Search results for: enzyme-linked immunosorbent assay

Authors: M. R. Vijayakumar, K. Priyanka, Ramoji Kosuru, Lakshmi, Sanjay Singh

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Trans resveratrol (RES) is a non-flavonoid poly-phenolic compound proved for its therapeutic and preventive effect against various types of cancer. However, the practical application of RES in cancer treatment is limited because of its higher dose (up to 7.5 g/day in humans), low biological half life, rapid metabolism and faster elimination in mammals. PEGylated core-shell type lipid polymer hybrid nanoparticles are the novel drug delivery systems for long circulation and improved anti cancer effect of its therapeutic payloads. Therefore, the main objective of this study is to extend the biological half life (long circulation) and improve the therapeutic efficacy of RES through core shell type of nanoparticles. D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS), a novel surfactant is applied for the preparation of PEGylated lipid polymer hybrid nanoparticles. The prepared nanoparticles were evaluated by various state of the art techniques such as dynamic light scattering (DLS) technique for particle size and zeta potential, TEM for shape, differential scanning calorimetry (DSC) for interaction analysis and XRD for crystalline changes of drug. Entrapment efficiency and invitro drug release were determined by ultracentrifugation method and dialysis bag method, respectively. Cancer cell viability studies were performed by MTT assay, respectively. Pharmacokinetic studies after i.v administration were performed in sprague dawley rats. The prepared NPs were found to be spherical in shape with smooth surfaces. Particle size and zeta potential of prepared NPs were found to be in the range of 179.2±7.45 to 266.8±9.61 nm and -0.63 to -48.35 mV, respectively. DSC revealed absence of potential interaction. XRD study revealed presence of amorphous form in nanoparticles. Entrapment efficiency was found to be 83.7 % and drug release was found to be in controlled manner. MTT assay showed low MEC and pharmacokinetic studies showed higher AUC of nanoformulaition than its pristine drug. All these studies revealed that the RES loaded PEG modified core-shell type lipid polymer hybrid nanoparticles can be an alternative tool for chemopreventive and therapeutic application of RES in cancer.

Keywords: trans resveratrol, cancer nanotechnology, long circulating nanoparticles, bioavailability enhancement, core shell nanoparticles, lipid polymer hybrid nanoparticles

Procedia PDF Downloads 472

Authors: Ali Bahri Lubis

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Tryptophan oxidation by Heme-dioxygenase enzyme is initial important stepTryptophan oxidation by Heme-dioxygenase enzyme is initial important step in kynurenine pathway implicating to several severe diseases such as Parkinson’s Disease, Huntington Disease, poliomyelitis and cataract. It is crucial to comprehend the oxidation mechanism with the hope to find decent treatment upon abovementioned diseases. The mechanism has been debatable since no one has been yet proved the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) using various substrates: L-tryptophan, L-tryptophan (indole-ring-2-13C), L-fully-labelled13C-tryptophan, L-N-methyl-tryptophan, L-tryptophan and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR, and HSQC. We also successfully synthesized enzyme products as our control in NMR measurements. The result exhibited that the distinct substrates produced N-formyl kynurenine (NFK) and hydroxypyrrolloindoleamine carboxylate acid (HPIC) in different concentrations and isomers, correlated to the proposal of considered mechanism reaction in kynurenine pathway implicating to several severe diseases such as Parkinson’s Disease, Huntington Disease, poliomyelitis and cataract. It is crucial to comprehend the oxidation mechanism with the hope to find decent treatment for the abovementioned diseases. The mechanism has been debatable since no one has yet proven the mechanism obviously. In this research we have attempted to prove mechanistic steps of tryptophan oxidation via human indoleamine dioxygenase (h-IDO) using various substrates: L-tryptophan, L-tryptophan (indole-ring-2-13C), L-fully-labelled13C-tryptophan, L-N-methyl-tryptophan, L-tryptophan and 2-amino-3-(benzo(b)thiophene-3-yl) propanoic acid. All enzyme assay experiments were measured using a UV-Vis spectrophotometer, LC-MS, 1H-NMR and HSQC. We also successfully synthesized enzyme products as our control in NMR measurements. The result exhibited that the distinct substrates produced N-formyl kynurenine (NFK) and hydroxypyrrolloindoleamine carboxylate acid (HPIC) in different concentrations and isomers, correlated to the proposal of considered mechanism reaction.

Keywords: heme-dioxygenase enzyme, tryptophan oxidation, kynurenine pathway, n-formyl kynurenine

Procedia PDF Downloads 79

Authors: Abdelaaty A. Shahat, Mansour S. Alsaid

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Four Acetylated flavonol glycosides were isolated from Centaurium spicatum (L.) Fritsch (Gentianaceae). Structure elucidation, especially the localization of the acetyl groups, and complete 1H and 13C NMR assignments of these biologically active compounds were carried out using one- and two-dimensional NMR methods, including CNMR, DEPT-135 and DEPT-90 and gradient-assisted experiments such as DQF-COSY, TOCSY, HSQC and HMBC experiments. The antioxidant activities of the new acetylated flavonoid glycosides using DPPH• assay were determined. The compounds tested showed a good DPPH• activity compared with control, but their activity was lower than that of their corresponding aglycone, quercetin.

Keywords: Centaurium spicatum, flavonoids, biological activity, isolation, glycosides

Procedia PDF Downloads 407

Authors: Mayuva Youngsabanant, Chanikarn Srinark, Supanyika Sengsai, Soratorn Kerdkriangkrai, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The follicular fluid proteins of healthy medium size follicles (4-6 mm in diameters) and large size follicles (7-8 mm in diameter) of large white pig ovaries were collected by using sterile technique. They were used for testing the effect on primary in vitro cell culture growth of porcine oviductal epithelial cells (pOEC). Porcine oviductal epithelial cells of luteal phase was culture in M199 and added with 10% fetal calf serum 2.2 mg/mL, NaHCO₃, 0.25 mM pyruvate, 15 µg/mL and 50 µg/mL, gentamycin sulfate at high humidified atmosphere with 5% CO₂ in 95% air atmosphere at 37°C for 96 h before testing. The optimized concentration of pFF of two follicle sizes (at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins) in culture medium was observed for 24 h using MTT assay. Results were analyzed with a one-way ANOVA in SPSS statistic. Moreover, pOEC was also studied in morphological characteristic on long-term culture. The results of long-term study revealed that pOEC showed 70-80 percentage of healthy morphology on epithelial-like character and contained 30 percentage of an elongated shape (fibroblast-like morphology) at 4 weeks of culture time. MTT assay reviewed an increase in the percentage of viability of pOEC in 2 treated of follicular fluid groups. Two treatment concentration groups were higher than control group (p < 0.05) but not in positive control group. Interestingly, at 200 µg protein of 2 treated follicular fluid groups were reached the highest cell viability which is higher than a positive control and it is significantly different form control group (P < 0.05). These cells are developed and had fibroblast elongate shape which is longer than the cells in control group and positive control group. This report implies that pFF of medium follicle size at 200 µg proteins and large follicle size at 200 and 500 µg proteins could be optimized concentration for using as a supplement in culture medium to promote cell growth and development instead of growth hormone from fetal calf serum. It could be applied in cell biotechnology researches. Acknowledgements: The project was funded by a grant from Silpakorn University Research and Development Institute (SURDI) and Faculty of Science, Silpakorn University, Thailand.

