Search results for: luciferase assay

Authors: Aussara Panya, Nunghathai Sawasdee, Mutita Junking, Chatchawan Srisawat, Kiattawee Choowongkomon, Pa-Thai Yenchitsomanus

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Dengue virus (DENV) infection is a global public health problem with approximately 100 million infected cases a year. Presently, there is no approved vaccine or effective drug available; therefore, the development of anti-DENV drug is urgently needed. The clinical reports revealing the positive association between the disease severity and viral titer has been reported previously suggesting that the anti-DENV drug therapy can possibly ameliorate the disease severity. Although several anti-DENV agents showed inhibitory activities against DENV infection, to date none of them accomplishes clinical use in the patients. The surface envelope (E) protein of DENV is critical for the viral entry step, which includes attachment and membrane fusion; thus, the blocking of envelope protein is an attractive strategy for anti-DENV drug development. To search the safe anti-DENV agent, this study aimed to search for novel peptide inhibitors to counter DENV infection through the targeting of E protein using a structure-based in silico design. Two selected strategies has been used including to identify the peptide inhibitor which interfere the membrane fusion process whereby the hydrophobic pocket on the E protein was the target, the destabilization of virion structure organization through the disruption of the interaction between the envelope and membrane proteins, respectively. The molecular docking technique has been used in the first strategy to search for the peptide inhibitors that specifically bind to the hydrophobic pocket. The second strategy, the peptide inhibitor has been designed to mimic the ectodomain portion of membrane protein to disrupt the protein-protein interaction. The designed peptides were tested for the effects on cell viability to measure the toxic to peptide to the cells and their inhibitory assay to inhibit the DENV infection in Vero cells. Furthermore, their antiviral effects on viral replication, intracellular protein level and viral production have been observed by using the qPCR, cell-based flavivirus immunodetection and immunofluorescence assay. None of tested peptides showed the significant effect on cell viability. The small peptide inhibitors achieved from molecular docking, Glu-Phe (EF), effectively inhibited DENV infection in cell culture system. Its most potential effect was observed for DENV2 with a half maximal inhibition concentration (IC50) of 96 μM, but it partially inhibited other serotypes. Treatment of EF at 200 µM on infected cells also significantly reduced the viral genome and protein to 83.47% and 84.15%, respectively, corresponding to the reduction of infected cell numbers. An additional approach was carried out by using peptide mimicking membrane (M) protein, namely MLH40. Treatment of MLH40 caused the reduction of foci formation in four individual DENV serotype (DENV1-4) with IC50 of 24-31 μM. Further characterization suggested that the MLH40 specifically blocked viral attachment to host membrane, and treatment with 100 μM could diminish 80% of viral attachment. In summary, targeting the hydrophobic pocket and M-binding site on the E protein by using the peptide inhibitors could inhibit DENV infection. The results provide proof of-concept for the development of antiviral therapeutic peptide inhibitors to counter DENV infection through the use of a structure-based design targeting conserved viral protein.

Keywords: dengue virus, dengue virus infection, drug design, peptide inhibitor

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Authors: Patience Orobosa Olajide

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Five hydrocarbon degrading bacterial strains isolated from contaminated environment were investigated with respect to polyhydroxybutyrate (PHB) biosynthesis. Screening for bioplastic production was done on assay mineral salts agar medium containing 0.2% poly (3-hydroxybutyrate) as the sole carbon source. Two of the test bacteria were positive for PHB biosynthesis and were identified based on gram staining, biochemical tests, 16S rRNA gene sequence analysis as Pseudomonas aeruginosa and Bacillus licheniformis which grew at 37 and up to 65 °C respectively, thus suggesting the later to be thermotolerant. In this study, the effects of different carbon and nitrogen sources on PHB production in these strains were investigated. Maximum PHB production was obtained in 48 hr for the two strains and amounted to yields of 72.86 and 62.22 percentages for Bacillus licheniformis and Pseudomonas aeruginosa respectively. In these strains, glycine was the most efficient carbon sources for the production of PHB compared with other carbon (glucose, lactose, sucrose, Arabinose) and nitrogen (L- glycine, L-cysteine, DL-Tryptophan, and Potassium Nitrate) sources. The screening of microbial strains for industrial PHB production should be based on several factors including the cell’s capability to mineralize an inexpensive substrate, rate of growth and the extent of polymer accumulation.

Keywords: bacteria, poly-3-hydroxybutyrate (PHB), hydrocarbon, thermotolerant

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Authors: Zakieh Rostamzadeh Khameneh, Nariman Sepehrvand, Khalkhali-Zahra Shirmohamadi

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Background: Although the hepatitis E virus mostly causes a self-limited disease in general population, the disease is more severe in pregnant women. Hepatitis E accounts for about 10% of pregnancy-associated deaths in southern Asia. Methods: 136 pregnant women who referred to urban health centers of Urmia for pursuing pregnancy-related health services were selected randomly and enrolled in a descriptive, cross-sectional study. Each subject was tested for the presence of anti-HEV IgG antibody using an enzyme-linked immunosorbent assay (ELISA, Dia.Pro). Results: The mean age among 136 pregnant women was 25.12±4.91 years old (range of 14-39 years). Only five cases (3.6%) among all 136 subjects were demonstrated to be seropositive for anti-HEV IgG using ELISA method. There was no significant difference between age (P=0.88), income level (P=0.19) of two seropositive and seronegative groups. All seropositive cases were from urban areas. Conclusion: The seroprevalence of anti-HEV IgG is low in the population of pregnant women in Urmia, Iran. Because of limited sample size in this study, we recommend to perform further studies with larger sample size in other regions of Iran in order to be able to systematically generalize the findings of studies to the population of Iranian pregnant women.

