Influence of galactosidases on glycosaminoglycan removal Vis-à-Vis opening up of skin matrix to enable complete rehydration.

Enzymatic interventions in animal skin processing are increasingly being considered as safe and benign technology options due to the reduction and replacement of potential harmful chemicals. In this study, galactosidases have been employed for rehydration of preserved skins and hides to improve the process efficiency and minimize hazardous sodium sulfide. The purpose of rehydration is to ensure the skin is hydrated uniformly to facilitate subsequent physico-chemical processes of leather making. Improper rehydration leads to reduction in the quality and value of the leather. The efficacy of the enzymatic process was studied using histological images and scanning electron microscopic analysis. Pollution load changes and the extent of carbohydrate removal were also quantified. The study indicates possibility for substantial reduction in process duration and water input (up to 30%) during rehydration of preserved animal skins when galactosidases are used as rehydration aid without affecting the quality of the leather. Thus use of galactosidases in rehydration ensures uniform accelerated rehydration and provides significant environmental benefits to tanning industry, by reducing harmful substances in subsequent operations.

Alkaline protease for an efficacious rehydration of skin matrix by a novel Bacillus crolab MTCC 5468 in sustainable leather production: a green approach.

OBJECTIVES: The utilization of biotechnology in leather sector has more extensive in modern years; more particular to proteolytic enzymes and employed in several steps of the leather making such as soaking, dehairing, bating, solid waste management etc. The current study evaluates the performance of alkaline protease from Bacillus crolab MTCC 5468 in single soaking of goat skins matrix by comparing with the conventional multiple soaking processes. RESULTS: According to the obtained results, the optimum concentration for maximum rehydration of goat skins was accomplished at 1.0% (v/w) of alkaline protease at duration of 3 h over traditional rehydration method (4-6 h). The moisture level, total protein, chloride content and total organic carbon of enzymatic rehydration was superior to that of conventional rehydration and it was also used to measure the effectiveness of rehydration process. Scanning electron microscopic images of enzymatically processed leather exhibits enhanced opening of fiber bundles and smooth grain surface than conventional method. Furthermore, the alkaline protease treated leather exhibited improved moisture uptake, removal of chlorides and suppleness because of hydrolysis of non-collagenous proteins as indicated by well opened up fiber bundles in histological analysis. CONCLUSIONS: The application of alkaline protease in rehydration operation of leather production confirmed scope for diminishing water quantity around 66.6%, soaking duration at 50%, minimizing use of harmful dehairing chemicals at 50-60%, thereby, eliminating the bating operation during pre-tanning. These outcomes suggest that alkaline protease have potential application in rehydration of skins for immense environmental concerns of leather tanning sectors.

Green processing: minimising harmful substances in leather making.

Conventional leather processing poses serious threat to the environment due to its numerous chemical treatments which include hazardous chemicals such as sodium sulphide and lime. To minimise the pollutants and harmful substances during leather processing, an enzymatic rehydration-dehairing-fibre-opening process has been achieved in shortest possible time compared to conventional process. The physicochemical characteristics of experimental leathers were found to be comparable with those of conventionally processed leathers. The releases of sugar and proteoglycans were found to be in congruence with the scanning electron micrographs and histology. TGA and DSC results ascertained the stability of enzymatically processed leathers. Pollution load in terms of TOC, BOD, COD, and TDS was reduced up to 80% compared to that of the conventional process. The present work provides immense potential for a new approach in leather making with environmental safeguards. Graphical abstract.

Bioactive compound 1,8-Cineole selectively induces G2/M arrest in A431 cells through the upregulation of the p53 signaling pathway and molecular docking studies.

