IRSPAS 2016
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/15651
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Item Effect of different storage conditions on the nitrite levels of human saliva(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Sewwandi, A.L.S.; Jayathilaka, N.; Seneviratne, K.N.Nitric oxide (NO) is an intracellular messenger molecule that plays an important role in biological systems as a physiological and pathophysiological mediator. Therefore, levels of NO in biological fluids reflect physiological aspects of diagnostic and therapeutic significance. Relatively stable end products of NO, nitrite and nitrate, is commonly measured to evaluate the production of NO in biological fluids such as serum, saliva and urine. Salivary nitrite and nitrate levels have been reported to reflect a spectrum of the health and disease states serving as non-invasive, clinically informative and effective source for prognosis, laboratory or clinical diagnosis in humans. However, detection of salivary nitrite levels in resource limited settings present several challenges such as availability of analytical equipment and stability of nitrite levels during sample storage and transportation. Hence, the aim of this study was to detect the effect of different storage conditions on the salivary nitrite levels to evaluate the stability of salivary nitrite during storage. Saliva samples were collected from six healthy females between the age of 20-30 using the spit method. Salivary nitrite levels were between 8 - 46 μM (23.3 ± 10.4 μM) for samples that were analyzed directly after sample collection by Griess colorimetric reaction following stabilization with NaOH and deproteination with ZnSO4. Samples from each individual was sampled twice. Similarly, the nitrite levels of the saliva samples were measured following storage for one hour at room temperature (RT) and at 4OC, and after storage overnight at RT and at -80 OC. Sample storage for one hr at RT (21.5 ± 5.1 μM) and at 4OC (18.1 ± 4.6 μM) and overnight at -80 OC (22.0 ± 5.2 μM), prior to sample analysis did not show statistically significant difference in salivary nitrite levels from the direct sample analysis. Storage of samples overnight, at RT (3.6 ± 0.7 μM) prior to sample analysis, on the other hand, showed statistically significant difference (P <0.005) in salivary nitrite levels compared to the nitrite levels detected during direct sample analysis based on student’s t-test. The study reveals that the levels of nitrite changes during prolonged storage at room temperature while storage at ultralow temperatures is suitable for prolonged sample storage for subsequent analysis for salivary nitrites.Item Use of miR-33a in human plasma as a potential biomarker for severe Dengue infection(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Hapugaswatta, H.; Seneviratne, K.N.; Jayathilaka, N.MicroRNAs (miRNAs) are small noncoding RNAs about 22 nucleotides long that can regulate the gene expression at mRNA level by RNA silencing. As such, differential expression of miRNA leading to regulation of gene expression during various infections has been investigated as potential biomarkers for many diseases. Perturbations in lipid homeostasis and cellular imbalance of cholesterol and fatty acid metabolism has been implicated in Dengue infections. Intronic microRNA, miR33a, located within the sterol regulatory element-binding protein-2 and -1 genes, has been shown to regulate cholesterol homeostasis in concert with their host genes. In fact, miR33a has shown differential expression in cultured cells infected with Dengue virus. Therefore, we evaluated the expression level of miR33a in human plasma to evaluate its potential as a biomarker for severe Dengue infection. Total RNA was purified from plasma from six EDTA blood samples collected with consent from healthy people using mirVana microRNA isolation kit (Applied Biosciences). Plasma was separated within one hour of sample collection and was stored at -80 °C. Purified RNA was used for subsequent 3’polyadenylation of the mature microRNA and cDNA synthesis with oligo-dT primers with a universal tag sequence using miScript II RT Kit (Qiagen). Similarly, negative control experiments for genomic DNA were carried out with same amount of RNA without any reverse transcriptase in the cDNA synthesis reaction (-RT). Presence of miR33a in plasma was confirmed by quantitative real-time PCR using the mature miRNA33a nucleotide sequence with thiamidine in place of uracils as the forward primer and a universal primer with universal tag sequence in the oligo-dT primer as the reverse primer. Target specificity of the miR33a specific primer was confirmed by NCBI