Process optimization for green synthesis of silver nanoparticles using Rubus discolor leaves extract and its biological activities against multi-drug resistant bacteria and cancer cells.

Multi-drug resistant (MDR) bacteria are considered a serious public health threat. Also, increasing rate of resistance to anticancer drugs, as well as their toxicity, is another point of concern. Therefore, the new antibacterial and anticancer agents are always needed. The synthesizing silver nanoparticles (AgNPs) using medicinal plants, is an effective approach for developing novel antibacterial and anticancer agents. Rubus discolor, a native species of the Caucasus region, produces leaves that are typically discarded as a by-product of raspberry production. The present study has focused on optimizing the green synthesis of AgNPs using R. discolor leaves extract through response surface methodology. The optimal values for AgNPs synthesis were an AgNO3 concentration of 7.11 mM, a time of 17.83 h, a temperature of 56.51 °C, and an extract percentage of 29.22. The production of AgNPs was confirmed using UV-visible spectroscopy (λmax at 456.01 nm). TEM analysis revealed well-dispersed AgNPs (an average size of 37 nm). The XRD analysis confirmed the crystalline structure. The EDX detected a strong peak at 3 keV corresponded to Ag. The zeta potential value (- 44.2 mV) indicated the stability of nanoparticles. FT-IR spectra showed the presence of various functional groups from plant compounds, which play an important role in the capping and bio-reduction processes. The AgNPs revealed impressive antibacterial activities against MDR Escherichia coli and Pseudomonas aeruginosa (MIC ranging from 0.93 to 3.75 mg ml-1). The phytochemical analysis indicated the presence of phenolics, tannins, and flavonoids on the surface of AgNPs. They also showed significant cytotoxic effects on A431, MCF-7, and HepG2 cells (IC50 values ranging from 11 to 49.1 µg ml-l).

Inhibitory effect of temozolomide on apoptosis induction of cinnamaldehyde in human glioblastoma multiforme T98G cell line.

Glioblastoma is the most common primary malignant brain tumor in adults. Temozolomide (TMZ) is an FDA-approved drug used to treat this type of cancer. Cinnamaldehyde (CIN) is a derivative of cinnamon extract and makes up 99% of it. The aim of this study was to investigate the in vitro combined effect of CIN and TMZ on human glioblastoma multiforme T98G cell line viability. In this study, we used 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl-tertazolium bromide (MTT) method to evaluate the extent of IC50, acridine orange, Giemsa and Hoechst staining to evaluate the manner of apoptosis and the Western blotting method to examine the expression change of apoptotic proteins. Our results show that TMZ has an inhibitory effect on CIN when both used in combination at concentrations of 300 and 100 µM (P<0.05) and has a cytotoxic effect when used alone at the same concentrations (P<0.05). The western blotting result showed that TMZ at concentrations of 2,000 and 1,000 µM significantly increased Bax expression and decreased Bcl2 expression (P<0.05), indicating that TMZ induced apoptosis through the mitochondrial pathway. However, CIN had no effect on Bax and Bcl2 expressions, thus causing apoptosis from another pathway. Also, the Bax:Bcl2 expression ratio at concentrations combined was lower than that for TMZ 1,000 µM and higher than that for CIN 150 and 100 µM (P<0.05), which confirms the inhibitory effect of TMZ on CIN. From the present study, we conclude that TMZ in combination with CIN has an inhibitory effect on increasing the cytotoxicity rate.

SPION based magnetic PLGA nanofibers for neural differentiation of mesenchymal stem cells.

Recently, magnetic platforms have been widely investigated in diagnostic, therapeutic and research applications due to certain properties, such as cell and tissue tracking and imaging, thermal therapy and being dirigible. In this study, the incorporation of magnetic nanoparticles (MNPs) in nanofibers has been proposed to combine the advantages of both nanofibers and MNPs to induce neural differentiation of mesenchymal stem cells. Magnetic poly (lactic-co-glycolic acid) nanofibers (containing 0%, 5% and 10% SPION) were fabricated and utilized as the matrix for the differentiation of mesenchymal stem cells (MSCs). Morphological, magnetic and mechanical properties were analyzed using FESEM, VSM and tensile test, respectively. The expression of neural markers (TUJ-1, NSE, MAP-2) was assessed quantitative and qualitatively utilizing RT-PCR and immunocytochemistry. Results confirmed the incorporation of MNPs in nanofibrous scaffold, presenting a saturation magnetization of 9.73 emu g-1. Also, with increase in magnetic particle concentration (0%-10%), tensile strength increased from 4.08 to 5.85 MPa, whereas the percentage of elongation decreased. TUJ-1 expression was 3.8 and 1.8 fold for 10% and 5% magnetic scaffold (versus non-magnetic scaffold) respectively, and the expression of NSE was 6.3 and 1.2-fold for 10% and 5%, respectively. Consequently, it seems that incorporation of magnetic biomaterial can promote the neural differentiation of MSCs, during which the augmentation of super paramagnetic iron oxide concentration from 0% to 10% accelerates the neural differentiation process.

