Hypotonic Research Articles

The peptide Phα1β, from spider venom, acts as a TRPA1 channel antagonist with antinociceptive effects in mice.

Peptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1β, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel. Cultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice. Phα1β selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1β did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1β (up to 300pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1β abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1β was reproduced by a recombinant form of the peptide, CTK 01512-2. Phα1β and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1β and CTK 01512-2 for pain treatment.

Enteral Fluid Therapy in Horses: Effects of Maintenance Hypotonic Electrolyte Solutions Containing Maltodextrin, Sucrose, or Dextrose Administered in Continuous Flow

To evaluate the effects of maintenance hypotonic electrolyte solutions containing carbohydrates administered by nasoesophageal tube in continuous flow in horses during 12 hours on body weight, waist circumference, water intake, aspect and moisture content of the stool, frequency of defecation, urine density and urinary glucose, sodium, potassium, chloride, urea and creatinine, and urinary volume in horses. We used six mares randomly divided into three treatments in two Latin squares 6 × 3, concurrent, in mixed model. All solutions had the same composition (5 g of NaCl, 0.5 g of KCl, 200 mg of magnesium pidolate, and 1 g of calcium gluconate, diluted in 1,000 mL of water), only differing in power source and osmolarity, ESMaltodextrin—electrolyte solution plus 15 g of maltodextrin (measured osmolarity of 203 mmol L−1); ESSucrose—electrolyte solution plus 15 g of sucrose (measured osmolarity of 234 mmol L−1); and ESDextrose—electrolyte solution plus 15 g of dextrose (measured osmolarity of 264 mmol L−1). The electrolyte solutions were administered at a dose of 15 mL kg−1 h−1 for 12 hours in continuous flow. Maintenance hypotonic electrolyte solutions containing maltodextrin, dextrose, and sucrose were effective in promoting diuresis and decreasing urinary density, without presenting adverse effects. ESSucr was more effective in softening the stool.

Should We Perform Semen Analysis, DNA Fragmentation, and Hypo-osmotic Swelling Tests together?

Semen analysis, sperm DNA fragmentation (SDF) and hypo-osmotic swelling test (HOST) are usually performed for the evaluation of sperm fertilizing ability. There are some debates over the necessity of SDF and HOST incorporation in male infertility work-up.Semen of 77 men was evaluated by SDF and HOST through three semen analyses. Sperm parameters were arranged into different categories: <5%, 5-15%, >15% for normal morphology; <50%, 50-70%, >70 % for motility; and <10, 10-20, 21-34, 35-50, >50 million/ml for concentration. SDF analysis was performed and values under 30% were assumed to be normal. Normal range of HOST was considered to be >60%.Only normal sperm morphology had significant relationship with DF rate (P<0.001). Normal morphology, motility, and concentration of sperms had significant relationship with HOST (P<0.001, 0.05, and <0.003,respectively). There was a significant negative correlation between sperm morphology and DF rate. The correlations between sperm parameters and percentage of HOST were significantly positive (r: 0.44, 0.19, and 0.32 for morphology, motility, and concentration, respectively). Receiver operating characteristic curve (ROC) showed that sperm morphology is a strong predictor of the rate of DF and HOST (accuracy: 0.74' and 0.81, respectively). The best sperm morphology cut off point for DF and HOST rate prediction was 4.5% and 5.5%, respectively.Sperm morphology had significant correlation with DF rate and HOST and is supposed to be a predictor for these tests. Performing these three tests collectively for evaluation of semen samples would not be necessarily required in all cases.

Lipophilic but not hydrophilic statin functionally inhibit volume-activated chloride channels by inhibiting NADPH oxidase in monocytes