Keywords: pregnancy, hepatitis E, women, ELISA

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Authors: Supakan Rattanakon, Jakkrapan Boonpimon, Akkaragiat Bhuangsaeng, Aphiwat Ratriphruek

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Rice bran oil (RBO) has been found to lower the level of serum cholesterol, has antioxidant and anti-carcinogenic property, and attenuate allergic inflammation. These properties of RBO are due to antioxidant compositions, especially, phenolic compounds. The higher amount of these active compounds in RBO, the greater value of RBO is. Thermal process of rice bran before solvent RBO extraction has been found to have a higher phenolic contents. Therefore, the purpose of this study is to using different heating methods on rice bran before the solvent extraction. Then, % yield of RBO, total phenolic content (TPC), and antioxidant property of two white Thai rice; KDML105 and RD6 were determined. The Folin-Ciocalteu colorimetric assay was used to determine TPC and scavenging of free radicals (DPPH) was used to determine antioxidant property expressed as EC50. The result showed that thermal process did not increase % yield of RBO but increase the TPC with 1.41 mg gallic acid equivalent (GAEmg-1). The highest TPC was found in KDML105 by using sonicator. The highest antioxidant activity was found in RD6 using autoclave. The EC50 of RBO was 0.04 mg/mL. Further study should be performed on different pretreatments to increase the TPC and antioxidant property.

Keywords: antioxidant, rice bran oil, total phenol content, white rice

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Authors: Aferdita Dinaku, Jonida Canaj

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Aflatoxins are potentially toxic metabolites produced by certain kinds of fungi (molds) that are found naturally all over the world; they can contaminate food crops and pose a serious health threat to humans by mutagenic and carcinogenic effects. Several types of aflatoxin (14 or more) occur in nature. In Albanian nutrition, cereals (especially wheat and corn) are common ingredients in some traditional meals. This study aimed to investigate the presence of aflatoxins in the flour of wheat and maize that are consumed in Albania’s markets. The samples were collected randomly in different markets in Albania and detected by the ELISA method, measured in 450 nm. The concentration of total aflatoxins was analyzed by enzyme-linked immunosorbent assay (ELISA), and they were ranged between 0.05-1.09 ppb. However, the screened mycotoxin levels in the samples were lower than the maximum permissible limits of European Commission No 1881/2006 (4 μg/kg). The linearity of calibration curves was good for total aflatoxins (B1, B2, G1, G2, M1) (R²=0.99) in the concentration range 0.005-4.05 ppb. The samples were analyzed in two replicated measurements and for each sample, the standard deviation (statistical parameter) is calculated. The results showed that the flour samples are safe, but the necessity of performing such tests is necessary.

Keywords: aflatoxins, ELISA technique, food contamination, flour

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Authors: Satya Eswari Jujjavarapu, Swasti Dhagat, Lata Upadhyay, Reecha Sahu

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Silver nanoparticles have a broad range of antimicrobial and antifungal properties ranging from soaps, pastes to sterilization and drug delivery systems. These can be synthesized by physical, chemical and biological methods; biological methods being the most popular owing to their non-toxic nature and reduced energy requirements. Microbial surfactants, produced on the microbial cell surface or excreted extracellularly are an alternative to synthetic surfactants for the production of silver nanoparticles. Hence, they are also called as green molecules. Microbial lipopeptide surfactants (biosurfactant) exhibit anti-tumor and anti-microbial properties and can be used as drug delivery agents. In this study, biosurfactant was synthesized by using a strain of acillus subtilis. The biosurfactant thus produced was analysed by emulsification assay, oil spilling test, and haemolytic test. Biosurfactant-mediated silver nanoparticles were synthesised by microwave irradiation of the culture supernatant and further characterized by UV–vis spectroscopy for a range of 400-600 nm. The UV–vis spectra showed a surface plasmon resonance vibration band at 410 nm corresponding to the peak of silver nanoparticles.

Keywords: biosurfactant, Bacillus subtilis, silver nano particle, lipopeptide

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Authors: Mahmoud Mabrok, Channarong Rodkhum

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Flavobacterium columanre is one of the devastating pathogen that causes noticeable economic losses in freshwater cultured fish. Like other filamentous bacteria, F. columanre tends to aggregate and fluctuate to all kind of media, thus revealing obstacles in recognition of its colonies. Since the molecular typing is the only fundamental tool for rapid and precise detection of this pathgen. The present study developed a species-specific PCR assay based on cslA unique gene of F. columnare. The cslA gene sequences of 13 F. columnare, strains retrieved from gene bank database, were aligned to identify a conserved homologous segment prior to primers design. The new primers yielded amplicons of 287 bp from F. columnare strains but not from relevant or other pathogens, unlike to other published set that showed no specificity and cross-reactivity with F. indicum. The primers were sensitive and detected as few as 7 CFUs of bacteria and 3 pg of gDNA template. The sensitivity was reduced ten times when using tissue samples. These primers precisely defined all field isolates in a double-blind study, proposing their applicable use for field detection.

