Proteomic Characterization of Human Placenta: Insights into Potential Therapeutic Applications for Osteoarthritis.

Biologics have become increasingly prominent as therapeutics in recent years due to their innate immune-privileged nature, biocompatibility, and high levels of protein biofactors. The aim of the study is to characterise the biologic, lyophilized human placenta (LHP) and explore its therapeutic potential for osteoarthritis (OA). The presence of six bioactive constituents that regulate cell-extracellular matrix interaction was identified by liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS). Metalloproteinase inhibitor 3 (TIMP3), alpha-1 anti-trypsin (a1AT), basic fibroblast growth factor (bFGF), and transforming growth factor ß1 (TGFß1) were detected and quantified using ELISA. The total protein content present in LHP by Bradford assay was found to be 409.35 ± 0.005 µg/ml. The analytical techniques such as Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR), solid state carbon-13 Nuclear Magnetic Resonance (ssC13 NMR) spectroscopy, and Differential Scanning Calorimetry (DSC) revealed the secondary structure and conformational stability of LHP. X-Ray diffraction (XRD) studies showed its amorphous nature. Bioactivity assessment of LHP was performed in human keratinocytes (HaCaT) and human dermal fibroblasts (HDF) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The LHP was highly proliferative against skin cells and non-toxic, based on the findings of the bioactivity assay. LHP has the potential to be used as a therapeutic agent for OA, as its characterisation unveiled its physical stability, significant concentration of bioactive components that are pertinent to cartilage repair and its conformational stability.

Extraction of fleshing oil from waste limed fleshings and biodiesel production.

The aim of the study was focused on extraction of fleshing oil from limed fleshings with different neutralization process by ammonium chloride (NH4Cl) and hydrochloric acid (HCl) followed by solvent extraction. The production of fatty acid methyl esters (FAMEs) from limed fleshing oil by two stage process has also been investigated. The central composite design (CCD) was used to study the effect of process variables viz., amount of flesh, particle size and time of fleshing oil extraction. The maximum yield of fleshing oil from limed fleshings post neutralization by ammonium chloride (NH4Cl) and hydrochloric acid (HCl) was 26.32g and 12.43g obtained at 200g of flesh, with a particle size of 3.90mm in the time period of 2h. Gas chromatography analysis reveals that the biodiesel (FAME) obtained from limed fleshings is rich in oleic and palmitic acids with weight percentages 46.6 and 32.2 respectively. The resulting biodiesel was characterized for its physio-chemical properties of diesel as per international standards (EN14214).

Quantitation of serrapeptase in formulations by UV method in the microplate format.

Serrapeptase is an anti-inflammatory, proteolytic enzyme isolated from the microorganism, Serratia sp. HY-6. Very few methods are available for the quantification of serrapeptase. The activity of the enzyme is determined by an ELISA assay, colorimetric method using casein as substrate or by HPLC method. These methods are lengthy, time consuming and require a number of reagents and solvents. Therefore an attempt was made to develop a simple alternative method for regular estimation of drug in formulations. Serrapeptase enzyme was estimated in formulations by using microplate readers which uses the principle of vertical photometry. Further this method was validated and the robustness of this method was checked by estimating the drug in various formulations including liposomes and marketed tablet formulations. A linear relationship between drug concentration and absorbance was observed between 1-4 microg/ml at 230 nm (R(2)=0.9911). The percentage recovery values of the drug in serrapeptase liposomes were found to lie within the standard limit (97-98%) which confirms the method is accurate and free from any positive or negative interference of the excipient. The low value of standard deviation obtained confirms the precision of the method. (+/-0.020 - +/-0.044). The drug content values in marketed tablets values obtained matched the label claim. The proposed microplate UV-method for determination of serrapeptase in formulations is novel, simple, inexpensive, fast, specific and robust. Thus this method could be a better alternative for regular estimation of drug in the various marketed formulations of serrapeptase.