Phenylpropanoids and Polyacetylenes from Ligusticum mutellina (Apiaceae) of Tyrolean Origin

Roots of Ligusticum mutellina (L.) CRANTZ afforded five major compounds, the phenylpropanoids trans-isoelemicin (I), sarisan (2), and trans-isomyristicin (3), and the polyacetylenes falcarindiol (4) and falcarindiol-3-0-acetate (5). Structures were assigned by 1 Dand 2D-NMR spectroscopy. Close inspection of the NMR spectra of falcarindiol-3-O-acetate (5) and comparison with the literature revealed that published NMR data for this compound are most probably attributable to cis or trans A2-isofalcarindiol-1-0-acetate (6sl6b). 'H and "C NMR data for falcarindiol-3-0-acetate are given and trans-isoelemicin (I), sarisan (2), and trans-isomyristicin (3), falcarindiol (4), and falcarindiol-3-0-acetate (5) are reported for the first time from Ligusticum mutellina. Chemosystematic and ethnopharmacological implications of the findings are discussed


Introduction
Ligusticum mutellina L. (German name: Alpen-Muttenvurz, Tyrolean folk names: Madaun, Mataun, Medaun, Muttern, Padaun, Roter ~iirenfenche1)'-3 is an aromatic umbelliferous herb of 10-50 cm height, distributed over Central and Southern Europe from Southern Central France to the Carpathians and Southern ~ul~aria.'In the Alps it is widespread and grows mainly in alpine and subalpine meadows.*Leaves, flowers and roots were formerly used in folk medicine against colds, obstipation, liver disorders, and kidney and bladder diseases.Furthermore "Muttenvurz" was used as a spice like parsley (Petroselinum crispum, German: ~etersilie),~ to flavor goat ~h e e s e , ~ and to brew liquor similar to that made from Meum athamanticum (German name: ~iirwurz).'Analyses of the essential oils of L. mutellina roots from the Bavarian Forest revealed ligustilide as the main compound (> 20 %), other main compounds with a portion of over 5 % each were the monoterpenes a-phellandrene, P-phellandrene, and carnphene as well as the CII-substance ~iridene.~The phenylpropanoids myristicin and dillapiol were also detected in reasonably high amounts (3.7 and 13.1 %, respectively).8Analyses of plants collected in the Black Forest showed pronounced differences between root, herb and fruit oils.9Phenylpropanoids were amongst the main compounds in all three oils, while phtalides were detectable in the underground parts only.9Non-volatile compounds of Ligusticum mutellina have not been investigated yet.

Results and Discussion
The HPL-chromatogram of the dichloromethane extract of roots of Ligusticum mutellina showed five major peaks in the apolar region (Figure 1).After isolation by repeated silica gel column chromatography (CC) and subsequent Lobar and Sephadex LH-20 CC, the appertaining compounds were identified as trans-isoelemicin (I), sarisan (2), and trans-isomyristicin (3), falcarindiol (4) and falcarindiol-3-0-acetate (5).'H NMR and I3c NMR data of trans-isoelemicin  However, when compared with our data some significant differences were observed (Table 1).0 The shift differences between falcarindiol (Table 1) and the compound isolated by Lund et al.
are not explainable by a simple substitution of the oxygen in position 3 with an acetyl moiety.As ~u n d ' ~ pointed out in a later publication, falcarindiol-3-0-esters tend to isomerize to their A2-1-0acyl isomeres.Therefore, data published earlier for falcarindiol-3-0-acetate" are most probably attributable to A2-isofalcarindiol-1-0-acetate (6al6b).The fact that the compound isolated from Ligusticum was indeed falcarindiol-3-0-acetate was also supported by the HSQC data, which showed signals for a terminal methylene group (6* = 5.53 and 5.34; Ljc = 119.8).Conclusively this is the first reliableverified by HSQC and HMBC spectroscopyreport about the NMR properties of falcarindiol-3-0-acetate 5.
The HPLC data (Figure 1) from our collection of subaerial parts of Ligusticum mutellina from the Hahntennjoch (TyrolIAustria) showed that compounds 1-5 are the prevalent compounds in that extract.Brandt and schultze9 used GC analysis for their investigation of essential oils from subaerial parts of L. mutellina from Southern German low mountain ranges.Therefore, it is not surprising that these authors did not observe any polyacetylenes.

Figure 1 .
Figure 1.HPL chromatogram of a dichloromethane extract from roots of Ligusticum mutellina.

