and 92 non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from German lakes Langersee, Melangsee and Scharmützelsee.As demonstrated by LC-MS/MS, ATX concentrations in L. Stolpsee were undetectable in 2007 and ranged from 0.01 to 0.12 μg l⁻¹ in 2008. Fifty-nine of the 61 strains isolated were classified as Aphanizomenon gracile and two as Aphanizomenon issatschenkoi. One A. issatschenkoi strain was found to produce ATX at concentrations of 2354 ± 273 μg g⁻¹ fresh weight, whereas the other A. issatschenkoi strain and A. gracile strains tested negative. The polyketide synthase gene putatively involved in ATX biosynthesis was found in the ATX-producing A. issatschenkoi strain from L. Stolpsee but not in the non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from lakes Stolpsee, Langersee, Melangsee, and Scharmützelsee.This study is the first confirming A. issatschenkoi as an ATX producer in German water bodies.     

In vitro endocrine disruption and TCDD-like effects of three novel brominated flame retardants: TBPH,TBB, & TBCO

The novel brominated flame retardants (NBFRs), 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (TBB), Bis(2-ethylhexyl)-2,3,4,5-tetrabromophtalate (TBPH), and 1,2,5,6-tetrabromocyclooctane (TBCO) are components of flame retardant mixtures including Firemaster 550 and Saytex BC-48. Despite the detection of these NBFRs in environmental and biotic matrices, studies regarding their toxicological effects are poorly represented in the literature. The present study examined endocrine disruption by these three NBFRs using the yeast YES/YAS reporter assay and the mammalian H295R steroidogenesis assay. Activation of the aryl hydrocarbon receptor (AhR) was also assessed using the H4IIE reporter assay. The NBFRs produced no TCDD-like effects in the H4IIE assay or agonistic effects in the YES/YAS assays. TBB produced a maximal antiestrogenic effect of 62% at 0.5 mg L⁻¹ in the YES assay while TBPH and TBCO produced maximal antiandrogenic effects of 74% and 59% at 300 mg L⁻¹ and 1500 mg L⁻¹, respectively, in the YAS assay. Significant effects were also observed in the H295R assay. At 0.05 mg L⁻¹, 15 mg L⁻¹, and 15 mg L⁻¹ TBB, TBPH, and TBCO exposures, respectively resulted in a 2.8-fold, 5.4-fold, and 3.3-fold increase in concentrations of E2. This is one of the first studies to demonstrate the in vitro endocrine disrupting potentials of TBB, TBPH, and TBCO.     

Antioxidant activity and ultra-performance LC-electrospray ionization-quadrupole time-of-flight mass spectrometry for phenolics-based fingerprinting of Rose species: Rosa damascena, Rosa bourboniana and Rosa brunonii

Roses are one of the most important groups of ornamental plants and their fruits and flowers are used in a wide variety of food, nutritional products and different traditional medicines. The antioxidant activity of methanolic extracts from fresh flowers of three rose species (Rosa damascena, Rosa bourboniana and Rosa brunonii) was evaluated by 1,1-diphenyl-2-picryl hydrazyl (DPPH) free-radical method. The ability to scavenge DPPH radical was measured by the discoloration of the solution. The methanolic extract from R. brunonii exhibited maximum free-radical-scavenging activity (64.5 ± 0.38%) followed by R. bourboniana (51.8 ± 0.46%) and R. damascena (43.6 ± 0.25%) at 100 μg/ml. Simultaneously, ultra-performance liquid chromatography coupled with electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) was used to study phenolic composition in the methanolic extracts from the fresh flowers of rose species. The phenolic constituents were further investigated by direct infusion-ESI-QTOF-MS/MS in negative ion mode. Characteristic Electrospray ionization tandem mass spectrometry (ESI-MS/MS) spectra with other diagnostic fragment ions generated by retro Diels–Alder (RDA) fragmentation pathways were recorded for the flavonoids. Distinct similarities were observed in the relative distribution of polyphenolic compounds among the three species. The dominance of quercetin, kaempferol and their glycosides was observed in all the three species.     