Keywords: in vitro, porcine follicular fluid protein (pFF), porcine oviductal epithelial cells (pOEC), MTT

Procedia PDF Downloads 145

Authors: Shohei Matsuo, Saki Mikai, Hiroshi Morita

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Objectives: Shochu is a popular Japanese distilled spirits. In the production of shochu, the filamentous fungus Aspergillus kawachii has traditionally been used. A. kawachii produces two types of starch hydrolytic enzymes, α-amylase (enzymatic liquefaction) and glucoamylase (enzymatic saccharification). Liquid culture system is a relatively easy microorganism to ferment with relatively low cost of production compared for solid culture. In liquid culture system, acid-unstable α-amylase (α-A) was produced abundantly, but, acid-stable α-amylase (Aα-A) was not produced. Since there is high enzyme productivity, most in shochu brewing have been adopted by a solid culture method. In this study, therefore, we investigated production of Aα-A in liquid culture system. Materials and methods: Microorganism Aspergillus kawachii NBRC 4308 was used. The mold was cultured at 30 °C for 7~14 d to allow formation of conidiospores on slant agar medium. Liquid Culture System: A. kawachii was cultured in a 100 ml of following altered SLS medium: 1.0 g of rice flour, 0.1 g of K2HPO4, 0.1 g of KCl, 0.6 g of tryptone, 0.05 g of MgSO4・7H2O, 0.001 g of FeSO4・7H2O, 0.0003 g of ZnSO4・7H2O, 0.021 g of CaCl2, 0.33 of citric acid (pH 3.0). The pH of the medium was adjusted to the designated value with 10 % HCl solution. The cultivation was shaking at 30 °C and 200 rpm for 72 h. It was filtered to obtain a crude enzyme solution. Aα-A assay: The crude enzyme solution was analyzed. An acid-stable α-amylase activity was carried out using an α-amylase assay kit (Kikkoman Corporation, Noda, Japan). It was conducted after adding 9 ml of 100 mM acetate buffer (pH 3.0) to 1 ml of the culture product supernatant and acid treatment at 37°C for 1 h. One unit of a-amylase activity was defined as the amount of enzyme that yielded 1 mmol of 2-chloro-4-nitrophenyl 6-azide-6-deoxy-b-maltopentaoside (CNP) per minute. Results and Conclusion: We experimented with co-culture of A. kawachii and lactobacillus in order to get control of pH in altered SLS medium. However, high production of acid-stable α-amylase was not obtained. We experimented with yoghurt or milk made an addition to liquid culture. The result indicated that high production of acid-stable α-amylase (964 U/g-substrate) was obtained when milk made an addition to liquid culture. Phosphate concentration in the liquid medium was a major cause of increased acid-stable α-amylase activity. In liquid culture, acid-stable α-amylase activity was enhanced by milk, but Fats and oils in the milk were oxidized. In addition, Tryptone is not approved as a food additive in Japan. Thus, alter SLS medium added to skim milk excepting for the fats and oils in the milk instead of tryptone. The result indicated that high production of acid-stable α-amylase was obtained with the same effect as milk.

Keywords: acid-stable α-amylase, liquid culture, milk, shochu

Procedia PDF Downloads 284

Authors: D. ChobfroushKhoei, S. K. Heidari , Sh. Dariadel

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Carbon nanotubes were used in medical sciences especially in drug delivery system and cancer therapy. In this study, we functionalized carboxylated single-wall carbon nanotubes (SWNT-COOH) with 2-en 4-hydroxy cyclo 1-octanon. Synthesized sample was characterized by FT-IR, Raman spectroscopy, SEM, TGA and cellular investigations. The results showed well formation of SWNT-Ester. Cell viability assay results and microscopic observations demonstrated that cancerous cells were killed in the sample. The synthesized sample can be used as a toxic material for cancer therapy.

Keywords: MWNT-COOH, functionalization, phenylisocyanate, phenylisothiocyanate, 1, 4-phenylendiamine, toxicity investigation

Procedia PDF Downloads 453

Authors: Balgabay Sadepovich Maikanov, Laura Tyulegenovna Auteleyeva, Seidenova Simbat Polatbekovna

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In this article reduced nutritional value of the rabbits’ meat at 1, 1 dimethylhydrazine experimental toxicosis is shown. The assay was performed on liquid chromatograph SHIMADZU LC-20 Prominence (Japan) with fluorometric and spectrophotometric detector. This research has revealed that samples of rabbit meat of the experimental group had significant differences from the control group:in amino acids concentration from 1.2% to 9.1%; vitamin concentration from 11.2% to 60.5%, macro – minerals concentration from 17.4% to 78.1% and saturated fatty acids concentration from 17,1% to 34.5%, respectively. The decrease in the chemical composition of rabbits’ meat at 1,1 dimethylhydrazine toxicosis may be due to changes in the internal processes associated with impaired metabolic homeostasis of animals.

Keywords: 1, 1-dimethylhydrazine, metabolic homeostasis, nutritional value, rabbit meat

