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   Table of Contents - Current issue
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March-April 2021
Volume 16 | Issue 2
Page Nos. 118-226

Online since Friday, March 5, 2021

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ORIGINAL ARTICLES  

Using carboxymethyl gum from Tamarindus indica and Cassia fistula seeds with Chromolaena odorata leaf extract to develop antibacterial gauze dressing with hemostatic activity p. 118
Wancheng Sittikijyothin, Bussaba Phonyotin, Tanikan Sangnim, Kampanart Huanbutta
DOI:10.4103/1735-5362.310519  
Background and purpose: Gauze dressing is a barrier against microbial infection in wounds. The seed gums of Tamarindus indica and Cassia fistula are abundant in tropical countries; we used them as a coating material of cotton gauzes to improve the liquid absorption ability. Moreover, Chromolaena odorata leaf extract was loaded on the gums for antibacterial gauze dressing with hemostatic activity. Experimental approach: Crude gums were extracted from T. indica and C. fistula seeds and carboxymethyl gums were then derived and chracterized. C. Odorata ethanolic extract was also prepared by maceration and its antimicrobial and blood clotting activities were determine coated gauze dressing containing different concentrations of carboxymethyl gum was prepared in the presence of various concentrations of C. odorata extract. Then, the physical properties, antibacterial activity, and skin-irritating effects of the coated gauze were analyzed. Findings/Results: The results indicated that the amount of carboxymethyl gum affected the physical properties and absorption capacity of the coated gauze. C. odorata extract exhibited better bactericidal activity against Staphylococcus aureus than against Escherichia coli. The blood clotting effects of C. odorata extract indicated that it had dramatic hemostatic efficacy. The coated gauze exhibited bactericidal activity against S. aureus. In the human skin irritation test, the coated gauze caused no adverse effects on human skin. Conclusion and implication: Coated gauze has the potential to serve as a prototype for primary hemostasis in first aid for opened wounds such as abrasions and lacerations.
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The effects of Benjakul extract and its isolated compounds on cell cycle arrest and apoptosis in human non-small cell lung cancer cell line NCI-H226 p. 129
Arunporn Itharat, Ruchilak Rattarom, Pintusorn Hansakul, Intouch Sakpakdeejaroen, Buncha Ooraikul, Neal M Davies
DOI:10.4103/1735-5362.310520  
Background and purpose: Benjakul, a traditional Thai formulation for cancer treatment, is composed of five plants. This study aimed to assess the cytotoxicity of Benjakul, its five plants, and its isolated compounds against non-small cell lung cancer (NSCLC) by the sulforhodamine B (SRB) assay. Experimental approach: Analyses of cell cycle and membrane asymmetry changes were performed with different fluorescent dyes and analyzed by flow cytometry in NCI-H226 cells. Activation of caspase-3 was measured using a caspase-3 colorimetric assay kit. The pan-caspase inhibitor Z-VAD-FMK was used in analyses of cell cycle and caspase-3 activity. Findings/Results: Benjakul exhibited cytotoxicity against NSCLC with IC50 between 5.56-5.64 μg/mL. Among its five ingredients, Benjakul displayed the highest selectivity with selectivity index values ranging from 2.93 to 6.88, with the exception of Plumbago indica, indicating its protective effects. Plumbagin and 6- shogaol displayed the highest cytotoxicity and underwent molecular studies in NCI-H226 cells. Flow cytometry analysis revealed that Benjakul and 6-shogaol dose-dependently induced G2/M phase arrest, and plumbagin dose-dependently induced S-G2/M phase arrest with the highest percentage in early incubation time (12-24 h). At the highest doses, Benjakul extract, 6-shogaol, and plumbagin time-dependently increased the population of sub-G1 apoptotic cells with the highest percentage in longer incubation time (60-72 h). Similarly, membrane asymmetry changes showed time-dependent increases in the percentage of early and late apoptotic cells. Moreover, the apoptosis-inducing effect of Benjakul, 6-shogaol, and plumbagin at the highest dose, via the caspase cascade was confirmed by time-dependent induction of caspase-3 activity, followed by its complete reduction and abolished sub-G1 peaks upon addition of Z-VAD-FMK. Conclusion and implication: Our findings demonstrated for the first time the effects of Benjakul and its compounds on S-G2/M or G2/M phase arrest and caspase-dependent apoptosis in lung cancer cells.