Chemical profile,antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sinensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistillation and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their 1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The EOs and their combination completely inhibited aflatoxin B₁ (AFB₁) production at 500 ppm, whereas, dl-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC₅₀ for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 μl ml⁻¹, respectively. The EOs were found non-mammalian toxic showing high LD₅₀ for mice (oral, acute). The oils may be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life of food commodities by checking their fungal infestation, aflatoxin production as well as lipid peroxidation.     

Mthfr gene ablation enhances susceptibility to arsenic prenatal toxicity

Background

In utero exposure to arsenic is known to adversely affect reproductive outcomes. Evidence of arsenic teratogenicity varies widely and depends on individual genotypic differences in sensitivity to As. In this study, we investigated the potential interaction between 5,10-methylenetetrahydrofolate reductase (Mthfr) genotype and arsenic embryotoxicity using the Mthfr knockout mouse model.

Methods

Pregnant dams were treated with sodium arsenate, and reproductive outcomes including: implantation, resorption, congenital malformation and fetal birth weight were recorded at E18.5.

Results

When the dams in Mthfr^+/− × Mthfr^+/− matings were treated with 7.2 mg/kg As, the resorption rate increased to 43.4%, from a background frequency of 7.2%. The As treatment also induced external malformations (40.9%) and significantly lowered the average fetal birth weight among fetuses, without any obvious toxic effect on the dam. When comparing the pregnancy outcomes resulting from different mating scenarios (Mthfr^+/+ × Mthfr^+/−, Mthfr^+/− × Mthfr^+/− and Mthfr^−/− × ^Mthfr+/−) and arsenic exposure; the resorption rate showed a linear relationship with the number of null alleles (0, 1 or 2) in the Mthfr dams. Fetuses from nullizygous dams had the highest rate of external malformations (43%) and lowest average birth weight. When comparing the outcomes of reciprocal matings (nullizygote × wild-type versus wild-type × nullizygote) after As treatment, the null dams showed significantly higher rates of resorptions and malformations, along with lower fetal birth weights.

Conclusions

Maternal genotype contributes to the sensitivity of As embryotoxicity in the Mthfr mouse model. The fetal genotype, however, does not appear to affect the reproductive outcome after in utero As exposure.     

First report of (homo)anatoxin-a and dog neurotoxicosis after ingestion of benthic cyanobacteria in The Netherlands

In April and May 2011, three dogs died and one dog became ill after swimming in Lake IJmeer (The Netherlands). At the time, the lake was infested with the benthic cyanobacterial species Phormidium. A Eurasian Coot (Fulica atra) and a Black-headed Gull (Chroicocephalus ridibundus) also died near Lake IJmeer in the same period. One of the dogs and both birds were subjected to a pathological investigation. Furthermore, the Phormidium mat; algal samples from the dikes; contents of the animals’ digestive systems and organ tissues were analysed for the following cyanobacterial toxins: (homo)anatoxin-a; (7-deoxy-)cylindrospermopsin; saxitoxins and gonyautoxins by LC-MS/MS. Samples were also analysed for the nontoxic (homo)anatoxin-a metabolites dihydro(homo)anatoxin-a and epoxy(homo)anatoxin-a. The dog necropsy results indicated neurotoxicosis and its stomach contained Phormidium filaments. Anatoxin-a was detected in the Phormidium mat (272 μg g⁻¹ dry weight, stdev 65, n = 3) and in the dog’s stomach contents (9.5 μg g⁻¹ dry weight, stdev 2.4, n = 3). Both samples also contained the anatoxin-a metabolite dihydroanatoxin-a, and a trace of homoanatoxin-a was detected in the Phormidium mat. The birds were in bad nutritive condition at the time of necropsy and their stomachs and intestines did not contain any cyanobacterial material. Furthermore, no cyanobacterial toxins were detected in their stomachs, intestines and organs and they both had lesions that are not associated with cyanobacterial intoxication. This is the first report of anatoxin-a and homoanatoxin-a occurrence in The Netherlands, these toxins have likely caused the deaths of three dogs. The birds probably died of other causes. Dutch recreational waters are at this moment only screened for pelagic cyanobacterial species, the current bathing water protocol therefore does not protect humans and animals from negative effects of blooms of benthic cyanobacteria.     

