Search results for: In vitro cytotoxicity

Authors: Rekaya A. Shabbir, Marco Mingarelli, Glenn Flux, Ananya Choudhury, Tim A. D. Smith

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Introduction: Heterogeneity of dose distributions across a tumour is problematic for targeted radiotherapy. Gold nanoparticles (AuNPs) enhance dose-distributions of targeted radionuclides. The aim of this study is to demonstrate if tumour dose-distribution of targeted AuNPs radiolabelled with either of two radioisotopes (¹⁷⁷Lu and ⁹⁰Y) in breast cancer cells produced homogeneous dose distributions. Moreover, in vitro and in vivo studies were conducted to study the importance of receptor level on cytotoxicity of EGFR-targeted AuNPs in breast and colorectal cancer cells. Methods: AuNPs were functionalised with DOTA and OPPS-PEG-SVA to optimise labelling with radionuclide tracers and targeting with Erbitux. Radionuclides were chelated with DOTA, and the uptake of the radiolabelled AuNPs and targeted activity in vitro in both cell lines measured using liquid scintillation counting. Cells with medium (HCT8) and high (MDA-MB-468) EGFR expression were incubated with targeted ¹⁷⁷Lu-AuNPs for 4h, then washed and allowed to form colonies. Nude mice bearing tumours were used to study the biodistribution by injecting ¹⁷⁷Lu-AuNPs or ⁹⁰Y-AuNPs via the tail vein. Heterogeneity of dose-distribution in tumours was determined using autoradiography. Results: Colony formation (% control) was 81 ± 4.7% (HCT8) and 32 ± 9% (MDA-MB-468). High uptake was observed in the liver and spleen, indicating hepatobiliary excretion. Imaging showed heterogeneity in dose-distributions for both radionuclides across the tumours. Conclusion: The cytotoxic effect of EGFR-targeted AuNPs is greater in cells with higher EGFR expression. Dose-distributions for individual radiolabelled nanoparticles were heterogeneous across tumours. Further strategies are required to improve the uniformity of dose distribution prior to clinical trials.

Keywords: cancer cells, dose distributions, radionuclide therapy, targeted gold nanoparticles

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Authors: Lamees A. Ben Saad, Kah Hwi Kim, Chin Chew Quah, Mustafa Shahimi

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Background: Punica granatum (pomegranate) was used as a traditional medicine in different parts of the world. It has been used in the treatment of pain and inflammatory conditions such as peptic ulcer. The numerous risks associated with nonsteroidal anti-inflammatory drugs (NSAIDs) for the treatment of pain and inflammation give rise to using medicinal herbs as alternative therapies. This study aimed to evaluate the anti-inflammatory effect of the ethyl acetate pomegranate fraction (EtOAc) by determination of its inhibitory effects on lipopolysaccharide (LPS), stimulated nitric oxide (NO), prostaglandin E2 (PGE-2), interleukin-6 (IL-6) and cyclooxxgenase-2 (COX2) release from RAW264.7cells. Methods: The inhibitory effect of EtOAc was evaluated on (LPS) induced NO production, PGE2, and IL-6 quantified by immunoassay kit and prostaglandin E2 competitive ELISA kit. COX2 production is an in vitro indication of possible anti-inflammatory activity and was estimated by Western blotting. Results: EtOAc potentially inhibited LPS-induced nitric oxide, prostaglandin, and IL-6 production. With these findings, it was evident that the EtOAc could reduce the LPS-induced cyclooxygenase-2 (COX-2) at the protein level in a dose-dependent manner as determined by Western blotting. Conclusion: The results emphasize potential therapeutic applications of Punica granatum in the treatment of inflammation.

Keywords: inflammation, Punica granatum, cytotoxicity, cytokines

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Authors: Gyati Shilakari Asthana, Abhay Asthana

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Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1 and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

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Authors: N. P. D. Tran, H. Q. D. Nguyen, M. C. Yang

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In this study, contact lenses were prepared through the polymerization of tris-(trimethyl-silyl-propyl-methacrylate) (TRIS), N,N-dimethylacrylamide (DMA), N-vinylpyrrolidone (NVP), and cross-linked with ethylene glycol dimethylacrylate (EGDMA). The equilibrium water content (EWC), oxygen permeability (Dk), light transmittance, and in vitro cytotoxicity of TRIS-DMA-NVP with various ratios were measured. The results showed that the EWC increased while the Dk decreased with the increase of NVP content. For the sample with 25 wt% NVP, the EWC attained 53% whereas the Dk decreased to 46 barrers. All these lenses exhibited light transmittance over than 95%. In addition, all these lenses exhibited no inhibition to the growth of L292 fibroblasts. Thus, this study showed that TRIS-DMA-NVP can be applicable for making contact lens.

Keywords: DMA, TRIS, NVP, silicone hydrogel, contact lens

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Authors: Babak Qasemi-Panahi, Gholamali Moghaddam, Seyed-Abbas Rafat, Hossein Daghigh Kia, Mansoureh Movahedin, Reza Hadavi

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The objective of this study was to determine the effects of IGF-I on ovine oocytes maturation and subsequent development of embryos derived from in vitro fertilization (IVF). In vitro maturation (IVM) of oocytes and in vitro culture (IVC) of embryos was conducted with or without 100 ng/mL IGF-1. In the IGF-I treated group, mean percentage of oocyte maturation was significantly higher than the control group (57.67 ± 3.04 versus 49.81 ± 3.04%, respectively, P < 0.05). However, in comparison with control group, there was no significant effect of IGF-1 on rates of cleavage, morula, and blastocyst formation (85% versus 84%; 63% versus 65%, and 40% to 39%, respectively). These data demonstrate that IGF-I has a positive effect on ovine oocyte maturation rate, but it has not the significant outcome on embryo development.

