1696
C. Zidorn et al. / Phytochemistry 66 (2005) 1691–1697
of the methanolic extract (165 g) of freeze-dried subaer-
ial parts (791 g) of T. porrifolius by silica gel 60 (230–400
mesh) column chromatography (CC) using a gradient of
CH2Cl2 and MeOH and Sephadex LH-20 CC using
MeOH as an eluant.
Steinheim, Germany). The final DPPH concentration
was 40 mg/l. Compounds 1–6 were tested in final con-
centrations of 1, 2, 5, 10, 20, 50, 100, and 200 lg/ml,
respectively. After incubation in 96 well-plates, the reac-
tion mixture (250 ll) was kept in the dark at ambient
temperature (25 °C) for 30 min. Then, the optical den-
sity of the test mixtures in comparison to DPPH and
pure methanol was measured using a Hidex Chameleon
plate reader at 515 nm. IC50 values for each replicate
were calculated using the following formula:
3.2.4. Isolation of compound 5 from Italian plant material
Scorzocreticoside I 5 (21.3 mg) was isolated from the
EtOAc phase (3.31 g) of the methanolic extract (43.9 g)
of air-dried subaerial parts (129 g) of T. porrifolius by
silica gel 60 (230–400 mesh) column chromatography
(CC) using a gradient of CH2Cl2 and MeOH and Sepha-
dex LH-20 CC using MeOH as an eluant.
IC50 = [(50 ꢀ LP)/(HP ꢀ LP) (HC ꢀ LC)] + LC. LP =
*
low percentage, i.e., highest percent inhibition less than
50%; HP = high percentage, i.e., lowest percent inhibi-
tion greater than 50%; HC = high concentration, i.e.,
concentration of test substance at the high percentage,
LC = low concentration, i.e., concentration of test sub-
stance at the low percentage. All compounds and con-
centrations were assayed in triplicate and mean values
were calculated for each compound. Ascorbic acid
(Merck, Darmstadt, Germany), caffeic acid (Fluka,
Buchs, Switzerland), chlorogenic acid (Roth, Karlsruhe,
Germany), and DPPH (Sigma-Aldrich, Steinheim, Ger-
many) were obtained commercially.
3.3. Physical data of new compounds
3.3.1. 2-Carboxyl-3,40-dihydroxy-5-b-D-
xylopyranosyloxybibenzyl (1)
Amorphous yellowish compound, glass transition
20
above 205 °C; ½aꢁ ꢀ22ꢂ (MeOH; c 1.32); FTIR
D
ZnSe
max
m
cmꢀ1: 2361, 2341, 1919, 1869, 1844, 1829, 1792,
1772, 1750, 1734, 1717, 1699, 1684, 1670, 1653, 1636,
1617, 1576, 1569, 1559, 1541, 1521, 1507, 1497, 1489,
1473, 1457, 1436, 1419, 1396, 1387, 1374, 1363, 1340,
1105; HRMS: m/z = 429.11787 [M + Na]+; calculated
for C20H22O9 Na: 429.11615.
Acknowledgements
3.3.2. 5,40-Dihydroxy-3-a-L-rhamnosyl-(100!30)-b-D-
xylopyranosyloxybibenzyl (2)
Amorphous yellowish compound, glass transition
The authors thank C. Kreutz and R. Micura for mea-
suring CD spectra, J. M. Rollinger for IR spectra, and
K. and S. Grass and R. Spitaler for helping collect the
plant material. This work was supported by the Fonds
zur Fo¨rderung der wissenschaftlichen Forschung
(FWF, P15594) and the Fonds zur Fo¨rderung der wis-
senschaftlichen Forschung und des wissenschaftlichen
Nachwuchses in Tirol (Uni-5404/65).
20
above 179 °C; ½aꢁ ꢀ39ꢂ (MeOH; c 0.235); FTIR
D
ZnSe
max
m
cmꢀ1: 2921, 2536, 2361, 2341, 2149, 1943, 1919,
1890, 1868, 1844, 1829, 1792, 1772, 1749, 1734, 1717,
1699, 1684, 1670, 1653, 1636, 1576, 1559, 1541, 1521,
1507, 1498, 1489, 1473, 1457, 1437, 1419, 1375, 1292,
1251, 1203, 1177, 1078, 1038. HRMS: m/z = 509.20444
[M + H]+; calculated for C25H33O11: 509.20229.
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20
D
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Methanolic solutions of test compounds were mixed
with a methanolic solution of DPPH (Sigma-Aldrich,