US 6,782,891 B2 · Process for the isolation of a major harmful oxidant from cigarette smoke

Abstract

The components of cigarette smoke, known until now, do not explain the overwhelming hazardous effects of smoking; this invention describes the isolation, identification and procedures for determination of the structure, properties and assay of a relatively stable major harmful oxidant (cs-oxidant) present in the cigarette smoke, the content of which is about 190±10 μg per cigarette; the cs-oxidant alone almost quantitatively accounts for the oxidative damage of proteins produced by the aqueous extract of whole cigarette smoke, it is also responsible for the oxidative damage of DNA; since the cs-oxidant is relatively stable, it further explains the deleterious effects of the side stream smoke and passive smoking; a number of chemical compounds/agents including vitamin C have been found to prevent the cs-oxidant induced protein oxidation in vitro.

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Claims18

Dependent claims

A process for the isolation of p-benzosemiquinone of formula 1

a major harmful oxidant from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process comprising the steps of
(a) collecting tar or cs (cigarette smoke) solution from lighted conventional filtered tipped cigarettes,
(b) collecting tar by lighting conventional filter-tipped cigarettes having a tar content of 20-30 mg per cigarette in a glass flask dipped in a mixture of ice and salt and allowing the tar to condense and settle at the bottom of the flask,
(c) keeping the above said flask at room temperature and extracting the said tar with 30-60 mM potassium phosphate buffer at a pH ranging between 7.4 to 7.8, filtering the above solution through 0.45 μm Millipore filter and adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain the desired tar solution,
(d) extracting the above said tar solution thrice with equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow coloured aqueous layer termed as aqueous extract of cigarette smoke,
(e) extracting the above said aqueous extract of cigarette smoke twice with equal volume of water saturated n-butanol, lyophilizing the pooled yellow butanol extract in a lyophilizer at a temperature ranging between −50° C. to −60° C. under vacuum followed by extraction of the lyophilized material twice with HPLC grade acetone and drying the acetone solution under vacuum and dissolving the said acetone extract with HPLC grade methanol,
(f) subjecting the above said methanol solution to band TLC using non-fluorescent silica plates, developing the said silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the said plate and drying it at about 25-30° C. using a drier, cutting small strips containing the 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 material with HPLC grade acetone followed by collection of the acetone layer and drying it under vacuum,
(g) dissolving the above said acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water, extracting the resultant aqueous solution with equal volume of HPLC grade water saturated n-butanol followed by drying upper n-butanol layer in small glass tubes under vacuum to obtain the major cigarette smoke (cs) oxidant with a purity of 98-99% and yield of about 18-22 μg per cigarette,
(h) purifying the above said cs oxidant as obtained in step (g) 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. and at a pressure of about 29 kgf/cm2 followed by collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of 8.4% of the total cs oxidant present in the parent tar solution.

6 more elements

A process for the quantitative determination of p-benzosemiquinone of formula 1, a major harmful oxidant isolated from cigarette smoke responsible for the oxidative damage of proteins and DNA, the said process comprising the steps of

(a) collecting tar or cs (cigarette smoke) solution from lighted conventional filtered tipped cigarettes,
(b) collecting tar by lighting conventional filter-tipped cigarettes having a tar content of 20-30 mg per cigarette in a glass flask dipped in a mixture of ice and salt and allowing the tar to condense and settle at the bottom of the flask,
(c) keeping the above said flask at room temperature and extracting the said tar with 30-60 mM potassium phosphate buffer at a pH ranging between 7.4 to 7.8, filtering the above solution through 0.45 μm Millipore filter and adjusting the pH of the filtrate ranging between 7.4 to 7.6 by adding NaOH solution to obtain the desired tar solution,
(d) extracting the above said tar solution thrice with equal volume of methylene chloride, discarding the lower methylene chloride layer and collecting the upper yellow coloured aqueous layer termed as aqueous extract of cigarette smoke,
(e) extracting the above said aqueous extract of cigarette smoke twice with equal volume of water saturated n-butanol, lyophilizing the pooled yellow butanol extract in a lyophilizer at a temperature ranging between −50° C. to −60° C. under vacuum followed by extraction of the lyophilized material twice with HPLC grade acetone and drying the acetone solution under vacuum and dissolving the said acetone extract with HPLC grade methanol,
(f) subjecting the above said methanol solution to band TLC using non-fluorescent silica plates, developing the said silica plates using a mixture of toluene and ethyl acetate in a ratio of 80:20, taking out the said plate and drying it at about 25-30° C. using a drier followed by cutting small strips containing the 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 material with HPLC grade acetone followed by collection of the acetone layer and drying it under vacuum,
(g) dissolving the above said acetone extract which appeared as pale yellow needles by adding equal volume of milli Q water, extracting the resultant aqueous solution with equal volume of HPLC grade water saturated n-butanol followed by drying upper n-butanol layer in small glass tubes under vacuum to obtain the major cigarette smoke (cs) oxidant with a purity of 98-99% and yield of about 18-22 μg per cigarette, and
(h) purifying the above said cs oxidant as obtained in step (g) 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. and at a pressure of about 29 kgf/cm2 followed by collecting the effluent which appears as a single peak at a retention time of 8.808 min with a purity of 100% and yield of 8.4% of the total cs oxidant present in the parent tar solution.