Keywords: Columnaris infection, cslA gene, Flavobacterium columnare, PCR

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Authors: Shahid-ul-Islam, Faqeer Mohammad

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The present study was conducted to investigate the effect of annatto, teak and flame of the forest natural dyes on color, fastness, and antimicrobial property of protein based textile substrate. The color strength (K/S) of wool samples at various concentrations of dyes were analysed using a Reflective Spectrophotometer. The antimicrobial activity of natural dyes before and after application on wool was tested against common human pathogens Escherichia coli, Staphylococcus aureus, and Candida albicans, by using micro-broth dilution method, disc diffusion assay and growth curve studies. The structural morphology of natural protein fibre (wool) was investigated by Scanning Electron Microscopy (SEM). Annatto and teak natural dyes proved very effective in inhibiting the microbial growth in solution phase and after application on wool and resulted in a broad beautiful spectrum of colors with exceptional fastness properties. The results encourage the search and exploitation of new plant species as source of dyes to replace toxic synthetic antimicrobial agents currently used in textile industry.

Keywords: annatto, antimicrobial agents, natural dyes, green textiles

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Authors: Sana Abbasa, Shahzad Bhattiab, Nadir Khan

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Sargassum siliquastrum is brown seaweed with traditional claims for some medicinal properties. This research was done to investigate the methanol extract of S. siliquastrum for antiproliferative activity against human cervical cancer cell line, HeLa and its mode of cell death. From methylene blue assay, S. siliquastrum exhibited antiproliferative activity on HeLa cells with IC50 of 3.87 µg/ml without affecting non-malignant cells. Phase contrast microscopy indicated the confluency reduction in HeLa cells and changes on the cell shape. Nuclear staining with Hoechst 33258 displayed the formation of apoptotic bodies and fragmented nuclei. S. siliquastrum also induced early apoptosis event in HeLa cells as confirmed by FITC-Annexin V/propidium iodide staining by flow cytometry analysis. Cell cycle analysis indicated growth arrest of HeLa cells at G1/S phase. Protein study by flow cytometry indicated the increment of p53, slight increase of Bax and unchanged level of Bcl-2. In conclusion, S. siliquastrum demonstrated an antiproliferative activity in HeLa cell by inducing G1/S cell cycle arrest via p53-mediated pathway.

Keywords: sargassum siliquastrum, cervical cancer, P53, antiproleferation

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Authors: Drashti G. Daraji, Rosa E. Moo-Puc, Hitesh D. Patel

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Substituted 2-Thioimidazole analogues have been synthesized and confirmed by advanced spectroscopic techniques. Among them, ten compounds have been selected and evaluated for their in vitro anti-cancer activity at the National Cancer Institute (NCI) for testing against a panel of 60 different human tumor cell lines derived from nine neoplastic cancer types. Furthermore, synthesized compounds were tested for their in vitro antiprotozoal activity, and none of them exhibited significant potency against antiprotozoans. It was observed that the tested all compounds seem effective on the UACC-62 melanoma cancer cell line as compared to other cancer cell lines and also exhibited the least potent in the Non-Small Cell Lung Cancer cell line in one-dose screening. In silico studies of these derivatives were carried out by molecular docking techniques and Absorption, Distribution, Metabolism, and Excretion (ADME) using Schrödinger software to find potent B-Raf kinase inhibitor (PDB ID: 3OG7). All the compounds have been performed for docking study; Compound D4 has a good docking score for melanoma cancer as compared with other.

Keywords: anticancer activity, cancer cell line, 2-thio imidazole, one-dose assay, molecular docking

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Authors: Achraf Al Faraj, Asma Sultana Shaik, Baraa Al Sayed

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Specific and effective targeting of drug delivery systems (DDS) to cancerous sites remains a major challenge for a better diagnostic and therapy. Recently, SWCNTs with their unique physicochemical properties and the ability to cross the cell membrane show promising in the biomedical field. The purpose of this study was first to develop a biocompatible iron oxide tagged SWCNTs as diagnostic nanoprobes to allow their noninvasive detection using MRI and their preferential targeting in a breast cancer murine model by placing an optimized flexible magnet over the tumor site. Magnetic targeting was associated to specific antibody-conjugated SWCNTs active targeting. The therapeutic efficacy of doxorubicin-conjugated SWCNTs was assessed, and the superiority of diffusion-weighted (DW-) MRI as sensitive imaging biomarker was investigated. Short Polyvinylpyrrolidone (PVP) stabilized water soluble SWCNTs were first developed, tagged with iron oxide nanoparticles and conjugated with Endoglin/CD105 monoclonal antibodies. They were then conjugated with doxorubicin drugs. SWCNTs conjugates were extensively characterized using TEM, UV-Vis spectrophotometer, dynamic light scattering (DLS) zeta potential analysis and electron spin resonance (ESR) spectroscopy. Their MR relaxivities (i.e. r1 and r2*) were measured at 4.7T and their iron content and metal impurities quantified using ICP-MS. SWCNTs biocompatibility and drug efficacy were then evaluated both in vitro and in vivo using a set of immunological assays. Luciferase enhanced bioluminescence 4T1 mouse mammary tumor cells (4T1-Luc2) were injected into the right inguinal mammary fat pad of Balb/c mice. Tumor bearing mice received either free doxorubicin (DOX) drug or SWCNTs with or without either DOX or iron oxide nanoparticles. A multi-pole 10x10mm high-energy flexible magnet was maintained over the tumor site during 2 hours post-injections and their properties and polarity were optimized to allow enhanced magnetic targeting of SWCNTs toward the primary tumor site. Tumor volume was quantified during the follow-up investigation study using a fast spin echo MRI sequence. In order to detect the homing of SWCNTs to the main tumor site, susceptibility-weighted multi-gradient echo (MGE) sequence was used to generate T2* maps. Apparent diffusion coefficient (ADC) measurements were also performed as a sensitive imaging biomarker providing early and better assessment of disease treatment. At several times post-SWCNT injection, histological analysis were performed on tumor extracts and iron-loaded SWCNT were quantified using ICP-MS in tumor sites, liver, spleen, kidneys, and lung. The optimized multi-poles magnet revealed an enhanced targeting of magnetic SWCNTs to the primary tumor site, which was found to be much higher than the active targeting achieved using antibody-conjugated SWCNTs. Iron-loading allowed their sensitive noninvasive tracking after intravenous administration using MRI. The active targeting of doxorubicin through magnetic antibody-conjugated SWCNTs nanoprobes was found to considerably decrease the primary tumor site and may have inhibited the development of metastasis in the tumor-bearing mice lung. ADC measurements in DW-MRI were found to significantly increase in a time-dependent manner after the injection of DOX-conjugated SWCNTs complexes.

