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Hair waste is one of the solid substances rejected by the leather industry. This waste finds its way into the surroundings causing serious environmental pollution. This hair waste may be utilized for effective extraction of keratin, thereby generating value-added products with numerous applications. Thus we focusing on utilizing red sheep’s hair waste for extracting keratin by the application of different chemical treatments such as sodium hydroxide, sodium sulfide, mercaptoethanol, cysteine, sodium metabisulfite with urea (SMB), and SMB with SDS (SMBS). CD spectrum and FTIR results of the keratin samples indicated a predominance of the helical conformation along with β sheets. SDS-PAGE confirmed the molecular weight of the keratin samples to be in the range of 40–60 kDa. DSC and TGA analysis exhibited the extracted keratin to have a higher denaturation temperature (>200 °C) and thermal stability. The keratin samples obtained using varied chemical treatments were compared in terms of yield, protein content, and cost-effectiveness, and the sample obtained using SMBS was preferred for in vitro studies. It is indicated that keratin extracted using SMBS effectively involved for fibroblast cell growth. Thus, we suggest that these keratin could produce biomaterials that can serve as a valuable material for biomedical applications.

Hybrid silver (Ag)–gold (Au) nanoparticles (NPs) with different sizes and compositions were synthesized. Ag/Au alloy and Ag@Au core–shell type NPs were prepared from Ag and Au with various ratios using the COCO gemini surfactant, 1,6-bis (N,N-hexadecyldimethylammonium) adipate (COCOGS), 16-6-16 as a stabilizer. The formation of the Ag/Au alloy and Ag@Au core–shell was confirmed by UV-visible absorption spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray spectroscopy (EDX) and selected area electron diffraction (SAED) patterns. Depending on the composition of the Ag/Au alloy NPs, the λmax values varied from 408 nm to 525 nm. FTIR measurements were used to evaluate the adsorption of the COCO gemini surfactant (16-6-16) on the Ag/Au alloy and Ag@Au core–shell surface. In this present work, we study how to achieve the stability and activity of the COCO gemini surfactant (16-6-16) capped Ag/Au alloy and Ag@Au core–shell NPs for developing novel anti-cancer agents by evaluating their potentials in the Hep-2 cell line model. Thus the developed core–shell NPs were possibly involved in inducing cytotoxicity followed by inhibition of cell proliferation to the cancer cells with apoptosis induction. The developed core–shell NPs might serve as highly applicable agents in the development of next-generation cancer chemotherapeutic agents.

Intensifying researches in leather sector are focusing on eco-benign tanning chemicals with less environmental impact processing route. The present study aims to design an eco-acceptable shoe upper leather process that could potentially eliminate use of chrome from the leather industry. Possible conversion of Cr3+ to Cr6+ in suitable oxidant environment during leather manufacture is always a threat to the ecosystem. Water load for leather processing is very high, also becoming a precious resource that needs to be reduced for industrial applications. Further without water usage for a process, generation of wastewater could be averted. With this context, a combination tanning using wattle and aluminium has been developed in a pickle-less and waterless medium. The experimental leather possesses a shrinkage temperature of 84 °C with a fully loaded compact fibre structure indicating that tannin penetration is not affected in waterless medium. Experimental leathers exhibit better physical strength and comparable organoleptic characteristics. The process enjoys environmental benefits as it shows significant pollution load reduction in Biological Oxygen Demand (BOD), chemical oxygen demand (COD), Total Dissolved Solids (TDS) by 88, 89, and 97%, respectively. Hence, a viable option is made available to manufacture eco-acceptable leather without water usage and chrome salts in tanning.

Bioactive compounds are extracted from mekmeko and characterized for antioxidant, phytochemical, and antibacterial activities. The phytochemical studies of the extracts revealed the presence of condensed tannins, flavonoid, and phenolic compounds. Radical scavenging activity of the extract demonstrates that the ethanol extract has more significant antioxidant activity than the aqueous extract. Ethanolic extract of mekmeko is more effective against gram-positive bacteria compared to gram-negative. A Box–Behnken-design was used to study the response pattern and to determine the optimum combination of variables for maximization of extraction yield. Experiments were carried out to evaluate the four independent variable effects (particle size, temperature, time, and solid–solvent-ratio) on the maximum extraction yield of bioactive compounds. Second order polynomial mathematical model was developed with a high coefficient of determination ( R 2 = 0.99) from the experimental data. A maximum of 18.85% total extraction yield was achieved at the optimal conditions of particle size of 350 mesh, extraction temperature at 41 °C, solid–solvent-ratio of 0.07, and extraction time of 24 h. The extracted constituents are predominantly phenolics (39%) and condensed tannins (21%). Under theses optimal conditions, the experimental values agreed well with the predicted values and indicated the suitability of the developed models.

