Browsing by Author "Jayathilaka, N."

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Antioxidant and Nutritional Properties of Domestic and Commercial Coconut Milk Preparations
(International Journal of Food Science, 2020) Karunasiri, A.N.; Gunawardane, M.; Senanayake, C.M.; Jayathilaka, N.; Senevirathne, K.N.
The aqueous extract of scraped coconut kernel is known as coconut milk. Coconut milk preparations are also commercially available in the form of desiccated powders or liquids. While these various coconut milk preparations are heavily used in cooking in the Asian countries as a major source of dietary fat, limited studies have been conducted on their chemical and nutritional composition. In this study, we have determined the chemical composition and nutritional effects of both domestic preparations of coconut milk and the commercially available counterparts. The results indicate that the phenolic compounds of all coconut milk preparations provide protection against oxidative damage on lipids and inhibit oxidative damage of both proteins and DNA. The lipid profiles are not significantly affected by the consumption of the three coconut milk preparations despite their different fat contents.
Antioxidant Effect of Coconut Milk on Oxidative Damage in Commensal Lactobacilli in the Gastrointestinal Tract
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Karunasiri, A.N.; Gunawardena, M.; Seneviratne, K.; Jayathilaka, N.
Coconut milk (CM) is the aqueous extract of coconut endosperm which is rich in polyphenols. Coconut milk is commonly used in culinary applications in South Asia. In this study, protective effect of phenolic antioxidants (PA) extracted from CM was tested in lactobacilli which are the prominent probiotic forms in the human gut. These commensals help to maintain the immune and metabolic homeostasis. The intestinal environment can be altered by many factors that generate reactive oxygen species (ROS) resulting in oxidative stress. Enteric bacteria have been reported to mediate redox homeostasis through the regulation of ROS production. However, oxidative damage to the gut microbiota has been suggested to contribute to several diseases including intestinal and neurodegenerative disorders. Here, the activity of phenolic antioxidants extracted from CM on oxidative damage in Lactobacillus acedophillus, L. plantarum, L. lactis, L. casei and L. fermentum under aerobic conditions were evaluated based on the amount of products of macromolecular damage. The total polyphenol content of aqueous extract of CM was 8.21±0.13 mg/L as determined by the Folin Ciocalteu method. Bacterial cultures at optical density of 0.5 at 620 nm were incubated at 37°C under aerobic conditions to induce the oxidative damage. The cells were cultured overnight with a concentration series of PA (0, 0.4, 0.5, 0.6, 0.7, 0.8 and 0.9 mg/mL) to assess the protective effect on oxidative damage. The concentration of PA was kept below the concentration that affect cell viability as determined by 2,3,5-triphenyltetrazolium chloride assay at 620 nm. Lipid peroxide levels (μg/mL) and protein carbonyl levels (nmol/mL) were detected with thiobarbituric acid and 2,4-DNPH respectively. Growth under aerobic conditions affected the cell viability and induced significant (P<0.05) damage to proteins and lipids in the lactobacilli under investigation. Treatment with increasing concentrations of PA from CM showed a corresponding increase in the cell viability and a corresponding decrease in the amount of lipid peroxides and protein carbonyls under aerobic conditions. Therefore, PA from CM protect gut microbiota from oxidative damages in lipid and proteins without affecting their viability.
Application of rolling circle amplification (RCA) to detect direct amplification of dengue virus in patient serum samples.
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Manilgama, T.; Seneviratne, K. N.; Jayathilaka, N.
