\
Pergamon
PII] S9920Ð8311"87#99986Ð0
Phytochemistry\ Vol[ 38\ No[ 4\ pp[ 0218Ð0221\ 0887
Þ 0887 Elsevier Science Ltd[ All rights reserved
Printed in Great Britain
9920Ð8311:87:,*see front matter
SECOIRIDOID GLUCOSIDES FROM FRAXINUS ORNUS BARK
TANYA IOSSIFOVA\ BERNHARD VOGLER$ and IVANKA KOSTOVA
Institute of Organic Chemistry with Centre of Phytochemistry\ Bulgarian Academy of Sciences\ So_a 0002\ Bulgaria^
$ Department of Chemistry\ University of Hohenheim\ Stuttgart 69482\ Germany
"Received in revised form 08 January 0887#
Key Word Index*Fraxinus ornus^ Oleaceae^ secoiridoid glucosides^ hydroxyframoside A^ hyd!
roxyframoside B^ framoside^ hydroxyornoside^ ligstroside^ oleuropein^ l!hydroxypinoresinol
glucoside[
Abstract*Two novel secoiridoid glucosides\ hydroxyframoside A and hydroxyframoside B\ were isolated as
a mixture from the ethanolic extract of Fraxinus ornus bark\ together with the known secoiridoids hyd!
roxyornoside\ ligstroside\ framoside and oleuropein\ and the lignan l!hydroxypinoresinol glucoside[ The struc!
tures of the new compounds were elucidated on the basis of spectral data[ Þ 0887 Elsevier Science Ltd[ All
rights reserved
INTRODUCTION
Our previous studies ð0\ 1Ł on the chemical com!
position of Fraxinus ornus bark resulted in the iso!
lation of ligstroside "0# and the structurally new type
macrocylic secoiridoids insularoside and hyd!
roxyornoside "1#[ The occurrence of these interesting
compounds prompted us to continue our inves!
tigations on the same plant species[ Here\ we describe
the isolation and structure elucidation of two new
secoiridoid glucosides named hydroxyframoside A
"2a# and hydroxyframoside B "2b# together with the
secoiridoids oleuropein "3# and framoside "4#\ and the
lignan l!hydroxypinoresinol glucoside "5#[
RESULTS AND DISCUSSION
A fraction of the ethanolic extract of F[ ornus bark
was worked up as described in the Experimental to
give ligstroside "0#\ hydroxyornoside "1#\ the two new
compounds hydroxyframoside A "2a# and hyd!
roxyframoside B "2b#\ and oleuropein "3#\ framoside
"4#\ and the l!hydroxypinoresinol glucoside "5# ð2Ł[
This is the _rst report for the presence of 3Ð5 in F[
ornus[
The new secoiridoid glucosides hydroxyframoside
A "2a# and hydroxyframoside B "2b# were isolated as
a mixture 2a\ b in a ratio 1]0 as suggested by their
0
H and 02C NMR spectra[ The molecular formula
C21H27O03 was established for 2a and 2b on the basis
Author to whom correspondence should be addressed[
of their 0H and 02C NMR data "Tables 0 and 1# and
the negative ESIMS\ where only one peak at m:z 534
was found for the ðMÐHŁ− ion in both compounds[
The structure elucidation of 2a and 2b\ and the com!
plete assignment of all carbons and protons in their
molecules were achieved by detailed 0D "0H\ TOCSY\
02
C# and 1D "COSY\ HH!LR!COSY\ GHSQC\
HMBC\ NOESY# NMR experiments "Tables 0 and
1\ Fig[ 0\ and Experimental#[ The two compounds
exhibited the same NMR pattern\ almost identical
coupling constants for all protons of the phenethoxy
and oleoside moieties\ but small di}erences for the
corresponding chemical shifts[ No di}erences for the
glucosidic signals were observed[ Except for the aro!
matic systems\ the data were very similar to those of
framoside "4# "Tables 0 and 1# ð3Ł and jasmultiside "6#
ð4Ł[
The 0H NMR spectrum of 2a\ b revealed the pres!
ence of four methylene groups\ one A1B1 and one
AMX aromatic system for hydroxyframoside A as
well as for hydroxyframoside B[ This suggested that
each of the isomeric compounds had one 3!hyd!
