Three new acetylenic compounds, compounds I, II and III were isolated from the cultured cells of Asparagus officinalis L. (Liliaceae) and their structures identified as 1-methoxy-4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]-benzene, 4-[5-(4-methoxyphenoxy)-3-penten-1-ynyl]phenol and 4-[5-(4-hydroxyphenoxy)-3-penten-1-ynyl]phenol, respectively, from chemical and spectral analysis.

Cigarette smoke contains many harmful chemicals, which contribute to the pathogenesis of smokingrelated diseases such as chronic obstructive pulmonary disease, cancer and cardiovascular disease. The cytotoxicity of cigarette smoke is well documented, but the definitive mechanism behind its toxicity remains unknown. Ingredients in cigarette smoke are known to deplete intracellular glutathione (GSH), the most abundant cellular thiol antioxidant, and to cause oxidative stress. In the present study, we investigated the mechanism of cigarette smoke extract (CSE)-induced cytotoxicity in B16-BL6 mouse melanoma (B16-BL6) cells using liquid chromatography-tandem mass spectrometry. CSE and ingredients in cigarette smoke, methyl vinyl ketone (MVK) and crotonaldehyde (CA), reduced cell viability in a concentration-dependent manner. Also, CSE and the ingredients (m/z 70, each) irreversibly reacted with GSH (m/z 308) to form GSH adducts (m/z 378) in cells and considerably decreased cellular GSH levels at concentrations that do not cause cell death. Mass spectral data showed that the major product formed in cells exposed to CSE was the GSH-MVK adduct via Michael-addition and was not the GSH-CA adduct. These results indicate that MVK included in CSE reacts with GSH in cells to form the GSH-MVK adduct, and thus a possible reason for CSE-induced cytotoxicity is a decrease in intracellular GSH levels. Key words cigarette smoke extract (CSE); glutathione (GSH); methyl vinyl ketone (MVK); B16-BL6 mouse melanoma (B16-BL6) cell; LC/MS; LC/MS/MSCigarette smoking has been known as a major risk factor implicated in increased incidence of cardiovascular disease, cancer and chronic obstructive pulmonary disease.1) Cigarette smoke contains more than 4800 identified chemical compounds, many of which are toxic and harmful to the human body.2) Oxidants and aldehydes, major constituents in the gaseous phase of cigarette smoke, are thought to mediate oxidative stress which has been implicated in the pathogenesis of smoking-related diseases. [3][4][5] Reactive α,β-unsaturated carbonyl compounds such as acrolein and crotonaldehyde, are abundant in the gas phase of cigarette smoke and are major mediators of cigarette smokeinduced macrophage activation. 6) These compounds are also thought to contribute to oxidative stress-induced inflammation and vascular dysfunction. 7) Furthermore, acrolein and crotonaldehyde have been reported to directly react with the thiol groups, 8) especially glutathione (GSH), via a Michael-type addition reaction, resulting in the formation of nonreducible GSH-aldehyde derivatives, thereby depleting the total available GSH pool.9,10) Because GSH plays a key role in cellular antioxidant defense against oxidant injury, GSH depletion leads to cigarette smoke-induced cytotoxicity.11,12) Similarly, methyl vinyl ketone (MVK), an α,β-unsaturated carbonyl compound, has been shown to react with free GSH in nucleophilic Michael-type addition.13) The MVK-induced cell apoptosis was associated with depletion of GSH, disruption of the mi...

Comparison of the 'H and I3C chemical shift data of bryonolic acid (D: C-friedoolean-8-en-3~-01-2Y-oic acid) and its derivatives (13 species) revealed that the conformational features of these compounds in CDCI, solution depend on whether the functional groups at C-29 are trigonal, such as COOCH3, or tetrahedral, such as CH,OH. In order to study the conformational features of these two groups of compounds, vicinal coupling constants between the protons on the E rings of the methyl ester of D : C-friedoolean-8-en-29-oic acid, D : C-friedoolean-8-en-29-01 and its acetate were measured. Comparison of the results with the steric geometries to be expected from the molecular mechanics calculation for the E-ring moieties of these derivatives in the S form (D-E rings: boat-boat form) and the F form (D-E rings: chair-chair form) led to the conclusion that the compounds having a trigonal functional group at C-29 mainly adopt the S form, and those having a tetrahedral functional group at C-29 are present as mixtures of the S and F form in equilibrium in the solutions.

Cigarette smoke, a collection of many toxic chemicals, contributes to the pathogenesis of smokingrelated diseases such as chronic obstructive pulmonary disease and cancer. Much work has been done on the chemical analysis of ingredients in cigarette smoke, but there are few reports on the active ingredients that can modify biomolecules. We used a sensitive liquid chromatography-mass spectrometry (LC/MS) and LC/ MS/MS method to show that l-tyrosine (Tyr), an amino acid with a highly reactive hydroxyl group, readily reacts with cigarette smoke extract (CSE) at body temperature (37°C) to form various Tyr derivatives. Among these derivatives were N-(3-oxobutyl)-Tyr and two acetylated compounds, N-acetyl-Tyr and O-acetylTyr, which were synthesized by reaction of Tyr with methyl vinyl ketone and acetic anhydride, respectively, at 37°C. The presence of methyl vinyl ketone and acetic anhydride in CSE was confirmed by gas chromatography-mass spectrometry (GC/MS). These results indicate that Tyr can easily react with active ingredients in CSE. The present analytical methods should aid the search for active ingredients in cigarette smoke.

The -C NMR spectra of some derivatives of bryonolic acid ( diacetyl-3P,29-diols (3,29-diacetyl-DC-friedoolean-8-en-3P,29-diol) 5, 5a, 5b and 5c, and 3-acetyl-3P,29-diols 6,6a and 6b were prepared from 1, la, l b and lc, and their -C NMR spectra were recorded. The -C concentration of the 13C-enriched species was high enough to exhibit the satellite peaks clearly, and the analysed data were very useful for this study. Thns, total assignments for 2 , 3 , 4 , 5 and 6 were established. It was found that conversion of the methoqaubonyl group at C-29 into an acetoxymethyl group cawed complex changes in the chemical shifts of the C, D-and E-ring carbons and those of the methyl carbons linked to these rings.