Browsing by Author "Paranagama, P. A."

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Antibacterial polyketide from Lasiodiplodia theobromae, inhabiting the lichen host, Pyrenula bahiana on mangrove ecosystems in Puttalam lagoon, Sri Lanka
(Asian Journal of Chemistry, 2022) Paranagama, P. A.; Santhirasegaram, S.; Fathima, M. M. A.; Wickramarachchi, S. R.; Attanayake, R. N.; Weerakoon, G.; Maduranga, K.
Lasiodiplodia theobromae is one of the frequently isolated fast growing endolichenic fungus. This fungus was isolated from the lichen host, Pyrenula bahiana collected from the mangrove ecosystems in Puttlam lagoon and identity was confirmed based on rDNA-ITS sequence homology. Secondary metabolites of L. theobromae were extracted into ethyl acetate and subjected to antibacterial assay against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051). Crude extract at a concentration of 6.8 μg/ml showed good anti-bacterial activity against the bacterial strain S. aureus compared with the activity of the standard Azithromycin at a concentration of 5.0 μg/ml. Active crude extract was partitioned to obtain methanol,
Anticancer activity of Trichoderma harzianum extract against NCI-H292 lung cancer cells.
(International Research Symposium on Pure and Applied Sciences, 2017 Faculty of Science, University of Kelaniya, Sri Lanka., 2017) Sinthujah, S.; Samarakoon, S. R.; Tennakoon, K. H.; Attanayake, R. N.; Weerakoon, G.; Gunasegara, D. S.; Paranagama, P. A.
Cancer is one of the leading causes of death worldwide. Chemotherapy has been the choice of cancer treatment for many years however, it can also affect normal cells and create many undesirable side effects and have the potential to develop resistance. Therefore, investigators must reassess their approach to translate discovery research into greater clinical success and impact aiming to find novel compounds. Endolichenic fungi (ELF) are potential source of producing many bioactive compounds. Preparations of ELFs extracts are commonly used to search for anticancer activity. Based on the fact that fungal extracts provide evidence to develop anticancer drugs, this study was conducted to evaluate the anticancer activity of an ELF, Trichoderma harzianum, (strain No: MF029755) extract against NCI-H292 lung cancer cells. Organ specific in-vitro assays are imperative in large scale screening of natural products with useful clinical activity. Among many such assays, sulforhodamine B (SRB) assay employs a protein binding aminoxanthene dye, to provide a quantitative analysis of viable cells in a culture following the introduction of the compound. Preliminary investigations revealed that crude ethylacetate extract of an endolichenic fungus, T. harzianum, and chloroform fractions of crude extract (12.5 mgL-1, 25 mgL-1, 50 mgL-1, 100 mgL-1 and 200 mgL-1) obtained by partition were positive for the SRB assay. IC50 values of crude extract and the chloroform fraction were 68.48 mgL-1 and 38.44 mgL-1 respectively. The chloroform fraction was chromatographed over silica gel column to obtain seven fractions. Cytotoxicity of the seven fractions obtained from the crude extract of the fungus was determined using SRB assay against lung cancer cell line NCI-H292 following standard protocols. The cell suspension in Dulbecco’s Modified Eagle Medium (DMEM) was aliquoted into 96-well plate. After incubation cells were treated with two concentrations (100 mgL-1 and 200 mgL-1) of fractions obtained by column chromatography. SRB dye was added to each well and acetic acid was used to remove unbound dye. Absorbance was measured at 540 nm using microplate reader. Survival percentage of the cells was calculated. If no viable cells present pink color of the medium turns colorless. In the current assay control wells and 1st fraction remained pink and all the other treatments turned pink into colorless. Seventh fraction showed the highest activity and further purification, SRB assays and structure elucidation will be carried out.
Assessment of in vitro antioxidant activity and flavonoid content Vernonia cinerea
(4th International Research Symposium on Pure and Applied Sciences, Faculty of Science, University of Kelaniya, Sri Lanka, 2019) Weerasinghe, P.; Abeysinghe, T.; Paranagama, P. A.
Leafy greens are an essential part of a healthy diet. Eating a diet rich in leafy greens can offer numerous health benefits including reduced risk of obesity, heart diseases, high blood pressure and mental decline. In Sri Lanka, Kola kanda or herbal porridge is a traditional breakfast made up of green leaves or herbs. Out of many herbs used for Kola kanda, Vernonia cinerea (Monarakudumbiya) is used to treat various diseases on folklore levels in Sri Lanka. However, no much scientific validation is found for V. cinerea for its medicinal uses. The present study was carried out to access the in vitro antioxidant activities and flavonoid content of leaf, root, stem and flower parts of V. cinerea. In vitro antioxidant potential of methanolic extracts of V. cinerea was evaluated by means of total phenolics by the Folin-Ciocalteu assay and the DPPH radical scavenging assay using standard procedures with slight modifications. The highest phenolic content of 101.80 ± 7.74 mg GAE/g was observed in flowers and the stem having the least phenolic content (71.95 ± 5.72 mg GAE/g). The flavonoid content ranged from 17.67 mg GAE/g to 12.76 mg GAE/g in all parts. DPPH radical scavenging activity of extracts of V. cinerea increased in a dose dependent manner with IC50 values ranging from 800 μg/mL to 2350 μg/mL in different parts. The leafy green, V. cinerea under this study contains a significant amount of essential phytochemicals which possess anti-oxidant properties supporting its use as a medicinal herb.