roxyphenethoxy and one 2\3!dihydroxyphenethoxy
unit[ The exact position of their attachment to the
oleoside nucleus of 2a and 2b was deduced on the
basis of HMBC\ HH!LR!COSY and NOESY spectra
"see Experimental and Fig[ 0#[ The heteronuclear long
range correlations from the methylene protons at d
3[09 "0H\ dt\ J 09[60 and 6[96 Hz# and d 3[10 "0H\
dt\ J 09[60 and 6[96 Hz# of isomer 2a to the carbon
signal at d 061[34 "assigned to C!6#\ and from the
methylene protons at d 3[98 "0H\ dt\ J 09[69 and
6[06 Hz# and d 3[11 "0H\ dt\ J 09[69 and 6[06 Hz#
of isomer 2b to the carbon signal at d 061[49 "C!6#
0218
T[ IOSSIFOVA et al[
0229
Fig[ 0[ Important NOEs observed for 2a and 2b[
Table 0[ 0H NMR data of hydroxyframoside A "2a#\ hydroxyframoside B "2b# and framoside "4# in CD2OD
H
2a
2b
4
0
2
4
5a
5b
7
09
0?
1?
2?
3?
4?
5?a
5?b
0ýa
0ýb
1ý
3ý
4ý
6ý
7ý
01a
01b
11
31
41
61
71
4[89 brs
6[36 s
2[83 dd "8[39\ 3[34#
1[28 dd "03[11\ 8[39#
1[53 dd "03[11\ 3[34#
5[97 qd "6[09\ 0[99#
0[55 dd "6[09\ 0[37#
3[79 d "6[71#
2[1Ð2[3 obscured by MeOH
2[30 t "7[72#
2[1Ð2[3 obscured by MeOH
2[1Ð2[3 obscured by MeOH
2[57 dd "00[76\ 4[49#
2[78 dd "00[76\ 0[69#
3[16 dt "09[79\ 5[63#
3[20 dt "09[79\ 5[63#
1[76 1H\ t "5[63#
6[96 d "7[59#
5[60 d "7[59#
5[60 d "7[59#
6[96 d "7[59#
3[09 dt "09[60\ 6[96#
3[10 dt "09[60\ 6[96#
1[66 1H t "6[96#
5[56 d "1[98#
*
5[69 d "7[91#
5[44 dd "7[91\ 1[98#
4[89 brs
6[37 s
2[83 dd "8[39\ 3[50#
1[28 dd "03[11\ 8[39#
1[54 dd "03[11\ 3[50#
5[96 qd "6[94\ 0[99#
0[53 dd "6[94\ 0[37#
3[79 d "6[71#
2[1Ð2[3 obscured by MeOH
2[30 t "7[72#
2[1Ð2[3 obscured by MeOH
2[1Ð2[3 obscured by MeOH
2[57 dd "00[76\ 4[49#
2[78 dd "00[76\ 0[69#
3[16 dt "09[79\ 5[63#
3[20 dt "09[79\ 5[63#
1[70 1H\ t "5[60#
5[57 d "1[91#
*
5[58 d "7[91#
5[45 dd "7[91\ 1[91#
3[98 dt "09[69\ 6[06#
3[11 dt "09[69\ 6[06#
1[71 1H t "6[06#
6[94 d "7[44#
5[61 d "7[44#
5[61 d "7[44#
6[94 d "7[44#
4[89 brs
6[35 s
2[83 dd "8[34\ 3[19#
1[27 dd "03[05\ 8[34#
1[52 dd "03[05\ 3[19#
5[96 qd "5[84\ 9[86#
0[54 dd "6[99\ 0[24#
3[79 d "6[70#
2[1Ð2[3 obscured by Me
2[31 t "7[80#
2[1Ð2[3 obscured by Me
2[1Ð2[3 obscured by Me
2[57 dd "01[98\ 4[63#
2[78 dd "01[98\ 0[72#
3[16 dt "09[71\ 5[60#
3[29 dt "09[71\ 5[60#
1[76 1H\ t "5[60#
6[96 d "7[43#
5[60 d "7[43#
5[60 d "7[43#
6[96 d "7[43#
3[09 dt "09[64\ 6[97#
3[12 dt "09[64\ 6[97#
1[71 1H t "6[97#
6[94 d "7[44#
5[61 d "7[44#
5[61 d "7[44#
6[94 d "7[44#
"J in Hz#
placed the corresponding CH1 groups at position 01
in 2a and 2b\ respectively[
In the COSY spectrum the CH1!01 protons of 2a
and 2b showed coupling cross peaks to the proton
resonances of the methylene groups at d 1[66 "1H\ t\
J 6[96 Hz# for 2a and d 1[71 "1H\ t\ J 6[06
Hz# for 2b\ assigned to the respective CH1!11 in both
compounds[ The presence of the cross peaks d 1[66
"CH1!11#:d 5[56 "0H\ d\ J 1[98 Hz# and d 1[66 "CH1!