Keywords: single-walled carbon nanotubes, nanomedicine, magnetic resonance imaging, cancer diagnosis and therapy

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Authors: H. Aksas, S. Boughrara, K. Louhab

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The goal of this study was the use of life cycle assessment (LCA) methodology to assess the environmental impact of pharmaceutical product (four kinds of syrup form and tree kinds of pomade form), which are produced in one leader manufactory in Algeria town that is SAIDAL Company. The impacts generated have evaluated using SimpaPro7.1 with CML92 Method for syrup form and EPD 2007 for pomade form. All impacts evaluated have compared between them, with determination of the compound contributing to each impacts in each case. Data needed to conduct Life Cycle Inventory (LCI) came from this factory, by the collection of theoretical data near the responsible technicians and engineers of the company, the practical data are resulting from the assay of pharmaceutical liquid, obtained at the laboratories of the university. This data represent different raw material imported from European and Asian country necessarily to formulate the drug. Energy used is coming from Algerian resource for the input. Outputs are the result of effluent analysis of this factory with different form (liquid, solid and gas form). All this data (input and output) represent the ecobalance.

Keywords: pharmaceutical product, drug residues, LCA methodology, environmental impacts

Procedia PDF Downloads 247

Authors: Purnima Rawat, Divya Vohora, Sarika Gupta, Farhan J. Ahmad, Sushama Talegaonkar

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Nanoparticles (NPs) that target bone tissue were developed using PLGA–mPEG (poly(lactic-co-glycolic-acid)–polyethylene glycol) diblock copolymers by using pamidronate as a bone-targeting moieties. These NPs are expected to enable the transport of hydrophilic drugs. The NP was prepared by in situ polymerization method, and their in- vitro characteristics were evaluated using dynamic light scattering, transmission electron microscopy (TEM) and in phosphate-buffered solution. The bone targeting potential of the NP was also evaluated on in-vitro pre-osteoblast MCT3E1 cell line using ALP activity, degree of mineralization and RT-PCR assay. The average particle size of the NP was 101.6 ± 3.7nm, zeta potential values were negative (-25±0.34mV) of the formulations and the entrapment efficiency was 93± 3.1 % obtained. The moiety of the PLGA–mPEG–pamidronate NPs exhibited the best apatite mineral binding ability in-vitro MCT3E1 pre-osteoblast cell line. Our results suggested that the developed nanoparticles may use as a delivery system for Pamidronate in bone repair and regeneration, warranting further evaluation of the treatment of bone disease.

Keywords: nanoparticle, pamidronate, in-situ polymerization, osteoblast

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Authors: Ashish Kumar Jain, Deepak Mishra

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The objective of the present investigation was to prepare and evaluate a vesicular dual drug delivery system for effective management of mucosal ulcer. Inner encapsulating and Double liposomes were prepared by glass bead and reverse phase evaporation method respectively. The formulation consisted of inner liposomes bearing Ranitidine Bismuth Citrate (RBC) and outer liposomes encapsulating Amoxicillin trihydrate (AMOX). The optimized inner liposomes and double liposomes were extensively characterized for vesicle size, morphology, zeta potential, vesicles count, entrapment efficiency and in vitro drug release. In vitro, the double liposomes demonstrated a sustained release of AMOX and RBC viz 91.4±1.8% and 77.2±2.1% respectively at the end of 72 hr. Furthermore binding specificity and targeting propensity toward H. pylori (SKP-56) was confirmed by agglutination and in situ adherence assay. Reduction of the absolute alcohol induced ulcerogenic index from 3.01 ± 0.25 to 0.31 ± 0.09 and 100% H. pylori clearance rate was observed. These results suggested that double liposomes are potential vector for the development of dual drug delivery for effective treatment of H. pylori-associated peptic ulcer.

