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Item In Silico and In Vitro Analysis of Inhibition of Rice Bran Lipase to Extend the Shelf Life(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Weerakoon, W.M.T.D.N.; Munaweera, R.; Chandula, S.; Dodampe, N.; Seneviratne, K.N.; Jayathilaka, N.Rice bran is a byproduct of the rice (Oryza sativa) milling process. The hard, outer layer removed from the starchy endosperm of the rice grains is known as rice bran. Rice bran is a rich source of nutrients. Rice bran is used to produce rice bran oil and as animal feed. Oxidation of fatty acids in lipids is one of the main causes for the spoilage of rice bran during storage. Lipase enzymes catalyze the hydrolysis of ester bond in triglycerides (lipids) releasing free fatty acids, which are more prone to oxidation than fatty acids in triglycerides. Therefore, inhibition of lipases can be a possible solution to restrict the lipid oxidation. The active site of lipases contains a characteristic GXSXG pentapeptide sequence (where X = any amino acid) which plays a major role in the lipase enzymatic activity. Orlistat is a lactone known to act as a potent inhibitor of human lipase. Therefore, in the present study, we evaluated the lipase inhibitory potential of lactones that are present in Psidium guineense. Homology-modelling of rice bran lipase and molecular docking studies (SWISSDOCK) were carried out to identify compounds with high affinity at the binding site. The top 20 docking poses with the lowest estimated Gibbs free energy values (ΔG) were considered from the molecular docking study. Low ΔG values of lactones show preferable binding at the binding site of Oryza sativa lipase. Close proximity of electrophilic carbon of lactones to the nucleophilic oxygen of the serine residue indicates the possibility of a nucleophilic attack by the oxygen of the serine residue to the electrophilic carbon of lactones leading to a covalent bond formation inhibiting the lipase enzyme. This suggests that lactones present in guava may be capable of inhibiting the lipases present in Oryza sativa. The in silico data were validated using lipase purified from rice bran. Rice bran lipase was purified by ion exchange chromatography followed by size exclusion chromatography. Inhibition of lipase activity was assessed using phenyl acetate assay. Percentage inhibition of lipase activity by guava leaf extract and Orlistat were 74.1% and 58.8% respectively. This indicates that guava extract contains compounds with inhibitory action towards lipase enzyme and they are more effective in inhibiting lipase than Orlistat. Even though the rice bran is one of the richest and cheapest sources of antioxidants, easy oxidative spoilage makes the shelf life of rice bran short. However, when the antioxidants stripped from rice bran re mixed with rice bran, a concentration dependent inhibition of the formation of oxidation products in rice bran was observed suggesting that bioavailability of the antioxidants present in the rice bran is low. While the docking studies provide evidence of inhibition of rice bran lipase activity, empirical evidence require analysis using purified lactones from guava on the inhibition of rice bran lipase. Our findings suggest antioxidants and lactones inhibit rancidity of rice bran during storage by inhibiting oxidation of lipids and inhibiting the lipase activity.Item Protective effect of coconut oil meal phenolic antioxidants on mitochondrial DNA damage in HEp-2 human epithelial cells(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Nisansala, A.; Hapugaswatte, H.; Seneviratne, K.N.; Jayathilaka, N.Coconut oil meal is a rich source of phenolic antioxidants. In most of the reported research, antioxidant activities of phenolic extracts have been tested using chemical systems. However, similar studies conducted in biological systems is relatively less common. In this study, glutathione oxidation and mitochondrial DNA (mt-DNA) damage in HEp-2 cells were used as biological reactions to assess protective effect of coconut oil meal phenolic antioxidants (CMPA). CMPA were extracted with ethanol water solvent system (70% v/v) and the total phenolic content was measured by Folin Ciocalteu method. HEp-2 cells at 80% confluence were treated with 0.5 mg/ml CMPA overnight. Oxidative stress in HEp-2 cells was induced by exposing cells to H2O2 (10, 50, 100, 250 and 500 μM) for 1hr. The maximum concentration of H2O2 that does not affect the cell viability (>99%) was 100 μM as measured by Cell-Titer Glo Luminescent Cell viability assay (Promega). Glutathione (GSH) to oxidized glutathione (GSSG) ratio (GSH/GSSG) was measured by GSH/GSSG Glo assay (Promega). To evaluate the mt-DNA damage, total DNA of high purity (A260nm/ A280nm >1.8) was extracted from stressed and unstressed cells pretreated with CMPA using HiPurATM Blood Genomic DNA Miniprep Purification Kit. Two primer sets that amplify a short and long fragment of mt-DNA in the D-loop region were selected to assess damage in the D-loop region that is known to be more prone to DNA damage. Quantitative Real time PCR was carried using the QuantiTect SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions and the amplification was monitored with StepOne quantitative