Rolling Circle Amplification (RCA) is an isothermal amplification process that can be utilized for rapid amplification of target nucleic acids. In contrast to PCR, which uses thermocycling to mediate denaturation, annealing, and subsequent extension, RCA can be performed at a single reaction temperature making RCA an attractive solution for disease diagnosis based on amplification of pathogen nucleic acids at resource limited settings. In addition, PCR-based detection of pathogenic RNA involves the additional steps required to make complementary DNA copies of the target for amplification. Dengue is a mosquito vector borne viral RNA infection that largely affects urban and semi-urban, sub-tropical and tropical areas. While majority of dengue fever patients recover with careful hospital monitoring some patients may develop severe complications that result in mortality. Therefore, early diagnosis is critical for screening the patients that require hospital management and to prevent exceeding hospital capacity during a dengue outbreak. We developed a direct RCA of dengue virus RNA in serum samples from dengue virus positive patients using Phi 29 DNA Polymerase for the disease diagnosis at resource limited settings. Serum samples were collected from patients suffering from dengue virus infection based on a positive NS 1 antigen test within four days from fever onset with informed consent (n=3). Serum samples collected from healthy individuals were used as the controls (n=3). Multiple Displacement Amplification (MDA) was used to increase the amplification efficiency. Positive control reactions were carried out using a circularized 66 bp linear 5´phosphorylated probe that contained a complementary sequence to all four dengue serotypes and a forward primer against the conserved target region on the probe at 30 oC overnight. The product formation was confirmed by gel electrophoresis following restriction enzyme digestion of the RCA/MDA products with EcoRI. The RCA/MDA products were quantified using a ssDNA dye. Direct isothermal amplification of dengue virus from serum samples collected from dengue infected subjects confirmed that RCA/MDA reaction specifically amplifies dengue virus in patients while no amplification was detected for the serum samples collected from healthy volunteers. Since RCA/MDA can be used for direct gene expression analysis of mRNA and micro RNA in resource limited settings, this novel method can be used for simultaneous disease diagnosis and early prognosis of severe dengue based on differential expression in resource limited settings.
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.
Comparison of the basic nutritional characteristics of the first extract and second extract of coconut milk
(2015) Nadeeshani, R.; Wijayaratna, U.N.; Prasadani, W.C.; Ekanayake, S.; Seneviratne, K.N.; Jayathilaka, N.
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.
Determination of Antioxidant and Antimicrobial Activities of Psidium guineense Sw. Leaf Extracts Fractioned Based on Polarity
(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Munaweera, R.R.K.W.; Senanayake, C.; Algama, H.; Seneviratne, K.; Jayathilaka, N.
Psidium guineense Sw. is a guava species distributed in South America, some parts of Africa and South Asia including Sri Lanka. Our previous studies have shown that Psidium guineense Sw. leaves contain 195.25±9.56 mg g-1 phenolic substances and 70 % ethanolic extract of P. guineense Sw. Leaves (PGLE) improve the oxidative stability and microbial shelf life of vanilla cake. PGLE may contain highly polar as well as medium and low polar phenolic substances. Therefore, antioxidant activity and antimicrobial activity of the further fractionated portions by chloroform and hexane of PGLE on food spoilage bacteria were determined. For this purpose, PGLE was obtained by solvent extraction and solvents of PGLE and different fractions were evaporated and reconstituted in 10 % ethanol. The antioxidant activities of solvent fractions, BHT and PGLE measured using DPPH radical scavenging assay are given in Figure 1. Figure 1. DPPH radical scavenging activity The antimicrobial activities of PGLE, chloroform fraction of PGLE and hexane fraction of PGLE were determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for four microbial strains, two gram negative; Escherichia coli and Salmonella typhimurium and two gram positive; Bacillus cereus and Staphylococcus aureus. Ethanol from the extracts was maintained at 1% in the antimicrobial assays. PGLE showed the higher antimicrobial activity towards gram positive bacteria with LD50 values of 190.4 ± 20.2 mg/L for Staphylococcus aureus and 305.4 ± 22.4 mg/L for Bacillus cereus than gram negative bacteria with LD50 of 444.9 ± 13.0 mg/L for Escherichia coli and 508.6 ± 64.7 mg/L for Salmonella typhi. Streptomycin and chloramphenicol were used as positive controls. No antimicrobial activity was observed for chloroform and hexane fractions of PGLE in the four bacterial strains tested. The results of the present study suggest that phenolic compounds with medium polarity may be mainly responsible for antioxidant activity while phenolic compounds with high polarity may be responsible for antimicrobial activity.