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Evaluation of the effect of hydroalcoholic and flavonoid-enriched extracts of Dracocephalum kotschyi on indomethacin-induced gastric ulcer in rats p. 141
Mohsen Minaiyan, Hassan Sadraei, Iman Yousefi, Sayed-Ebrahim Sajjadi, Ardeshir Talebi
DOI:10.4103/1735-5362.310521  
Background and purpose: Dracocephalum kotschyi (Zaringiah) is a fragrant wild medicinal plant found in Iran. Traditionally, it is used for the treatment of rheumatism, asthma, and gastrointestinal ailments. So far no investigation has been done on the beneficial or side effects of D. kotschyi on peptic ulcer. Therefore, this research was performed to find out whether D. kotschyi extract would induce peptic ulcer or could alleviate existing peptic ulcer. Experimental approach: Effect of hydroalcoholic (DKHE) and flavonoid extracts (DKFE) of D. kotschyi were determined in normal or indomethacin-induced gastric ulcer rats (n = 6) and compared with the vehicle and ranitidine treated controls. All the treatments were carried out orally and 24 h later the stomach mucus was visually examined for peptic ulcers. A section of the stomach was taken for microscopic histopathological examinations while another section of the stomach was used for measurement of myeloperoxidase (MPO) and malondialdehyde (MDA) activities. Findings/Results: Oral administration of the DKHE and DKFE alone, did not cause any sign of gastric ulcer induction. The D. kotschyi extracts not only didn’t aggravate the induced ulcer but also significantly prevented the severity of gastric ulcer induction by indomethacin. In addition, DKHE and DKFE inhibited MPO (up to 58.2%) and MDA (up to 44.2%) activities indicating their anti-inflammatory and antioxidant potential action on the stomach-induced ulcer. Conclusion and implication: Usage of D. kotschyi extracts is not associated with gastric ulcer induction and its co-administration with NSAIDs would be beneficial for controlling both the inflammation and preventing gastric ulcer in diseases such as rheumatism.
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Statistical optimization of culture conditions for expression of recombinant humanized anti-EpCAM single-chain antibody using response surface methodology p. 153
Aidin Behravan, Atieh Hashemi
DOI:10.4103/1735-5362.310522  
Background and purpose: The epithelial cell adhesion molecule (EpCAM), is one of the first cancer- associated markers discovered. Its overexpression in cancer stem cells, epithelial tumors, and circulating tumor cells makes this molecule interesting for targeted cancer therapy. So, in recent years scFv fragments have been developed for EpCAM targeting. Experimental approach: In this study, an scFv against EpCAM extracellular domain (EpEX) derived from 4D5MOC-B humanized mAb was expressed in Escherichia coli k12 strain, and in order to obtain the optimum culture conditions in chemically defined minimal medium, response surface methodology (RSM) was employed. According to the RSM-CCD method, a total of 30 experiments were designed to investigate the effects of various parameters including isopropyl-b-D-thiogalactopyranoside (IPTG) concentration, cell density before induction, post-induction time, and post-induction temperature on anti EpEX-scFv expression level. Findings/Results: At the optimum conditions (induction at cell density 0.8 with 0.8 mM IPTG for 24 h at 37 °C), the recombinant anti EpEX-scFv was produced at a titer of 197.33 μg/mL that was significantly consistent with the prediction of the model. Conclusion and implication: The optimized-culture conditions obtained here for efficient production of anti EpEX-scFv in shake flask cultivation on a chemically defined minimal medium could be applied to large- scale fermentation for the anti EpEX-scFv production.
