Search results for: In vitro cytotoxicity

Authors: Maryam Nejat Dehkordi, Per Lincoln, Hassan Momtaz

Abstract:

Interaction of Schiff base complexes of iron and manganese (iron [N, N’ Bis (5-(triphenyl phosphonium methyl) salicylidene) -1, 2 ethanediamine) chloride, [Fe Salen]Cl, manganese [N, N’ Bis (5-(triphenyl phosphonium methyl) salicylidene) -1, 2 ethanediamine) acetate) with DNA were investigated by spectroscopic and isothermal titration calorimetry techniques (ITC). The absorbance spectra of complexes have shown hyper and hypochromism in the presence of DNA that is indication of interaction of complexes with DNA. The linear dichroism (LD) measurements confirmed the bending of DNA in the presence of complexes. Furthermore, isothermal titration calorimetry experiments approved that complexes bound to DNA on the base of both electrostatic and hydrophobic interactions. Furthermore, ITC profile exhibits the existence of two binding phases for the complex. Antibacterial activity of ligand and complexes were tested in vitro to evaluate their activity against the gram positive and negative bacteria.

Keywords: Schiff base complexes, ct-DNA, linear dichroism (LD), isothermal titration calorimetry (ITC), antibacterial activity

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Authors: Serier Bouchenak NORA, Bouguerni ABDELMADJID

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Algeria is a Mediterranean country which provides an ideal habitat for a wide range of species of medicinal plants. The objective of this current work is to extract the gel contained in the leaves of Aloe vera in order to formulate an oral gel as a prebiotic and see its effects on probiotics (lactic and pseudo lactic bacteria and bifido bacterium). Aloe vera polysaccharid extract is a matrix mainly composed of non-digestible oligosaccharids or slow-fermentation polysaccharids, as this produces a lower pH. The behavior of Aloe vera during in vitro fermentation of the colon was similar to that of lactulose, indicating the possibility of using Aloe vera and its polysaccharids extracts as a prebiotic. The microbiological control of the two kinds of bacteria (bifidobacteria and staphylococci) has demonstrated the gel capacity to stimulate them by these bioactive compounds. The generation time of Bifidobacteria in fermented milk with added prebiotic Aloe vera gel is 80.408 min with a µ growth rate equal to 0.012 min -1. The doubling time is 61.459 min with a growth rate µ equal to 0.016 min -1 for the Streptococcus sp. species.

Keywords: aloe vera, probiotics, prebiotics, growth rate, bifidobacteria

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Authors: Sandeep Kumar Agrawal, Aditi Bhattacharya, Janvie Manhas, Krushna Bhatt, Yatin Kholakiya, Nupur Khera, Ajoy Roychoudhury, Sudip Sen

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Autologous cultured explants of human oral mucosal epithelial cells (OMEC) are a potential therapeutic modality for limbal stem cell deficiency (LSCD). Injury or inflammation of the ocular surface in the form of burns, chemicals, Stevens Johnson syndrome, ocular cicatricial pemphigoid etc. can lead to destruction and deficiency of limbal stem cells. LSCD manifests in the form of severe ocular surface diseases (OSD) characterized by persistent and recurrent epithelial defects, conjuntivalisation and neovascularisation of the corneal surface, scarring and ultimately opacity and blindness. Most of the cases of OSD are associated with severe dry eye pertaining to diminished mucin and aqueous secretion. Mycophenolate mofetil (MMF) has been shown to upregulate the mucin expression in conjunctival goblet cells in vitro. The aim of this study was to evaluate the effects of MMF on mucin expression in primary cultures of oral mucosal epithelial cells. With institutional ethics committee approval and written informed consent, thirty oral mucosal epithelial tissue samples were obtained from patients undergoing oral surgery for non-malignant conditions. OMEC were grown on human amniotic membrane (HAM, obtained from expecting mothers undergoing elective caesarean section) scaffold for 2 weeks in growth media containing DMEM & Ham’s F12 (1:1) with 10% FBS and growth factors. In vitro dosage of MMF was standardised by MTT assay. Analysis of stem cell markers was done using RT-PCR while mucin mRNA expression was quantified using RT-PCR and q-PCR before and after treating cultured OMEC with graded concentrations of MMF for 24 hours. Protein expression was validated using immunocytochemistry. Morphological studies revealed a confluent sheet of proliferating, stratified oral mucosal epithelial cells growing over the surface of HAM scaffold. The presence of progenitor stem cell markers (p63, p75, β1-Integrin and ABCG2) and cell surface associated mucins (MUC1, MUC15 and MUC16) were elucidated by RT-PCR. The mucin mRNA expression was found to be upregulated in MMF treated primary cultures of OMEC, compared to untreated controls as quantified by q-PCR with β-actin as internal reference gene. Increased MUC1 protein expression was validated by immunocytochemistry on representative samples. Our findings conclude that OMEC have the ability to form a multi-layered confluent sheet on the surface of HAM similar to a cornea, which is important for the reconstruction of the damaged ocular surface. Cultured OMEC has stem cell properties as demonstrated by stem cell markers. MMF can be a novel enhancer of mucin production in OMEC. It has the potential to improve dry eye in patients undergoing OMEC transplantation for bilateral OSD. Further clinical trials are required to establish the role of MMF in patients undergoing OMEC transplantation.