Keywords: ovine, IGF-I, IVM, ICSI

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Authors: Nancy M. El-Baz, Laila Ziko, Rania Siam, Wael Mamdouh

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Cancer is one of the most devastating diseases, and has over than 10 million new cases annually worldwide. Despite the effectiveness of chemotherapeutic agents, their systemic toxicity and non-selective anticancer actions represent the main obstacles facing cancer curability. Due to the effective enhanced permeability and retention (EPR) effect of nanomaterials, nanoparticles (NPs) have been used as drug nanocarriers providing targeted cancer drug delivery systems. In addition, several inorganic nanoparticles such as silver (AgNPs) nanoparticles demonstrated a potent anticancer activity against different cancers. The present study aimed at formulating core-shell inorganic NPs-based combinatorial therapy based on combining the anticancer activity of AgNPs along with doxorubicin (DOX) and evaluating their cytotoxicity on MCF-7 breast cancer cells. These inorganic NPs-based combinatorial therapies were designed to (i) Target and kill cancer cells with high selectivity, (ii) Have an improved efficacy/toxicity balance, and (iii) Have an enhanced therapeutic index when compared to the original non-modified DOX with much lower dosage The in-vitro cytotoxicity studies demonstrated that the NPs-based combinatorial therapy achieved the same efficacy of non-modified DOX on breast cancer cell line, but with 96% reduced dose. Such reduction in DOX dose revealed that the combination between DOX and NPs possess a synergic anticancer activity against breast cancer. We believe that this is the first report on a synergic anticancer effect at very low dose of DOX against MCF-7 cells. Future studies on NPs-based combinatorial therapy may aid in formulating novel and significantly more effective cancer therapeutics.

Keywords: nanoparticles-based combinatorial therapy, silver nanoparticles, doxorubicin, breast cancer

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Authors: Debasmita Dubey, Rajesh Kumar Meher, Smruti Pragya Samal, Pradeep Kumar Naik

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Background: To determine antioxidant properties and anticancer activity by ROS and mitochondrial transmembrane potential mediated apoptosis against MCF7, MDA-MB-231, cell line. Methods: Leaf sample was extracted using methanol by microwave digestion technique. The antioxidant properties of the methanolic extract were determined by a DPPH scavenging assay. In vitro anticancer activity, mitochondrial transmembrane potential, apoptosis activity and DNA fragmentation study, as well as intracellular ROS activity of most potential leaf extract, were also determined by using the MDA-MB-231cell line. In vivo animal toxicity study was carried out using mice model. Results: Methanolic leaf extract has shown the highest antioxidant, as well as anticancer activity, is based on the assay conducted. For the identification of active phytochemicals from methanolic extract, High-resolution mass spectroscopy-LCMS was used. In vitro cytotoxicity study against MCF-7 and MDA-MB-231 cell line and IC 50 value was found to be 37.5µg/ml. From histopathological studies, no toxicity in liver and kidney tissue was identified. Conclusion: This plant tuber can be used as a regular diet to reduce the chance of breast cancer. Further, more studies should be conducted to isolate and identify the responsible compound.

Keywords: human breast adenocarcinoma, ROS, mitochondrial transmembrane, apoptosis

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Authors: Zelikha Labbani

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The In Vitro isolated micro spore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a micro spore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the micro spore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of micro spore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, micro spore became a strategy to achieve various objectives particularly in genetic engineering. In this study we would show the most recent advances in the producing haploid embryos via In Vitro isolated micro spore culture.

Keywords: haploid cells, In Vitro isolated microspore culture, success

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Authors: Thilivhali Emmanuel Tshikalange, Darky Cheron Modishane, Frederick Tawi Tabit

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The fruit pulp extracts of twelve selected ethnobotanical wild edible fruits from Mutale local municipality in Venda (Limpopo Province, South Africa) were investigated for their antimicrobial, antioxidant and cytotoxicity activities. Methanol extracts were prepared and tested against six micro-organisms (Salmonella typhi, Streptococcus pyogenes, Bacillus cereus, Klebsiella pneumoniae, Prevotella intermedia and Candida albicans). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the micro-dilution method, while for antioxidant activity the 2,2-diphenyl-1-picrylhydrazyl method was used. Of the 12 extracts tested, Adonsonia digitata, Berchemia discolor, Manilkara mochisia, Xanthocercis zambesiaca, Landolphia kirkii and Garcinia livingstonei showed antimicrobial activity, with MIC values ranging from 12.5 to 0.4 mg/ml. Gram negative bacteria were more resistant to the extracts in comparison to Gram positive bacteria. Antioxidant activity was only detected in Adonsonia digitata extract and the IC50 (substrate concentration to produce 50% reduction) was found to be 16.18µg/ml. The cytotoxicity of the extracts that showed antimicrobial and antioxidant activities was also determined. All plant extracts tested were non-toxic against human kidney cells (HEK293), with IC50 values of >400 µg/ml. The results presented in this study provide support to some traditional uses of wild edible fruits.

