Inhibition of Advanced Glycation End-Products by Tamarindus indica and Mitragyna inermis Extracts and Effects on Human Hepatocyte and Fibroblast Viability.

Tamarindus indica and Mitragyna inermis are widely used by herbalists to cure diabetes mellitus. The aim of this study is to investigate the inhibitory potential of aqueous and various organic solvent fractions from both plants and some isolated compounds against advanced glycation end-products (AGEs). For this purpose, an in vitro BSA-fructose glycation model was used to evaluate the inhibition of AGE formation. Furthermore, the effects of the fractions on mouse fibroblast (NIH-3T3) and human hepatocyte (HepG2) survival were evaluated. The leaf, stem, and root fractions of both plants exhibited significant inhibition of AGEs formation. The IC50 values appeared to be less than 250 µg/mL; however, all fractions presented no adverse effects on NIH-3T3 up to 500 µg/mL. Otherwise, our phytochemical investigation afforded the isolation of a secoiridoid from the Mitragyna genus named secoiridoid glucoside sweroside (1), along with three known quinovic acid glycosides: quinovic acid-3ß-O-ß-d-glucopyranoside (2), quinovic acid-3-O-ß-d-6-deoxy-glucopyranoside, 28-O-ß-d-glucopyranosyl ester (3), and quinovic acid 3-O-α-l-rhamnopyranosyl-(4→1)-ß-d-glucopyranoside (4). In particular, 1-3 are compounds which have not previously been described in Mitragyna inermis roots. However, the isolated compounds did not exhibit AGE inhibitory activity. Further investigation on these potent antiglycation fractions may allow for the isolation of new antidiabetic drug candidates.

Synthesis of [1-8-NαC]-zanriorb A1, alanine-containing analogues, and their cytotoxic and anti-inflammatory activity.

The synthesis of the orbitide[1-8-NαC]-zanriorb A1, isolated from the medicinal plant Zanthoxylum riedelianum, was investigated by solution-phase macrocyclization of a linear peptide and on-resin solid-phase macrocyclization with an acylsulfonamide safety-catch linker. The solution-phase route produced a mixture of proline rotamers, and the main component was assigned as the trans, cis rotamer, identical to the natural product. The on-resin cyclization was less successful, producing mainly a linear peptide, and minor cyclic products as rotameric mixtures. Although the natural product was reported to be significantly cytotoxic against Jurkat leukemia T cells, our synthetic peptides were inactive, suggesting the presence of other rotamers or impurities in the naturally isolated material. Additional analogues of zanriorb A1 were synthesized in which proline and glycine residues were replaced with alanine. While these analogues were not cytotoxic, several of them inhibited the production of nitric oxide in lipopolysaccharide (LPS)-stimulated macrophages. The most active compound, cyclic[Ala5,6,8 ]-zanriorb A1 had an IC50 of 22 µM and was more potent compared with the standard NG-monomethyl-l-arginine acetate (L-NMMA) with an IC50 of 98 µM, indicating their strong anti-inflammatory potential.

Discovery of stylissatin A analogs exhibiting potent nitric oxide inhibition.

The cyclic peptide stylissatin A(STA) was obtained from the Papua New Guinean marine spongeStylissamassaas a potent nitric oxide (NO) inhibitor.Among its reported analogs,cyclo-{Glu6, Ala2}-STA1potentlyinhibited theinterleukin-2 and proliferation of T-cells indicating position 2 of sequence playing important part in biological activities of this compound.In current studies, second generation analogs of STAwere synthesizedaround its most active analog1by screening position 2 of analog1with different amino acid. All analogs2-6were identified by mass, and NMR techniques.The synthesized analogswere also evaluated against NO generation by lipopolysaccharide (LPS)-stimulated murine J774.2macrophages, ROS inhibition from whole blood phagocytes, and T-cell proliferation from Jurkat cells.All analogswere found to be inactive towards interleukin-2, T-cells proliferation, and ROS inhibition. The analog2showed a potent suppression of NO (IC50 = 46.0 ± 2.2 µM) that was superior to the activityreported for natural product STA.Further attempts to optimizeanalog2afforded new nitric oxide inhibitors2a-2fwhich were found less active than2.The analog2also downregulated the transcription of pro-inflammatory molecules, tumor necrosis factor-α, interlukin-1ß, caspase-1 and ASC which further highlights its anti-inflammatory and possible therapeutic potential. Analog2was non-toxic to BJ and Vero cell lines of normal mammalian origin.