Assessment of the antioxidant, anti-diabetic, and anti-obesity activities of a Sri Lankan "spice" mixture at different storage conditions used for treating obesity
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Jayawantha, M. L. D. C.; Manuha, M. I.; Paranagama, P. A.
Obesity, diabetes, and hypertension have become prevalent health issues in Asian countries, affecting individuals at young age. Systemic oxidative stress and adipose tissue contribute significantly to the development of insulin resistance associated with obesity and type II diabetes. Due to the possible toxicity and carcinogenicity of synthetic antioxidants, there is a global need to discover safe antioxidants to use as food additives. To address these concerns, clinical trials were conducted in Sri Lanka using a spice mixture consisting of commonly used spices in Sri Lankan cuisine: Cinnamomum zeylanicum, Cuminum cyminum, Piper nigrum, Murraya koenigii, and Allium sativum. This study aimed to evaluate the antioxidant, anti-obesity, and anti-diabetic potential of a methanol extract derived from this Sri Lankan spice mixture, as well as to evaluate the above activity properties of five glass bottle samples stored at different storage conditions: the positive control (STD), the initial spice mixture sample (INT), the sample after three months at room temperature (RT), the sample after three months in a dark environment (BLK), and the sample after three months in a refrigerator (COOL). The IC50 value of the methanol extract of INT demonstrated significant antioxidant activity (0.08 ± 0.03x10-2 mg/mL, p < 0.05) in the DPPH assay compared to the positive control, BHT (0.07 ± 0.05x10-2 mg/mL). Similarly, the lower IC50 value of INT indicated higher antioxidant activity (0.17 ± 0.14x10-1 mg/mL) in the ABTS assay when compared to the positive control BHT (0.36 ± 0.15x10-1 mg/mL). In both assays, the IC50 values followed the order of INT < COOL < BLK < RT, signifying decreasing antioxidant activity in the same order. The methanol extract samples (1.0 mg/mL) in the FRAP assay showed increasing relative % reducing power in the order of RT < BLK < COOL < INT, with values of 30.37%, 32.14%, 32.74%, and 37.05%, respectively, which was lower compared to the positive control (BHT). The α-amylase inhibition assay revealed that the IC50 of the methanol extract of the INT was 0.29 ± 0.21x10-1 mg/mL compared to the positive control Acarbose (IC50, 0.06 ± 0.02x10-1 mg/ml). The IC50 of the methanol extract of INT in the antilipase assay was 0.14 ± 0.05x10-1 mg/mL, while the positive control (Orlistat) had an IC50 of 0.09 ± 0.01x10-1 mg/mL. These results revealed that the IC50 value increased in the order of INT < COOL < BLK < RT, when each glass bottle sample was stored after three months according to their distinct conditions, suggesting a negative correlation between assay activity with light intensity, temperature, and storage time. Overall, the study concluded that the spice mixture used for reducing obesity exhibits significant antioxidant potential. Additionally, the results from the anti-lipase and α-amylase inhibition assays suggest that the bioactive constituents present in the methanol extract have the potential to inhibit lipase and α-amylase enzymes, providing antiobesity and anti-diabetic benefits.
Bioactive properties and metabolite profile of an endolichenic fungus, Hypoxylon lividipigmentum
(Faculty of Science, University of Kelaniya, Sri Lanka., 2021) Weerasinghe, W. R. H.; Shevkar, C. D.; Silva, R. S. De; Attanayake, R. N.; Weerakoon, G.; Kate, A. S.; Kalia, K.; Paranagama, P. A.
Endolichenic fungi (ELF) serve as a novel source of secondary metabolites. Hypoxylon lividipigmentum is an ELF isolated from the lichen Opegrapha medusulina, collected from mangrove plant Xylocarpus granatum from Negombo lagoon, Sri Lanka. The fungus was identified to the species level using morphological and DNA barcoding techniques. Ethyl acetate extract of the fungus was subjected to in vitro assays to determine antioxidant, anti-inflammatory, tyrosinase inhibitory and antibacterial potency. Liquid Chromatography-Mass Spectrometry (LCMS) dereplication was conducted on the crude extract in order to detect the secondary metabolites present. The extract reported a IC50 value of 18.34±1.37 µg/ml on par with the positive control BHT, in DPPH radical scavenging assay. It also exhibited moderate anti-inflammatory activity with an IC50 value of 81.08±1.05 μg/ml. Tyrosinase inhibitory activity was fairly comparable with an IC50 value of 121.20±2.55 μg/ml. Agar well diffusion assay was conducted to determine antibacterial activity against aerobic bacterial species Escherichia coli, Bacillus subtilis, Staphylococcus aureus and the anaerobic bacterial species Streptococcus mutans. Suppression of growth was shown only against B. subtilis. Five major mass peaks were observed during the study of LCMS profile of the extract. After a thorough dereplication process, two masses could be presumed to be from novel scaffolds. Since none of the mass peaks could be dereplicated within the species or genus level, it could be speculated that the chemical profile of Hypoxylon lividipigmentum was previously poorly explored in literature thus making it an interesting organism to study further for novel metabolites.
