Browsing by Author "Seneviratne, K.N."

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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.
Chemical investigation of the properties of four traditional Sri Lankan oils
(Sri Lanka Association for the Advancement of Science, 2006) Seneviratne, K.N.; Jayawardena, B.M.; Kotuwegedara, R.T.; Manoj, R.P.K.
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.
Comparison of the in vivo antioxidant activity of traditional coconut oil, virgin coconut oil and soya oil
(Sri Lanka Association for the Advancement of Science, 2008) Seneviratne, K.N.; Ekanayake, S.; Hapuarachchi, C.D.
When the nutritional quality of cooking oils is considered, it is extremely important to evaluate the contribution of cooking oils to the antioxidant activity in blood. In the present study, the in vivo antioxidant potentials of three cooking oils are compared. Male Wistar rats were fed with a special diet containing traditional coconut oil (TCO, prepared by boiling coconut milk), virgin coconut oil (VCO) and soya oil (SO). The effect of the consumption of these oils on the total antioxidant activity in blood serum was analyzed and compared. The decolorization of ABTS•+ (radical cation of 2,2'-azinobis-(3-ethylbenzothiazoline- 6-sulfonic acid) was used as a measure of antioxidant activity and the antioxidant activity was expressed as trolox equivalent antioxidant capacity (TEAC).
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.
Determination of thermal stabilities of guava leaf, coconut cake, rice bran and sesame cake extracts
(Sri Lanka Association for the Advancement of Science, 2015) Senanayake, C.M.; Seneviratne, K.N.; Jayawardena, B.M.; Prasadani, W.C.
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 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 three edible oils on the intestinal absorption of caffeic acid: An in vivo and in vitro study.
(PLoS ONE 12(6), 2017) Prasadani, W.C.; Senanayake., C.M; Jayathilaka., N; Ekanayake., S; Seneviratne, K.N.
Polyphenolic antioxidants are mainly absorbed through passive paracellular permeation regulated by tight junctions. Some fatty acids are known to modulate tight junctions. Fatty acids resulting from the digestion of edible oils may improve the absorption of polyphenolic antioxidants. Therefore, we explored the effect of three edible oils on the intestinal absorption of caffeic acid. Rats were fed with soybean oil and caffeic acid dissolved in distilled water. Caffeic acid contents in the plasma collected up to 1 hr were quantified. The experiment was repeated with coconut oil and olive oil. Component fatty acids of the oils were individually tested in vitro for their effect on permeability of caffeic acid using Caco-2 cell monolayers. Highest absorption of caffeic acid was observed in animals fed with coconut oil. In vitro transport percentages of caffeic acid in 2.5 mmol/L solutions of fatty acids were 22.01±0.12 (lauric), 15.30 ± 0.25 (myristic acid), 13.59 ± 0.35 (linoleic acid), 3.70 ± 0.09 (oleic acid) and 0.10±2.0 (all other fatty acids). Lauric acid and myristic acid are the two major fatty acids present in coconut oil. Therefore, these fatty acids may contribute to the higher absorption of caffeic acid in the presence of coconut oil.
Enzyme assisted extraction, quantification and antioxidant activity of phenolic compounds of coconut cake
(Sri Lanka Association for the Advancement of Science, 2013) Prasadani, W.C.; Seneviratne, K.N.; Jayawardena, B.M.
Establishment of chemical parameters to distinguish between Sri Lankan teas of different geographical origins
(Sri Lanka Association for the Advancement of Science, 2008) Seneviratne, K.N.; Seneviratne, C.A.
Unblended Sri Lankan teas are classified as high grown (HG), medium grown (MG) and low grown (LG) based on their geographical origin. In the quality control purposes, it is an added advantage if suitable chemical data that are helpful to distinguish between different teas are available. Present study explains the identification of the geographical origin of teas based on the total phenol, caffeine and catechin contents. Even though the quantities of these substances have been reported, the origin, whether the tea is pure or blended and the particle size of teas have not been specified in such studies. Total phenol contents (TPC) were determined by Folin-Denis colorimetric method. Caffeine contents (CFT) and catechin contents (CTC) were determined by the comparison of the signal areas of these compounds in HPLC chromatograms using calibration plots. The results indicate that there is no significant difference in the total phenols, caffeine and catechin contents in tea samples collected within a geographical area. However, the compositions of these compounds varied significantly among the tea samples from different geographical origins (HG, MG and LG).
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
Identification and Quantification of Phenolic Antioxidants in Some Selected Traditional Sri Lankan Medicinal Oils
(University of Kelaniya, 2007) Seneviratne, K.N.; Kotuwegedara, R.T.
ABSTRACTSeveral medicinal properties of seed oils are known to originate from the nonsaponifiable compounds present in the oils. Among the nonsaponifiable compounds. phenolic fraction includes phenolic acids and flavonoids. These compounds are known to render several beneficial health effects. In the present study, phenolic compounds of the seed oils of Brassica juncea (Aba), Madhuca nerifolia (Mee), Sessamwn indicum (Thala), Calophyllum inophyllum (Dhomba), Canerium zeylanicum (Kekuna) and Ricinus calamus (Endaru) were identified by high performance liquid chromatography (HPLC) technique and individual phenolic antioxidants were quantified by the integration of the signal areas of chromatograms. The results arc given in the Table 1. Table 1. Phenolic antioxidants in selected traditional Sri Lankan medicinal oils -- ---- -----·---- -··------ ··----- Phenolic compound HPLC Amount of phenolic compound (mg/ kg of oil) retention ----.. ---- time Do mba Aha Mee Kekuna Thala (min) 3,4- DHBA 12.8 0.43 ± 0.21 ± 1.29 ± 0.02 0.02 0.20 CH 14.4 1.82 ± 1.33 :1:: 1.24 ± 0.12 0.30 0.10 PHBA 16.6 0.10 ± 0.32 ± 1.04± 1.0 L 0.01 0.03 0.20 0.02 Vanillic 18.5 Caffeic acid 18.9 1.61 1 0.44 ± 0.83 + 0.10 0.04 0.03 Syringic acid 19.7 0.56 ± 0.06 Vanillin 21.3 0.24 ± 0.13 ± 0.31 ± 0.04 0.02 0.02 Ellagic acid 28.9 1.54 ± 2.12 L 1.55 ± 0.30 0.30 0.20 Cinnamic acid 37.9 1.93 ± 0.32 ± 0.20 0.02 Each data point represents the mean of three replicates± S.E Endaru 2.33 ± 0.30 0.90± 0.10 0.74 ± 0.04 1.30 ± 0.10 3, 4- DHBA - 3, 4-Dihydroxybenzoic acid PHBA - P-hydroxybenzoic acid CH -Catechin hydrate The results indicate that these medicinal oils contain several phenolic acids and lavonoids whose beneficial health effects and antioxidant properties are already known. Financial assistance of IFS E/3652-1 and NSF /RG/2005/ AGIO 1 grants is highly acknowledged.
Improving the nutritional quality of coconut oil by incorporating phenolic substances of coconut cake
(Sri Lanka Association for the Advancement of Science, 2009) Seneviratne, K.N.; Hapuarachchi, C.D.; Ekanayake, S.
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.
Inhibition of lipid peroxidation in tissue homogenates by selected medicinal oils and their antioxidant activity
(Sri Lanka Association for the Advancement of Science, 2006) Jayawardena, B.M.; Jayakody, C.H.; Seneviratne, K.N.