BLASTN suite. Quantitative real-time PCR using miScript SYBR Green PCR Kit (Qiagen) was performed using diluted cDNA at an annealing temperature of 58 °C for 40 cycles followed by melting curve analysis. Negative control reactions were carried out in parallel with same volume of water and -RT as template. miR33a in human plasma was detected above threshold 1.0 at cycle number (Ct) 35. The Ct number is higher due to the low abundance of RNA in plasma. miR33a was consistently detected above threshold at the given Ct values while no amplification was detected in the negative control experiments containing water or -RT as template, indicating the absence of primer dimer formation and genomic DNA. Presence of a single peak in the melting curve confirmed single product amplification. Therefore, plasma is a good source for detection of miR33a to evaluate differential expression in severe Dengue.Item Antioxidant activities in extracts of five plant sources on stabilization of stripped sunflower oil and egg yolk homogenate(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Senanayake, C.M.; Seneviratne, K.N.out to evaluate the antioxidant potential of five natural sources namely, coconut cake (A), Psidium guajava L. leaf (Guava) (B), Psidium guineense Sw. leaf (Ambul guava) (C), rice bran (D) and sesame cake (E) in both chemical and food model systems (stripped sunflower oil and egg yolk homogenate). Phenolic substances from the test plant materials were extracted using ethanol:water (70:30) solvent system. Total phenolic content (TPC) was determined using Folin-Ciocalteu method and expressed as gallic acid equivalents (GAE) per kilogram of sample. Antioxidant activities of extracts and butylated hydroxytoluene (BHT) were evaluated using deoxyribose degradation assay after adjusting the concentration to 30 μg mL-1. Antioxidant activities of phenolic extracts on stripped (antioxidant free) sunflower oil were determined by comparing the induction time (IT) using the Rancimat Apparatus at 100 0C. Effect of phenolic antioxidants on the inhibition of thiobarbituric acid reactive substances (TBARS) formation was evaluated using egg yolk homogenate as the food model system. Results of TPC as GAE vary in the order, C (195.25±9.56 g/kg) > B (68.83±3.74 g/kg) > D (4.14±0.46 g/kg) > E (2.11±0.29 g/kg) > A (0.77±0.03 g/kg). Phenolic extract of C showed a significantly (p<0.05) higher percentage inhibition of deoxyribose degradation (76.5±1.5 %) than other phenolic extracts and BHT. Inhibition percentages obtained for A, B, D, E and BHT were 39.5±1.4 %, 71.0±2.7 %, 46.1±3.1 %, 42.1±2.5 % and 32.6±2.1 % respectively. Results of IT of stripped sunflower oil and inhibition % of TBARS formation were stated in Table 1.Item Determination of polyphenol content in coconut milk by modified Folin-Denis assay(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Nadeeshani, R.; Seneviratne, K.N.; Jayathilaka, N.Polyphenols are micronutrients which has nutritional value owing to their antioxidant activity. Polyphenol content is usually determined by the standard Folin-Denis Assay. Water soluble compounds commonly present in biological samples, such as proteins, ascorbic acid, DNA and RNA may interfere with this assay. Therefore, it is difficult to determine whether the antioxidant capacity of such samples are owing to these interfering compounds or other polyphenols present in the aqueous extracts (AE) like coconut milk (CM). In order to overcome these drawbacks, a modified extraction method was employed to remove proteins from the AE of CM to determine the polyphenol content in first (FE) and second (SE) extracts of both domestic and commercial preparations of CM using Folin-Denis assay. The results were reported as Gallic acid equivalents (mg/mL). Proteins/peptides present in the AE of CM (1.00 mL) was removed by organic extraction with chloroform (1.00 mL), distilled water (4.00 mL) and methanol (4.00 mL). Samples were mixed at 30 Hz for 01 min followed by centrifugation at 6000 rpm for 05 min. The methanolic layer was used for the Folin-Denis assay. The methanolic extracts (ME) were confirmed free of proteins by Bradford assay. Results showed significantly low polyphenol content in the ME compared to the AE indicating interference in the assay from proteins/peptides present in the AE of CM. Corresponding antioxidant activity of the ME of both FE and SE of domestic CM preparations were significantly higher compared to the commercial counterparts regardless of the presence of high polyphenol content in the AE. Therefore, the modified Folin-Denis assay reported here determine the polyphenol content in AE of food preparations that may contribute to their antioxidant potential.