Bioassay-guided isolation and antioxidant properties of phytochemicals present in methanolic extract of Halosarcia indica from salt marsh ecosystems in Sri Lanka
(Faculty of Science, University of Kelaniya Sri Lanka, 2024) Siriwardhana, U. L. I. G.; Perera, D.; Attanayake, R. N.; Paranagama, P. A.
Halosarcia indica, belonging to the family Amaranthaceae, is a halophyte that exhibits unique adaptations to saline environments. Halophytes contain various bioactive chemicals, including plant polyphenols, which have garnered interest due to their antioxidant properties. Isolation of pure compounds responsible for the antioxidant activity is also very important in nutraceuticals, cosmetics, and pharmaceutical industries. The aim of the present study was to isolate and identify bioactive compounds from H. indica samples collected from Sri Lanka. Fresh plant materials were collected from salt marshes in Mannar district, Sri Lanka. Phytochemicals from finely powdered freeze-dried areal parts of H. indica were extracted using cold extraction and sequentially partitioned with methanol, dichloromethane (DCM), and hexane. The antioxidant activity of these fractions was evaluated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay, and the Ferric Reducing Antioxidant Power (FRAP) assay, with butylated hydroxytoluene (BHT) serving as the positive control. The total phenolic content (TPC) was assessed using the Folin-Ciocalteu method, while the determination of total flavonoid content (TFC) was conducted through the aluminum chloride assay. Additionally, the total tannin content (TTC) was examined using the vanillin/HCl method. Finally, methanolic and DCM extractions were subjected to column chromatography, thin layer chromatography, and preparative thin layer chromatography techniques to isolate compounds responsible for antioxidant activity. The methanolic fraction exhibited the highest radical scavenging activity, with IC50 values of 69.78 ± 3.48 µg mL-1 in the DPPH assay and 81.38 ± 1.89 µg mL-1 in the ABTS assay, while the positive control BHT showed IC50 values of 20.91 ± 0.48 µg mL-1 and 22.93 ± 0.65 µg mL-1, respectively. In the FRAP assay, the methanolic fraction presented higher mean absorbance values of 0.598 ± 1.81 compared to that of the BHT 0.821 ± 1.82. Methanolic extracts of the H. indica had higher TPC (184.77 ± 3.21 mg GAE/g), TFC (190.69 ± 5.84 mg CE/g), and TTC (201.33 ± 4.21 mg GAE/g) values compared with DCM and hexane extracts. Methanolic extracts showed higher antioxidant activity followed by DCM and hexane extracts. Therefore, two pure compounds with antioxidant activities were isolated in methanolic (IC50 - 78.69 ± 1.24 µg mL-1) and DCM extracts (IC50 - 192.84 ± 1.65 µg mL-1) respectively. The study identified two antioxidant compounds from H. indica extracts, with methanolic extracts showing the highest activity. Further analysis will be done using spectroscopic methods. These two compounds will be identified using spectroscopic techniques, including 1H NMR, 13C NMR, HPLC, and mass spectrometry.
Biodeterioration of low density polyethylene by Montagnula scabiosae
(Faculty of Science, University of Kelaniya, Sri Lanka, 2021) Perera, T. W. N. K.; Attanayake, R. N.; Paranagama, P. A.
Endolichenic Fungi (ELF) are a group of fungi adapted to live within lichen thalli. Present study investigates the ability of Montagnula scabiosae, an ELF, for biodeterioration of low density polyethylene (LDPE) by determining the activity of three extracellular fungal enzymes, reported to have potential roles in depolymerizing polyethylene, using enzymatic assays. Liquid medium screening assays, using four potato dextrose agar (PDA) plugs (0.5 cm diameter) of ELF cultures, inoculated into Erlenmeyer flasks containing sterile mineral salt medium broth (MSMB). Eight sterile LDPE strips (10 cm x 3 cm) were added into each flask and incubated at room temperature (28 ± 2°C) for 21 days. MSMB flasks with plain PDA plugs and sterile LDPE strips were used as the controls. Solid medium biodeterioration studies also were conducted using three PDA plugs of the ELF cultures inoculated into mineral salt medium agar (MSMA) plates. Eight sterile LDPE strips were aseptically placed on the surface of each MSMA plate and incubated at room temperature for 45 days. MSMA plates with plain PDA plugs and sterile LDPE strips were used as the controls. Both liquid and solid media assays were carried out in four replicates. LDPE biodeterioration was analyzed based on reductions in weights and tensile properties, changes in peaks of Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectra, changes in carbonyl index (CI) and Scanning Electron Microscopic (SEM) images. A distinguishable LDPE deterioration ability was shown by Montagnula scabiosae, isolated from the host lichen Pyrenocarp sp. Results showed that isolate reduced weights of strips by 13.22% in liquid medium and by 12.06% in solid medium. Tensile strength of liquid medium treated strips, reduced by 6.64% and solid medium treated strips, by 3.00%. The control LDPE strips on ATR-FTIR analysis showed four prominent peaks at 2916 cm-1, 2848 cm-1, 1463 cm-1 and 720 cm-1 and a few minor peaks. Changes visible in the ATR-FTIR spectra of treated strips, manifested new peaks, fluctuations in percentage transmittance and deletions of existing peaks. CI value of the LDPE strips in liquid medium were found to be increased by 2.70% and in solid medium increment was 2.31%. SEM images of treated strips showed erosion zones on LDPE matrix. Qualitative enzymatic assays using Agar plate-based screening methods, indicated the presence of laccase and peroxidases, in this ELF isolate. In the quantitative enzymatic assays, M. scabiosae showed laccase, lignin peroxidase and manganese peroxidase activities as 1.07×10-7 kat L-1, 1.22×10-8 kat L-1 and 2.10×10-8 kat L-1 respectively. Remarkable LDPE deterioration ability shown by this ELF isolate indicates that it has a high potential in degrading LDPE waste.
Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea
(Journal of Chemistry, 2022) Weerasinghe, R. H.; Shevkar, C. D.,; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.
Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.
Bioprospecting of an Endolichenic Fungus Phanerochaete sordida Isolated from Mangrove-Associated Lichen Bactrospora myriadea
(Journal of Chemistry,, 2022) Weerasinghe, R. H.; Shevkar, C. D.; Maduranga, K.; Pandey, K. H.; Attanayake, R. N.; Kate, A. S.; Weerakoon, G.; Behera, S. K.; Kalia, K. S.; Paranagama, P. A.
Bioassay-guided fractionation of the ethyl acetate extract of Phanerochaete sordida, an endolichenic fungus (ELF) isolated from the host lichen Bactrospora myriadea, collected from Negombo lagoon, Sri Lanka, led to the isolation of a bioactive compound. Following the identification of the fungus using morphological and DNA barcoding techniques, the pure compound was isolated using column chromatography, preparative TLC, and semipreparative HPLC. The structure elucidation was carried out using IR, HR-ESI-MS and 1H, 13C & 2D NMR spectroscopic methods. The in vitro bioassays conducted revealed that compound 1 has a high antioxidant activity with ABTS•+ (IC5058:91 ± 1:35 μM), moderate anti-inflammatory activity (IC50254:79 ± 1:41 μM), comparable antibacterial activity against the oral-bacterial strain Streptococcus mutans (MIC 898.79 μM and MLC 1797.58μM), moderate tyrosinase inhibition (IC501713:69 ± 8:65 μM), and moderate cytotoxicity against oral cancer (IC5013:65 ± 0:02 μM), in comparison with respective positive controls. The in silico experiments conducted for tyrosinase inhibition and cytotoxicity using Schrödinger revealed results in line with the in vitro results, thus confirming the bioactivities. The molecule also satisfies the key features of drug likeliness according to pharmacokinetic studies.
Chemical characterization of endolichenic fungi, Talaromyces pinophilus residing in the lichen, Arthonia sp. collected from mangroves of Sri Lanka
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Shevkar, C.; Armarkar, A.; Maduranga, K.; Weerasinghe, R.; Pandey, K.; Kalia, K.; Paranagama, P. A.; Kate, A.
Lichens are one of the interesting symbiotic organisms comprising of algae, fungi and other microbiota. The complex miniature ecosystem of lichen provides a competitive environment to endolichenic fungi, which makes it suitable for the production of structurally diverse molecules. Mangroves of Sri Lanka are well-known for the presence of various type of lichens and hence, in this study, 32 specimens of lichen were collected from mangroves of Puttalam Lagoon of Sri Lanka. LCMS based dereplication study was carried out for the isolated 70 cultures of endolichenic fungi from lichens. Talaromin B and Ergochrome BB were present in the ethyl acetate extract of Talaromyces pinophilus along with probable novel compounds having m/z [M+H]+ 663.2321, 778.4128, 401.3029 with “no hit” in Dictionary of Natural Products (DNP). The mentioned extract showed promising anti-cancer activity having IC50 of 50.32 μg/mL tested against MCF-7 cell line (Human breast cancer cell line) by Alamar blue assay. The extract was then scaled up for further isolation of masses showing no hits in DNP. Here, 2 known and 2 new compounds were isolated with 95-98% purity from the scaled up fermentation batch including [NIPER20099_83_7/30_1] Peniazaphilin B and a macrocyclicpolyester [NIPER20099_83_7/30_2] (15G25α). Structural elucidation of two novel molecules is in process. All four molecules were tested in vitro against MCF-7 cell line and only one molecule [NIPER20099_83_7/30_3] having molecular weight 560.2045 showed promising activity with the IC50 value of 22.15μM. The study suggest Talaromyces pinophilus might serve as a source of novel cytotoxic hit. Investigating secondary metabolites of the endolichenic fungi might help us to unravel the complex ecosystems of lichen and help us understand its further therapeutic benefits.