11#:d 5[44 "0H\ dd\ J 7[91 and 1[98 Hz# for 2a and
d 1[71 "CH1!11#:d 6[94 "1H\ d\ J 7[44 Hz# for 2b in
the HH!LR!COSY spectrum of 2a\ b indicated the
linkage of one 2\3!dihydroxybenzene ring to CH1!11
in 2a and one 3!hydroxybenzene ring to the same
Secoiridoid glucosides from Fraxinus Ornus
0220
Table 1[ 02C NMR data of hydroxyframoside "2a# hyd!
roxyframoside "2b# and framoside "4# in CD2OD
C
2a
2b
4
0
2
3
4
5
6
7
8
09
00
0?
1?
2?
3?
4?
5?
0ý
1ý
2ý
3ý
4ý
5ý
6ý
7ý
01
11
21
31
41
51
61
71
83[40
043[28
097[73
20[93
39[36
061[34
013[03
029[91
01[77
056[36
099[08
63[95
66[60
69[66
66[14
51[92
54[56
23[46
018[61
029[14
004[43
045[23
004[43
029[14
55[06
23[69
018[41
005[25
034[42
033[12
004[64
019[51
83[34
043\25
097[75
20[96
39[36
061[49
013[03
029[91
01[75
056[36
099[04
63[95
66[63
69[79
66[14
51[95
54[52
23[35
018[65$
005[18
034[42
033[11
004[55
019[47
55[06
23[71
018[22$
029[18
004[59
045[23
004[59
029[18
84[03
044\09
098[42
20[64
30[05
062[04
013[74
029[31
02[47
057[03
099[72
63[67
66[84
60[49
67[34
51[66
55[36
24[16$
029[10%
029[86&
005[29
046[06
005[29
029[86&
55[76
24[06$
029[90%
020[99&
005[29
046[06
005[29
020[99&
\ $\ %\ & Values with the same superscript are inter!
changeable[
position in 2b[ These data unambiguously proved the
substitution at C!6 in the two compounds] one 2\3!
dihydroxyphenethoxy unit in 2a and one 3!hyd!
roxyphenethoxy unit in 2b[
Furthermore\ the HMBC\ COSY and HH!LR!
COSY spectra of 2a\ b gave evidence for the attach!
ment of one 3!hydroxphenethoxy unit in 2a and one
2\3!dihydroxyphenethoxy unit in 2b to C!00 of their
oleoside moieties[
The proposed arrangements of 2a and 2b were fur!
ther con_rmed by the following cross peaks in the
NOESY spectrum of 2a\ b] for 2a] H!2:H!1ý and H!
1ý:H!3ý\ 7ý "d 6[96\ 1H\ d\ J 7[59 Hz#\ and for 2b]
H!2:H!1ý and H!1ý:H!3ý "d 5[57\ 0H\ d\ J 1[91 Hz#\
and H!1ý:H!7ý "d 5[45\ 0H\ dd\ J 7[91 and 1[91 Hz#[
Therefore\ the structures of the isomeric hyd!
roxyframoside A and hydroxyframoside B have been
con_rmed to be 6!"2\3!dihydroxyphenethoxy#!00!"3!
hydroxyphenethoxy#!oleoside "2a# and 6!"3!hyd!
roxyphenethoxy# ! 00 ! "2\3 ! dihydroxyphenethoxy# !
oleoside "2b#\ respectively[
EXPERIMENTAL
General experimental procedures
0
H and 02C NMR] 499 MHz "0H NMR# and 014
MHz "02C NMR#[ All experiments\ HH!DQFCOSY\
HH!LR!COSY\ GHSQC\ HMBC\ NOESY were
implemented using the standard Varian pulse library[
Spectra were obtained under temperature control at
13[4> or 14> with a 4 mm!PFG gradient inverse detec!
tion probe[ GHSQC] 017 experiments\ 1937 data
points\ FT size 7081×1937[ HMBC] optimised for
long!range couplings of 4 Hz and 01 Hz[ 017 experi!
