فهرست مطالب

Pharmaceutical Research - Volume:18 Issue: 1, 2019
  • Volume:18 Issue: 1, 2019
  • تاریخ انتشار: 1397/11/22
  • تعداد عناوین: 50
  • Sukhbir Khokra, Kanika Arora, Shah Alam Khan* , Pawan Kaushik, Reetu Saini, Asif Husain Pages 1-15
    We report herein the synthesis of ¾ substituted benzene sulfonamides linked via phenyl ring to a benzothiazole moiety. The title compounds in the two series namely N-(4-(benzothiazole-2-yl) phenyl) 4- substituted benzene sulfonamides and N-(4-(benzothiazole-2-yl) phenyl) 3- substituted benzene sulfonamides were synthesized by condensing 2-(3/4-aminophenyl) benzothiazole with various substituted sulfonyl chlorides. The synthesized compounds were subjected to neurotoxicity screening, computational studies, and evaluation of their anticonvulsant potential. Amongst all the synthesized compounds, compound 9 emerged as the most potent anticonvulsant agent in maximal electroshock (MES) model (standard: phenytoin) in mice and showed three hydrogen bond interactions with the nicotinic acetylcholine ion gated receptors (PDB ID: 2BG9). Interestingly, compound 13 showed five hydrogen bond interactions with the target protein and thus excellent binding affinity upon computational analysis but was found to be neurotoxic.
    Keywords: Anticonvulsant, Benzenesulfonamide, Benzothiazole, Computational analysis, MES
  • Sima Golmakaniyoon, Vahid Reza Askari, Khalil Abnous, Afshin Zarghi, Razieh Ghodsi* Pages 16-29
    Quinones such as 1,4-naphthoquinones are abundant in nature and naphthoquinone based natural products are known to possess anticancer activity. This pharmacophore is known to convey anticancer activity to some drugs such as streptonigrin, mitomycin A, etc. We synthesized and characterized different classes of naphthoquinone derivatives including bis naphthoquinone, 2-arylaminonaphthoquinone, benzoxantene-6,11-dione and benzoacridine-5,6-dione derivatives instead of the expected 2-hydroxy-3-(substituted phenyl(aryl amino)methyl)naphthalene-1,4-dione derivatives from the reaction of 2-hydroxy1,4-naphthoquinone (lawson) with different benzaldehydes and aryl amines. Benzoacridine-5,6-dione derivatives and related imines showed potent anti-breast cancer activity in MCF-7 cancer cells. The in-vitro results revealed that five compounds benzoacridinedione derivatives (6b and 7b) and imines (13, 14 and 15) by the IC50 range of 5.4-47.99 μM are the most potent anti-breast cancer structures.
    Keywords: Synthesis, 2-hydroxynaphthalene-1, 4-dione, Naphthoquinone, Bis naphthoquinone, Benzoxantene-6, 11-dione, Benzoacridine-5, 6-dione, Anticancer
  • Amirali Delnavaz Shahr, Fazel Nasuhi Pur *, Karim Akbari Dilmaghani Pages 30-33
    In this paper, the synthesis and free-radical scavenging capacity of novel calix[4]arene-based cluster of paracetamol was reported. The phenolic structures of acetaminophen and calix[4]arene prompted us for designing a synthetic route for calix[4]arene-based cyclic tetramer of paracetamol. The present chalice-shaped cluster is the first example of calixarene/acetaminophen hybrid and paracetamol can be considered as ¼ of the synthetic cyclic tetramer. Free-radical scavenging tests were determined by the 2,2-diphenyl-1-picrylhydrazyl radical in methanol. The results of antiradical-testing showed the enhanced free-radical scavenging capacity (~ 10-fold) for the prepared chaliced-shaped cluster with respect to the corresponding single therapeutic drug unit (acetaminophen). It is maybe attributed to the multivalency, spatial preorganization, and synergistic effect of four impacted drug units in the cluster structure (clustering effect).
    Keywords: Acetaminophen, Paracetamol, Calixarene, Cluster, Antiradical, Radical scavenger
  • Aliasghar Jarrahpour *, Roghayeh Heiran, Véronique Sinou, Christine Latour, Lamia Djouhri Bouktab, Jean Michel Brunel, Javed Sheikh, Taibi Ben Hadda Pages 34-48
    Some new b-lactams bearing biologically important morpholine ring have been synthesized by acylation of amino b-lactams in the presence of morpholine-4-carbonyl chloride. These novel β-lactams were prepared under mild reaction conditions without any solvent in short reaction times. Their biological activities have been examined against microbial agents such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and fungi such as Candida albicans (C. albicans) and Candida glabrata (C. glabrata). They have been also tested against Plasmodium falciparum K14 resistant strain and showed moderate to good IC50 values.
    Keywords: b-Lactam, 2-Azetidinones, Acylation, Antimalarial activities, POM analyses
  • Massoud Amanlou, Elham Hashemi, Mohammad Ali Oghabian, Mehdi Shafiee Ardestani * Pages 49-60
    Cancer detection in early stage using a powerful and noninvasive tool is of high global interest. In this experiment, a small-molecular-weight glucose based derivative of Gd3+-1-(4-isothiocyanatobenzyl) diethylene tri amine penta acetic acid (Gd3+-p-SCN-Bn‐DTPA‐DG) as a novel potential MR imaging contrast agents was synthesized. Gd3+-p-SCN-Bn‐DTPA‐DG was synthesized with reacting of Glucosamine and 1-(4-isothiocyanatobenzyl) diethylene tri amine penta acetic acid then loaded by gadolinium to make novel agent of functional MR imaging. The relaxivity, T1, T2 relaxation times, and cell toxicity of this contrast agent were studied. The results demonstrated that the sugar moieties linked to Gd3+-p-SCN-Bn‐DTPA efficiently increase its cellular uptake. The Gd3+-p-SCN-Bn‐DTPA‐DG significantly decreased MCF-7 tumor cell numbers without any significant toxicity on normal human kidney cells. Finally, it displayed an intense signal on T1 weighted with respect to the unlabeled cells. Based on the findings from the present research Gd3+-p-SCN-Bn‐DTPA‐DG be a potential breast molecular imaging. However, further investigations by anticancer studies are in the pipeline.
    Keywords: Cancer diagnosis, Contrast agent, Gd3+-1-(4-isothiocyanatobenzyl) diethylene tri amine penta acetic acid, Magnevist, MRI
  • Zahra Bangaleh, Hayedeh Bagheri Sadeghi *, Soltan Ahmad Ebrahimi, Parvaneh Najafizadeh Pages 61-71
    Methods routinely utilized for detection of phenylalanine in new-born blood consist of enzymatic assays, lacking sensitivity and HPLC assays which are expensive and time-consuming to conduct. We, here, report for the first time, the construction of a phenylalanine sensitive electrode, on the basis of a selective molecularly imprinted polymer, offering sensitivity, economy and ease of use for the measurement of phenylalanine .The sensor was constructed of a graphite-rod electrode which was coated by MIP embedded polymer base made from polyvinyl chloride and plasticizer mixture, dissolved in THF. At optimized conditions the electrode revealed a Nernstian response 29.73 ± 1.0 mV decade-1 in a concentration range of 1 × 10⁻⁸ to 1 × 10-4 M with detection limit of 5 × 10⁻⁹ M. The potential response of the electrode was constant in the pH range of 4.0–7.5. The electrode unfolded a response time of ~20 sec. The selectivity coefficient of the sensor towards a number of different amino acids with molecular similarities and some metal ions was evaluated. The sensor was successfully used for determination of phenylalanine in blood serum and the results were in good compatibility with HPLC method.
    Keywords: Phenylalanine, Potentiometric sensor, Molecular electrochemistry, MIPs
  • Elahehnaz Parhizkar, Hadi Saeedzadeh, Fatemeh Ahmadi, Mohammad Ghazali, Amirhossein Sakhteman* Pages 72-79
    Iron is an essential element used as supplement in different dosage-forms. Different time and expenditure-consuming methods introduced for detection and determination of elemental ions such as atomic absorption. In this research, two different and routine methods containing ATR-IR and atomic absorption were applied to define the amount of iron in 198 samples containing different concentrations of commercial iron drops and syrups and the output data of the methods was transferred to chemometric model to compare the accuracy and robustness of the methods. By applying this mathematical model, in addition to the confirmation of ATR-IR (a time and energy-saving method) as a replacement of AAS to produce the same results, chemometrical model was used to evaluate the output data in a faster and easier method.
    At first, ATR-IR spectra data converted to normal matrix by SNV preprocessing approach. Then, a relationship between iron concentrations achieved by AAS and ATR-IR data was established using PLS-LS-SVM. Consequently, model was able to predict ~99% of the samples with low error-values (root mean square-error of cross-validation equal to 0.98). Y-permutation test performed to confirm that the model was not assessed accidentally.
    Although, chemometric methods for detection of some heavy metals have been reported in the literature, combination of PLS-LS-SVM with ATR-IR was not cited. In this study a fast and robust method for iron assay was suggested.
    As a result, ATR-IR can be a suitable method in detection and qualification of iron-content in pharmaceutical dosage with less energy-consumption but similar accuracy.