Detailed evaluation of methylated and acetylated products of cinnamon leaf oil against Aedes aegypti
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Medagedara, H. K.; Paranagama, P. A.; Gunaratna, Medha J.
Aedes aegypti is a known vector of several viruses including Yellow fever, Dengue, Chikungunya and Zika virus. Two potential Aedes aegypti mosquito repellents; methyl eugenol and acetyl eugenol were semi-synthesized using eugenol extracted from cinnamon leaf, which is the main constituent in cinnamon leaf as well as in other plant leaf extracts like maduruthala and clove oil. Methyl eugenol was semi-synthesized using methyl iodide as a methylation precursor and acetyl eugenol was semi-synthesized using acetic anhydride, triethylamine base and catalytic amount of DMAP. Synthesis reactions were monitored using TLC and products were purified using flash silica columns. Formations of desired products were confirmed using GC-MS analysis. A diluted series (0.6-8 mg/ mL) was prepared for these semi-synthesized compounds and for the cinnamon leaf extract using ethanol and tested separately for their mosquito repellency using arm-in-cage tests followed by standard WHO methods. Experiments were performed on twenty volunteers in triplicates. Effective dose (ED99) for each repellent was calculated using beta regression analysis. Current study suggests that all compounds tested were effective mosquito repellents. Their ED99 varied as; cinnamon leaf extract (5.80) > methyl eugenol (5.41) > acetyl eugenol (5.13). Acetyl eugenol could be a potential safe repellent as its ED99 was achieved at a lower concentration and due to the absence of any side effects on skin when applied. Both maduruthala and clove oil contained significant contents of eugenol and clove oil had slight amounts of acetyl eugenol which could be extracted to synthesize repellent compounds or use directly in future.
Development of a herbal fairness cream using four medicinal plants
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Wimalasekera, H. N.; Paranagama, P. A.; Harapathdeniya, S.
There are reports in the public domain which state that artificially synthesized fairness creams have shown side effects on humans. The purpose of a fairness cream is to get a fairer skin by blocking harmful Ultraviolet (UV) rays which can activate the melanin biosynthesis in the body. When there is an excess production of melanin, it can lead to several hyperpigmentation diseases and finally result in a darker or tanned skin. This excess melanin formation can be inhibited by avoiding exposure to UV light, using tyrosinase inhibitors and anti-oxidants, which can reduce melanin proliferation. In this research study, a herbal fairness cream was prepared using four traditional medicinal plants found in Sri Lanka to inhibit excess melanin production in the body. The four plants chosen for this study were, Hemidesmus indicus (Heen Iramusu), Ixora coccinea (Rathmal), Hibiscus rosa sinensis (Pokuru Wadamal) and Citrus reticulata (Heen Naran). The plant extracts and the cream were separately subjected to anti-oxidant (DPPH and ABTS) assays and anti-tyrosinase assay. In addition, the anti-bacterial activity of this cream was studied. The methanol extracts of these four plant species displayed a high antioxidant activity and a significant tyrosinase inhibitory activity. The herbal cream was formulated using equal proportions of these extracts. Anti-tyrosinase studies of the cream revealed that it has a good anti-tyrosinase activity compared to the positive standard, Kojic acid. In addition, anti-bacterial studies revealed that the cream can inhibit bacteria Staphylococcus aureus and Bacillus subtilis. The ABTS assay showed that this polyherbal cream can act as an excellent antioxidant (IC50: 0.54±3.86) with respect to the control BHT (IC50:9.99±1.76). The cream was prepared using 100% natural ingredients which helped to enhance the therapeutic benefits of this cream. Thus, this cream can be used as a substitute for the synthetic fairness creams already available in the market.
Development of herbal mosquito coil formulations using mosquito repellent plant materials
(Faculty of Science, University of Kelaniya Sri Lanka, 2022) Rathnayaka, T. M. T. N.; Paranagama, P. A.
