Quantitation of Polydeoxyribonucletides (PDRNs) in Human Placental Extract by Fluorescence Spectroscopy Using Ethidium Bromide

Abstract

Characterization of an aqueous extract of human placenta, used as a licensed drug for wound healing, leads to the identification of several bioactive components including polydeoxyribonu-cleotides (PDRNs). PDRNs are mixture of DNA fragments of different molecular weight. A spectro-fluorimetric method of quantitation of PDRNs in the aqueous extract of human placenta by using ethidium bromide (EtBr) has been described here. It has been demonstrated by thin layer chromatography (TLC) followed by reversed phase HPLC that EtBr binds specifically with the PDRN fraction of the multi-component extract. The binding specificity of EtBr has been verified by the analysis of emission spectra of the extract. A concentration of 0.29 μg/ml EtBr exhibits a linear range of standard CT-DNA from 0.5 - 5 μg/ml of buffer (R 2 = 0.992). The same concentration of EtBr shows a linear range of measurements of placenta extract from 5 - 35 μl/ml of buffer (R 2 = 0.976). The points of the curve were the average of three sets where maximum variation observed was ±3%. PDRN content of the extract has been estimated based on the resultant fluorescence emission (after background correction) with respect to the standard calibration curve of calf thymus DNA (CT-DNA). Estimation of PDRN in a large number of batches of placenta extract (n = 100) has been done. The statistical analysis of the estimation was found to be significant and the lower and upper levels of PDRN were 158.30 and 239.03 μg/ml of the extract respectively. This easy-to-use method of estimation of PDRN in multi-component biological extract is reported for the first time. This will help in quantitation of PDRNs for other biological extracts.