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Rapamycin attenuates gene expression of programmed cell death protein-ligand 1 and Foxp3 in the brain; a novel mechanism proposed for immunotherapy in the brain p. 165
Mostafa Khedri, Hamid Kooshki, Ramezan Ali Taheri
DOI:10.4103/1735-5362.310523  
Background and purpose: Programmed cell death protein-1 (PD1) expresses on the cell surface of the activated lymphocytes and at least a subset of Foxp3+ regulatory T cells. The binding of PD1 to its ligands including PD-L1 and PD-L2 leads to deliver an inhibitory signal to the activated cells. Although PD1/PD-L signal deficiency can lead to failure in the self-tolerance and development of autoimmunity disorders, PD1 blockade with monoclonal antibodies is considered an effective strategy in cancer immunotherapy. Determining effective environmental factors such as stress conditions on the expression of PD1 and PD-L1 genes can provide an immunotherapeutic strategy to control PD1 signaling in the patients Mammalian target of rapamycin signaling is a stress-responsive pathway in the cells that can be blocked by rapamycin. In this study, the effects of rapamycin on the expression of immunoregulatory genes were investigated in the stress condition. Experimental approach: Daily administration of rapamycin (1.5 mg/kg per day) was used in the mouse model of restraint stress and the relative expression of PD1, PD-L1, and Foxp3 genes in the brain and spleen were evaluated using quantitative real-time polymerase chain reaction method. Findings/Results: With our observation, daily restraint stress ceased rapamycin to decrease the expression of Foxp3 in the brain significantly. These findings would be beneficial in developing tolerance to autoimmune diseases and finding immunopathology of stress in the CNS. In another observation, daily administration of rapamycin decreased the expression of PD-L1 in the brain cells of mice. In the spleen samples, significant alteration in genes of interest expression was not detected for all groups of the study. Conclusion and implications: Downregulation of the PD-L1 gene in the brain induced by rapamycin can be followed in future experiences for preventing immunosuppressive effects of PD/PD-L1 signal in the brain.
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Mutual assistance of nucleus accumbens cannabinoid receptor-1 and orexin receptor-2 in response to nicotine: a single-unit study p. 173
Reza Fartootzadeh, Hojjatallah Alaei, Parham Reisi
DOI:10.4103/1735-5362.310524  
Background and purpose: The nucleus accumbens (NAc) express both orexin-2 receptor (OX2R) and cannabinoid receptor type 1 (CB1R). Orexin and cannabinoid regulate the addictive properties of nicotine. In this study, the effect of the CB1R blockade on the electrical activity of NAc neurons in response to nicotine, and its probable interaction with the OX2R in this event, within this area, were examined via the single-unit recording. Experimental approach: The spontaneous firing rate of NAc was initially recorded for 15 min, and then 5 min before subcutaneous injection of nicotine (0.5 mg/kg)/saline, AM251 and TCS-OX2-29 were injected into the NAc. Neuronal responses were recorded for 70 min, after nicotine administration. Findings/Results: Nicotine excited the NAc neurons significantly and intra-NAc microinjection of AM251 (25 and 125 ng/rat), as a selective CB1R antagonist, prevented the nicotine-induced increases of NAc neuronal responses. Moreover, microinjection of AM251 (125 ng/rat), before saline injection, could not affect the percentage of change of the neuronal response. Finally, simultaneous intra-NAc administration of the effective or ineffective doses of AM251 and TCS-OX2-29 (a selective antagonist of OX2R) prevented the nicotine- induced increases of NAc neuronal responses, so that there was a significant difference between the group received ineffective doses of both antagonists and the AM251 ineffective dose. Conclusion and implications: The results suggest that the CB1R can modulate the NAc reaction to the nicotine, and it can be concluded that there is a potential interplay between the OX2R and CB1R in the NAc, in relation to nicotine.