Keywords: limbal stem cell deficiency, mycophenolate mofetil, mucin, ocular surface disease

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Authors: V. Hakimzadeh, M. Noori, M. maleki

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The aim of this study was to determine the composition and antimicrobial effect of Mentha piperita essential oil in "in-vitro" condition. The chemical composition of the essential oil obtained by hydro-distillation was examined by GC/MS and the antimicrobial effect was studied on the growth of seven microbial species including Bacillus cereus, Pseudomonas aeruginosa and Proteus vulgaris using micro-dilution method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Chemical composition analysis identified a total of 28 compounds in which the main components were menthol (32%), mentone (13.4), menthyl acetate (12%), 1,8-cineole (8.2%) and neomenthol (4%) representing 69.6 % of the total oil. Other separated components accounted for less than 30.4% of the oil. Results of antimicrobial analysis showed that the MIC values for Bacillus cereus, Pseudomonas aeruginosa and Proteus vulgaris was respectively 50, 200 and 100 µg/ml and the MBC was determined at 200, 400 and 200 µg/ml respectively. The results of the present study indicated that Mentha piperita essential oil had significant antimicrobial activity.

Keywords: antimicrobial activity, essential oil composition, Mentha piperita

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Authors: Ernest Moles, Patricia Urbán, María Belén Jiménez-Díaz, Sara Viera-Morilla, Iñigo Angulo-Barturen, Maria Antònia Busquets, Xavier Fernàndez-Busquets

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Bearing in mind the absence of an effective vaccine against malaria and its severe clinical manifestations causing nearly half a million deaths every year, this disease represents nowadays a major threat to life. Besides, the basic rationale followed by currently marketed antimalarial approaches is based on the administration of drugs on their own, promoting the emergence of drug-resistant parasites owing to the limitation in delivering drug payloads into the parasitized erythrocyte high enough to kill the intracellular pathogen while minimizing the risk of causing toxic side effects to the patient. Such dichotomy has been successfully addressed through the specific delivery of immunoliposome (iLP)-encapsulated antimalarials to Plasmodium falciparum-infected red blood cells (pRBCs). Unfortunately, this strategy has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here, we show that encapsulation efficiencies reaching >96% can be achieved for the weakly basic drugs chloroquine (CQ) and primaquine using the pH gradient active loading method in liposomes composed of neutrally charged, saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the intracellular delivery of drugs not affecting the erythrocytic metabolism. Using this strategy, we have obtained unprecedented nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Polyethylene glycol-coated liposomes conjugated with monoclonal antibodies specific for the erythrocyte surface protein glycophorin A (anti-GPA iLP) were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5 μM total lipid in the culture, with >95% of added iLPs retained into the cells. When exposed for only 15 min to P. falciparum in vitro cultures synchronized at early stages, free CQ had no significant effect over parasite viability up to 200 nM drug, whereas iLP-encapsulated 50 nM CQ completely arrested its growth. Furthermore, when assayed in vivo in P. falciparum-infected humanized mice, anti-GPA iLPs cleared the pathogen below detectable levels at a CQ dose of 0.5 mg/kg. In comparison, free CQ administered at 1.75 mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement in drug antimalarial efficacy is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.

Keywords: immunoliposomal nanoparticles, malaria, prophylactic-therapeutic polyvalent activity, targeted drug delivery

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Authors: P. I. Bobyleva, E. R. Andreeva, I. V. Andrianova, E. V. Maslova, L. B. Buravkova

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This work studied the ability of adipose tissue-derived mesenchymal stromal cells (MSCs) to form stroma for expansion of cord blood hematopoietic cells. We showed that 72-hour interaction of MSCs with cord blood mononuclear cells (MNCs) in vitro at atmospheric (20%) and low (5%) O2 conditions increased the expression of ICAM-1, HCAM (at the beginning of interaction) on MSCs. Viability of MSCs and MNCs were maintained at high level. Adhesion of MNCs to MSCs was faster at 20% O2. MSCs promoted the proliferation of adhered MNCs to form the suspension containing great number of hematopoietic colony-forming units, and this effect was more pronounced at 5% O2. Thus, adipose-derived MSCs supplied sufficient stromal support to cord blood MNCs both at 20% and 5% О2, providing their adhesion with further expansion of new generation of different hematopoietic lineages.

Keywords: hematopoietic stem and progenitor cells, mesenchymal stromal cells, tissue-related oxygen, adipose tissue

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Authors: Shweta Sinha, Mukesh Doble, Manju S. L.

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Pyrazole scaffold is an important group of compound in heterocyclic chemistry and is found to possess numerous uses in chemistry. Pyrazole derivatives are also known to possess important biological activities including antitumor, antimicrobial, antiviral, antifungal, anticancer and anti-inflammatory. Inflammation is associated with pain, allergy and asthma. Leukotrienes are mediators of various inflammatory and allergic disorders. 5-Lipoxygenase (5-LOX) is an important enzyme involved in the biosynthesis of leukotrienes and metabolism of arachidonic acid (AA) and thus targeted for anti-inflammation. In vitro inhibitory activity of pyrazol-3-yl thiazole 4-carboxylic acid derivatives is tested against enzyme 5-LOX. Most of these compounds exhibit good inhibitory activity against this enzyme. Binding mode study of these compounds is determined by computational tool. Further experiments are being done to understand the mechanism of action of these compounds in inhibiting this enzyme. To conclude, these compounds appear to be a promising target in drug design against 5-LOX.

