IRSPAS 2016
Permanent URI for this collectionhttp://repository.kln.ac.lk/handle/123456789/15651
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Item A preliminary study of lip moisturizer rich in antioxidants produced using coffee leaf extract(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Gunasinghe, K.G.; Nadeeshani, R.; Jayathilaka, N.As the body’s first environmental defense, the skin is exposed to various sources of free radical damage including the sun. In addition, to maintain healthy skin, it is important to maintain the moisture content not only in the deeper dermal and epidermal layers but also at the surface. As such, there are numerous skin moisturizers commercially available specially formulated to not only moisturize the body, face and the lips but also block the harmful rays from the sun to protect the skin surface. The composition of the lip moisturizers available in the market varies from brand to brand. These products often contain castor oil, carnauba wax and chemicals/ preservatives such as propyl paraben, methyl paraben, retinyl palmitate, tocopheryl acetate etc. as well as different agents to block the harmful rays from the sun. Antioxidants can be added to these products to neutralize the free radicals that can cause damage to the skin. Plant polyphenols are known to have high antioxidant activity. In this study, we have formulated a lip moisturizer with aqueous extracts from coffee leaves rich with polyphenols in an effort to develop a product that can neutralize free radical damage on the surface skin. The product was developed using bees wax, vaseline, coffee leaves and water (1: 2: 1: 11.5) with no other additional chemicals to formulate a natural healthy cosmetic. Polyphenols in the water extract was extracted in to methanol by removing the proteins using chloroform. The polyphenol content in the aqueous extract (0.18 ± 0.01 mg/ml) was measured by Folin-Denis assay as Gallic acid equivalent, using water as the control. The antioxidant activity of the extract was measured by DPPH radical scavenging assay. The percentage inhibition of DPPH radical scavenging activity of the aqueous extract of the coffee leaves measured using water as the blank gave 83.46 ± 0.11% of inhibition. Each sample was assayed three times for three biological replicates. The polyphenol content and the percentage inhibition of DPPH radical scavenging activity of the aqueous extracts, extracted from the formulated lip moisturizer were 0.14 ± 0.01 mg/ml and 83.44 ± 0.43% respectively. There is no statistically significant difference in the polyphenol content and the antioxidant activity between the aqueous extracts (p< 0.01). Lip moisturizer produced without the additon of coffee leaf extract was used as the control. According to the DPPH assay 99.97± 0.27% of percentage inhibition of DPPH radical scavenging activity was retained. Therefore, the lip moisturizer formulated with the coffee leaf extract retained the antioxidant properties.Item Design and construction of low cost petri dish incubator(Faculty of Science, University of Kelaniya, Sri Lanka, 2016) Wanigasekara, G.; Perera, N.W.; Abeysinghe, D.; Geegamage, S.S.; Wijekoon, D.; Jayathilaka, N.Petri dish incubators are used in laboratories to keep petri dish samples at a stable and optimal temperature of 37 °C. Incubators are one of the frequently needed equipment. These incubators are expensive due to the use of complex systems. Many of the local universities do not have the necessary financial resources to purchase this equipment. Therefore, undergraduate students usually do not have access to incubators for academic learning. In order to surmount this challenge, it is necessary to look at a low cost, simple design for petri dish incubators. Hence, we have designed an incubator utilizing low cost microcontroller boards and sensors. Both microcontrollers and sensors were selected to provide adequate accuracy for the incubation at 37 °C. The incubator is constructed of three major components; sensors, controller and temperature regulation system. The incubator uses three LM35 temperature sensors to monitor the temperature with 0.5 °C accuracy and the system is controlled by Arduino Uno board with 16 MHz ATmega328P microcontroller. The microcontroller regulates the temperature of the incubation chamber utilizing 200W Nichrome heating element and two exhaust fans. Three temperature sensor readings were taken to acquire chamber temperature by averaging three values. Microcontroller uses these data to control the heating element, the fan for heating and the fan for cooling. The controller uses a PID (Proportional–Integral–Derivative) algorithm to stabilize the temperature. The sensor input wiring is highly shielded to avoid interference from the main powerline magnetic noise. The incubator body is shielded with porcelain to avoid fire hazards. The average temperature recorded by the incubator sensor and the chamber temperature as recorded with a thermometer was monitored at 2 hr intervals over a 16 hr period at 37.6 ± 0.5 °C and 37.6 ± 0.5 °C respectively indicating the accuracy of temperature regulation in the petri dish incubator over an extended period of incubation.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 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.