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Expression of cyclin D1, p21, and estrogen receptor alpha in aflatoxin G1-induced disturbance in testicular tissue of albino mice p. 182
Toraj Zamir-Nasta, Mona Pazhouhi, Ali Ghanbari, Amir Abdolmaleki, Cyrus Jalili
DOI:10.4103/1735-5362.310525  
Background and purpose: Aflatoxin (AF) is a mycotoxin produced by various strains of the Aspergillus family. AFG1 as one of the most important types is highly found in cereals and grains. AF affects sperm production or even its quality. This study was designed to test the effects of AFG1 on mice testicular tissue. Experimental approach: Twenty-four Albino mice were divided into four groups of 6 each; a control group (0.2 mL corn oil and ethanol), three treatment groups with different periods (20 μg/kg AFG1 for 7, 15, and 35 consecutive days). All treatments were applied intraperitoneally. Biosynthesis of cyclin D1, p21, and estrogen receptor alpha (ERα) proteins was evaluated by immunohistochemistry (IHC) staining. Levels of cyclin D1, p21, and ERa mRNA were evaluated by the real-time polymerase chain reaction (RT-PCR) technique. Tubular differentiation index (TDI), reproductive index (RI), and spermiogenesis indices were also analyzed. Findings/Results: AFG1 increased the percentage of seminiferous tubules with negative TDI, RI, and SPI compared to the control group (P < 0.05). RT-PCR and IHC analyses illustrated time-dependent enhancement in p21 expression and cyclin D1 biosynthesis in AFG1-treated groups significantly (P < 0.05). While the protein and mRNA levels of ERa were significantly (P < 0.05) decreased in a time-dependent manner. Conclusion and implications: The chronic exposure to AFG1 reduced the expression and synthesis of ERa, increased the expression and synthesis of p21 and cyclin D1, impaired apoptosis, which in turn could impair spermatogenesis.
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Hypoglycemic, hepatoprotective, and hypolipidemic effects of hydroalcoholic extract of Eryngium billardieri root on nicotinamide/streptozotocin-induced type II diabetic rats p. 193
Samira Khani, Maasoume Abdollahi, Zeynab Asadi, Mohamad Nazeri, Mohammad Amin Nasiri, Hossein Yusefi, Abbas Moghadam, Hamid Heidari
DOI:10.4103/1735-5362.310526  
Background and purpose: Erynginum billardieri has been used to control diabetes in traditional medicine. This research was performed to study the antidiabetic, hepatoprotective, and hypolipidemic effects of E. billardieri root extract (EBRE) on streptozotocin/nicotinamide-induced type 2 diabetic male rats. Experimental approach: Type two diabetic animals were treated by three different doses of EBRE (100, 200, and 400 mg/kg), orally administered for 4 weeks. Ultimately, after anesthesia, the glucose, insulin, lipid profiles, hepatic enzyme levels in the blood and liver, and pancreas tissues of the animals were analyzed. Findings/Results: Induction of diabetes caused a diminution in insulin level, high-density lipoprotein (HDL), and significantly enhanced the level of other lipid profiles, glucose, and liver enzymes (P < 0.05). Administration of the EBRE to diabetic-male rats significantly reduced glucose level, lipid profiles, and liver enzymes, and increased the level of HDL to near normal. Conclusion and implications: The results of the present study showed that E. billardieri had a positive effect on diminishing the lipid profiles, liver enzymes, and controlling diabetes. The most effective dose was found to be 100 mg/kg.