Keywords: inflammation, inhibition, 5-lipoxygenase, pyrazole

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Authors: Mthokozisi B. C. Simelane

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Despite the various efforts by governmental and non-governmental organizations aimed at eradicating the disease, malaria is said to kill a child every 30 seconds. Traditional healers use different concoctions prepared from medicinal plants to treat malaria. In the quest to bio-prospect plant-derived triterpenes for anti-malaria activity, we report here the in vivo antiplasmodial activity of ursolic acid acetate (ursolic acid isolated from dichloromethane extract of Mimusops caffra was chemically modified to its acetate derivative). The transdermal administration of ursolic acid acetate (UAA) dose dependently showed complete inhibition of the parasites’ growth at the highest concentration of 400 mg/kg after 15 days of Plasmodium berghei infection. UAA prevented the in vitro aggregation of MDH but did not prevent the expression of PfHsp 70 in E. coli XL1 blue cells. It, however, enhanced PfHsp70 ATPase activity with the specific activity of 65 units (amount of phosphate released 73.83 nmolPi/min.mg). Ursolic acid acetate prevented the formation of hemozoin (60 ± 0.02% at 6 mg/ml). The results suggest that Ursolic acid acetate possesses potential anti-malaria properties.

Keywords: Mimusops caffra, ursolic acid acetate, hemozoin, Malaria

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Authors: Hanan Moawad, Naglaa M. Abd EL-Rahman

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Erwinia carotovora var. carotovora is the main cause of soft rot in potatoes. Hyphaene thebaica was studied for biocontrol of E. carotovora which inhibited growth of E. carotovora on solid medium, a comparative study of classical and ultrasound-assisted extractions of Hyphaene thebaica fruit. The use of ultrasound decreased significant the total time of treatment and increase the total amount of crude extract. The crude extract was subjected to determine the in vitro, by a bioassay technique revealed that the treatment of paper disks with ultrasound extraction of Hyphaene thebaica reduced the growth of pathogen and produced inhibition zones up to 38mm in diameter. The antioxidant activity of ultrasound-ethanolic extract of Doum fruits (Hyphaene thebaica) was determined. Data obtained showed that the extract contains the secondary metabolites such as Tannins, Saponin, Flavonoids, Phenols, Steroids, Terpenoids, Glycosides and Alkaloids.

Keywords: ultrasound, classical extract, biological control, Erwinia carotovora, Hyphaene thebaica

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Authors: Desta Gebretekle Shiferaw, Balakrishna Kalluraya

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Pyrazole is an aromatic five-membered heterocycle with two nitrogen and three carbon atoms in its ring structure. According to the literature, pyrazoline, pyrazole, and thiazole-containing moieties are found in various drug structures and are responsible for nearly all pharmacological effects. The pyrazoline linked to pyrazole moiety carbothioamides was synthesized via the reaction of pyrazole-bearing chalcones (3-(5-chloro-3-methyl-¹-phenyl-1H-pyrazol-4-yl)-¹-(substituted aryl) prop-2-ene-¹-one derivatives) with a nucleophile thiosemicarbohyrazide by heating in ethanol using fused sodium acetate as a catalyst. Then the carbothioamide derivatives were converted into the pyrazoline hybrid to pyrazole and thiazole derivatives by condensing with substituted phenacyl bromide in alcohol in a basic medium. Next, the chemical structure of the newly synthesized molecules was confirmed by IR, 1H-NMR, and mass spectral data. Further, they were screened for their in vitro antioxidant activity. Compared to butylated hydroxy anisole (BHA)., the antioxidant data showed that the synthesized compounds had good to moderate activity.

Keywords: pyrazoline-pyrazole carbothioamide derivatives, pyrazoline-pyrazole-thiazole derivatives, spectral studies, antioxidant activity

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Authors: Yuping Liu, Li Tao, Yin Lu

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Accumulating evidence has been shown that diallyl trisulfide (DATS) from garlic may reduce the risk of developing several types of cancer. In view of the dynamic crosstalk interplayed by tumor cells and platelets in hematogenous metastasis, we demonstrate the effectiveness of DATS on the metastatic behaviors of MDA-MB-435s human breast cancer cell line co-incubated with activated platelets. Indeed, our data identified that DATS significantly blocked platelets fouction induced by PAF, followed by the decreased production of TXB2. DATS was found to dose-dependently suppressed MDA-MB-435s cell migration and invasion in presence of activated platelets by PAF in vitro. Furthermore, the expression, secretion and enzymatic activity of matrix metalloproteinase (MMP)-2/9, as well as the luciferase activity of upstream regulator NF-κB in MDA-MB-435s, were obviously diminished by DATS. In parallel, DATS blocked upstream NF-κB activation signaling complexes composed of extracellular signal-related kinase (ERK) as assessed by measuring the levels of the phosphorylated forms.

Keywords: DATS, ERK, metastasis, MMPs, NF-κB, platelet

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Authors: Tatjana Kadifkova Panovska, Svetlana Kulevanova, Blagica Jovanova

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Biological systems possess the ability to neutralize the excess of reactive oxygen species (ROS) and to protect cells from destructive alterations. However, many pathological conditions (cardiovascular diseases, autoimmune disorders, cancer) are associated with inflammatory processes that generate an excessive amount of reactive oxygen species (ROS) that shift the balance between endogenous antioxidant systems and free oxygen radicals in favor of the latter, leading to oxidative stress. Therefore, an additional source of natural compounds with antioxidant properties that will reduce the amount of ROS in cells is much needed despite their broad utilization; many plant species remain largely unexplored. Therefore, the purpose of the present study is to investigate the antioxidant activity of twenty-five selected medicinal and aromatic plant species. The antioxidant activity of the ethanol extracts was evaluated with in vitro assays: 2,2’-diphenyl-1-pycryl-hydrazyl (DPPH), ferric reducing antioxidant power (FRAP), non-site-specific- (NSSOH) and site-specific hydroxyl radical-2-deoxy-D-ribose degradation (SSOH) assays. The Folin-Ciocalteu method and AlCl3 method were performed to determine total phenolic content (TPC) and total flavonoid content (TFC). All examined plant extracts manifested antioxidant activity to a different extent. Cinnamomum verum J.Presl bark and Ocimum basilicum L. Herba demonstrated strong radical scavenging activity and reducing power with the DPPH and FRAP assay, respectively. Additionally, significant hydroxyl scavenging potential and metal chelating properties were observed using the NSSOH and SSOH assays. Furthermore, significant variations were determined in the total polyphenolic content (TPC) and total flavonoid content (TFC), with Cinnamomum verum and Ocimum basilicum showing the highest amount of total polyphenols. The considerably strong radical scavenging activity, hydroxyl scavenging potential and reducing power for the species mentioned above suggest of a presence of highly bioactive phytochemical compounds, predominantly polyphenols. Since flavonoids are the most abundant group of polyphenols that possess a large number of available reactive OH groups in their structure, it is considered that they are the main contributors to the radical scavenging properties of the examined plant extracts. This observation is supported by the positive correlation between the radical scavenging activity and the total polyphenolic and flavonoid content obtained in the current research. The observations from the current research nominate Cinnamomum verum bark and Ocimum basilicum herba as potential sources of bioactive compounds that could be utilized as antioxidative additives in the food and pharmaceutical industries. Moreover, the present study will help the researchers as basic data for future research in exploiting the hidden potential of these important plants that have not been explored so far.

