S.-i. Kurimoto et al. / Phytochemistry Letters 5 (2012) 729–733
731
one disubstituted olefin, two tetrasubstituted olefins, one
carbonyl group and
glucosyl moiety. The 1H–1H-COSY
indicated
data, the structure of 8 was elucidated as shown in Fig. 1.
All the isolated compounds had a cis-fused -lactone ring at C-6
and C-7, which was also seen in sesquiterpene lactones isolated
from the EtOAc-soluble fraction. F. varia may characteristically
a-orientation of these protons. On the basis of these
a
correlations of H-2–H-3, and of H-6–H-7–(H-11)–H2-8–H2-9,
together with the HMBC correlations of H-3 with C-1, H-6 with
C-1, H-9 with C-1 and C-10, Me-13 with C-12, H2-14 with C-10,
and of H2-15 with C-3, C-4 and C-5 showed that 6 was a
guaiane-type sesquiterpene lactone glucoside, possessing three
olefins at C-2(C-3), C-4(C-5), C-10(C-1) and two oxygenated
functional groups at C-14, C-15. The location of the glucosyl
moiety was assigned to be at C-15 from the HMBC correlation of
g
contain sesquiterpene lactone derivatives with
-lactone.
a cis-fused
g
3. Experimental
H-10 with C-15, and its
coupling constant value of H-10 (J = 7.7 Hz). The NOESY correla-
tions of H-7 with H-6, H-8 and H-11 disclosed the
-orientations of these protons and the cis-relationship of
-lactone ring. The -orientation of Me-13 was elucidated
from the NOESY cross peak of Me-13 with H-8 From
was determined as
b
-linkage was determined from the
3.1. General experimental procedures
a
Optical rotations were measured with a JASCO DIP-370 digital
polarimeter. MS were obtained on a Waters LCT PREMIER 2695.
NMR spectra were measured on Bruker AVANCE-400 Fourier
transform spectrometers (1H NMR: 400 MHz, 13C NMR:
100 MHz) using TMS (0.03%, v/v) as an internal standard.
a
g
b
b
.
these observations, the structure of
shown (Fig. 1).
6
Column chromatography: silica gel 60 N (63–210
Kagaku, Japan), Diaion HP-20 (Mitsubishi Chemical, Japan),
Sephadex LH-20 (25–100 m; GE Health Care, UK), MCI gel CHP
20P (75–150 m; Mitsubishi Chemical, Japan), YMC-pack ODS-A
(S-50 m; YMC Co., Ltd., Japan). Preparative HPLC: CAPCELL PAK
C18 SG120 (250 mm ꢁ 20 mm; 5 m; Shiseido, Japan), COSMO-
SIL Cholester (250 mm ꢁ 20 mm; 5 m; Nacalai Tesque), COS-
MOSIL m; Nacalai Tesque), GPC
mm, Kanto
The molecular formula of 7 was assigned to be C21H28O9 by
the HRESIMS. The 1H and 13C NMR data were quite similar to
m
those of deacetylmatricarin 8-O-
b
-glucopyranoside (I) reported
m
by Michalska and Wanda Kisiel (2003). The 1H–1H COSY and
HMBC spectroscopic analyses showed that 7 possessed the same
planar structure as I. The NOESY correlations of H-7 with H-6, H-
m
m
m
m
9
a
and H-11 indicated that they were located mutually on the
same -side, while the relative configurations of Me-13 , H-5
and H-8 were concluded from the NOESY cross-peaks of Me-13
p
NAP (250 mm ꢁ 20 mm; 5
a
b
b
,
(Gel-Permeation Chromatography) [Asahi pack GS-310 2G
(MeOH, SHOWA DENKO)]. TLC: silica gel 60 F254 (Merck,
Germany).
b
with H-5 and H-8. Thus, the structure of 7 was assigned as
shown in Fig. 1.
A pseudo-molecular ion peak at m/z 451.1961 (calcd for
451.1944 [M+Na]+), observed in the positive-ion HRESIMS of 8,
indicated a molecular formula of C21H32O9. The 1H and 13C NMR
spectra displayed the signals due to one tert-methyl, two vinyl
methyls, three sp3 methylenes, one sp3 methine, two oxygen-
bearing sp3 methines, one oxygen-bearing sp3 quaternary
carbon, two trisubstituted olefins, one carbonyl group and one
glucosyl moiety. The connectivity of C-1 to C-3, and of C-5 to C-9
was provided by the analysis of the 1H–1H COSY spectrum of 8.