thermal cycler (Applied Bio). The ratio of intact DNA was calculated according to the following equation; Lesion rate [Lesions per 10kb DNA] = (1-2-( Δ long- Δ short)) x (10000 bp/Size of long fragment bp). Total CMPA was 1892 ± 51 mg/kg as gallic acid equivalents . Two sample t-test was carried out for the determination of significant differences (p ≤ 0.05) between the mean values .The GSH/GSSG ratio of the samples pretreated with CMPA, subjected to H2O2 oxidative stress (100 μM) (5.01±0.08 (was significantly higher )P<0.05 (compared to that of samples subjected to H2O2 oxidative stress without pretreatment )2.19±0.04) while GSH/GSSG ratio of control cells without any treatment was 5.19±0.20. In mt-DNA damage assay, the samples pretreated with CMPA, subjected to H2O2 oxidative stress (100 μM) showed significantly less (P<0.05) lesions (7.65±0.06 lesions/10 kb DNA) compared to samples subjected to H2O2 oxidative stress without pretreatment (9.38±0.60 lesions/10 kb DNA). Thus, CMPA can inhibit glutathione oxidation and mt-DNA damage in HEp-2 cells suggesting that CMPA may help reduce health conditions associated with oxidative stressItem Expression Changes in Putative Target Genes of Differentially Expressed miRNA as Early Biomarkers for Severe Dengue(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Hapugaswatta, H.; Seneviratne, K.N.; Perera, H.S.S.; Premaratna, R.; Jayathilaka, N.Dengue fever is caused by a flavivirus transmitted by mosquitoes. Primary infection of dengue mostly causes mild dengue fever (DF) characterized by headache, retro orbital pain, body pain, nausea, vomiting, joint pains and weakness. Severe manifestations of dengue, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) also shows similar symptoms during the early stages of infection. After 3-5 days from fever onset, DHF patients manifest plasma leakage, elevated hematocrit and pleural effusions. Lack of proper medication or vaccines for dengue fever and inability to distinguish severe dengue from DF during the early stages of infection renders this disease life threatening. Early diagnosis and disease management can alleviate DHF related complications. Therefore, biomarkers that distinguish DHF during the acute phase of infection can help reduce mortality. In our previous studies, we evaluated the differential expression of five miRNAs during the acute phase of infection including hsa-miR-150, which showed significant (p<0.05) expression changes with the disease severity. Since the main function of miRNA is to regulate target gene expression at post-transcriptional level, we evaluated the expression levels of four target genes of those miRNA in peripheral blood cells (PBC) collected from 20 DF (male-70% and female-30%) and 20 DHF (male-85% and female-15%) patients (based on evidence of plasma leakage by ultrasonography) who tested positive for NS1 antigen within four days of fever onset (acute phase) by qRT-PCR. Relative expression of EZh2, ABCA1, DNMT3a and RIP140 were evaluated against GAPDH as the reference gene. EZh2 showed over 2-fold downregulation (P<0.05) in DHF patients compared to DF patients. Based on logistic regression analysis of ΔCq values, EZh2 expression within 4 days from fever onset may be useful as a biomarker for progression from DF to DHF with an area under the receiver operating characteristic curve (AUC) of 0.76, sensitivity of 0.80 and specificity of 0.65 at 2.69 (P<0.05). DNMT3a, RIP140 and ABCA1 did not show significant differential expression during the acute phase of infection between DF and DHF patient samples. EZh2 also showed significant (P<0.05) downregulation within 4 days from fever onset in patients with platelet count <100,000 cells/mm3 (n=31) compared to those with platelet count >100,000 cells/mm3 (n=9) during the course of infection. Therefore, EZh2 expression may also serve as a biomarker for disease severity marked by low platelet count. This analysis is limited by relatively small sample size and a disproportionate number of male subjects. However, the calculated sample size with 95% CI at 80% power for EZh2 expression as a marker to predict disease outcome is 34 (17 each). The data was confirmed normally distributed based on q-q plot and Shapiro-Wilk test (P>0.05).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.Item Preliminary Studies on the Acidity of Coconut Oil(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2002) Dissanayake, D.M.S.; Dhannaraja, K.D.L.S.; Seneviratne, K.N.Item Effect of Repeated Heating on The Oxidative Degradation of White Coconut Oil and Soy Bean Oil(Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2016) Senanayake, C.M.; Jayathilaka, N.; Seneviratne, K.N.Repeated heating of cooking oils is a common practice used mainly to save the cost in food preparations. The aim of the present study was to investigative the effect of repeated heating on the oxidative degradation of frying oils (white coconut oil and soy bean oil). Initially, fresh potatoes were peeled off and sliced into uniform thickness (4×0.3×0.3 cm3). Sliced potatoes (batches of 25 g) were fried in 100 mL portions of white coconut oil (WCO) and soy bean oil (SO) separately at 180±5 °C for 10 minutes. The oils were reused for 2 more frying cycles over a span of 3 days (1 frying