    Keywords: Attenuate Total Reflectance Mid-infrared, atomic absorption spectroscopy, iron, partial least squares- least squares, support vector machine model
  • Hadi Beitollahi *, Fariba Garkani, Nejad, Somayeh Tajik, Mohammad Reza Ganjali Pages 80-90
    A high sensitive electrochemical nanostructure sensor based on graphene oxide/Fe3O4@SiO2 nanocomposite modified graphite screen printed electrode (GO/Fe3O4@SiO2/SPE) has been developed for trace analysis of acetaminophen. The electrochemical study of the modified electrode, as well as its efficiency for simultaneous voltammetric oxidation of acetaminophen and tryptophan is described. Compared with bare SPE the GO/Fe3O4@SiO2/SPE exhibited excellent electrocatalytic activity toward the oxidation of acetaminophen. The plot of catalytic current versus acetaminophen concentration showed a linear segment in the concentration range 0.5 to 100.0 µM. The detection limit of 0.1 µM was obtained using calibration plot. Also the anodic peaks of acetaminophen and tryptophan in their mixture can be well separated. The GO/Fe3O4@SiO2/SPE has been successfully applied and validated by analyzing acetaminophen and tryptophan in urine and pharmaceutical samples.
    Keywords: Acetaminophen, Tryptophan, Nanocomposite, Graphene, Graphite screen printed electrode
  • Fatemeh Farjami *, Farshid Fasihi, Forough Alimohammadi, Seyed Esmaeil Moradi Pages 91-101
    In this manuscript, the electrocatalytic oxidation of doxepin (DOX) was studied at a carbon ionic liquid electrode, fabricated using graphite, and the ionic liquid 1-octylpyridinium hexaflourophosphate (OPFP). The surface of the proposed electrode was characterized by scanning electron microscopy. Differential pulse voltammetry was applied as an analytical technique for quantification of sub-micromolar concentration of doxepin. Various parameters were optimized for practical application. Under the optimal conditions, the proposed electrode exhibited interesting sensitivity toward determination of doxepin compared to the other conventional electrodes and the anodic peak current versus doxepin concentration was linear in the ranges of 0.05-24 µM. The detection limit of 21 nM was achieved. Cyclic voltammetry (CV) was also applied to acquire information about the reaction mechanism and calculating the kinetic parameters. The electroxidation process was irreversible and revealed adsorption controlled behavior. The method was successfully applied for determination of doxepin content in pharmaceuticals and blood serum samples.
    Keywords: Carbon ionic liquid electrode, Doxepin, Tricyclic antidepressants, Voltammetry
  • Morteza Yaghoobian, Azadeh Haeri, Noushin Bolourchian, Soraya Shahhosseini, Simin Dadashzadeh* Pages 102-110
    The present study aimed at exploring the potential of the P-glycoprotein (P-gp) transporters as a barrier to the repaglinide (REG) epithelial permeability. In-vitro intestinal absorption models, the everted gut sac, and Caco-2 cell line, were used to study the possible role of P-gp in intestinal transport of REG. In the everted gut sacs, apparent permeability coefficients showed cargo concentration dependency transport over the concentration of 40 µM, indicating involvement of a saturable mechanism in REG absorption (Papp were 1.23 × 10 -5and 3.29 × 10 -5at drug concentrations of 40 and 100 μM, respectively). Adding verapamil (100 μM), valspodar (5 μM) and ketoconazole (10 μM) significantly enhanced the permeability of REG across mucosal to serosal in the rat jejunum (P < 0.05) suggesting role of CYP 3A4 and/or efflux transporters in oral bioavailability of REG. However, the results of Caco-2 cell experiments indicated low efflux ratios (less than 2) and insignificant involvement of P-gp efflux pumps in REG intestinal transport. Given that Caco-2 cells do not express adequate level of CYP 3A4, the current study suggests that the presystemic metabolism by cytochrome P450 (and not ejection by P-gp) may play a significant role in limiting the oral absorption of REG in small intestine.
    Keywords: Repaglinide, Caco-2 Cells, Everted gut sac, P-glycoprotein, Permeability
  • Fatemeh Rasti Boroojeni, Shohreh Mashayekhan* , hojjat allah abbaszadeh Pages 111-124
    In this study, a system of dexamethasone sodium phosphate (DEXP)-loaded chitosan nanoparticles embedded in poly-ε-caprolacton (PCL) and gelatin electrospun nanofiber scaffold was introduced with potential therapeutic application for treatment of the nervous system. Besides anti-inflammatory properties, DEXP act through its glucocorticoid receptors, which are involved in the inhibition of astrocyte proliferation and microglial activation. Bovine serum albumin (BSA) was used to improve the encapsulation efficiency of DEXP within chitosan nanoparticles and to overcome its initial burst release. BSA incorporation within the chitosan nanoparticles increased the encapsulation efficiency of DEXP from 30% to 77%. The comparison between DEXP release profile from PCL/gelatin scaffold with and without chitosan nanoparticles revealed that the system of DEXP-BSA-loaded chitosan nanoparticles embedded in electrospun PCL nanofiber scaffold provided a more controlled release pattern of the loaded drug. The scaffolds properties in terms of structure, hydrophilicity, cell compatibility, mechanical property and biodegradability were further investigated, which might show its potential application for the repair of spinal cord injury.
    Keywords: Spinal cord injury, Dexamethasone, Electrospinning, Nanofiber scaffold, Controlled release
  • Amin Amani , Toraj Khabiri, Samira Shafiee, Aliasghar Hamidi* Pages 125-141
    Tri-block poly (lactide) poly(ethylene glycol) poly(lactide) (PLA–PEG–PLA) copolymers are among the most attractive nano-carriers for gene delivery into mammalian cells, due to their biocompatibility and biodegradability properties. However, the low efficiency of the gene delivery by these copolymers is an obstacle to gene therapy. Here, we have investigated nanoparticles formulated using the polyethylenimine (PEI) associated with PLA-PEG-PLA copolymer for efficient DNA encapsulation and delivery. PLA-PEG-PLA/DNA and PLA-PEG-PLA/PEI/DNA nanoparticles with different concentrations of PEI were prepared by the double emulsion-solvent evaporation technique. PLA-PEG-PLA/PEI/DNA were characterized for particle size, zeta potential, morphology, biocompatibility, DNA protection, DNA release, and their ability for gene delivery into MCF-7 cells. We found that enhancing the mass ratio of PEI: (PLA-PEG-PLA) (w/w %) in the PLA-PEG-PLA/PEI/DNA nanoparticles results in an increase in particles size, zeta potential, encapsulation efficiency, and DNA release. The electrophoretic analysis confirmed that the PLA-PEG-PLA and PLA-PEG-PLA/PEI could protect DNA from ultrasound damage and nuclease degradation. MTT assay showed that the PLA-PEG-PLA/PEI/DNA had low cytotoxicity than PEI complexes. The potential of PLA-PEG-PLA/PEI/DNA nanoparticles with different concentrations of PEI as a non-viral gene delivery vector for transferring pEGFP-N1 to MCF-7 cells was examined by fluorescent microscopy and flow cytometry. The flow cytometry analysis revealed that by increasing the mass ratio of PEI: (PLA-PEG-PLA) (w/w %) in PLA-PEG-PLA/PEI/DNA nanoparticles, the efficiency of the gene delivery into MCF-7 cells was improved. The results also demonstrated that PLA-PEG-PLA/PEI/DNA nanoparticles in the serum medium improved the efficiency of gene delivery more than two-fold, compared to PEI/DNA complex.
    Keywords: PLA-PEG-PLA, Polyethylenimine, Gene delivery, Cytotoxicity, DNA release
  • Elham Khodaverdi, Zahra Tayarani, Najaran, Elham Minbashi, Mona Alibolandi, Javad Hosseini, Samaneh Sepahi, Hossein Kamali, Farzin Hadizadeh * Pages 142-155
    Microwave irradiation was used to synthesize PEG-PCL and PEG-PLA copolymers that are composed of biodegradable polymers including PEG, PLA, and PCL. These copolymers were used for loading docetaxel in nanoparticles. Single emulsion-solvent evaporation technique was applied for preparing the PEG-PLA and PEG-PCL mixed nanoparticles (micelles and polymersomes) with different proportions, including 0:1, 1:1, 3:1, 1:3, and 1:0. The unimodal gel permeation chromatography curve showed low polydispersity of the di-block copolymers. The in vitro drug release curves of formulations were compared. Micelles and polymersomes of 75% PEG-PCL and 25% PEG-PLA (P5 and M5) have the lowest burst release (5%) at the same period compared to other copolymers. The dynamic light scattering and TEM results clarified that the size and shape of the formulations are uniform. The cytotoxicity effect of P5 and M5 was evaluated in different cell lines. The best one was found to P5 with IC50 between 1.48-11.79 g/ml. The pro-apoptotic effect of P5 was confirmed with flow cytometry study. These mixed micelles (M5) and polymersomes (P5) was found to be superior formulations than non-mixed ones.
