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   Table of Contents - Current issue
September-October 2018
Volume 13 | Issue 5
Page Nos. 385-475

Online since Monday, July 30, 2018

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Improvement of dermal delivery of tetracycline using vesicular nanostructures Highly accessed article p. 385
Azam Hasanpouri, Farzaneh Lotfipour, Saeed Ghanbarzadeh, Hamed Hamishehkar
The objective of this investigation was to study the potential use of nanoliposomes and nanotransfersomes in dermal delivery of tetracycline hydrochloride (TC) for acne treatment. Vesicular nanostructures were prepared by thin film hydration method and evaluated for their size, zeta potential, morphology, and entrapment efficiency. Minimal inhibitory concentration values of TC-loaded vesicles were evaluated and compared with TC aqueous solution against Staphylococcus epidermis. In vitro drug release and ex vivo drug permeation through the excised rat skin were performed to assess drug delivery efficiency. Particle size, zeta potential, and entrapment efficiency of prepared nanoliposomes and nanotransfersomes were found to be 75 and 78 nm, 17 and 7 mV, and 45 and 55%, respectively. Antimicrobial analysis indicated that there was no difference between vesicular formulations and aqueous solution of TC. In vitro drug release study indicated that nanoliposomes could release TC 2.6 folds more than nanotransfersomes, and skin permeation study showed that the permeability of TC-loaded nanotransfersomes was 1.6 times higher than nanoliposomes which was also confirmed by fluorescence microscope imaging. These findings concluded that nanoliposomal and especially nanotransfersomal formulations could be proposed as the potential approach for better therapeutic performance of TC against acne.
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Computational comparison of two new fusion proteins for multiple sclerosis p. 394
Nasrin Payab, Karim Mahnam, Mostafa Shakhsi-Niaei
Multiple sclerosis (MS), as one of the human autoimmune diseases, demyelinates the neurons of the central nervous system (CNS). Activation of the T cells which target the CNS antigens is the first autoimmune event in MS. Myelin oligodendrocyte glycoprotein (MOG) and myelin basic protein (MBP) are two proteins of the myelin sheath and have been shown to be among the high antigens contributing to the pathogenesis of MS. Production of the drugs with high specificity for the immune system diseases is a concern for various researchers. Therefore, tolerogenic vaccines are considered as a new strategy for the treatment of MS by presenting specific antigens. This study aimed to design and compare two fusion proteins by a combination of two neuroantigens linked to interleukin-16 (IL-16) (MOG-Linker-MBP-IL16 and MBP-Linker-MOG-IL16) as vaccines for MS. In this study, at first two models MOG (aa 11-30) linked to MBP (aa 13-32) was made by Modeler 9.10 and simulated for 20 ns via Gromacs 5.1.1 package. Then simulated antigen domains connected to the N-terminal domain of IL-16 and obtained structures simulated for 50 ns. The results revealed that both constructs had stable structures and the linker could keep two antigenic fragments separate enough, preventing undesired interactions. While MOG-Linker-MBP-IL16 showed better solubility, more accessible surface areas, more flexibility of its IL-16 domain, and better functionality of its IL-16 domain as well as more specific cleavage of its related epitopes after endocytosis lead to a better presentation of its antigenic property.
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Cytotoxic effect of methanolic extract, alkaloid and terpenoid fractions of Stachys pilifera against HT-29 cell line p. 404
Esmaeel Panahi Kokhdan, Hossein Sadeghi, Hossin Ghafoori, Heibatollah Sadeghi, Nazanin Danaei, Hamedreza Javadian, Mahmoud Reza Aghamaali
Stachys pilifera (S. pilifera) Benth (Lamiaceae) is used in traditional medicine to treat a variety of diseases. Despite some reports on the antitumor effects of some species of this genus, anticancer activity of S. pilifera has not been yet reported. Here, we examined the cytotoxic effect and cell death mechanisms of methanolic extract of S. pilifera and its alkaloid and terpenoid fractions on the HT-29 colorectal cell line. HT-29 cells were cultivated and then incubated in the methanolic extract of S. pilifera and its fractions at various concentrations for 24 h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Morphology of cells was evaluated by contrast microscopy. Furthermore, effects of the tested extract and fractions were tested on some regulators of cell death and proliferation such as caspase-8, caspase-9, nuclear factor-κB (NF-κB), and nitric oxide (NO). Cisplatin was used as positive control. The estimated IC50 values of the methanolic extract, alkaloid and terpenoid fractions, and cisplatin against HT29 cell after 24 h were determined to be 612, 48.12, 46.44, and 4.02 μg/mL, respectively. Morphological changes such as plasma membrane blebbing, cell size reduction, and apoptotic bodies were observed in cells faced with the extract and fractions. S. pilifera extract and its fractions induced apoptosis through inhibition of NF-κB, NO, and activation of caspase-8 and caspase-9. Data showed considerable cytotoxic and antiproliferative effects of S. plifera on colorectal cell line through induction of apoptosis. These findings provide a basis for the therapeutic potential of S. pilfera in the treatment of colon cancer.
