003 1 9422.‘90 $3.00 + 0.00
i” 1990 PergamonPressplc
Phytochemistry,
Vol. 29, No. 5, pp. 1696-1699, 1990.
Printedin Great Britain.
TERPENOID
GLYCOSIDES
MATTEO
ADINOLFI,
FROM
OPHIOPOGON
MICHELANGELO
PARRILLI*
JAPONICUS
zyxwvutsr
ROOTS
and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJI
YONGXIN
ZHU?
Dipartimento di Chimica Organica e Biologica, Universitd di Napoli, Via Mezzocannone 16, 80134 Napoli, Italy; *Istituto di
Chimica, Universita della Basilicata, Via N. Sauro 85, 85100 Potenza, Italy; tNationa1 Institute for the Control of Pharmaceutical
and Biological Products, Temple of Heaven, Beijing 100050, P. R. China
(Received
Key Word
Index-Ophiopogon
17 July 1989)
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
japonicus; Liliaceae; roots; ophiogenin glycosides; borneol glycosides.
Abstract-The
isolation
and the structure
elucidation
of ophiogenin
3-0-r-L-rhamnopyranosyl-(
glucopyranoside
and bornyl 7-0-ol-L-arabinofuranosy1(1-*6)-/_(-D-glucopyranoside
through spectral
FABMS) and chemical study is reported.
The roots of Ophiopogon japonicus
(Thunb) Ker-Gawl
(known as Maidong in China) are among the constituents
of Sheng Mai Sen, a Chinese drug also containing Panax
ginseng
and Schisandra
chinensis
and used in the traditional medicine of that country for its pharmacological
effects on the cardiovascular
system. The roots have been
RO
R=
‘OHa
3
R=H
4
R=
OH
2
and
shown to contain, inter alia. homoisoflavanones
[l, 21,
two borne01 glycosides 1 and 2 [l] and a steroidal
sapogenin, ophiogenin
(3) [3]. In connection
with our
interest in both homoisoflavanones
and glycosides from
Liliaceae [2, 4, 51 we report the isolation and the structure elucidation
of two new compounds.
ophiogenin
glycoside (4) and bornyl glycoside (5), from 0. japonicus
roots.
INTRODUCTION
1
1 -&/ho-
(NMR
R= zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
6
1696
I
1697 zyxwvutsr
Short Reports
RESULTS AND DISCUSSION
The material extracted from 0. japonicus roots with
ethanol was dissolved in water and treated with nbutanol. The butanol extract was subjected to a combination of column, thin-layer and high-performance-liquid
chromatography
(see Experimental) to yield compounds
4 and 5.
The ‘H NMR spectrum of compound 4 (Table 1) displayed a group of signals in the 6 3.5-5 zone, indicative of
the presence of several CHOH protons in the molecule.
This, in addition to two tertiary (6 1.09 and 1.12) and two
secondary (6 0.66 and 1.26) methyl signals, suggested the
compound to be a spirostane steroidal saponin. This was
supported by the 13C NMR data which, when compared
to those of ophiogenin 3 [3], clearly indicated the latter as
the aglycone moiety. Thus, the low-field signals at 6 5.38
(lH, m, II’,,, = 5Hz),6.37(1H,d,J=1.5Hz)and5.02(1H,
d, J = 7.9 Hz) may be assigned to H-6 of the aglycone and
to two anomeric protons, respectively.
The positive FAB mass spectrum
of compound
4
exhibited pseudomolecular
ion peaks at m/z 793 [M
+K]+ and 777 [M +Na]+ that, together with the 13C
and ‘HNMR
data, suggested the molecular formula
C39Hs2014 and, therefore, a glycone chain constituted by
one hexose and one deoxyhexose unit. Accordingly, the
FABMS also displayed peaks at m/z 719 [M + Na - 58]+,
573 [M+Na-58-146]+
and 411 [M+Na-58-146
- 162]+,
due
to
the
subsequent
losses
of a
(27)Me(25)CH<26)CHZ-0
fragment from the spiranic
ring [6], of one desoxyhexose
and one hexose, thus
revealing the sequence of the two monose units in the
chain.
Acid hydrolysis of compound
4 gave rhamnose and
glucose, identified by TLC and HPLC of the watersoluble fraction in the hydrolysate through comparison
with authentic samples of the monosaccharides.
Isolation
of the two monoses by HPLC and determination
of the
sign of their rotations established the absolute configuration D of the glucose unit and L of the rhamnose unit of 4.