Procedia PDF Downloads 215

Authors: Elena Maria Scalisi

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The increasing production and the use of TiO₂ nanoparticles (NPs) have inevitably led to their release into the environment, thereby posing a threat to organisms and also for human. Human exposure to TiO₂-NPs may occur during both manufacturing and use. TiO₂-NPs are common in consumer products for dermal application, toothpaste, food colorants, and nutritional supplements, then oral exposure may occur during use of such products. Into the body, TiO₂-NPs thanks to their small size (<100 nm), can, through testicular blood barrier inducing effect on testis and then on male reproductive health. The nanoscale size of TiO₂ increase the surface-to-volume ratio making them more reactive in a cell, then TiO₂ NPs increase their ability to produce reactive oxygen species (ROS). In male germ cells, ROS may have important implications in maintaining the normal functions of mature spermatozoa at physiological levels, moreover, in spermatozoa they are important signaling molecules for their hyperactivation and acrosome reaction. Nevertheless, an excess of ROS by external inputs such as NPs can increased the oxidative stress (OS), which results in damage DNA and apoptosis. The aim of our study has been investigate the impact of TiO₂ NPs on human spermatozoa, evaluating DNA damage and the expression of proteins involved in cell stress. According WHO guidelines 2021, we have exposed human spermatozoa in vitro to TiO₂ NP at concentrations 50 ppm, 100 ppm, 250 ppm, and 500 ppm for 1 hour (at 37°C and CO₂ at 5%). DNA damage was evaluated by Sperm Chromatin Dispersion Test (SCD) and TUNEL assay; moreover, we have evaluated the expression of biomarkers of oxidative stress like Heat Shock Protein 70 (HSP70) and Metallothioneins (MTs). Also, sperm parameters as motility viability have been evaluated. Our results not report a significant reduction in motility of spermatozoa at the end of the exposure. On the contrary, the progressive motility was increased at the highest concentration (500 ppm) and was statistically significant compared to control (p <0.05). Also, viability was not changed by exposure to TiO₂-NPs (p <0.05). However, increased DNA damage was observed at all concentrations, and the TUNEL assay highlighted the presence of single strand breaks in the DNA. The spermatozoa responded to the presence of TiO₂-NPs with the expression of Hsp70, which have a protective function because they allow the maintenance of cellular homeostasis in stressful/ lethal conditions. A positivity for MTs was observed mainly for the concentration of 4 mg/L. Although the biological and physiological function of the metallothionein (MTs) in the male genital organs is unclear, our results highlighted that the MTs expressed by spermatozoa maintain their biological role of detoxification from metals. Our results can give additional information to the data in the literature on the toxicity of TiO₂-NPs and reproduction.

Keywords: human spermatozoa, DNA damage, TiO₂-NPs, biomarkers

Procedia PDF Downloads 144

Authors: Stephanie Newman

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Niemann-Pick Type C disease is a rare, usually fatal lysosomal storage disorder. Although clinically characterized by progressive neurodegeneration, there is also evidence of altered innate immune responses such as neuroinflammation that promote disease progression. We have initiated an investigation into whether phagocytosis, an important innate immune activity and the process by which particles are ingested is defective in NPC. Using an in vitro assay, we have shown that NPC macrophages have a deficiency in the phagocytosis of different particles. We plan to investigate the mechanistic basis for impaired phagocytosis, the contribution that this deficiency makes to disease pathology, and whether therapies that have shown in vivo benefit are able to restore phagocytic activity.

Keywords: Niemann Pick Disease C, phagocytosis, innate immunity, lysosomal storage disorder

Procedia PDF Downloads 392

Authors: Michael R. Shurin, Galina Shurin, Vladimir A. Kirichenko

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Background. Visibility of hepatic malignancies is poor on non-contrast imaging for daily verification of liver malignancies prior to radiation therapy on MRI-guided Linear Accelerators (MR-Linac). Ferumoxytol® (Feraheme, AMAG Pharmaceuticals, Waltham, MA) is a SPION agent that is increasingly utilized off-label as hepatic MRI contrast. This agent has the advantage of providing a functional assessment of the liver based upon its uptake by hepatic Kupffer cells proportionate to vascular perfusion, resulting in strong T1, T2 and T2* relaxation effects and enhanced contrast of malignant tumors, which lack Kupffer cells. The latter characteristic has been recently utilized for MRI-guided radiotherapy planning with precision targeting of liver malignancies. However potential radiotoxicity of SPION has never been addressed for its safe use as an MRI-contrast agent during liver radiotherapy on MRI-Linac. This study defines the radiomodulating properties of SPIONs in vitro on human monocyte and macrophage cell lines exposed to 60Go gamma-rays within clinical radiotherapy dose range. Methods. Human monocyte and macrophages cell line in cultures were loaded with a clinically relevant concentration of Ferumoxytol (30µg/ml) for 2 and 24 h and irradiated to 3Gy, 5Gy and 10Gy. Cells were washed and cultured for additional 24 and 48 h prior to assessing their phenotypic activation by flow cytometry and function, including viability (Annexin V/PI assay), proliferation (MTT assay) and cytokine expression (Luminex assay). Results. Our results reveled that SPION affected both human monocytes and macrophages in vitro. Specifically, iron oxide nanoparticles decreased radiation-induced apoptosis and prevented radiation-induced inhibition of human monocyte proliferative activity. Furthermore, Ferumoxytol protected monocytes from radiation-induced modulation of phenotype. For instance, while irradiation decreased polarization of monocytes to CD11b+CD14+ and CD11bnegCD14neg phenotype, Ferumoxytol prevented these effects. In macrophages, Ferumoxytol counteracted the ability of radiation to up-regulate cell polarization to CD11b+CD14+ phenotype and prevented radiation-induced down-regulation of expression of HLA-DR and CD86 molecules. Finally, Ferumoxytol uptake by human monocytes down-regulated expression of pro-inflammatory chemokines MIP-1α (Macrophage inflammatory protein 1α), MIP-1β (CCL4) and RANTES (CCL5). In macrophages, Ferumoxytol reversed the expression of IL-1RA, IL-8, IP-10 (CXCL10) and TNF-α, and up-regulates expression of MCP-1 (CCL2) and MIP-1α in irradiated macrophages. Conclusion. SPION agent Ferumoxytol increases resistance of human monocytes to radiation-induced cell death in vitro and supports anti-inflammatory phenotype of human macrophages under radiation. The effect is radiation dose-dependent and depends on the duration of Feraheme uptake. This study also finds strong evidence that SPIONs reversed the effect of radiation on the expression of pro-inflammatory cytokines involved in initiation and development of radiation-induced liver damage. Correlative translational work at our institution will directly assess the cyto-protective effects of Ferumoxytol on human Kupfer cells in vitro and ex vivo analysis of explanted liver specimens in a subset of patients receiving Feraheme-enhanced MRI-guided radiotherapy to the primary liver tumors as a bridge to liver transplant.

Keywords: superparamagnetic iron oxide nanoparticles, radioprotection, magnetic resonance imaging, liver

