Browsing by Author "Seneviratne, K. N."

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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.
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 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.
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.
Identification of potential inhibitors against prostate cancer metastasis drug target, human fatty acid binding protein-12: An in silico study
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Seneviratne, K. N.; Jayathilaka, N.
Fatty acid-binding proteins (FABPs) play a vital role in fatty acid metabolism, cell growth and proliferation and cancer development in humans. Recent studies have revealed that FABP-12 can promote prostate cancer through activation of peroxisome proliferator-activated receptor gamma (PPAR-) which has previously been reported as a driver of metastasis in prostate cancer. Hence targeting the human FABP-12 might be a therapeutic strategy to control prostate cancer metastasis. Due to the absence of crystal structure of this protein, a 3D homology model of FABP-12 was generated using crystal structure of human myelin protein P2 (PDB ID: 2WUT, Resolution: 1.85 Å) as the template by Modeller 9.23 software. The hypothetical model showed the backbone root mean square deviation (RMSD) value of 0.128 Å after superimposition with the template. Further, the structural quality of the model was validated through QMEAN, VERIFY3D, ERRAT, PROCHECK and PROSA tools. Structure based drug discovery of the FABP-12 protein was performed using AutoDock4.2 software with a library of ligands consisting experimentally known FABP family inhibitors (BMS309403 and SBFI-26) and anti-prostate cancer phytochemicals that have been reported earlier. Molecular interactions were explored to understand the nature of intermolecular bonds between ligand and the protein binding site residues using BIOVIA Discovery Studio Visualizer. The in silico hepatoxicity, mutagenicity and cytotoxicity end points for experimentally known FABP family inhibitors and top docked phytochemicals were examined using ProTox-II web server. The BMS309403 showed the highest binding affinity of -10.02 kcal/mol closely followed by celastrol, SBFI-26 and glycyrrhetinic acid with binding affinities of -9.39 kcal/mol, - 9.24 kcal/mol and -9.39 kcal/mol respectively. The inhibition constant (Ki) of BMS309403, celastrol, SBFI-26 and glycyrrhetinic acid were 44.94 nM, 131.65 nM, 167.52 nM and 200.62 nM respectively. Moreover, the in-silico toxicity results revealed that BMS309403 has a weak hepatotoxic potential. Notably, other three compounds obtained negative results for all toxicity descriptors, implying no severe human side effects. These computational findings indicate that celastrol, SBFI-26, and glycyrrhetinic acid have the ability to suppress FABP-12 activity and hence could be utilized as a starting point for future drug development to treat prostate cancer metastasis.
In silico evaluation of coconut milk phenolic antioxidants and their metabolites by human gut bacteria
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Chamikara, V; Perera, S. M.; Seneviratne, K. N.; Jayathilaka, N.
Coconut milk (CM) or the aqueous extract of grated coconut meat is used in culinary applications when preparing both vegetarian and non-vegetarian foods in many Asian countries. Despite CM is traditionally known to be beneficial for gut health, scientific evidence supporting such claims is extremely limited. The beneficial properties of phenolic antioxidants on gut microbiota suggest that phenolic-rich CM may also benefit gut health. Human intestinal gut microbiota plays a vital role in metabolizing dietary components that enter the intestinal tract. These metabolites possess molecular characteristics as well as biological activities, pharmacokinetic and toxicity properties that are different from their parent compounds. Due to a lack of experimental data to examine the gut microbial metabolism of phenolic compounds of CM, a computational study was designed to predict its phenolic metabolites. Our previous high-performance liquid chromatography (HPLC) study reported seven phenolic acids in the CM extract. BioTransformer 3.0 web-based tool was used to predict metabolic transformations of the seven phenolic acids by human gut bacteria. In silico studies were conducted to predict the antioxidant, intestinal anti-inflammatory, antibacterial activities and toxicity of the phenolic compounds and their metabolites using PASS and ProTox-II web servers respectively. The safety of the phenolic compounds of CM and their metabolites were also evaluated based on the endocrine-disrupting effect and the probability of interaction with multiple human receptors using Endocrine Disruptome web server. The in silico analysis of human gut microbial biotransformation predicted the formation of 41 metabolites from 7 parent phenolic compounds present in CM. Most of the parent phenolic acids and the predicted metabolites of CM were shown to have moderate to high antioxidant, intestinal anti-inflammatory activity and antibacterial activity with Probable activity (Pa) > Probable inactivity (Pi) values. Most of the metabolites had a low probability of binding to human nuclear receptors, causing small risks to the endocrine system and posing minimal risk to human health. Moreover, the results revealed that only a few compounds have a weak mutagenic and hepatotoxic potential, while all compounds were devoid of cytotoxicity. However, further in vitro and in vivo testing is required to confirm the impact of these parent phenolic acids and their metabolites on diverse gut microbes and human health and in promoting CM as a functional food as well as a vegan replacement for cow’s milk.
