Search results for: enzyme assay

Authors: Duanghathai Saipinta, Tanittian Panyamongkol, Witaya Suriyasathaporn

Abstract:

Animal management aims to provide a suitable environment for animals allowing maximal productivity in those animals. Prevention of disease is an important part of animal management. Foot-and-mouth disease (FMD) is a highly contagious viral disease in cattle and is an economically important animal disease worldwide. Monitoring the FMD virus in farms is useful management for the prevention of the FMD outbreak. A recent publication indicated collection samples from nasal swabs can be used for monitoring FMD in symptomatic cows. Therefore, the objectives of this study were to determine the FMD virus in asymptomatic dairy cattle using nasal swab samples during the absence of an FMD outbreak. The study was conducted from December 2020 to June 2021 using 185 asymptomatic signs of FMD dairy cattle in Chiang Mai Province, Thailand. By random cow selection, nasal mucosal swabs were used to collect samples from the selected cows and then were to evaluate the presence of FMD viruses using the real-time rt-PCR assay. In total, 4.9% of dairy cattle detected FMD virus, including 2 dairy farms in Mae-on (8 samples; 9.6%) and 1 farm in the Chai-Prakan district (1 sample; 1.2%). Interestingly, both farms in Mae-on were the outbreak of the FMD after this detection for 6 months. This indicated that the FMD virus presented in asymptomatic cattle might relate to the subsequent outbreak of FMD. The outbreak demonstrates the presence of the virus in the environment. In conclusion, monitoring of FMD can be performed by nasal swab collection. Further investigation is needed to show whether the FMD virus presented in asymptomatic FMD cattle could be the cause of the subsequent FMD outbreak or not.

Keywords: cattle, foot-and-mouth disease, nasal swab, real-time rt-PCR assay

Procedia PDF Downloads 232

Authors: F. Ambreen, A.Naheed

Abstract:

Osteoarthritis (OA) is a degenerative disorder affecting millions of people worldwide. Nitric oxide (NO) was found to play a catabolic role in the development of osteoarthritis. It is a toxic free radical gas generated during the metabolism of L-arginine by the enzyme Nitric oxide synthase (NOS). Inducible Nitric Oxide Synthase (iNOS) is one of the isoform of NOS, and its overexpression leads to the excessive formation of NO that results in pathophysiological joint conditions. Several synthetic anti-inflammatory drugs and inhibitors are present to date, but all showed side effects and complications. Therefore, the pursuit of natural disease-modifying drugs remains a top priority. Curcumin is an active component of turmeric, and the past few decades have witnessed intense research devoted to the antioxidant and anti-inflammatory properties of curcumin. The present study focused on curcumin and its derivatives in the search for new iNOS inhibitors for the treatment of osteoarthritis. We conducted a molecular docking study on curcumin and its four derivatives; cyclocurcumin, tetrahydrocurcumin, demethoxycurcumin and curcumin monoglucoside with iNOS using CLC Drug discovery work bench 3.02. We selected two co-crystallized ligands for this study; tetrahydrobiopterin and N-omega-propyl-L-arginine present in complex with the enzyme iNOS. Results showed the best binding affinity of N-omega-propyl-L-arginine with cyclocurcumin and curcumin monoglucoside that exhibit binding energies of -65.2 kcal/mol and -68 kcal/mol respectively. Whereas with tetrahydrobiopterin, best binding scores of -64.7 kcal/mol and -62.2 kcal/mol were found with tetrahydrocurcumin and demethoxycurcumin respectively. This information could open doors of research for the designing of novel drugs using herbs such as curcumin for the treatment of inflammatory joint diseases.

Keywords: curcumin, iNOS, molecular docking, osteoarthritis

Procedia PDF Downloads 129

Authors: Salma Baig, Bakrudeen Ali Ahmad, Ainnul Hamidah Syahadah Azizan, Hapipah Mohd Ali, Elham Rouhollahi, Mahmood Ameen Abdulla

Abstract:

Free radical damage induced by reactive oxygen species (ROS) contributes to etiology of many chronic diseases, cancer being one of them. Recent studies have been successful in ROS targeted therapies via antioxidants using mouse models in cancer therapeutics. The present study was designed to scrutinize anticancer activity, antioxidant activity of 5 different extracts of Thymus serpyllum in MDA-MB-231, MCF-7, HepG2, HCT-116, PC3, and A549. Identification of the phytochemicals present in the most active extract of Thymus serpyllum was conducted using gas chromatography coupled with mass spectrophotometry and antioxidant activity was measured by using DPPH radical scavenging and FRAP assay. Anticancer impact of the extract in terms of IC50 was evaluated using MTT cell viability assay. Results revealed that the hexane extract showed the best anticancer activity in HepG2 (Liver Carcinoma Cell Line) with an IC50 value of 23 ± 0.14 µg/ml followed by 25 µg/ml in HCT-116 (Colon Cancer Cell Line), 30 µm/ml in MCF-7 (Breast Cancer Cell Line), 35 µg/ml in MDA-MB-231 (Breast Cancer Cell Line), 57 µg/ml in PC3 (Prostate Cancer Cell Line) and 60 µg/ml in A549 (Lung Carcinoma Cell Line). GC-MS profile of the hexane extract showed the presence of 31 compounds with carvacrol, thymol and thymoquione being the major compounds. Phenolics such as Vitamin E, terpinen-4-ol, borneol and phytol were also identified. Hence, here we present the first report on cytotoxicity of hexane extract of Thymus serpyllum extract in HepG2 cell line with a robust anticancer activity with an IC50 of 23 ± 0.14 µg/ml.

Keywords: Thymus serpyllum L., hexane extract, GC-MS profile, antioxidant activity, anticancer activity, HepG2 cell line

Procedia PDF Downloads 517

Authors: Abbas Jafarizad, Duygu Ekinci

Abstract:

In this work targeted drug delivery is proposed to decrease adverse effect of drugs with concomitant reduces in consumption and treatment outgoings. Nanoparticles (NPs) can be prepared from a variety of materials such as lipid, biodegradable polymer that prevent the drugs cytotoxicity in healthy cells, etc. One of the most important drugs used in chemotherapy is mitoxantrone (MTX) which prevents cell proliferation by inhibition of topoisomerase II and DNA repair; however, it is not selective and has some serious side effects. In this study, mentioned aim is achieved by using several nanocarriers like magnetite (Fe3O4) and their composites with magnetic graphene oxide (Fe3O4@GO). Also, cytotoxic potential of Fe3O4, Fe3O4-MTX, and Fe3O4@GO-MTX nanocomposite were evaluated on mesenchymal stem cells (MSCs). In this study, we reported the synthesis of monodisperse Fe3O4 NPs and Fe3O4@GO nanocomposite and their structures were investigated by using field emission scanning electron microscope (FESEM), Fourier transform infrared (FTIR) spectra, atomic force microscopy (AFM), Brauneur Emmet Teller (BET) isotherm and contact angle studies. Moreover, we used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate cytotoxic effects of MTX, Fe3O4 NPs, Fe3O4-MTX and Fe3O4@GO-MTX nanocomposite on MSCs. The in-vitro MTT results indicated that all concentrations of MTX and Fe3O4@GO-MTX nanocomposites showed cytotoxic effects while all concentrations of Fe3O4 NPs and Fe3O4-MTX NPs did not show any cytotoxic effect on stem cells. The results from this study indicated that using Fe3O4 NPs as anticancer drug delivery systems could be a trustworthy method for cancer treatment. But for reaching excellent and accurate results, further investigation is necessary.