Keywords: antimicrobial, antioxidant, cytotoxicity, ethnobotanical, fruits

Procedia PDF Downloads 361

Authors: Gyati Shilakari Asthana, Abhay Asthana, Dharm Veer Kohli, Suresh Prasad Vyas

Abstract:

Purpose: The therapeutic potential of oligonucleotide (ODN) is primarily dependent upon its safe and efficient delivery to specific cells overcoming degradation and maximizing cellular uptake in vivo. The present study is focused to design low molecular weight chitosan nanoconstructs to meet the requirements of safe and effectual delivery of ODNs. LMW-chitosan is a biodegradable, water soluble, biocompatible polymer and is useful as a non-viral vector for gene delivery due to its better stability in water. Methods: LMW chitosan ODN nanoparticles (CHODN NPs) were formulated by self-assembled method using various N/P ratios (moles ratio of amine groups of CH to phosphate moieties of ODNs; 0.5:1, 1:1, 3:1, 5:1, and 7:1) of CH to ODN. The developed CHODN NPs were evaluated with respect to gel retardation assay, particle size, zeta potential and cytotoxicity and transfection efficiency. Results: Complete complexation of CH/ODN was achieved at the charge ratio of 0.5:1 or above and CHODN NPs displayed resistance against DNase I. On increasing the N/P ratio of CH/ODN, the particle size of the NPs decreased whereas zeta potential (ZV) value increased. No significant toxicity was observed at all CH concentrations. The transfection efficiency was increased on increasing N/P ratio from 1:1 to 3:1, whereas it was decreased with further increment in N/P ratio upto 7:1. Maximum transfection of CHODN NPs with both the cell lines (Raw 267.4 cells and Hela cells) was achieved at N/P ratio of 3:1. The results suggest that transfection efficiency of CHODN NPs is dependent on N/P ratio. Conclusion: Thus the present study states that LMW chitosan nanoparticulate carriers would be acceptable choice to improve transfection efficiency in vitro as well as in vivo delivery of oligonucleotide.

Keywords: LMW-chitosan, chitosan nanoparticles, biocompatibility, cytotoxicity study, transfection efficiency, oligonucleotide

Procedia PDF Downloads 824

Authors: Zelikha Labbani

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Since its creation in 1964 by Guha and Maheshwari in India on Datura innoxia Mill, in vitro androgenesis has become the method of choice in the production of doubled haploid in many species. However, in durum wheat, the Doubled haploid plant breeding programs remained limited due to the low production of androgenetic embryos and converting them into fertile green plants. We describe here an efficient method for inducing embryos and regenerating green plants directly from isolated microspores of durum wheat.

Keywords: durum wheat, haploid embryos, on in vitro, pretreatment

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Authors: Gayatri Nahak, Satyajit Kanungo, Rajani Kanta Sahu

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Holarrhena antidysenterica Linn. (Apocynaceae) is a typical Indian medicinal plant popularly known as “Indrajav”. Traditionally the plant has been considered a popular remedy for the treatment of dysentery, diarrhea, intestinal worms and the seeds of this plant are also used as an anti-diabetic remedy. In the present study axillary shoot multiplication, callus induction and shoot regeneration from callus culture were obtained on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. Then in vivo and in vitro grown healthy plants were selected for study of antioxidant activity through DPPH and OH methods. Significantly higher antioxidant activity and phenol contents were observed in vitro raised plant in comparison to in vivo plants. The findings indicated the greater amount of phenolic compounds leads to more potent radical scavenging effect as shown in in vitro raised plant in comparison to in vivo plants which showed the ability to utilize tissue culture techniques towards development of desired bioactive metabolites from in vitro culture as an alternative way to avoid using endangered plants in pharmaceutical purposes.

Keywords: Holarrhena antidysenterica, in vitro, in vivo, antioxidant activity

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Authors: Faraz Zarghami, Elham Anari, Nosratollah Zarghami, Yones Pilehvar-Soltanahmadi, Abolfazl Akbarzadeh, Sepideh Jalilzadeh-Tabrizi

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The development of nanotherapy has presented a new method of drug delivery targeted directly to the neoplasmic tissues, to maximize the action with fewer dose requirements. In the past two decades, poly(lactic-co-glycolic acid) (PLGA) has frequently been investigated by many researchers and is a popular polymeric candidate, due to its biocompatibility and biodegradability, exhibition of a wide range of erosion times, tunable mechanical properties, and most notably, because it is a FDA-approved polymer. Chrysin is a natural flavonoid which has been reported to have some significant biological effects on the processes of chemical defense, nitrogen fixation, inflammation, and oxidation. However, the low solubility in water decreases its bioavailability and consequently disrupts the biomedical benefits. Being loaded with PLGA-PEG increases chrysin solubility and drug tolerance, and decreases the discordant effects of the drug. The well-structured chrysin efficiently accumulates in the breast cancer cell line (T47D). In the present study, the structure and chrysin loading were delineated using proton nuclear magnetic resonance (HNMR), Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM), and the in vitro cytotoxicity of pure and nanochrysin was studied by the MTT assay. Next, the RNA was exploited and the cytotoxic effects of chrysin were studied by real-time PCR. In conclusion, the nanochrysin therapy developed is a novel method that could increase cytotoxicity to cancer cells without damaging the normal cells, and would be promising in breast cancer therapy.