    Keywords: Micelles, polymersomes, di-block copolymer, docetaxel, Cytotoxicity
  • Parisa Amir Kalvanagh, Masoumeh Ebtekar *, Parviz Kokhaei , Hoorieh Soleimanjahi Pages 156-167
    During the 15 years since the discovery of type III human interferons [IFN-λ1(IL-29), IFN-λ2(IL-28A), and IFN-λ3(IL-28B)], numerous biological properties such as anticancer, antiviral, and immunomodulatory activities of this new IFN family have been investigated. Several studies have shown that the encapsulation of pcDNA with PLGA nanoparticles (NPs) protects them against DNase enzyme action and increases the efficiency of gene delivery to the cells. The purpose of this study was to encapsulate pcDNA encoding IFN-λ1 (pIFN-λ1) with a simple and cost-effective method using PLGA NPs. The pIFN-λ1-loaded PLGA NPs were produced by a double-emulsion-solvent evaporation method and characterized in terms of size, size distribution, and zeta potential by DLS and morphologically by SEM and TEM. The bioactivity of NPs was also examined by fluorescent microscopy. The results showed that IFN-λ1 expressed by HEK293T cells could protect HepC-2 cells from the cytopathic effects of EMCV. The NPs were spherical in shape with a mean diameter of 380 ± 3 nm, a zeta potential of −3.3 ± 7.6 mV, an encapsulation efficiency of 75 ± 5%, and a loading capacity of 0.83 ± 0.06. The NPs were also bioactive and easily engulfed by RAW264.7 cells. The pIFN-λ1 could be sustainably released from NPs. Due to the facility and affordability of encapsulation of pIFN-λ1 in the PLGA NPs proposed in this study and the advantages of encapsulation by PLGA, it appeared rational to use pIFN-λ1-loaded NPs instead of naked pIFN-λ1 to determine other unexplained activities of this new cytokine or to use it as an alternative or adjunct to current IFN-α therapy.
    Keywords: IFN-?1, PLGA, Non-viral gene delivery, Therapy
  • Yannian Huang, Xiuhua Zhao *, Yuangang Zu, Lu Wang, Yiping Deng, Mingfang Wu, Huimei Wang Pages 168-182
    In this study, a novel mesoporous silica nanoparticles drug carrier contributes to improving the solubility, dissolution, and the oral bioavailability of apigenin (AP). The apigenin of solid dispersion of mesoporous silica nanoparticles (AP-MSN) was prepared by physical absorption method and also, in-vitro drug release and in-vivo bioavailability performance were evaluated. Based on its solubility, the AP-MSN solid dispersion was prepared at the weight ratio of 1:1 to obtain the optimum solubility. The loading efficiency (LE), encapsulation efficiency (EE), and solubility of AP-MSN solid dispersion were 29.71%, 42.27%, and 25.11 µg/mL, respectively. SEM, TEM, BET, FTIR, XRD, DSC, and TG were also carried out. These results demonstrated that AP was good absorbed into the pores of MSN through physical absorption effect of MSN. The DMF residues of AP-MSN solid dispersion meet the ICH requirements. It was vital that the AP-MSN solid dispersion behaved well in-vitro and the accumulative release of AP-MSN solid dispersion was 2.37 times higher than that of raw AP. In-vivo study, the AP area under curve0-t was 8.32 times higher for the AP-MSN solid dispersion than that of raw AP, which indicated that the bioavailability of AP-MSN solid dispersion was greatly improved. Therefore, the prepared AP-MSN solid dispersion presents potential as a novel oral therapeutic agent formulation for clinical application.
    Keywords: Apigenin, Mesoporous silica nanoparticles, Solid dispersion, Solubility, Dissolution, Bioavailability
  • Rasoul Rahimnia, Zeinab Salehi *, Mehdi Shafiee Ardestani, Hamid Doosthoseini Pages 183-197
    In this work, we investigate the loading and conjugation of Curcumin on oleic acid (OA) and citric acid (CA) functionalized iron oxide nanoparticles and its applications in improving contrast in MRI. Magnetic iron oxide nanoparticles (Fe3O4, MNPs) synthesized using the co-precipitation method and characterized by XRD, DLS, FT-IR, VSM and SEM. FT-IR results confirmed functionalization with oleic acid and citric acid. Curcumin was loaded and conjugated with the Nano-Systems and the amount of Curcumin loaded was quantified using spectrophotometry at 419 nm wavelength. The impact of solvent on the loading of Curcumin studied. the wt% of loaded Curcumin was found to be 0.189 wt% using dimethylformamide (DMF) whereas using a combination of water-ethanol (15% v/v), this increased to 56.149 wt%. T2 relaxation time was determined using a 1.5 Tesla MRI machine; results showed that the MNPs reduced T2. Cytotoxicity of Nano-Systems (NS) in MTT assay showed that concentrations higher than 80 μg/mL (CNS > 80 μg/mL) can lead to cancer cell death and low concentrations, up to 40 μg/mL (CNS < 40 μg/mL) could be evaluated for diagnostic purposes.
    Keywords: Magnetic nanoparticles, Curcumin, MRI, Citric acid, Functionalization MNPs
    Essential oil of Citrus family plant is known to have repellent effect against mosquito. Unfortunately, due to its high volatility effect, its repellency effect was compromised. The incorporation of essential oil in a microencapsulation formulation has been shown to help improve the stability and potency of the repellent. In this study, Citrus grandis peel oil (CGPO) was encapsulated by using the interfacial precipitation chemistry technique. The microencapsulated CGPO was then formulated into lotion form to produce topical repellent formulation. This study includes the characterization of microcapsules with regards to the morphology, size distribution, zeta potential, Fourier Transmission Infrared spectrophotometer (FTIR) and Thermogravity analysis (TGA). The effectiveness of the microencapsulated CGPO-lotion formulation against mosquitoes was evaluated in the laboratory setting. Results indicated that CGPO have been successfully encapsulated with 6.5 µm in diameter and zeta potential values, -47.9 mV. The FTIR analysis spectrum indicated the presence of interaction between the wall materials in microcapsules. The TGA analysis demonstrated that microencapsulation improved the thermal stability of CGPO. Repellency assay revealed that microencapsulated CGPO- based formulation possessed excellent effect compared with pure CGPO. In conclusion, CGPO was successfully encapsulated and the microencapsulation aid to improve the repellency effect of CGPO against mosquito bites.
    Keywords: Microencapsulation, essential oil, DEET, Citrus grandis, Aedes aegypti
  • Zohreh Rezvani Amin , Zahra Khashyarmanesh, Bibi Sedigheh Fazly Bazzaz *, Zahra Sabeti Noghabi Pages 210-221
    Control of size and shape is a challenge in nanoparticle synthesis. Synthetic and biosynthetic (both extracellular and intracellular) methods are used to prepare silver nanoparticle (SNP). In this study, the behavior of three strains of Staphylococcus aureus (S. aureus) was investigated in the presence of silver nitrate intra- and extracellularly. S. aureus strains biosynthesized SNPs intracellularly, while in the method of the extracellular biosynthesis, none of the strains could produce the SNP under different conditions (dark, bright light, and the presence of nitrate ion). Intracellular SNPs were purified. The results of this study and previous results were used to compare different properties of the biosynthetic (intra- and extracellular) and synthetic SNPs in terms of shape, size, zeta potential, stability, and toxicity. The results confirmed lower toxicity of biosynthetic SNPs in-vitro assays, and their more stability with less aggregation compared to the synthetic ones. Also, the biosynthetic nanoparticles were found uniform and small. These nanoparticles may be useful for being employed as biosensors.
    Keywords: Biosynthesis, Purification, Silver nanoparticles, Staphylococcus aureus, Toxicity
  • Tayebe Ramezani *, Mohammad Nabiuni, Javad Baharara, Kazem Parivar, Farideh Namvar Pages 222-231
    Today, drug resistance is one of the major problems in fight against cancer. Therefore, combination of therapeutic strategies was raised to effectively improve disease prognosis. In this regard, silver nanoparticles (AgNPs) are considered significant due to their anticancer properties. This study aimed to return sensitivity to cisplatin to A2780 cisplatin-resistance cell lines in the presence of biogenic synthesis curcumin-coated silver nanoparticles (cAgNPs). Synergic cellular effects of cAgNPs and cisplatin on ovarian carcinoma 2780 resistant to cisplatin cells were assessed using MTT assay, Acridine orange (AO)/propidium iodide (PI), DAPI staining, Annexin V/PI assay, and caspase 3/9 activation assay. Finally, expression of p53 and MMP-9 genes were evaluated using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). According to the results, 8 μg/mL and 62 μg/mL of cAgNPs and cisplatin led to 50% cell death in 48 h, respectively. Therefore, we combined non-toxic concentration of nanoparticles (1-5 μg/mL) with cisplatin (2.5 μg/mL). Decreased proliferation rate was about 50% for synergic use of cisplatin (2.5 μg/mL) and cAgNPs (2 μg/mL). According to the results, cell death induction significantly increased by AO/PI, DAPI staining and Annexin V/PI assay in the combined group. Moreover, activity of caspase 3/9 significantly increased in the mentioned group. The combined use of cAgNPs and cisplatin resulted in upregulated expression of p53 gene and downregulated expression of MPP-9 gene. As observed in this study, a combination of cAgNPs and cisplatin increased the efficiency of apoptosis induction in A2780 cells, compared to the independent use of cisplatin or cAgNPs.