Antibiofilm, antiquorum sensing and antioxidant activity of secondary metabolites from seeds of Annona senegalensis, Persoon.

The increasing resistance of bacteria to antibiotics has motivated the interest in potent natural compounds capable of disrupting bacterial cell-to-cell communication. Column chromatography of seed extract of Annona senegalensis afforded N-cerotoyltryptamine (1), asimicin (2) and ent-19-carbomethoxykauran-17-oic acid (3). The compounds were tested for their antimicrobial, antibiofilm, and anti-quorum sensing activities. The minimum inhibitory concentrations (MIC) values ranged from 50 µg/mL to 100 µg/mL for C. albicans ATCC 10239 and S. aureus ATCC 25923 E. coli ATCC 25922, C. violaceum CV026 and C. violaceum CV12472. All the compounds inhibited biofilm formations of all microorganisms tested in various percentages at MIC and MIC/2. Compound 2 also exhibited the highest antibiofilm activity against C. albicans (yeast) and E. coli with percentage inhibitions ranging from 6.3 ± 4.1 (MIC/4) to 37.9 ± 4.5 (MIC) for C. albicans and from 18.8 ± 1.1 (MIC/4) to 43.2 ± 0.5 (MIC) for E. coli. Compound 1, however, showed highest biofilm inhibition on S. aureus as the percentage inhibition varied from 26.7 ± 3.6 (MIC/4) to 43.8 ± 2.1 (MIC). Compound 2 showed highest percentage violacein inhibition on C. violaceum CV12472 ranging from 10.2 ± 0.5 (MIC/8), 65.76 ± 1.3 (MIC/2) and 100 (MIC). Compound 1 and 3 had percentage violacein formation inhibitions on C. violaceum CV12472 ranging from 9.66 ± 1.1 (MIC/4) to 100 (MIC), and from 17.4 ± 2.4 (MIC/4) to 100 (MIC), respectively. Swimming and swarming motility of P. aeruginosa PA01 strain was evaluated at three concentrations of 50, 75 and 100 µg/mL. The compounds inhibited the P. aeruginosa swimming and swarming motility at the three tested concentrations (50, 75 and 100 µg/ml) in a dose-dependent manner. The extents of inhibition of motility migration was relatively higher in the swimming model than in the swarming model for all compounds. Compound 1 exhibited the highest percentage inhibition of motility of 41.50 ± 3.5 and 39.73 ± 1.5 in swimming model and swarming model respectively at 100 µg/ml. Compound 3 showed the lowest percentage inhibition of 30.36 ± 2.0 and 23.50 ± 2.5 in swimming and swarming respectively at 100 µg/ml. At the lowest tested concentration of 50 µg/ml, it was compound 2 showing the highest inhibition of motility of 17.49 ± 0.5 and 14.29 ± 1.0 in swimming and swarming respectively. Compound 1 showed highest quorum sensing (QS) activity with QS inhibition zone of 20.0 ± 1.5 mm at MIC and 11.0 ± 1.0 mm at MIC/8 while compound 2 had the highest antimicrobial (AM) zone diameter amongst the compounds at MIC. Compound 3 was the QS inhibitory sample and did not show any QS inhibition at MIC/8 while showing its highest QS inhibition zone of 13.0 ± 1.6 mm at MIC. For antioxidant assays, no sample showed better activity than the standards. Compound 2 had highest activity with IC50 values of 87.79 ± 2.70 and 42.77 ± 1.53 µg/mL in DPPH and ß-carotene-linoleic acid assay respectively and was more active (IC50 of 97.69 ± 1.40 µg/mL) than standard quercetin (IC50 250.09 ± 0.87 µg/mL) in metal chelation assay.

Effect of a dianthin G analogue in the differentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes.