Keywords: MTT assay, chrysin, flavonoids, nanotherapy

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Authors: Saravanan Govindaraju, Kyusik Yun

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The purpose of this study is to develop the chitosan doped curcumin gold cluster nanofiber for wound healing and skin cancer drug delivery applications. Chitosan is a typical marine polysaccharide composed of glucosamine and n-acetyl glucosamine biodegradable and biocompatible polymer. Curcumin is a natural bioactive molecule obtained from Curcuma longo, it mostly occurs in some Asian countries like India and China. It has naturally antioxidant, antimicrobial, wound healing and anticancer property. Due to this advantage, we prepared a combination of natural polymer chitosan with Curcumin and gold nanocluster nanofiber (CH-CUR-AuNCs nanofibers). The prepared nanofiber was characterized by using Fourier transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). Antibacterial studies were performed with E.coli and S.aureus. Antioxidant assay, drug release test, and cytotoxicity will be evaluated. Prepared nanofiber emits low intensity of red fluorescent. The FTIR confirm the presence of chitosan and Curcumin in the nanofiber. In vitro study clearly shows the antibacterial activity against the gram negative and gram positive bacteria. Particularly, synthesised nanofibers provide better antibacterial activity against gram negative than gram positive. Cytotoxicity study also provides better killing rate in cancer cell, biocompatible with normal cell. Prepared CH-CUR-AuNCs nanofibers provide the better killing rate to bacterial strains and cancer cells. Finally, prepared nanofiber can be possible to use for wound healing dressing, patch for skin cancer and other biomedical applications.

Keywords: curcumin, chitosan, gold clusters, nanofibers

Procedia PDF Downloads 238

Authors: Ibrahim A. H. Barakat, Ahmed R. Al-Himaidi

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The present study was conducted to determine the role and optimum concentration of fenugreek seed extract during in-vitro maturation on in-vitro maturation and developmental competence of Neaimi sheep oocytes following in-vitro fertilization. The Cumulus Oocyte Complexes (COCs) collected from sheep slaughterhouse ovaries were randomly divided into three groups, and they were matured for 24 hrs. in maturation medium containing fenugreek seed extract (0, 1 and 10 µg ml-1). Oocytes of a control group were matured in a medium containing 1 µg ml-1 estradiol 17β. After maturation, half of oocytes were fixed and stained for evaluation of nuclear maturation. The rest of oocytes were fertilized in vitro with fresh semen, then cultured for 9 days for the assessment of the developmental capacity of the oocytes. The results showed that the mean values of oocytes with expanded cumulus cells percentage were not significantly different among all groups (P < 0.05). But nuclear maturation rate of oocytes matured with 10 µg ml-1 fenugreek seed extract was significantly higher than that of the control group. The maturation rate and development to morula and blastocyst stage for oocytes matured at 10 µg ml-1 fenugreek seed extract was significantly higher than those matured at 1µg ml-1 of fenugreek seed extract and the control group. In conclusion, better maturation and developmental capacity rate to morula and blastocyst stage were obtained by the addition of 10 µg ml-1 fenugreek seed extract to maturation medium than addition of 1 µg ml-1 estradiol-17β (P < 0.05).

Keywords: fenugreek seed extract, in vitro maturation, sheep oocytes, in vitro fertilization, embryo development

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Authors: Miroslava Rysová, Zdena Syrová, Tomáš Zajíc, Petr Exnar

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Currently, attention of tissue engineers has been attracted to novel nanofibrous materials having advanced properties and ability to mimic extracellular matrix (ECM) by structure which makes them interesting candidates for application in regenerative medicine as scaffolding and/or drug delivering material. Throughout the last decade, more than 200 synthetic and natural polymers have been successfully electrospun leading to the formation of nanofibres with a wide range of chemical, mechanical and degradation properties. In this family, inorganic nanofibres represent very specific group offering an opportunity to manufacture inert to body, well degradable and in properties tunable material. Aim of this work, was to reveal unique properties of silica (SiO2, CAS 7631-86-9) nanofibres and their potential in field of regenerative medicine. Silica nanofibres were prepared by sol-gel method from tetraethyl orthosilicate (TEOS, CAS 78-10-4) as a precursor and subsequently manufactured by needleless electrospinning on NanospiderTM device. Silica nanofibres thermally stabilized under 200°C were confirmed to be fully biodegradable and soluble in several simulated body fluids. In vitro cytotoxicity tests of eluate (ES ISO 10993-5:1999) and in direct contact (ES ISO 10993-5:2009) showed no toxicity - e.g. cell viabilities reached values exceeding 80%. Those results were obtained equally from two different cell lines (Vero, 3T3). Non-toxicity of silaca nanofibres´ eluate was additionally confirmed in real time by testing on xCelligence (ACEA Biosciences, Inc.) device. Both cell types also showed good adhesion to material. To conclude, all mentioned results lead to resumption that silica nanofibres have a potential as material for regenerative medicine which opens door to further research.