The comparison of the “C chemical shifts of 4 with those
of reference methyl glycosides indicated that the terminal
unit was cc-rhamnopyranose,
which is linked to the 2carbon of a /I-glucopyranose
unit, that carbon being
shifted by only + 3.1 ppm as expected for rhamnosylation
[7]. The J, _ r,u _ 2 values measured for the two anomeric
protons (1.5 and 7.9 Hz for the rhamnopyranose
and the
glucopyranose
unit, respectively) supported the configuration of the anomeric centres as they were deduced from
the 13C data. Based on the glycosylation
shift of the 3carbon signal of the aglycone moiety (+ 8.1 ppm), the
disaccharide
chain was established to be linked to that
carbon. The structure of compound
4 was thus determined to be ophiogenin
3-O-a-L-rhamnopyranosyl(1+2)-/l-D-glucopyranoside.
The comparison
of the r3CNMR spectrum of compound
5 with
that
of bornyl
7-O-b-D-apio-Dfuranosyl( 1+6)-/I-D-glucopyranoside
(2) [ 11 revealed
that the only noteworthy differences detected concerned
the carbons of the terminal pentose unit. A DEPT
experiment carried out to establish the substitution
degree of each carbon and the comparison
with the
13C NMR data of reference methyl pentosides indicated
a-arabinofuranose
to be the terminal monose of the
disaccharide
chain. Accordingly, in the ‘HNMR
spectrum, two anomeric-proton
signals were displayed at
6 5.79, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML
d, J = 2.1 Hz, (H-l of a-arabinofuranose
unit) and
at 6 4.84, zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP
d, J= 7.9 Hz (H-l of j%glucopyranose
unit).
Finally, upon acid hydrolysis of 5, arabinose and glucose
were identified (TLC, HPLC) in the hydrolysate. Therefore, compound
5 may be confidently
identified
as
bornyl
7-O-cl-L-arbinofuranosyl(
1+6)-I-D-glucopyranoside.
EXPERIMENTAL
General. The ‘H (400.13 MHz) and i3C (100.62 MHz) NMR
spectra were taken on a Fourier transform Bruker instrument
WM400 with an Aspect 3ooO computer.
Two-dimensional
proton-proton
correlated spectroscopy
(COSY-45) was executed with 90-45” pulses. The prep. and analytical
TLC were
carried out on precoated silica gel plates (Merck F,,,, 2 mm and
0.25 mm, respectively). Merck silica gel 60H was used for CC.
PIant material. The roots of Ophiopogonjaponics (Thunb) KerGawl were collected in Cixi country, Zhejiang province (R. P.
China), and authenticated
by Prof. Xu Guojun, China Pharmaceutical University, Naijing.
Isolation of gly cosides. Roots of 0. japonicus
(0.5 kg) were
extracted with 85% EtOH. The residue from the evapn of the
EtOH was dissolved in H,O, washed with Et,0 and extracted
with n-BuOH. Removal of n-BuOH yielded a gum (4 g). By CC
on silica gel using CHCl,--MeOH-H,O
mixtures of increasing
polarities 16 fractions were collected. TLC (CHCI,-MeOH-Hz0
3O:lO: 1, 2 runs) of fr. 8 gave compound
4 (9mg). CC
(CHCl,-MeOH-H,O
mixtures
of increasing
polarities)
and
HPLC (Lichrosphere
RP-18, 7 pm, 7:3 MeOH-H,O,
0.8 ml/
min) of fraction 12 gave compounds
2 (8 mg) and 5 (6 mg).
LIornyl
7-0-fi-D-apio-D-@anosyl(l+6)-/?-D-glucopyranoside
(2) was identified
on the basis of ‘H and “C NMR data [l].
Ophiogenin
3-0-a-L-rhamnopyranosyL( l~2)- /?- D- g~ucopy -
ranoside (4). ‘H
see Table 1. FABMS: m/z
and “CNMR:
777
[M+Na]+,
719
[M+Na-58
573 [M+Na-58-146]+
411 [M+Na
393 [M+Na-58-146-162-18]+.
793
[M+K]+,
(CH3CHCHZO)]+,
-58-146-162]+,
Bornyl
(5). ‘HNMR
7- O- a- L- arabinofuranosy l(1+6)- j- D- gly copy ranoside
(pyridine-d,):
6 0.76 (3H, s, H,-9 or H,-lo), 0.82
(3H, s, Ha-10 or H,-9), 1.04 (3H, s, Ha-l), 4.84 (lH, d, _I=7.9 Hz,
H-l’), 5.79 (IH, d, 5=2.1 Hz, H-l”). i3C NMR (pyridine-d,):
6
14.1 (Me-l), 18.9 (Me-9), 19.9 (Me-lo), 27.2 (CH,-3), 28.6 (CH,4), 38.2 (CH,-6), 45.4 (CH-5), 47.6 (C-8), 49.9 (C-2), 62.7 (CH,-5”),
68.4 (CH,-6’), 72.3 (CH-4’), 75.5 (CHJ.‘), 77.0 (CH-5’) 78.6 (CH3’), 78.9 (CH-3”), 83.4 (CHZ),
86.1 (CH-7), 86.3 (CH-4”), 106.3
(CH-I’), 110.1 (CH-1”).