The effect of isopropyl alcohol (IPA) and n-propyl alcohol (PA) on collagen, the main component of the various connective tissues in animals, was analyzed to explore the solvent-induced polarity stress. Collagen precipitates in the presence of IPA and PA at concentrations (>50 %, v/v). The properties of precipitated collagen, after re-dissolving them in acetic acid solution, was investigated to understand (i) whether collagen precipitates induced by IPA and PA is in native or denatured form and (ii) the role of alkyl chain (linear and branched) in the solvent on the precipitation of collagen. Results demonstrate that precipitated collagen in both PA and IPA not only retains its triple helical structure but also maintains its thermal stability, fibrillation, and fluorescence properties as that of native collagen. The results also demonstrate that the nature of alkyl chain in the solvent has minor influence on the structural characteristics of collagen during precipitation.

Excess accumulation of collagen (fibrosis) is the hallmark of many fibrotic diseases such as keloids, hypertrophic scars, etc. The inhibition of collagen fibrillation during its accumulation is a therapeutic way to limit the fibrosis. Herein, the effect of Ferulic acid (FA), a natural phenolic acid compound, on collagen fibrillation is studied using biophysical methods. Optical density (OD) and microscopic analysis indicate that FA inhibits collagen self-association, and the inhibitory efficiency depends on the concentration and temperature. The absence of an increase in OD for matured collagen fibrillar solution upon addition of FA followed by collagen solution indicates that FA could also terminates the progression of preformed collagen fibrils. Spectroscopic measurements indicate that collagen retains its unique triple helical structure in the presence of FA. Saturation Transfer Difference NMR suggests that FAs are in proximity to collagen while fluorescence quenching upon addition of FA proposes that FA most likely binds to the telopeptide regions of collagen. Enzymatic studies suggest that FA protects collagen from enzymatic degradation. The current study demonstrates that FA is a potential inhibitor of collagen fibrillation and its propagation and thus it can be considered for therapeutic studies to make FA as a remedy for the plaques related to collagen deposition.

Keratin has gained much attention in the recent past as a biomaterial for wound healing owing to its biocompatibility, biodegradability, intrinsic biological activity and presence of cellular binding motifs. In this paper, a novel biomimetic scaffold containing keratin, chitosan and gelatin was prepared by freeze drying method. The prepared keratin composite scaffold had good structural integrity. Fourier Transform Infrared (FTIR) spectroscopy showed the retention of the native structure of individual biopolymers (keratin, chitosan, and gelatin) used in the scaffold. Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) results revealed a high thermal denaturation temperature of the scaffold (200-250°C). The keratin composite scaffold exhibited tensile strength (96 kPa), compression strength (8.5 kPa) and water uptake capacity (>1700%) comparable to that of a collagen scaffold, which was used as control. The morphology of the keratin composite scaffold observed using a Scanning Electron Microscope (SEM) exhibited good porosity and interconnectivity of pores. MTT assay using NIH 3T3 fibroblast cells demonstrated that the cell viability of the keratin composite scaffold was good. These observations suggest that the keratin-chitosan-gelatin composite scaffold is a promising alternative biomaterial for tissue engineering applications.

In this paper, we report the synthesis of gold nanoparticles (AuNPs) using the aqueous neem (Azadirachta indica) fruit extract as a reducing agent and capped by two different capping agents (monomeric surfactant: hexadecyltrimethylammonium chloride (CTAC) and dimeric counterion coupled gemini surfactant (COCOGS): 1, 6-Bis (N,N-hexadecyl dimethylammonium) adipate (16-6-16)). The synthesized AuNPs have been characterized by different modern techniques such as, UV–Visible spectroscopy, HR-TEM, DLS, XRD, FTIR, and XPS analyses, etc. The FTIR measurements were used to evaluate the adsorption of CTAC and COCOGS on the AuNPs surface. A significant shift in the stretching vibrations of CTAC and COCOGS head functional groups suggested their orientation towards the AuNPs surface and was further supported by the high-resolution XPS spectra of CTAC and COCOGS on the AuNPs surface. The main objective of the work is to investigate how to achieve the stability and activity of COCOGS-AuNPs for developing novel anticancer agents against triple negative breast cancer cells (TNBC). Hence, we have synthesized CTAC-AuNPs and COCOGS-AuNPs and evaluated theirs in vitro cytotoxicity against TNBC cell line (MDA-MB-231) as well as in normal NIH3T3 cells. The synthesized COCOGS-AuNPs displayed the excellent cytotoxicity against selected TNBC cell lines without affecting normal cells. In addition to the analysis of cellular apoptosis with mitochondrial membrane potential ( Δ ψ m ) loss demonstrated an increased apoptotic cell death after 24 h at their dose-dependent concentrations. Thus we conclude that the synthesized AuNPs may directly inhibit the TNBC cell growth and induced the tumor cell autophagy which consequently increasing cellular apoptosis.

