Pre-treatment of extracellular water soluble pigmented secondary metabolites of marine imperfect fungus protects HDF cells from UVB induced oxidative stress.

The present study explores the UVB-induced oxidative stress protective efficacy of the pigmented fungal metabolite (identified as DHICA: 5,6-dihydroxyindole-2-carboxylic acid - a melanin precursor) using human dermal fibroblast (HDF) cells. DHICA is a water soluble pigment of the marine Aspergillus nidulans strain SG 28. Preliminary compatibility studies revealed 95% HDF cell viability with 600 µM concentration of DHICA. HDF cells were exposed to UVB irradiation with and without DHICA pre-treatment and the morphological, physiological and molecular level changes were observed accordingly. The results suggested that UVB exposure increases reactive oxygen species (ROS) generation and subsequent DNA damage in HDF cells, whereas DHICA pre-treatment appreciably reduces ROS generation and DNA damage. DHICA pre-treatment upregulates the antioxidant enzyme expressions and reduces the number of cells in the sub-Go/G1 phase. Gene expression analysis of TNF-α, IL-6, COX-2, NF-κB, Bax and Caspase 3 suggested that pre-treatment with DHICA downregulates the above-mentioned genes and simultaneously upregulates Bcl2 expression. In vivo experiments with BALB/c mice suggested that the topical application of DHICA protected mice skin from UVB-induced oxidative stress (which increases the epidermal thickness as evidenced in the skin sectioning). Thus, DHICA application protects the cells from UVB induced oxidative stress and may find applications in sunscreen cosmetic preparations.

Marine fungal DHICA as a UVB protectant: Assessment under in vitro and in vivo conditions.

The present study explores UVB protective role of a melanin precursor namely DHICA (5,6- Dihydroxyindole-2-carboxylic acid) expressed by the marine imperfect fungus Aspergillus nidulans. In brief, A. nidulans grown in a modified growth medium for the period of 5 days at 25 °C under shaking conditions and the extracellular medium free from fungal biomass used for the extraction of DHICA. The extracted DHICA further exposed to partial purification and subjected to UVB protection studies using HaCaT cells and Balb/c mice independently. DHICA obtained in the present study found soluble in water. Experiments on HaCaT cell compatibility revealed nil cell death up to 500 µM concentration of DHICA. UVB protection studies under in vitro conditions emphasizes DHICA significantly protect HaCaT cells from UVB exposure by quenching the generated ROS, reducing cell apoptosis, maintain the cellular integrity and sequentially down regulating the LPO (Lipid peroxidation) and up-regulating the antioxidant enzyme (SOD (Superoxide Dismutase), Catalase, GPx (Glutathione peroxidase)) respectively. Further, experiments on cell cycle arrest analysis, gelatin zymography, and western blot analysis on COX-2 and TNF-alpha, IHC (Immunohistochemistry) on apoptotic markers (Bax, Bcl2) substantiate the protective role of DHICA. Furthermore, in vivo studies on BALB/c mice carried out and compared with the sunscreen cream with sun protective factor (SPF) of 20. Analysis of skin sections of experimental samples revealed that an appreciable reduction in the epidermal thickness of the skin samples of mice pre-exposed to DHICA followed by UVB exposure compared to UVB exposure alone. RT-PCR results on various inflammatory apoptotic markers also suggested that DHICA has UVB protective potential. The observations made in the present study explore the possible application of DHICA alone as a sun-protective agent for skin care.

Exploring the UVB-protective efficacy of melanin precursor extracted from marine imperfect fungus: Featuring characterization and application studies under in vitro conditions.

The present study explores the production, characterization, and application of a potentially stable melanin precursor from marine imperfect non-spore-forming Aspergillus nidulans sp. strain SG 28. Growth of the culture in artificial seawater with galactose and monosodium glutamate, pH at 7.2-7.5, and temperature at 35 ± 0.5 °C exhibits pigment production with the yield of 0.700-0.800 g/L. The characterization studies revealed that the pigment is orange reddish pink in color, soluble in water, insoluble in both polar and non-polar organic solvents, and decolorized when exposed to H2O2, KMnO4, and K2Cr2O7. UV-visible spectrum exhibits maximum absorption peak at 330 nm and mild shoulders at 400 and 500 nm. The pigment displayed both antioxidant and reducing power properties when exposed with ABTS and ferricyanide, respectively. The sun protection factor of the pigment was determined as 9.9. The cytocompatibility of the obtained pigment studied using HaCaT cells revealed that the cells are viable up to 500 µM. Experiments on UVB protection of the pigment using HaCat cells demonstrated the appreciable protective effect by reducing the ROS generation upon UVB exposure. In conclusion, the pigment obtained from the marine imperfect fungus showed structural similarity with DHICA which is confirmed by LC-MS. The presence of metal ions in the growth medium mediates the reaction to proceed towards DHICA formation. The SPF analysis, antioxidant property, and the UVB protection studies authenticate the potential use of pigment for skin care.

Induced oxidative stress management in wounds through phenolic acids engineered fibrous protein: An in vitro assessment using polymorphonuclear (PMN) cells.

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level.