Keywords: ethanol extracts, radical scavenging activity, reducing power, total polyphenols.

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Authors: Sibel Daglilar, Isil Kerti, Murat Karagoz, Fatih Dumludag, Oguzhan Gunduz, Faik Nuzhet Oktar

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Hydroxyapatite (HA) materials have common use in bone repairing due to its ability to accelerate the bone growth around the implant. In spite of being a biocompatible and bioactive material, HA has a limited usage as an implant material because of its weak mechanical properties. HA based composites are required to improve the strength and toughness properties of the implant materials without compromising of biocompatibility. The excellent mechanical properties and higher biocompatibilities are expected from each of biomedical composites. In this study, HA composites were synthesized by using bovine bone reinforced doped with different amount of (wt.%) Li2O. The pressed pellets were sintered at various sintering temperatures between 1000ºC and 1300°C, and mechanical, electrical properties of the obtained products were characterized. In addition to that, in vitro stimulated body fluid (SBF) tests for these samples were conducted. The most suitable composite composition for biomedical applications was discussed among the composites studied.

Keywords: biocomposites, sintering temperature, biocompatibility, electrical property, conductivity, mechanical property

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Authors: D. Zujur, J. Moret, D. Rodriguez, L. Cruz, J. Lira, L. Gil, E. Dominguez, J. F. Alvarez-Barreto

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In this work, chitosan (CH) has been used to produce a novel coating for Ti6Al4V, the most widely used alloy in orthopedic implants, so as to improve the biological tissue response at the metallic surface. The Ti6Al4V surface was sandblasted with alumina particles and observed by SEM. Chitosan was chemically modified, via crodiimide chemistry, with lactobionic and 4-azidebenzoic acid to make it soluble at physiological pH and photo-crosslinkable, respectively. The reaction was verified by FTIR, NMR, and UV/vis spectroscopy. Ti6Al4V surfaces were coated with solutions of the modified CH and exposed to UV light, causing the polymer crosslinking, and formation of a hydrogel on the surface. The crosslinking reaction was monitored by FTIR at different exposure times. Coating morphology was observed by SEM. The coating´s cytocompatibility was determined in vitro through the culture of rat bone marrow´s mesenchymal stem cells, using an MTT assay. The results show that the developed coating is cytocompatible, easy to apply and could be used for further studies in the encapsulation of bioactive molecules to improve osteogenic potential at the tissue-implant interface.

Keywords: chitosan, photo-crosslinking, Ti6Al4V, bioactive coating, hydrogel

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Authors: Sachin Mulmi Shrestha, Xin Fang, Hui Ye, Lihua Ren, Qinghua Ji, Ruihua Shi

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Background: Esophageal squamous cell carcinoma (ESCC) is a tumor arising from esophageal epithelial cells and is one of the major disease subtype in Asian countries, including China. Esophageal cancer is the 7th highest incidence based on the 2020 data of GLOBOCAN. The pathogenesis of cancer is still not well understood as many molecular and genetic basis of esophageal carcinogenesis has yet to be clearly elucidated. Circular RNAs are RNA molecules that are formed by back-splicing covalently joined 3′- and 5′-endsrather than canonical splicing, and recent data suggest circular RNAs could sponge miRNAs and are enriched with functional miRNA binding sites. Hence, we studied the mechanism of circular RNA, its biological function, and the relationship between microRNA in the carcinogenesis of ESCC. Methods: 4 pairs of normal and esophageal cancer tissues were collected in Zhongda hospital, affiliated to Southeast University, and high-throughput RNA sequencing was done. The result revealed that circ_0032746 was upregulated, and thus we selected circ_0032746 for further study. The backsplice junction of circRNA was validated by sanger sequence, and stability was determined by RNASE R assay. The binding site of circRNA and microRNA was predicted by circinteractome,mirandaand RNAhybrid database. Furthermore, circRNA was silenced by siRNA and then by lentivirus. The regulatory axis of circ0032746/miR4270 was validated by shRNA, mimic, and inhibitor transfection. Then, in vitro experiments were performed to assess the role of circ0032746 on proliferation (CCK-8 assay and colon formation assay), migration and invasion (Transewell assay), and apoptosis of ESCC. Results: The upregulated circ0032746 was validated in 9 pairs of tissues and 5 types of cell lines by qPCR, which showed high expression and was statistically significant (P<0.005) ). Upregulated circ0032746 was silenced by shRNA, which showed significant knockdown in KYSE 30 and TE-1 cell lines expression compared to control. Nuclear and cytoplasmic mRNA fraction experiment displayed the cytoplasmic location of circ0032746. The sponging of miR4270 was validated by co-transfection of sh-circ0032746 and mimic or inhibitor. Transfection with mimic showed the decreased expression of circ_0032746, whereas inhibitor inhibited the result. In vitro experiments showed that silencing of circ_0032746 inhibited the proliferation, migration, and invasion compared to the negative control group. The apoptosis was seen higher in a knockdown group than in the control group. Furthermore, 11 common mircoRNA target mRNAs were predicted by Targetscan, MirTarbase, and miRanda database, which may further play role in the pathogenesis. Conclusion: Our results showed that novel circ_0032746 is upregulated in ESCC and plays role in itsoncogenicity. Silencing of circ_0032746 inhibits the proliferation and migration of ESCC whereas increases the apoptosis of cancer cells. Hence, circ0032746 acts as an oncogene in ESCC by sponging with miR4270 and could be a potential biomarker in the diagnosis of ESCC in the future.