Safety evaluation of proanthocyanidin polymer-rich fraction obtained from stem bark of Stryphnodendron adstringens (BARBATIMÃO) for use as a pharmacological agent

The widespread use of medicinal plants among the Brazilian population warrants an assessment of the potential risks associated with their intake. Stryphnodendron adstringens (barbatimão) is one of the most frequently used medicinal plants in Brazil, and the risks associated with its use have yet to be investigated. This study evaluated the genotoxic safety of the use of the proanthocyanidin polymer-rich fraction (F2) of stem bark of S. adstringens. The micronucleus test with 750, 1500, and 2250 mg kg⁻¹ of F2 administered in Mus musculus (Swiss) outbred mice, showed respectively, 5.0 ± 0.8 (Mean ± S.D.), 9.1 ± 1.7, and 10.6 ± 1.9 micronucleated polychromatic erythrocytes (MNPCE). A positive control with cyclophosphamide resulted in 21.0 ± 3.8 MNPCE. Antimutagenicity was also evaluated, by adding 750 mg kg⁻¹ to cyclophosphamide; the result of 8.7 ± 1.4 showed a protective cytotoxic effect. For the Artemia salina test, 10, 100, and 1000 mg L⁻¹ of F2 showed, respectively, 8.7 ± 0.6, 7.7 ± 0.6, and 5.7 ± 1.2 survival, i.e., F2 did not inhibit 50% of the population when compared to the control (9.7 ± 0.6). These results indicated that F2 obtained from stem bark of S. adstringens has no genotoxic activity.     

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-d-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC₅₀ around 1 μM at −70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca²⁺ responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells.Memantine was confirmed to be a moderate affinity (IC₅₀ at −70 mV of 0.79 ± 0.02 μM, Hill = 0.92 ± 0.02), strongly voltage-dependent (δ = 0.90 ± 0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 μM - onset τ_fast = 273 ± 25 ms (weight 69%), onset τ_slow = 2756 ± 296 ms, offset τ_fast = 415 ± 82 ms (weight 38%) offset τ_slow = 5107 ± 1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC₅₀ at −70 mV of 0.71 ± 0.03 μM, Hill = 0.84 ± 0.02) but somewhat less pronounced voltage-dependency (δ = 0.79 ± 0.04), slower, single exponential kinetics (ketamine: k_on = 0.15 ± 0.05 × 10⁶ M⁻¹ s⁻¹, k_off = 0.22 ± 0.05 s⁻¹ c.f. memantine following normalization k_on = 0.32 ± 0.11 × 10⁶  M⁻¹ s⁻¹, k_off = 0.53 ± 0.10 s⁻¹) and was fully trapped.The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.     

Neuroprotective effect of Bacopa monniera on ischemia induced brain injury

Purpose

Brain stroke is a leading cause of death without effective treatment. B. monniera, an Indian herbal medicine, exerts antioxidant activity and antistress activity by modulating the antioxidative defence system. We wanted to test if B. monniera could alleviate the ischemia induced brain injury and cognitive dysfunction in Wistar rats.

Procedure

We studied the effect of B. monniera (120 mg kg^− 1, 160 mg kg^− 1 and 240 mg kg^− 1 P.O.) on transient intracarotid artery (ICA) occlusion induced ischemia by testing the neurobehavioral and biochemical parameters on treated and control rats.

Findings

B. monniera attenuated the reduced transfer latency in ischemic rats in a step through test and showed a protective effect on ischemia induced memory impairment in the plus maze task. It also showed a marginal improvement in neurodeficit score and fore limb muscle grip strength. B. monniera reduced the infarct size in the ischemic brain. It also decreased nitrite, nitrate and lipid peroxidation and significantly improved catalase activity.

Conclusion

These observations suggest the neuroprotective and antioxidant activity of B. monniera on ischemia induced brain injury and pave the way for future investigations.