The current study is to develop a natural drug carrier with seaweed derived polymers namely κ-Carrageenan (κ-Car) for drug delivery applications. κ-Car is a natural polysaccharide which derived from edible red seaweeds, they are easily available, non-toxic, cost effective, biodegradable and biocompatible nature. Curcumin (Cur) is a yellow-orange polyphenol existing in turmeric, which is predominantly used as spice and food coloring agent. The ultimate use of polymeric composites, especially those composed of natural polymers, has become a very interesting approach in recent drug delivery applications, due to their non-toxicity and biological origin. In this study the primary approach which depends on the loading of Curcumin into κ-Carrageenan was accomplished, and which (κ-Car-Cur) an active drug carrier was developed for drug delivery against selected lung cancer cells (A549). Thus, the κ-Car-Cur was synthesized by solvent evaporation method followed by freeze drying, and it was further characterized. From this study, it has been reported that the high encapsulation efficiency, good stability, and successful release of Cur from the carrier (κ-Car) was achieved. The drug release was more active at acidic pH 5.0 with the cumulative release of 78%, which is the favorable condition present in tumor microenvironments. The in vitro cellular applications studies of κ-Car-Cur demonstrated that, κ-Car-Cur composites induced higher cytotoxicity against selected cancer cells than free Cur and effectively involved to trigger cellular apoptosis in A549 cancer cells. Further, it was also possessed that inhibition of cell growth and changes in metabolic activity of cancer cells are the unique characteristic features of cellular apoptosis, through reactive oxygen species (ROS) generation. It also observed that there was a decrease in mitochondrial membrane potential (ΔψmΔψm) which leads to a cellular apoptosis during treatment with κ-Car-Cur. Hence, the study outcomes may provide the potential outline for the use of κ-Car-Cur as a promising tool to deliver drugs at intracellular level.

Missense mutations, which replace one Gly with a larger residue in the repeating sequence of the type I collagen triple helix, lead to the hereditary bone disorder osteogenesis imperfecta (OI). Previous studies suggest that these mutations may interfere with triple-helix folding. NMR was used to investigate triple-helix formation in a series of model peptides where the residue replacing Gly, as well as the local sequence environment, was varied. NMR measurement of translational diffusion coefficients allowed the identification of partially folded species. When Gly was replaced by Ala, the Ala residue was incorporated into a fully folded triple helix, whereas replacement of Gly by Ser or Arg resulted in the presence of some partially folded species, suggesting a folding barrier. Increasing the triple-helix stability of the sequence N-terminal to a Gly-to-Ser replacement allowed complete triple-helix folding, whereas with the substitution of Arg, with its large side chain, the peptide achieved full folding only after flexible residues were introduced N-terminal to the mutation site. These studies shed light on the factors important for accommodation of Gly mutations within the triple helix and may relate to the varying severity of OI.

Concern about pollution related problems in the global scenario are persuading all the processing industries to adopt cleaner manufacturing practices. Thus, the leather industry is also under pressure to look for effective alternative tanning materials for chromium. Natural products like vegetable tannins are regaining importance. However, there are limitations in the use of vegetable tanning materials because of its high organic load in the effluent, which are difficult to degrade leading to high chemical oxygen demand (COD). Moreover, conventional vegetable tanning process requires partial pickling that involves the use of sodium chloride, to suppress osmotic swelling. This results in very high amount of total dissolved solid (TDS) content in wastewaters. In this investigation, an attempt has been made to design an eco-friendly vegetable tanning process combining pickle-free tanning and application of proteolytic enzymes to improve the exhaustion of vegetable tannins. Such an approach has resulted in more than 95% tannin exhaustion in the case of the experimental process, an increase of 10% compared with the conventional vegetable tanning process. The tanned leathers showed slight improvement in hydrothermal stability. Physical and tactile evaluation of experimental leathers has been better than conventionally tanned leathers. Surface colour values illustrated negligible variation in colour and shade between control and experimental leathers. The resultant leather showed opened up, split compact fibre structure that has been well coated, indicating that the enzyme assisted tanning process did not bring about any major change or destruction on the fibre structure of the leathers. The optimized system has been field tested in a commercial tannery. The results showed that the enzyme assisted tanning process is efficient in terms of improved quality of leather and also led to reduction in total solids (TS), chlorides and COD loads. The enzyme assisted tanning system presented appears to be a viable option for combating pollution arising from the conventional vegetable tanning system.