Keywords: circRNA, esophageal squamous cell carcinoma, microRNA, upregulated

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Authors: Mahboubeh Davoudi Pahnekolayi

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Begonia rex cv. DS-EYWA is a rare, unique cultivar of begonia rex with curly colorful leaves. Optimization of an in vitro efficient regeneration protocol by focusing on transverse Thin Cell Layer (tTCL) petiole explants for high-scale production of such a beautiful cultivar was considered as our main purpose in this experiment. Thus, various concentrations of Plant Growth Regulators (PGRs) including 6-Benzylaminopurine (BAP), Thidiazuron (TDY), and –Naphthaleneacetic Acid (NAA), were selected in a Completely Randomized Design (CRD) to establish and optimize the direct organogenesis efficiency of this cultivar. Cultivation of 1 mm tTCL petiole explants in noted treatments showed that 1.5 mgl-1 BAP + 0.5 mgl-1 NAA can induce the highest number of direct regenerated shoots and lower concentration of BAP (0.5 mgl-1) can be suggested for shoot elongation before rooting stage. Elongated shoots were successfully rooted in MS free basal medium and acclimatized in 1:1 peat moss: perlite sterilized pot mixture.

Keywords: begonia rare cultivar, direct organogenesis, explant type, regeneration, thin cell layering (TCL)

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Authors: Qaiser Akram, Ahsan Naeem, Hafiz Muhammad Rizwan, Waqas Ahmad, Rubeena Yasmeen

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Aspergillus has good secretory potential for phytases. Morphologically and microscopically identified Aspergillus terreus (A. terreus) (n=20) were screened for phytase production and non-toxicity. Phytases produced by non-toxigenic A. terreus under optimum conditions were quantified. Phytases of highest producer A. terreus were evaluated for stability after exposure to temperature (35, 55, 75 and 95ºC) and pH (2, 4, 6 and 8). Effect of metal ions (Fe⁺³, Ba⁺², Ca⁺², Cu⁺², Mg⁺², Mn⁺², K⁺¹ and Na⁺¹) was assessed on phytase activity. Log reduction in phytase activity was calculated. The highest activity units of phytase produced by A. terreus were 271.49 ± 8.14 phytase unit / mL (FTU/ mL). The lowest reduction in phytase activity was 50.20 ± 7.36 (18.5%) and 68.22 ± 10.3 FTU/mL (25.13%) at 35ºC and pH 6, respectively for 15 minutes. The highest reduction 259 ± 0.84 (95.5%) and 211.99 ± 4.39 FTU/mL (78.1%) was recorded at 95ºC for 60 minutes and pH 2.0 for 45 minutes exposure, respectively. All metal ions negatively affected phytase activity. Phytase activity was inhibited minimum (45.32 ± 28.54 FTU/mL, 16.69%) by K⁺¹(1 mM) and maximum (231.48 ± 3.68 FTU/mL, 80.8%) by Cu⁺² (10 mM). It was concluded that A. terreus phytase stability and activity was dependent on physio-chemical factors.

Keywords: stability, phytase, aspergillus terreus, physio-chemical factors and metal ions

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Authors: Abdulrahman T. Essa, Ahmed Aied, Omar Hamid, Felicity R. A. J. Rose, Kevin M. Shakesheff

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Biodegradable poly(ester amide)s are promising polymers for biomedical applications such as drug delivery and tissue engineering because of their optimized chemical and physical properties. In this study, we developed a biodegradable polyester amide elastomer poly(serinol sebacate) (PSS) composed of crosslinked networks based on serinol and sebacic acid. The synthesized polymers were characterized to evaluate their chemical structures, mechanical properties, degradation behaviors and in vitro cytocompatibility. Analysis of proton nuclear magnetic resonance and Fourier transform infrared spectroscopy revealed the structure of the polymer. The PSS exhibit excellent solubility in a variety of solvents such as methanol, dimethyl sulfoxide and dimethylformamide. More importantly, the mechanical properties of PSS could be tuned by changing the curing conditions. In addition, the 3T3 fibroblast cells cultured on the PSS demonstrated good cell attachment and high viability.