    Keywords: Cisplatin, Silver, Nanoparticles, Curcumin, Resistance
  • Peyman Kheirandish Zarandi, Abbas Zare Mirakabadi *, Fattah Sotoodehnejadnematalahi Pages 232-240
    ICD-85 (venom derived peptides) has anti-proliferative effect and anti-angiogenesis activity on cancer cells. This study was performed to test the effect of ICD-85, on Human breast adenocarcinoma (MCF-7) and normal Human Dermal Fibroblasts (HDF) cell lines. In this experimental study, Mitochondrial activity, Neutral red uptake, Lactate dehydrogenase (cell necrosis), and cell morphology were assessed under unexposed and ICD-85 exposed conditions. Caspase-9 colorimetric assay kit was used to determine caspase protease activity.
    Morphological changes in MCF-7 cells on treatment with ICD-85 compared with untreated MCF-7 cells are consistent with characterizing the features of apoptosis such as granulation and cell rounding which finally results in the generation of apoptotic bodies. In contrast, this difference was not observed in normal cells. In MTT assay, ICD-85 induced dose dependent manner cytotoxic effects on MCF-7 cells which were confirmed by neutral red assay. The results showed that inhibitory concentration 50% (IC50) value of ICD-85 for MCF-7 cells at 24 h was 36.45 ± 0.38 μg/mL. However, when HDF cells were exposed to ICD-85, no significant elevation of LDH release were observed at concentrations below 20 μg/mL. The apoptosis-induction of ICD-85 on MCF-7 cell was found to be through activation of caspase-9 which was 13 fold greater than unexposed cell.
    This study showed that ICD-85 induced apoptosis in MCF-7 cell line through caspase activation and hence it can be considered for further investigation to use ICD-85 as a potential therapy for breast cancer.
    Keywords: ICD-85, MCF-7, HDF, Caspase-9, MTT assay
  • Atefeh Safarpour , Marzieh Ebrahimi *, Seyed Abolhassan Shahzadeh Fazeli , Mohammad Ali Amoozegar Pages 241-253
    Halophilic archaea are known as the novel producers of natural products and their supernatant metabolites could have cytotoxic effects on cancer cells. In the present study, we screened the anticancer potential of supernatant metabolites from eight native haloarchaeal strains obtained from a culture collection in Iran. Five human cancer cell lines including breast, lung, prostate and also human fibroblast cells as the normal control were used in the present study. Moreover, to evaluate the anti-tumor effect of the selected supernatant, inhibition of sphere formation and tumor development was assessed in-vitro and in-vivo, respectively. Among all strains, supernatant metabolites from Halobacterium salinarum IBRC M10715 had the most potent cytotoxic effect on prostate cancer cell lines (IC50 = 0.5 mg/mL) without any effects on normal cells. It significantly increased both early and late apoptosis (about 11% and 9%, respectively) in the androgen-dependent PC3 cell line, reduced sphere formation ability of DU145 and PC3 cells with down-regulation of SOX2 gene expression. Furthermore, our results revealed that tumors developed in nude mice significantly shrank post intratumor injection of metabolites of the haloarchaeal strain. In conclusion, we suggested here for the first time that supernatant metabolites from Halobacterium salinarum IBRC M10715 could be a novel component against prostate cancer in-vitro and in-vivo with remarkable reduction in stem-like properties of tumor.
    Keywords: Prostate cancer, Supernatant metabolites, Nude mice, Archaea, Halophile
  • Ameneh Eslamparast, , Reza Abbasgholizadeh, Seyed Nasser Ostad, Mehdi Gharghabi, Mohammad Hossein Ghahremani * Pages 254-262
    Fragile histidine triad (FHIT) serves a critical function as a tumor suppressor that inhibits p53 degradation by mouse double minute 2 (MDM2). The functional domains of FHIT involved in tumor inhibition was interpreted.
    In-silico screening data were employed to construct truncated forms of FHIT to assess their cytotoxic effects on the HT1080 cell line. Full FHIT expression was confirmed by western blotting and expression of two FHIT truncates were confirmed by RT-PCR. Transfection of these truncated forms into HT1080 cells showed that the N-terminal truncated form (amino acids 17-102) better inhibited proliferation than the full-length FHIT. The combined effects of these truncated forms augmented doxorubicin-induced cytotoxicity. Functional analysis demonstrated that these fragments and their combination with doxorubicin can arrest cells in the G2 phase of the cell cycle as specified by flow cytometry.
    The FHIT functional domains can be used as lead compounds for development of drug designs and gene transfer for cancer therapy.
    Keywords: Fragile histidine triad, HT1080, doxorubicin, Combination therapy, MTT assay, Flowcytometry
  • Fatemeh Zangeneh, Amir Vazirizadeh, Mohammadreza Mirshamsi, Amir Fakhri, Mehrdad Faiziorcid , Jalal Pourahmad * Pages 263-274
    Despite recent improvements in treatment, ovarian cancer is still the leading cause of death from gynaecological malignancies. Today, marine mollusks are considered as natural source of new biologically and pharmacologically active compounds by scientists and the pharmaceutical industries. The aim of this study is to investigate the selective apoptotic effects of Turbo coronatus crude extract fractions on human epithelial ovarian cancer (EOC) cells and mitochondria. Cells and mitochondria were isolated from cancerous and non-cancerous ovarian tissues and exposed to IC50 concentration of F1 fraction for evaluation of mitochondrial and cellular parameters. Our results showed that F1 fraction of T. coronatus crude extract significantly induced toxic effects only in the cancerous ovarian mitochondria, including; increased reactive oxygen species (ROS) formation, mitochondrial membrane depolarization, mitochondrial swelling and cytochrome c release. Flow-cytometry analysis demonstrated that F1 fraction of T. coronatus progressively induced apoptosis and necrosis only on EOC but not non-cancerous cells. We eventually concluded that F1 fraction of T. coronatus crude extract selectively induces apoptosis in EOC through a ROS- mediated pathway.
    Keywords: Turbo coronatus, Epithelial ovarian cancer, Gel filtration chromatography, F1 fraction, Apoptosis
  • Zahra Hadian *, Samira Eslamizad, Hassan Yazdanpanah Pages 275-285
    Pesticide residues in fruits and vegetables are one of the highest concerns of consumers who need food safety. In this study, forty-eight pesticide residues from different chemical structure including organochlorine, organophosphorus, organonitrogen, dicarboximides, strobilurin, triazine, pyrethroids and other chemical groups. in 85 fruits and vegetables were determined and confirmed by GC-MS. The pesticide was extracted with ethyl-acetate, then, the extracts cleaned using high performance gel permeation column chromatography (GPC) and solid phase column (SPE). The mean recoveries of the pesticides were between 81 and 136%. The reproducibility of the relative standard deviation values was 2.1 and 14.8%. Pesticide residues were more frequently found in vegetables (65.5%) than in fruits (26.7%).
    The limits of detection and quantification of pesticide residues for the method were ranged from 0.003 to 0.06 μg g-1 and between 0.01 to 0.1 μg g-1 respectively. The analyzed samples did not contain residues from the monitored pesticides that were higher than the accepted maximum residue limits (MRLS) as adapted by the FAO/WHO Codex Alimentarius Commission.
    Keywords: Gas Chromatography-Mass Spectrometry, Pesticide residues, Fruit, Vegetables, Iran
  • Leila Zare, Hossein Baharvand , Mohammad Javan* Pages 286-295
    The generation of oligodendrocyte progenitor cells (OPCs) offers tremendous opportunities for cell replacement therapy in demyelinating diseases such as multiple sclerosis (MS) and spinal cord injury. Recently, the prospect of reprogramming terminally differentiated adult cells towards another mature somatic cell or progenitor cells without an intermediate pluripotent state has been of interest. Trichostatin A is a histone deacetylase inhibitor which opens the chromatin and facilitates the transcription of silence genes. In this study, we have treated human astrocytes line U87 and primary culture of mouse astrocytes with TSA for 12 hours, prior their transfer to OPC induction medium. Then we evaluated the morphology and the fate of the treated astrocytes at post-treatment days. Both cell lines acquired OPC morphology and expressed OPC specific markers. Following transfer to differentiation medium, U87-derived iOPCs differentiated to oligodendrocyte like cells and expressed PLP as a mature oligodendrocyte marker. Our results introduce TSA as an inducer for production of OPCs from astrocytes and can be considered a potential way for the treatment of demyelinating diseases.
    Keywords: Human astrocytes, Oligodendrocyte progenitors, Trichostatin A, Cell fate conversion, Myelin repair, Multiple sclerosis
  • Mehdi Moslemi, Fereshteh Motamedi, Sareh Asadi, Fariba Khodagholi * Pages 296-307
    Peroxisomes are single membrane cell organelles with a diversity of metabolic functions. Here we studied the peroxisomal dysfunction and oxidative stress after 3-nitropropionic acid (3-NP) induced neurotoxicity and the possible protective effects of oxytocin. Adult male and female rats were subjected to Oxt and/or 3-NP treatment. The antioxidant enzymes, Superoxide dismutase (SOD) and Catalase (CAT) activities as well as expression level of Peroxin 14 (Pex14), a marker for peroxisomal number and Peroxisomal membrane protein of 70 kDa (PMP70), a metabolic transporter in peroxisome in different brain regions of both sexes were studied. The results indicated that 3-NP significantly decreased the expression level of Pex14 and PMP70 in various studied areas in male and female rats. In addition, 3-NP reduced the SOD and CAT activity in different brain regions in both sexes. OXT treatment increased the expression level of peroxisomal proteins Pex14 and PMP70 which are representative of peroxisome performance improvement. Besides, it ameliorated the antioxidant system capability through increasing the activity of the SOD and CAT in all studied brain regions including Striatum, Hippocampus, Prefrontal Cortex and Amygdala with no differences in male and female rats. This study demonstrated that toxin 3-NP, could ultimately cause peroxisomal malfunction and so determines the contribution of peroxisomal dysfunction in the etiology of HD pathology. OXT significantly increased peroxisomal function and antioxidant system defense capability, therefore illustrates that OXT might be an alternate treatment approach for the neurodegenerative diseases like HD.