In silico study of physicochemical, pharmacokinetic, toxicity, metabolism and molecular docking of aristolactam E, a constituent of Aristolochia elegans Mast
(Faculty of Graduate Studies, University of Kelaniya Sri Lanka, 2022) Wadanambi, P. M.; Seneviratne, K. N.; Jayathilaka, N.
Aristolochia elegans Mast, also known as Calico flower, is an ornamental plant found around the world including Asia. In traditional medicine, the plant has been used to treat a variety of disorders by employing its broad spectrum of pharmacological properties such as antibacterial, antitumoral, antidiarrheal, anti-snake venom, and anti-scorpion venom. It is also rich in aristolactam alkaloids, which have a variety of biological effects including anti-inflammatory, antibacterial, and anticancer activities. Aristolactam E (AE) is one of the less studied aristolactam alkaloids found in Aristolochia elegans Mast. Therefore, this computational study was designed to investigate (a) physicochemical properties and drug likeness, (b) pharmacokinetic and toxicity, (c) mode of antibacterial, anticancer and antiinflammatory action, (d) sites of metabolism mediated by cytochrome P450 (CYP) 3A4 isoform and (e) probable metabolites of AE compound. The physicochemical attributes and drug likeness were assessed using molinspiration server. SwissADME, LASAR, and Pred-hERG 4.2 servers were used to estimate pharmacokinetic and toxicological characteristics. Molecular docking was performed using AutoDock 4.2 software to determine the binding affinity and molecular interactions of AE with protein targets namely, dihydropteroate synthase (DHPS), dihydroneopterin aldolase (DHNA), phospholipase A2 (PLA2) and tankyrase 2 (TNK2). SOMP, SMARTcyp, and RS-WebPredictor webservers were used to predict the sites of AE metabolism mediated by the CYP 3A4 isoform. Furthermore, the BioTransformer and GLORYx online tools were used to forecast the potential AE metabolites. AE demonstrated favorable physicochemical qualities and met the requirements for oral bioavailability and druggability by following both Lipinski's rule of five and the Veber rule. It also showed a high rate of human intestinal absorption and no blood-brain barrier permeability. Furthermore, the toxicity predictions revealed that AE was a mutagenic chemical, but it was not carcinogenic or cardiotoxic in the mouse model. The AE showed binding affinity of -5.45 kcal/mol, -8.79 kcal/mol, -7.32 kcal/mol, -8.30 kcal/mol and -8.41 kcal/mol with DHPS, DHNA, PLA2, TNK2 (nicotinamide binding site) and TNK2 (adenosine binding site) respectively. The AE exhibited a stronger binding affinity than the control compounds of DHNA and PLA2 while showing close binding affinity to the control of TNK2 (nicotinamide binding site). The overall evaluation identified two sites of metabolism of AE based on a consensus of different metabolism site predictions by CYP3A4 using SOMP, SMARTcyp and RS-WebPredictor webservers. Two probable metabolites of AE were proposed based on the consensus of the results of BioTransformer and GLORYx web tools. This computational analysis can aid in the development of AE derivatives with improved pharmacokinetic and toxicological profiles, hence accelerating drug discovery against microbial infections and inflammation.