Lund et a1 l 7
reported shift values for carbons C-1 1 to C-16 in increasing order from C-1 1 to C-16 without actually assigning the respective positions of each signal.This fact might be explained by the unavailability of 2D-hetero experiments at the time of the publication.Further shift differences occur in the polyine part (C-4 to C-7) of the molecule.However, the main difference was observed in the I3c NMR shift value given for carbon C-1 (Ljc = 1 10.6 versus 6~ = 1 19.8).There are no other signals in this region, so this difference can not be explained by a mix-up of signal assignments.

a 3 .
, lH, ddd (17.0, 1.5, 1.0) 5.26, lH, ddd (10.0, 1.5, 1.0) 5.94, 1H dd (17.0, 10.0, 5.0) 4.94, lH, br d (5Measured in CDCI3 at 300 and 75 MHz, respectively.Exchangeable signals.* Signals not resolved.n.g.not given.n.0.not observed.However, the phenylpropanoid spectrum observed in the German samples also differed considerably from our data.While Brandt and schultze9 observed myristicin and phtalides as the main phenylpropanoids, extracts of Tyrolean origin mainly contained the myristicin isomeres Phenylpropanoids and Polyacetylenes from Ligusticum mutellina (Apiaceae) of . . .107 sarisan (1) and trans-isomyristicin (2) and no major amounts of myristicin or phtalides were detectable (Figure1).Apparently different chemotypes of Ligusticum mutellina are inhabiting the German low mountain ranges (Black Forest, Bavarian Forest) and the Tyrolean alps.Phenylpropanoids possess pronounced biological activity, e.g. as anti~arcino~enic;~' antiplatelet and hypolipidemic;'2 and psychotropic22 compounds.The activity profiles of the particular phenylpropanoids differ considerably and therefore the qualitative and quantitative differences observed between extraalpine and Tyrolean samples of Ligusticum mutellina imply a different spectrum and degree of biological activity of the extracts of plant samples collected in different areas.Further comparative chemosystematic studies of plants collected in different areas and analyzed by the same technique (GC andlor HPLC) might therefore solve ethnopharmacological and botanical questions alike.Experimental General.-NMR spectra were recorded in CDC13 at 300 and 75 MHz, respectively.'H NMR spectra were referenced to solvent residual signals of CHC13 at 7.25 ppm and "C NMR spectra were referenced to solvent signals at 77.16 ppm.23 Plant material.-Ligusticum mutellina was collected in July 1999 at the Faselfeiljochl N of the Anhalter Hiitte near the HahntennjocNTyrol/Austria (altitude: 1980 m; coordinates (WGS84): N 47" 1 8'43"; E 1 0°40'1 4").A voucher specimen (CZ-99-003 34) was deposited in the herbarium of the Institut fiir Pharmazie, Abteilung Pharmakognosie.Extraction and isolation.-172g of air dried were ground and extracted exhaustively with dichloromethane.The crude extract (1 1.9 g) obtained after evaporating the solvent in vactro was fractionated by silica gel column chromatography (CC) using a gradient from petrol ether to dichloromethane and from dichloromethane to methanol.Fractions containing 1 and 2 (885 mg) were further fractionated by silica gel CC employing a gradient from petrol ether and dichloromethane.Enriched fractions of 1 (383 mg) were finally purified by RP18 Lobar CC using a gradient from H20 and methanol to yield 17.9 mg of pure compound 1.Enriched fractions of 2 (226 mg) were purified by Sephadex LH-20 CCusing the eluant methanolyielding 23.4 mg of compound 2.More polar silica gel CC fractions containing prevalently compound 3 (207 mg) were further fractionated by repeated Sephadex LH-20 CC (eluant methanol) to give 21.7 mg of compound Silica gel fractions containing 4 and 5 (1.45 g) were fractionated by silica gel CC again using a gradient of petrol ether, dichloromethane, and methanol.Enriched fractions of 4 (482 mg) were hrther purified by silica gel CC employing a gradient of dichloromethane and acetone.Further enrichment of impure compound 4 (63.5 mg) was performed by Lobar RP18 CC using a gradient of H20 and MeCN to yield a fraction of 23.9 mg containing prevalently compound 4.This was finally purified by Sephadex LH-20 CC (eluant methanol) to yield 17.0 mg of pure 4. Enriched silica gel fractions of compound 5 were purified by Sephadex LH-20 CC (eluant methanol) yielding 1 1.2 mg of compound 5.