Loss of cardiomyocytes due to myocardial infarction results in ventricular remodeling which includes non-contractile scar formation, which can lead to heart failure. Stem cell therapy aims to replace the scar tissue with the functional myocardium. Mesenchymal stem cells (MSCs) are undifferentiated cells capable of self-renewal as well as differentiation into multiple lineages. MSCs can be differentiated into cardiomyocytes by treating them with small molecules and peptides. Here, we report for the first time, the role of a cyclic peptide, an analogue of dianthin G, [Glu2]-dianthin G (1) in the in vitro cardiac differentiation of rat bone marrow MSCs. In this study, [Glu2]-dianthin G (1) was synthesized using solid-phase total synthesis and characterized by NMR spectroscopy. MSCs were treated with two different concentrations (0.025 and 0.05 mM) of the peptide separately for 72 h and then incubated for 15 days to allow the cells to differentiate into cardiomyocytes. Treated cells were analyzed for the expression of cardiac-specific genes and proteins. Results showed significant upregulation of cardiac-specific genes GATA4, cardiac troponin T (cTnT), cardiac troponin I (cTnI), cardiac myosin heavy chain, and connexin 43 in the treated MSCs compared to the untreated control. For cardiac-specific proteins, GATA4, cTnT, and Nkx2.5 were analyzed in the treated cells and were shown to have significant upregulation as compared to the untreated control. In conclusion, this study has demonstrated the cardiac differentiation potential of [Glu2]-dianthin G (1)-treated rat bone marrow MSCs in vitro both at the gene and at the protein levels. Transplantation of pre-differentiated MSCs into the infarcted myocardium may result in the efficient regeneration of cardiac cells and restoration of normal cardiac function.

In vivo anticonvulsant activity of 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine in pilocarpine and strychnine induced-seizure models.

An imbalance between inhibitory (GABA) and excitatory (Glutamate) neurotransmission contribute to the development of epilepsy. Earlier studies reported that dysregulation of GABA and glutamatergic activities resulted in status epilepticus (SE) and ultimately support the development of temporal lobe epilepsy (TLE), a type of resistant epilepsy. In the earlier work, 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine demonstrated anticonvulsant activity against pentylenetetrazole (PTZ)-induced seizures. Apart from the PTZ-induced TLE, the dysregulation muscaranic receptors and glycine receptors are also widely reported phenomena in the development of temporal lobe epilepsy. Keeping the role of these two receptors in epilepsy, the present work investigated the effect of 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine in pilocarpine-induced and strychnine-induced seizure models. Our results demonstrated that 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine significantly delayed the onset of seizure with maximum protection from SE in pilocarpine-induced seizure model. However, the test compound did not revealed any effect on strychnine-induced seizures in mice. Based on these observations, we suggest that 2-propanone-1,3,5,5-trimethyl-2-cyclohexen-1-ylidine could be a potential candidate in reduction of SE and treatment of temporal lobe epilepsy (TLE) in future.

Potent leishmanicidal and antibacterial metabolites from Olea ferruginea.

Inspired from the leishmanicidal and antibacterial potential of the fractions obtained from the crude extract of Olea ferruginea stem, the anti-leishmanial ethyl acetate fraction was subjected to chromatographic separation, leading to the isolation of a new compound ferruginan (1) and a known compound (+)- cycloolivil (2). The structures of 1 and 2 were determined by various spectroscopic techniques and were assayed for their in vitro antibacterial and leishmanicidal potential. Compound 1 showed 75% inhibition after 24 h of incubation and 98% inhibition after 48 h of incubation against Leishmania tropica KWH23 promastigotes at 100 µg/mL concentration, while compound 2 exhibited 73% and 96% inhibition at the same concentration and incubation time. Compound 1 also showed good activity against various bacterial pathogens.

Inhibitory Effects of Myrtucommuacetalone 1 (MCA-1) from Myrtus communis on Inflammatory Response in Mouse Macrophages.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.

Norditerpenoid alkaloids of Delphinium denudatum as cholinesterase inhibitors.

Three new norditerpenoids alkaloids, 1ß-hydroxy,14ß-acetyl condelphine (1), jadwarine-A (2), jadwarine-B (3) along with two known alkaloids isotalatizidine hydrate (4) and dihydropentagynine (5) were isolated from medicinal plant Delphinium denudatum. The structures of natural products 1-5 were established on the basis of HR-EIMS, 1H and 13C NMR (1D & 2D) spectroscopic data as well as by comparison from literature data. The structures of compound 1 and 4 were also confirmed by single crystal X-ray diffraction studies. In-vitro AChE and BChE enzyme inhibitory activities of compounds 1-5 and molecular docking studies were performed to investigate the possible molecular inhibitory mechanism of the isolated natural products. Compound 2, 4 and 5 showed competitive inhibitory effects by inhibiting AChE and BChE, respectively, while 1 and 3 showed non-competitive inhibition. This work is the first report that provides a supporting evidence about the use of constituents of Delphinium denudatum in cerebral dementia and Alzheimer diseases.