In vitro antioxidant, anti-inflammatory and anti-cancer activities of plant extracts used in Ayurvedic medicine
(Faculty of Science, University of Kelaniya, Sri Lanka, 2021) Munasinghe, C. C. J.; Hapugaswatta, H.; Seneviratne, K. N.; Jayathilaka, N.
Traditional medicine may provide leads for potential new therapeutics. Sri Lanka is home to numerous plant species with reported anti-cancer properties according to traditional medical practices. Further, polyphenolic compounds extracted from various plant products have been reported to exhibit antioxidant, anti-inflammatory and anticancer activity. In this study we assessed the antioxidant, anti-inflammatory and anti-cancer potential against acute myelogenous leukemia (AML) in several ayurvedic plant species; Nigella sativa (Kaluduru) seeds, Hemidesmus indicus (Iramusu) roots, Adenanthera pavonina (Madatiya) seeds and Murraya koenigii (Curry leaves). These plant species have already reported of possessing anti-cancer activity against different cancer types. The main objective of this study is to determine the anti-cancer activity of theses plant extracts against AML. All the plant parts were thoroughly cleaned, dried, grounded into fine powder and bioactive compounds were fractionated into different solvents. The Total phenolic content (TPC), antioxidant activity, anti-inflammatory activity and cytotoxicity against AML were evaluated using Folin-Ciocalteu assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, Ferric reducing power antioxidant assay, protein denaturation inhibition assay and Cell TiterGo viability assay using THP-1 cells as a model for AML. N. sativa (kaluduru) aqueous extract showed the highest TPC; 3.14 ± 0.01 mg GAE/g dry weight (after tannin removal) and 2.92 ± 0.01 mg GAE/g dry weight (after protein removal), H. indicus (iramusu) aqueous extract recorded the highest DPPH radical scavenging activity and ferric reducing power and N. sativa (aqueous extract), A. pavonina (madatiya) ethanol extract and M. koenigii (curry leaves) methanol extract showed significantly high values (p<0.05) for the anti-inflammatory activity compared to other tested extracts. H. indicus (aqueous extract) showed the highest cytotoxicity against THP-1 cells with the lowest IC50 value (0.37 ± 0.01 mg/mL). All the tested extracts showed more than 50% inhibition of THP-1 cells at the highest tested concentration (15 mg/mL). All the tested plant extracts showed presence of polyphenols with antioxidant, anti-inflammatory and cytotoxic properties. However, the TPC as determined by Folin Ciocalteu assay does not explain the resulting cytotoxicity against AML cells. Further studies are needed to assess whether the observed anti-leukemic activity is due to cytotoxicity that would affect healthy cells and to assess whether the observed activities are specific against AML.
In vivo and in silico analysis of inhibition of rice (Oryza sativa) bran lipase with guava leaf (Psidium guineense swartz) extract
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Sooriyawansha, A. M. S. C.; Munaweera, R.; Jayathilaka, N.; Seneviratne, K. N.