Procedia PDF Downloads 72

Authors: Michael Russelle S. Alvarez, Noel S. Quiming, Francisco M. Heralde

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This study aims to: a) obtain ethanolic (95% EtOH) and aqueous extracts of Selaginella elmeri, Christella dentata, Elatostema sinnatum, Curculigo capitulata, Euphorbia hirta, Murraya koenigii, Alpinia speciosa, Cymbopogon citratus, Eucalyptus globulus, Jatropha curcas, Psidium guajava, Gliricidia sepium, Ixora coccinea and Capsicum frutescens and screen them for larvicidal activities against Aedes aegypti (Linn.) and Aedes albopictus (Skuse) larvae; b) to fractionate the most active extract and determine the most active fraction; c) to determine the larvicidal properties of the most active extract and fraction against by computing their percentage mortality, LC50, and LC90 after 24 and 48 hours of exposure; and d) to determine the nature of the components of the active extracts and fractions using phytochemical screening. Ethanolic (95% EtOH) and aqueous extracts of the selected plants will be screened for potential larvicidal activity against Ae. aegypti and Ae. albopictus using standard procedures and 1% malathion and a Piper nigrum based ovicide-larvicide by the Department of Science and Technology as positive controls. The results were analyzed using One-Way ANOVA with Tukey’s and Dunnett’s test. The most active extract will be subjected to partial fractionation using normal-phase column chromatography, and the fractions subsequently screened to determine the most active fraction. The most active extract and fraction were subjected to dose-response assay and probit analysis to determine the LC50 and LC90 after 24 and 48 hours of exposure. The active extracts and fractions will be screened for phytochemical content. The ethanolic extracts of C. citratus, E. hirta, I. coccinea, G. sepium, M. koenigii, E globulus, J. curcas and C. frutescens exhibited significant larvicidal activity, with C. frutescens being the most active. After fractionation, the ethyl acetate fraction was found to be the most active. Phytochemical screening of the extracts revealed the presence of alkaloids, tannins, indoles and steroids. A formulation using talcum powder–300 mg fraction per 1 g talcum powder–was made and again tested for larvicidal activity. At 2 g/L, the formulation proved effective in killing all of the test larvae after 24 hours.

Keywords: larvicidal activity screening, partial purification, dose-response assay, capsicum frutescens

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Authors: Oseyemi Omowunmi Olubomehin, Olufemi Michael Denton

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Diabetes mellitus is a disease that is related to the digestion of carbohydrates, proteins and fats and how this affects the blood glucose levels. Various synthetic drugs employed in the management of the disease work through different mechanisms. Keeping postprandial blood glucose levels within acceptable range is a major factor in the management of type 2 diabetes and its complications. Thus, the inhibition of carbohydrate-hydrolyzing enzymes such as α-amylase is an important strategy in lowering postprandial blood glucose levels, but synthetic inhibitors have undesirable side effects like flatulence, diarrhea, gastrointestinal disorders to mention a few. Therefore, it is necessary to identify and explore the α-amylase inhibitors from plants due to their availability, safety, and low costs. In the present study, extracts from the leaves of Rauvolfia vomitoria and seeds of Picralima nitida which are used in the Nigeria traditional system of medicine to treat diabetes were tested for their α-amylase inhibitory effect. The powdered plant samples were subjected to phytochemical screening using standard procedures. The leaves and seeds macerated successively using n-hexane, ethyl acetate and methanol resulted in the crude extracts which at different concentrations (0.1, 0.5 and 1 mg/mL) alongside the standard drug acarbose, were subjected to α-amylase inhibitory assay using the Benfield and Miller methods, with slight modification. Statistical analysis was done using ANOVA, SPSS version 2.0. The phytochemical screening results of the leaves of Rauvolfia vomitoria and the seeds of Picralima nitida showed the presence of alkaloids, tannins, saponins and cardiac glycosides while in addition Rauvolfia vomitoria had phenols and Picralima nitida had terpenoids. The α-amylase assay results revealed that at 1 mg/mL the methanol, hexane, and ethyl acetate extracts of the leaves of Rauvolfia vomitoria gave (15.74, 23.13 and 26.36 %) α-amylase inhibitions respectively, the seeds of Picralima nitida gave (15.50, 30.68, 36.72 %) inhibitions which were not significantly different from the control at p < 0.05, while acarbose gave a significant 56 % inhibition at p < 0.05. The presence of alkaloids, phenols, tannins, steroids, saponins, cardiac glycosides and terpenoids in these plants are responsible for the observed anti-diabetic activity. However, the low percentages of α-amylase inhibition by these plant samples shows that α-amylase inhibition is not the major way by which both plants exhibit their anti-diabetic effect.

Keywords: alpha-amylase, Picralima nitida, postprandial hyperglycemia, Rauvolfia vomitoria

Procedia PDF Downloads 191

Authors: A. Mulry, C. Kealey, D. B. Brady

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Planktonic bacteria can form biofilms which are microbial aggregates embedded within a matrix of extracellular polymeric substances (EPS). They can be found attached to abiotic or biotic surfaces. Biofilms are responsible for oral diseases such as dental caries, gingivitis and the progression of periodontal disease. Biofilms can resist 500 to 1000 times the concentration of biocides and antibiotics used to kill planktonic bacteria. Biofilm development on oral surfaces involves four stages, initial attachment, early development, maturation and dispersal of planktonic cells. The Minimum Inhibitory Concentration (MIC) was determined using a range of saturated and unsaturated fatty acids using the resazurin assay, followed by serial dilution and spot plating on BHI agar plates to establish the Minimum Bactericidal Concentration (MBC). Log reduction of bacteria was also evaluated for each fatty acid. The Minimum Biofilm Inhibition Concentration (MBIC) was determined using crystal violet assay in 96 well plates on forming and pre-formed S. mutans biofilms using BHI supplemented with 1% sucrose. Saturated medium-chain fatty acids Octanoic (C8.0), Decanoic (C10.0) and Undecanoic acid (C11.0) do not display strong antibiofilm properties; however, Lauric (C12.0) and Myristic (C14.0) display moderate antibiofilm properties with 97.83% and 97.5% biofilm inhibition with 1000 µM respectively. Monounsaturated, Oleic acid (C18.1) and polyunsaturated large chain fatty acids, Linoleic acid (C18.2) display potent antibiofilm properties with biofilm inhibition of 99.73% at 125 µM and 100% at 65.5 µM, respectively. Long-chain polyunsaturated Omega-3 fatty acids α-Linoleic (C18.3), Eicosapentaenoic Acid (EPA) (C20.5), Docosahexaenoic Acid (DHA) (C22.6) have displayed strong antibiofilm efficacy from concentrations ranging from 31.25-250µg/ml. DHA is the most promising antibiofilm agent with an MBIC of 99.73% with 15.625µg/ml. This may be due to the presence of six double bonds and the structural orientation of the fatty acid. To conclude, fatty acids displaying the most antimicrobial activity appear to be medium or long-chain unsaturated fatty acids containing one or more double bonds. Most promising agents include Omega-3-fatty acids Linoleic, α-Linoleic, EPA and DHA, as well as Omega-9 fatty acid Oleic acid. These results indicate that fatty acids have the potential to be used as antimicrobials and antibiofilm agents against S. mutans. Future work involves further screening of the most potent fatty acids against a range of bacteria, including Gram-positive and Gram-negative oral pathogens. Future work will involve incorporating the most effective fatty acids onto dental implant devices to prevent biofilm formation.