Keywords: biodegradable, biomaterial, elastomer, mechanical properties, poly(serinol sebacate)

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Authors: Saman Khan, Imrana Naseem

Abstract:

Copper is an essential trace element required for pro-angiogenic co-factors including vascular endothelial growth factor (VEGF). Elevated levels of copper are found in various types of cancer including prostrate, colon, breast, lung and liver for angiogensis and metastasis. Therefore, targeting copper via copper-specific chelators in cancer cells can be developed as effective anticancer treatment strategy. In continuation of our pursuit to design and synthesize copper chelators, herein we opted for a reaction to incorporate di-(2-picolyl) amine in 3-(bromoacetyl) coumarin (parent backbone) for the synthesis of complex 1. We evaluated lipid peroxidation, protein carbonylation, ROS generation, DNA damage and consequent apoptosis by complex 1 in exogenously added Cu(II) in human peripheral lymphocytes (simulate malignancy condition). Results showed that Cu(II)-complex 1 interaction leads to cell proliferation inhibition, apoptosis, ROS generation and DNA damage in human lymphocytes, and these effects were abrogated by cuprous chelator neocuproine and ROS scavengers (thiourea, catalase, SOD). This indicates that complex 1 cytotoxicity is due to redox cycling of copper to generate ROS which leads to pro-oxidant cell death in cancer cells. To further confirm our hypothesis, using the rat model of diethylnitrosamine (DEN) induced hepatocellular carcinoma; we showed that complex 1 mediates DNA breakage and cell death in isolated carcinoma cells. Membrane permeant copper chelator, neocuproine, and ROS scavengers inhibited the complex 1-mediated cellular DNA degradation and apoptosis. In summary, complex 1 anticancer activity is due to its copper chelation capability. These results will provide copper chelation as an effective targeted cancer treatment strategy for selective cytotoxic action against malignant cells without affecting normal cells.

Keywords: cancer treatment, copper chelation, ROS generation, DNA damage, redox cycling, apoptosis

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Authors: Li Chen, Feng Zhang, Shizhong Zheng

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SaikosaponinD (SSD) is a major component of saikosaponins isolated from Bupleurumfalactum. Our current study was to examine the toxic effect of SSD on liver cells and explore the possible mechanism. The results demonstrated that SSD induced mouse liver injury and led to apoptosis in LO2 cells. HE staining and TUNEL analyses showed that SSD stimulated liver injury and hepatocyte apoptosis in vivo. Subsequent experiments showed that SSD down-regulated Bcl-2 but up-regulated Bax. In vitro, SSD-treated LO2 cells exhibited apparent down-regulated expression of p-p38. Moreover, PDGF-βR agonist PDGF-BB alone significantly upregulated p38 phosphorylation, while combined with SSD, p38 phosphorylation expression was reduced. Furthermore, shRNA-mediated PDGF-βR knockdown augmented the inactivation of p-p38 and Bcl2 but abrogated the activation of Bax, these results were more obvious when shRNA combined with SSD. These data indicated that SSD stimulated liver injury and apoptosis in hepatocytes and PDGF-βR /p38 pathway may be the potential mechanistic.

Keywords: saikosaponin D, hepatotoxicity, liver injury, apoptosis, platelet-derived growth factor-β receptor, p38

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Authors: Mohd Adil, Rosina Khan, Asad U. Khan, Vasantha Rupasinghe HP

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The study examined the effects of Syzygium aromaticum extracts on the virulence properties of Streptococcus mutans. The activity of glucosyltransferases in the presence of crude and diethylether fraction was reduced to 80% at concentration 78.12μg/ml and 39.06μg/ml respectively. The glycolytic pH drop by S. mutans cells was also disrupted by these extracts without affecting the bacterial viability. Microscopic analysis revealed morphological changes of the S. mutans biofilms, indicating that these plant extracts at sub-MICs could significantly affect the ability of S. mutans to form biofilm with distorted extracellular matrix. Furthermore, with the help of quantitative RT-PCR, the expression of different genes involved in adherence, quorum sensing, in the presence of these extracts were down regulated. The crude and active fractions were found effective in preventing caries development in rats. The data showed that S. aromaticum holds promise as a naturally occurring source of compounds that may prevent biofilm-related oral diseases.

Keywords: biofilm, quorum sensing, Streptococcus mutans, Syzygium aromaticum extract

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Authors: Sema Şenoğlu, Sevgi Karakuş

Abstract:

Hydrazone scaffold is important to design new drug groups and is found to possess numerous uses in pharmaceutical chemistry. Besides, hydrazone derivatives are also known for biological activities such as anticancer, antimicrobial, antiviral, and antifungal. Hydrazone derivatives are promising anticancer agents because they inhibit cancer proliferation and induce apoptosis. Human carbonic anhydrase IX has a high potential to be an antiproliferative drug target, and targeting this protein is also important for obtaining potential anticancer inhibitors. The protein construct was retrieved as a PDB file from the RCSB protein database. This binding interaction of proteins and ligands was performed using Discovery Studio Visualizer. In vitro inhibitory activity of hydrazone derivatives was tested against enzyme carbonic anhydrase IX on the PyRx programme. Most of these molecules showed remarkable human carbonic anhydrase IX inhibitory activity compared to the acetazolamide. As a result, these compounds appear to be a potential target in drug design against human carbonic anhydrase IX.

Keywords: cancer, carbonic anhydrase IX enzyme, docking, hydrazone

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Authors: Chuan Yin

Abstract:

Cancer stem cells (CSCs) represent a subpopulation of cancer cells that possess the characteristics associated with normal stem cells. CD133 is a phenotype of melanoma CSCs responsible for melanoma metastasis and drug resistance. Although adriamycin (ADR) is commonly used drug in melanoma therapy, but it is ineffective in the treatment of melanoma CSCs. In this study, we constructed CD133 antibody conjugated ADR immunoliposomes (ADR-Lip-CD133) to target CD133+ melanoma CSCs. The results showed that the immunoliposomes possessed a small particle size (~150 nm), high drug encapsulation efficiency (~90%). After 72 hr treatment on the WM266-4 melanoma tumorspheres, the IC50 values of the drug formulated in ADR-Lip-CD133, ADR-Lip (ADR liposomes) and ADR are found to be 24.42, 57.13 and 59.98 ng/ml respectively, suggesting that ADR-Lip-CD133 was more effective than ADR-Lip and ADR. Significantly, ADR-Lip-CD133 could almost completely abolish the tumorigenic ability of WM266-4 tumorspheres in vivo, and showed the best therapeutic effect in WM266-4 melanoma xenograft mice. It is noteworthy that ADR-Lip-CD133 could selectively kill CD133+ melanoma CSCs of WM266-4 cells both in vitro and in vivo. ADR-Lip-CD133 represent a potential approach in targeting and killing CD133+ melanoma CSCs.