Rice (Oryza sativa) bran (RB) is one of the most important co-products in the rice milling industry. It is frequently utilized in animal feed or discarded as waste. After milling, the majority of RB cells are ruptured, and the bran layer is removed from the endosperm. Due to that, the RB lipids encounter highly reactive rice bran lipase (RBL) enzymes which hydrolyze the RB lipids into free fatty acids. Hence RB has a short shelf life. This is the major drawback of using RB as an animal feedstock. This study focused on inhibiting the RBL activity using natural potent inhibitors and determining the mechanism of inhibition to increase the shelf life of RB. The RBLs were isolated by ion-exchange chromatography at pH 10.0 buffer. Guava (Psidium guineense swartz) leaf ethanol: water extracts were tested for inhibition of RBLs. Orlistat, a human gastric and pancreatic lipase inhibitor was used as a positive control. The percent RBLs inhibitory activities were assessed using phenyl acetate assay and the guava leaf ethanol: water extract inhibited the RBL activity (47.81% ± 9.44%). The in vivo data were validated by computational analysis. Homology-modeling of RBLs size in 40 kDa and 32 kDa, and molecular docking (AUTODOCK4) were carried out with the lactone orlistat to determine the best docking poses with the lowest estimated Gibbs free energy values (ΔG) for the mechanism of inhibition. Orlistat lactone ring carbon bound covalently to the serine residue in the characteristic GXSXG pentapeptide sequence (where X=any amino acid) in the catalytic site of the RBLs enzyme. Therefore, lactones present in guava leaf ethanol: water extract has the potential to inhibit the activity of RBLs with a similar mechanism of orlistat. Our finding suggests that guava leaf ethanol: water extract can be applied as a natural solution to increase the shelf life of RB.
Metagenomic analysis of the effect of coconut milk on the colon microbiota
(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.
The main source of fat in the diet affects the gut microbiome composition. Coconut milk (CM) has a high percentage of medium chain fatty acids (MCFA). A portion of MCFA reaches the colon and is fermented by the microbiota. This study was conducted with Wistar rats to study the effect of CM on colonic microbial diversity. Twelve-week-old female Wistar rats were randomly assigned to two experimental groups (12 rats/group). Ad libitum access to water and food was provided throughout the study. The control group was fed with a WHO-recommended diet containing 4.2 % total fat; of that 3% fat from soybean oil (SOD). The other group was fed a diet in which the fat component was replaced with CM (CMD). After 28 days, six rats from each group were fasted for 10–12 h and treated with ethanol (20%, 6 g/kg body weight) by oral gavage (SODE and CMDE). Mean ± standard deviation (SD) feed Intakes were 900.50±4.93 g, 899.50±9.31 g, 818.00±6.57 g, 820.00±6.57 g and body weight gains were 52.83±1.83 g, 52.33±1.75 g, 45.50±2.43 g, 47.33±2.34 g in CMD, CMDE, SOD and SODE groups respectively. Feed conversion rates were approximately equal in the four groups (average 0.0580±0.002). 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. All experimental procedures were approved by the Ethics Review Committee, University of Kelaniya. Microbial DNA was isolated from cecal wash samples using DNeasy blood and tissue kit (Qiagen). The 16S rRNA gene libraries were prepared and sequenced according to the protocols recommended by Ion Torrent (Ion GeneStudio S5 prime system, Thermo Fisher Scientific). Trimmed sequences were clustered into operational taxonomic units (OTUs) with a hierarchical cutoff of 97.0% similarity using Ion Reporter v5.16. Taxonomic annotation was conducted against Curated MicroSEQ 16S Reference Library v2013.1 and Curated Greengenes v13.5 databases. Alpha-diversity and beta-diversity analyzes were performed using the QIIME2 platform. Bacteroidetes and Firmicutes phyla represent 90% of the cecal bacterial community across dietary groups. Other microbial phyla in the cecal wash samples were Actinobacteria, Proteobacteria, and Tenericutes. The cecal microbiota of CMD-fed rats was characterized by a significant increase (P<0.05) in the relative abundance of Desulfovibrionaceae, Eubacteriaceae, Erysipelotrichaceae, Lachnospiraceae Porphyromonadaceae, Ruminococcaceae bacterial families, and a decreased relative abundance of Bacteroidaceae, Clostridiaceae and Lactobacillaceae compared to the control diet. Studies have shown that alcohol promotes both dysbiosis and bacterial overgrowth. According to the two factor ANOVA, there was a significant difference (P<0.05) in colonic-microbiota between the four groups. Family level rarefaction plots were varied CMD>CMDE>SOD>SODE and CMD>SODE>CMDE>SOD according to Chao1 index and Simpson’s indexes respectively. Principle component analysis revealed four distinct clusters, suggesting that both diet and alcohol-induced oxidative stress affected gut microbiota. The elevated bacterial families have an impact on microbial-mediated saccharolytic functions, lipophilic functions, vitamin synthesis, and protection against intestinal infections. Thus, the intestinal microbiota in Wistar rats varies significantly with dietary fat source and oxidative stress conditions.