    Keywords: Oxytocin, Huntington disease, 3-NP, Peroxisome, Oxidative Stress, Pex14
  • Davoud Farajzadeh, Sadigheh Karimi, Gharigh, Parisa Jalali, Kondori, Siavoush Dastmalchi* Pages 308-319
    The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of inflammatory diseases, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized scFv mAb against human TNF-α (hD2). Cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.
    Keywords: TNF-?, Single chain antibody, affinity chromatography, Pull down, MTT assay
  • Mohammad zavarshani, Malahat Ahmadi *, Habib Dastmalchi Saei, Ali Asghar Tehrani, Bahram Dalir Naghadeh Pages 320-327
    Pseudomonas aeruginosa is one of the most common causes of keratitis. The current study was done to evaluate the therapeutic effects of antibacterial combinations with Silver nanoparticles (Ag-NPs) and Ciprofloxacin in experimental Pseudomonas keratitis. Sixty four New Zealand rabbits were prepared. All rabbits were randomly categorized into eight groups (each group containing eight rabbits): Control +, Control −, Ciprofloxacin, Ag-NPs, Ciprofloxacin plus Betamethasone, Ag-NPs plus Betamethasone, Ciprofloxacin plus Ag-NPs and Ciprofloxacin plus Ag-NPs plus Betamethasone. Twelve hours after bacterial inoculation into the cornea, the eyes were examined daily to evaluate the number of days of ocular discharge and blepharospasm. Also, after 108 and 204 h, first grading of corneal opacity was done and then four rabbits of each groups were euthanized for bacterial count. The results showed that the means of days of blepharospasm, ocular discharge and bacterial counts (log CFU ml-1) were significantly different in the treatment groups at 108 and 204 hours (P<0.0005, ANOVA). According to Tukey’s test, Ciprofloxacin plus Ag-NPs plus Betamethasone group was significantly less than Control +, Ag-NPs and Ag-NPs plus Betamethasone groups for current variables (P<0.05). The mean rank of opacity scores were significantly different between treatment groups (P=0.01, Kruskal-Wallis). Mann-Whitney U-test revealed that Ciprofloxacin plus Ag-NPs plus Betamethasone group had significantly better score than Control +, Ag-NPs and Ag-NPs plus Betamethasone groups (P<0.05). It seems Ag-NPs can be an appropriate adjuvant for Ciprofloxacin, but due to the results they can’t be an alternative for Ciprofloxacin to treat Pseudomonas keratitis.
    Keywords: Ciprofloxacin, Keratitis, Pseudomonas, Rabbit, Silver
  • Farzaneh Sadat Naghavi, Parastoo Namvar, Fatemeh Sadeghzadeh, Abbas Haghparast* Pages 328-338
    The activity of dopamine (DA)-containing neurons in the ventral tegmental area (VTA) is a key mechanism in mesolimbic reward processing that has modulatory effects on different diencephalic structures like hippocampus (HIP), and receives inhibitory feedback and excitatory feed forward control. In addition, within the hippocampus, DA receptors are mostly located in the dorsal part (CA1) and dopaminergic innervations are predominant in this sub-region. The current study aimed to examine the effect of intra-hippocampal CA1 administration of SCH23390 and Sulpiride as D1- and D2-like receptor antagonists on the acquisition of orexin-induced conditioned place preference (CPP), respectively. Cannulas were unilaterally implanted into the VTA and HIP of adult male albino Wistar rats weighing 200-250 g. For induction of CPP, orexin A (10 ng/0.3 µL saline) was daily microinjected into the VTA during a three-day conditioning phase. Thereafter, various doses of SCH23390 and Sulpiride (0.25, 1 and 4 µg) were unilaterally injected into the CA1 during this 3-day conditioning phase after intra-VTA administration. The conditioning score was then calculated. Results revealed that intra-CA1 administration of D1- and D2-like receptor antagonists during the 3-day conditioning phase attenuated the acquisition of place preference by orexin A in a dose-dependent manner. It seems the effect of D2-like receptor antagonist within the CA1 region of hippocampus on this phenomenon was found to be more considerable than that of D1-like receptor antagonist. It is concluded that orexin-induced CPP may be mediated, at least in part, by stimulation of DA receptors in the CA1.
    Keywords: Reward, Dopaminergic system, Orexin, Hippocampus, Ventral tegmental area, Rat
  • Laleh Khodaie *, Abbas Delzar, Hossein Nazemiyeh Pages 339-347
    This research paper presents findings of an investigation about antibacterial effect of methanol/water fractions as well as cytotoxic activity of the extracts obtained from Pedicularis wilhelmsiana (Scrophulariaceae) which grows in Azarbaijan/Iran by agar well diffusion method and brine shrimp lethality test successively. Phytochemical study of this plant was determined as well. A combination of solid-phase extraction (SPE), preparative reversed-phase HPLC analysis and spectroscopic means were applied for fractionation, purification, and identification of ingredients respectively. Antimicrobial test demonstrated that 40% and 60% methanol/water fractions were more active than methanolic extract and other SPE fractions. No cytotoxic effect was detected from the extracts of this plant by brine shrimp lethality assay. Phytochemical study of aerial parts of P. wilhelmsiana afforded two phenylethanoids (verbascoside and martynoside), three iridoids (Aucubin, ipolamiid, 5-hydroxy-8-epi-loganin) and two flavonoids (luteolin, luteolin-7-glucoside) along with mannitol on the basis of spectral evidences (UV, 1H and 13CNMR) as well as comparison with literature data. The findings of this research supported further studies related to antibiotic potential of methanolic extract of P. wilhelmsiana.
    Keywords: Pedicularis wilhelmsiana, Scrophulariaceae, Phytochemistry, Antimicrobial
  • Sima Seifabadi, Golnaz Vaseghi, Mustafa Ghannadian, Shaghayegh Haghjooy Javanmard * Pages 348-357
    Melanoma is a challenging disease to treat. Punica granatum L. has a potential anticancer effect. This study determined the antiproliferative and antiangiogenic potential of the extract from pomegranate peel (PPE) in melanoma. Melanoma cells (1 × 106) were injected to C57BL6 mice subcutaneously. On 8th day, mice were randomly divided into 9 groups. Group 1 was considered as control and received distilled water. Groups 2 to 5 received 50, 100, 200 or 400 mg/kg of standardized PPE, orally. Group 6 received 400 mg/kg PPE and PPAR-γ antagonist (T0070907, 5 mg/kg/day). Group 7 received 400 mg/kg PPE and PPAR-α antagonist (GW6471, 10 mg/kg/day). Groups 8 and 9 received PPAR antagonists alone. On the 16th day, mice were euthanized and the tumor samples were analyzed by immunohistochemistry staining for Ki-67 and CD31. Vascular endothelial growth factor (VEGF) plasma level was determined by ELISA. PPE at the doses of 50, 100, 200 and 400 mg/kg decreased tumor weight to 1.28, 1.03, 0.82 and 0.58 g, respectively, in comparison with 1.46 g in control group. Tumor volume reduced to 2.1, 1.7, 1.35 and 0.95 cm3 at the mentioned doses, in comparison with 2.4 cm3 in control group (P < 0.05 for all groups). VEGF, Ki-67 and CD31 were decreased dose dependently in the treatment groups (P < 0.05). PPARα and PPARγ antagonists significantly reduced the extract effects (P < 0.05). It was concluded that PPE may have a potential implication in melanoma treatment through activation of PPARα and PPARγ receptors.
    Keywords: Pomegranate Peel, Tumor Growth, Vascular Endothelial Growth Factor, Tumor Proliferation, Angiogenesis
  • Shideh Montasser Kouhsari *, Ehsan Gharib Pages 358-368
    Pancreatic β-cells dysfunction and impairment of insulin action usually leads to hyperglycemia. Punica granatum L. is a well-known traditional herbal remedy in Iran due to its positive effects on ameliorating blood glucose homeostasis. In this study, we aimed to investigate and compare the pomegranate fruits aqueous extract (PE) on glucose and lipid metabolisms in normal and Alloxan-induced diabetic male Wistar rats. Therefore, normal (N) and diabetic (D) rats were each divided into four groups (control, a, b and c) which were treated orally with PE at doses of 0, 100, 200 and 350 mg/kg (bw), respectively. Oral glucose tolerance test (OGTT) followed by short-term and long-term glycemic controls were performed on the experimental groups. Also, plasma insulin, free fatty acids and triglyceride levels and tissues contents of glycogen and triglyceride were evaluated. By using Real-time PCR, the possibility of modulations of the Insulin receptor substrate 1 (IRS-1), Protein kinase B (Akt), Glucose transporter 2 and 4 (Glut-2, 4) mRNAs expression levels in treated rats were investigated. The obtained data showed noticeable reduction in fasting blood glucose (FBG) by 28.1% and 67.9% in short-term and long-term treatment models, respectively, in the PE+Dc group. Also, there were marked increase in the mRNAs expression levels of IRS-1, Akt, Glut-2 and Glut-4, which results in improvement of glucose uptake and storage. Taking together, it is suggested that PE administration contributed to the modulation of both hyperglycemia and hyperlipidemia in Alloxan-diabetic Wistar rats.