Keywords: mitoxantrone, magnetite, magnetic graphene oxide, MTT assay, mesenchymal stem cells

Procedia PDF Downloads 272

Authors: Changhan Ouyang, Zhonglin Xie

Abstract:

In response to proatherosclerotic factors such as oxidized lipids, or to therapeutic interventions such as angioplasty, stents, or bypass surgery, vascular smooth muscle cells (VSMCs) migrate from the media to the intima, resulting in intimal hyperplasia, restenosis, graft failure, or atherosclerosis. These proatherosclerotic factors also activate autophagy in VSMCs. However, the functional role of autophagy in vascular health and disease remains poorly understood. In the present study, we determined the role of autophagy in the regulation of VSMC migration. Autophagy activity in cultured human aortic smooth muscle cells (HASMCs) and mouse carotid arteries was measured by Western blot analysis of microtubule-associated protein 1 light chain 3 B (LC3B) and P62. The VSMC migration was determined by scratch wound assay and transwell migration assay. Ex vivo smooth muscle cell migration was determined using aortic ring assay. The in vivo SMC migration was examined by staining the carotid artery sections with smooth muscle alpha actin (alpha SMA) after carotid artery ligation. To examine the relationship between autophagy and neointimal hyperplasia, C57BL/6J mice were subjected to carotid artery ligation. Seven days after injury, protein levels of Atg5, Atg7, Beclin1, and LC3B drastically increased and remained higher in the injured arteries three weeks after the injury. In parallel with the activation of autophagy, vascular injury-induced neointimal hyperplasia as estimated by increased intima/media ratio. The en face staining of carotid artery showed that vascular injury enhanced alpha SMA staining in the intimal cells as compared with the sham operation. Treatment of HASMCs with platelet-derived growth factor (PDGF), one of the major factors for vascular remodeling in response to vascular injury, increased Atg7 and LC3 II protein levels and enhanced autophagosome formation. In addition, aortic ring assay demonstrated that PDGF treated aortic rings displayed an increase in neovessel formation compared with control rings. Whole mount staining for CD31 and alpha SMA in PDGF treated neovessels revealed that the neovessel structures were stained by alpha SMA but not CD31. In contrast, pharmacological and genetic suppression of autophagy inhibits VSMC migration. Especially, gene silencing of Atg7 inhibited VSMC migration induced by PDGF. Furthermore, three weeks after ligation, markedly decreased neointimal formation was found in mice treated with chloroquine, an inhibitor of autophagy. Quantitative morphometric analysis of the injured vessels revealed a marked reduction in the intima/media ratio in the mice treated with chloroquine. Conclusion: Autophagy activation increases VSMC migration while autophagy suppression inhibits VSMC migration. These findings suggest that autophagy suppression may be an important therapeutic strategy for atherosclerosis and intimal hyperplasia.

Keywords: autophagy, vascular smooth muscle cell, migration, neointimal formation

Procedia PDF Downloads 314

Authors: Nadhiya N. Subramony, Jenifer Vaughan, Lesley E. Scott

Abstract:

In South Africa, the World Health Organisation estimated 454000 new cases of Mycobacterium tuberculosis (M.tb) infection (MTB) in 2015. Disseminated tuberculosis arises from the haematogenous spread and seeding of the bacilli in extrapulmonary sites. The gold standard for the detection of MTB in bone marrow is TB culture which has an average turnaround time of 6 weeks. Histological examinations of trephine biopsies to diagnose MTB also have a time delay owing mainly to the 5-7 day processing period prior to microscopic examination. Adding to the diagnostic delay is the non-specific nature of granulomatous inflammation which is the hallmark of MTB involvement of the bone marrow. A Ziehl-Neelson stain (which highlights acid-fast bacilli) is therefore mandatory to confirm the diagnosis but can take up to 3 days for processing and evaluation. Owing to this delay in diagnosis, many patients are lost to follow up or remain untreated whilst results are awaited, thus encouraging the spread of undiagnosed TB. The Xpert® MTB/RIF (Cepheid, Sunnyvale, CA) is the molecular test used in the South African national TB program as the initial diagnostic test for pulmonary TB. This study investigates the optimisation and performance of the Xpert® MTB/RIF on bone marrow aspirate specimens (BMA), a first since the introduction of the assay in the diagnosis of extrapulmonary TB. BMA received for immunophenotypic analysis as part of the investigation into disseminated MTB or in the evaluation of cytopenias in immunocompromised patients were used. Processing BMA on the Xpert® MTB/RIF was optimised to ensure bone marrow in EDTA and heparin did not inhibit the PCR reaction. Inactivated M.tb was spiked into the clinical bone marrow specimen and distilled water (as a control). A volume of 500mcl and an incubation time of 15 minutes with sample reagent were investigated as the processing protocol. A total of 135 BMA specimens had sufficient residual volume for Xpert® MTB/RIF testing however 22 specimens (16.3%) were not included in the final statistical analysis as an adequate trephine biopsy and/or TB culture was not available. Xpert® MTB/RIF testing was not affected by BMA material in the presence of heparin or EDTA, but the overall detection of MTB in BMA was low compared to histology and culture. Sensitivity of the Xpert® MTB/RIF compared to both histology and culture was 8.7% (95% confidence interval (CI): 1.07-28.04%) and sensitivity compared to histology only was 11.1% (95% CI: 1.38-34.7%). Specificity of the Xpert® MTB/RIF was 98.9% (95% CI: 93.9-99.7%). Although the Xpert® MTB/RIF generates a faster result than histology and TB culture and is less expensive than culture and drug susceptibility testing, the low sensitivity of the Xpert® MTB/RIF precludes its use for the diagnosis of MTB in bone marrow aspirate specimens and warrants alternative/additional testing to optimise the assay.

Keywords: bone marrow aspirate , extrapulmonary TB, low sensitivity, Xpert® MTB/RIF

Procedia PDF Downloads 171

Authors: Jyoti Kumari, Pavuluri Srinivasa Rao

Abstract:

Jackfruit (ArtocarpusheterophyllusL.) is an underutilized yet highly nutritious fruit with unique flavour, known for its therapeutic and culinary properties. Fresh jackfruit bulb has a very short shelf life due to high moisture and sugar content leading to microbial and enzymatic browning, hindering its consumer acceptability and marketability. An attempt has been made for the preservation of the ripe jackfruit bulbs, by the application of high pressure (HP) over a range of 200-500 MPa at ambient temperature for dwell times ranging from 5 to 20 min. The physicochemical properties of jackfruit bulbs such as the pH, TSS, and titrable acidity were not affected by the pressurization process. The ripening index of the fruit bulb also decreased following HP treatment. While the ascorbic acid and antioxidant activity of jackfruit bulb were well retained by high pressure processing (HPP), the total phenols and carotenoids showed a slight increase. The HPP significantly affected the colour and textural properties of jackfruit bulb. High pressure processing was highly effective in reducing the browning index of jackfruit bulbs in comparison to untreated bulbs. The firmness of the bulbs improved upon the pressure treatment with longer dwelling time. The polyphenol oxidase has been identified as the most prominent oxidative enzyme in the jackfruit bulb. The enzymatic activity of polyphenol oxidase and peroxidase were significantly reduced by up to 40% following treatment at 400 MPa/15 min. HPP of jackfruit bulbs at ambient temperatures is shown to be highly beneficial in improving the shelf stability, retaining its nutrient profile, color, and appearance while ensuring the maximum inactivation of the spoilage enzymes.