ments\ 1937 data points\ FT size 7081×1937[
NOESY] mixing time 0 s\ 259 experiments\ 1937 data
points\ FT size 3985×3985[ HH!DQFCOSY] 229
experiments\ 1937 data points\ FT size 3985×3985[
MS] ESI negative ion\ Finnigan TSQ 699\ 2[4 kV\ ~ow
4 ml:min\ solvent MeOH[ CC] silica gel 59\ Merck[
LVC] silica gel LS 4Ð39 m "Chemapol#[ Prep[ HPLC]
RP!07 "09 mm#\ LiChrospher 099 "149×05 mm#\ UV
detector at 125 nm\ ~ow 4[9 ml:min[
0221
T[ IOSSIFOVA et al[
Plant material
A commercial sample of F[ ornus L bark collected
in 0880 in the region of Dragoman\ Bulgaria\ was
investigated[ The plant material was authenticated by
Dr[ A[ Mitrev and a voucher specimen was deposited
in the Herbarium of the Institute of Botany\ BAS\
So_a[
Extraction and isolation
Dried and well!ground bark "0 kg# was extracted
with hot EtOH "2×6 l#[ The insoluble material was
removed by _ltration and the extract was concentrated
under reduced pressure to a small vol[ After _ltration
of the deposited esculin "29[99 g#\ the mother liquor
was concentrated under reduced pressure and sub!
jected to a solvent!solvent partitioning using petrol
and EtOAc to a}ord R!0 "19[90 g# and R!1 "49[91 g#\
respectively[ R!1 "5[19 g# was further worked up by
LVC over 69 g silica gel\ using dichloroethane "DCE#
and DCEÐMeOH with increasing polarity "09]0\ 4]0\
2]0#[ Fractions eluted with DCEÐMeOH "4]0# were
combined and concentrated under reduced pressure
to give residues R!2 "9[73 g#\ R!3 "9[83 g# and R!4
"9[21 g#[
Fraction R!3 was subjected to silica gel CC with
CHCl2ÐMeOH "8]0#[ The _rst fraction A!0 "9[17 g#
was worked up on silica gel CC\ with EtOAcÐtolueneÐ
EtOH "3]0]0# to give one enriched in secoiridoids
subfraction B!0 "9[07 g# and 5 "9[914 g#[ B!0 was
subjected to prep[ HPLC RP!07 using a MeOHÐH1O
gradient "29 min 39) MeOH\ 04 min to 59) MeOH#
to obtain 3 "39 mg#\ 0 "8[7 mg#\ 1 "04[6 mg#\ 2a\ b "6
mg# and 4 "8 mg#[
Mixture of hydroxyframoside A and hydroxy!
KBr
framoside B "2a\ b#[ Powder^ IR nmax
cm−0] 2308\ 0699\
MeOH
0573\ 0517\ 0405^ UV "MeOH# nmax nm] 139\ 171\
215^ ESI!MS] m:z 534 ðM−0Ł−^ 0H NMR] Table 0^
02
C NMR] Table 1[ Signi_cant HMBC data of 2a and
2b "d in ppm#*for 2a] C!00 "056[36#:H!0ýa "3[16#\ H!
0ýb "3[20#\ H!2 "6[36#^ C!6 "061[34#:H!5a "1[28#\ H!5b
"1[53#\ H!01a "3[09#\ H!01b "3[10#^ C!71 "019[51#:H!
11 "1[66#^ for 2b] C!00 "056[36#:H!0ýa "3[16#\ H!0ýb
"3[20#\ H!2 "6[37#^ C!6 "061[49#:H!5a "1[28#\ H!5b
"1[54#\ H!01a "3[98#\ H!01b "3[11#^ C!7ý "019[47#:H!
1ý "1[70#[ Important proton long range correlations
for 2a and 2b "d in ppm#*for 2a] H!1ý "1[76#:H!3ý\7ý
"6[96#^ H!11 "1[66#:H!31 "5[56#\ H!71 "5[44#^ for 2b]
H!1ý "1[70#:H!3ý "5[57#\ H!7ý "5[45#^ H!11 "1[71#:H!
31\71 "6[94#[
Acknowled`ements*T[ Iossifova thanks the German
Academic Exchange Service "DAAD# for a schol!
arship and Prof[ W[ Kraus\ University of Hohenheim\
Stuttgart\ Germany\ for the kind support of these
investigations[ We are obliged to Ms[ I[ Klaiber and
Ms[ S[ Reeb from the same university for the mass
spectrometrical and NMR measurements[
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