With the increasing cases of mosquito-borne diseases such as dengue, malaria, yellow fever, Japanese encephalitis, and chikungunya, it has been a global concern to control the transmission of these diseases by controlling the mosquito population and protecting from mosquito bites using repellent products. Currently, more studies are being focused on developing safe, efficient, and eco-friendly herbal mosquito repellents by combining herbal plant materials due to the health and environmental concerns of overuse of chemical repellents for a long period. Since the studies on the mosquito repellent activity of plants seem to be quite low, through this study, the collaborating mosquito repellent activity of several selected plants is explored and assessed. The objectives of this study are to develop herbal mosquito coil formulations using mosquito repellent plant materials and to evaluate the bio-efficacy of developed coil formulations using preliminary laboratory studies and field studies. In this study, Piper betel (Betel) leaves, Azadirachta indica (Neem) leaves, Ocimum tenuiflorum (Maduruthala) leaves, Cinnamomum zeylanicum (Cinnamon) leaf oil, and Cymbopogon nardus (Citronella) oil were assessed for their potential mosquito repellent activity. This study was approved by the Ethics Review Committee, University of Kelaniya, Sri Lanka, and ethical clearance was obtained before commencing the study. All of the bio-efficacy tests were conducted according to the World Health Organization (WHO) regulations and guidelines for efficacy testing of household insecticide products. In field studies, a total of 12 developed coil formulations were tested in six different locations using adult human volunteers against a free-flying mosquito population, and efficacy was assessed in terms of biting inhibition. Preliminary laboratory studies were conducted in a glass chamber (20 cm х 20 cm х 20 cm in size) using sucrose-fed female Aedes aegypti mosquitoes (2-7 days old) with three replicates for each coil formulation. The number of knocked-down mosquitoes was counted at regular intervals for 60 minutes, and knock-down time (KDT50 & KDT90) and mortality of the mosquitoes were recorded after post-exposure to the smoke. The results from the field studies showed that five coil formulations are effective in repelling mosquitoes for 6 hours, with low to average smoke visibility, good- satisfactory odour, and no irritability. From the laboratory studies, the same coil formulations could be identified as having fast mean knock-down times and higher 24-hour post-mortality values above 80%. Therefore, through this study, it can be concluded that these coil formulations with neem: maduruthala: betel: citronella: cinnamon ratios, respectively, 1:3:1:1:1, 1:1:3:1:1, 1:1:1:3:1, and 1:1:1:1:3 are more efficient in repelling mosquitoes compared to other formulations and the plant materials used in this study have components capable of repelling mosquitoes individually or collaboratively. Further laboratory studies and field studies of a larger study population at different geographical locations with a higher number of replicates are required for the commercialization of these mosquito coil formulations as herbal mosquito repellents.
Evaluation of phytochemical profile and in -vitro antioxidant activity Suaeda maritima
(Faculty of Science, University of Kelaniya Sri Lanka, 2023) Pathirana, K. P. S. S.; Herath, H. M. N. P.; Perera, D.; Attanayake, R. N.; Paranagama, P. A.
Suaeda maritima, a halophytic plant species from the family Amaranthaceae, possesses unique adaptations that enable it to thrive in high-salt environments. Halophytes contain various bioactive chemicals, including plant polyphenols, which have garnered interest due to their antioxidant properties. This study aims to investigate how the polyphenolic content and antioxidant activity of extracts change across the polar, mid-polar, and non-polar fractions of crude extracts from S. maritima. These extracts are obtained from both the aerial parts and the roots of the plant, which were harvested from Seguwantivu area of Puttalam, Sri Lanka. Each dried and powdered plant part was sequentially extracted into hexane, dichloromethane (DCM), and methanol at ambient temperature using cold maceration. The Antioxidant activity of each concentrated extract was determined by 2,2-diphenyl-1-1-picrylhydrazyl(DPPH) assay, and 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid (ABTS) assay , phenolic content by (TPC) by FolinCiocalteau method, flavonoid content (TFC) by aluminum chloride assay and condense tannin content (CTC) by vanillin/HCl method. Six sequential extracts of S. maritima (from aerial parts and roots) showed antioxidant activity. The results indicated that S. maritima shoot extracts have significant levels of polyphenolic compounds (p=0.05), with methanolic extracts exhibiting the highest TPC (31.55±1.54 mg GAE/g dw), TFC (18.41±0.91 mg CE/g dw) and CTC (3.66±0.19 mg CE/g dw). In contrast, root extracts showed lower polyphenolic content (23.69±5.63 mg CE/g dw, methanol extract) but still possessed appreciable antioxidant activity (IC50, 0.40±0.07×10-1 mg/mL DPPH and IC50, 0.69±0.02×10-1 mg/mL ABTS). The ABTS assays revealed the radical scavenging ability of the extracts, with the methanolic aerial parts extract exhibiting the most potent antioxidant activity (IC50, 0.17±0.01×10-1 mg/mL) exhibiting it has more water-soluble antioxidants. Overall, Solvent extracts of the S. maritima possess antioxidant properties at varying levels. The fraction of polar compounds in the areal parts of S. maritima contains a relatively high amount of phenolic compounds and potent antioxidant properties. Moreover, identifying antioxidant properties from halophytes could contribute significantly to developing of natural antioxidants for combating oxidative stress-related diseases.
Exploring bioactive compounds in the endolichenic fungus, Xylaria feejeensis, inhabiting the lichen, Graphis librata, collected from Negombo lagoon, Sri Lanka
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Gunawardhana, M. H. A. Y.; Wickramarachchi, P. A. S. R.; Weerasinghe, W. R. H.; Paranagama, P. A.