Selective dual cholinesterase inhibitors from Aconitum laeve.

New lycoctonine-type dual cholinesterase inhibitor, swatinine-C (1), along with three known norditerpenoid alkaloids, hohenackerine (2), aconorine (5) and lappaconitine (6) and two synthetically known but phytochemically new benzene derivatives, methyl 2-acetamidobenzoate (3) and methyl 4-[2-(methoxycarbonyl)anilino]-4-oxobutanoate (4), was isolated from the roots of A. laeve. Structures of new and known compounds (1-6) were established on the basis of latest spectroscopic techniques and by close comparison with the data available in literature. In vitro, compounds (1-6) were tested against AChE and BChE inhibitory activities. Compounds 1 and 2 showed competitive inhibition against AChE (IC50 = 3.7 µM, 4.53 µM) and BChE (IC50 = 12.23 µM, 9.94 µM), respectively. Compounds 5 and 6 showed promising noncompetitive type of inhibitory profile against AChE (IC50 = 2.51 and 6.13 µM) only. Compounds 3 and 4 showed weak inhibitory profile against both AChE and BChE.

Antioxidant and anticholinesterase potential of diterpenoid alkaloids from Aconitum heterophyllum.

Extensive chromatographic separations performed on the basic (pH=8-10) chloroform soluble fraction of Aconitum heterophyllum resulted in the isolation of three new diterpenoid alkaloids, 6ß-Methoxy, 9ß-dihydroxylheteratisine (1), 1α,11,13ß-trihydroxylhetisine (2), 6,15ß-dihydroxylhetisine (3), and the known compounds iso-atisine (4), heteratisine (5), hetisinone (6), 19-epi-isoatisine (7), and atidine (8). Structures of the isolated compounds were established by means of mass and NMR spectroscopy as well as single crystal X-ray crystallography. Compounds 1-8 were screened for their antioxidant and enzyme inhibition activities followed by in silico studies to find out the possible inhibitory mechanism of the tested compounds. This work is the first report demonstrating significant antioxidant and anticholinesterase potentials of diterpenoid alkaloids isolated from a natural source.

Isolation, crystal structure determination and cholinesterase inhibitory potential of isotalatizidine hydrate from Delphinium denudatum.

CONTEXT: Delphinium denudatum Wall (Ranunculaceae) is a rich source of diterpenoid alkaloids and is widely used for the treatment of various neurological disorders such as epilepsy, sciatica and Alzheimer's disease. OBJECTIVE: The present study describes crystal structure determination and cholinesterase inhibitory potential of isotalatazidine hydrate isolated from the aerial part of Delphinium denudatum. MATERIALS AND METHODS: Phytochemical investigation of Delphinium denudatum resulted in the isolation of isotalatazidine hydrate in crystalline form. The molecular structure of the isolated compound was established by X-ray diffraction. The structural data (bond length and angles) of the compound were calculated by Density Functional Theory (DFT) using B3LYP/6-31 + G (p) basis set. The cholinesterase inhibitory potential of the isolated natural product was determined at various concentrations (62.5, 125, 250, 500 and 1000 µg/mL) followed by molecular docking to investigate the possible inhibitory mechanism of isotalatazidine hydrate. RESULTS: The compound crystallized in hexagonal unit cell with space group P65. Some other electronic properties such as energies associated with HOMO-LUMO, band gaps, global hardness, global electrophilicity, electron affinity and ionization potential were also calculated by means of B3LYP/6-31 + G (p) basis set. The compound showed competitive type inhibition of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with IC50 values of 12.13 µM and 21.41 µM, respectively. DISCUSSION AND CONCLUSION: These results suggest that isotalatazidine hydrate is a potent dual cholinesterase inhibitor and can be used as a target drug in Alzheimer diseases. This is first report indicating isotalatazidine hydrate with anticholinesterase potential.