Keywords: cytotoxicity, electrospinning, nanofibres, silica, tissue engineering

Procedia PDF Downloads 398

Authors: Mthokozisi Simelane

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Background: Malaria remains a life-threatening disease in tropical regions despite the advances in the treatment of this disease, it still remains a significant burden as some parasites have become resistant to the currently available drugs. This has created a necessity for the development of alternative, more efficient antimalarial drugs. Warburgia salutaris is a traditional medicinal plant used in malaria treatment by Zulu traditional healers. Materials and methods: The W. salutaris stem-bark was extracted with dichloromethane and the compound was isolated through column chromatography. The compound was identified and characterized by spectroscopic analysis (1H NMR, 13C NMR, IR and MS) and the structure was also confirmed by x-ray crystallography. The anti-plasmodial activity (in vitro) was studied on NF54 Plasmodium falciparum strain (CQS). Cytotoxicity was measured using the MTT assay on HEK239 and HEPG2 cell lines. Docking of Mukaadial acetate was conducted in AutoDock Vina. Structural modifications were conducted in UCSF Chimera and molecular interactions examined in LigPlot. Results: The compound, Mukaadial Acetate showed appreciable inhibition (IC50 0.44±0.10 µg/ml) of the parasite growth and cytotoxicity activity of 0.124±0.109 and 0.199±0.083 (µg/ml) on HEK293 and HEPG2 cells respectively. Molecular docking revealed that Mukaadial Acetate binds to the purine, pyrophosphate and ribose binding sites of the PfHGXPRT with an optimum binding conformation and forms hydrogen bond, steric and hydrophobic interactions with the residues inhabiting the respective binding sites. Conclusion: It is apparent that W. salutaris contains components (including Mukaadial Acetate) that exhibit antimalarial activity. This study scientifically validates the use of this plant in folk medicine.

Keywords: plasmodium falciparum, molecular docking, antimalarial activity, PfHGXPRT, Warburgia salutaris, mukaadial acetate

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Authors: Bello Gonzalez, Setten Van M., Brouwer M.

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The chicken small intestine represents a dynamic and complex organ in which the enzymatic digestion and absorption of nutrients take place. The development of an in vitro fermentation chicken small intestinal model could be used as an alternative to explore the interaction between the microbiota and nutrient metabolism and to enhance the efficacy of targeting interventions to improve animal health. In the present study we have developed an in vitro fermentation chicken ileum microbiota model for unrevealing the complex interaction of ileum microbial community under physiological conditions. A two-vessel continuous fermentation process simulating in real-time the physiological conditions of the ileum content (pH, temperature, microaerophilic/anoxic conditions, and peristaltic movements) has been standardized as a proof of concept. As inoculum, we use a pool of ileum microbial community obtained from chicken broilers at the age of day 14. The development and validation of the model provide insight into the initial characterization of the ileum microbial community and its dynamics over time-related to nutrient assimilation and fermentation. Samples can be collected at different time points and can be used to determine the microbial compositional structure, dynamics, and diversity over time. The results of studies using this in vitro model will serve as the foundation for the development of a whole small intestine in vitro fermentation chicken gastrointestinal model to complement our already established in vitro fermentation chicken caeca model. The insight gained from this model could provide us with some information about the nutritional strategies to restore and maintain chicken gut homeostasis. Moreover, the in vitro fermentation model will also allow us to study relationships between gut microbiota composition and its dynamics over time associated with nutrients, antimicrobial compounds, and disease modelling.

Keywords: broilers, in vitro model, ileum microbiota, fermentation

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Authors: Chen-En Lin, Lih-Rou Rau, Jiunn-Woei Liaw, Shiao-Wen Tsai

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The unique optical properties of plasmon resonance metallic particles have attracted considerable applications in the fields of physics, chemistry and biology. Metal-Enhanced Fluorescence (MEF) effect is one of the useful applications. MEF effect stated that fluorescence intensity can be quenched or be enhanced depending on the distance between fluorophores and the metal nanoparticles. Silver nanoparticles have used widely in antibacterial studies. However, the major limitation for silver nanoparticles (AgNPs) in biomedical application is well-known cytotoxicity on cells. There were numerous literatures have been devoted to overcome the disadvantage. The aim of the study is to evaluate the cytotoxicity and biocompatibility of silica coated AgNPs against NIH-3T3 cells. The results were shown that NIH-3T3 cells started to detach, shrink, become rounded and finally be irregular in shape after 24 h of exposure at 10 µg/ml AgNPs. Besides, compared with untreated cells, the cell viability significantly decreased to 60% and 40% which were exposed to 10 µg/ml and 20 µg/ml AgNPs respectively. The result was consistent with previously reported findings that AgNPs induced cytotoxicity was concentration dependent. However, the morphology and cell viability of cells appeared similar to the control group when exposed to 20 µg/ml of silica coated AgNPs. We further utilized the dark-field hyperspectral imaging system to analysis the optical properties of the intracellular nanoparticles. The image displayed that the red shift of the surface plasmonic resonances band of the enclosed AgNPs further confirms the agglomerate of the AgNPs rather than their distribution in cytoplasm. In conclusion, the study demonstrated the silica coated of AgNPs showed well biocompatibility and significant lower cytotoxicity compared with bare AgNPs.