Keywords: double liposomes, H. pylori targeting, PE liposomes, glass-beads method, peptic ulcers

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Authors: Izdihar Ismail, Alona C. Linatoc, Maryati Mohamed

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The tropical rainforest of Malaysia is known for its rich biological diversity and high endemicity. The potential for these forests to hold the cure for many diseases and illnesses is high and much is yet to be discovered. This study explores the richness of the tropical rainforest of Endau-Rompin National Park in Johor, Malaysia in search of plants traditionally used by the indigenous people in the treatment of malaria and malaria-like symptoms. Seven species of plants were evaluated and tested for antiplasmodial activities. Different plant parts were subjected to methanolic and aqueous extractions. A total of 24 extracts were evaluated by histidine-rich protein II (HRP2) assay against K1 strain of Plasmodium falciparum chloroquine-resistant. Ten extracts showed significant inhibition of the growth of P. falciparum. Phytochemical screening of the same extracts revealed the presence of alkaloids, flavonoids, terpenoids and anthraquinones. This study affirms that tropical rainforests may still hold undiscovered cures for many diseases and illnesses that have inflicted millions of people worldwide. The species studied herein have not known to have been studied elsewhere before.

Keywords: Endau-Rompin, malaria, Malaysia, tropical rainforest, traditional knowledge

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Authors: Meziani Samira, Menadi Noreddine, Labga Lahouaria, Chenni Fatima Zohra, Toumi Asma

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A comparative study between two varieties of soya beans was carried out in this work. The method consists of studying and proceeding to prepare a by-product (Flour) from two varieties of soybeans, a Chinese variety imported and marketed in Algeria. The chemical composition of ash, protein and fat was determined in this study. The minerals, namely potassium and sodium, were measured by flame spectrophotometer. In addition, the estimation of the polyphenol content and evaluation of the antioxidant activity Ferric Reducing Antioxidant Power assay (FRAP) f the methanol extracts of the flours were also carried out. The result revealed that soy flour from two cultivars, on average, contained 8% moisture, more than 50% protein, 1.58-1.87g fat, and 0.28-0.30g of ash. A slight difference was found for contents of 489 mg/ml of K + and 20 mg/ml of NA +. In addition, the phenolic content of the methanolic extracts gives a value of almost 37 mg EAG / g for both cultivars of soy flour. The estimated Reductive Antioxidant Iron (FRAP) potency of soy flour might be related to its polyphenol richness, which is similar to the variety of China. The flour Soya varieties tested contained a significant amount of protein and phenolic compounds with good antioxidant properties.

Keywords: soye beans, soya flour, protein, total polyphenols

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Authors: Abdelkrim Cheriti, Mebarka Belboukhari, Nasser Belboukhari

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Launaea Cass. is a small genus of the family Asteraceae (tribe Lactuceae, subtribe Sonchinae), consisting of 54 species, of which 9 are presented in the flora of Algeria and is mainly distributed in the South Mediterranean, Africa and SW Asia. Plants in the Launaea genus have been used ethnobotanically as bitter stomachic, for treating diarrhea, gastrointestinal tracts, as anti-inflammatory, for skin diseases, treatment of infected wounds, hepatic pains, children fever, as soporific, lactagogue, diuretic and as insecticidal. Antioxidants are vital substances, which possess the ability to protect the body from damages caused by free radical induced oxidative stress. A variety of free radical scavenging antioxidants is found in a number of dietary sources. The main objective of this study focused on the screening of antioxidant activity of Launaea nudicaulis (Asteraceae) extracts. The in vitro antioxidant activity was investigated with DPPH radical scavenging assay. The quantitative evaluation of DPPH scavenging activity showed that n-BuOH and EtOAc extracts are the most active extracts with a percentage of antiradical activity of 89,62% and 71,57% respectively.

Keywords: Launaea, phytochemical, South Algeria, Sahara, endemic specie

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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: Manal Kamel, Sara Maher

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Development of sensitive non-invasive tests for detection of SARS-CoV-2 antigens is imperative to manage the extent of infection throughout the population, yet, it is still challenging. Here, we designed and optimized a sandwich enzyme-linked immunosorbent assay (ELISA) for SARS-CoV-2 S1 antigen detection in saliva. Both saliva samples and nasopharyngeal swapswere collected from 170 PCR-confirmed positive and negative cases. Gold nanoparticles (AuNPs) were conjugated with S1protein receptor binding domain (RBD) nanobodies. Recombinant S1 monoclonal antibodies (S1mAb) as primery antibody and gold conjugated nanobodies as secondary antibody were employed in sandwich ELISA. Our developed system were optimized to achieve 87.5 % sensitivity and 100% specificity for saliva samples compared to 89 % and 100% for nasopharyngeal swaps, respectively. This means that saliva could be a suitable replacement for nasopharyngeal swaps No cross reaction was detected with other corona virus antigens. These results revealed that our developed ELISAcould be establishedas a new, reliable, sensitive, and non-invasive test for diagnosis of SARS-CoV-2 infection, using the easily collected saliva samples.

