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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.
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.
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.
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.
Volatiles from host plant brinjal attract the brinjal Fruit and Shoot Borer -Leucinodes orbonalis Guenee
(Journal of Asia-Pacific Entomology, 2021) Nusra, M. S. F.; Udukala, D. N.; Amarasinghe, L. D.; Paranagama, P. A.
Brinjal Fruit and Shoot Borer- Leucinodes orbonalis Guenee is a major insect pest on brinjal- Solanum melongena worldwide. An effective strategy used in developing pest controlling agents is the synergism between insect pheromones and host plant volatiles, which can increase the attraction of insect pest. The present study was aimed at investigating the chemical constituents and attractant effects of the volatiles extracted from different parts of the host plant brinjal on the behavior of adult L. orbonalis. Bioassay using Y-shaped olfactometer revealed that the one-day old virgin female, gravid female and male insects respond positively to the host plant volatiles extracted from fruits, leaves and shoots but not to that of flowers. It was shown that the gravid females were significantly attracted to all three volatiles (p < 0.05). Bioassay using X-shaped olfactometer identified that all three types of insects highly preferred the volatiles from fruits (p < 0.05). Gas chromatography-mass spectrometry analysis of volatiles indicated that brinjal plant produces volatile secondary metabolites, which include 2,2′ -(Ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl) dibenzoate (12.11%), 3,7-dimethylocta-1,6-dien-3-ol (22.38%), Benzyl alcohol (22.9%) and Benzyl alcohol (27.06%) as major constituents from fruits, shoots, leaves and flowers respectively. Responses of insects to the volatiles from host plant in the absence of visual cues direct us to focus on the importance of host plant volatiles to locate the plant. Results of this study emphasize the major role that host plant volatiles play in the attraction of insect pests towards the plant.
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.
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,
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.
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.

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