The accelerating pharmaceutical problem of bacteria growing resistant to existing antibiotics forces the scientific community to search for new antibacterial compounds for antibiotic drug development. Literature reveals that Sri Lankan mangrove inhabiting endolichenic fungal (ELF) population is rich in many such bioactive compounds. Previously isolated and cultured ELF, Xylaria feejeensis; from the lichen Graphis librata inhabiting in the mangrove plant Rhizophora mucronata was cultured on 60 large potato dextrose agar medium containing petri dishes and incubated for 14 days at room temperature. After extracting secondary metabolites to ethyl acetate (EA) the resulted crude extract was tested for its antibacterial and antioxidant activity. EA crude extract showed negligible antioxidant activity in DPPH radical scavenging assay hence this assay was not proceeded further. The activity of crude extract (5 mg/ml) against Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923) and Bacillus subtilis (ATCC6051) was tested using agar well diffusion antibacterial assay. An inhibition zone diameter of 1.9 cm, 2.2 cm, and 2.2 cm against three bacterial strains with Azithromycin positive control (5 mg/ml) 2.2 cm, 2.2 cm and 2.3 cm was observed respectively. By partitioning of EA crude extract resulted two antibacterial active hexane and chloroform fractions and one antibacterial inactive 60% methanol in water fraction. Hexane fraction showed the highest antibacterial activity with inhibition diameter zone of 2.6 cm against E.coli with 2.6 cm diameter of inhibition zone for positive control. Further purification of hexane fraction was performed by normal phase column chromatography and 4 fractions were resulted. The highest polar fraction showed 1.4, 2.1 and 1.7 cm diameter inhibition zones of growth of E.coli, S. aureus and B. subtilis comparable with 2.2 cm Azithromycin. The other 3 fractions showed no antibacterial activity against these bacterial strains. Results suggest that further purification of active fraction and structure elucidation might result in new antibiotic lead compounds.
Identification of Novel Bioactive Compounds, Neurosporalol 1 and 2 from an Endolichenic Fungus, Neurospora ugadawe Inhabited in the Lichen Host, Graphis tsunodae Zahlbr. from Mangrove Ecosystem in Puttalam Lagoon, Sri Lanka
(ASSIIAN JOURNAL OF CHEMIISSTRY, 2021) Maduranga, H.; Weerasinghe, W.; Attanayake, R.; Santhirasegaram, S.; Shevkar, C.; Kate, A.; Weerakoon, G.; Samanthi, K.; Kalia, K.; Paranagama, P. A.
Endolichenic fungi (ELF) have been recognized as a promising source of bioactive secondary metabolites. Sri Lanka, a biodiversity hotspot, harbours a remarkable diversity in mangrove ecosystems. In the present study, endolichenic fungi obtained from mangrove lichens of Puttalam lagoon were studied for their secondary metabolites. An endolichenic fungus, Neurospora ugadawe, isolated from the lichen host Graphis tsunodae Zahlbr. was identified up to the species level. Secondary metabolites of N. ugadawe were extracted into ethyl acetate and crude extract showed promising antioxidant and anti-inflammatory activities. Bioassay guided separation on silica gel column chromatography and preparative TLC were performed to isolate the bioactive pure compounds. Two novel bioactive pure compounds were identified as Neurosporalol 1 and Neurosporalol 2. Neurosporalol 1 showed the highest antioxidant activity compared with the positive control BHT. Further, this compound showed very low anti-inflammatory activity compared to that of the positive control aspirin. Neurosporalol 2 showed comparable antioxidant and moderate anti-inflammatory activities.
Identification of Novel Bioactive Compounds, Neurosporalol 1 and 2 from anEndolichenic Fungus, Neurospora ugadawe Inhabited in the Lichen Host,Graphis tsunodae Zahlbr. from Mangrove Ecosystem in Puttalam Lagoon, Sri Lanka
(Asian Journal of Chemistry, 2021) Maduranga, H.; Weerasinghe, W.; Attanayake, R.; Santhirasegaram, S.; Shevkar, C.; Kate, A.S.; Weerakoon, G.; Samanthi, K.; Kalia, K.; Paranagama, P. A.
Endolichenic fungi (ELF) have been recognized as a promising source of bioactive secondary metabolites. Sri Lanka, a biodiversity hotspot, harbours a remarkable diversity in mangrove ecosystems. In the present study, endolichenic fungi obtained from mangrove lichens of Puttalam lagoon were studied for their secondary metabolites. An endolichenic fungus, Neurospora ugadawe, isolated from the lichen host Graphis tsunodae Zahlbr. was identified up to the species level. Secondary metabolites of N. ugadawe were extracted into ethyl acetate and crude extract showed promising antioxidant and anti-inflammatory activities. Bioassay guided separation on silica gel column chromatography and preparative TLC were performed to isolate the bioactive pure compounds. Two novel bioactive pure compounds were identified as Neurosporalol 1 and Neurosporalol 2. Neurosporalol 1 showed the highest antioxidant activity compared with the positive control BHT. Further, this compound showed very low anti-inflammatory activity compared to that of the positive control aspirin. Neurosporalol 2 showed comparable antioxidant and moderate anti-inflammatory activities.
A Novel Cytotoxic Compound From the Endolichenic Fungus, Xylaria psidii Inhabiting the Lichen, Amandinea medusulina
(Natural Product Communications, 2020) Santhirasegaram, S.; Wickramarachchi, S. R.; Attanayake, R. N.; Weerakoon, G.; Samarakoon, S.; Wijeratne, K.; Paranagama, P. A.
