Macrofilaricidal Activity, Acute and Biochemical Effects of Three Lichen Species Found on Mount Cameroon

Onchocerciasis is a parasitic infection affecting a relatively small population globally but has very devastating pathological outcomes. Ivermectin and recently moxidectin are the only drugs approved for clinical management of the disease, both of which have several limitations. In particular, they are efficacious against microfilariae (microfilaricidal) with no activity against adult worms (nonmacrofilaricidal). Promising anthelmintic activity has been reported in some lichens. This study investigated three lichens, Usnea articulata, Parmotrema tinctorum, and Heterodermia obscurata, found on Mount Cameroon, for potential macrofilaricidal activity. Organic extracts were screened for anti-Onchocerca activity against Onchocerca ochengi isolated from cattle skin using worm motility and MTT formazan assays. Toxicity of highly active extracts was investigated on monkey kidney epithelial (LLCMK2) cells and in BALB/c mice (2000 mg/kg body weight) including effects on liver enzymes. The methanol extract of P. tinctorum (Pammet) was the most active against adult male worms ( I C 50 = 8.1 μg/mL) with the highest selectivity index ( SI = 21.3 ). U. articulata was the most active against the adult female ( I C 50 = 36.3 μg/mL) but had a low SI value (3.4). No mortality and no adverse effects were recorded in the acute toxicity test. These two most active extracts had no significant effect on liver enzymes, alanine aminotransferase, and aspartate ( P values < 0.05), but a high AST : ALT ratio (2.59) for Pammet indicates likely reversible adverse hepatic toxicity. The high macrofilaricidal activity and selectivity of P. tinctorum suggest it is a potential source of new macrofilaricides which should be further investigated to identify its bioactive constituents.

TOXICOLOGICAL AND BEHAVIOR ANALYSES OF A FOLIOSE LICHEN PARMOTREMA TINCTORUM (DELISE EX NYL.) HALE EXTRACT USING ZEBRAFISH (DANIO RERIO) MODEL

Objective: The objective of the study was to evaluate the toxicity and behavior analysis with the extract of foliose lichen Parmotrema tinctorum against the adult zebrafish. Methods: The zebrafishes were exposed to different concentrations of P. tinctorum extract such as 50, 100, and 200 mg/ml for 7 days of exposure, in which the mortality and behavioral responses of zebrafishes were recorded. The standard histopathological examination was conducted with the lichen extracts of P. tinctorum. Results: The results revealed that P. tinctorum extract did not show any prominent behavior abnormalities in zebrafishes even at a high concentration of 200 mg/ml. The extract was found to have dose-dependent toxic to zebrafish and the number of neutrophil cells in the muscle bundles reduced at a high concentration. The results of the inflammatory marker gene expression using polymerase chain reaction results suggested that the dose-dependent suppression of tumor necrosis factor gene by the P. tinctorum lichen extract. Conclusion: Overall, concluded that the extract might contain anti-inflammatory induction properties and further tests are required to prove apoptosis and anticancer activity using other in vivo or in vitro techniques.

Chemical Composition and Antimicrobial Activity of Two Sri Lankan Lichens, Parmotrema rampoddense, and Parmotrema tinctorum against Methicillin-Sensitive and Methicillin-Resistant Staphylococcus aureus

Introduction. Medicinal utility of lichens is ascribed to the presence of various secondary metabolites of low molecular weight and they have been used in traditional medicine including Ayurveda in the treatment of wounds and skin disorders. Despite the urgent need to effectively address the antibiotic resistance worldwide, the discovery of new antibacterial drugs has declined in the recent past. This emphasizes the increasing importance of investigating and developing new classes of antibiotics that can withstand antibiotic resistance. Aims of the study. The present study was conducted to investigate the chemical composition and the antibacterial activity of hexane, ethanol, and aqueous extracts of Parmotrema rampoddense and Parmotrema tinctorum, two lichens collected from Belihuloya, Sri Lanka, against Gram-negative and Gram-positive bacteria including twenty clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Materials and methods. Phytochemical analysis, thin layer chromatography (TLC), and Gas Chromatography Mass Spectrometry (GC-MS) were performed to determine the chemical composition of the two lichens. Hexane, ethanol, and aqueous extracts of both lichens were tested against clinical isolate of Gram-negative and Gram-positive bacteria including twenty clinical isolates of MRSA. Bacterial susceptibility was tested using a disc diffusion assay. Minimum inhibitory concentration (MIC) was determined by a broth microdilution method. Vancomycin was used as the positive control. Results. Alectorialic acid, atranorin, atraric acid, orcinol, and O-orsellinaldehyde were among the secondary metabolites identified by the TLC and GC-MS analysis. None of the lichen extracts were active against Gram-negative bacteria but both lichens showed a concentration-dependent activity against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA. Ethanol extract of P. rampoddense showed the highest activity against MSSA with the MIC, 0.0192 mg/ml, but all MRSA isolates investigated showed MIC between 0.096 and 2.4 mg/ml for the same extract. Conclusion. Both lichens, P. rampoddense and P. tinctorum, represent potentially important sources of future antimicrobial drugs. Further investigation on the ethanol extract of P. rampoddense will enable us to determine the most active phytoconstituents responsible for the activity, their mechanism of action against bacterial pathogens, and also their cytotoxicity against normal cells.