Determination of Nutrient composition of domestic and commercially available coconut milk preparations
(Faculty of Graduate Studies, University of Kelaniya, 2015) Nadeeshani, D.L.W.R.; Seneviratne, K.N.; Jayathilaka, N.
This study evaluated the nutrient composition of coconut milk (CM) prepared by blending (pressing) the grated coconut (Cocos nucifera L.) kernel and commercially available powdered or liquid CM. Nine randomly selected coconuts from ordinary tall coconut trees, three each from three regions in Kurunegala district were analyzed using standard methods. First extract (FE) of CM was prepared by blending a mixture of water and grated coconut kernel 1:1 (w:w) in a household blender. The strained pulp was used similarly, to prepare the Second Extract (SE). Commercial CM was prepared according to instructions on the packages. The results are given in Table 1.
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.
Differential expression of microRNA, miR-150 and Enhancer of Zeste Homolog 2 (EZH2) in peripheral blood cells as early prognostic markers of severe forms of dengue
(Karger Medical and Scientific Publishers., 2020) Hapugaswatta, H.; Amarasena, P.; Premaratna, R.; Seneviratne, K.N.; Jayathilaka, N.
BACKGROUND: Dengue presents a wide clinical spectrum. Most patients recover following a self-limiting non-severe clinical course. A small proportion of patient’s progress to severe disease, mostly characterized by plasma leakage with or without hemorrhage. Early symptoms of severe dengue (SD) are similar to those of non-severe dengue fever (DF). Severe symptoms manifest after 3-5 days of fever, which can be life threatening due to lack of proper medications and inability to distinguish severe cases during the early stages. Early prediction of SD in patients with no warning signs who may later develop severe infection is very important for proper disease management to alleviate related complications and mortality. microRNA are small non-coding RNA molecules that regulate post-transcriptional gene expression. Due to the remarkable stability and the role of microRNA in gene expression, altered expression of microRNA was evaluated to explore clinically relevant prognostic markers of severe dengue. METHODS: The relative expression of microRNA hsa-let-7e (let-7e), hsa-miR-30b-5p (miR-30b), hsa-miR-30e-3p (miR-30e), hsa-miR-33a (miR-33a), and hsa-miR-150-5p (miR-150) and several putative target genes in peripheral blood cells (PBC) collected from 20 DF and 20 SD positive patients within 4 days from fever onset was evaluated by quantitative reverse transcription PCR (qRT-PCR). RESULTS: miR-150 showed significant (P < 0.01) up regulation in PBC of SD patients compared to DF patients during the acute phase of infection. Expression of enhancer of zeste homolog 2 (EZH2) was significantly (P < 0.01) down regulated indicating that genes involved in epigenetic regulation are also deferentially expressed in SD patients during the early stage of infection. CONCLUSIONS: Differential expression of microRNA miR-150 and the putative target gene EZH2 may serve as reliable biomarkers of disease severity during early stages of dengue infection. KEYWORDS: Acute dengue biomarkers; Dengue; Severe dengue; microRNA.
Effect of chain length and saturation of the fatty acids in dietary triglycerides on lipid metabolism in Wistar rats
(Journal of Food Biochemistry, 2021) Senanayake, C. M.; Hapugaswatta, H.; Samarawickrama, G. R.; Jayathilaka, N.; Seneviratne, K. N.
We investigated the effect of the chain length and the degree of saturation of fatty acids in dietary triglycerides on serum lipid profiles and hepatic lipid metabolism in Wistar rats. Fat component of the basal diet (soybean oil) was replaced with fats with fatty acids of different chain lengths and saturation and the serum lipids were monitored for 150 days. Principal component (PC) analysis of serum lipid components was related to chain length and saturation. The combined effect of chain length and saturation on PC 1 scores was evaluated by multiple regression analysis. The results indicated that average chain length of the fatty acids of triglycerides has a higher influence on the quality of serum lipid parameters than the average degree of saturation. Expression of selected genes responsible for lipid metabolism showed similar trends in medium chain saturated and long chain polyunsaturated diet groups. Practical applications Dietary lipids contain a wide range of saturated and unsaturated fatty acids with different chain lengths. Overall contribution of these different fatty acids decides the health effects of the lipids in the diet. Present study shows that the fats with medium chains and higher degree of saturation and fats with long chains and higher degree of unsaturation (lower degree of saturation) affect serum lipid parameters and expression of hepatic genes involved in the lipid metabolism in a similar manner. Such information is important for physicians to plan dietary schemes to improve the nutritional health and manage the noncommunicable diseases.