Keywords: cancer stem cells, melanoma, immunoliposomes, CD133

Procedia PDF Downloads 382

Authors: Akbar Esmaeili, Mahnoosh Aliahmadi

Abstract:

This study synthesized a modified imaging of gallium@deferoxamine/folic acid/chitosan/polyaniline/polyvinyl alcohol (Ga@DFA/FA/CS/PANI/PVA). It contains Morus nigra extract by selenium nanoparticles prepared from Lactobacillus acidophilus. Using the impregnation method, Se nanoparticles were then deposited on (Ga@DFA/FA/ CS/PANI/PVA). The modified contrast agents were mixed with M. nigra extract, and investigated their antibacterial activities by applying to L929 cell lines. The influence of variable factors, including 1. surfactant, 2. solvent, 3. aqueous phase, 4. pH, 5. buffer, 6. minimum Inhibitory concentration (MIC), 7. minimum bactericidal concentration (MBC), 8. cytotoxicity on cancer cells., 9. antibiotic, 10. antibiogram, 11. release and loading, 12. the emotional effect, 13. the concentration of nanoparticles, 14. olive oil, and 15. they have investigated thermotical methods. The structure and morphology of the synthesized contrast agents were characterized by zeta potential sizer analysis (ZPS), X-Ray diffraction (XRD), Fourier-transform infrared (FT-IR), energy dispersive X-ray (EDX), ultraviolet–visible (UV–Vis) spectra, and scanning electron microscope (SEM). The experimental section was conducted and monitored by response surface methods (RSM), MTT, MIC, MBC, and cancer cytotoxic conversion assay. Antibiogram testing of NCs on Pseudomonas aeruginosa bacteria was successful and obtained MIC = 2 factors with less harmful effect. All experimental sections confirmed that our synthesized particles have potent antioxidant properties. Antibiogram testing revealed that NPS could kill P. aeruginosa and P. aeruginosa. A variety of synthetic conditions were done by diffusion emulsion method by varying parameters, the optimum state of DFA release Ga@DFA/FA/CS/PANI/PVA NPs (6 ml) with pH = 5.5, time = 3 h, NCs and DFA (3 mg), and achieved buffer (20 ml). DFA in Ga@DFA/FA/ CS/PANI/PVA was released and showed an absorption peak at 378 nm by applying a 300-rpm magnetic rate. In this report, Ga decreased the harmful effect on the human body.

Keywords: nanocapsules, technolgy, biology, nano

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Authors: D. R. Rama Brahma Reddy, A. Vijaya Sarada Reddy

Abstract:

Nanoparticles represent a promising drug delivery system of controlled and targeted drug release. They are specially designed to release the drug in the vicinity of target tissue. The aim of this study was to prepare and evaluate polymethacrylic acid nanoparticles containing Zidovudine in different drug to polymer ratio by nanoprecipitation method. SEM indicated that nanoparticles have a discrete spherical structure without aggregation. The average particle size was found to be 120 ± 0.02 - 420 ± 0.05 nm. The particle size of the nanoparticles was gradually increased with increase in the proportion of polymethacrylic acid polymer. The drug content of the nanoparticles was increasing on increasing polymer concentration up to a particular concentration. No appreciable difference was observed in the extent of degradation of product during 60 days in which, nanoparticles were stored at various temperatures. FT-IR studies indicated that there was no chemical interaction between drug and polymer and stability of drug. The in-vitro release behavior from all the drug loaded batches was found to be zero order and provided sustained release over a period of 24 h. The developed formulation overcome and alleviates the drawbacks and limitations of Zidovudine sustained release formulations and could possibility be advantageous in terms of increased bio availability of Zidovudine.

Keywords: nanoparticles, zidovudine, biodegradable, polymethacrylic acid

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Authors: Hirowati Ali, Aisyah Ellyanti, Dewi Rusnita, Septelia Inawati Wanandi

Abstract:

Stem cell has been known for its potency to be differentiated in many cells. Recently stem cell has been used for many treatment of degenerative medicine. It is still controversy whether stem cell can be used for therapy or these cells can activate cancer stem cell. SCUBE2 is a novel secreted and membrane-anchored protein which has been reported to its role in better prognosis and inhibition of cancer cell proliferation. Our study aims to observe whether stem cell can up-regulate SCUBE2 gene in MCF7 breast cancer cell line. We used in vitro study using MCF-7 cell treated with stem cell derived from placenta Wharton's jelly which has been known for its stemness and widely used. Our results showed that MCF-7 cell line grows up rapidly in 6-well culture dish. Stem cell was cultured in 6-well dish. After 50%-60% MCF-7 confluence, we co-cultured these cells with stem cells for 24 hours and 48 hours. We hypothesize SCUBE2 gene which is previously known for its higher expression in better prognosis of breast cancer, is up-regulated after stem cells addition in MCF7 culture dishes.

