## 3 Results and Discussion
### Antiproliferative Activity  
Hydrodistillate (HD), CH\({}_{2}\)Cl\({}_{2}\) extract following autolysis (EXT), as well as isolates obtained through microwave hydrodiffusion and gravity (MHG) and microwave distillation (MAD), were evaluated for their antiproliferative activity against the human lung cancer cell line A549 and the human bladder cancer cell line T24 (Figure 3, Table 4).  
EXT can be considered highly active on both cell lines, with an IC\({}_{50}\) of 1.46 \(\mathrm{\SIUnitSymbolMicro mL}\) for the A549 cell line and 1.83 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for the T24 cell line achieved after 72 h of incubation. These high antiproliferative activities of EXT can be attributed to the presence of 3-(methylsulfonyl)propyl ITC (identified as 3-(methylsulfonyl)propyl ITC and allyl ITC comprising over 85% of total volatiles (Table 3, Figure 2). Samples obtained by HD and MAD, both containing more than 99% of 3-(methylsulfonyl)propyl ITC (Table 2), displayed comparably high activity with IC\({}_{50}\) values of 8.45 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 9.42 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for A549 and 0.61 \(\mathrm{\SIUnitSymbolMicro g}\)/mL and 0.62 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for T24 cell lines, respectively. On the other hand, MHG exhibited slightly lower activity on both cell lines compared to the other isolates and was the only isolate containing 4-(methylsulfonyl)butanenitrile (37%), along with the corresponding ITC (60%).  
Previously, volatile isolates of different plants containing ITCs were tested against these two cell lines: bladder T24 (_Armonacia rusticana_, _Sysphimurium officinal_) and lung A549 (_A. rusticana_, _Lunaria annua_, _S. officinal_, _Lobularia lyclica_) [14; 31; 32; 34]. The major ITCs found in these volatile isolates can be categorized into arylaliphatic and aliphatic (olefinic, branched, and/or sulfur-containing) types. Volatile isolates from _A. rusticana_ containing 2-phenylethyl ITC and allyl ITC as the major constituents as well as the tested standards showed high activity against the T24 cancer cell line, while isopropyl ITC, as the major volatile in _S. officinal_, had moderate activity [14; 34]. High activities against  
Figure 2: Degradation of 3-(methylsulfonyl)propyl GSL (**73**) to corresponding methylsulfonylalkyl isothiamate (ITC) and his thermolysis product during GC-MS analysis (detected in the autolysis volatile).  
the A549 cancer cell line were observed for volatile isolates from _A. rusticana_ (containing 2-phenylethyll ITC and allyl ITC) and for volatile isolates from _L. annuau_ (containing 5-(methylsulfonyl)pentyl ITC and 6-(methylsulfonyl)hexylITC). On the other hand, the isolates containing isopropyl ITC (from _L. annuau_ and _S. officinal_) and 3-(methylsulfonyl)propyl ITC (from _L. lybica_), as the major ones, had moderate to low activity for the same cell line [14; 32; 33; 35]. The difference in antiproliferative activity against the A549 cell line of the ITCs with different oxidation states of sulfur atoms in the side chain was also observed by [PERSON] et al. They showed that the factor in ITCs' cytotoxicity is associated with the negative charge populations, delocalization of the border molecular orbital, and energy gaps of the HOMO-LUMO [35]. Since the charge of the oxygen atom is more negative than the charge of the sulfur atom, the cytotoxicity of 4-(methylsulfonyl)butyl ITC (sulforabane) and 3-(methylsulfonyl)propyl ITC was higher than that of 3-(methylsulfonyl)propyl ITC. The A549 cells treated with 3-(methylsulfonyl)propyl ITC and 3-(methylsulfonyl)propyl ITC displayed typical morphological signs of apoptosis, with 3-(methylsulfonyl)propyl ITC generating higher rates of cellular apoptosis than 3-(methylsulfonyl)propyl ITC [35].