An investigation of the anti-inflammatory effects of gabapentin on acetic acid-induced colitis in rats.

Inflammatory bowel disease (IBD) is considered a chronic inflammatory gastrointestinal disease with treatment options which exhibit low efficacies and lead to considerable side effects. Hence, the challenge to alleviate IBD complications is remained to be resolved. The purpose of this study is evaluating anti-inflammatory impacts of gabapentin on acetic acid-induced colitis in rats. Colitis was induced by the instillation of 2 mL of 3% acetic acid solution into rat's colons. Rats were randomly allocated into six groups including normal group, colitis control group, gabapentin-treated groups (25, 50, and 100 mg/kg; i.p.), and dexamethasone-treated group (1 mg/kg; i.p.). Based on the macroscopic assessment besides histological and biochemical findings [myeloperoxidase (MPO), pro-inflammatory cytokines], the efficacy of gabapentin was investigated. Gabapentin (50 and 100 mg/kg), and dexamethasone considerably reduced macroscopic and microscopic colonic lesions induced by acetic acid in rats in comparison with colitis control group. These results were confirmed by reduced levels of MPO activity and colonic concentrations of interleukin-6, interleukin-1 beta, and tumor necrosis factor-alpha, in inflamed colon tissue. Our data demonstrated that gabapentin exerts profitable impacts in experimental colitis that might be ascribed to its anti-inflammatory features and thus can be a potential therapeutic agent for IBD treatment.

Effects of melatonin on the toxicity and proliferation of human anaplastic thyroid cancer cell line.

BACKGROUND: Thyroid carcinoma is the most common endocrine malignancy and anaplastic thyroid carcinoma (ATC) is a rare but most aggressive cancer. Melatonin has enhanced or induced apoptosis in many different cancer cells, however, there has not been any study on the effects of melatonin in the treatment of ATC. In this study, we examined the effect of melatonin on cytotoxicity in the human ATC cell line. MATERIALS AND METHODS: Cultured ATC cells were treated at melatonin concentrations 0.6, 1, 4, 16, 28 mM for 24 h. The MTT assay was performed to examine cell viability. Cytotoxicity was assayed with the determination of lactic dehydrogenase (LDH) activity. Apoptosis was detected by acridine orange/ethidium bromide and Hoechst 33342 staining. Giemsa staining is considered for evaluating the morphological changes of ATC cells. The reproductive ability of cells to form a colony was evaluated by the clonogenic assay. RESULTS: Results showed that melatonin could significantly decrease cell viability and the lowest cell viability was observed at 28 mM, 10.26 % ± 0.858 versus control. Similar results were obtained when analyzing LDH activity. The highest LDH levels were observed at 16 and 28 mM (546.08 ± 4.66, 577.82 ± 3.14 munit/mL versus control) that confirmed the occurrence of late apoptosis. The clonogenic assay showed that cells at the high concentration of melatonin (16 and 28 mM) don't enable to form the colony that approved the occurrence of reproductive death. CONCLUSION: Our results showed a dose-dependent cytotoxic effect of melatonin on ATC cells that significantly decreased cell viability and induced cell reproductive death at the concentration greater than 1 mM and findings suggested that MLT might be useful as an adjuvant in ATC therapy.

Anti-proliferative and apoptotic effects of hull-less pumpkin extract on human papillary thyroid carcinoma cell line.

Papillary thyroid carcinoma (PTC) is one of the most common cancers of the endocrine system. Previous studies have shown that the extract of hull-less pumpkin seed (HLPS) has a significant anti-cancer effect. The aim of this study was to evaluate the effect of this plant extract on the proliferation of PTC cells. In this study, an extract of this plant was prepared by soxhlet extraction method and analyzed by Gas Chromatography-Mass Spectrometry. The cytotoxicity of PTX and plant extract was investigated using the methylthiazol tetrazolium (MTT) method. For careful investigation of morphological alteration, we used hematoxylin and eosin and Giemsa stinging. Based on MTT assay test, the IC50 value of paclitaxel (PTX) was significantly less than the hydro-alcoholic extract of HLPS at all of the incubation time. Our results of histological staining showed that HLPS and PTX induced significant morphological alteration in the PTC cultured cell that consistent with cell death. Comparing the groups treated by PTX or HLPS with control group showed significant differences. It seems that HLPS extract has an apparent effect on treatment of PTC, at least in laboratory condition, albeit for realistic decision about the effect of HLPS on PTC, more molecular investigations are necessary.