Total synthesis, structural, and biological evaluation of stylissatin A and related analogs.

The natural product cyclic peptide stylissatin A (1a) was reported to inhibit nitric oxide production in LPS-stimulated murine macrophage RAW 264.7 cells. In the current study, solid-phase total synthesis of stylissatin A was performed by using a safety-catch linker and yielded the peptide with a trans-Phe(7) -Pro(6) linkage, whereas the natural product is the cis rotamer at this position as evidenced by a marked difference in NMR chemical shifts. In order to preclude the possibility of 1b being an epimer of the natural product, we repeated the synthesis using d-allo-Ile in place of l-Ile and a different site for macrocyclization. The resulting product (d-allo-Ile(2) )-stylissatin A (1c) was also found to have the trans-Phe(7) -Pro(6) peptide conformations like rotamer 1b. Applying the second route to the synthesis of stylissatin A itself, we obtained stylissatin A natural rotamer 1a accompanied by rotamer 1b as the major product. Rotamers 1a, 1b, and the epimer 1c were separable by HPLC, and 1a was found to match the natural product in structure and biological activity. Six related analogs 2-7 of stylissatin A were synthesized on Wang resin and characterized by spectral analysis. The natural product (1a), the rotamer (1b), and (d-allo-Ile(2) )-stylissatin A (1c) exhibited significant inhibition of NO(.) . Further investigations were focused on 1b, which also inhibited proliferation of T-cells and inflammatory cytokine IL-2 production. The analogs 2-7 weakly inhibited NO(.) production, but strongly inhibited IL-2 cytokine production compared with synthetic peptide 1b. All analogs inhibited the proliferation of T-cells, with analog 7 having the strongest effect. In the analogs, the Pro(6) residue was replaced by Glu/Ala, and the SAR indicates that the nature of this residue plays a role in the biological function of these peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A cyclic peptide accelerates the loading of peptide antigens in major histocompatibility complex class II molecules.

Major histocompatibility complex (MHC)-loading enhancers (MLE) have recently attracted attention because of their ability to enhance the efficacy of peptide immunotherapeutics. As small molecular weight compounds, they influence the loading of peptides in MHC molecules by converting them from a non-receptive to a receptive state. Herein, we report a 14-mer cyclic peptide 1 (CP-1) as a new class of MLE-peptide. This peptide was used to investigate its loading on human leukocyte antigen (HLA)-DR molecules. It was found that CP-1 strongly accelerates peptide-loading on both soluble and cell surface HLA-DR molecules in a dose-dependent manner. The effect was evident for all subsets of HLA-DR tested, including HLA-DRB1*1501, indicating that it acts independently of P1-pocket size, which is the canonical MLE-binding site. Importantly, increased peptide-loading by CP-1 was correlated with improved CD4(+) T cell responses in vitro, while propidium iodide staining indicated low peptide-induced cytotoxicity. Thus, this study revealed a new class of peptide-based enhancers that catalyze peptide-loading by allosteric interactions with MHC molecules. Because of its low cellular cytotoxicity and high MLE activity, it may be useful in stimulating antigen-specific T cell responses for therapeutic purposes.

Antiseizure Activity of Mitragyna inermis in the Pentylenetetrazol-Induced Seizure Model in Mice: Involvement of Flavonoids and Alkaloids.

