IPRC - 2017
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Item Nutritional Effect of Consumption of Domestic and Commercially Available Coconut Milk Preparations in Wistar Rats.(In: Proceedings of the International Postgraduate Research Conference 2017 (IPRC – 2017), Faculty of Graduate Studies, University of Kelaniya, Sri Lanka., 2017) Senanayake, C.M.; Seneviratne, K.N.; Jayathilaka, N.; Ekanayaka, S.The use of both domestic coconut milk (CM) preparations and commerciallyavailable CM preparations in cooking has become popular. The present study involves evaluating In vivo effect of domestic CM prepared by blending (BCM), commercially available powdered CM (PCM) and liquid CM (LCM) on serum lipid profiles and serum antioxidant capacity using Wistar rats. Seven weeks old male Wistar rats were randomly assigned into treatment groups. Control group was fed with a semisynthetic diet recommended by WHO. Second, third and fourth groups were fed with semi synthetic diet which contains 12 mL BCM, PCM or LCM per kg of feed respectively. Blood was drawn on day before feeding experimental diets (Day 0), 30 days, 90 days, 120 days and 150 days after feeding experimental diets. Serum total cholesterol (TC), high density lipoprotein (HDL) and triglycerides (TG) were analyzed using a test kit. Low density lipoprotein (LDL) was determined using Friedewald equation. Antioxidant activity of serum was determined by ABTS assay and DPPH radical scavenging assay. TC levels of all groups were significantly (p<0.05) increased after 150 days of feeding compared to their day 0 levels. TC levels (mg/dL) of rats fed with BCM (80±4), PCM (80±5) and LCM (81±3) were similar to control group (77±7). However, rats fed with LCM showed a statistically significant (p<0.05) increase in TC compared to control group. Although, TG levels of CM diet groups indicated significant (p<0.05) increase on day 150 compared to their day 0 levels, these levels were similar to that of control group. Both HDL and LDL levels of CM diet groups remained same compared to control group at day 150. All CM diet groups showed a significantly (p<0.05) increased activity on day 150 compared to their day 0 levels.CM fat contains nearly 90 % saturated fat. However, majority of the saturated fat in CM fatis composed of short and medium chain fatty acids, which are beneficial to health. As such, adding CM to diet did not affect average levels of serum TC, LDL and TG of Wistar rats suggesting that none of the CM preparations under investigation contribute to detrimental changes in lipid profiles. All CM preparations, on the other hand, appear to increase serum antioxidant activity which may contribute in retarding oxidative damage to biomolecules. Financial assistance from National Research Council (12-012) and University Research Grant (RP/03/02/06/05/2015) areacknowledged.Item Comparison of Methods for miRNA Extraction from Plasma and Peripheral Blood Mononuclear Cells.(In: Proceedings of the International Postgraduate Research Conference 2017 (IPRC – 2017), Faculty of Graduate Studies, University of Kelaniya, Sri Lanka., 2017) Hapugaswatta, H. P. H.; Seneviratne, K.N.; Jayathilaka, N.miRNAs are small non-coding RNA that are known to regulate gene expression at transcription level. Altered expression levels of miRNAs due to the infections can serve as clinically relevant biomarkers. Reproducible and efficient recovery of miRNA from biological samples is important for their reliable quantification. Therefore, we compared the recovery of miRNAs from plasma and PBMC using several commercially available RNA isolation kits in the presence and absence of carrier molecules to enhance the yield, by quantification of hsa-mir-103-5p, hsa-let-7e and hsa-mir-30b-5p with RT-qPCR. Organic extraction and precipitation of total RNA with or without the addition of tRNA from brewer’s yeast or glycogen as carrier molecules, mirVana microRNA isolation kit (Applied Biosciences), and miRNeasy Serum/Plasma Kit (Qiagen) with or without tRNA were evaluated for RNA recovery from plasma. mirVana kit and miRNeasy kit were also evaluated for RNA recovery from PBMC. RNA isolations were performed from either plasma or PBMC isolated from whole blood collected from healthy volunteers with informed consent. Total RNA was used for subsequent 3’polyadenylation of the miRNA followed by cDNA synthesis. Presence of target miRNAs in plasma and PBMC were confirmed by RT-qPCR using target specific primers. Primer specificity was confirmed using NCBI blastn suite. All three miRNA targets were detectable in PBMC using the two commercial kits, without the addition of a carrier molecule. PBMC samples processed with miRNeasy extraction kits showed earlier target amplification due to concentration of total RNA in smaller elution volumes compared to the mirVana extraction method. Addition of low amount of carrier RNA (1 μg/mL) yielded more RNA. Adding high amount of carrier RNA (10 μg/mL) during RNA extraction with mirVana kit and organic extraction showed selective effect on RNA recovery. Using glycogen as the carrier for organic extraction also yielded higher amount of miRNA from plasma. Therefore, addition of limited amount of carrier molecules can enhance the miRNA recovery.Item Protective Effect of Coconut Cake Phenolic Antioxidants on Oxidative Stress Induced Macromolecular Damage in HEp-2 Cells.(In: Proceedings of the International Postgraduate Research Conference 2017 (IPRC – 2017), Faculty of Graduate Studies, University of Kelaniya, Sri Lanka., 2017) Karunasiri, M. G. A. N.; Seneviratne, K.N.; Jayathilaka, N.Coconut cake, a by-product of the coconut oil manufacturing is a rich source of phenolic antioxidants. The majority of research dealing with phenolic antioxidants is primarily focused on the extraction of phenolic substances from plant materials and assessment of antioxidant properties in chemical systems. However, such assays in chemical systems do not guarantee the antioxidant properties of phenolic substances in biological systems. In this study, inhibition of H2O2 induced oxidative damage on lipids and proteins by coconut cake phenolic antioxidants (CCPA) was studied in HEp-2 cells as the biological system. CCPA were extracted with 70 % ethanol and the total polyphenol content was measured by Folin Ciocalteu method. The CCPA content, calculated as gallic acid equivalents was 182.81 ± 28.73 mg/kg. The o-diphenols content, calculated as caffeic acid equivalent using a method reported by Gutfinger was 66.83 ± 16.50 mg/kg. Oxidative damage in HEp-2 cells was induced by adding H2O2in PBS for 1 hr. The maximum concentration of H2O2 that does not affect the cell viability (>99 %) was determined as 100 µM using Cell-Titer Glo Luminescent Cell Viability Assay. Formationof thiobarbituric acid reactive species (TBARS) due to lipid peroxidationin HEp-2 cells (0.010±0.000 µM/mL) compared to the control (0.007±0.000 µM/mL) without H2O2was inhibited with 0.5mg/mLCCPA (0.007±0.000µM/mL). Protein oxidation (3.05±0.06nmol/mL) compared to the control (2.14±0.06nmol/mL) without H2O2 as assessed by protein carbonyl formation assay with 2, 4-dinitophenylhydrazine was alsoinhibited by treating the HEp-2 cells with 0.5mg/mL CCPA (2.41±0.06 nmol/mL). Thus, CCPA caninhibit oxidative stress-induced macromolecular damage of lipids and proteins in biological systems.