Volume-activated Cl− channels (VACCs) can be activated by hypotonic solutions and have been identified in many cell types. Here, we investigated the effects of different statins on VACCs in monocytes. Whole-cell patch clamp recordings demonstrated that a hypotonic solution induced 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB)- and 4,4′-diisothiocyanatostilbene-2, 2′-disulfonic acid (DIDS)-sensitive VACC currents in human peripheral monocytes and RAW 264.7 cells. The VACC currents were inhibited by the lipophilic statin (simvastatin) but not by the hydrophilic simvastatin acid and pravastatin. A low-molecular-weight superoxide anion scavenger (tiron, 1 mM) and inhibitor of NADPH oxidase (DPI 10 μM) was able to abolish the VACC currents. A hypotonic solution increased the reactive oxygen species (ROS) detected by the fluorescence of dichlorodihydrofluorescein (DCF), which was abolished by tiron and DPI. NPPB, DIDS, and simvastatin but not pravastatin decreased the fluorescence of DCF. Simvastatin could not further decrease VACC currents when pretreated with tiron or DPI, whereas exogenous H2O2 (100 μM), increased the VACC currents and overcame the blockade of VACC currents by simvastatin. Functionally, hypotonic solution increased the TNF-α mRNA expression, which could be decreased by tiron, DPI, NPPB, DIDS and simvastatin but not pravastatin. However, simvastatin could not decrease the TNF-α expression further when pretreatment with tiron, DPI, NPPB or DIDS. We conclude that lipophilic (simvastatin) rather than hydrophilic statin inhibit VACCs and decrease hyposmolality induced inflammation in monocytes by inhibiting NADPH oxidase.

Experimental Estimation of Membrane Tension Induced by Osmotic Pressure

Osmotic pressure (Π) induces the stretching of plasma membranes of cells or lipid membranes of vesicles, which plays various roles in physiological functions. However, there have been no experimental estimations of the membrane tension of vesicles upon exposure to Π. In this report, we estimated experimentally the lateral tension of the membranes of giant unilamellar vesicles (GUVs) when they were transferred into a hypotonic solution. First, we investigated the effect of Π on the rate constant, kp, of constant-tension (σex)-induced rupture of dioleoylphosphatidylcholine (DOPC)-GUVs using the method developed by us recently. We obtained the σex dependence of kp in GUVs under Π and by comparing this result with that in the absence of Π, we estimated the tension of the membrane due to Π at the swelling equilibrium, σosmeq. Next, we measured the volume change of DOPC-GUVs under small Π. The experimentally obtained values of σosmeq and the volume change agreed with their theoretical values within the limits of the experimental errors. Finally, we investigated the characteristics of the Π-induced pore formation in GUVs. The σosmeq corresponding to the threshold Π at which pore formation is induced is similar to the threshold tension of the σex-induced rupture. The time course of the radius change of GUVs in the Π-induced pore formation depends on the total membrane tension, σt; for small σt, the radius increased with time to an equilibrium one, which remained constant for a long time until pore formation, but for large σt, the radius increased with time and pore formation occurred before the swelling equilibrium was reached. Based on these results, we discussed the σosmeq and the Π-induced pore formation in lipid membranes.

Calcium is not required for triggering volume restoration in hypotonically challenged A549 epithelial cells.

Maintenance of cell volume is a fundamental housekeeping function in eukaryotic cells. Acute cell swelling activates a regulatory volume decrease (RVD) process with poorly defined volume sensing and intermediate signaling mechanisms. Here, we analyzed the putative role of Ca2+ signaling in RVD in single substrate-adherent human lung epithelial A549 cells. Acute cell swelling was induced by perfusion of the flow-through imaging chamber with 50% hypotonic solution at a defined fluid turnover rate. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and cell volume were monitored simultaneously with ratiometric Fura-2 fluorescence and 3D reconstruction of stereoscopic single-cell images, respectively. Hypotonic challenge caused a progressive swelling peaking at ∼20min and followed, during the next 20min, by RVD of 60±7% of the peak volume increase. However, at the rate of swelling used in our experiments, these processes were not accompanied by a measurable increment of [Ca2+]i. Loading with intracellular Ca2+ chelator BAPTA slightly delayed peak of swelling but did not prevent RVD in 82% of cells. Further, electrophysiology whole-cell patch-clamp experiments showed that BAPTA did not block activation of volume-regulated anion channel (VRAC) measured as swelling-induced outwardly rectifying 5-nitro-2-(3-phenylpropyl-amino) benzoic acid sensitive current. Together, our data suggest that intracellular Ca2+-mediated signaling is not essential for VRAC activation and subsequent volume restoration in A549 cells.