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Effect of buffer additives on solubilization and refolding of reteplase inclusion bodies p. 413
Iman Esmaili, Hamid Mir Mohammad Sadeghi, Vajihe Akbari
Reteplase is a non-glycosylated and recombinant form of tissue type plasminogen activator, which is produced in Escherichia coli. However, its overexpression usually leads to formation of inactive aggregates or inclusion bodies. In the present study, we report on the development of optimized processes for isolation, solubilization, and refolding of reteplase inclusion bodies to recover active protein. After protein overexpression in E. coli BL21 (DE3) inclusion bodies were isolated by cell disruption and repeated wash of pellet with buffer containing Triton X-100. To solubilize the inclusion bodies, different types, concentrations, pHs, and additives of denaturing agents were used. Rapid micro dilution method was applied for refolding of solubilized reteplase. Different chemical additives including sugars, alcohols, polymers, detergents, amino acids, kosmotropic, and chaotropic salts, reducing agents, and buffering agents were used in the refolding buffer. To evaluate the biological activity of refolded reteplase, an indirect chromogenic assay was performed. The best solubilizing agent for dissolving reteplase inclusion bodies was 6 M urea at pH 12. The optimized buffer for refolding of solubilized reteplase was found to be 1.15 M glucose, 9.16 mM imidazole, and 0.16 M sorbitol which resulted in high yield of biologically active protein. Our results indicate type, concentration, and pH of solvent and type, concentration, and combination of chemical additives can significantly influence the yield of inclusion bodies solubilization and refolding.
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Flavone constituents of Phlomis bruguieri Desf. with cytotoxic activity against MCF-7 breast cancer cells p. 422
Seyed Ebrahim Sajjadi, Zeinab Delazari, Mahmoud Aghaei, Mustafa Ghannadian
Phlomis bruguieri (P. bruguieri) is a large genus in the Lamiaceae family, with a wide variety distributed in Euro-Asia, Central Asia, Iran, and China. Phlomis flowers have been used as herbal tea for gastrointestinal disturbances, protection of liver and cardiovascular systems. The aim of this study was to analyse phytochemical of flavonoid constituents in semi polar fraction of P. bruguieri. Methanol extract of plant material (4 kg) yielded 361 g dark green concentrated extract gum. After preliminary fractionation by normal column chromatography on silica gel, Fr. 2 eluted with chloroform: methanol (90:10) selected as semi polar fraction and was more purified using different chromatography columns on silica gel, polyamide SC6 and Sephadex LH-20 adsorbents. Finally one new and three known flavonoids (1-4) were characterized in semi polar fraction. Isolated structures were identified using 1H-NMR, 13C-NMR, 31P-NMR, HSQC, HMBC, negative ESI mass, and UV spectra using different shift reagents. Using standard MTT assay, cytotoxicity of isolated new compound was done against michigan cancer foundation-7 (MCF-7) breast cancer cells. Phytochemical analysis of P. bruguieri resulted in identification of one new 4’-methoxy-luteolin-7-phosphate and three known flavones including luteolin, apigenin, and tricin for the first time in this plant. In MTT cytotoxicity test, 4’-methoxy-luteolin-7-phosphate showed cytotoxicity with IC50 value of 43.65 ± 8.56 μM agasint MCF-7 breast cancer cells.
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Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface p. 430
Maryam Boshtam, Seddigheh Asgary, Ilnaz Rahimmanesh, Shirin Kouhpayeh, Jamal Naderi, Zahra Hejazi, Hoda Mohammad-Dezashibi, Ina Laura Pieper, Hossein Khanahmad
Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F’ was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.
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Atorvastatin mitigates cyclophosphamide-induced hepatotoxicity via suppression of oxidative stress and apoptosis in rat model p. 440
Maedeh Hamzeh, Seyed Jalal Hosseinimehr, Ali Reza Khalatbary, Hamid Reza Mohammadi, Ayat Dashti, Fereshteh Talebpour Amiri
Cyclophosphamide (CP), as a chemotherapy drug, induces hepatotoxicity through causing oxidative stress. Atorvastatin (ATV) at a low dose has antioxidant and anti-inflammatory properties. The present study was designed to investigate the protective effects of ATV against CP-induced hepatotoxicity in rat. In this experimental study, 32 rats were treated with ATV orally at a dose of 10 mg/kg for 10 consecutive days, 5 days before and 5 days after the administration of a single intraperitoneal injection of CP (150 mg/kg). The hepatoprotective effect of ATV was evaluated by measuring liver function markers, oxidative markers, histological and immunohistochemical assays. The biochemical results showed that administration of CP increased hepatic biomarkers enzymes as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) levels. CP increased malondialdehyde (MDA), protein carbonyl (PC) and decreased glutathione (GSH) content in rats. Moreover, administration of CP was associated with periportal leucocyte infiltration, dilation sinusoids, hepatocyte vacuolation, congestion and hemorrhage in livers of rats. CP significantly increased immunoreactivity of caspase-3 as a marker of apoptosis in liver tissue. ATV markedly mitigated liver injury through reduction in oxidative stress biomarkers, histopathological findings and apoptosis. The antioxidant and anti-apoptotic activities of ATV are main proposed mechanisms involved in its hepatoprotective effects against CP-induced hepatic injury.