Acid hy droly sis of gly cosides 4 and 5. A sample of each
glycoside (2-4 mg) was hydrolysed (90”, 3 hr) with 7% H,SO,
(1 ml). The reaction mixture was cooled, washed with CHCl, and
filtered through an Amberlite IR-4B(OH)column.
The eluate was
freeze-dried to give a residue, which was analysed for monosaccharides
by HPLC (Aminex Ion Exclusion HPX-87H, BioRad, 300x 7.8 mm, 0.005 M H,SO,,
0.4 ml/min) and TLC
(16:4: 1 CHCI,-MeOH-H,O).
In the hydolysate from 4 glucose
and rhamnose were identified. In the hydolysate from 5 glucose
and arabinose were identified.
The monose mixture obtained
from 4 was submitted
to
semiprep. HPLC with the Aminex column. The sign of the
rotation was found to be positive for both separated glucose and
inamnose.
Short Reports
1698
Table
Position
I
2
3
4
1. ‘H and 13C NMR chemical
Reference
substances
3: 37.3
31.6
71.6
42.3
C*
37.8
32.2
79.7
40.4
shifts of compound
DEPT
CH,
CH,
CH
CH,
5
6
7
8
9
10
11
12
13
14
15
140.2
121.6
25.8”
35.5
42.8
36.X
19.6
25.7;
47.3
87.7
39.2
140.3
122.3
26.2
36.3
43.6
37.4
20.1
26.6
48.4
87.8
39.0
C
CH
16
17
18
19
20
21
22
23
24
25
26
27
1’
2’
3’
4’
5’
6’
90.5
91.0
20.8
19.3
44.6
8.1
109.9
30.8
28.1
30.0
66.9
! 7.0
A 105.4
74.8
78.1
7 I .4
78. I
62.5
90.5
91.1
20.7
19.4
45. I
9.8
109.9
30.3
2x.9
30.4
66.8
17.3
102.1
77.9
77.9
71.5
78.3
62.7
CH
C
Me
Me
CH
Me
c
CH,
4 in pyridine-d,
(6 relative to TMS)
Ht
2.10; 1.91
3.90 m
2.81 AB of ABX
J,, = 12.8
5.38 M, U’,,,=5
Hz
CH,
CH
CH
C
CH,
CH,
C
C
CH,
CH,
CH
CH,
Me
CH
CH
CH
CH
CH
CH*
2.54 (H,,,), 1.85 Wls&
AB of ABX. J,5z,,5B= 13.4.
-83.J
J 151.16I s~.,e= 5.8
4.8
1.09 Sh
1.12 Sh
2.39 q zyxwvutsrqponmlkjihgfedcbaZYXWVU
J 20.21 =7.3
1.26d J *,,.Ll--73
1.60
3.5 m
0.66 d J x.*7=5.8
5.02d .J,.,=7.9
4.28
4.26
4.16m
3.88 m
4.35 I J,,,, = 5.86
J h.hb = ’ 1.7
= 2.4
4.49 dd J,,,,
1”
2”
3”
4”
5”
6”
B 102.4
71.9
72.5
73.6
69.4
18.4
110.3
72.6
72.9
74.2
69.5
18.7
CH
CH
CH
CH
CH
Me
J h”.hh= 11.7
6.37 d ,I,,? = 1.5
4.80
4.63 dd J,,,=3.4.
J,.,=9.3
4.35 I J .,,b=Ja.fi=9.3
5.0
1.77 d J 5,6=6.3
*Chemical shifts were assigned on the basis of a DEPT experiment and of comparison with
reference substances: ophiogenin 3131. A = methyl b-D-glucopyranoside
181 and B = methyl aL-rhamnopyranoside
[S].
tChemica1 shifts were assigned on the basis of proton-proton
correlations
inferred from
COSY-45 and decoupling
experiments.
Detectable signal multiplicities
and coupling constants (Hz) are indicated.
$Measured
in chloroform-d.
“,“Interchangeable
values.
Short Reports
1699
3. Nakanishi,
Acknowledgements-We
thank the IMIB-CNR,
Napoli, for the
FAB mass spectra; the Centro Interdipartimentale
di Metodologie Chimico-Fisiche,
Universita di Napoli, for NMR Spectra;
and the Minister0 della Pubblica Istruzione, Roma, for financial
support of this work.
H. and Kaneda,
N. (1987) Yakugaku Zaasshi 107,
780.