Leather making involves conversion of a putrefiable skin or hide protein (collagen) into nonputrescible material. Environmental concerns have forced the leather industry to develop tanning systems based on natural products. Vegetable tanning is one of the oldest methods of tanning. However, there is limitation in the usage of vegetable tanning materials because of its high organic load in the effluent generated, which is difficult to degrade and leads to high biological and chemical oxygen demand. In this investigation an attempt has been made to design an eco-friendly tanning process of protein fibers using proteolytic enzymes to improve the exhaustion of vegetable tannins by increased uptake of vegetable tannins. The enzymatic treatment of the protein fibers brought in an exhaustion of 97% in the case of the optimized experimental process, compared to 85% in conventional vegetable tanning process. The enzyme treatment before tanning showed slight improvement in hydrothermal stability. Physical ...

The central role of collagen as the major structural fibrous protein in the mammalian extracellular matrix has motivated a significant effort toward the determi- nation of its mechanical properties at all levels, ranging from single monomers and long-chain polymers to a structural element within a biological tissue. However, the stabiliza- tion of collagen against collagenolytic degradation finds sig- nificance in biomedical and industrial applications. Tannins are plant-derived polyphenols that have the ability to inhibit the collagenase activity at minimum concentration. The in- hibitory effect of wattle (Acacia mollissima) and myrobalan (Terminalia chebula) on the action of collagenase against colla- gen was probed in this study. The kinetics of the inhibition of collagenase by wattle and myrobalan was deduced from the extent of hydrolysis of 2-furanacryloyl-L-leucyl-glycyl- L-prolyl-L-alanine. Both wattle and myrobalan tannin exhib- ited competitive modes of inhibition against collagenase. Circular dichroism studies of collagenase on treatment with wattle and myrobalan revealed changes in the secondary structure of collagenase. These results suggest that the tan- nins of A. mollissima and T. chebula extracts facilitated colla- gen stabilization through collagenase inhibition. 2007

All non-fibrillar collagens contain interruptions in the (Gly-X-Y)n repeating sequence, such as the more than 20 interruptions found in chains of basement membrane type IV collagen. Two selectively doubly labeled peptides are designed to model a site in type IV collagen with a GVG interruption in the α1(IV) and a corresponding GISLK sequence within the α2(IV) chain. CD and NMR studies on a 2:1 mixture of these two peptides support the formation of a single-component heterotrimer that maintains the one-residue staggering in the triple-helix, has a unique chain register, and contains hydrogen bonds at the interruption site. Formation of hydrogen bonds at interruption sites may provide a driving force for self-assembly and chain register in type IV and other non-fibrillar collagens. This study illustrates the potential role of interruptions in the structure, dynamics, and folding of natural collagen heterotrimers and forms a basis for understanding their biological role.

Keratin from the hoof is a less explored source for making valuable products. In this paper we present the extraction of pure keratin from bovine hooves and characterized them to better address the possible exploitation of this bio-resource as an alternative material for tissue engineering applications. The keratin protein from the pulverized hooves was extracted by reduction, which was observed to be pure, and two polypeptide chains of molecular weight in the range of 45–50 and 55–60 KDa were determined using SDS-PAGE assay. FTIR analysis complementing circular dichroism (CD) data, established that hoof keratin predominantly adopted α-helical conformation with admixture of β-sheet. The keratin was shown to have appreciably high denaturation temperature (215°C) as indicated by differential scanning calorimetric (DSC) analysis. Thermogravimetric analysis (TGA) also showed the retention of 50% of the original weight of the sample even at a temperature of 346°C. The keratin from the hoof had been observed to be biocompatible when analyzed with MTT assay using fibroblast cells, showing more than 90% cell viability. Hence, hoof keratin would be useful for high value biomedical applications.

Molecular modelling approaches have been used to understand the interaction of collagen-like peptides with gallic acid, which mimic vegetable tanning processes involved in protein stabilization. Several interaction sites have been identified and the binding energies of the complexes have been calculated. The calculated binding energies for various geometries are in the range 6–13 kcal/mol. It is found that some complexes exhibit hydrogen bonding, and electrostatic interaction plays a dominant role in the stabilization of the peptide by gallic acid. The π-OH type of interaction is also observed in the peptide stabilization. Molecular dynamics (MD) simulation for 600 ps revealed the possibility of hydrogen bonding between the collagen-like peptide and gallic acid.