Keywords: antioxidant capacity, ascorbic acid, carotenoids, color, HPP-high pressure processing, jackfruit bulbs, polyphenol oxidase, peroxidase, total phenolic content

Procedia PDF Downloads 174

Authors: Mohammad K. Parvez, Ahmed H. Arbab, Mohammed S. Al-Dosari, Adnan J. Al-Rehaily

Abstract:

We investigated ex vivo and in vivo antioxidative and hepatoprotective effect of Aerva javanica. Total ethanol extract of A. javanica aerial parts was prepared, and tested on DCFH-toxicated HepG2 cell in CCl4-injured Wistar rats. MTT-assay was used to determine cell viability, and serum biochemical markers of liver injury as well as histopathology were performed. In vitro DPPH and β-carotene free-radical scavenging assay and phytochemical screening of the extract was done. Furthermore, A. javanica total extract was standardized and validated by HPTLC method. While DCFH-injured cells were recovered to about 56.7% by 100 microg/ml of the extract, a 200 microg/ml dose resulted in hepatocytes recovery by about 90.2%. Oral administration of the extract (100 and 200 mg/kg.bw/day) significantly normalized the serum SGOT, SGPT, GGT, ALP, bilirubin, cholesterol, HDL, LDL, VLDL, TG and MDA levels, including tissue NP-SH and TP in CCl4-injured rats. In addition, the histopathology of dissected liver also revealed that A. javanica cured the tissue lesion compared to reference drug, Silymarin. In vitro assays revealed strong free-radical scavenging ability of the extract and presence of alkaloids, flavonoids, tannins, sterols and saponins where Rutin, a well-known antioxidant flavonoid was identified. Our finding therefore, suggests the therapeutic potential of A. javanica in various liver diseases. However, isolation of the active principles, their mechanism of action and other therapeutic contribution remain to be addressed.

Keywords: Aerva javanica, antioxidant, hepatoprotection, rutin

Procedia PDF Downloads 295

Authors: K. S. Zaytsev, Y. R. Nartsissov

Abstract:

Glycine is one of the key inhibitory neurotransmitters in Central nervous system (CNS) meanwhile glycinergic transmission is highly dependable on its appropriate reuptake from synaptic cleft. Glycine transporters (GlyT) of types 1 and 2 are the enzymes providing glycine transport back to neuronal and glial cells along with Na⁺ and Cl⁻ co-transport. The distribution and stoichiometry of GlyT1 and GlyT2 differ in details, and GlyT2 is more interesting for the research as it reuptakes glycine to neuron cells, whereas GlyT1 is located in glial cells. In the process of GlyT2 activity, the translocation of the amino acid is accompanied with binding of both one chloride and three sodium ions consequently (two sodium ions for GlyT1). In the present study, we developed a computer simulator of GlyT2 and GlyT1 activity based on known experimental data for quantitative estimation of membrane glycine transport. The trait of a single protein functioning was described using the probability approach where each enzyme state was considered separately. Created scheme of transporter functioning realized as a consequence of elemental steps allowed to take into account each event of substrate association and dissociation. Computer experiments using up-to-date kinetic parameters allowed receiving the number of translocated glycine molecules, Na⁺ and Cl⁻ ions per time period. Flexibility of developed software makes it possible to evaluate glycine reuptake pattern in time under different internal characteristics of enzyme conformational transitions. We investigated the behavior of the system in a wide range of equilibrium constant (from 0.2 to 100), which is not determined experimentally. The significant influence of equilibrium constant in the range from 0.2 to 10 on the glycine transfer process is shown. The environmental conditions such as ion and glycine concentrations are decisive if the values of the constant are outside the specified range.

Keywords: glycine, inhibitory neurotransmitters, probability approach, single protein functioning

Procedia PDF Downloads 119

Authors: V. Nigam, V. Nambiar

Abstract:

Aegle Marmelos leaf has been used as a remedy for various gastrointestinal infections and lowering blood sugar level in traditional system of medicine in India due to the presence of various constituents such as flavonoids, tannins and alkaloids (eg. Aegelin, Marmelosin, Luvangetin).The objective of the present study was to evaluate the total antioxidant activity, total and individual phenol content of the wild and cultivated variety of Aegle marmelos leaves to assess the role of this plant in ethanomedicine in India. The methanolic extracts of the leaves were screened for total antioxidant capacity through Ferric Reducing Antioxidant Potential (FRAP) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay; Total Phenol content (TPC) through spectrophotometric technique based on Folin Ciocalteau assay and for qualitative estimation of phenols, High performance Liquid Chromatography was used. The TPC of wild and cultivated variety was 7.6% and 6.5% respectively whereas HPLC analysis for quantification of individual polyphenol revealed the presence of gallic acid, chlorogenic acid and Ferullic acid in wild variety whereas gallic acid, Ferullic acid and pyrocatechol in cultivated variety. FRAP values and IC 50 value (DPPH) for wild and cultivated variety was 14.65 μmol/l and 11.80μmol/l; 437 μg/ml and 620μg/ml respectively and thus it can be used as potential inhibitor of free radicals. The wild variety was having more antioxidant capacity than the cultivated one it can be exploited further for its therapeutic application. As Aegle marmelos is rich in antioxidant, it can be used as food additives to delay the oxidative deterioration of foods and as nutraceutical in medicinal formulation against degenerative diseases like diabetes.

Keywords: antioxidant activity, aegle marmelos, antidiabetic, nutraceutical

Procedia PDF Downloads 373

Authors: Pravaree Phuneerub, Chanida Palanuvej, Nijsiri Ruangrungsi

Abstract:

Cymbopogon nardus Rendel (Family Gramineae) is commonly known as citronella grass. The dried root of C. nardus is used for antipyretic, anti-inflammation, anti-analgesic and anticancer in traditional Thai medicine. Transverse sectional and pulverized C. nardus root were illustrated. The volatile oil was extracted from oil gland by hydrodistillation and analysed by GC/MS. Cymbopogon nardus root was exhaustively extracted by continuously maceration in ethanol and water respectively. The mutagenic and antimutagenic properties of the ethanol extract and fractionated water extract of C. nardus root were evaluated by Ames assay using the S. typhimurium strains TA98 and TA100 as the models. The result indicated that the anatomical character of root transverse section displayed epidermis, parenchyma, oil gland, phloem, xylem vessel, endodermis and pith. Histological characters of root powder showed parenchyma containing oleoresin, parenchyma in longitudinal view, reticulate vessel, annular vessel, starch granules and fragment of fiber. The root volatile oil was rich in sesquiterpenes dominated by elemol (22.87%) and alpha-eudesmol (16.09%). For mutagenic activity, the both extracts of C. nardus were no mutagenic toward S. typhimurium strains TA98 and TA100. Furthermore, the ethanol extract and fractionated water extract of C. nardus root demonstrated strong antimutagenic effect against of nitrite treated 1-aminopyrene to S. typhimurium strains TA98 and TA100. This present investigation suggested that the dried root extract of C. nardus can be further developed as promising antimutagenic agent.

Keywords: Cymbopogon nardus, volatile oil analysis, mutagenic, antimutagenic effect, Ames Salmonella assay

Procedia PDF Downloads 346

Authors: Sarah Gaßmeyer, Nadine Hülsemann, Raphael Stoll, Kenji Miyamoto, Robert Kourist

Abstract:

Enzymatic racemization allows the smooth interconversion of stereocenters under very mild reaction conditions. Racemases find frequent applications in deracemization and dynamic kinetic resolutions. Arylmalonate decarboxylase (AMDase) from Bordetella Bronchiseptica has high structural similarity to amino acid racemases. These cofactor-free racemases are able to break chemically strong CH-bonds under mild conditions. The racemase-like catalytic machinery of mutant G74C conveys it a unique activity in the racemisation of pharmacologically relevant derivates of 2-phenylpropionic acid (profenes), which makes AMDase G74C an interesting object for the mechanistic investigation of cofactor-independent racemases. Structure-guided protein engineering achieved a variant of this unique racemase with 40-fold increased activity in the racemisation of several arylaliphatic carboxylic acids. By saturation–transfer–difference NMR spectroscopy (STD-NMR), substrate binding during catalysis was investigated. All atoms of the substrate showed interactions with the enzyme. STD-NMR measurements revealed distinct nuclear Overhauser effects in experiments with and without molecular conversion. The spectroscopic analysis led to the identification of several amino acid residues whose variation increased the activity of G74C. While single-amino acid exchanges increased the activity moderately, structure-guided saturation mutagenesis yielded a quadruple mutant with a 40 times higher reaction rate. This study presents STD-NMR as versatile tool for the analysis of enzyme-substrate interactions in catalytically competent systems and for the guidance of protein engineering.