Objective: This study aimed to investigate whether Mitragyna inermis (Willd.) Otto Kuntze organic and aqueous extracts are able to control seizures induced by pentylenetetrazol (PTZ) in mice based on flavonoid fingerprints and alkaloidal contents. Materials and Methods: Ethanolic extract and decoction-derived fractions from roots, leaves, and stems were subjected to chromatographic fingerprinting using AlCl3 and screening for their antiseizure effects using PTZ-induced acute seizure model. From the fractions that showed potent bioactivities, plausible antiseizure alkaloids were isolated using thin layer chromatography, and their structures were elucidated using 1H NMR, 2D NMR, 13C NMR, and FAB-HR (+ve or -ve). Results: All fractions, with the exception of the dichloromethane and hexane fractions, revealed remarkable flavonoid fingerprints. An acute PTZ-induced seizure test revealed that ethanolic extract of stem bark [500 mg/kg body weight (bw)], ethyl acetate extract of stem bark (500 mg/kg bw), and aqueous extract of leaves (300 mg/kg bw) significantly delayed the occurrence of hind limb tonic extension (HLTE); however, a non-significant delay was observed in the onset of first myoclonic jerk compared with control animals. Isolation yielded four main alkaloids: that are, pteropodine (1), isopteropodine (2), mitraphylline (3) and corynoxeine (4). Corynoxeine is a new compound derived from M. inermis. Conclusion: This study suggests that flavonoid fingerprints are tracers of M. inermis anticonvulsant ingredients. The stem bark ethanolic and ethyl acetate extracts and leaf aqueous extracts contain anticonvulsant bioactive principles that delay notifying the HLTE occurring in male naval medical research institute mice. Furthermore, alkaloidal contents also remain plausible bioactive anticonvulsant principles. All observations support the traditional use of M. inermis to manage epilepsy. However, further studies are needed to understand the effects of alkaloid fractions, flavonoids, and the isolated compounds as promising antiseizure agents derived from M. inermis in experimental animals.

A novel anticonvulsant modulates voltage-gated sodium channel inactivation and prevents kindling-induced seizures.

Here, we explore the mechanism of action of isoxylitone (ISOX), a molecule discovered in the plant Delphinium denudatum, which has been shown to have anticonvulsant properties. Patch-clamp electrophysiology assayed the activity of ISOX on voltage-gated sodium channels (VGSCs) in both cultured neurons and brain slices isolated from controls and rats with experimental epilepsy(kindling model). Quantitative transcription polymerase chain reaction (qRT-PCR) (QPCR) assessed brain-derived neurotrophic factor (BDNF) mRNA expression in kindled rats, and kindled rats treated with ISOX. ISOX suppressed sodium current (I(Na)) showing an IC50 value of 185 nM in cultured neurons. ISOX significantly slowed the recovery from inactivation (ISOX τ = 18.7 ms; Control τ = 9.4 ms; p < 0.001). ISOX also enhanced the development of inactivation by shifting the Boltzmann curve to more hyperpolarized potentials by -11.2 mV (p < 0.05). In naive and electrically kindled cortical neurons, the IC50 for sodium current block was identical to that found in cultured neurons. ISOX prevented kindled stage 5 seizures and decreased the enhanced BDNF mRNA expression that is normally associated with kindling (p < 0.05). Overall, our data show that ISOX is a potent inhibitor of VGSCs that stabilizes steady-state inactivation while slowing recovery and enhancing inactivation development. Like many other sodium channel blocker anti-epileptic drugs, the suppression of BDNF mRNA expression that usually occurs with kindling is likely a secondary outcome that nevertheless would suppress epileptogenesis. These data show a new class of anti-seizure compound that inhibits sodium channel function and prevents the development of epileptic seizures.

Correlates of preferences for home or hospital confinement in Pakistan: evidence from a national survey.

BACKGROUND: Despite the pregnancy complications related to home births, homes remain yet major place of delivery in Pakistan and 65 percent of totals births take place at home. This work analyses the determinants of place of delivery in Pakistan. METHODS: Multivariate Logistic regression is used for analysis. Data are extracted from Pakistan Demographic and Health Survey (2006-07). Based on information on last birth preceding 5 years of survey, we construct dichotomous dependent variable i.e. whether women deliver at home (Coded=1) or at health facility (coded=2). RESULTS: Bivariate analysis shows that 72% (p≤0.000) women from rural area and 81% women residing in Baluchistan delivered babies at home. Furthermore 75% women with no formal education, 81% (p≤0.000) women working in agricultural sector, 75% (p≤0.000) of Women who have 5 and more children and almost 77% (p≤0.000) who do not discussed pregnancy related issues with their husbands are found delivering babies at home. Multivariate analysis documents that mothers having lower levels of education, economic status and empowerment, belonging to rural area, residing in provinces other than Punjab, working in agriculture sector and mothers who are young are more likely to give births at home. CONCLUSION: A trend for home births, among Pakistani women, can be traced in lower levels of education, lower autonomy, poverty driven working in agriculture sector, higher costs of using health facilities and regional backwardness.