Keywords: silver nanoparticles, silica, cell viability, morphology

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Authors: Pakakrong Thongdeeying, Srisopa Ruangnoo, Arunporn Itharat

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The rhizomes of Smilax corbularia Kunth have long been used as common ingredients in anticancer preparations. Thus, the objective of this study is to investigate cytotoxicity of S. corbularia and its ingredients against cholangiocarcinoma cell line (KKU-M156) by SRB assay. Ethanolic and water extracts of S. corbularia rhizomes were obtained using the procedures followed by Thai traditional doctors. Bioassay guided isolation was used to isolate cytotoxic compounds. The results revealed that the ethanolic extract of S. corbularia exhibited activity against KKU-M156 cell line with an IC50 value of 84.53±1.62 µg/ml, but the water extract showed no cytotoxic activity. Three flavonoid compounds [astilbin (1), engeletin (2), and quercetin (3)] were isolated from the ethanolic extract. Compound 3 exhibited the strongest activity against KKU-M156 cell line (IC50 = 8.14 ± 1.15 µg/ml), but 1 and 2 showed no cytotoxic activity (IC50 > 100 µg/ml). In conclusion, quercetin showed the highest efficacy against cholangiocarcinoma. These results support the traditional use of this plant by Thai traditional doctors for cancer treatment.

Keywords: cholangiocarcinoma, cytotoxicity, flavonoid, Smilax corbularia

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Authors: A. Zuchowska, A. Buta, M. Mazurkiewicz-Pawlicka, A. Malolepszy, L. Stobinski, Z. Brzozka

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In recent years, graphene-based materials are finding more and more applications in biological science. As a thin, tough, transparent and chemically resistant materials, they appear to be a very good material for the production of implants and biosensors. Interest in graphene derivatives also resulted at the beginning of research about the possibility of their application in cancer therapy. Currently, the analysis of their potential use in photothermal therapy and as a drug carrier is mostly performed. Moreover, the direct anticancer properties of graphene-based materials are also tested. Nowadays, cytotoxic studies are conducted on in vitro cell culture in standard culture vessels (macroscale). However, in this type of cell culture, the cells grow on the synthetic surface in static conditions. For this reason, cell culture in macroscale does not reflect in vivo environment. The microfluidic systems, called Lab-on-a-chip, are proposed as a solution for improvement of cytotoxicity analysis of new compounds. Here, we present the evaluation of cytotoxic properties of graphene oxide (GO) on breast, liver and colon cancer cell line in a microfluidic system in two spatial models (2D and 3D). Before cell introduction, the microchambers surface was modified by the fibronectin (2D, monolayer) and poly(vinyl alcohol) (3D, spheroids) covering. After spheroid creation (3D) and cell attachment (2D, monolayer) the selected concentration of GO was introduced into microsystems. Then monolayer and spheroids viability/proliferation using alamarBlue® assay and standard microplate reader was checked for three days. Moreover, in every day of the culture, the morphological changes of cells were determined using microscopic analysis. Additionally, on the last day of the culture differential staining using Calcein AM and Propidium iodide were performed. We were able to note that the GO has an influence on all tested cell line viability in both monolayer and spheroid arrangement. We showed that GO caused higher viability/proliferation decrease for spheroids than a monolayer (this was observed for all tested cell lines). Higher cytotoxicity of GO on spheroid culture can be caused by different geometry of the microchambers for 2D and 3D cell cultures. Probably, GO was removed from the flat microchambers for 2D culture. Those results were also confirmed by differential staining. Comparing our results with the studies conducted in the macroscale, we also proved that the cytotoxic properties of GO are changed depending on the cell culture conditions (static/ flow).

Keywords: cytotoxicity, graphene oxide, monolayer, spheroid

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Authors: Ajay Pratap Singh Lodhi, Deepak Kumar

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Vegetable oil-based environmentally friendly metalworking fluids (MWFs) are formulated. The tribological performance, cytotoxicity, and corrosion resistance of the formulated fluids (FFs) are evaluated and benchmarked with commercial mineral oil-based MWFs (CF). Results show that FFs exhibited better machining characteristics (roughness, cutting forces, and surface morphology) during machining than CF. MTT assay and Live dead cell assay confirm the cytocompatibility nature of the FFs relative to the toxic CF. Electrochemical analysis shows that FFs and CF exhibited comparable corrosion current density.

Keywords: corrosion inhibitors, cytotoxicity, machining, MTT assay, Taguchi method, vegetable oil

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Authors: Amir Saber Gharamaleki, Beitollah Alipour, Zeinab Faghfoori, Ahmad YariKhosroushahi

Abstract:

Colorectal cancer (CRC) is one of the most prevalent cancers and intestinal microbial community plays an important role in colorectal tumorigenesis. Probiotics have recently been assessed as effective anti-proliferative agents and thus this study was performed to examine whether CRC undergo apoptosis by treating with isolated Iranian native dairy yeast, Kluyveromyces marxianus YAS, secretion metabolites. The cytotoxicity assessments on cells (HT-29, Caco-2) were accomplished through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as well as qualitative DAPI (4',6-diamidino-2-phenylindole staining) and quantitative (flow cytometry assessments) evaluations of apoptosis. To evaluate the main mechanism of apoptosis, Real time PCR method was applied. Kluyveromyces marxianus YAS secretions (IC50) showed significant cytotoxicity against HT-29 and Caco-2 cancer cell lines (66.57 % and 66.34 % apoptosis) similar to 5-Fluorouracil (5-FU) while apoptosis only was developed in 27.57 % of KDR normal cells. The prophylactic effects of Kluyveromyces marxianus (PTCC 5195), as a reference yeast, was not similar to Kluyveromyces marxianus YAS indicating strain dependency of bioactivities on CRC disease prevention. Based on real time PCR results, the main cytotoxicity is related to apoptosis phenomenon and the core related mechanism is depended on the overexpression of BAX, CASP 9, CASP 8 and CASP 3 inducing apoptosis genes. However, several investigations should be conducted to precisely determine the effective compounds to be used as anticancer therapeutics in the future.