Keywords: breast cancer cells, inhibition of cancer cells, mesenchymal stem cells, SCUBE2

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Authors: Rajapaksha Gedara Prasad Tharanga Jayasooriya, Matharage Gayani Dilshara, Yung Hyun Choi, Gi-Young Kim

Abstract:

Camptothecin (CPT) is a quinolone alkaloid which inhibits DNA topoisomerase I that induces cytotoxicity in a variety of cancer cell lines. We previously showed that CPT effectively inhibited invasion of prostate cancer cells and also combined treatment with subtoxic doses of CPT and TNF-related apoptosis-inducing ligand (TRAIL) potentially enhanced apoptosis in a caspase-dependent manner in hepatoma cancer cells. Here, we found that treatment with CPT caused an irreversible cell cycle arrest in the G2/M phase. CPT-induced cell cycle arrest was associated with a decrease in protein levels of cell division cycle 25C (Cdc25C) and increased the level of cyclin B and p21. The CPT-induced decrease in Cdc25C was blocked in the presence of proteasome inhibitor MG132, thus reversed the cell cycle arrest. In addition to that treatment of CPT-increased phosphorylation of Cdc25C was the resulted of activation of checkpoint kinase 2 (Chk2), which was associated with phosphorylation of ataxia telangiectasia-mutated. Interestingly CPT induced G2/M phase of the cell cycle arrest is reactive oxygen species (ROS) dependent where ROS inhibitors NAC and GSH reversed the CPT-induced cell cycle arrest. These results further confirm by using transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2) since it regulates the production of ROS. Our data reveal that treatment of siNrf2 increased the ROS level as well as further increased the CPT induce G2/M phase cell cycle arrest. Our data also indicate CPT-enhanced cell cycle arrest through the extracellular signal-regulated kinase (ERK) and the c-Jun N-terminal kinase (JNK) pathway. Inhibitors of ERK and JNK more decreased the Cdc25C expression and protein expression of p21 and cyclin B. These findings indicate that Chk2-mediated phosphorylation of Cdc25C plays a major role in G2/M arrest by CPT.

Keywords: camptothecin, cell cycle, checkpoint kinase 2, nuclear factor-erythroid 2-related factor 2, reactive oxygen species

Procedia PDF Downloads 441

Authors: Damandeep Kaur, O.P. Pandey, M.S. Reddy

Abstract:

In the present research, bio-synthesisation of silica particles has been carried out successfully. For this purpose, agriculture waste rice husk (RH) has been utilized. Among several types of agriculture waste, RH is considered to be cost-effective and easily accessible. In the present investigation, a chemical approach has been followed to extract silica nanoparticles. X-Ray Diffraction (XRD) patterns indicated the amorphous nature of silica at lower temperature range. Silica and other mineral contents have been found using energy dispersive spectroscopy (EDS). Morphological and structural studies have been carried out with the use of Field Emission Scanning Electron Microscopy (FE-SEM) and Fourier Transform Infrared Transmission (FTIR) spectroscopy. Further, extracted silica from RH has been used for preparation of the glasses. The appearance of broad humps in XRD patterns confirmed the amorphous nature of prepared glasses. These glasses exhibited enhanced antibacterial effect against both Gram-positive and Gram-negative bacteria. The as-synthesized glass samples can be further used for physical and structural studies for drug loading applications.

Keywords: rice husk, biosynthesized silica, bioactive glasses, antibacterial studies

Procedia PDF Downloads 114

Authors: Ravi J. Shah

Abstract:

The present study deals with the synthesis of novel spiro[azetidine-2, 3’-indole]-2’, 4(1’H)-dione derivative from the reactions of 3-(phenylimino)-1,3-dihydro-2H-indol-2-one derivatives with chloracetyl chloride in presence of triethyl amine (TEA). All the compounds were characterized using IR, 1H NMR, MS and elemental analysis. They were screened for their antibacterial and antifungal activities. Results revealed that, compounds (7a), (7b), (7c), (7d) and (7e) showed very good activity with MIC value of 6.25-12.5 μg/ml against three evaluated bacterial strains and the remaining compounds showed good to moderate activity comparable to standard drugs as antibacterial agents. Compounds (7c) and (7h) displayed equipotent antifungal activity in comparison to standard drugs. Structure-activity relationship study of the compounds showed that the presence of electron withdrawing group substitution at 5’ and 7’ positions of indoline ring and on ortho or para position of phenyl ring increases both antibacterial and antifungal activity of the compound. Henceforth, our findings will have a good impact on chemists and biochemists for further investigations in search of bromine containing spiro fused antimicrobial agents.

Keywords: antibacterial activity, antifungal activity, 2-Azetidinone, indoline

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Authors: Damião Sousa, Hasan Turkez, Ozlem Tozlu, Tamires Lima

Abstract:

Glioblastoma (GBM) is an aggressive cancer from the brain and with high prevalence and significant morbimortality. Therefore, it is necessary to investigate new therapeutic options against this pathology. Thus, the purpose of this study was to evaluate the antitumor activity from Mentha crispa essential oil (MCEO), its major constituent rotundifolone (ROT) and a series of six analogues on human U87MG glioblastoma cell line. The antitumor effects of the compounds on human U87MG-GBM cell line were assessed using in vitro cell viability assays. In addition, biosafety tests were performed on cultured human blood cells. The data show that MCEO, 1,2-perillaldehyde epoxide (EPER1) and perillaldehyde (PALD) were the most cytotoxic compounds against the U87MG cells, with IC50 values of 16.263, 15.087 and 14.888 μg/mL, respectively. The treatment with MCEO, EPER1 and PALD did not lead to damage in blood cells. These chemical analogues may be useful as prototypes for development of novel antitumor drugs due to their promising activities and toxicological safety.

Keywords: antitumor activity, cancer, natural products, terpenes

Procedia PDF Downloads 147