First report on the synthesis and structural studies of trans-Phakellistatin 18: a rotamer of marine natural product phakellistatin 18.

Phakellistatin peptides from marine organisms are the sources of proline-rich cyclic peptides with reported significant antitumor activities. Phakellistatin 18 (1), reported from marine sponge Phakellia fusca, contains three proline-peptide linkages in cis form. We attempted the total synthesis of natural product 1 through solution-phase macrocyclization approach, as a result, the synthetic cyclic peptide 2 was obtained as a rotamer of natural product having all three proline residues in trans-conformation. Here, we describe the synthesis, structural, and cytotoxicity studies of trans-Phakellistatin 18 (2), and its analog [Ala1,3,6]-Phakellistatin 18 (3). Detailed NMR studies were carried out to characterize the synthesized peptides, and anti-cancer screening was performed by using MTT assay. The synthetic trans-Phakellistatin 18 (2) (IC50=67.5 ± 2.938 µM) showed comparable cytotoxicity against HepG2 cancer cell line with standard drug doxorubicin (IC50=63.88 ± 6.48 µM). Here, the first synthetic and structural studies on trans-Phakellistatin 18 (2), and its anticancer screening against HepG2 cell line was reported.

Rac2 regulates immune complex-mediated granule polarization and exocytosis in neutrophils.

A key molecule for neutrophil degranulation is Rac2 guanosine triphosphatase. Neutrophils from Rac2 knockout mice (Rac2-/-) exhibit impaired primary granule exocytosis in response to cytochalasin B/f-Met-Leu-Phe, while secondary and tertiary granule release is unaffected. Coronin 1A, a protein involved in actin remodeling, is diminished in Rac2-/- neutrophils. However, primary granule exocytosis from Rac2-/- neutrophils has not been determined using more immunologically relevant stimuli. We sought to determine the role of Rac2 in degranulation and actin cytoskeleton rearrangement in response to immobilized immune complexes and relate this to intracellular coronin 1A localization. We used bone marrow neutrophils from wild-type and Rac2-/- mice stimulated with immobilized immune complexes. Secretion of primary (myeloperoxidase), secondary (lactoferrin), and tertiary granule (MMP-2 and MMP-9) products was evaluated. Subcellular colocalization of coronin 1A with actin and the primary granule marker CD63 was determined by deconvolution microscopy. We found major differences in myeloperoxidase, MMP-2, and MMP-9 but not lactoferrin release, along with diminished filopodia formation, CD63 polarization, and colocalization of coronin 1A with CD63 in immune complex-stimulated Rac2-/- bone marrow neutrophils. Rac2 and coronin 1A were found associated with granules in cytochalasin B/f-Met-Leu-Phe-activated human neutrophils. This report confirms a role for Rac2 in immunologically relevant stimulation of neutrophil granule exocytosis. Rac2 appears to attach to neutrophil granules, polarize CD63+ granules to the cell surface in a manner dependent on coronin 1A, and induce filopodia formation. Our studies provide insight into mechanisms of Rac2-mediated regulation of granule exocytosis.

Solid-phase total synthesis of cherimolacyclopeptide E and discovery of more potent analogues by alanine screening.

Cherimolacyclopeptide E (1) is a cyclic hexapeptide obtained from Annona cherimola, reported to be cytotoxic against the KB (human nasopharyngeal carcinoma) cell line. The solid-phase total syntheses of this cyclic peptide and its analogues were accomplished by employing FMOC/tert-butyl-protected amino acids and the Kenner sulfonamide safety-catch linker. The synthetic peptide 1 was found to be weakly cytotoxic against four cell lines (MOLT-4, Jurkat T lymphoma, MDA-MB-231, and KB). Analogues 3 and 7, where glycine at positions 2 and 6 of the parent compound was replaced by Ala, exhibited enhanced cytotoxicity against KB (3, IC50 6.3 µM; 7, IC50 7.8 µM) and MDA-MB-231 breast cancer cells (3, IC50 10.2 µM; 7, IC50 7.7 µM), thereby suggesting possible selective targeting of these cancer cells by these peptides. The spectral data of synthetic peptide 1 was found to be similar to that reported for the natural product. However, a striking difference in biological activity was noted, which warrants the re-evaluation of the original natural product for purity and the existence of conformational differences.