Keywords: COVID 19, diagnosis, ELISA, nanobodies

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Authors: Khaled M. Khleifat

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An extracellular methanol and ethanol tolerant lipase producing bacterial strain K5b4 was isolated from soil samples contaminated with hydrocarbon residues. It was identified by using morphological and biochemical characteristics and 16srRNA technique as Acinetobacter species. The immobilized lipase from Acinetobacter sp. K5b4 retained more than 98% of its residual activity after incubation with pure methanol and ethanol for 24 hours. The highest hydrolytic activity of the immobilized enzyme was obtained in the presence of 75% (v/v) methanol in the assay solution. In contrary, the enzyme was able to maintain its original activity up to only 25% (v/v) ethanol whereas at elevated concentrations of 50 and 75% (v/v) the enzyme activity was reduced to 10 and 40%, respectively. Maximum lipase activity of 31.5 mU/mL was achieved after 48 hr cultivation when the optimized medium (pH 7.0) that composed of 1.0% (w/v) olive oil, 0.2% (w/v) glycerol, 0.15% (w/v) yeast extract, and 0.05% (w/v) NaCl was inoculated with 0.4% (v/v) seed culture and incubated at 30°C and 150 rpm agitation speed. However, the presence of CaCl2 in the growth media did not show any inhibitory or stimulatory effect on the enzyme production as it compared to the control experiment. Meanwhile, the other mineral salts MgCl2, MnCl2, KCl and CoCl2 were negatively affected the production of lipase enzyme. The inhibition of lipase production from Acinetobacter sp. K5b4 in presence of glucose suggesting that lipase gene expression is prone to catabolic repression.

Keywords: K5B4, methanol and ethanol, acinetobacter, morphological

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Authors: Byeal-I Han, Michael Lee

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Ageing is associated with an increased risk of diseases such as cancer, and neurodegenerative disorders. Increased autophagy delays ageing and extends longevity. In this study, we investigated the role of autophagy in longevity using human foreskin fibroblast HS68 cells, in which a senescence-like growth arrest can be induced. In particular, cellular senescence is manifested by the irreversible cell cycle arrest, and may contribute to the ageing of organisms. The senescence state was measured with staining for senescence-associated β-galactosidase (SA-β-gal) activity that represents a sensitive and reliable marker to quantify senescent cells. We detected a significantly increased percentage (%) of SA-β-gal positive cells in HS68 cultures at passage 40 (63%) when compared with younger ones at passage 15 (0.5%). As expected, HS68 cells at passage 40 exhibited much lower proliferation rate than cells at passage 15. The basal levels of LC3 were measured by immunoblotting showing a comparison of LC3-I and LC3-II levels at 3 age-points in serially passaged HS68 cells. LC3-II/LC3-I ratio at different passage levels relative to β-actin levels of each band confirmed that cells at passage 34 showed lower conversion of non-autophagic LC3-I to autophagic LC3-II than the cells at passage 16. Furthermore, Cyto-ID autophagy assay also revealed that late passage cells showed lower autophagy than the early passage cells. Together, our findings suggest that senescence induction might be associated with decreased autophagy.

Keywords: ageing, autophagy, senescence, HS68

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Authors: Abdelkader Basli, Jean-Claude Delaunay, Eric Pedrot, Jean-Michel Mérillon, Jean-Pierre Monti, Khodir Madani, Mohamed Chibane, Tristan Richard

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The antioxidant and antimicrobial activities of Origanum glandulosum collected in Algeria have been studied. Extract was prepared from aerial part of endemic Algerian oregano. The produced extract has been characterized in terms of total phenols (using Folin method), total flavonoid, antioxidant activities (using the DPPH radical scavenging method and ORAC assay) and microbial activity against four bacteria: Streptococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae one yeast: Candida albicans and one fungi: Aspergillus niger. The results pointed the antioxidant activities of the extract of O. glandulosum and antimicrobial activities against all bacteria and C. Candida, but no effect on A. niger. High performance liquid chromatography combined with mass spectrometry (LC-MS) and nuclear magnetic resonance (LC-NMR) were used to separate and identify the major compounds present in the oregano extract. Rosmarinic acid, globoidnan A and B, lithospermic acid B and three flavonoids were identified.

Keywords: origanum glandulosum, antioxidant, microbial activity, polyphenol, LC-MS, LC-NMR

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Authors: Iyiola O. Otunniyi, Oluwayimika O. Oluokun

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The two-step leaching process of PCB dust will produce a first leaching stream containing assorted metals that still requires more demanding multistage processing afterward to recover base metals and precious metals. In this work, three-step selective complexations produce a clean gold solution from printed circuit board dust. After optimizing for temperature and concentrations, the first step under oxidative ammonia leaching recovered no gold, 90 % Cu and 50 % Zn. Second step acid leaching recovered no gold, 89 % Fe, 48 % Zn, 94 % Ni. The recoveries generally increased with reducing dust particle sizes, except for zinc under oxidative ammonia, and it was noted that its various alloy forms in PCB could be responsible for this. At the third leaching step using acidified thiourea with 0.1 M H₂O₂ at 25 OC, gold recovery was 99 %. The leaching rate was shown to be chemically controlled, implying that reagent dosage control will compensate for feed assay shifts in an operation design. Copper, zinc and nickel will be easily recoverable from leach solutions of the first two steps in this leaching scheme. The third step produced a clean gold solution for easy processing downstream.

Keywords: gold thiourea complexation, printed circuit board, step leaching, selective recovery

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Authors: Nootchanat Mairuae, Walaiporn Tongjaroenbuangam, Chalisa Louicharoen Cheepsunthorn, Poonlarp Cheepsunthorn

Abstract:

The aim of this study was to investigate the anti-oxidative effect, the anti-inflammatory effects, and the molecular mechanisms of okra (Abelmoschus esculentus Linn.) on lipopolysaccharide (LPS)-stimulated BV2 microglial cells. The BV2 cells were treated with LPS in the presence or absence of okra. Reactive oxygen species (ROS) and nitric oxide (NO) production were measured using the ROS detection reagent DCF-DA and the Griess reaction, respectively. The phosphorylation levels of nuclear factor-kappa B (NF-kB) p65 was detected by Western blot assay. Treatment of BV2 microglia cells with okra was found to significantly suppress the LPS-induced inflammatory mediator NO as well as ROS compared to untreated cells. The levels of LPS-induced NF-kB p65 phosphorylation were significantly decreased following okra treatment too. These results show that okra exerts anti-oxidative and anti-inflammatory effects in LPS-stimulated BV2 microglial cells by suppressing the NF-κB pathway. This suggests okra might be a valuable agent for treatment of anti-neuroinflammatory diseases mediated by microglial cells.