4. Adinolfi,
Lanzetta,
M. Barone, G., Corsaro,
M. M., Mangoni,
R. and Parrilli, M. (1988) Tetrahedron, 4981.
L.,
5. Adinolfi, M. Barone, G. Corsaro,
M. M., Mangoni, L.,
Lanzetta, R. and Parrilli, M. (1988) Can. J. Chem., 64, 2787.
6. Budzikiewicz, H., Djerassi, C. and Williams, D. H. (1964). in
Structure Elucidation of Natural Products by M ass Spectrometry, Vol. 2, p. 1 IO, Holden-Day,
San Francisco.
7. Mahato, S. B., Sahu, N. P., Ganguly, A. N., Kasai, R. and
Tanaka, 0. (1980) Phytochemistry 19, 2017.
8. Seo, S., Tomita, Y., Tori, K. and Yoshimura, Y. (1978) J. Am.
REFERENCES
1. Kaneda,
N., Nakanishi, H., Kuraishi, T. and Katori, T. (1983)
103, 1133.
2. Zhu, Y., Yan, K. and Tu, G. (1987) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Phytochemistry 26, 2873.
Chem. Sot. 100, 3331.
Yakugaku Zaasshi
Phytochemistry,Vol. 29, No. 5, pp. 1699 1701, 1990.
Printed in Great Britain.
A DEGRADED
0
LIMONOID
FROM
003 1 9422/90 $3.00 + 0.00
1990 Pergamon Press plc
FAGAROPSZS GLABRA
JOEL BOUSTIE, CLAUDE MOULIS, JACQUELINE GLEYE, IS&BELLE FOURASTE, PHILIPPE SERVIN* and MARYSE BON*
Laboratoire
de Pharmacognosie,
Faculte de Pharmacie, Universitt Toulouse III, 31 All&es Jules-Guesde F-31400, Toulouse,
*Laboratoire
des IMRCP, Universiti Toulouse III, 118 route de Narbonne
F-31062, Toulouse Cedex; France
(Received in reuised.form
Key Word Index- Fagaropsis
glabra; Rutaceae;
France;
I3 October 1989)
trunk bark; limonoids;
fraxinellonone;
fraxinellone;
isofraxinellone.
Abstract-The
structure and absolute configuration of fraxinellonone isolated from Fagaropsis glabra was established
as 3cr-(3’-furanyl)-3acc,7-dimethyl-4,5-dihydro-l,6-isobenzofurandione,
by NMR and CD comparison
with fraxinellone. Complete carbon assignments for fraxinellone and isofraxinellone were achieved by ’ %‘H
COSY experiments.
INTRODUCTION
Limonoids are polyoxygenated
triterpenoid
derivatives
and have been widely investigated in the Rutaceae family
for their chemotaxonomic
and commercial interest [l].
Fraxinellone (2) [ llSJ, isofraxinellone [4,5] and calodendrolide [4, 61 belong to a small group of degraded
limonoids from the Rutaceae. In this paper, we report the
complete elucidation of the structure of fraxinellonone
(l), a new natural relative of fraxinellone,
isolated from
the trunk bark of Fagaropsis glabra Capuron, a Madagascan Rutaceae.
tonated carbons as follows: 632.1 (C-6), 31.6 (C-4), 20.3
(C-9), 18.6 (C-8), 18.3 (C-5). The values for C-4 and C-6
have been interchanged
[S]. Similarly, the remaining
carbons for isofraxinellone
[S] were assigned as follows:
622.0 (C-8), 21.8 (C-5), 21.1 (C-9).
Initially, the relative stereochemistry
of 1 presented a
problem. The Me-9 protons of 1 and epifraxinellone
resonate at 61.07 [7], suggesting a trans-relationship.
However, the CD curve, [6]z33 - 13 100 [&,z
+25 500
(MeOH; c 0.015), indicated that the chirality of 1 was the
same as that of other known natural limonoids at the
corresponding
centres [6, 81. Therefore, the Me-9 and
RESULTS AND DISCUSSION
Enhanced absorption at 1669 cm-’ in the IR spectrum
of 1 and a bathochromic
shift in the IJV spectrum relative
to 2 suggested the presence of a second conjugated
carbonyl function. The mass spectral fragmentation
and
‘HNMR
spectrum
were consistent
with a C-7/C-7a
double bond and a carbonyl group at C-6, and the
molecular ion at m/z 246 corresponds
to the molecular
h4;
”
formula C,,H,,O,.
Structure zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
1 for fraxinellonone
is in
full accord with the carbon and proton assignments listed
2 R=H
in Table I. A i3C1H COSY experiment on fraxinellope
3 R=OH
(2) permitted the unambiguous
assignment of the pro-
zyxwvutsr
Michelangelo Parrilli