Keywords: racemase, rational protein design, STD-NMR, structure guided saturation mutagenesis

Procedia PDF Downloads 304

Authors: Carlos A. C. G. Neto, Natan C. G. e Silva , Thaís de O. Costa, Luciana R. B. Gonçalves, Maria V. P. Rocha

Abstract:

β-galactosidases (E.C. 3.2.1.23) are enzymes that have attracted by catalyzing the hydrolysis of lactose and in producing galacto-oligosaccharides by favoring transgalactosylation reactions. These enzymes, when immobilized, can have some enzymatic characteristics substantially improved, and the coating of supports with multifunctional polymers is a promising alternative to enhance the stability of the biocatalysts, among which polyethylenimine (PEI) stands out. PEI has certain properties, such as being a flexible polymer that suits the structure of the enzyme, giving greater stability, especially for multimeric enzymes such as β-galactosidases. Besides that, protects them from environmental variations. The use of chitosan support coated with PEI could improve the catalytic efficiency of β-galactosidase from Kluyveromyces lactis in the transgalactosylation reaction for the production of prebiotics, such as lactulose since this strain is more effective in the hydrolysis reaction. In this context, the aim of the present work was first to develop biocatalysts of β-galactosidase from K. lactis immobilized on chitosan-coated with PEI, determining the immobilization parameters, its operational and thermal stability, and then to apply it in hydrolysis and transgalactolisation reactions to produce lactulose using whey as a substrate. The immobilization of β-galactosidase in chitosan previously functionalized with 0.8% (v/v) glutaraldehyde and then coated with 10% (w/v) PEI solution was evaluated using an enzymatic load of 10 mg protein per gram support. Subsequently, the hydrolysis and transgalactosylation reactions were conducted at 50 °C, 120 RPM for 20 minutes, using whey supplemented with fructose at a ratio of 1:2 lactose/fructose, totaling 200 g/L. Operational stability studies were performed in the same conditions for 10 cycles. Thermal stabilities of biocatalysts were conducted at 50 ºC in 50 mM phosphate buffer, pH 6.6 with 0.1 mM MnCl2. The biocatalyst whose support was coated was named CHI_GLU_PEI_GAL, and the one that was not coated was named CHI_GLU_GAL. The coating of the support with PEI considerably improved the parameters of immobilization. The immobilization yield increased from 56.53% to 97.45%, biocatalyst activity from 38.93 U/g to 95.26 U/g and the efficiency from 3.51% to 6.0% for uncoated and coated support, respectively. The biocatalyst CHI_GLU_PEI_GAL was better than CHI_GLU_GAL in the hydrolysis of lactose and production of lactulose, converting 97.05% of lactose at 5 min of reaction and producing 7.60 g/L lactulose in the same time interval. QUI_GLU_PEI_GAL biocatalyst was stable in the hydrolysis reactions of lactose during the 10 cycles evaluated, converting 73.45% lactose even after the tenth cycle, and in the lactulose production was stable until the fifth cycle evaluated, producing 10.95 g/L lactulose. However, the thermal stability of CHI_GLU_GAL biocatalyst was superior, with a half-life time 6 times higher, probably because the enzyme was immobilized by covalent bonding, which is stronger than adsorption (CHI_GLU_PEI_GAL). Therefore, the strategy of coating the supports with PEI has proven to be effective for the immobilization of β-galactosidase from K. lactis, considerably improving the immobilization parameters, as well as, the catalytic action of the enzyme. Besides that, this process can be economically viable due to the use of an industrial residue as a substrate.

Keywords: β-galactosidase, immobilization, kluyveromyces lactis, lactulose, polyethylenimine, transgalactosylation reaction, whey

Procedia PDF Downloads 111

Authors: Amin Abbasi, Fariborz Shekari, Seyed Bahman Mousavi

Abstract:

Seed aging during storage is a complex biochemical and physiological processes that can lead to reduce seed germination. This phenomenon associated with increasing of total antioxidant activity during aging. To study the effects of hormones on seed aging, aged wheat seeds (control, 90 and 80% viabilities) were treated with GA3, Salicylic Acid, and paclobutrazol and antioxidant system were investigated as molecular biomarkers for seed vigor. The results showed that, seed priming treatment significantly affected germination percentage, normality seedling percentage, H2O2, MDA, CAT, APX, and GPX activates. Maximum germination percentage achieve in GA3 priming in control treatment. Germination percentage and normal seedling percentage increased in other GA3 priming treatment compared with other hormones. Also aging increased MDA, H2O2 content. MDA is considered sensitive marker commonly used for assessing membrane lipid peroxidation and H2O2result in toxicity to cellular membrane system and damages to plant cells. Amount of H2O2 and MDA declined in GA3 treatment. CAT, GPX and APX activities were reduced by increasing the aging time and at different levels of priming. The highest APX activity was observed in Salicylic Acid control treatment and the highest GPX and CAT activity was obtained in GA3 control treatment. The lowest MDA and H2O2 showed in GA3 control treatment, too. Hormone priming increased Antioxidant enzyme activity and decreased amount of reactive oxygen space and malondialdehyde (MDA) under aging treatment. Also, GA3 priming treatments have a significant effect on germination percentage and number of normal seedling. Generally aging seed, increase ROS and lipid peroxidation. Antioxidant enzymes activity of aged seeds increased after hormone priming.

Keywords: hormones priming, wheat, aging seed, antioxidant, lipid peroxidation

Procedia PDF Downloads 496

Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava

Abstract:

The tobacco caterpillar, Spodoptera litura Fab (Lepidoptera: Noctuidae) is a polyphagous pest various field and horticulture crops in India. Pest had virtually developed resistance to all commonly used insecticides. Enhanced detoxification is the prime mechanism that is dictated by detoxification different enzymes and carboxylesterase is one of the major enzyme responsible development of resistance. In India, insecticide resistance studies on S. litura are mainly deployed on detoxification enzymes activity and investigation at gene level alteration i.e. at nucleotide level is very merger. In the present study, we collected the S. litura larvae from three different cauliflower growing belt viz., IARI, New Delhi (Delhi), Palari, Sonepat (Haryana) and Varanasi (Uttar Pradesh) to study the role of carboxylesterase activity and its gene level variation The CarE activity was measured using UV-VIS spectrophotometer with 3rd instar larvae of S. litura. The elevated activity of CarE was observed in Sonepat strain (28.09 ± 0.09 µmol/min/mg of protein) followed by Delhi (26.72 ± 0.04 µmol/min/mg of protein) and Varanasi strain (10.00 ± 0.44 µmol/min/mg of protein) of S. litura. The genomic DNA was isolated from 3rd instar larvae and CarE gene was amplified using a primer sequence, F:5’tccagagttccttgtcaggcac3’; R:5’ctgcatcaagcatgtctc3. CarE gene, about 500bp was partially amplified, sequenced and submitted to NCBI (Accession No. KF835886, KF835887 and KF835888). The sequence data revealed polymorphism at nucleotide level in all the three strains and gene found to have 88 to 97% similarity with previous available nucleotide sequences of S. litura, S. littoralis and S. exiqua. The polymorphism at the nucleotide level could be a reason for differential activity of carboxylesterase enzymes among the strains. However, investigation at gene expression level would be useful to analyze the overproduction of carboxylesterase enzyme.