    Keywords: Alloxan monohydrate, Diabetes mellitus, Insulin expression, secretion, Insulin signaling mediators, Glucose uptake, storage, Pomegranate fruits aqueous extract
  • Naeem Erfani Majd *, Hajar Azizian, Mohammad Reza Tabandeh, Ali Shahriari Pages 369-382
    Okra (A.esculentus) is an antidiabetic plant that it's beneficial effects on ovarian dysfunction in diabetes condition has not been clarified. The present study was designed to examine the effect of Okra powder on serum oxidant/antioxidant status, ovarian structure and expression of apoptotic/antiapoptotic related genes in ovary of experimentally induced high fat diet diabetic rats. Diabetes was induced by 5 weeks feeding of Wistar rats with high fat diet (HFD) and subsequent i.p injection of STZ (35mg/kg). Diabetic animals (serum glucose above 250mg/dl) were treated with Okra powder (200mg/kg)supplemented in diet or metformin (200mg/kg) for 30days. Animals were euthanized and insulin resistance markers (insulin and glucose levels and HOMA-IR), ovarian expression of apoptotic/antiapoptotic genes (Bax,caspase3 and Bcl2) and serum oxidant/antioxidant levels (SOD,GPX and CAT activities and MDA level) were determined. The ovaries were also processed for histological study. Hyperglycemia and reduced body weight of diabetic rats were improved after administration of Okra for 30days. This effect was relatively similar to metformin. Okra resulted in reduction of follicular atresia in concomitant with down regulation of apoptotic related genes (Bax and caspase3) in ovary of diabetic rats. Okra could also diminished oxidative stress in diabetic rats by increasing of serum GPX and CAT activities and reducing the lipid peroxidation level. The results of the present study revealed that Okra powder could be useful intervention for improvement of ovaian dysfunction in diabetic rat by three probable mechanisms; attenuation of glucotoxicity, down regulation of ovarian apoptosis related genes and reduction of oxidative stress.
    Keywords: Diabetes, High Fat Diet, Abelmoschus esculentus, Apoptosis, Oxidative Stress, Ovary, Histology
  • Foroogh Namjoyan, Massoumeh Farasat, Mojtaba Alishahi, Alireza Jahangiri, Hamideh Mousavi * Pages 383-390
    Tyrosinase is a key enzyme in melanin production. Therefore, tyrosinase inhibitors are used in cosmetic and medicinal industries to prevent or treat overproduction of melanin such as melasma, solar lentigo and post inflammatory melanoderma. Due to safety of natural whitening agents, in the present study, in-vitro anti-tyrosinase and in-vivo anti-melanogenesis activities of some selected red macroalgae of the Persian Gulf were investigated. The effects of various concentrations (100, 250 and 500 µg/mL) of methanolic extracts of three red macroalgae including Digenea simplex, Laurencia papillosa, and Laurencia paniculata on the activity of diphenolase of mushroom tyrosinase were studied by using L-Dopa as substrate. Subsequently, the activity of macroalgae with high inhibitory effect on hydroxylation of L-tyrosine was investigated by mushroom tyrosinase and zebrafish model. Anti-melanogenesis effects of algae extracts were studied on zebrafish as an alternative in-vivo model. Kojic acid was used as a positive control.
    All the tested macroalgae showed significantly a lower inhibitory effect on activities of diphenolase and monophenolase (of mushroom tyrosinase) compared to kojic acid. D. simplex showed the most anti-tyrosinase activity in zebrafish model among the samples. D. simplex extract and Kojic acid inhibited tyrosinase activity by 43.18% and 50.45%, and decreased total melanin content of zebrafish by 47.27% and 50.21%, respectively.
    Keywords: Algae, Persian Gulf, Melanogenesis, Mushroom tyrosinase, Zebrafish
  • Leila Hosseinzadeh, Yalda Shokoohinia, Mehri Arab, Elnaz Allahyari, Mahdi Mojarrab * Pages 391-399
    Different types of Artemisia aucheri extracts were reported to have various biological activities including a cytotoxic effect on some cancer cell lines. We investigated the antiproliferative activity of isolated sesquiterpenoids from petroleum ether extract of Artemisia aucheri (A. aucheri)aerial parts on SK-N-MC, MCF-7, and A2780 cell lines. Phytochemicals from the petroleum ether cold macerated extract were isolated using normal phase vacuum liquid chromatography and high pressure liquid chromatography (VLC and HPLC) and the structures of the components were determined by spectroscopic means. Cell viability was determined by 3-(4,5- dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Activation of caspases-3 and -9 was evaluated using a spectrophotometer. Mitochondrial membrane potential (MMP) was measured using rhodamine 123 fluorescent dye. Two tetrahydrofuran- type sesquiterpenoids, hydroperoxide of davanone (1) and hydroxydavanone (2) were isolated and characterized. Between these compounds, compound 1 exhibited the most potent activity against the MCF-7, SK-N-MC and A2780 cell lines with IC50 value of 8.45 ± 0.81 µg/mL, 9.60 ± 1.32 µg/mL and 10.9 ± 2.03 µg/mL in A2780, MCF-7 and SK-N-MC cells, respectively. Compound 1 inhibited cell growth of human cancer cells by induction of apoptosis. To the best of our knowledge, this is the first comprehensive study on cytotoxic and apoptotic mechanism of two davanone derivatives isolated from A. aucheri in human cancer cells. Overall, our data suggest that hydroperoxide of davanone (1) should be further studied in-vivo as a potential antitumor agent.
    Keywords: Artemisia aucheri, Sesquiterpenoid, Cytotoxicity, Apoptosis, Davanone derivatives
  • Aynur Sar? *, Zeynep Kececi Pages 400-405
    Plants of the genus Taraxacum Wigg., have long been used as medicinal herbs. A phytochemical investigation of the aerial parts of Taraxacum bessarabicum (Hornem.) Hand.-Mazz. subsp. bessarabicum (Hornem.) Hand.-Mazz. (Asteraceae) yielded two coumarins [esculetin (1), cichoriin (2)], three flavonoids [ luteolin (3), luteolin 7-O-β-D-glucoside (4), gossypetin (5)] and six phenolic acids and their derivatives [ p-coumaric acid (6) , caffeic acid (7), ferulic acid (8), chlorogenic acid methyl ester (9), 3,5-di-O-caffeoylquinic acid (10), 3,5-di-O-caffeoylquinic acid methyl ester (11)]. Their structures were established conclusively by UV, ESI-MS, 1-D and 2-D NMR spectra analyses and comparison with literature data. The presence of these compounds has been shown for the first time from this species. This is the first report of the isolation of compound 5 from the genus Taraxacum.
    Keywords: Taraxacum bessarabicum subsp. bessarabicum, Asteraceae, coumarins, flavonoids, phenolic acids
  • Mahdi Moridi Farimani *, Mansour Miran, Samad Ebrahimi Pages 406-411
    The genus Salvia is a valuable origin of structurally diverse terpenoids. In a project directed at structurally interesting bioactive metabolites from Iranian Salvia species, we studied Salvia reuterana. Two new labdane diterpene, 6β,14α-dihydroxy-15-acetoxysclareol (1), and 14α,15- dihydroxy sclareol (2), were isolated from the aerial part of the plant. Their structures were established mainly by 1D and 2D NMR spectroscopic techniques, including 1H-1H COSY, HSQC and HMBC methods and HR-ESI-TOFMS spectral data. Compound 1 and 2 were tested for their inhibitory activity toward HeLa and MCF-7 cell lines. Our results showed that S. reuterana is a rich source of labdane diterpenoids. These compounds are rather rare in Salvia species, although they are frequently found in other genera of the Lamiaceae. S. reuterana is a new source of these diterpenoids.
    Keywords: Salvia reuterana, labdane diterpene, structure elucidation, NMR, cytotoxicity
  • Mahdieh Eftekhari, Mohammad Reza Shams Ardekani, mohsen Amin, Farideh Attar, Tahmineh Akbarzadeh, Maliheh Safavi, Elahe Karimpour, razkenari, Mohsen Amini, Murray Isman, Mahnaz Khanavi* Pages 412-421
    Oliveria decumbens is an aromatic plant traditionally used for treatment of infections and gastrointestinal diseases. In the present study, the volatile oil of the plant was obtained by hydrodistillation and analyzed by GC-MS. In addition, antibacterial and anti-Helicobacter pylori activities of this essential oil were determined using disc diffusion and agar dilution methods, respectively. Insecticidal activity was assessed through topical and fumigation application of the essential oil to cabbage looper larvae. Acetylcholinesterase (AChE) inhibition by the essential oil was examined using Ellman’s method. Furthermore, its cytotoxic potential against three different cancer cell lines was assessed using the MTT assay. The phenolic monoterpenoids, thymol (38.79%) and carvacrol (36.3%) were identified as major constituents of the essential oil. We observed significant antibacterial activity of the essential oil against H. pylori (MIC=20.4 µg /mL) as well as other tested bacteria, except for Pseudomonas aeruginosa. O. decumbens essential oil showed significant toxicity to cabbage looper larvae with LD50 value of 52.1 µg /larva following topical and fumigant administration. O. decumbens essential oil was considerably inhibitory to acetylcholinesterase activity (IC50=0.117 µg/ml). Cytotoxic assay of the volatile oil resulted in IC50 =0.065, 0.104 and 0.141 μg/mL for MCF-7, T47D and MDA- MB-231 cell lines, respectively. According to our data, this species with high concentrations of thymol and carvacrol could be considered as a natural source for pharmaceutical products.