The lichen host, Amandinea medusulina, collected from mangrove habitats in Sri Lanka, and its associated endolichenic fungi were isolated and identified by rDNA-ITS sequence analysis and morphological features. One of the fungal strains frequently isolated from the lichen thalli was identified as Xylaria psidii. This study aimed at the isolation and identification of the cytotoxic compounds present in this fungus. Secondary metabolites of X. psidii were first extracted into ethyl acetate and subsequently subjected to bioassay-guided fractionation to isolate the bioactive compounds. Sulforhodamine B assay against a lung cancer (NCI-H292) cell line was used to determine the differential cytotoxic activity. Bioassay-guided fractionation led to the isolation of an active compound, SS/02/29/08, showing moderate cytotoxicity (IC50 = 27.2 μg/mL). Its structure was elucidated by IR, 1D- and 2D-NMR, and 13C-NMR spectrophotometry and MS, in combination with HRMS, 13C NMR, HSQC, HMBC, and DQF-COSY. The structure of SS/02/29/08 was determined as (Z)-3-{(3-acetyl- 2-hydroxyphenyl) diazenyl}-2,4-dihydroxybenzaldehyde and identified as a new compound. This novel compound has promising differential cytotoxic activity against human lung cancer cell line (NCI-H292).
The OSMAC effect on the production of secondary metabolites by the endolichenic fungus, Curvularia trifolii
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Lekamwasam, C. J.; Paranagama, P. A.; Gunaratna, Medha J.
Fungi are a very diverse group of organisms that can produce a vast number of pharmacologically active secondary metabolites. Fungi also have the ability to produce numerous secondary metabolites from a single strain. In this study, the One Strain Many Compounds (OSMAC) approach was used to observe the biosynthetic potential of endolichenic fungus, Curvularia trifolii isolated from Usnea sp. in Sri Lanka. Under the OSMAC approach, the variation of the metabolic profile of the fungus when cultured in different culture media was investigated. Culturing in Potato Dextrose Broth (PDB) medium resulted in the elution of two peaks in High- Performance Liquid Chromatographic (HPLC) study while four major peaks resulted from Yeast Potato Dextrose Broth (YPDB) medium cultures. Five major peaks eluted from Oatmeal extract broth (OEB) medium cultures and two major peaks from Rice-Yeast Broth (RYB) medium cultures. The bioactivities of crude extracts from each culture media were investigated using antioxidant, antibacterial, and anti-inflammatory assays and analyzed using Graph Pad Prism software. The crude extract of OEB showed a comparatively high antioxidant activity with ABTS radical scavenging assay with an IC50 value of 0.501 ± 0.036 mg mL-1 while the crude extract of YPDB showed a comparatively high anti-inflammatory activity with human red blood cell stabilization assay with an IC50 value of 0.151 ± 0.026 mg mL-1. The crude extract of RYB showed the highest antibacterial activity against Escherichia coli, Staphylococcus aureus, and Bacillus subtilis bacterial strains upon analysis using agar-well diffusion method. The study confirms the variation of metabolic products and associated bioactivities of Curvularia trifolii with the variation of its culture medium.
Polyethylene degradation capability of Schizophyllum commune
(Institute of Chemistry Ceylon Adamantane House, Rajagiriya, Sri Lanka., 2020) Perera, T. W. N. K.; Attanayake, R. N.; Paranagama, P. A.
Tremendous increases in the manufacture and consumption of polyethylene, over recent decades have led to numerous environmental and economic concerns. This is the first comprehensive research on determination of Endolichenic Fungi (ELF), capable of degrading polyethylene substrates. The objective of this study was to identify the ability of various ELF, for the degradation of polyethylene in liquid media. Commercially available Low Density Polyethylene (LDPE) bags were used as the test material. ELF isolates cultured on Potato Dextrose Agar, were introduced into Mineral Salt Medium Broth containing LDPE strips. The isolate, Schizophyllum commune showed a positive growth response in the broth medium. The strips treated with this isolate, were tested for the reduction in dry weight, changes in peaks of Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy, reduction in tensile strain at break and changes in Scanning Electron Microscopic analyses. Results showed that isolate, reduced the weight of LDPE strip by 10.54%, after 21 days of incubation, while control showed 0% reduction. The control LDPE strip on ATR-FTIR analysis showed prominent peaks at 2916 cm-1, 2848 cm-1, 1463 cm-1 and 720 cm-1. Changes visible in sample infrared spectra after 21 days of incubation, were the fluctuations in the percentage transmittance of above peaks and formation of new peaks at 1740 cm-1, 1215 cm-1 and 1046 cm-1. The tensile strain at break, of treated strips, reduced by 27.41%, after 21 days of incubation while control showed 0% reduction. The presence of pits and cavities, in Scanning Electron Micrographic images, suggested the penetration of fungi, into the LDPE matrix, during degradation. The occurrence of several nonuniformly scattered whitened areas and erosion zones, indicated the surface erosion of LDPE strips, in degradation. These changes were not observed in the control LDPE strips. This study provides insight into the role of Schizophyllum commune towards solving the dilemma of polyethylene wastes, through biodegradation.