Effect of coconut milk on intestinal barrier function and management of oxidative stress
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Ambanpola, N.; Anjali, N. V. P.; Manilgama, T.; Gunawardane, M.; Seneviratne, K. N.; Jayathilaka, N.
Coconut milk (CM) is a major source of dietary fat in a Sri Lankan meal. It is rich in saturated, medium-chain fatty acids (MCFA) and various polyphenols. Some of the ingested fats and polyphenols are not absorbed in the small intestine and reach the colon. This study assessed the formation of metabolic products from CM and the influence of CM on intestinal barrier function. Twelve-week-old female Wistar rats were housed at 25 ± 1°C with a 12 h light and dark cycle. Rats were randomly assigned to two experimental groups (12 rats/ group). Ad libitum access to water and a diet containing 4.2 % total fat; from that 3% fat by means of soybean oil (SOD) control or CM (CMD) was provided for four weeks. Six rats from each group fasted for 10–12 h and were treated with ethanol (20%, 6 g/kg body weight) by oral gavage (SODM and CMDE groups were obtained). Blood (1 mL) was then drawn from the tail vein. Plasma antioxidant capacity, lipid peroxidation, and protein carbonyl content were determined by 2,2-diphenyl-1-picryl-hydrazy (DPPH) assay, ferric reducing antioxidant power (FRAP) assay, protein carbonyl assay, and thiobarbituric acid reactive substances (TBARS) assay according to previously reported methods. At the end of the feeding experiments, animals were subjected to barbiturate euthanasia and a transverse abdominal incision was made. The cecal wash samples with phosphate-buffered saline (pH 7.4) were stored at -80 °C. Liver and brain samples were also harvested. All experimental procedures were approved by the Ethics Review Committee, University of Kelaniya. Short-chain fatty acids (SCFAs) in the cecal wash, plasma, liver, and brain samples were quantified by Gas chromatography. SCFA levels were determined by the standard curves of each SCFA. Shapiro-Wilk normality test (P<0.05) and t-test was used for the statistical comparison. Acetate, propionate, and butyrate concentrations were 802.9±0.4 μg/mL, 156.3±2.1 μg/mL, 20.5±0.4 μg/mL and 802.8±0.4 μg/mL, 153.5±1.7 μg/mL, 19.9±0. μg/mL in CMD and SOD cecal wash samples respectively. Acetate, propionate, and butyrate concentrations were 236.2±0.1 μg/mL, 16.2±0.2 μg/mL, 1.3±0.0 μg/mL and 226.3±1.4 μg/mL, 14.4±0.2 μg/mL, 1.2±0.0 μg/mL in CMD and SOD plasma samples respectively. There was a significant (P<0.05) difference between plasma acetate and propionate levels in CMD compared to SOD. SCFAs were not detected in liver and brain samples. Saccharolytic microbes ferment oligo- and polysaccharides and produce SCFAs. Following their production SCFAs are rapidly absorbed by colonic cells and those not metabolized by colonic cells pass into the liver. Thus, only a small amount of the SCFAs reach systemic circulation and other tissues. Alcohol causes oxidative stress by releasing reactive oxygen species (ROS) during alcohol metabolism. Polyphenols serve as exogenous antioxidants, and they scavenge free radicals to control ROS. According to the four assays, there were no significant differences in the antioxidant capacity between the four groups suggesting no antioxidant effect of coconut milk over soy oil control. Thus, CM has a significant (P<0.05) impact on SCFAs passing through the intestinal barrier but no effect on the management of oxidative stress than soy oil.
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.
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).