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Effects of intrathecal and intracerebroventricular microinjection of kaempferol on pain: possible mechanisms of action p. 203
Sajjad Jabbari, Maryam Bananej, Mohammad Zarei, Alireza Komaki, Ramin Hajikhani
DOI:10.4103/1735-5362.310527  
Background and purpose: Kaempferol (KM), a flavonoid, has an anti-inflammatory and anticancer effect and prevents many metabolic diseases. Nonetheless, very few studies have been done on the antinociceptive effects of KM. This research aimed at assessing the involvement of opioids, gamma-aminobutyric acid (GABA) receptors, and inflammatory mediators in the antinociceptive effects of KM in male Wistar rats. Experimental approach: The intracerebroventricular and/or intrathecal administration of the compounds was done for examining their central impacts on the thermal and chemical pain by the tail-flick and formalin paw tests. For assessing the role of opioid and GABA receptors in the possible antinociceptive effects of KM, several antagonists were used. Also, a rotarod test was carried out for assessing motor performance. Findings/Results: The intracerebroventricular and/or intrathecal microinjections of KM (40 μg/rat) had partially antinociceptive effects in the tail-flick test in rats (P < 0.05). In the formalin paw model, the intrathecal microinjection of KM had antinociceptive effects in phase 1 (20 and 40 μg/rat; P < 0.05 and P < 0.01, respectively) and phase 2 (20 and 40 μg/rat; P < 0.01 and P < 0.001, respectively). Using naloxonazine and/or bicuculline approved the involvement of opioid and GABA receptors in the central antinociceptive effects of KM, respectively. Moreover, KM reduced the expression levels of caspase 6, interleukin-1β, tumor necrosis factor-α, and interleukin-6. The antinociceptive effects of KM were not linked to variations in the locomotor activity. Conclusion and implications: It can be concluded that KM has remarkable antinociceptive effects at a spinal level, which is associated with the presence of the inflammatory state. These impacts were undetectable following injections in the lateral ventricle. The possible mechanisms of KM antinociception are possibly linked to various modulatory pathways, including opioid and GABA receptors.
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Suppression of hypoxia and inflammatory pathways by Phyllanthus niruri extract inhibits angiogenesis in DMBA-induced breast cancer mice p. 217
Abu Hanifah Ramadhani, Ahmad Hafidul Ahkam, Aditya Ragil Suharto, Yoga Dwi Jatmiko, Hideo Tsuboi, Muhaimin Rifa'i
DOI:10.4103/1735-5362.310528  
Background and purpose: Angiogenesis has been one of the hallmarks of cancer. In recent years, Phyllanthus niruri extract (PNE) was reported to inhibit angiogenesis by decreasing the levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1a (HIF-1a) in breast cancer. However, the experimental results were confirmed in cancer cell lines only, whereas the anti-angiogenic activity in animal models has not been demonstrated. In this study, we tried to examine the anti-angiogenic activity of PNE on BALB/c strain mice models that were induced for breast cancer using the carcinogenic substance 7,12- dimethylbenz[a]anthracene (DMBA). Experimental approach: Experimental animals were divided into five different groups; vehicle, DMBA, PNE 500 mg/kg, PNE 1000 mg/kg; and PNE 2000 mg/kg. Mammary carcinogenesis was induced using a subcutaneous injection of 15 mg/kg of DMBA for 12 weeks. Afterward, oral PNE treatment was given for the following 5 weeks. VEGFA and HIF-1a were observed using immunohistochemistry. Endothelial cell markers CD31, CD146, and CD34 were observed using the fluorescent immunohistochemistry method. The levels of interleukin-6 (IL-6), IL-17, and C-X-C motif chemokine (CXCL12) were measured using flow cytometry. Findings/Results: The survival analysis indicated that PNE increased the survival rate of mice (P = 0.043, log-rank test) at all doses. The PNE treatment decreased the immunoreactive score of angiogenic factors (VEGF and HIF-1a), as well as the endothelial cell markers (CD31, CD146, and CD34). The PNE- treated groups also decreased the levels of inflammatory cytokines (IL-6, IL-17, and CXCL12) at all doses. Conclusion and implications: This finding suggests that PNE may inhibit the progression of angiogenesis in breast cancer mice by targeting the hypoxia and inflammatory pathways.
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