Effect of Isolation Conditions on Diversity of Endolichenic Fungal Communities from a Foliose Lichen, Parmotrema tinctorum

Endolichenic fungi (ELF) are emerging novel bioresources because their diverse secondary metabolites have a wide range of biological activities. Metagenomic analysis of lichen thalli demonstrated that the conventional isolation method of ELF covers a very limited range of ELF, and the development of an advanced isolation method is needed. The influence of four variables were investigated in this study to determine the suitable conditions for the isolation of more diverse ELF from a radially growing foliose lichen, Parmotrema tinctorum. Four variables were tested: age of the thallus, severity of surface-sterilization of the thallus, size of a thallus fragment for the inoculation, and nutrient requirement. In total, 104 species (1885 strains) of ELF were isolated from the five individual thalli of P. tinctorum collected at five different places. Most of the ELF isolates belong to Sordariomycetes. Because each part of lichen thallus (of different age) has unique ELF species, the whole thallus of the foliose lichen is needed to isolate diverse ELF. Moderate sterilization is appropriate for the isolation of diverse ELF. Inoculation of small fragment (1 mm2) of lichen thallus resulted in the isolation of highest diversity of ELF species compared to larger fragments (100 and 25 mm2). Moreover, ELF species isolated from the small thallus fragments covered all ELF taxa detected from the medium and the large fragments in this study. The use of two media—Bold’s basal medium (nutrient poor) and potato dextrose agar (nutrient rich)—supported the isolation of diverse ELF. Among the tested variables, size of thallus fragment more significantly influenced the isolation of diverse ELF than other three factors. Species composition and richness of ELF communities from different lichen thalli differed from each other in this study.

EMBRYOTOXICITY AND TERATOGENIC EFFECTS OF PARMOTREMA TINCTORUM (NYL) HALE ON ZEBRAFISH (DANIO RERIO) AND CYTOTOXIC ACTIVITY OF LICHEN EXTRACT ON HEK293T CELL LINE

Parmotrema tinctorum (Nyl) Hale an edible lichen, used as a spice and flavouring agent for meat and vegetable preparations by ethnic groups in India and Nepal. In Zebrafish embryos and larva, the therapeutic applications of P.tinctorum widely reported. However the teratogenic effects not reported. This study was aimed to examine the toxicity of P.tinctorum on the promising model for toxicity research, the Zebrafish, because their genetic structure is more similar to human beings. Additionally, we have demonstrated a toxic effect of P.tinctorum extract on the human normal cell line (HEK293T) by MTT assay. The methanol extract of P.tinctorum was extracted by Soxhlet and for embryotoxicity and teratogenicity activities, we use different concentrations (50µg/ml – 200µg/ml) of lichen extracts on twelve selected fertilized Zebrafish embryos. The extract did not exhibit any effect on Zebrafish Survival rate, hatching rate, heartbeat and teratogenicity which was similar to control groups. To conclude that, methanol extract of P.tinctorum is found to have a nontoxic property to zebrafish embryo and human normal cell line (HEK293T) and suggests that this might be safe for consumption

Phenolic compounds from the lichen Parmotrema tinctorum

Introduction: The metabolites of lichens concentrated depsidones, depsides, and diphenyl ethers were possessed antibiotic, antifungal, antiviral, antitumor, and anticancer activities. Parmotrema tinctorum (Despr. ex Nyl.) Hale, a species of foliose lichen, is widely distributed in Lam Dong province, Vietnam. Herein, this paper describes the isolation and structure elucidation of seven compounds isolated from this lichen. Methods: Phytochemical investigations of the ethyl acetate extract of the lichen P. tinctorum led to the isolation of seven pure compounds. Their chemical structures were elucidated by extensive HR-ESI-MS and NMR spectroscopic analysis and comparison with previously published data. Results: Seven compounds, namely orcinol (1), orsellinic acid (2), methyl orsellinate (3), methyl heamatomate (4), lecanorin (5), lecanoric acid (6), and gyrophoric acid (7). These compounds were determined the α-glucosidase inhibitory activity. Conclusions: Compound 7 was determined for the first time in P. tinctorum, and this was also the first time these compounds were determined the α-glucosidase inhibitory activity.

Photoprotective Activity of Lichen Extracts and Isolated Compounds in Parmotrema Tinctorum

Lichens are an excellent source for the discovery and development of new photoprotective compounds. However, investigation on their bioactivities remains limited, especially in Vietnam. In this study, six lichen extracts (Parmotrema poolii, P. sancti-angelii, P. tinctorum, Usnea baileyi, U. pulvinulata, U. undulata) and five isolated compounds from commonly distributed species P. tinctorum (methyl β-orcinol carboxylate (1), atranorin (2), lecanorol (3), salazinic acid (4), 1β-acetoxyhopan-3β,22-diol (5)) have been screened in vitro for their photoprotective activity. The photoprotective potential was investigated by determination of their absolute (UV-PF, c, UVA-PF) and relative indexes (SUI, ISP). The structures of isolated compounds were elucidated by spectroscopic methods and confirmed by X-ray diffraction. The results showed that P. poolii, P. sancti-angelii, P. tinctorum, U. baileyi, U. undulata, and compounds of methyl β-orcinol carboxylate (1) and salazinic acid (4) acted as strict UVB filters, and their photoprotective activities are comparable to that of the commercial UVB filter (4-methylbenzylidene camphor). Meanwhile, U. pulvinulata acted as a UVA booster. Compound (3) presented characteristics of a UVA filter, (2) protected well from both UVA and UVB while (5) exhibited a weak photoprotective activity. For the first time, this work successfully extracted several highly potential solar photoprotective compounds from Vietnam's lichens.