Keywords: carboxylesterase, CarE gene, nucleotide polymorphism, insecticide resistance, spodoptera litura

Procedia PDF Downloads 922

Authors: Harukuni Tokuda, Takanari Arai, Xu FengHao, Nobutaka Suzuki

Abstract:

In our continuous search for anti-tumor promoting, chemopreventive active potency from natural source material, a kind of healthy tea, Gromwell seed (Coix lachryma-jobi) ext., and including compounds Monoolein and Trilinolein have been screened using the in vitro synergistic assay indicated by inhibitory effects on the induction of Epstein-Barr virus early antigen (EBV-EA) by TPA. In assay, Gromwell seed aqueous extract and hot aqueous extract exhibited the potential inhibitory effects on EBV-EA activation without strong cytotoxicity on Raji cells. In our experimental system, the inhibitory effects of both Gromwell extracts and compounds were greater than that of beta-carotene, which is known anti-tumor promoting agent and/or chemopreventive agent. These compounds were evaluated for their in vitro inhibitory effect on EBV-EA activation induced by TPA. The percentages of the inhibition of TPA-induced EBV-EA activation for these materials were 60% and 30% at concentration 100 μg. Based on the results obtained in vitro, we studied the inhibitory effect of compounds, in an in vivo two-stage carcinogenesis test of mouse skin papilloma using DMBA as an initiator and TPA as a potential promoter. The control animals showed a 100% incidence of papilloma at 20 weeks after DMBA-TPA tumor promotion, while treatment with compounds reduced the percentage of number of tumor to 60 % after 20 weeks. Results from in vitro and in vivo studies showing chemopreventive activity against TPA promoting stage and these data support the effective potency of carcinogenic stage in clinical evaluation of integrative oncology.

Keywords: gromwell seed, medicinal plant, chemoprevention, pharmaceutical medicine

Procedia PDF Downloads 408

Authors: Yogesh Dalvi, Ruby Varghese, Nibu Varghese, C. K. Krishnan Nair

Abstract:

Introduction: The Genus Phellinus, locally known as Phansomba is a well-known traditional folk medicine. Especially, in Western Ghats of India, many tribes use several species of Phellinus for various ailments related to teeth, throat, tongue, stomach and even wound healing. It is one of the few mushrooms which play a pivotal role in Ayurvedic Dravyaguna. Aim: The present study focuses on to investigate phytochemical analysis, antioxidant, and antitumor (in vitro and in vivo) potential of Phellinus robinae from South India, Kerala Material and Methods: The present study explores the following: 1. Phellinus samples were collected from Ranni, Pathanamthitta district of Kerala state, India from Artocarpus heterophyllus Lam. and species were identified using rDNA region. 2. The fruiting body was shadow dried, powdered and extracted with 50% alcohol using water bath at 60°C which was further condensed by rotary evaporator and lyophilized at minus 40°C temperature. 3. Secondary metabolites were analyzed by using various phytochemical screening assay (Hager’s Test, Wagner’s Test, Sodium hydroxide Test, Lead acetate Test, Ferric chloride Test, Folin-ciocalteu Test, Foaming Test, Benedict’s test, Fehling’s Test and Lowry’s Test). 4. Antioxidant and free radical scavenging activity were analyzed by DPPH, FRAP and Iron chelating assay. 5. The antitumor potential of Water alcohol extract of Phellinus (PAWE) is evaluated through In vitro condition by Trypan blue dye exclusion method in DLA cell line and In vivo by murine model. Result and Discussion: Preliminary phytochemical screening by various biochemical tests revealed presence of a variety of active secondary molecules like alkaloids, flavanoids, saponins, carbohydrate, protein and phenol. In DPPH and FRAP assay PAWE showed significantly higher antioxidant activity as compared to standard Ascorbic acid. While, in Iron chelating assay, PAWE exhibits similar antioxidant activity that of Butylated Hydroxytoluene (BHT) as standard. Further, in the in vitro study, PAWE showed significant inhibition on DLA cell proliferation in dose dependent manner and showed no toxicity on mice splenocytes, when compared to standard chemotherapy drug doxorubicin. In vivo study, oral administration of PAWE showed dose dependent tumor regression in mice and also raised the immunogenicity by restoring levels of antioxidant enzymes in liver and kidney tissue. In both in vitro and in vivo gene expression studies PAWE up-regulates pro-apoptotic genes (Bax, Caspases 3, 8 and 9) and down- regulates anti-apoptotic genes (Bcl2). PAWE also down regulates inflammatory gene (Cox-2) and angiogenic gene (VEGF). Conclusion: Preliminary phytochemical screening revealed that PAWE contains various secondary metabolites which contribute to its antioxidant and free radical scavenging property as evaluated by DPPH, FRAP and Iron chelating assay. PAWE exhibits anti-proliferative activity by the induction of apoptosis through a signaling cascade of death receptor-mediated extrinsic (Caspase8 and Tnf-α), as well as mitochondria-mediated intrinsic (caspase9) and caspase pathways (Caspase3, 8 and 9) and also by regressing angiogenic factor (VEGF) without any inflammation or adverse side effects. Hence, PAWE serve as a potential antioxidant and antitumor agent.

Keywords: antioxidant, antitumor, Dalton lymphoma ascites (DLA), fungi, Phellinus robinae

Procedia PDF Downloads 304

Authors: Efri Mardawati, Ronny Purwadi, Made Tri Ari Penia Kresnowati, Tjandra Setiadi

Abstract:

EFB are mainly composed of cellulose (≈ 43%), hemicellulose (≈ 23%) and lignin (≈20%). The palm oil empty fruit bunches (EFB) is the lignosellulosic waste from crude palm oil industries mainly compose of (≈ 43%), hemicellulose (≈ 23%) and lignin (≈20%). Xylan, a polymer made of pentose sugar xylose and the most abundant component of hemicellulose in plant cell wall. Further xylose can be used as a raw material for production of a wide variety of chemicals such as xylitol, which is extensively used in food, pharmaceutical and thin coating applications. Currently, xylose is mostly produced from xylan via chemical hydrolysis processes. However, these processes are normally conducted at a high temperature and pressure, which is costly, and the required downstream processes are relatively complex. As an alternative method, enzymatic hydrolysis of xylan to xylose offers an environmentally friendly biotechnological process, which is performed at ambient temperature and pressure with high specificity and at low cost. This process is catalysed by xylanolytic enzymes that can be produced by some fungal species such as Aspergillus niger, Penicillium crysogenum, Tricoderma reseei, etc. Fungal that will be used to produce crude xylanase enzyme in this study is T. Viride ITB CC L.67. It is the purposes of this research to study the influence of pretreatment of EFB for the enzymatic hydrolysis process, optimation of temperature and pH of the hydrolysis process, the influence of substrate and enzyme concentration to the enzymatic hydrolysis process, the dynamics of hydrolysis process and followingly to study the kinetics of this process. Xylose as the product of enzymatic hydrolysis process analyzed by HPLC. The results show that the thermal pretreatment of EFB enhance the enzymatic hydrolysis process. The enzymatic hydrolysis can be well approached by the Michaelis Menten kinetic model, and kinetic parameters are obtained from experimental data.