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Synthesis and cytotoxic evaluation of novel quinazolinone derivatives as potential anticancer agents p. 450
Safoora Poorirani, Sedighe Sadeghian-Rizi, Ghadamali Khodarahmi, Marzieh Rahmani Khajouei, Farshid Hassanzadeh
Nitrogen-rich heterocyclic compounds represent a unique class of chemicals with especial properties and have been modified to design novel pharmaceutically active compounds. In this study, a series of novel quinazolinone derivatives with substituted quinoxalindione were synthesized in two parts. In the first part, 6-(4-amino-3-methylphenoxy)quinoxaline-2,3(1H,4H)-dione was prepared from para-amino -m-crozol in 5 steps. In the next part, 2-alkyl-4H-benzo[d][1,3]oxazin-4-one derivatives were obtained from antranilic acid. Then reaction of 6-(4-amino-3-methylphenoxy)quinoxaline-2,3(1H,4H)-dione with 2-alkyl-4H-benzo[d][1,3]oxazin-4-one derivatives resulted in the production of final componds. The structures of synthesized compounds were confirmed by IR and 1H-NMR. Cytotoxic activity of the compounds were evaluated at 0.1, 1, 10, 50 and 100 μM concentrations against MCF-7 and HeLa cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. Almost all new compounds showed cytotoxic activity in both cell lines. Among tested compounds, 11g displayed the highest cytotoxic activity against both cell lines.
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Effect of lansoprazole on human sperm motility, sperm viability, seminal nitric oxide production, and seminal calcium chelation p. 460
Saleem Ali Banihani, Falak Hisham Khasawneh
Lansoprazole is a proton-pump inhibitor that is commonly used to treat many gastric illnesses. However, little is known about its effect on sperm function. Here, we investigated the in vitro effect of LP on human sperm motility, viability, nitric oxide (NO) production, and the ability of LP to chelate seminal calcium. Seventy-two semen samples from normozoospermic men were tested in this study. The effects of LP at 0.375, 0.75, 1.5, and 3 μg/mL on sperm motility and viability as well as at 3 μg/mL on NO production and calcium chelation in semen were assessed. Lansoprazole at 3 μg/mL significantly decreased total and progressive sperm motility (P = 0.0021, P = 0.0256, respectively), but not sperm viability (P = 0.8763). In addition, semen samples supplemented with 3 μg/mL LP had insignificant changes (P = 0.9085) in nitrite concentrations. Moreover, LP exhibited a significant (P < 0.0001) calcium chelation effect in semen. In conclusion, LP reduced sperm motility, but not viability. The reduction in sperm motility may be due to the calcium chelating effect of LP and/or decreased Na+/K+-ATPase activity, but not an alteration in NO production. Besides, none of the tested parameters was found to be correlated with male age.
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Isolation of dioscin-related steroidal saponin from the bulbs of Allium paradoxum L. with leishmanicidal activity p. 469
Fatemeh Rezaee, Behzad Zolfaghari, Masoud Sadeghi Dinani
Alliums are rich sources of steroidal saponins, flavonoids, and sulphoric compounds of which steroidal saponins have recently received more attention due to their important pharmacological activities. Allium paradoxum L. is a common edible vegetable in north regions of Iran, especially in Mazandaran province, where it is named “Alezi” and considerably used as a raw vegetable, to make dishes, and as a medicinal plant. Phytochemical investigation of chloroform-methanol extract of the plant resulted in the isolation and identification of a dioscin related steroidal saponin, using comprehensive spectroscopic methods including 1D and 2D NMR, its chemical structure was determined as (25R)-spirost-5-en-3b-ol,3-O-α-rhamnopyranosyl-(1→4)-α-rhamnopyranosyl-(1→4)-[a-rhamnopyranosyl-(1→2)]-glucopyranoside. Investigation of in vitro antileishmanial activity of the isolated compound, in 10 and 50 μg/mL concentrations, exhibited significant leishmanicidal effects (P < 0.001) against the promastigotes of Leishmania major. The results established a valuable basis for further studies about A. paradoxum and anti-parasitic activity of steroidal saponins.
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