2.1. Profiling of S. martensii oxylipins

The aerial parts of S. martensii plants possessed the complex oxylipins patterns. The structural formulae of two major oxylipins groups, namely the divinyl ethers and cyclopentenones, are presented in Figs. 1 and 2, respectively. Oxylipins were extracted from S. martensii tissues, methylated, and trimethylsilylated. The resulting oxylipin derivatives were subjected to the GC–MS analyses. The observed total ion current (TIC) oxylipin profile is presented in Fig. 3 A. Major oxylipins 1–3 and 4–9 possessed M + at m / z 308 and 306, respectively (Fig. 3B) in their mass spectra. Products 1– 3 had nearly identical mass spectra, see the spectrum of compound 2 (Fig. 4A) as an example. These spectra fully corresponded to those of etheroleic acid (Me ester) and its geometric isomers (Grechkin et al., 1995, 1997 and Hamberg, 1998). Oxylipins 5–7 (M + at m / z 306) also exhibited the identical mass spectral data. The spectrum of product 7 is illustrated in Fig. 4B. The spectra of products 5–7 matched those of etherolenic acid (Me ester) and its geometric isomers (Grechkin et al., 1995, 1997; Hamberg, 1998). The equivalent chain length (ECL) values 19.03, 19.56 and 19.80 (respectively) of peaks 1, 2 and 3 were ca. 0.20 bigger than those of (ω5 Z)-etheroleic, etheroleic and (11 Z)-etheroleic acids (Me esters) (Hamberg, 1998, 2004), measured on a methylsiloxane GLC column. Peak 7 corresponded to (11 Z)-etherolenic acid (Me ester), ECL value 20.22. Etherolenic and (ω5 Z)-etherolenic acid Me esters (5 and 6) appeared as a single peak on 5% phenylmethylsiloxane columns (ECL 20.05). For final structural identification, the individual divinyl ethers were separated at micro-preparative scale and purified by HPLC. Then the NMR spectral data were recorded for each of isomers 1–3 and 5–7. These data are presented below.

Mass spectra of products 4, 8 and 9 also exhibited M + at m / z 306. Products 4 and 8 have nearly identical spectral patterns. The mass spectrum of compound 8 is presented in Fig. 5A. This spectrum matches that of cis -12-oxo-10,15(Z)-phytodienoic acid (12- oxo-PDA) Me ester (Chechetkin et al., 2008). Both the mass spectra and the retention times of products 4 and 8 corresponded to those of trans -12-oxo-PDA and cis -12-oxo-PDA (Me esters), respectively. Compound 9 possessed the distinct, recognizable mass fragmentation patterns (Fig. 5B) of 12-oxo-9(13),15(Z)-phytodienoic acid (Me ester) (Vick et al., 1979). Catalytic hydrogenation turned product 9 to a nearly equimolar mixture of trans and cis isomers of 12-oxophytonoic acid (Me). Their mass spectra exhibited M + at m / z 310 (0.1%), [M + — MeO] at m / z 279 (1%), [M + — Me(CH 2) 4 +H] at m / z 240 (4%), [M + — (CH 2) 7 COOMe] at m / z 153 (14%), 83 (100%). Similarly, the hydrogenation turned compounds 4 and 8 to trans and cis isomers of 12-oxophytonoic acid (Me), respectively. The obtained results allowed us to assign the structures of Me esters of trans - 12-oxo-PDA, cis -12-oxo-PDA and 12-oxo-9(13)-PDA to products 4, 8 and 9, respectively. Steric analysis of purified cis -12-oxo-PDA (8, Me ester) revealed that it was composed of 45% (R, R) and 55% (S, S) enantiomers.

Peak of one more related minor product 10 eluted shortly after the methyl stearate (not illustrated). Despite its relative minority, it is of substantial interest. Its mass spectrum is presented in Fig. 5C. It possessed M + at m / z 278 and characteristic fragmentation patterns shown in the scheme (Fig. 5, inset). Product 10 is the cis -2,3-dinor-12-oxo-PDA, a lower homologue of compound 8 (Weber et al., 1997). Catalytic hydrogenation turned product 10 to nearly equimolar mixture of trans and cis isomers of 2,3-dinor-12-oxophytonoic acid (Me). Their mass spectra exhibited [M + — MeO] at m / z 279 (1%), [M + — Me(CH 2) 4 +H] at m / z 212 (7%), [M + — (CH 2) 7 COOMe] at m / z 153 (15%), 83 (100%). The cis -2,3-dinor-12-oxophytonoic acid (Me) had the ECL value 18.22.

Along with the above mentioned DES and AOS products, some minor oxylipins have been detected. These are 9-oxononanoic acid, azelaic acid and (3 Z)-traumatic acid. Their physical data (not presented) were identical to those described in our recent paper (Mukhtarova et al., 2011). Appearance of these chain fragmentation products is apparently related to the HPL activity. Besides, the Oi- ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (Me/ TMS) was detected. However, it was much less abundant than the cyclopentenones 4, 8 and 9.