    Keywords: Oliveria decumbens, Antibacterial, Anti-Helicobacter pylori, insecticide, acetylcholinesterase, cytotoxic
  • Heba Handoussa *, Walid AbdAllah, Mona AbdelMohsen Pages 422-429
    Halocnemum strobilaceum is a halophyte present in the humid and arid bioclimatic regions of Egypt. The current study aimed at UPLC/PDA/ESI-MSn qualitative chemical profiling of the phytoconstituents underlining both antioxidant and cytotoxic activities of the bio-active fraction in comparison with the other fractions. It resulted in detection of several related compounds to prenylated flavonol icariin as a first report in this species. Results showed that ethyl acetate exhibits an appreciable antioxidant activity using in vitro DPPH assay with percentage of 82.35% and remarkable anticancer capacity against most common types of cancer in Egypt; breast (MCF-7), human prostate (PC-3) cancer cell lines, and human lung carcinoma (A-549) with IC50 43.1± 2 µg/ml, 115±5 µg/ml and 53.3±3 µg/ml respectively. These findings point out the appropriate solvent for extraction of the bio-phenolics with this halophyte which is a considerable source of remarkable potential secondary metabolites that exhibit original and interesting anticancer capacity.
    Keywords: Halocnemum strobilaceum, phenolics, UPLC-PDA-ESI-MSn, anticancer, antioxidant
  • Sepideh Salari, Sedigheh Esmaeilzadeh Bahabadi *, Alireza Samzadeh, Kermani, Forough Yousefzaei Pages 430-445
    Nowadays, green synthesis of metal nanoparticles has become a promising synthetic strategy in nanotechnology and materials sciences. In this research, biosynthesis of silver nanoparticles (AgNPs) was successfully accomplished in the presence of Prosopis farcta fruit extract as a reducing agent. Proceeding of the reaction was assessed by using UV–vis spectroscopy. Characterization of silver nanoparticles was carried out by X-ray Diffraction spectroscopy (XRD) and transmission electron microscopy (TEM). The influence of process variables such as temperature, reaction time and extract concentration was also investigated to optimize the biosynthesis of silver nanoparticles. The average size of synthesized AgNPs was 12.68 nm (10.26-14.65 nm). Furthermore, fruit extract and AgNPs were evaluated for total phenolic and flavonoid contents and were subjected to determine their antiradical scavenging activity using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assay and antimicrobial activity against Staphylococcus aureus, Streptococcus pneumonia, Escherichia, Salmonella typhi using the disk diffusion method. The total phenols and flavonoids in AgNPs-containing plant extract were 462.69 (mg GAE/g extract) and 386.94 (mg QE/g extract) respectively, which were significantly higher than fruit extract. Biosynthesized AgNPs showed a higher antioxidant and antibacterial activity compared to P. farcta fruit extract alone. It could be concluded that P. farcta fruit extract can be extensively used in the production of potential antioxidant and antibacterial AgNPs for biomedical application.
    Keywords: antioxidant activity, antibacterial properties, Prosopis farcta, Silver nanoparticles, green nano synthesis
  • Farzad Khademi, Arshid Yousefi, Avarvand, Mohammad Derakhshan, Mohammad Reza Abbaspour, Mohsen Tafaghodi *, Kayvan Sadri Pages 446-458
    Polymeric particles and liposomes are efficient tools to overcome the low immunogenicity of subunit vaccines. The aim of the present study was formulation and optimization of a new cationic lipid-modified PLGA nanoparticles (NPs) as a delivery system for Mycobacterium tuberculosis HspX/EsxS fusion protein. The cationic lipid-modified PLGA NPs containing HspX/EsxS fusion protein were prepared using a modified double emulsion solvent evaporation method. Scanning electron microscopy and dynamic light scattering (DLS) tools were used to determine physical properties of hybrid NPs. A multi-level full factorial design was used to evaluate the influence of two factors of PLGA:DDA ratio (w/w) and PVA concentration (%) on size, surface charge, polydispersity index, encapsulation efficiency and yield. Finally, the optimal formulation was achieved based on desired responses. Mathematical models were obtained to indicate the relation between the studied factors and responses. The DDA concentration showed an increasing effect on surface charge and also a decreasing effect on particle size, encapsulation efficiency and yield. Higher amounts of DDA, increased surface charge of NPs, however, the size, encapsulation efficiency and yield were decreased. The influence of various concentrations of PVA on different physical characteristics of PLGA:DDA hybrid NPs was variable. The optimal formulation was consisted of 0.91 (55:5, w/w) ratio of PLGA:DDA and 0.5% PVA. The hybrid NPs showed acceptable particle size distribution, strong positive surface charge, prolonged antigen release and good encapsulation efficiency in comparison to PLGA alone. However, further preclinical and clinical studies are needed.
    Keywords: Mycobacterium tuberculosis, PLGA:DDA hybrid nanoparticle, HspX-EsxS fusion protein, Experimental design, Optimization
  • Fatemeh Salimi , Javad Hamedi *, Elaheh Motevaseli, Fatemeh Mohammadipanah Pages 459-468
    Thrombotic disorders increase the risk of cardiovascular/cerebrovascular complications and represent a major health problem worldwide. Anticoagulants are extensively used in treatment of these disorders. Vitamin K antagonists, like Warfarin, are frequently used in medication. Vascular calcification (VC) is a significant side-effect of vitamin K antagonists especially Warfarin. There is an urgent need to find natural, efficient, non-toxic and cost effective anticoagulants without dangerous side-effect like VC. In the present study, we evaluated the potential of thirteen fermentation broth extracts of actinobacteria (FBEA) (200 µg ml-1) to prolong whole blood prothrombin time (PT)/international normalized ratio (INR) and activated partial thromboplastin time (APTT). The fractions of the most effective FBEA were further investigated for their inhibitory effect on VC. The results showed PT/INR of the healthy blood samples was sensitive to the presence of five FBEA. Their INR index fell in the 1.2 to 8.6 range and six FBEA prolonged both PT/INR and APTT parameters (1.7-5 INR, and 46-59 s for APTT). The fractions of Kribbella sp. UTMC 267 FBE (200 µg ml-1), as the most efficient FBE, only inhibited intrinsic and common pathways of coagulation (APTT). Under calcification condition, Kribbella sp. UTMC 267 fractions (20 µg ml-1) showed significant inhibitory effect on VC in alizarin red staining (13.3-76 %) and alkaline phosphatase activity of VSMCs (33-62%). They also inhibited osteopontin mRNA expression in treated vascular smooth muscle cells (VSMCs) under that situation. So, we can introduce Kribbella sp. UTMC 267 FBE as a good candidate for more investigation on thrombotic medication.
    Keywords: Anticoagulant, Actinobacterial metabolites, Coagulation, Vascular calcification, Warfarin
  • Razieh Malekian, Ali Jahanian, Najafabadi, Fatemeh Moazen, Reza Ghavimi, Elmira Mohammadi, Vajihe Akbari* Pages 469-478
    A novel strategy to increase protein expression yield is unintended induction of expression in complex media, called auto-induction. This method can be used to express proteins under control of the lac promoter without any need to monitor bacterial growth pattern, and addition of specific expression inducers such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) at proper time. In the present study, a codon optimized gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) was cloned and over-expressed in Escherichia coli BL21 (DE3) using both conventional inducer-based and auto-induction approaches in a shake flask scale and the yield of GM-CSF expression and biomass production was identified. Results showed higher biomass production and expression yield for GM-CSF in case of auto-induction comparing with IPTG-induction. The auto-induction approach was also performed in a fed batch fermentation process in a 2-L bioreactor scale. The feeding strategy yielded an amount of 300 mg/L GM-CSF within 20 h of induction. However, most of the over-expressed GM-CSF was produced as inclusion bodies and following purification and refolding, a final yield of 90 mg/L was achieved. These results suggest that auto-induction approach can be effectively applied in fed-batch fermentation for the large scale production of GM-CSF; however, further optimization of purification process is obligatory to increase the purification yield.
    Keywords: Granulocyte-macrophage colony-stimulating factor, Auto-induction, Inclusion bodies, Escherichia coli, Overexpression
  • Azam Mohamadloo, Saeed Zarein, Dolab, Ali Ramezankhani *, Jamshid Salamzadeh Pages 479-487
    Inappropriate request for health care services which are considered to be unnecessary for the patients has long been addressed by several writers. The hypothesis supplier induced demand refers to the induced demand initiated by the supplier who acts in his own economic self-interest rather than patient best interest. The purpose of the present qualitative study was to explore about induced demand and the relevant motivating factors associated with unnecessary prescriptions of medicine.