The Extracellular C-loop Domain Plays an Important Role in the Cell Adhesion Function of Aquaporin 0

ABSTRACTPurpose: Although aquaporin 0 (AQP0) is a member of the AQP family, it has limited water permeability compared with other members. AQP0 may also have cell adhesion-related functions, but the evidence is still limited. Here, we studied the relationship of AQP0 to cell adhesion and determined the region required for cell adhesion.Methods: L-cell fibroblasts stably expressing AQP0 or AQP1 (L-AQP0 or L-AQP1) were established. One group of cells was stained with CellTracker Red and cultured into a confluent monolayer, whereas the other group was loaded with CellTracker Blue and seeded over the monolayer. To study cell adhesion, the percentages of lower and upper layer cells were measured using flow cytometry. To determine the region of AQP0 required for adhesion, activity was done by pull-down assay using glutathione S-transferase fusion proteins. To study the water permeability, Xenopus laevis oocyte expressing AQP0 wild-type or AQP0 mutated in C-loop was transferred to a hypotonic solution and photographed, and the diameter was measured to calculate the volume.Results: More cells adhered to the lower cells in the L-AQP0 homotypic pair than other pairs such as L-AQP1 homotypic or L-AQP0/L-AQP1 heterotypic pairs. Pull-down assays revealed that AQP0 could bind to itself via the C-loop extracellular domain. Furthermore, we determined that 109Pro and 110Pro in the C-loop were important for cell adhesion. However, mutation of the C-loop in AQP0 did not affect its water permeability.Conclusions: AQP0 is known to bind lipids in the opposing membrane. Our data suggest that this cell-to-cell adhesion occurs not only in the AQP0/liquids but also via AQP0/AQP0 interaction through the C-loop domain. Mutations in the C-loop amino acids did not affect the water permeability of AQP0 but did affect its cell adhesion function. These independent dual functions of AQP0 are important for lens transparency.

PERUBAHAN BENTUK ERITROSIT DI GLOMERULONEFRITIS

In glomerulonephritis there are intraglomerular inflammation, cell proliferation, and hematuria. Hematuria is characterized by more than 3 (three) erythrocytes per high-power field in the urine, which indicates the pathological processes in kidney or urinary tract. The combination of mechanical damage of erythrocyte membrane through the damaged glomerular basement membrane followed by the osmotic damage when it passes through the tubular system in the hypotonic osmotic solutions causes dysmorphic morphology. Erythrocytes trapped in the Tamm-Horsfall protein will form erythrocyte casts. Dysmorphic erythrocytes and or erythrocyte casts in the urine indicate glomerular hematuria. Various forms of dysmorphic erythrocytes in the urine can be found. Acanthocytes (G1-cells) are specific for glomerular hematury. The examination of these urinary sediments can be done natively or by using automated urinalysis analyzers.

English

Crude methanolic extracts of Crinum latifolium plant (Leaves) was assayed to identify various pharmacological properties. Antimicrobial potential of crude methanolic extracts of C. latifolium was accomplished by most commonly used disc diffusion method against a wide range of Gram positive (+ve) and Gram negative(-ve) bacteria. Extracts showed slight antimicrobial activity against Gram positive (+ve) bacteria while surprisingly showed significant antimicrobial activity against Gram negative (-ve) bacteria Escherichia coli. In contrast to vincristine sulphate, the crude methanolic, n-hexane soluble, petroleum ether soluble and chloroform soluble extracts showed slight to moderate cytotoxic properties with LC50 value of 7.06, 48.978, 242.83 and 153.93 &micro;g/ml respectively. Plant extract showed significant (P&lt;0.05) anti-inflammatory properties, that is, 16.21 and 20.55%10 mg/ml for hypotonic solution and heat induced condition respectively. So, this plant extract demands further research for revealing all its potency to have new safe drug for the entire respective field of medical science. Key words: Crinum latifolium, zone of inhibition, brine shrimp lethality bioassay, anti-inflammatory.