Novel agonists of benzodiazepine receptors: design, synthesis, binding assay and pharmacological evaluation of 1,2,4-triazolo[1,5-a]pyrimidinone and 3-amino-1,2,4-triazole derivatives.

Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical practice. In spite of their benefits they have several side effects, so synthesis of new agonists of these receptors to get more specific effect and better profile of adverse drug reactions is still continued. Novel BZD agonists were designed based on the pharmacophore/receptor model of BZD binding site of GABAA receptor. Energy minima conformers of the designed compounds and estazolam, a known BZD receptor agonist, were well superimposed in conformational analysis. Docking studies revealed that the carbonyl group of the compound 4c, 3-(2-chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, was near the nitrogen moiety of triazole ring of estazolam providing the hydrogen bond acceptor in proper direction in the BDZ-binding site of GABAA receptor model (α1ß2ϒ2). The designed compounds were synthesized and their in vitro affinity for the central BZD receptor was determined. Most of the novel compounds had better affinity for the BZD site of action on GABAA receptor complex than diazepam. Finally, the novel compound 4c with the best affinity in radioligand receptor binding assay (Ki=0.42 nM and IC50=0.68 nM) was selected as candidate for in vivo evaluation. This compound showed significant hypnotic activity and weak anticonvulsant effect with no impairment on learning and memory performance in mouse. The pharmacological effects of the compound 4c were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects of the novel ligand.

Optimum conditions of radioligand receptor binding assay of ligands of benzodiazepine receptors.

To obtain drugs which are more selective at benzodiazepine (BZD) receptors, design and synthesis of functionally selective ligands for BZD receptors is the current strategy of our pharmaceutical chemistry department. The affinity of newly synthesized ligands is assessed by radioligand receptor binding assays. Based on our previous studies, 2-phenyl-5-oxo-7-methyl-1,3, 4-oxadiazolo[a,2,3]-pyrimidine (compound A) was chosen for design and synthesis of new triazole derivatives as GABAA BZD receptor agonist. The cortical membrane of male Sprague-Dawley rats was prepared as the source of the BZD receptors. Different concentrations of membrane protein and [3H]-flumazenil were incubated at room temperature at different time periods to reach the steady-state. To saturate the receptors, increased amounts of radioligand were incubated with membrane protein. The bound and un-bound ligands were separated by centrifugation. The affinity of compound A was measured in competition studies at optimum conditions by displacement of [3H]-Flumazenil from rat cortical membrane. Based on results, the optimum conditions of radioligand receptor binding assay of benzodiazepines were 35 min incubation of ligands with 100 µg cortical membrane protein and 8.6 × 10-5 nmole 3H-flumazenil in a final volume of 0.5 mL Tris-HCl buffer (50 mM, pH 7.4) at 30 °C. The binding parameters of [3H]-flumazenil, Bmax and Kd were determined through saturation studies as 0.638 ± 0.099 pmol/mg and 1.35 ± 0.316 nM respectively. The affinity of compound A was 1.9 nM comparable with diazepam (1.53nM). This finding makes the compound an interesting lead for further optimization. Starting from this compound, new ligands were synthesized and screened in-vitro by competitive binding assays.

Human neutrophil peptide-1 (HNP-1): a new anti-leishmanial drug candidate.

The toxicity of available drugs for treatment of leishmaniasis, coupled with emerging drug resistance, make it urgent to find new therapies. Antimicrobial peptides (AMPs) have a strong broad-spectrum antimicrobial activity with distinctive modes of action and are considered as promising therapeutic agents. The defensins, members of the large family of AMPs, are immunomodulatory molecules and important components of innate immune system. Human neutrophil peptide-1 (HNP-1), which is produced by neutrophils, is one of the most potent defensins. In this study, we described anti-parasitic activity of recombinant HNP-1 (rHNP-1) against Leishmania major promastigotes and amastigotes. Furthermore, we evaluated the immunomodulatory effect of rHNP-1 on parasite-infected neutrophils and how neutrophil apoptosis was affected. Our result showed that neutrophils isolated from healthy individuals were significantly delayed in the onset of apoptosis following rHNP-1 treatment. Moreover, there was a noteworthy increase in dying cells in rHNP-1- and/or CpG-treated neutrophils in comparison with untreated cells. There is a considerable increase in TNF-α production from rHNP-1-treated neutrophils and decreased level of TGF-ß concentration, a response that should potentiate the immune system against parasite invasion. In addition, by using real-time polymerase chain reaction (real-time PCR), we showed that in vitro infectivity of Leishmania into neutrophils is significantly reduced following rHNP-1 treatment compared to untreated cells.