Effect of human placental extract in the management of biofilm mediated drug resistance - A focus on wound management.

Management of infectious wounds, particularly chronic wounds and burn injuries, is a matter of global concern. Worldwide estimates reveal that, billions of dollars are being spent annually for the management of such chronic ailments. Evidently, bacterial biofilms pose a greater problem in the effective management of infection in chronic wounds, since most of the currently available antibiotics are unable to act on the microorganisms residing inside the protected environment of the biofilms. Accordingly, in the present study, we have attempted to evaluate the anti-biofilm properties of human placental extract (PLX) and also other virulence factors that are mediated via the quorum sensing (QS) signalling system. PLX is well known for its anti inflammatory action and it has been shown earlier some anti microbial and enzymatic activity also. PLX was found to produce significant inhibition of biofilm formation and also decreased the levels of pyoverdin and pyocyanin. The microscopic analysis (both light microscopy and atomic force microscopy) of biofilms was also used for substantiating the findings from spectrophotometric (crystal violet estimation) and fluorescence analysis (resazurin uptake). PLX pre-treatment decreased the hydrophobicity of gram-positive and gram negative cells, indicating the effect of placental extract on adherence property of planktonic cell, serving as an indicator for its antibiofilm effect on microorganisms. The reduced extracellular DNA (eDNA) content in biofilm matrix following treatment with PLX also indicates the effectiveness of placenta extract on bacterial adherence, which in turn serves as evidence substantiating the antibiofilm effects of the PLX. Furthermore, PLX was also found to be significantly effective in the in vitro wound biofilm model. Thus the present study, the first of its kind with PLX, establishes the therapeutic benefit of the same particularly in infected wounds, opening up newer avenue for further exploration.

Regulation of trypsin activity by peptide fraction of an aqueous extract of human placenta used as wound healer.

An aqueous extract of human placenta, used as wound healer, shows stabilization of trypsin against autodigestion as one of the peptides of the extract binds very strongly with the protease. Trypsin retains 40% of activity at constant level between 20 and 26 days in presence of the extract against complete inactivation in its absence. Inhibition of esterolytic activity and inability to react with p-nitrophenyl-p'-guanidinobenzoate, HCl, an active site directed reagent, by trypsin in presence of a peptide fraction of the extract indicated blocking of the catalytic site of the enzyme. Rayleigh scattering, size-exclusion HPLC, fluorescence resonance energy transfer, and surface plasmon resonance show that fibronectin type III-like peptide present in the extract interacts with trypsin. The peptide-trypsin complex is dissociated in presence of high concentration of substrates. Thus, regulation of trypsin activity by the placental extract is evident.

Analysis of free and bound NADPH in aqueous extract of human placenta used as wound healer.

NADPH is an important biomolecule involved in cellular regeneration. The distribution of free and bound NADPH in aqueous extract of human placenta used as a potent wound healer has been analyzed. Quantification from fluorescence and immuno-affinity chromatography indicates that 75.1+/-2.2% of NADPH present in the extract exists as free nucleotide or bound to very small peptides or amino acids whereas the rest remains bound to large peptides. Inability to dissociate the bound form of the nucleotide from the large peptides using urea or guanidium hydrochloride indicates that the binding is covalent. Identification of a fragmented mass of m/z 382.94 (nicotinamide+sugar+phosphate) from the NADPH-peptide conjugates supported the intactness of the nicotinamide moiety. Glutathione reductase assay indicated that 95.2+/-3.5% of the total NADPH pool of the extract can act as cosubstrate of the enzyme. This indicates that while a major fraction of free NADPH of the extract is easily available for cellular processes, the rest can also function locally where the conjugated peptides are deposited.

In vitro induction of nitric oxide by mouse peritoneal macrophages treated with human placental extract.

Nitric oxide (NO) is an important cellular mediator of tissue repair. It is produced in macrophages by the enzyme inducible nitric oxide synthase (iNOS) during wound healing. An aqueous extract of human placenta used as wound healer, has been investigated in terms of induction of NO by mouse peritoneal macrophages as well as human monocyte derived macrophages. NO production was estimated in macrophages culture supernatants. Incubation of 0.1 to 20 mg/ml of placental extract with 2x10(6) cells in vitro produced 10 to 100 microM of nitrite (n=4) in a dose dependent manner suggesting production of NO. With increase of NO production, NADPH present in the applied extract decreased proportionately. Application of L-NG monomethyl arginine (L-NMMA), an NO synthase (NOS) inhibitor, reduced the production of NO at the basal level. Dose dependent release of IFN-gamma with respect to placental extract by the mouse macrophages was observed. It has been observed that human monocytes derived macrophages also produced significant amount of NO by induction of the extract. Similar induction of NO by placental extract in presence and absence of polymyxin B suggested that this property is not likely to be mediated by the endotoxin/LPS.

Isolation of fibronectin type III like peptide from human placental extract used as wound healer.

A peptide of around 7.4 kDa has been purified from the aqueous extract of human placenta used as wound healer. Derived partial amino acid sequence from mass spectrometric analysis showed its homology with human fibronectin type III. Under nondenaturing condition, it formed aggregate, the elution pattern of which from reverse-phase HPLC was identical with that of fibronectin type III. Immuno-blot of the peptide with reference fibronectin type III-C showed strong cross reactivity. Since fibronectin type III plays important roles in wound healing, similar peptide in the extract is likely to take part in curing process.