Evaluation of &lt;i&gt;In Vitro&lt;/i&gt; Antimicrobial and Anti-Inflammatory Potentials of Crude Methanolic Extracts of &lt;i&gt;Excoecaria agallocha &lt;/i&gt;(Leaves)

Excoecaria agallochaplant (Leaves) was analyzed to exploreIn Vitroantimicrobial and membrane stabilizing activities as a crude methanolic extract. Antimicrobial activity was performed against a wide range of Gram positive(+Ve) and Gram negative(-Ve) bacteria by using disc diffusion method and various methanolic extracts of leaves ofE.agallochawas tested for determining membrane stabilizing activity at hypotonic solution and heat induce condition and standard acetyl salicylic acid (0.10 mg/mL) was employed as standard. the zones of inhibition created was found to be 1.3 cm at a concentration of 100 µL/ disc in case of Gram negative(-Ve) bacteria Salmonella typhi. The results obtained were compared with that of a standard Ampicillin (10 µL) and imipenem (10µL), penicillin (10µl), cefoxitine(30µl). Crude methanolic extract of 10mg/ml concentration showed maximum value of 17.67±0.0102 and 18.92±0.086% respectively under hypotonic solution and heat induced condition. Thus the result suggest that, crude methanolic extracts ofE.agallochapossessed slight to moderate antimicrobial and membrane stabilizing properties.

Development of a decellularization method to produce nerve allografts using less invasive detergents and hyper/hypotonic solutions

Here, we describe a novel method of processing decellularized nerve grafts using osmotic effects of hypotonic and hypertonic solutions and Triton X-100 (a nonionic detergent) and CHAPS (an amphoteric detergent). To evaluate decellularization, the devised method and Hudson's method were compared with respect to remaining cellular components (as assessed by H&E staining and S-100 immunoreactivity) and extracellular matrix structural integrity (as assessed by H&E staining and laminin immunoreactivity) by using rat sciatic nerves. In addition, a 1.5-cm sciatic nerve gap rat model was treated by implanting decellularized nerve grafts prepared using the devised method, Hudson's method, or an autograft to evaluate nerve regeneration. Nerve histomorphometry of distal stumps and wet muscle mass were evaluated at 12 weeks after implantation. The devised method produced outcomes similar to those of Hudson's method in terms of cellular component removal, but the devised method was significantly better in terms of ECM preservation. Histomorphometric study showed that the devised method produced significantly fewer nerve fiber and axonal densities than autografting, but much more than Hudson's method. The wet muscle mass of the devised method was also significantly lower than that of autografting, but much higher than that of Hudson's method. The described process for producing decellularized nerve grafts yielded better outcomes with respect to peripheral nerve regeneration than the established ionic detergent-based methods in a rat model. This study indicates that decellularized nerve grafts produced in this manner show favorable nerve regeneration used for bridging nerve gaps.

Analysis of cytogenetic differentiation in selected species of the family Camelidae: Vicugna pacos and Vicugna vicugna

The aim of this study was to analysis of cytogenetic differentation in two species: Vicugna pacos and Vicugna vicugna . Cytogenetic analysis based on blood culture technique. Blood culture were set up in culture medium Limphogrow and Lymphochrome. The cells growth, after 71 hours, was kept in metaphase using colcemid. Metaphase chromosomes were treated with hypotonic solution (KCl) for 30 minutes and fixed in a mixture of methanol and glacial acetic acid (3:1). After that, chromosomes were stained with Giemsa. Chromosomes were analyzed under microscope using Optica Vision pro-3 and MultiScan programs. Both species have 2n=74 and showed close homology between autosomes and sex chromosomes. Analysed karyotypes consist of 19 pairs acrocentric, 9 pairs metacentric, 5 pairs subtelocentric and 3 pairs submetacentric autosomes. Sex chromosomes X was metacentric and Y was acrocentric.

ANTI-INFLAMMATORY STUDY OF ANREDERA CORDIFOLIA LEAVES AND CENTELLA ASIATICA HERBS AND ITS COMBINATIONS USING HUMAN RED BLOOD CELL-MEMBRANE STABILIZATION METHOD