Keywords: anticancer, anti-proliferative, apoptosis, cytotoxicity, yeast

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Authors: A. N.Ukwuani, A. K. Abdulfatah

Abstract:

G. crenata is used locally for the treatment of fractured bones, wound healing and inflammatory conditions. In vitro and in vivo antioxidant activity of hydromethanolic extracts of the leaves of G. crenata were assessed. The phytochemical analysis shows the presence of phenols, flavonoids, saponins, cardiac glycosides and tannins. An in vitro quantitative analysis of phenols, flavonoids and tannins respectively were (164±1.20, 199±0.88 and 88.67±0.88 mg/100g FW). In vivo studies of hydromethanolic extract demonstrated a dose dependent increase in hepatic superoxide dismutase (1.14±0.14, 2.13±0.11, 2.55±0.11 U/mg Protein) with improvement in hepatic glutathione (6.98±0.42, 8.91±0.37, 11.07±0.46 µM/mg Protein) and Catalase (4.47±0.05, 6.24±0.02, 7.17±0.04 U/mg Protein) and Total protein (6.18±0.08, 6.69±0.18, 7.27±0.16 mg/ml) respectively at 100-300mg/kg body weight Grewia crenata leaves when compared to the control and standard drug. It can be concluded from the present findings of that G. crenata leaves possess antioxidant potential.

Keywords: Grewia crenata, antioxidant, hydromethanolic extract, in vivo, in vitro

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Authors: Sobhy El-Sohaimy, Abduvali Toshev, Hanem Mansour

Abstract:

(1) Objective: The main objective of the current study was to evaluate in an In Vitro and In Vivo, the potential activity of olive leaves extract (OLE) in the treatment and/or preventing the diabetes mellitus type II and related implications; (2) Methodology: Five groups of male rats were used in the current study: group (1)- negative control (normal); group (2)- positive control, streptozotocin (STZ) induced rats; group (3)-diabetic rats treated with metformin (200 mg/kg) plus OLE (200 mg/kg); group 4- diabetic rats treated with metformin (200 mg/kg); group 5- diabetic rats treated with OLE (200 mg/kg). A four-week regime of oral treatment was administered once daily; (3) Results: Diabetic rats treated with metformin + OLE clearly showed normal blood glucose level (121.67 ± 5.49 mg/dl), and glycated hemoglobin (HbA1c) (3.70 ± 0.10%). The combination of metformin + OLE obviously showed a superior improvement in the lipid profile (TG, TC, HDL and LDL) compared to both metformin and OLE individually. The histological examination revealed that the combination of metformin + Olive leaves extract successfully repaired of the liver, kidneys, and pancreatic tissues in diabetic rats to be near to the normal status; (4) Conclusion: Finally, it can be concluded that, the combination of metformin and OLE exhibited a superior improvement than metformin and OLE individually which emphasized the promising adjuvant role of the OLE in the treatment protocol of diabetes mellitus type II.

Keywords: olive (olea europaea) leaves extracts, hypoglycemic agents, cytotoxicity, nitic oxide scavenging activity, α-glucose oxidase inhibitor

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Authors: Vijayakameswara Rao Neralla, Jueun Jeon, Jae Hyung Park

Abstract:

To develop a potential nanocarrier for diagnosis and treatment of rheumatoid arthritis (RA), we prepared a hyaluronic acid (HA)-5β-cholanic acid (CA) conjugate with an acid-labile ketal linker. This conjugate could self-assemble in aqueous conditions to produce pH-responsive HA-CA nanoparticles as potential carriers of the anti-inflammatory drug methotrexate (MTX). MTX was rapidly released from nanoparticles under inflamed synovial tissue in RA. In vitro cytotoxicity data showed that pH-responsive HA-CA nanoparticles were non-toxic to RAW 264.7 cells. In vivo biodistribution results confirmed that, after their systemic administration, pH-responsive HA-CA nanoparticles selectively accumulated in the inflamed joints of collagen-induced arthritis mice. These results indicate that pH-responsive HA-CA nanoparticles represent a promising candidate as a drug carrier for RA therapy.

Keywords: rheumatoid arthritis, hyaluronic acid, nanocarrier, self-assembly, MTX

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Authors: Onmanee Prajuabjinda, Pakakrong Thondeeying, Jipisute Chunthorng-Orn, Bhanuz Dechayont, Arunporn Itharat

Abstract:

A Thai medicinal preparation has been used for cancer treatment more than ten years ago in Khampramong Temple. Tectona grandis stem is one ingredient of this Thai medicinal remedy. The ethanolic extract of Tectona grandis stem showed the highest cytotoxic activities against human breast adenocarcinoma (MCF-7), but was less cytotoxic against large cell lung carcinoma (COR-L23) (IC50 = 3.92 and 7.78 µg/ml, respectively). It was isolated by bioassay-guided isolation method. Tectoquinone, a anthraquinone compound was isolated from this plant. This compound showed high specific cytotoxicity against human breast adenocarcinoma (MCF-7), but was less cytotoxic against large cell lung carcinoma (COR-L23)(IC50 =16.15 and 47.56 µg/ml or 72.67 and 214.00 µM, respectively). However, it showed less cytotoxic activity than the crude extract. In conclusion, tectoquinone as a main compound, is not the best cytotoxic compound from Tectona grandis, so there are more active cytotoxic compounds in this extract which should be isolated in the future. Moreover, tectoquinone displayed specific cytotoxicity against only human breast adenocarcinoma (MCF-7) which is a good criterion for cancer treatment.