Effect of the wet extraction methods on the phenolic profile of coconut oil
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Anjali, N. V. P.; Algama, C. H.; Seneviratne, K. P.; Jayathilaka, N.; Seneviratne, K. N.; Sakalasuriya, D. D.; Silva, C. D.
There are multiple methods for producing virgin coconut oil, which can broadly be divided into wet and dry processes. In the wet methods, coconut oil is directly extracted from the coconut milk, an aqueous emulsion is prepared using freshly grated coconut kernel. The method used to extract oil can affect the quality parameters and the phenolic profile of each coconut oil. Therefore, the phenolic profile, and the antioxidant capacity of coconut oil produced using four wet extraction methods, namely, boiling method (BM), fermentation method (FM), chilling and thawing method (CTM) and centrifugation method (CM) were quantified using previously reported methods. The shelf life of each oil sample at 28 ℃ was analyzed based on the induction time of each oil sample using a Rancimat apparatus. Phenolic profiles and unsaponifiable matter were analyzed qualitatively and quantitatively using HPLC. Shelf life at 28 ℃ (2.9±0.0 years), 𝛼-tocopherol (78.9±0.4 mg/Kg), total phenolic content (660±1 gallic acid equivalent mg/oil Kg) and antioxidant activity (19.4± 1.0%) are significantly (P<0.05) higher in the oil prepared by BM compared to the other wet extraction methods. The phenolic profile of CM and CTM included p-hydroxybenzoic acid, epigallocatechin gallate (EGCG), and epicatechin. The phenolic profile of coconut oil prepared by FM included gallic acid, p-hydroxybenzoic acid, EGCG, epicatechin, and epigallocatechin (EGC). In addition to the p-hydroxybenzoic acid and gallic acid, gallocatechin gallate (GCG), and catechin were found in significantly (P<0.05) higher amounts in coconut oil extracted using BM as a result of epimerization of EGCG and epicatechin to GCG and catechin under the heating conditions used in the BM. Hydrolysis of EGCG was found to be responsible for the observed low levels of EGCG (0.01±0.00 mg/oil Kg) and the presence of gallic acid and EGC in the coconut oil prepared by FM compared to the other two cold extraction methods (CTM, CM). Therefore, the extraction method has a significant impact on the phenolic profile of coconut oil.
Epigenetic modifications in human disease
(University of Kelaniya, 2015) Jayathilaka, N.; Weeraratne, K.S.; Buwaneka, P.A.; Dilhani, G.R.S.
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).
Expression of Nitric Oxide synthase and Nitric Oxide levels in peripheral blood cells and oxidized low-density lipoprotein levels in saliva as early markers of severe dengue.
(Hindawi, 2021) Hapugaswatta, H.; Ruwani, L.; Wimalasekara; Perera, S.S.; Premaratna, R.; Seneviratne, K.N.; Jayathilaka, N.
BACKGROUND: Severe dengue (SD), experienced by only a fraction of dengue patients, can be lethal. Due to the lack of early markers that can predict the evolution of SD, all dengue patients have to be monitored under hospital care. We discovered early oxidative stress markers of SD to identify patients who can benefit from early intervention before the symptoms appear. METHODS: The expression of inducible nitric oxide synthase (iNOS) in peripheral blood cells (PBC), nitric oxide (NO), and oxidized lowdensity lipoprotein (oxLDL) levels in plasma and saliva collected at early stages of dengue infection from 20 nonsevere dengue fever (DF) patients and 20 patients who later developed SD were analyzed in a retrospective nested case-control study. RESULTS: The expression of iNOS is significantly (P < 0:05) lower in patients who developed SD than in DF patients at admission within 4 days from fever onset. Median plasma NO concentration within 4 days from fever onset is also significantly (P < 0:05) lower in patients who developed SD (17:9±1:6 μmol/L) than DF (23:0±2:1 μmol/L). Median oxLDL levels in plasma within 3 days from fever onset is significantly (P < 0:05) lower in patients who developed SD (509:4 ± 224:1 ng/mL) than DF (740:0 ± 300:0 ng/mL). Median salivary oxLDL levels are also significantly (P < 0:05) lower in patients who developed SD (0:8±0:5 ng/mL) than DF (3:6±2:6 ng/mL) within 4 days from fever onset. CONCLUSIONS: These findings suggest that the expression of iNOS (73% sensitivity, 86% specificity) and plasma NO (96% sensitivity, 61% specificity at 22.3 μmol/L; P < 0:05) may serve as early markers of SD within 3 days from fever onset. Salivary oxLDL levels may serve as early noninvasive markers of SD with a sensitivity and specificity, respectively, of 57% and 91% at 0.9 ng/mL; 76% and 55% at 2.3 ng/mL; and 100% and 50% at 4.6 ng/mL (P < 0:05) within 4 days from fever onset
Host gene expression analysis in Sri Lankan melioidosis patients.