A preliminary study on the utilization of coconut paring residue as an antioxidant and a food preservative
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Thilakarathna, G. T. M.; Jayathilaka, N.; Seneviratne, K. N.
Coconut paring is the brownish outer layer of the coconut kernel, which is removed when desiccated coconut is produced. Coconut Paring Residue (CPR) is the residue remaining after expulsion of coconut oil from the coconut parings. The present study was conducted to evaluate the potential of the ethanolic extract of CPR as an antioxidant mixture capable of preserving edible oils and milk. Phenolic compounds of CPR were extracted using ethanol: water (70:30 v/v) solvent system as 70% ethanol has shown the highest oxygen radical absorption capacity for the phenolic mixture. Total phenolic content and antioxidant activity of CPR extracts were measured using Folin-Ciocalteu and α, α-diphenyl-β-picrylhydrazyl (DPPH) free radical scavenging assays respectively. The potential of the phenolic extract to inhibit lipid oxidation in soybean oil, sesame oil, sunflower oil and palm oil was determined by monitoring peroxides using ferric thiocyanate method. Microbial count was used to evaluate the ability of the phenolic extract to inhibit or delay the action of microorganisms in milk. All the tests were conducted in duplicates. Total phenolic content of the coconut paring residue extract was 1947 ± 12 mg/kg as gallic acid equivalents. The phenolic extract showed higher antioxidant activity between the concentration range 5-90 µg/mL and lower IC50 value (10.40 ± 0.36 μg/mL) compared to the ascorbic acid standard. Peroxide values of the phenolic-added oil samples were lower than those of the control with no added extract during the storage time period of 15 days. Colony Forming Units of yeast and molds per mL of milk (CFU/mL) were estimated using test samples with phenolic extract at 200 μg/mL). Milk without phenolic extract served as the control. The initial CFU count in both control and test samples was 4.5 × 104 ± 2.1 x 104 CFU/mL and the CFUs on the day two were 4.6 x 109 ± 1.7 x 109 CFU/mL and 2 x 108 ± 1.41 x 108 CFU/mL, respectively. The results suggest that the phenolic extracts of CPR can be used for improving the shelf life of edible oils and milk.
Soybean oil and coconut oil enhance the absorption of chlorogenic acid in humans
(Journal of Food Biochemistry, 2021) Weerakoon, W. N. M. T. D. N.; Anjali, N. V. P.; Jayathilaka, N.; Seneviratne, K. N.
The effect of a mainly polyunsaturated oil (soybean oil) and a mainly medium chain triglyceride oil (coconut oil) on the absorption of the phenolic antioxidant chlorogenic acid (5-caffeoylquinic acid) was investigated using 90 healthy volunteers. Serum concentrations and the absorbed percentages of chlorogenic acid of volunteers who received chlorogenic acid without oils (0.006 ± 0.001 mg/ml, 5.7 ± 0.2%), chlorogenic acid with soybean oil (0.012 ± 0.001 mg/ml, 11.8 ± 1.3%), and chlorogenic acid with coconut oil (0.067 ± 0.014 mg/ml, 65.6 ± 18.1%) were significantly different from each other (p < .05). There was a strong positive correlation between the increase in serum and plasma antioxidant capacity and the absorption of chlorogenic acid. The major fatty acid of each of soybean oil and coconut oil also improved the permeability of chlorogenic acid in Caco-2 cell monolayers. The results suggest that the tested edible oils may improve the nutritional value of chlorogenic acid-containing foods by improving the absorption of chlorogenic acid.