    In-depth interviews were used for data generation with a purposive sample of 20 participants who were selected according to their experience. The interviews were transcribed and analyzed. The key themes were identified, named and coded with a sample of quotation. In general, 24 sub-themes or factors were identified and classified into personal, community and institutional themes. Some important factors are asymmetric information, patient expectation, patient poor health literacy, physician's inadequate knowledge in medicine, neglecting patient rights, financial incentives, barriers in health insurance companies, reimbursement mechanism, marketing and advertising by pharmaceutical companies, poor financial condition of pharmacies and social interactions.
    Our results showed that the induced demand for medicine is multifactorial in a health system. Addressing these factors could lead to decrease unnecessary prescription of medicine by a multi-faceted strategy, including curriculum revision, health promotion, and policy making.
    Keywords: patients, physicians, prescriptions, qualitative research, health services, Medicine
  • Mohammad, Mehdi Kooshyar, Anoosheh Marouzi, Azar Fani, Pakdel, Sepideh Elyasi *, Ali Taghizadeh, Kermani, Mahdi Akbarzadeh, Seyyed Amir Aledavood Pages 488-495
    Using standardized forms for prescription and administration of medications is one of the main solutions for reducing medication errors in the chemotherapy process. Considering the high prevalence and mortality rate of colorectal cancer, in this study we tried to design and validate a standard printed form and evaluate oncologists’ and nurses’ adherence to this form. This cross-sectional study was performed in Omid hospital, Mashhad, Iran from January 2015 to October 2015. A Chemotherapy form including various demographic and clinical parameters and approved chemotherapy regimens for colorectal cancer was designed by the clinical pharmacist and validated by clinical oncologists working in this center. All eligible patients admitted in this center during this period of time were included in the study. Adherence of the oncologists and nurses to this form and probable medication errors were identified by the pharmacy student.
    Sixty-seven patients with colorectal cancer and a total of 251 chemotherapy courses were evaluated. All patients received regimens compatible with developed form but in 206 courses (98.56%) of chemotherapy dosing error happened and in most of cases patients received lower than calculated dose (37.8%). Three errors occurred in administration step by nurses which they infused the medication in shorter than recommended duration. In general, oncologists’ adherence with developed form for chemotherapy of colorectal cancer was relatively high, except in dose calculation. Avoiding from rounding the calculated medications’ doses and precise calculation of patients’ body surface area can prevent most of medication errors and reduce risk of adverse drug reaction occurrence.
    Keywords: Colorectal cancer, Chemotherapy, Medication error, Standard form, Teaching hospital
  • Farzad Shidfar , Seyedeh Neda Mousavi *, Hamid lorvand Amiri, Shahram Agah, Sharieh Hoseini, Seyed Javad Hajimiresmail Pages 496-505
    The role of non-alcoholic fatty liver disease (NAFLD) as a potential independent cardiovascular disease (CVD) risk factor has recently gained considerable attention because CVD is the common cause of death in NAFLD patients. We aimed to estimate the effects of vitamin D supplementation alone or in combination with calcium on atherogenic indices, liver function tests and grade of disease in patients with NAFLD. One-hundred twenty NAFLD patients were randomized in a double-blind, placebo-controlled clinical trial as follows: D (1000 IU vitamin D), CaD (500 mg as calcium carbonate plus 1000 IU vitamin D) or P (placebo), once daily with meals over 12 weeks. Adjusted for all the baseline measures, reduction in serum ALT, AST, LDL-C/HDL-C, TC/HDL-C and non-HDL-C were significantly higher in the CaD compared with the P group (p
    Keywords: Calcium, Vitamin D, atherogenic indices, non-alcoholic fatty liver, supplementation
  • Hossein Esmaeilzadeh , Shirin Farjadian *, Soheila Alyasin, Hamid Nemati, Hesamodin Nabavizadeh , Elmira Esmailzadeh Pages 506-522
    Severe cutaneous adverse drug reaction (SCAR) is considered to be a multifactorial drug side effect. This study was designed to investigate the epidemiology and human leukocyte antigen (HLA)-A and -B gene polymorphisms in pediatric patients with SCAR admitted in tertiary referral center, southwestern of Iran from 2013 to 2017. Demographic data, past allergy and autoimmune history, clinical presentations, drugs confirmed to be the cause of SCAR as well as its therapy were reviewed for each patient. HLA-A and -B allele frequencies were determined in 40 of the patients using polymerase chain reaction based on sequence specific primers (PCR-SSP) and compared with 40 healthy individuals as control group.Sixty-one patients with mean age of 6 years old and boy to girl ratio was 1.2/1 in this study. The most common type of SCAR in our patients was Steven Johnson Syndrome (SJS)/Toxic Epidermal Necrosis (TEN) mainly caused by beta-lactam antibiotics. Carbamazepine was the second cause of drug–induced SCAR. Moreover, HLA-A*02:01 and A*51:01 were related to the increased risk of SCAR while A*11:01 seemed to be protective against SCAR. HLA-A*02:01, HLA-A*24:02, and HLA-B*51:01 showed associations to the increased risk of SJS. Based on our results, beta-lactam antibiotics and antiepileptic drugs are the most common causes of severe adverse drug reaction in southwestern Iranian pediatric patients. Moreover, some HLA-A alleles can influence risk of SCAR.
  • Safeer Khan *, Wajahat Mahmood Pages 523-530
    The role of Pharmacist in making the therapeutic decisions for safe and effective therapy is increasing all over the world. However, there are many aspects of drugs in making these decisions that are less commonly studied such as the correlation of cardiac output with pharmacokinetics of drugs. The cardiac output, besides the other factors, is also affected by drugs like atenolol. Therefore, the objective of the present open labeled study was to know the effect of reduced cardiac output induced by atenolol on its own excretion parameters. After taking the informed consent, five healthy volunteers were selected for the study. Atenolol tablet at a dose of 50 mg, 75 mg and 100 mg for three consecutive days were given to all the volunteers. The echocardiography and renal function clinical tests were conducted prior and 5 h after dosing and the urine samples were collected at 5 and 10 h post dosing. The prepared samples were analyzed for atenolol by High-Performance Liquid chromatography. For comparison of atenolol excretion for three days, One-way repeated measure Analysis of Variance statistical test was used as Wilks’ Lambda = 0.2, F (2, 3) = 5.986, p < 0.1, multivariate partial squared = 0.8. These results showed that atenolol affects its own pharmacokinetics by prolonging its excretion half-life.
    Keywords: Cardiac Output, Atenolol, Urine Analysis, Excretion, Half-life
  • Seyyed Abolfazl Abolfazli , Mehdi Mohammadzadeh *, Farzad Peiravian, Jafar Zonoozi, Hesam Sharifnia, Ali Vashegani Pages 531-545
    This study investigated the relationship between main dimensions in the strategy-making process, environmental complexity, and performance in pharmaceutical companies of Iran. Results argue that pharmaceutical companies like other industries place differing emphasis on strategy-making and employ different modes of strategy-making. It offers a typology of different modes of strategy-making that most likely exist in pharmaceutical companies and hypothesizes how this typology relates to performance. It then describes the results of an empirical study of the strategy-making processes of pharmaceutical companies. The structural equation analysis of the data from 31 pharmaceutical companies indicates that there are simplistic, adaptive, entrepreneurial, participative, and SPACE (Strategy making Process According to Complexity Environment) modes of strategy-making in pharmaceutical companies; of these modes, SPACE mode exhibited a significant relationship with performance.
    Keywords: Strategy-making, Partial least square structural equation modeling, Smart PLS, pharmaceutical companies, SPACE mode
  • Hosein Shabaninejad, Hasan Yusefzadeh, GholamHossein Mehralian, Bahlol Rahimi * Pages 546-555
    Iran`s pharmaceutical products market as one of the major markets of the country has been faced with fluctuations over time. Suitable market selection is necessary for stability of pharmaceutical exports. Study of the global market structure of this product leads to policy makers in development, production, and trade of pharmaceuticals and finally increases country’s share of the world pharmaceutical market. Moreover, This study aimed to determine the structure of the world pharmaceutical market as well as the pharmaceutical export of Iran to its trade partners and to identify its target markets. Concentration ratios and Herfindahl index were used to determine the structure of the world market and pharmaceutical exports of Iran in the years 2001-2012. Also, a composite index was used to identify the target markets of Iran’s pharmaceutical industry.
    The results showed that the export side of world pharmaceutical trade has shifted to open oligopoly, thereby decreasing the monopolistic power of exporters. The import side, however, follows monopolistic competition. It has been observed that the structure of Iran's pharmaceutical export is shifting to open oligopoly; though, pharmaceutical importers from Iran have not been stable. Moreover, 27 countries were identified as target markets. Due to significant differences between the current and potential export destinations of Iran’s pharmaceutical products, exporters must choose suitable strategies in order to diversify export markets. Such mechanisms as setting preferential tariffs on the basis of bilateral agreements, effective advertisment, and paying attention to global consumers’preferences can be used to develop Iran’s pharmaceutical export to target countries.
    Keywords: Pharmaceuticals, Concentration ratio, World market structure, Herfindahl index, Import, export, Target markets