Patent ID: 6929012
Filing Date: 2005-08-16
Classification: A61P,G01N

Abstract:
1. A process for the isolation of p-benzosemiquinone of formula 1, a major harmful oxidant from cigarette smoke responsible for the oxidative damge of proteins and DNA, said process comprising the steps of: (a) passing whole cigarette smoke solution collected from conventional filter tipped cigarettes having a tar content of 20 mg-to 30 mg per cigarette into a 30 mM-to 60 mM potassium buffer at pH 7.4-to 7.8, filtering the solution through a 0.45 μm Millipore filter to obtain a filtrate, adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain a desired cigarette smoke solution (cs solution); (b) extracting the cs solution thrice with equal volume of methylene chloride to obtain a lower methylene chloride layer and an upper yellow colored aqueous layer, discarding the lower methylene chloride layer and collecting the upper yellow colored aqueous layer termed as aqueous extract of cigarette smoke; (c) extracting the aqueous extract of cigarette smoke twice with equal volume of water saturated n-butanol to obtain a pooled yellow butanol extract, lyophilizing the pooled yellow butanol extract in a Lyolab lyophilizer at a temperature ranging between −50° C. to −60° C. under vacuum followed by extraction of the lyophilized butanol extract twice with HPLC grade acetone to obtain an acetone solution, and drying the acetone solution under vacuum to obtain an acetone extract, and dissolving the acetone extract with HPLC grade methanol to obtain a methanol solution; (d) subjecting the methanol solution to band TLC using non-fluorescent silica plates, developing the silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the plate and drying at about 25° C. to 30° C. using a drier, cutting small strips containing developed material from both sides of the plates and keeping them in an iodine chamber for the location of the band corresponding to Rf 0.26, scraping the band and extracting the band with HPLC grade acetone to form a supernatant acetone layer, followed by collection of the acetone layer and drying it under vacuum; (e) dissolving the acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water to obtain an aqueous solution, extracting the aqueous solution with equal volume of HPLC grade water saturated n-butanol to form an upper n-butanol layer, followed by drying the upper n-butanol layer in small glass tubes under vacuum to obtain a major cs oxidant with a purity of 98-to 99% and yield of 18-to 22 μg per cigarette; and (f) purifying the cs oxidant as obtained in step (e) by dissolving it in a mobile solvent comprising a mixture of methylene chloride and methanol in a ratio of 90:10(v/v) and injecting it in a HPLC instrument with a normal phase 25 cm silica column using a uv detector at 294 nm at a flow rate of 0.5 ml/min, at a temperature of about 25° C., at a pressure of about 29 kgf/cm