5 more elements

Dependencies are inferred from the claim text.

Description12,293 words

Summary3,647 words

#The present invention relates to a process for the isolation of p-benzosemiquinone of formula 1:

#a major harmful oxidant from cigarette smoke. More particularly the present invention provides a process for the isolation of p-benzosemiquinone, a major harmful oxidant from cigarette smoke, which is responsible for the oxidative damage of proteins and DNA

#Exposure to cigarette smoke is a major cause of life-threatening diseases like bronchitis, emphysema, other diseases of the respiratory tract, coronary heart diseases, lung cancer and other malignancies [1-5]. In fact, cigarette smoke is the overwhelming cause of lung cancer, now the most common cancer globally. Since approaches to cessation of smoking by public health campaigns and anti-smoking laws passed by local Governments have had limited success, the most practicable approach is the prevention of the hazardous effects caused by cigarette smoke. Cigarette smoke in known to contain about 4000 components, out of which about 3000 components are present in the gas phase and about 1000 components in the tar phase [6]. The oxidants in the gas phase, such as O2−, H2O2, NO, peroxy radical are extremely unstable [7]. If the gas phase is passed into phosphate buffer and the resultant solution is added to albumin solution, no protein oxidation occurs (7). Apparently, any damage caused by the gas phase is expected to be restricted to the buccal cavity and upper respiratory tract [8]. On the other hand, the oxidant(s) present in the tar are quite stable and these are apparently responsible for producing oxidative damage in the lung, heart and other organs [7,9]. About 48 percent of the tar components are water soluble [10] and the aqueous extract of tar is known to produce oxidative damage of biological macromolecules including proteins and DNA [7,11,12]. However, it is perplexing to conceive how many of the components present in the aqueous extract of tar are responsible for producing oxidative damage in the biological system. Uptil now, among the many components of cigarette smoke, three classes of compounds have been suggested to be implicated as causative agents in the development of cancer and degenerative diseases, namely, (i) polycyclic aromatic hydrocarbons (ii) nitrosamines and (iii) free radicals.

Brief description of the drawings38 figures

#FIG. 1 shows Mesomeric forms of p-benzosemiquinone, a) anionic, b) and c) neutral and d) cationic;

#FIG. 2 Band thin layer chromatography of the methanol solution after lyophilization—indicates the band of the cs-oxidant

#FIG. 3 HPLC profile of the butanol extract after TLC The cs-oxidant (step 6) eluted as a major peak at the retention time of 8.808 min. The amount of cs-oxidant eluted was ˜12 μg.

Detailed description7,733 words

#Table 1. Estimation of oxidative potency at different stages of purification of the cs-oxidant

#Table 2. Oxidation of ascorbic acid by the cs-oxidant as measured by HPLC analysis

#Table 3. BSA oxidation by fractions of CS-solution at different stages of purification

Process for the isolation of a major harmful oxidant from cigarette smoke

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Process for the isolation of a major harmful oxidant from cigarette smoke

Life of this patent

Life of this patent

Abstract

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Claims18

Description12,293 words

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Life of this patent

Description12,293 words