Keywords: oil palm empty fruit bunches (EFB), xylose, enzymatic hydrolysis, kinetic modelling

Procedia PDF Downloads 389

Authors: Morteza Elsa, Amirhossein Moghanian

Abstract:

The major aim of this study was to evaluate the effect of CaO content on in vitro hydroxyapatite formation, MC3T3 cells cytotoxicity and proliferation as well as antibacterial efficiency of sol-gel derived SiO₂–CaO–P₂O₅ ternary system. For this purpose, first two grades of bioactive glass (BG); BG-58s (mol%: 60%SiO₂–36%CaO–4%P₂O₅) and BG-68s (mol%: 70%SiO₂–26%CaO–4%P₂O₅)) were synthesized by sol-gel method. Second, the effect of CaO content in their composition on in vitro bioactivity was investigated by soaking the BG-58s and BG-68s powders in simulated body fluid (SBF) for time periods up to 14 days and followed by characterization inductively coupled plasma atomic emission spectrometry (ICP-AES), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscopy (SEM) techniques. Additionally, live/dead staining, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and alkaline phosphatase (ALP) activity assays were conducted respectively, as qualitatively and quantitatively assess for cell viability, proliferation and differentiations of MC3T3 cells in presence of 58s and 68s BGs. Results showed that BG-58s with higher CaO content showed higher in vitro bioactivity with respect to BG-68s. Moreover, the dissolution rate was inversely proportional to oxygen density of the BG. Live/dead assay revealed that both 58s and 68s increased the mean number live cells which were in good accordance with MTT assay. Furthermore, BG-58s showed more potential antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) bacteria. Taken together, BG-58s with enhanced MC3T3 cells proliferation and ALP activity, acceptable bioactivity and significant high antibacterial effect against MRSA bacteria is suggested as a suitable candidate in order to further functionalizing for delivery of therapeutic ions and growth factors in bone tissue engineering.

Keywords: antibacterial, bioactive glass, hydroxyapatite, proliferation, sol-gel processes

Procedia PDF Downloads 147

Authors: Stephen Daniel Iduh, Evans Chidi Egwin, Oluwatosin Kudirat Shittu

Abstract:

The process for the development of reliable and eco-friendly metallic Nanoparticles is an important step in the field of Nanotechnology for biomedical application. To achieve this, use of natural sources like biological systems becomes essential. In the present work, extracellular biosynthesis of gold Nanoparticles using aqueous leave and stembark extracts of K. senegalensis has been attempted. The gold Nanoparticles produced were characterized using High Resolution scanning electron microscopy, Ultra Violet–Visible spectroscopy, zeta-sizer Nano, Energy-Dispersive X-ray (EDAX) Spectroscopy and Fourier Transmission Infrared (FTIR) Spectroscopy. The cytotoxicity of the synthesized gold nanoparticles on MCF-7 cell line was evaluated using MTT assay. The result showed a rapid development of Nano size and shaped particles within 5 minutes of reaction with Surface Plasmon Resonance at 520 and 525nm respectively. An average particle size of 20-90nm was confirmed. The amount of the extracts determines the core size of the AuNPs. The core size of the AuNPs decreases as the amount of extract increases and it causes the shift of Surface Plasmon Resonance band. The FTIR confirms the presence of biomolecules serving as reducing and capping agents on the synthesised gold nanoparticles. The MTT assay shows a significant effect of gold nanoparticles which is concentration dependent. This environment-friendly method of biological gold Nanoparticle synthesis has the potential and can be directly applied in cancer therapy.

Keywords: biosynthesis, gold nanoparticles, characterization, calotropis procera, cytotoxicity

Procedia PDF Downloads 490

Authors: Chukwuma S. Ezeonu, Ikechukwu N. E. Onwurah, Uchechukwu U. Nwodo, Chibuike S. Ubani, Chigozie M. Ejikeme

Abstract:

Trichophyton soudanense and Trichophyton mentagrophyte were isolated from the rice mill environment, cultured and used singly and as di-culture in the treatment of measure quantities of preheated rice husk. Optimized conditions studied showed that carboxymethylcellulase (CMCellulase) activity of 57.61 µg/ml/min was optimum for Trichophyton mentagrophyte heat pretreated rice husk crude enzymes at 50oC and 80oC respectively. Duration of 120 hours (5 days) gave the highest CMcellulase activity of 75.84 µg/ml/min for crude enzyme of Trichophyton mentagrophyte heat pretreated rice husk. However, 96 hours (4 days) duration gave maximum activity of 58.21 µg/ml/min for crude enzyme of Trichophyton soudanense heat pretreated rice husk. Highest CMCellulase activities of 67.02 µg/ml/min and 69.02 µg/ml/min at pH of 5 were recorded for crude enzymes of monocultures of Trichophyton soudanense (TS) and Trichophyton mentagrophyte (TM) heat pretreated rice husk respectively. Biomass components showed that rice husk cooled after heating followed by treatment with Trichophyton mentagrophyte gave 44.50 ± 10.90 (% ± Standard Error of Mean) cellulose as the highest yield. Maximum total lignin value of 28.90 ± 1.80 (% ± SEM) was obtained from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM). The hemicellulose content of 30.50 ± 2.12 (% ± SEM) from pre-heated rice husk treated with Trichophyton soudanense (TS); lignin value of 28.90 ± 1.80 from pre-heated rice husk treated with di-culture of Trichophyton soudanense and Trichophyton mentagrophyte (TS+TM); also carbohydrate content of 16.79 ± 9.14 (% ± SEM) , reducing and non-reducing sugar values of 2.66 ± 0.45 and 14.13 ± 8.69 (% ± SEM) were all obtained from for pre- heated rice husk treated with Trichophyton mentagrophyte (TM). All the values listed above were the highest values obtained from each rice husk treatment. The pre-heated rice husk treated with Trichophyton mentagrophyte (TM) fermented with palmwine yeast gave bio-ethanol value of 11.11 ± 0.21 (% ± Standard Deviation) as the highest yield.

Keywords: Trichophyton soudanense, Trichophyton mentagrophyte, biomass, bioethanol, rice husk

Procedia PDF Downloads 679

Authors: Massimo Mozzon, Nadia Raffaelli

Abstract:

Proteases from herbs, woody plants, and trees are exploited for cheesemaking in several countries, especially in South Europe and West Africa. Particularly, “thistles” belonging to several genera within the Asteraceae family (Cynara, Silybum, Centaurea, Carlina, Cirsium, Onopordum) are traditionally used in Mediterranean countries for clotting raw ewe’s and goat’s milk. For the first time, the clotting performance of an aqueous extract from flowers of Onopordum tauricum Willd. (Taurian thistle, bull cottonthistle) were tested in milk of different origin (cow, goat, ewe). The vegetable material was collected in the Central Apennines range, between the Marche and Umbria regions. A response surface methodology (RSM) approach was used to study the effect of the curdling variables (temperature, pH, amount of enzymatic extract) on the technological performance of the thistle extract. A three-step procedure for the purification of the enzyme (ammonium sulphate precipitation, gel filtration and ion-exchange chromatography) was also carried out. The milk clotting activity (MCA) of O. tauricum crude extracts was strongly affected by temperature, pH and by the interaction between these two variables, according to a second-order response surface model, while the milk/coagulant ratio did not affect in a significant way the clotting properties. Experimental data showed that the addition of 10 mM CaCl2 reduced the clotting time of ewe’s, goat’s, and cow’s milk by about 3-fold, 8-fold, and 14-fold, respectively, at 35°C and pH 6.7-6.8. After purification, an enzymatic preparation very close to homogeneity was obtained, which showed a major band at about 30 kDa when analyzed by SDS-PAGE. The identity of the enzyme as an aspartic protease was confirmed by inhibition studies. Cheese-making trials were carried out to check the scale-up (1 to 5 L of milk; 37 °C; 10 mM CaCl2 fortification) and set the recipe: 35-45% of curd yields were recorded, according to curd cutting and pressing.