Keywords: antibiofilm, biofilm, fatty acids, S. mutans

Procedia PDF Downloads 159

Authors: Indu Gaur, Neha Bhadauria, Shilpi Shilpi, Susmita Goswami, Prem D. Sharma, Prabir K. Paul

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The concept of organic agriculture has been accepted as novelty in Indian society, but there is no data available on the human pathogens colonizing plant parts due to such practices. Also, the pattern and mechanism of their colonization need to be understood in order to devise possible strategies for their prevention. In the present study, human pathogenic bacteria were isolated from organically grown tomato plants and five of them were identified as Klebsiella pneumoniae, Enterobacter ludwigii, Serratia fonticola, Stenotrophomonas maltophilia and Chryseobacterium jejuense. Tomato plants were grown in controlled aseptic conditions with 25±1˚C, 70% humidity and 12 hour L/D photoperiod. Six weeks old plants were divided into 6 groups of 25 plants each and treated as follows: Group 1: K. pneumonia, Group 2: E. ludwigii, Group 3: S. fonticola, Group 4: S. maltophilia, Group 5: C. jejuense, Group 6: Sterile distilled water (control). The inoculums for all treatments were prepared by overnight growth with uniform concentration of 108 cells/ml. Leaf samples from above groups were collected at 0.5, 2, 4, 6 and 24 hours post inoculation for the colony forming unit counts (CFU/cm2 of leaf area) of individual pathogens using leaf impression method. These CFU counts were used for the in vivo colonization assay and adherence assay of individual pathogens. Also, resistance of these pathogens to at least 12 antibiotics was studied. Based on these findings S. fonticola was found to be most prominently colonizing the phylloplane of tomato and was further studied. Tomato plants grown in controlled aseptic conditions same as mentioned above were divided into 2 groups of 25 plants each and treated as follows: Group 1: S. fonticola, Group 2: Sterile distilled water (control). Leaf samples from above groups were collected at 0, 24, 48, 72 and 96 hours post inoculation and homogenized in suitable buffers for surface and cell wall protein isolation. Protein samples thus obtained were subjected to isocratic SDS-gel electrophoresis and analyzed. It was observed that presence of S. fonticola could induce the expression of at least 3 additional cell wall proteins at different time intervals. Surface proteins also showed variation in the expression pattern at different sampling intervals. Further identification of these proteins by MALDI-MS and bioinformatics tools revealed the gene(s) involved in the interaction of S. fonticola with tomato phylloplane.

Keywords: cell wall proteins, human pathogenic bacteria, phylloplane, solanum lycopersicum

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Authors: Reshmi Vijayan

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Alkaline protease is one of the most important enzymes in the biological world. Microbial production of alkaline protease is getting more attention from researchers due to its unique properties and substantial activity. Microorganisms are the most common sources of commercial enzymes due to their physiological and biochemical properties. The study was conducted on Ayiramthenghu mangrove sediments to isolate protease producing bacteria. All the isolates were screened for proteolytic activity on a skim milk agar plate at 37˚C for 48hrs. Protease activities were determined by the formation of a clear zone around the colonies on Skim milk agar medium. The activity of the enzyme was measured by the tyrosine standard curve, and it was found to be 0.186285 U/ml/min.

Keywords: protease, protease assay, skim milk agar medium, mangrove ecosystem

Procedia PDF Downloads 100

Authors: Seok Woo Chang, Kee Yeon Kum, Kwang Shik Bae, WooCheol Lee

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Aim: The objective of this study was to compare the biocompatibilities and mineralization potential of ProRoot MTA and newly developed calcium phosphate based cement, Capseal. Materials and Methods: The biocompatibilities and mineralization-related gene expressions (Bone sialoprotein (BSP) and osteocalcin (OCN)) of ProRoot MTA and Capseal were also compared by a methylthiazol tetrazolium (MTT) assay and reverse transcription-polymerization chain reaction (RT-PCR) analysis on 1, 3, and 7 days, respectively. Empty rings were used as control group. The results were statistically analyzed by Kruskal-Wallis test with a Bonferroni correction. P-value of < 0.05 was considered significant. Results: The biocompatibilities of ProRoot MTA and Capseal were equally favorable. ProRoot MTA and Capseal affected the messenger RNA expression of osteocalcin and osteonectin. Conclusions: Based on the results, both ProRoot MTA and Capseal could be a useful biomaterial in clinical endodontics.

Keywords: biocompatibility, calcium silicate cement, MTT, RT-PCR

Procedia PDF Downloads 391

Authors: Mashayekh Amir Shahriar, Akhlaghi Morteza, Rajaee Hajar, Khani Mohammad Reza, Shokri Babak

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A new and novel approach in medicine is the use of cold plasma for various applications such as sterilization blood coagulation and cancer cell treatment. In this paper a pin-to-hole plasma jet suitable for biological applications is investigated, characterized and the possibility and feasibility of cancer cell treatment is evaluated. The characterization includes power consumption via Lissajous method, thermal behavior of plasma using Infra-red camera as a novel method, Optical Emission Spectroscopy (OES) to determine the species that are generated. Treatment of leukemia cancer cells is also implemented and MTT assay is used to evaluate viability.

Keywords: Atmospheric Pressure Plasma Jet (APPJ), Plasma Medicine, Cancer cell treatment, leukemia, Optical Emission

Procedia PDF Downloads 659

Authors: Christi Jackson, Chuka Onaga

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Introduction: We report a case of delayed diagnosis of extra-pulmonary INH-mono-resistant Tuberculosis (TB) in a South African patient with drug-resistant HIV. Case Presentation: A 36-year old male was initiated on 1st line (NNRTI-based) anti-retroviral therapy (ART) in September 2009 and switched to 2nd line (PI-based) ART in 2011, according to local guidelines. He was following up at the outpatient wellness unit of a public hospital, where he was diagnosed with Protease Inhibitor resistant HIV in March 2016. He had an HIV viral load (HIVVL) of 737000 copies/mL, CD4-count of 10 cells/µL and presented with complaints of productive cough, weight loss, chronic diarrhoea and a septic buttock wound. Several investigations were done on sputum, stool and pus samples but all were negative for TB. The patient was treated with antibiotics and the cough and the buttock wound improved. He was subsequently started on a 3rd-line ART regimen of Darunavir, Ritonavir, Etravirine, Raltegravir, Tenofovir and Emtricitabine in May 2016. He continued losing weight, became too weak to stand unsupported and started complaining of abdominal pain. Further investigations were done in September 2016, including a urine specimen for Line Probe Assay (LPA), which showed M. tuberculosis sensitive to Rifampicin but resistant to INH. A lymph node biopsy also showed histological confirmation of TB. Management and outcome: He was started on Rifabutin, Pyrazinamide and Ethambutol in September 2016, and Etravirine was discontinued. After 6 months on ART and 2 months on TB treatment, his HIVVL had dropped to 286 copies/mL, CD4 improved to 179 cells/µL and he showed clinical improvement. Pharmacy supply of his individualised drugs was unreliable and presented some challenges to continuity of treatment. He successfully completed his treatment in June 2017 while still maintaining virological suppression. Discussion: Several laboratory-related factors delayed the diagnosis of TB, including the unavailability of urine-lipoarabinomannan (LAM) and urine-GeneXpert (GXP) tests at this facility. Once the diagnosis was made, it presented a treatment dilemma due to the expected drug-drug interactions between his 3rd-line ART regimen and his INH-resistant TB regimen, and specialist input was required. Conclusion: TB is more difficult to diagnose in patients with severe immunosuppression, therefore additional tests like urine-LAM and urine-GXP can be helpful in expediting the diagnosis in these cases. Patients with non-standard drug regimens should always be discussed with a specialist in order to avoid potentially harmful drug-drug interactions.