We recently experimented with collagen coating on the surface of quercetin loaded polycaprolactone microspheres by simple adsorption technique to mimic extra cellular matrix and reduce immune or inflammatory responses at the site of implants. The collagen immobilization on polymeric scaffold surfaces through various surface modification techniques was the current scenario to improve bio-integration of the polymers with the in vivo system. Nevertheless, it requires other chemicals or processing methods to modify the surface of polymers to immobilize the collagen covalently. Here protein adsorption principle is used for the coating of collagen onto the surface of solid microspheres and characterized. Optical, ATR-FTIR, SEM analysis confirm collagen coating. The reduction in burst release of the quercetin from the PCL microspheres further confirms its presence and role in the controlled release. The results indicate that the adsorption technique can be the simple strategy to coat collagen on the surface of polyester implants to develop stealth implant in shorter time with low cost technology.

NMR spectroscopy is used to investigate the heterotrimeric nature of a collagen model peptide. Two distinct peptide chains (A and B) were synthesized to model a site in heterotrimeric basement membrane type IV collagen. For NMR studies, four amino acids in the B chain were labeled with 15N/13C. Circular dichroism spectroscopy and differential scanning calorimetry thermal stability results on a solution with both A and B peptides (molar ratio 2A:1B) are consistent with the presence of one heterotrimeric triple-helical molecular species. Heteronuclear single quantum coherence experiments on homotrimers of the B peptide show trimer peaks which disappear at temperatures higher than 10 degrees C, while the 2A:1B mixture has trimer peaks with increased stability and altered chemical shifts. The reduction in the number of Leu trimer peaks from three to one and the increased stability of trimer resonances confirm the participation of B chains in an AAB heterotrimer molecule.

Osteogenesis imperfecta (OI), or brittle bone disease, often results from missense mutation of one of the conserved glycine residues present in the repeating Gly-X-Y sequence characterizing the triple-helical region of type I collagen. A composite model was developed for predicting the clinical lethality resulting from glycine mutations in the alpha1 chain of type I collagen. The lethality of mutations in which bulky amino acids are substituted for glycine is predicted by their position relative to the N-terminal end of the triple helix. The effect of a Gly --> Ser mutation is modeled by the relative thermostability of the Gly-X-Y triplet on the carboxy side of the triplet containing the substitution. This model also predicts the lethality of Gly --> Ser and Gly --> Cys mutations in the alpha2 chain of type I collagen. The model was validated with an independent test set of six novel Gly --> Ser mutations. The hypothesis derived from the model of an asymmetric interaction between a Gly --> Ser mutation and its neighboring residues was tested experimentally using collagen-like peptides. Consistent with the prediction, a significant decrease in stability, calorimetric enthalpy, and folding time was observed for a peptide with a low-stability triplet C-terminal to the mutation compared to a similar peptide with the low-stability triplet on the N-terminal side. The computational and experimental results together relate the position-specific effects of Gly --> Ser mutations to the local structural stability of collagen and lend insight into the etiology of OI.

Peptides have been an integral part of the collagen triple-helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence-structure-function relationships. High resolution structures of triple-helical peptides have confirmed the basic Ramachandran triple-helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple-helix stability on the residues in its (Gly-X-Y)(n) repeating sequence has been investigated by measuring melting temperatures of host-guest peptides and an on-line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly-X-Y)(n) amino acid sequence pattern are found in the triple-helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod-like triple-helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple-helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple-helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self-association to the supramolecular structures found in tissues.

Stabilization of type I rat tail tendon (RTT) collagen by various aldehydes, viz. formaldehyde, gluteraldehyde, glyoxal and crotanaldehyde was studied to understand the effect of each on the thermal, enzymatic and conformational stability of collagen. The aldehydes have been found to increase the heat stability of rat tail tendon collagen fibres from 62 to 77-86 degrees C. The increase in thermal stability was found to be in a species dependent manner. The variation in the thermal stability of collagen brought about by aldehydes was in the order of formaldehyde > gluteraldehyde > glyoxal > crotanaldehdye. The aldehydes also impart a high degree of stability to collagen against the activity of the degrading enzyme, collagenase. The order of enzymatic stability brought about by aldehydes follows the same trend as the thermal stability brought about by them. This shows that the number of cross-links formed influence both the thermal and enzymatic stability in the similar manner. The effect of various aldehydes on the secondary structure of collagen was studied using circular dichroism and it was found that the aldehydes lead to changes in the amplitude of the circular dichroic (CD) spectrum but did not alter the triple helical conformation of collagen. The secondary structure of collagen is not significantly altered on interaction with different aldehydes.