ABSTRACTObjective: Inflammation is body reactions in response to tissue injury and infection. In 2011, non-steroidal anti-inflammatory drug (NSAID) was thehighest demand drug in Indonesia. However, long-term treatment using NSAID can cause several side effects to cardiovascular and digestive system.This research aimed to investigate anti-inflammatory properties of binahong leaves (Anredera cordifolia) and pegagan herbs (Centella asiatica).Methods: Ethyl alcohol extract of A. cordifolia leaves and C. asiatica herbs was evaluated for its anti-inflammatory properties using human redblood cell (RBC) – membrane stabilization assay. The extract concentrations used in this study was 100, 200, 400, and 800-ppm, and apigenin andasiaticoside concentration were 1, 2, 3, 4, 5, 6, 10, and 100 ppm. Diclofenac natrium (DN) was used as a standard drug.Results: The results showed that A. cordifolia extract (ACE) alone, C. asiatica extract (CAE) alone, and the combination of ACE and CAE could inhibit thehemolysis of RBC in hypotonic solution. The optimum concentration for ACE alone was 100 ppm; for CAE alone was 400 ppm; and for the combinationof ACE and CAE was 50 ppm and 50 ppm, respectively. Apigenin and asiaticoside in concentration of 1-10 ppm showed more than 97% inhibition ofhemolysis. DN as a standard drug showed optimum inhibition at concentration of 400 ppm.Conclusion: The ethyl alcohol extract of A. cordifolia leaves and C. asiatica herbs showed anti-inflammatory activity, both as a single treatment or ascombinations, and apigenin and asiaticoside were responsible for anti-inflammatory activity in C. asiatica.Keywords: Anti-inflammation, Human red blood cell – membrane stabilization, Anredera cordifolia, Centella asiatica.

In vitro Antioxidant and RBC membrane Stabilization Activity of Euphorbia wallichii

Background: Euphorbia wallichii belongs to family Euphorbiaceae is used to treat various diseases on folklore levels in Kashmir valley. Objective of the study is to explore antioxidant potential and anti-inflammatory activities of Euphorbia wallichii. Materials and Methods: Antioxidant potential of extracts was evaluated by means of total phenolics, DPPH, reducing power, microsomal LPO, and hydroxyl radical scavenging activity by using standard procedures. Anti-inflammatory activity was assessed using hypotonic solution induced human erythrocyte haemolysis. Results: The highest phenolic content of 465 mg GAE/g was observed in methanol extract followed by ethyl acetate (359 mg GAE/g) and aqueous extract (291 mg GAE/g). At concentration of 700 μg/mL, DPPH radical scavenging activity of methanol extract was (94.85%) IC50 (160 μg/ml), ethyl acetate (92.68%) IC50 (200 μg/ml) and aqueous (90%) IC50 (250 μg/ml). The reducing power of the extracts increased in a concentration dependent manner. At concentration of 70 μg/mL, 92.72, 80.74 and 75.75% inhibition was observed with methanol, ethyl acetate and aqueous extract on microsomal LPO with IC50 values 31.5, 34.5 and 42 gμ/ml. Superoxide radical scavenging activity of Euphorbia wallichii extracts increased in a dose dependent manner with IC50 values 36.05 μg/ml (methanol), 45 μg/ml (ethyl acetate) and 34.5 μg/ml (aqueous extract). Euphorbia wallichii extracts exhibited antioxidant effects on Calf thymus DNA damage. At the higher concentration of plant extracts (12 μg/ml), 90, 86 and 78% increase in RBC membrane stabilization was observed with methanol, ethyl acetate and aqueous extracts of Euphorbia wallichii. Conclusion: These results clearly indicate that Euphorbia wallichii extracts possesses the free radical savaging activity as such can be employed as potential antioxidant and anti-inflammatory agent against various oxidative stress related pathological conditions.

In vitro Membrane Stabilizing and In vivo Analgesic Activities of Boehmeria glomerulifera Miq. in Swiss-Albino Mice Model

Boehmeria glomerulifera Miq. is an indigenous plant of Bangladesh. Traditional healers use this plant to treat various diseases. The crude methanol extract of whole plant of B. glomerulifera and its Kupchan fractions were screened for in vitro membrane stabilizing and only the crude extract at doses of 200- and 400- mg/kg body weight were screened for analgesic activity in mice model in order to establish the medicinal values of this plant. The membrane stabilizing activity of the extractives was evaluated by the ability of test materials to inhibit hypotonic solution- and heat-induced haemolysis of human erythrocytes. Moreover, the analgesic activity of methanol extract was evaluated by acetic acid induced writhing method and tail immersion method in mice. In hypotonic solution- and heat-induced conditions, the crude methanol extract showed inhibition of haemolysis by 64.80±0.34% and 21.63±0.76%, respectively as compared to 77.74±0.67% and 40.41±0.69% demonstrated by the standard, acetyl salicylic acid. On the other hand, the analgesic activity was determined for its peripheral and central pharmacological responses using acetic acid-induced writhing test and tail immersion method at doses of 200- and 400- mg/kg body weight. The extract reduced the acetic acid-induced writhings significantly (p &lt; 0.05) in a dose dependent manner with the highest activity observed at 400 mg/kg (46.0%) body weight when compared to that of Diclofenac sodium (64.0%), as the standard drug. A significant increase (p &lt; 0.01) of latency period was also found in tail immersion method. Therefore, the present study established that the methanol extract of B. glomerulifera possesses moderate membrane stabilizing and significant analgesic activities.Bangladesh Pharmaceutical Journal 19(2): 185-189, 2016