Keywords: drug-resistance, HIV, line probe assay, tuberculosis

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Authors: Praskovya Britskaya, Fatima Shaizadina, Alua Omarova, Nessipkul Alysheva

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Planned preventive vaccination, which is carried out in the Republic of Kazakhstan, promoted permanent decrease in the incidence of measles and viral hepatitis B. In the structure of VHB patients prevail people of young, working age. Monitoring of infectious incidence, monitoring of coverage of immunization of the population, random serological control over the immunity enable well-timed identification of distribution of the activator, effectiveness of the taken measures and forecasting. The serological blood analysis was conducted in indicator groups of the population of Central Kazakhstan for the purpose of identification of antibody titre for vaccine preventable infections (measles, viral hepatitis B). Measles antibodies were defined by method of enzyme-linked assay (ELA) with test-systems "VektoKor" – Ig G ('Vektor-Best' JSC). Antibodies for HBs-antigen of hepatitis B virus in blood serum was identified by method of enzyme-linked assay (ELA) with VektoHBsAg test systems – antibodies ('Vektor-Best' JSC). The result of the analysis is positive, the concentration of IgG to measles virus in the studied sample is equal to 0.18 IU/ml or more. Protective level of concentration of anti-HBsAg makes 10 mIU/ml. The results of the study of postvaccinal measles immunity showed that the share of seropositive people made 87.7% of total number of surveyed. The level of postvaccinal immunity to measles in age groups differs. So, among people older than 56 the percentage of seropositive made 95.2%. Among people aged 15-25 were registered 87.0% seropositive, at the age of 36-45 – 86.6%. In age groups of 25-35 and 36-45 the share of seropositive people was approximately at the same level – 88.5% and 88.8% respectively. The share of people seronegative to a measles virus made 12.3%. The biggest share of seronegative people was found among people aged 36-45 – 13.4% and 15-25 – 13.0%. The analysis of results of the examined people for the existence of postvaccinal immunity to viral hepatitis B showed that from all surveyed only 33.5% have the protective level of concentration of anti-HBsAg of 10 mIU/ml and more. The biggest share of people protected from VHB virus is observed in the age group of 36-45 and makes 60%. In the indicator group – above 56 – seropositive people made 4.8%. The high percentage of seronegative people has been observed in all studied age groups from 40.0% to 95.2%. The group of people which is least protected from getting VHB is people above 56 (95.2%). The probability to get VHB is also high among young people aged 25-35, the percentage of seronegative people made 80%. Thus, the results of the conducted research testify to the need for carrying out serological monitoring of postvaccinal immunity for the purpose of operational assessment of the epidemiological situation, early identification of its changes and prediction of the approaching danger.

Keywords: antibodies, blood serum, immunity, immunoglobulin

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Authors: Shashi Bala, Neha A. Chugh, Subhash C. Bansal, Mohal L. Garg, Ashwani Koul

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Purpose: The present study was designed to evaluate the radioprotective efficacy of Aloe vera from whole body X-ray exposure in rodents. Materials and Methods: For this purpose, after on week’s acclimatization, male balb/c mice procured from Central Animal House, Panjab University, Chandigarh (India), were divided into four groups: Group I mice served as control. Group II mice were orally administrated Aloe vera pulp extract (50 mg/ kg body weight) on alternate days for 30 days. Group III mice were subjected to whole body X-ray irradiation to cumulative dose of 2Gy (0.258Gy twice a day for four days in the last week). Group IV animals were pretreated with Aloe vera pulp extract on alternate days as in Group II and in the last week of the study, they were exposed to X-ray as in Group III. Results: Spleen of X-ray irradiated mice showed histopathological alterations accompanied with enhanced activity of lactate dehydrogenase (LDH) in serum. Elevated levels of reactive oxygen species (ROS), lipid peroxidation (LPO), enhanced activities in Glutathione based enzymes such as Glutathione peroxidase (GSH-Px), Glutathione reductase (GR), Catalase (CAT), Superoxide dismutase (SOD) associated with depletion in reduced Glutathione (GSH) concentration were observed after X-ray exposure in blood plasma and spleen.. Pro-inflammatory cytokines like tumor necrosis factors (TNF-α) and Inteleukin-6 (IL-6) levels were also found to be enhanced in serum of irradiated mice. Irradiation-induced significant elevation in Total leucocyte counts (TLC), neutrophil counts and decline in platelet counts, associated with unaltered levels of red blood cell counts (RBC’s) and haemoglobin (Hb) in various treatment groups. Clastogenic damage and apoptosis was also found to be increase in splenic tissue of X-ray exposed mice as assessed by micronucleus and TUNEL assay. However, X-ray irradiated animals administered with Aloe vera revealed significant improvement in levels of ROS/ LPO, LDH activity, and antioxidant mechanism. Aloe vera pretreated animals exhibited less severe damage, and early recovery in micronucleated cells, hematological parameters, apoptotic cells and inflammatory markers as compared to X-ray exposed mice. Conclusion: These results indicate that the radioprotective potential of Aloe vera against X-ray induced damage. This may be due to its free radical scavenging, antioxidant, anti-apoptotic and anti-inflammatory properties.

Keywords: aloe vera, antioxidant defense system, lactate dehydrogenase (LDH), micronucleus assay, x-ray

Procedia PDF Downloads 192

Authors: İsmail Çeli̇k, Gülgün Ayhan-Kılcıgi̇l, Arzu Onay-Beşi̇kçi̇

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In this study, some 2-(2-phenyl/substitutedphenyl)- lH-benzo[d]'imidazol-l-yl)-N'-(alkylthiosemicarbazide were designed and prepared. Firstly, 2-phenyl/ suhstitutedphenyl-lH-Benzo[d]imidazole was prepared via oxidative condensation of o-phenylenediamine, benzaldehyde and sodium metabisulfite. Treatment of the benzimidazole compound with ethyl chloroacetate in KOH/DMSO gave the ester compound ethyl 2-(2-substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetate. Hydrazine hydrate and the ester in ethanol were refluxed for 4 h to give 2-(2-phenyl/substitutedphenyl)-1H-benzo[d]imidazol-l-yl)acetohydrazide. Thiosemicarbazides were obtained by condensing acyl hydrazide with the alkylisothiocyanate in ethanol. Following the structure elucidation, benzimidazole compounds were tested for their EGFR kinase inhibitory activities by using ADP-GloTM Kinase Assay.