Keywords: Tectona grandis, SRB assay, cytotoxicity, tectoquinone

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Authors: Kehinde T. Kareem

Abstract:

The need to increase the production of cucumber has led to the use of inorganic fertilizers. This chemical affects the ecological balance of nature by increasing the nitrogen and phosphorus contents of the soil. Surface runoffs into rivers and streams cause eutrophication which affects aquatic organisms as well as the consumers of aquatic animals. Therefore, this study was carried out in the screenhouse to investigate the use of a plant growth-promoting fungus; Trichoderma longibrachiatum for the growth promotion of conventional and in-vitro propagated Ashley and Marketmoor cucumber. Before planting of cucumber, spore suspension (108 cfu/ml) of Trichoderma longibrachiatum grown on Potato dextrose agar (PDA) was inoculated into the soil. Fruits were evaluated for the presence of Trichoderma longibrachiatum using a species-specific primer. Results revealed that the highest significant plant height produced by in-vitro propagated Ashley was 19 cm while the highest plant height of in-vitro propagated Marketmoor was 19.67 cm. The yield of the conventional propagated Ashley cucumber showed that the number of fruit/plant obtained from T. longibrachiatum-fertilized plants were significantly more than those of the control. The in-vitro Ashely had 7 fruits/plant while the control produced 4 fruits/plant. In-vitro Marketmoor had ten fruits/plant, and the control had a value of 4 fruits/plant. There were no traces of Trichoderma longibrachiatum genes in the harvested cucumber fruits. Therefore, the use of Trichoderma longibrachiatum as a plant growth-promoter is safe for human health as well as the environment.

Keywords: biofertilizer, cucumber, genes, growth-promoter, in-vitro, propagation

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Authors: A. Reum Son, Jin Seon Kwon, Seung Hun Park, Hai Bang Lee, Moon Suk Kim

Abstract:

These days, stem cell therapy is focused on for promising source of treatment in clinical human disease. As a supporter of stem cells, in situ-forming hydrogels with growth factors and cells appear to be a promising approach in tissue engineering. To examine osteogenic differentiation of hTMSCs which is one of mesenchymal stem cells in vivo in an injectable hydrogel, we use a methoxy polyethylene glycol-polycaprolactone blockcopolymer (MPEG-PCL) solution with osteogenic factors. We synthesized MPEG-PCL hydrogel and measured viscosity to check sol-gel transition. In order to demonstrate osteogenic ability of hTMSCs, we conducted in vitro osteogenesis experiment. Then, to confirm the cell cytotoxicity, we performed WST-1 with hTMSCs and MPEG-PCL. As the result of in vitro experiment, we implanted cell and hydrogel mixture into animal model and checked degree of osteogenesis with histological analysis and amount of expression genes. Through these experimental data, MPEG-PCL hydrogel has sol-gel transition in temperature change and is biocompatible with stem cells. In histological analysis and gene expression, hTMSCs are very good source of osteogenesis with hydrogel and will use it to tissue engineering as important treatment method. hTMSCs could be a good adult stem cell source for usability of isolation and high proliferation. When hTMSCs are used as cell therapy method with in situ-formed hydrogel, they may provide various benefits like a noninvasive alternative for bone tissue engineering applications.

Keywords: injectable hydrogel, stem cell, osteogenic differentiation, tissue engineering

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Authors: Genesis Julyus T. Agcaoili, Esperanza C. Cabrera

Abstract:

Thirty (30) isolates of lactic acid bacteria (LAB) from traditionally-prepared fermented products specifically fermented soy-bean paste, fermented mustard and fermented rice-fish mixture were studied for their in vitro antimicrobial and cytotoxic activities. Seventeen (17) isolates were identified as Lactobacillus plantarum, while 13 isolates were identified as Enterococcus spp using 16s rDNA sequences. Disc diffusion method was used to determine the antibacterial activity of LAB against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922), while the modified agar overlay method was used to determine the antifungal activity of LAB isolates on the yeast Candida albicans, and the dermatophytes Microsporum gypseum, Trichophyton rubrum and Epidermophyton floccosum. The filter-sterilized LAB supernatants were evaluated for their cytotoxicity to mammalian colon cancer cell lines (HT-29 and HCT116) and normal human dermal fibrolasts (HDFn) using resazurin assay (PrestoBlueTM). Colchicine was the positive control. No antimicrobial activity was observed against the bacterial test organisms and the yeast Candida albicans. On the other hand, all of the tested LAB strains were fungicidal for all the test dermatophytes. Cytotoxicity index profiles of the supernatants of the 15 randomly picked LABs and negative control (brain heart infussion broth) suggest nontoxicity to the cells when compared to colchicine, whereas all LAB supernatants were found to be cytotoxic to HT-29 and HCT116 colon cancer cell lines. Results provide strong support for the role of the lactic acid bacteria studied in antimicrobial treatment and anticancer therapy.

Keywords: antimicrobial, fermented products, fungicidal activity, lactic acid bacteria, probiotics

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