Foundations of identifying individual chromosomes by imaging flow cytometry with applications in radiation biodosimetry

Biodosimetry is an important tool for triage in the case of large-scale radiological or nuclear emergencies, but traditional microscope-based methods can be tedious and prone to scorer fatigue. While the dicentric chromosome assay (DCA) has been adapted for use in triage situations, it is still time-consuming to create and score slides. Recent adaptations of traditional biodosimetry assays to imaging flow cytometry (IFC) methods have dramatically increased throughput. Additionally, recent improvements in image analysis algorithms in the IFC software have resulted in improved specificity for spot counting of small events. In the IFC method for the dicentric chromosome analysis (FDCA), lymphocytes isolated from whole blood samples are cultured with PHA and Colcemid. After incubation, lymphocytes are treated with a hypotonic solution and chromosomes are isolated in suspension, labelled with a centromere marker and stained for DNA content with DRAQ5. Stained individual chromosomes are analyzed on the ImageStream®X (EMD-Millipore, Billerica, MA) and mono- and dicentric chromosome populations are identified and enumerated using advanced image processing techniques. Both the preparation of the isolated chromosome suspensions as well as the image analysis methods were fine-tuned in order to optimize the FDCA. In this paper we describe the method to identify and score centromeres in individual chromosomes by IFC and show that the FDCA method may further improve throughput for triage biodosimetry in the case of large-scale radiological or nuclear emergencies.

Evaluation of Thrombolytic, Membrane Stabilizing, Antidiarrhoeal and Analgesic Activities of Leaves of Triumfetta pilosa

The aim of the research has been focused on the phytochemical investigation of the plants which have ethno-botanical and folkloric importance for drug discovery. The widespread availability and folkloric use of Triumfetta pilosa leaves led us to determine the pharmacological potential of the plant through in vitro and in vivo experiments. The crude ethanolic extract (EE) of T. pilosa leaves were partitioned successively using solvent of different polarities. Then these fractions were subjected to qualitative and quantitative phytochemical screening by standard procedures. The extract of T. pilosa and its fractions were evaluated for their possible thrombolytic, membrane stabilizing, antidiarrhoeal and analgesic activities by using standard drugs streptokinase, acetyl salicylic acid, loperamide and diclofenac-Na, respectively. In the study for thrombolytic activity, among all partitionates, the ethyl acetate soluble fraction (ESF) showed the highest percent of clot lysis (58.67%) as compared to standard streptokinase (69.23%) and water (3.77%). Also, in case of membrane stabilizing activity, ESF significantly inhibited the haemolysis of human erythrocyte membrane both induced by hypotonic solution (65.33 ± 0.50%) and heat (56.22 ± 0.69%), as compared to standard acetyl salicylic acid (ASA) (71.12 ± 26%) and (75.92 ± 0.29%), respectively. In the antidiarrheal assay, the crude ethanolic extract inhibited the mean number of defecation by 45.71% and 63.18% at 200 and 400-mg/kg body weight, respectively. During assay for central and peripheral analgesic activity at dose of 400-mg/kg, the extract showed reaction times of 5.11 (p &lt; 0.001) and 1.96 (p &lt; 0.001) min in the tail-flick and tail-immersion models, while the normal and reference groups exhibited reaction times of 11.66, 1.46; (p &lt; 0.001) and 4.91, 1.16; (p &lt; 0.001) in the tail flick and tail immersion method, respectively. At 400 mg/kg, the extract showed 49.22% inhibition of acetic acid induced writhing in mice model.Bangladesh Pharmaceutical Journal 19(2): 226-232, 2016

Relationship between TMEM16A/anoctamin 1 and LRRC8A.