Keywords: benzimidazole, EGFR kinase inhibitor, synthesis, thiosemicarbazide

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Authors: Roihatul Mutiah, Sukardiman, Aty Widyawaruyanti

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Calotropis gigantiea (L.) R. Br (Apocynaceae) commonly called as “Biduri” or “giant milk weed” is a well-known weed to many cultures for treating various disorders. Several studies reported that C.gigantea roots has anticancer activity. The main aim of this research was to isolate and identify an active marker compound of C.gigantea roots for quality control purpose of its extract in the development as anticancer natural product. The isolation methods was bioactivity guided column chromatography, TLC, and HPLC. Evaluated anticancer activity of there substances using MTT assay methods. Identification structure active compound by UV, 1HNMR, 13CNMR, HMBC, HMQC spectral and other references. The result showed that the marker active compound was identical as Calotropin.

Keywords: calotropin, Calotropis gigantea, anticancer, marker active

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Authors: F. Bouchouka, S. Benamara

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The present work is an attempt to prepare a fresh cheese using lemon juice and lemon juice / orange juice mixture as acidifying / clotting agents. A reference cheese is obtained by acidification with commercial vinegar. The analysis performed on the final product (fat, cheese yield, sensory analysis, rheological and bacteriological properties) confirmed the technical feasibility of a natural cheese, using a lemon juice and / or lemon juice / orange juice mixture as acidifying / clotting agents. In addition, a general acceptance test allowed to select the cheese sample acidified with lemon juice as the best, compared to the two other samples (lemon juice/orange juice acidification and commercial vinegar acidification).

Keywords: clotting agent, fresh cheese, juice, lemon, orange

Procedia PDF Downloads 251

Authors: Sarwar Beg, Gajanand Sharma, O. P. Katare, Bhupinder Singh

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The current studies entail development of self-nano emulsifying drug delivery systems (SNEDDS) of rosuvastatin, employing rational QbD-based approach for enhancing its oral bioavailability. SNEDDS were prepared using the blend of lipidic and emulsifying excipients, i.e., Peceol, Tween 80, and Transcutol HP. The prepared formulations evaluated for in vitro drug release, ex vivo permeation, in situ perfusion studies and in vivo pharmacokinetic studies in rats, which demonstrated 3-4 fold improvement in biopharmaceutical performance of the developed formulations. Cytotoxicity studies using MTT assay and histopathological studies in intestinal cells revealed the lack of cytotoxicity and thereby safety and efficacy of the developed formulations.

Keywords: SNEDDS, bioavailability, solubility, Quality by Design (QbD)

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Authors: B. T. Naveen Kumar, Anuj Tyagi, Niraj Kumar Singh, Visanu Boonyawiwat, A. H. Shanthanagouda, Orawan Boodde, K. M. Shankar, Prakash Patil, Shubhkaramjeet Kaur

Abstract:

Early mortality syndrome (EMS)/Acute Hepatopancreatic Necrosis Disease (AHPND) has emerged as a major obstacle for the shrimp farming around the world. It is caused by a strain of Vibrio parahaemolyticus. The possible preventive and control measure is, early and rapid detection of the pathogen in the broodstock, post-larvae and monitoring the shrimp during the culture period. Polymerase chain reaction (PCR) based early detection methods are good, but they are costly, time taking and requires a sophisticated laboratory. The present study was conducted to develop a simple, sensitive and rapid diagnostic farmer level kit for the reliable detection of AHPND in shrimp. A panel of monoclonal antibodies (MAbs) were raised against the recombinant Pir B protein (rPirB). First, an immunodot was developed by using MAbs G3B8 and Mab G3H2 which showed specific reactivity to purified r-PirB protein with no cross-reactivity to other shrimp bacterial pathogens (AHPND free Vibrio parahaemolyticus (Indian strains), V. anguillarum, WSSV, Aeromonas hydrophila, and Aphanomyces invadans). Immunodot developed using Mab G3B8 is more sensitive than that with the Mab G3H2. However, immunodot takes almost 2.5 hours to complete with several hands-on steps. Therefore, the flow-through assay (FTA) was developed by using a plastic cassette containing the nitrocellulose membrane with absorbing pads below. The sample was dotted in the test zone on the nitrocellulose membrane followed by continuos addition of five solutions in the order of i) blocking buffer (BSA) ii) primary antibody (MAb) iii) washing Solution iv) secondary antibody and v) chromogen substrate (TMB) clear purple dots against a white background were considered as positive reactions. The FTA developed using MAbG3B8 is more sensitive than that with MAb G3H2. In FTA the two MAbs showed specific reactivity to purified r-PirB protein and not to other shrimp bacterial pathogens. The FTA is simple to farmer/field level, sensitive and rapid requiring only 8-10 min for completion. Tests can be developed to kits, which will be ideal for use in biosecurity, for the first line of screening (at the port or pond site) and during monitoring and surveillance programmes overall for the good management practices to reduce the risk of the disease.

Keywords: acute hepatopancreatic necrosis disease, AHPND, flow-through assay, FTA, farmer level, immunodot, pond site, shrimp

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Authors: Özlem Arpacı, Zeliha Soysal, Nuran Diril

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Benzoxazoles are heterocyclic compounds that have a fused benzene and an oxazole ring. These heterocyclic compounds are reported to have antibacterial, antitubercular, antifungal, antiviral, antioxidant and anticancer activities. In this study, some benzoxazole derivatives that have broad antimicrobial and potent antitumoral activities, are investigated their mutagenic activities with using the Ames Test. The Ames test was conducted using Salmonella typhimurium TA98, TA100 and TA102 tester strains in the standard plate incorporation assay in the absence of liver S9 fraction. The results are evaluated using SPSS and none of the benzoxazole derivatives showed mutagenic activity using the Ames test in the absence of S9 liver fraction.

