IN VITRO TOXICITY STUDIES ON THE EXTRACT OF MEDICINAL PLANT EVOLVULUS NUMMULARIUS AS A POTENT MICROBICIDAL CANDIDATE

The herb Evolvulus nummularius (L). L generally grown as an ornamental plant. This herb has found many applications in traditional folk medicine. There was however insufficient scientific data to back its safety to be used on humans. Methanolic extract of E. nummularius was used to check for its safety as a vaginal microbicide through various safety tests such as cell viability using MTT assay on three female genital tract epithelial cell lines, vaginal (VK2/E6E7), endocervical (End1/E6E7) and endometrial (HEC-1-A). Quantification of hemolytic activity was done on human red blood cells (RBCs). Determination of cellular integrity was checked by transepithelial electrical resistance (TER) assay and permeability by fluorescent microsphere assay. At 24 hours, application of the extract for cell viability assay showed extensive cell death with cell disruption. IC50 of VK2/E6E7 and HEC-1-A cells were found to be 2 mg/ml, IC50of End1/E6E7 was 1 mg/ml. For hemolytic assay, with treatment of the extract for one hour did not show hemolysis till the concentration of 2.5mg/ml. In TER and microsphere permeability assays, polarized HEC-1-A monolayer 24 hours post treatment had significant drop in TER and enhanced fluorescence from passage of microspheres implying disruption of the epithelial monolayer. The study revealed the crude methanolic extract appeared to be toxic towards human RBCs and female genital tract epithelial cells. Due to its toxic nature, its direct applications to the human vaginal tissue in vivo should be done with caution. Keywords: Medicinal plants; Microbicide; Evolvulus nummularius (L). L; MTT assay; Transepithelial electrical resistance; Fluorescent microsphere assay.   Â&nbsp

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

Oct-4: More than a Pluripotent Marker?

Oct-4/ Pou5f1/ Oct-3, a POU domain family protein acts as a crucial transcription factor duringembryonic development. It helps in maintenance of self renewal as well as pluripotentialstate of embryonic stem (ES) cells. Its expression starts right from 2 cell stage especiallyprior to 8 cell stage till the blastocyst stage where it is strongly expressed in inner cell mass(ICM). Thereafter, it is located predominantly in primordial germ cells (PGCs) till the birth. Ittargets particularly those genes which bear an octameric motif ATGCAAAT in promoter orenhancer region. Most of the target genes of Oct-4 are expressed in undifferentiated ES cellsand knockdown of Oct-4 results in ES cell differentiation as a result of down regulation oftargets of Oct-4 which are expressed in ES cells. Since, Oct-4 is crucial for embryo survivalits expression needs tight regulation. Oct-4 is carefully regulated epigenetically as well asby several other factors. DNA methylation and histone modification play an important role inexpression of Oct-4 while proximal promoter, enhancer and distal enhancer are the crucialregulatory elements present on Oct-4 upstream region. There is increasing evidence thatOct-4 is expressed in adult stem cells and these stem cells can get converted to cancer stemcells. Since, it is expressed in germ cells, immunohistochemical localization of Oct-4 andthereby its role as a marker for germ cell tumor detection is increasing. Thus, role of Oct-4 isnot only restricted as a marker for undifferentiated cells; it is also proving to be crucial factorwhich targets several genes involved in ES cell survival as well as help in establishing germcell origin of metastatic tumors

Interaction of contraceptive antimicrobial peptide nisin with target cell membranes: implications for use as vaginal microbicide

Background: Nisin, a naturally occurring antimicrobial peptide (AMP), is currently the focus of clinical trials as an intravaginal microbicide. Therefore its mechanism of interaction with various cell membranes was studied. Study Design: Flow cytometry was used for quantitative estimation of membrane damage by nisin which was further determined by scanning electron microscopy (SEM). Affinity of nisin for different unilamellar liposome vesicles was determined spectroflurometrically and confirmed using laser scanning confocal microscopy (LSCM). Results: Propidium iodide (PI) staining by flow cytometry exhibited selective membrane permeabilizing effect of nisin on sperm and bacterial membranes which correlated with ultrastructural changes. In vitro interaction of nisin with liposome model vesicles revealed significant leakage of calcein from liposomes composed of phosphatidylcholine/phosphatidylglycerol (POPC/POPG) (e.g., bacteria) and phosphatidylcholine/phosphatidylserine (POPC/POPS) (e.g., spermatozoa) as compared to phosphatidylcholine/phosphatidylethanolamine (POPC/POPE) vesicles (e.g., red blood corpuscles). LSCM results were in complete agreement with cell membrane affinity studies. Conclusion: This unique property of nisin can be exploited in the development of a safe and effective vaginal microbicide for the prevention of sexually transmitted infections/acquired immunodeficiency syndrome (STIs/AIDS) and unplanned pregnancies. (C) 200

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