cycle per day). In each day, an amount of fresh oil was added to make the volume of frying oil in to 100 mL. After each frying cycle, oil samples were collected from the frying pan and by extraction of fat with n-hexane from potato chips. Level of oxidation of frying oils and lipid extracted from potato chips were assessed by measuring the peroxide value (PV) and thiobarbituric acid reactive substances (TBARS). Table 01 states the results of PV and TBARS. Both PV and TBARS of frying oils and lipid extracted from potato chips increased as the number of frying cycles were increased (Table 01). Fried SO (FSO) and lipid extracted from potato chips fried in SO (PSO) showed higher PV and TBARS values than that of fried WCO (FWCO) and lipid extracted from potato chips fried in WCO (PWCO) in every frying cycle (Table 01).Item Thermal stability of phenolic compounds in coconut cake and its stabilizing effect on stripped sunflower oil(Faculty of Graduate Studies, University of Kelaniya, 2015) Prasadani, W.C.; Jayawardena, B.M.; Seneviratne, K.N.Coconut cake possesses phenolic compounds which are antioxidatively active in chemical and food model systems. However, the thermal stability of these phenolic compounds has not yet been investigated. In this study, the thermal stability of phenolic compounds in coconut cake (PCCC) was compared with that of synthetic antioxidants, butylated hydroxy toluene (BHT), butylated hydroxy anisole (BHA) and tert butyl hydroxy quinone (TBHQ) using two food model systems. PCCC were extracted using ethanol:water (70:30 v/v) and the phenolic concentration was determined using the Folin-Dennis method. Thermal stability was tested by heating PCCC and other synthetic antioxidants at 180 oC up to two hours. In 30 min intervals, the activity of heated antioxidants were tested by evaluating their ability to inhibit thiobarbituric acid reactive substances (TBARS) formation in egg yolk emulsion (EYEM). The percentage inhibition of TBARS formation was calculated against a control EYEM sample prepared without added antioxidants. Protective effect of antioxidants on stripped sunflower oil (SSO) was also evaluated. For this purpose, PCCC and synthetic antioxidants were heat treated at 180 oC for two hours and these antioxidants were incorporated into SSO. The induction time (IT) of SSO was determined at 100 oC in the Rancimat apparatus. The percentage inhibition of TBARS formation in EYEM by BHT, BHA, TBHQ and PCCC decreased with heating time and the percentage inhibition of all antioxidants decreased below 40 % after two hours of heating at 180 C. However, at 30 min of heating, inhibition percentage of TBARS formation by PCCC (72±4 %) and TBHQ (68±2 %) is considerably higher compared to BHT (54±2 %) and BHA (42±2%). The IT of SSO varied in the order, control (1.85±0.14 h) < BHT (2.06±0.08 h) < BHA (2.14±0.06 h) < PCCC (2.18±0.03 h) < TBHQ (2.44±0.10 h). The results of these experiments suggest that PCCC can be used to stabilize foods under high temperature cooking conditions.Item Phenolic content and shelf life of commercial virgin coconut oil and copra oil(Faculty of Graduate Studies, University of Kelaniya, 2015) Wijayaratna, U.N.; Jayathilaka, N.; Seneviratne, K.N.There are two main types of coconut oil, virgin coconut oil (VCO) and copra oil (CO), based on their production process. VCO is extracted from fresh, mature coconut kernel by wet or dry methods of extraction, whereas CO is extracted by the dry method of pressing copra. Temperature exceeds 80 C during the extraction of CO while lower temperatures around 50C are maintained during the extraction of VCO. Shelf life is an important parameter of cooking oils and oils become rancid quickly when the shelf life is short. Minor polar compounds such as phenolic substances are known to improve the shelf life. The objective of this study was to determine total phenolic contents (TPC) and shelf life of commercially available VCO and CO, in order to see whether TPC and shelf life are correlated. Two samples from each were used for the analysis. Phenolic compounds of the oils were extracted using methanol:water (80:20 v/v) and TPC was determined using Folin-Denis colorimetric assay and expressed as gallic acid equivalents. Oxidative stability was determined using the Rancimat apparatus at 120, 130, 140 and 150 °C temperatures and extrapolated using Q10 temperature coefficient to obtain the shelf life at 30 °C. The TPC was significantly (p<0.05) higher in CO (13.28±3.13 mg/kg oil) than in VCO (0.52±0.22 mg/kg oil). The induction times (hours) of VCO at 120,130,140 and 150 °C were 51.89±0.08, 26.39±3.44, 13.29±1.84 and 6.26±0.54 respectively, while that of CO were 16.22-41.71, 7.40-18.59, 3.89-14.18 and 1.89-8.43 respectively. The results indicate that induction times of commercial CO samples varied remarkably for different samples, showing the variable quality of CO in the market. The shelf life of VCO deduced was 4.75±1.07 years, while that of CO varied in the range 0.87–1.26 years. The results indicate that CO with higher TPC has a shorter shelf life compared to VCO with lower TPC, suggesting that non-phenolic antioxidants which may be destroyed or inactivated at higher temperatures may be preserved in VCO to improve its shelf life.