(PLoS Negl Trop Dis 11(6), 2017) Krishnananthasivam, S.; Jayathilaka, N.; Sathkumara, H.D.; Corea, E.; Natesan, M.; De Silva, A.D.
Melioidosis is a life threatening infectious disease caused by the gram-negative bacillus Burkholderia pseudomallei predominantly found in southeast Asia and northern Australia. Studying the host transcription profiles in response to infection is crucial for understanding disease pathogenesis and correlates of disease severity, which may help improve therapeutic intervention and survival. The aim of this study was to analyze gene expression levels of human host factors in melioidosis patients and establish useful correlation with disease biomarkers, compared to healthy individuals and patients with sepsis caused by other pathogens.
Identification of carbazole alkaloids from Murraya koenigii as potential main protease inhibitors of SARS-CoV-2 Omicron variant
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Jayathilaka, N.; Seneviratne, K. N.
Despite of COVID-19 vaccination, immune escape of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has created an urgent priority to identify additional antiviral drugs. In the short span of two years, SARS-CoV-2 has evolved to raise five variants of concern out of which Omicron has currently become the dominant variant all over the world. Targeting main protease (Mpro) expressed by SARS-CoV-2 Omicron variant, is a therapeutic strategy for drug development due to its prominent role in viral replication cycle. Leaves of Murraya koenigii are used in various traditional medicinal applications and this plant is known as a rich source of carbazole alkaloids. Previous research reports have shown that the leaves, roots and bark of this plant are high in carbazole alkaloids. Many drug compounds containing a carbazolic core have been discovered, and some have demonstrated antiviral action. Thus, this computational study was designed to investigate the inhibitory potential of carbazole alkaloids from Murraya koenigii against Mpro. Molecular docking was performed using AutoDock Vina software to determine the binding affinity and molecular interactions of carbazole alkaloids and the reference inhibitor (3WL) in the active site of SARS-CoV-2 Omicron variant Mpro (PDB ID: 7TLL). The top scoring compounds were further assessed for physicochemical properties and drug likeness, pharmacokinetic and toxicity (ADME/T) properties, antiviral activity, pharmacophore modeling and molecular dynamics (MD). Two carbazole alkaloids namely, koenine (-7.8 kcal/mol) and girinimbine (-7.6 kcal/mol) displayed a unique binding mechanism that shielded the catalytic dyad (His41 and Cys145) of Mpro with stronger binding affinities and molecular interactions than 3WL (-7.2 kcal/mol). Furthermore, the two compounds with high affinity displayed favorable physicochemical and ADME/T properties that satisfied the criteria for oral bioavailability and druggability. The pharmacophore modeling study showed shared pharmacophoric features (aromatic ring, hydrophobic area, hydrogen bond donor/acceptor and positively ionizable region) of those compounds for their biological interaction with Mpro. During the molecular dynamics simulation of 100 ns, the MD simulation trajectories of root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (Rg) of top two complexes exhibited high stability. Therefore, koenine and girinimbine from Murraya koenigii, may have the potential to restrict SARS-CoV-2 replication by inactivating the Mpro catalytic activity thus offering potential hits that may be further structurally modified and evaluated in vitro and in vivo for the discovery of novel SARS-COV-2 Mpro inhibitors.