Keywords: milk clotting activity, Onopordum tauricum, plant proteases, vegetable rennet

Procedia PDF Downloads 159

Authors: Anik Barua, Rabiul Hossain, Labonno Barua, Rashadul Hossain, Nurul Absar

Abstract:

Background: Globally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Hence, scientists are trying to seek noble compounds from natural sources to treat cancer. Objectives: The objectives of the present study are to evaluate the phytochemicals, in vitro antioxidants, and in vivo and in silico anticancer study of various solvent fractions of Tinospora cordifolia (Willd.). Methodology: In this experiment, standard quantitative and qualitative assay methods were used to analyze the phytochemicals. The antioxidant activity was measured using the DPPH and ABTS scavenging methods. The in vivo antitumor activity is evaluated against Ehrlich ascites carcinoma (EAC) cell bearing in Swiss albino mice. In-silico ADME/T and molecular docking study were performed to assess the potential of stated phytochemicals against Transcription Factor STAT3b/DNA Complex of adenocarcinoma. Findings: Phytochemical screening confirmed the presence of flavonoids, alkaloids, glycosides, tannins, and carbohydrates. A significant amount of phenolic (20.19±0.3 mg/g GAE) and flavonoids (9.46±0.18 mg/g GAE) were found in methanolic extract in quantitative screening. Tinospora cordifolia methanolic extract showed promising DPPH and ABTS scavenging activity with the IC50 value of 1222.99 µg/mL and 1534.34 µg/mL, respectively, which was concentration dependent. In vivo anticancer activity in EAC cell-bearing mice showed significant (P < 0.05) percent inhibition of cell growth (60.12±1.22) was found at the highest dose compared with standard drug 5-Fluorouracil (81.18±1.28). Forty-two phytochemicals exhibit notable pharmacokinetics properties and passed drug-likeness screening tests in silico. In molecular docking study, (25S)-3Beta-acetoxy-5-alpha-22-beta-spirost-9(11)-en-12-beta-ol showed docking score (-8.5 kJ/mol) with significant non-bonding interactions with target enzyme. Conclusions: The results were found to be significant and confirmed that the methanolic extract of Tinospora cordifolia has remarkable antitumor activity with antioxidant potential. The Tinospora cordifolia methanolic extract may be considered a potent anticancer agent for advanced research.

Keywords: anticancer, antioxidant, Tinospora cordifolia, EAC cell

Procedia PDF Downloads 129

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

Abstract:

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

Procedia PDF Downloads 250

Authors: T. S. Desak Ketut, A.n. Isna, A.a. Ayu, D. P. Ririn, Suharjono Hadiatullah

Abstract:

The requirement of paper from year to year rise rapidly. The raising of cellulose requirement in paper production caused increasing of wood requirement with the effect that limited forest areal because of deforestation. Alternative cellulose that can be used for making paper is microbial cellulose. The objective of this research are to know the effectivity fermentation media Spirogyra sp. by Gluconacetobacter xylinum for cellulose production as material for the making of paper and to know effect composition bacterial cellulose composite product of Gluconacetobacter xylinum in Spirogyra sp. The method, was used, is as follow, 1) the effect assay from variation composition of fermentation media to bacterial cellulose production by Gluconacetobacter xylinum. 2) The effect assay of composition bacterial cellulose fermentation producted by Gluconacetobacter xylinum in extracted Spirogyra media to paper quality. The result of this research is variation fermentation media Spirogyra sp. affect to production of cellulose by Gluconacetobacter xylinum. Thus, result showed by the highest value and significantly different in thickness parameter, dry weight and wet weight of nata in sucrose concentration 7,5 % and urea 0,75 %. Composition composite of bacterial cellulose from fermentation product by Gluconacetobacter xylinum in media Spirogyra sp. affect to paper quality from wet nata and dry nata. Parameters thickness, weight, water absorpsion, density and gramatur showed highest result in sucrose concentration 7,5 % and urea concentration 0,75 %, except paper density from dry nata had highest result in sucrose and urea concentration 0%.

Keywords: cellulose, fermentation media, , Gluconacetobacter xylinum, paper, Spirogyra sp.

Procedia PDF Downloads 343

Authors: Gulcin Yildiz

Abstract:

Drying (dehydration) is the process of removing water from food in order to preserve the food and an alternative to reduce post-harvest loss of fruits. Different pre-treatment methods have been developed for fruit drying, such as ultrasound. If no pre-treatment is done, the fruits will continue to darken after they are dried. However, the effects of ultrasound as pre-treatment on drying of apples has not been well documented. This study was undertaken to investigate the effect of ultrasound as pre-treatment before oven drying of red delicious and golden delicious apples. Red delicious and golden delicious apples were dried in different temperatures. Before performing drying experiments in an oven at 50, 75 and 100 °C, ultrasound as pretreatment was applied in 5, 10, and 15 minutes. Colors of the dried apples were measured with a Minolta Chroma Meter CR-300 (Minolta Camera Co. Ltd., Osaka, Japan) by directly holding the device vertically to the surface of the samples. Content of total phenols was determined spectrophotometrically with the FolinCiocalteau assay, and the antioxidant capacity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The samples (both red delicious and golden delicious apples) with longer ultrasound treatment produced higher weight loss due to the changes in tissue structure. However less phenolic content and antioxidant capacity were observed for the samples with longer ultrasound pre-treatment. The highest total phenolic content (TPC) was determined in dried apples at 75 °C with 5 minutes pre-treatment ultrasound and the lowest TPC was determined in dried apples at 50 °C with 15 minutes pre-treatment ultrasound which was subjected to the longest ultrasound pre-treatment and drying. The combination of 5 min of ultrasound pre-treatment and 75 °C of oven-drying showed to be the best combination for an energy efficient process. This combination exhibited good antioxidant properties as well. The present study clearly demonstrated that applying ultrasound as pre-treatment for drying of apples is an effective process in terms of quality of dried products, time, and energy.

Keywords: golden delicious apples, red delicious apples, total phenolic content, Ultrasound

Procedia PDF Downloads 296

Authors: Engy Shokry, Giancarlo La Marca, Maria Luisa Della Bona

Abstract:

Newborn screening for Congenital Adrenal Hyperplasia (CAH) relies on quantification of 17α-hydroxyprogesterone using enzyme immunoassays. These assays, in spite of being rapid, readily available and easy to perform, its reliability was found questionable due to lack of selectivity and specificity resulting in large number of false-positives, consequently family anxiety and associated hospitalization costs. To improve specificity of conventional 17α-hydroxyprogesterone screening which may experience false transient elevation in preterm, low birth weight or acutely ill neonates, steroid profiling by LC-MS/MS as a second-tier test was implemented. Unlike the previously applied LC-MS/MS methods, with the disadvantage of requiring a relatively high number of blood drops. Since newborn screening tests are increasing, it is necessary to minimize the sample volume requirement to make the maximum use of blood samples collected on filter paper. The proposed new method requires just one 3.2 mm dried blood spot (DBS) punch. Extraction was done using methanol: water: formic acid (90:10:0.1, v/v/v) containing deuterium labelled internal standards. Extracts were evaporated and reconstituted in 10 % acetone in water. Column switching strategy for on-line sample clean-up was applied to improve the chromatographic run. The first separative step retained the investigated steroids and passed through the majority of high molecular weight impurities. After the valve switching, the investigated steroids are back flushed from the POROS® column onto the analytical column and separated using gradient elution. Found quantitation limits were 5, 10 and 50 nmol/L for 17α-hydroxyprogesterone, androstenedione and cortisol respectively with mean recoveries of between 98.31-103.24 % and intra-/ inter-assay CV% < 10 % except at LLOQ. The method was validated using standard addition calibration and isotope dilution strategies. Reference ranges were determined by analysing samples from 896 infants of various ages at the time of sample collection. The method was also applied on patients with confirmed CAH. Our method represents an attractive combination of low sample volume requirement, minimal sample preparation time without derivatization and quick chromatography (5 min). The three steroid profile and the concentration ratios (17OHP + androstenedione/cortisol) allowed better screening outcomes of CAH reducing false positives, associated costs and anxiety.