Keywords: benzoxazoles, ames test, mutagenic activity, antimutagenic activity, antitumoral activity

Procedia PDF Downloads 345

Authors: Görkem Cemali̇, Avram Aruh, Gamze Torun Köse, Erde Can ŞAfak

Abstract:

In order to heal bone disorders, the conventional methods which involve the use of autologous and allogenous bone grafts or permanent implants have certain disadvantages such as limited supply, disease transmission, or adverse immune response. A biodegradable material that acts as structural support to the damaged bone area and serves as a scaffold that enhances bone regeneration and guides bone formation is one desirable solution. Poly(propylene fumarate) (PPF) which is an unsaturated polyester that can be copolymerized with appropriate vinyl monomers to give biodegradable network structures, is a promising candidate polymer to prepare bone tissue engineering scaffolds. In this study, hydroxyl-terminated PPF was synthesized and thermally cured with vinyl phosphonic acid (VPA) and diethyl vinyl phosphonate (VPES) in the presence of radical initiator benzoyl peroxide (BP), with changing co-monomer weight ratios (10-40wt%). In addition, the synthesized PPF was cured with VPES comonomer at body temperature (37oC) in the presence of BP initiator, N, N-Dimethyl para-toluidine catalyst and varying amounts of Beta-tricalcium phosphate (0-20 wt% ß-TCP) as filler via radical polymerization to prepare composite materials that can be used in injectable forms. Thermomechanical properties, compressive properties, hydrophilicity and biodegradability of the PPF/VPA and PPF/VPES copolymers were determined and analyzed with respect to the copolymer composition. Biocompatibility of the resulting polymers and their composites was determined by the MTS assay and osteoblast activity was explored with von kossa, alkaline phosphatase and osteocalcin activity analysis and the effects of VPA and VPES comonomer composition on these properties were investigated. Thermally cured PPF/VPA and PPF/VPES copolymers with different compositions exhibited compressive modulus and strength values in the wide range of 10–836 MPa and 14–119 MPa, respectively. MTS assay studies showed that the majority of the tested compositions were biocompatible and the overall results indicated that PPF/VPA and PPF/VPES network polymers show significant potential for applications as bone tissue engineering scaffolds where varying PPF and co-monomer ratio provides adjustable and controllable properties of the end product. The body temperature cured PPF/VPES/ß-TCP composites exhibited significantly lower compressive modulus and strength values than the thermal cured PPF/VPES copolymers and were therefore found to be useful as scaffolds for cartilage tissue engineering applications.

Keywords: biodegradable, bone tissue, copolymer, poly(propylene fumarate), scaffold

Procedia PDF Downloads 166

Authors: Rachid Kacem, Sara Talbi, Yasmina Hemissi, Sofia Bouguattoucha

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The aim of the present work is the evaluation of the antioxidant activity of the Peganum harmala (P. harmala) seeds extracts. The antioxidant activity was evaluated by applying two methods, the method of ß-carotene bleaching and DPPH (2, 2-Diphenyl-1-Picryl-Hydrazyl). Using Folin-Ciocalteu assay, these results revealed that the concentration of polyphenols in EthOH E. (122.28 ± 2.24 µg GAE/mg extract) is the highest. The antiradical activity of the P. harmala seeds extracts on DPPH was found to be dose dependent with polyphenols concentration. The E. EthOH extract showed the highest antioxidant activity (IC = 252.10 ± 11.18 μg /ml). The test of β-carotene bleaching indicates that the E. EthOH of P. harmala showed the highest percentage of the antioxidant activity (49.88 %).

Keywords: antioxidant activity, Peganum harmala, polyphenols, flavonoids

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Authors: Hairin Taha, P. Narrima, M. A. Hapipah, A. M. Mustafa

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The chemical constituents and antioxidant activity of the leaf essential oils of Pseuduvaria monticola and Pseuduvaria macrophylla from the Annonaceae family were investigated. GC-TOFMS analyses identified 46 compounds from Pseuduvaria monticola and 11 compounds from Pseuduvaria macrophylla. The major constituents in the leaf essential oil of Pseuduvaria monticola were a-cadinol (13.0%), calamenene-cis (6.9%), alfa copaene (4%), and epizonarene (3.8%), while in the leaf essential oil of Pseuduvaria macrophylla were caryophyllene oxide (29.7%) and elimicin (28%). The antioxidant activity of both the essential oils were determined using the 2,2'-diphenyl-1-picrylhydrazyl assay (DPPH). The present study suggests that both essential oils demonstrated good antioxidant activity.

Keywords: Pseuduvaria monticola, Pseuduvaria macrophylla, leaf essential oils, GC-MSTOF, antioxidant activity

Procedia PDF Downloads 381

Authors: Patrícia Severino, Luciana Nalone, Daniele Martins, Marco Chaud, Classius Ferreira, Cristiane Bani, Ricardo Albuquerque

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Hydrogels produced with polymers have been used in the development of dressings for wound treatment and tissue revitalization. Our study on polymer nanocomposites containing silver nanoparticles shows antimicrobial activity and applications in wound healing. The effects are linked with the slow oxidation and Ag⁺ liberation to the biological environment. Furthermore, bacterial cell membrane penetration and metabolic disruption through cell cycle disarrangement also contribute to microbial cell death. The silver antimicrobial activity has been known for many years, and previous reports show that low silver concentrations are safe for human use. This work aims to develop a hydrogel using natural polymers (sodium alginate and gelatin) combined with silver nanoparticles for wound healing and with antimicrobial properties in cutaneous lesions. The hydrogel development utilized different sodium alginate and gelatin proportions (20:80, 50:50 and 80:20). The silver nanoparticles incorporation was evaluated at the concentrations of 1.0, 2.0 and 4.0 mM. The physico-chemical properties of the formulation were evaluated using ultraviolet-visible (UV-Vis) absorption spectroscopy, Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), and thermogravimetric (TG) analysis. The morphological characterization was made using transmission electron microscopy (TEM). Human fibroblast (L2929) viability assay was performed with a minimum inhibitory concentration (MIC) assessment as well as an in vivo cicatrizant test. The results suggested that sodium alginate and gelatin in the (80:20) proportion with 4 mM of AgNO₃ in the (UV-Vis) exhibited a better hydrogel formulation. The nanoparticle absorption spectra of this analysis showed a maximum band around 430 - 450 nm, which suggests a spheroidal form. The TG curve exhibited two weight loss events. DSC indicated one endothermic peak at 230-250 °C, due to sample fusion. The polymers acted as stabilizers of a nanoparticle, defining their size and shape. Human fibroblast viability assay L929 gave 105 % cell viability with a negative control, while gelatin presented 96% viability, alginate: gelatin (80:20) 96.66 %, and alginate 100.33 % viability. The sodium alginate:gelatin (80:20) exhibited significant antimicrobial activity, with minimal bacterial growth at a ratio of 1.06 mg.mL⁻¹ in Pseudomonas aeruginosa and 0.53 mg.mL⁻¹ in Staphylococcus aureus. The in vivo results showed a significant reduction in wound surface area. On the seventh day, the hydrogel-nanoparticle formulation reduced the total area of injury by 81.14 %, while control reached a 45.66 % reduction. The results suggest that silver-hydrogel nanoformulation exhibits potential for wound dressing therapeutics.

Keywords: nanocomposite, wound healing, hydrogel, silver nanoparticle

Procedia PDF Downloads 101