The HMBC correlation of Me-13 with C-7, C-11 and C-12, Me-14
with C-1, C-9 and C-10, and of Me-15 with C-4, C-5 and C-6
indicated the structure of a germacrane-type sesquiterpene
lactone glucoside with two hydroxyl groups at C-2 and C-11. The
location of the glucosyl group at C-2 was concluded from
the HMBC correlations of H-10 with C-2, while the J-value of H-10
3.2. Plant material
The roots of F. varia were collected in Kyzylkum, Uzbekistan, in
April 2001. Herbarium specimens (ESM-01ky-10) are deposited in
the herbarium of the Graduate School of Pharmaceutical Sciences,
Kyoto University.
3.3. Extraction and isolation
The dried roots of F. varia (1.4 kg) were crushed and extracted
with MeOH for 4 h at 60 8C three times. The MeOH extracts were
concentrated in vacuo to give
a gum (227 g), which was
partitioned between EtOAc and H2O. The H2O-soluble fraction
(115 g) was subjected to chromatography over Diaion HP-20
[MeOH–H2O (0:1 ! 1:0)] to give 6 fractions. Fr.2 was fractionated
by Sephadex LH-20 (H2O) to afford fractions 2.1–2.8. Fr.2.3 was
applied to a YMC ODS-A column [MeOH–H2O (0:1 ! 1:0)] to yield
fraction 2.3.1–2.3.13. Fr.2.3.2 was separated by GPC on HPLC
(MeOH) to yield fr.2.3.2.1–2.3.2.8. Fr.2.3.2.3 and fr.2.3.2.4 were
purified by SiO2CC [CHCl3–MeOH–H2O (8:2:0.2 ! 6:4:1)] to give
1 (18 mg) and 5 (17 mg), respectively. Fr.2.3.7 was subjected to a
MCI gel CHP 20 column [MeOH–H2O (1:4 ! 1:0)] to afford 5
fractions (2.3.7.1–2.3.7.5). Fr.2.3.7.2 was separated by GPC on
HPLC (MeOH) to give fr.2.3.7.2.1–2.3.7.2.7. Compound 4 (1 mg)
was isolated from fr.2.3.7.2.3 by HPLC COSMOSIL Cholester
(J = 7.7 Hz) proved its
trisubstituted olefin at C-1(C-10) was confirmed from the
NOESY correlations of Me-14 with H-1, and of H-9 with H-2,
while the NOESY cross peaks of Me-15 with H-6, and of H-5 with
H-3 disclosed the E configuration of trisubstituted olefin at
C-4(C-5). Furthermore, the configuration of Me-13 was
concluded from the NOESY correlation of Me-13 with H-8
whereas the configuration of the hydroxyl group at C-2 was
determined from the NOESY correlations of Me-15 with H-2, H-6
b-linkage. The Z configuration of
a
b
b
b
,
b
and H-9
a
. In contrast, the NOE correlation of H-6 with H-7
[MeOH:H2O = 3:7], and then HPLC COSMOSIL
pNAP
[MeOH:H2O = 2:3]. Fr.2.3.7.2.4 was purified by HPLC COSMOSIL
Cholester [MeOH:H2O = 3:7] to afford 2 (3 mg). Fr.2.3.7.4 was
purified by GPC on HPLC (MeOH), followed HPLC COSMOSIL pNAP
[MeOH:H2O = 9:11] to give 7 (1 mg). Fr.2.4 was fractionated by
YMC ODS-A [MeOH–H2O (0:1 ! 1:0)] to give 9 fractions (2.4.1–
2.4.9). Fr.2.4.7 was separated by Sephadex LH-20 CC
(CHCl3:MeOH = 1:1) and MCI gel CHP 20 CC [MeOH–H2O
(1:1 ! 1:0)] to yield fr.2.4.7.1–2.4.7.6. Repeated CC of fraction
2.4.7.3 on YMC ODS-A [MeOH–H2O (1:1 ! 1:0)], silica gel CC
[CHCl3–MeOH–H2O (6:1:0.1 ! 6:4:1)] and HPLC CAPCELL PAK
C18 SG120 (MeOH:H2O = 1:1) gave 3 (5 mg). Fr.2.4.7.4 was
Fig. 2. Dd values of (S)- and (R)-MTPA esters of 1ap.