TMEM16A/anoctamin 1/ANO1 and VRAC/LRRC8 are independent chloride channels activated either by increase in intracellular Ca(2+) or cell swelling, respectively. In previous studies, we observed overlapping properties for both types of channels. (i) TMEM16A/ANO1 and LRRC8 are inhibited by identical compounds, (ii) the volume-regulated anion channel VRAC requires compartmentalized Ca(2+) increase to be fully activated, (iii) anoctamins are activated by cell swelling, (iv) both channels have a role for apoptotic cell death, (v) both channels are possibly located in lipid rafts/caveolae like structures, and (vi) VRAC and anoctamin 1 currents are not additive when each are fully activated. In the present study, we demonstrate in different cell types that loss of LRRC8A expression not only inhibited VRAC, but also attenuated Ca(2+) activated Cl(-) currents. Moreover, expression of LRRC8A enhanced Ca(2+) activated Cl(-) currents, and both LRRC8A and ANO1 could be coimmunoprecipitated. We found that LRRC8A becomes accessible to biotinylation upon exposure to hypotonic bath solution, while membrane capacitance was not enhanced. When intracellular Ca(2+) was increased in ANO1-expressing cells, the membrane capacitance was enhanced and increased binding of FM4-64 to the membrane was observed. As this was not seen in cells lacking ANO1 expression, a role of ANO1 for exocytosis was suggested. We propose that ANO1 and LRRC8A are activated in parallel. Thus, ionomycin or purinergic stimulation will not only activate ANO1 but also LRRC8 currents. Cell swelling will not only activate LRRC8/VRAC, but also stimulate ANO1 currents by enhancing compartmentalized Ca(2+) increase and/or through swelling induced autocrine release of ATP.

Antioxidant and Membrane Stabilizing Activities of Bark of Sonneratia apetala

This study was carried out to investigate the antioxidant activity in terms of free radical scavenging capacity and membrane stabilizing ability of methanol extract of bark of Sonneratia apetala. The total phenol content of the extract was measured by Folin-Ciocalteu reagent. The antioxidant potential was investigated using 2,2 diphenylpicrylhydrazyl (DPPH), reducing power assay and chelating power determination. The membrane stabilizing capacity was assessed by monitoring the hypotonic solution - and heat-induced haemolysis of human erythrocytes. The total phenolic content was found 50.75 mg/gm of gallic acid equivalent. The extract exhibited significant antioxidant activity in DPPH free radical scavenging assay with IC50 value of 81.42 µg/ml as compared to the standard, BHT (IC50=42.56 µg/ml), Fe2+ ion reducing power assay and chelating power ability. In case of assay for chelating ability, EDTA represented 98.76% chelation while the plant extract showed 82.83% at concentration of 100 ?g/ml. In addition, the methanol extract of bark of this plant was found to moderately inhibit the haemolysis of human erythrocyte. In conclusion, the methanol extract of S. apetala could be valuable candidate for future development for antioxidant activity.Bangladesh Pharmaceutical Journal 19(2): 147-151, 2016

Correlation of Thrombolytic and Membrane Stabilizing Activities with Total Flavonoid Content Aerial Parts of Spondias pinnata

Spondias pinnata is a medicinal herb belonging to the family of Anacardiaceae. Locally it is known as Amra and has a history of ethno-medicinal properties. The crude ethanolic extract along with different partitionates of the leaves of S. pinnata were evaluated for their possible thrombolytic and membrane stabilizing activities. The activities of the leaf extract were evaluated by using standard drugs; streptokinase for thrombolytic and acetyl salicylic acid for anti-inflammatory activities. In this study, among all partitionates the ethyl acetate soluble fraction showed highest % of clot lysis (58.06%) as compared to (69.23%) and (3.03%) exhibited by the standard streptokinase and water. On the other hand, ethyl acetate soluble fractionates also significantly inhibits the haemolysis of human erythrocyte membrane both in induced by hypotonic solution (65.33 ± 0.50%) and by heat (56.22 ± 0.69%), respectively as compared to (75.92 ± 29%) and (71.12 ± 0.26%) demonstrated by acetyl salicylic acid. Our study revealed that satisfactory amount of flavonoid content (132.27 ± 0.25) showed a significant and positive correlation between total flavonoid content with thrombolytic and membrane stabilizing activities.Bangladesh Pharmaceutical Journal 19(1): 48-52, 2016