Keywords: Abelmoschus esculentus Linn, microglia, neuroinflammation, reactive oxygen spicy

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Authors: Lourena M. Veríssimo, Lucas A. Machado, Renata Rutckeviski, Francisco H. Xavier Júnior, Éverton N. Alencar, Andreza R. V. Morais, Teresa R. F. Dantas, Christian M. Oliveira, Arnóbio A. Silva Júnior, Eryvaldo S. T. Egito

Abstract:

This study aimed to obtain emulsion systems based on bullfrog oil (BO). The BO was extracted at 80ºC and analyzed by Gas Chromatography-Mass Spectrometry (GC/MS). The critical Hydrophilic-Lipophilic Balance (HLBc) Assay of the BO was performed through BO, Tween® 20, Span® 80 and deionized water mixtures using an Ultra-Turrax® and determined using dynamic light scattering, pH, electrical conductivity and creaming rate. Then, a pseudoternary phase diagram (PPD) was constructed by water titration. The GC/MS analysis of BO suggested Methyl Oleate (9.26%) as major compound. The HLBc was 12.1, wherein the correspondent emulsion showed a pH of 4.83±1.29, electrical conductivity of 103.65 µS, creaming rate of 2.51±0.54%, droplet size of 207.07±8.31 nm and polydispersity index of 0.212±0.005. The PPD showed different formulations characterized as O/W emulsions. Thus, the PPD proved to be a useful tool to produce BO emulsions, in which their constituents may vary within the range of the desired system.

Keywords: bullfrog (Rana catesbeiana Shaw) oil, emulsion production, hydrophilic-lipophilic balance, gas chromatography/mass spectrometry analysis

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Authors: Chao Wu

Abstract:

Thioredoxin reductase 2 is a mitochondrial enzyme that belongs to the cellular defense against oxidative stress. We deleted mitochondrial Txnrd2 in a KrasG12D-driven pancreatic tumor model. Despite an initial increase in precursor lesions, tumor incidence decreased significantly. We isolated cancer cell lines from these genetically engineered mice and observed an impaired proliferation and colony formation. Reactive Oxygen Species, as determined by DCF fluorescence, were increased. We detected a higher mitochondrial copy number in Txnrd2-deficient cells (KTP). However, measurement of mitochondrial bioenergetics showed no impairment of mitochondrial function and comparable O₂-consumption and extracellular acidification rates. In addition, the mitochondrial complex composition was affected in Txnrd2 deleted cell lines. To gain better insight into the role of Txnrd2, we deleted Txnrd2 in clones from parental KrasG12D cell lines using Crispr/Cas9 technology. The deletion was confirmed by western blot and activity assay. Interestingly, and in line with previous RNA expression analysis, we saw changes in EMT markers in Txnrd2 deleted cell lines and control cell lines. This might help us explain the reduced tumor incidence in KrasG12D; Txnrd2∆panc mice.

Keywords: PDAC, TXNRD2, epithelial-to-mesenchymal-transition, ROS

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Authors: Borokini Funmilayo Boede

Abstract:

B. alba L., commonly called ‘Amunututu’ and Solanecio biafrae called ‘Worowo’ among the Yoruba tribe in the southwest part of Nigeria are reported to be of great ethnomedicinal importance but are among many underutilized green leafy vegetables in the country. Many studies have established the nutritional values of these vegetables, utilization are very poor and indepth information on their chemical profiles is scarce. The aqueous, methanolic and ethanolic extracts of these vegetables were subjected to phytochemical screening and phenolic profiles of the alcoholic extracts were characterized by using high-performance liquid chromatography coupled with diode array detector (HPLC-DAD). Total phenol and flavonoid contents were determined, antioxidant activities were evaluated using five in vitro assays to assess DPPH, nitric oxide and hydroxyl radical-scavenging abilities, as well as reducing power with ferric reducing antioxidant assay and phosphomolybdate method. The antibacterial activities of the extracts against Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhi were evaluated by using agar well diffusion method and the antifungal activity evaluated against food-associated filamentous fungi by using poisoned food technique with the aim of assessing their nutraceutical potentials to encourage their production and utilization. The results revealed the presence of saponnin, steroids, tannin, terpenoid and flavonoid as well as phenolic compounds: gallic acid, chlorogenic acid, caffeic acid, coumarin, rutin, quercitrin, quercetin and kaemferol. The vegetables showed varying concentration dependent reducing and radical scavenging abilities from weak to strong compared with gallic acid, rutin, trolox and ascorbic acid used as positive controls; the aqueous extracts which gave higher concentrations of total phenol displayed higher ability to reduce Fe (lll) to Fe (ll) and stronger inhibiting power against hydroxyl radical than the alcoholic extracts and in most cases exhibited more potency than the ascorbic acids used as positive controls, at the same concentrations, whereas, methanol and / or ethanol extracts were found to be more effective in scavenging 2, 2-diphenyl-1-picryl hydrazyl radical and showed higher ability to reduce Mo (VI) to Mo (V) in total antioxidant assay than the aqueous extracts. However, the inhibition abilities of all the extracts against nitric oxide were comparable with the ascorbic acid control at the same concentrations. There were strong positive correlations with total phenol (mg GAE/g) and total flavonoid (mg RE/g) contents in the range TFC (r=0.857- 0999 and r= 0.904-1.000) and TPC (r= 0.844- 0.992 and r= 0.900 -0.999) for Basella alba and Senecio biafrae respectively. Inhibition concentration at 50 % (IC50) for each extract to scavenge DPPH, OH and NO radicals ranged from 32.73 to 1.52 compared with control (0.846 - -6.42) mg/ml. At 0.05g/ml, the vegetables were found to exhibit mild antibacterial activities against Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi compared with streptomycin sulphate used as control but appreciable antifungi activities against (Trichoderma rubrum and Aspergillus fumigates) compared with bonlate antibiotic positive control. The vegetables possess appreciable antioxidant and antimicrobial properties for promoting good health, their cultivation and utilization should be encouraged especially in the face of increasing health and economic challenges and food insecurity in many parts of the world.