BACKGROUND: Callistemon citrinus has been traditionally known for its medicinal property. Recently, our research group identified 1,8-Cineole, as one of the predominant compound present in the hexane extract (HE-C), whose leaves have potent anticancer activity. HYPOTHESIS/PURPOSE: The present study was designed to isolate 1,8-Cineole from Callistemon citrinus plant and to determine their role in anticancer effects in in vitro using skin carcinoma cells. Moreover, the molecular mechanism of apoptosis and molecular docking studies were also investigated. STUDY DESIGN/METHODS: In vitro cytotoxicity test was performed with HE-C fractionates 1F, 2F, and 3F against A431 and HaCaT cell lines. MTT and AB assay demonstrated that 1F was toxic to cancer cells with no adverse effect to non-malignant cells and it was subjected to 1H NMR, 13C NMR spectroscopy and further characterized by FTIR and GC-MS analysis. On the basis of spectroscopic data, the metabolite was confirmed as 1,8-Cineole. RESULTS: Based on the cytotoxicity results, the well-characterized metabolite 1,8-Cineole was investigated upon to understand the mechanism that caused cancer cell death. In this process, the changes in mitochondrial membrane potential (ΔΨm) were confirmed by Rh-123/DAPI staining; the ultra structure was observed by TEM and quantified by flow cytometric analysis. These results proved that the compound effectively induced the apoptosis and G2/M phase arrest in A431 cells by increasing the expression of p53 and that it was monitored by FACS. Further, the expression of apoptotic proteins, such as Bax/Bcl-2, Cyt-c, caspase-9, and caspase-3 was confirmed by western blot. The molecular docking simulations predicted the hydrophobic interaction between 1,8-cineole with Bcl-2 and PARP1 receptor. CONCLUSIONS: 1,8-Cineole is a potential candidate for skin carcinoma, which is possible by regulating the p53 apoptotic signaling pathway.

Evaluation of in vitro anticancer activity of 1,8-Cineole-containing n-hexane extract of Callistemon citrinus (Curtis) Skeels plant and its apoptotic potential.

Plants are the source of a variety of secondary metabolites, which are often used in the anticancer activity. Discovering new anticancer drug from herbal source is more important in both biological and pharmacological activities. Hence, the objective of this study is to identify the anticancer agent in Callistemon citrinus (Curtis) Skeels (CC) for the treatment of cancer. Very recently we have reported an increased antioxidant activity in the ethanolic and methanolic extracts (EE and ME) of CC but significantly reduced activity (rather increased cytotoxicity), in the n-hexane extract (HE). In this study, the cytotoxicity of all the three solvent extracts was tested against A431, MG-63 and HaCaT cell lines by MTT assay. Interestingly HE has showed increased anti-proliferative effect against the cancer cells but was resisted by non-malignant cells. HPLC and GC-MS analysis revealed the presence of 1,8-Cineole as a predominant compound in HE, the semi-purified bioactive extract. Henceforth, this would be called HE-C and be used for further analyses to understand its mode of action on induced apoptosis/necrosis. Alamar blue assay of HE-C showed cytotoxicity and change in morphological characteristics, which was confirmed by AO/EB staining using fluorescence microscopy, ultra-structural features of apoptosis using SEM and TEM. HE-C induced cell death was also detected by FACS using FITC-labelled Annexin-V and Propidium iodide. ROS generation was monitored using DCF-DA by flow cytometry. The overall results suggested that the selective extract (HE-C) containing 1,8-Cineole has shown potential anti-cancer activity in a dose-dependent manner, and cell death was induced through ROS-mediated apoptosis. Our findings provide an insight into the potential of 1,8-Cineole as a novel drug for killing cancer cells.

Statistical medium optimization of an alkaline protease from Pseudomonas aeruginosa MTCC 10501, its characterization and application in leather processing.

Proteases are shown to have greener mode of application in leather processing for dehairing of goat skins and cow hides. Production of protease by submerged fermentation with potent activity is reported using a new isolate P. aeruginosa MTCC 10501. The production parameters were optimized by statistical methods such as Plackett-Burman and response surface methodology. The optimized production medium contained (g/L); tryptone, 2.5; yeast extract, 3.0; skim milk 30.0; dextrose 1.0; inoculum concentration 4%: initial pH 6.0; incubation temperature 30 degrees C and optimum production at 48 h with protease activity of 7.6 U/mL. The protease had the following characteristics: pH optima, 9.0; temperature optima 50 degrees C; pH stability between 5.0-10.0 and temperature stability between 10-40 degrees C. The protease was observed to have high potential for dehairing of goat skins in the pre- tanning process comparable to that of the chemical process as evidenced by histology. The method offers cleaner processing using enzyme only instead of toxic chemicals in the pre-tanning process of leather manufacture.