Keywords: congenital adrenal hyperplasia (CAH), 17α-hydroxyprogesterone, androstenedione, cortisol, LC-MS/MS

Procedia PDF Downloads 439

Authors: Dalita G. S. M. Cavalcante, Elton A. P. dos Reis, Andressa S. Gomes, Caroline S. Danna, Leandra Ernest Kerche-Silva, Eidi Yoshihara, Aldo E. Job

Abstract:

In order to minimize environmental impacts, a composite was developed from mixture of leather shavings (LE) with natural rubber (NR), which patent is already deposited. The new material created can be used in applications such as floors e heels for shoes. Besides these applications, the aim is to use this new material for the production of products for the textile industry, such as boots, gloves and bags. But the question arises, as to biocompatibility of this new material. This is justified because the structure of the leather shavings has chrome. The trivalent chromium is usually not toxic, but the hexavalent chromium can be highly toxic and genotoxic for living beings, causing damage to the DNA molecule and contributing to the formation of cancer. Based on this, the objective of this study is evaluate the possible genotoxic effects of the new composite, using as system - test two cell lines (MRC-5 and CHO-K1) by comet assay. For this, the production of the composite was performed in three proportions: for every 100 grams of NR was added 40 (E40), 50 (E50) or 60 (E60) grams of LE. The latex was collected from the rubber tree (Hevea brasiliensis). For vulcanization of the NR, activators and accelerators were used. The two cell lines were exposed to the new composite in its three proportions using elution method, that is, cells exposed to liquid extracts obtained from the composite for 24 hours. For obtaining the liquid extract, each sample of the composite was crushed into pieces and mixed with an extraction solution. The quantification of total chromium and hexavalent chromium in the extracts were performed by Optical Emission Spectrometry by Inductively Coupled Plasma (ICP-OES). The levels of DNA damage in cells exposed to both extracts were monitored by alkaline version of the comet assay. The results of the quantification of metals in ICP-OES indicated the presence of total chromium in different extracts, but were not detected presence of hexavalent chromium in any extract. Through the comet assay were not found DNA damage of the CHO-K1 cells exposed to both extracts. As for MRC-5, was found a significant increase in DNA damage in cells exposed to E50 and E60. Based on the above data, it can be asserted that the extracts obtained from the composite were highly genotoxic for MRC-5 cells. These biological responses do not appear to be related to chromium metal, since there was a predominance of trivalent chromium in the extracts, indicating that during the production process of the new composite, there was no formation of hexavalent chromium. In conclusion it can infer that the leather shavings containing chromium can be reused, thereby reducing the environmental impacts of this waste. Already on the composite indicates to its incorporation in applications that do not aim at direct contact with the human skin, and it is suggested the chain of composite production be studied, in an attempt to make it biocompatible so that it may be safely used by the textile industry.

Keywords: cell line, chrome, genotoxicity, leather, natural rubber

Procedia PDF Downloads 196

Authors: Jae Bok Heo, Yun Mi Lee, Hee Rang Yun

Abstract:

Several GTPases are required for ribosome biogenesis and assembly. We recently characterized rice (Oryza sativa) nuclear/nucleolar GTPase 2 (OsNug2), belonging to the YlqF/YawG family of GTPases, as playing a role in pre-60S ribosomal subunit maturation. To investigate the potential factors involved in regulating the function of OsNug2, yeast two-hybrid screens were carried out using OsNug2 as bait. Rice serine/threonine kinase 1 (OsSTK1) was identified as a potential interacting protein candidate. In vitro pull down and bimolecular fluorescence complementation assays confirmed the interaction between OsNug2 and OsSTK1, and like green fluorescent protein-tagged OsNug2, green fluorescent protein-tagged OsSTK1 was targeted to the nucleus of Arabidopsis protoplasts. OsSTK1 was not found to affect the GTP-binding activity of OsNug2; however, when recombinant OsSTK1 was included in OsNug2 assay reaction mixtures, OsSTK1 increased the GTPase activity of OsNug2. To test whether OsSTK1 phosphorylates OsNug2 in vitro, a kinase assay was performed. OsSTK1 was found to have weak autophosphorylation activity and strongly phosphorylated serine 209 of OsNug2. Yeast complementation testing resulted in a GAL::OsNug2(S209N) mutant-harboring yeast strain exhibiting a growth-defective phenotype on galactose medium at 39°C, divergent from that of a yeast strain harboring GAL::OsNug2. The intrinsic GTPase activity of mutant OsNug2(S209N) was found to be similar to that of OsNug2, was not fully enhanced upon weak binding of OsSTK1. Our findings reported here indicate that OsSTK1 functions as a positive regulator protein of OsNug2 by enhancing the GTPase activity of OsNug2, and that the phosphorylation of serine 209 of OsNug2 is essential for the complete function of OsNug2 in ribosome biogenesis.

Keywords: OsSTK1, OsNug2, GTPase activity, GTP binding activity, phosphorylation

Procedia PDF Downloads 371

Authors: Tomás Sobrino, Lara García-Varela, Marta Aramburu-Núñez, Mónica Castro, Noemí Gómez-Lado, Mariña Rodríguez-Arrizabalaga, Antía Custodia, Juan Manuel Pías-Peleteiro, José Manuel Aldrey, Daniel Romaus-Sanjurjo, Ángeles Almeida, Pablo Aguiar, Alberto Ouro

Abstract:

Introduction: Alzheimer’s disease (AD) and other tauopathies are primary causes of dementia, causing progressive cognitive deterioration that entails serious repercussions for the patients' performance of daily tasks. Currently, there is no effective approach for the early diagnosis and treatment of AD and tauopathies. This study suggests a theragnostic approach based on the importance of phosphorylated tau protein (p-Tau) in the early pathophysiological processes of AD. We have developed a novel theragnostic monoclonal antibody (mAb) to provide both diagnostic and therapeutic effects. Methods/Results: We have developed a p-Tau mAb, which was doped with deferoxamine for radiolabeling with Zirconium-89 (89Zr) for PET imaging, as well as fluorescence dies for immunofluorescence assays. The p-Tau mAb was evaluated in vitro for toxicity by MTT assay, LDH activity, propidium iodide/Annexin V assay, caspase-3, and mitochondrial membrane potential (MMP) assay in both mouse endothelial cell line (bEnd.3) and cortical primary neurons cell cultures. Importantly, non-toxic effects (up to concentrations of p-Tau mAb greater than 100 ug/mL) were detected. In vivo experiments in the tauopathy model mice (PS19) show that the 89Zr-pTau-mAb and 89Zr-Fragments-pTau-mAb are stable in circulation for up to 10 days without toxic effects. However, only less than 0.2% reached the brain, so further strategies have to be designed for crossing the Brain-Blood-Barrier (BBB). Moreover, an intraparenchymal treatment strategy was carried out. The PS19 mice were operated to implement osmotic pumps (Alzet 1004) at two different times, at 4 and 7 months, to stimulate the controlled release for one month each of the B6 antibody or the IgG1 control antibody. We demonstrated that B6-treated mice maintained their motor and memory abilities significantly compared with IgG1 treatment. In addition, we observed a significant reduction in p-Tau deposits in the brain. Conclusions /Discussion: A theragnostic pTau-mAb was developed. Moreover, we demonstrated that our p-Tau mAb recognizes very-early pathology forms of p-Tau by non-invasive techniques, such as PET. In addition, p-Tau mAb has non-toxic effects, both in vitro and in vivo. Although the p-Tau mAb is stable in circulation, only 0.2% achieve the brain. However, direct intraventricular treatment significantly reduces cognitive impairment in Alzheimer's animal models, as well as the accumulation of toxic p-Tau species.

Keywords: alzheimer's disease, theragnosis, tau, PET, immunotherapy, tauopathies

Procedia PDF Downloads 70