Keywords: antimicrobial, antioxidants, extracts, phytochemicals

Procedia PDF Downloads 327

Authors: V. Yurina, H. Sujuti, E. Rahmani, A. R. Nopitasari

Abstract:

Breast cancer has the highest prevalence cancer in women. Moringa leaves (M. oleifera) contain quercetin, kaempferol, and benzyl isothiocyanate which can enhance induction of apoptosis. This research aimed to study the role of the leaf extract of Moringa to increase apoptosis in breast cancer cell line, MCF-7 cells. This research used in vitro experimental, post-test only, control group design on breast cancer cells MCF-7 in vitro. Moringa leaves were extracted by maceration method with ethanol 70%. Cells were treated with drumstick leaves extract on 1100, 2200, and 4400 μg/ml for Hsp27 and caspase-9 expression (immunocytochemistry) and apoptosis (TUNEL assay) test. The results of this study found that the IC50 2200 µg/ml. Moringa leaves extract can significantly increase the expression of caspase-9 (p<0.05) and decreased Hsp 27 expression (p<0.05). Moreover it can increase apoptosis (p<0.05) significantly in MCF-7 cells. The conclusion of this study is Moringa leaves extract is able to increase the expression of caspase-9, decrease Hsp27 expression and increase apoptosis in breast cancer cell-line MCF-7.

Keywords: apoptosis, breast cancer, caspase-9, Hsp27, Moringa oleifera

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Authors: B. Loukil, L. Mallem, M. S. Boulakoud

Abstract:

Inorganic fertilizers consist mainly of salts of ammonium nitrate, phosphate and potassium, the combination of primary nutrients NPK including secondary and micro nutrients are essential for plant growth, used for intensive agriculture, ranching, and horticultural crops, to increase soil fertility and ensure sustainable crop production. The manufacture of fertilizers is generally at a high temperature and high pressure, in the presence of several highly hazardous chemicals, dust and gases. These products are absorbed high in the airway, increasing the airway resistance thereby adversely affecting the pulmonary functions of workers. A study was conducted on 34 employees, especially exposed to nitrate derivatives. A questionnaire was prepared and distributed to all employees in the unit. The workers were divided into two groups according to age. Several hormonal parameters Assay were measured. The results of the questionnaire have detected a fertility problem, Concerning the hormones a significant reduction in the concentration of testosterone in both groups and LH in the group aged 30 to 40 year were noted compared to the control. However, an increase in the concentration of prolactin in both groups compared to the control. There was a significant decrease in FSH in the group aged 30 to 40 always in compared with the control group.

Keywords: fertilizers, healthy worker, risk, fertility

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Authors: Isil Gazioglu, Abdulselam Ertas

Abstract:

Aflatoxins (AFs) are secondary toxic metabolites of Aspergillus flavus and A. parasiticus. AFs can be absorbed through the skin. Potent carcinogens like AFs should be completely absent from cosmetics, this can be achieved by careful quality control of the raw plant materials. Regulatory limits for aflatoxins have been established in many countries, and reliable testing methodology is needed to implement and enforce the regulatory limits. In this study, ten medicinal plant samples (Bundelia tournefortti, Capsella bursa-pastoris, Carduus tenuiflorus, Cardaria draba, Malva neglecta, Malvella sharardiana, Melissa officinalis, Sideritis libanotica, Stakys thirkei, Thymus nummularius) were investigated for aflatoxin (AF) contaminations by employing an HPLC assay for the determination of AFB1, B2, G1 and G2. The samples were extracted with 70% (v/v) methanol in water before further cleaned up with an immunoaffinity column and followed by the detection of AFs by using an electrochemically post-column derivatization with Kobra-Cell and fluorescence detector. The extraction procedure was optimized in order to obtain the best recovery. The method was successfully carried out with all medicinal plant samples. The results revealed that five (50%) of samples were contaminated with AFs. The association between particular samples and the AF contaminated could not be determined due to the low frequency of positive samples.

Keywords: aflatoxin B1, HPLC-FLD, KOBRA-Cell, mycotoxin

Procedia PDF Downloads 606