Lignan glucosides and serotonin phenylpropanoids from the seeds of Leuzea carthamoides

Article abstract of DOI:10.1135/cccc20070334

Two lignan glucosides, the known tracheloside and the new carthamoside, together with their two known aglycones, trachelogenin and (7E,8'S)-7,8- didehydroarctigenin (cf. carthamogenin), were isolated from the seeds of Leuzea carthamoides. Additional four phenyl-propanoid derivatives of tryptamine, the E and Z isomers of N-feruloylserotonin and N-isoferuloylserotonin, were also isolated and identified. Their structure determination and Identification were performed by spectroscopic, mainly NMR analysis.

Full text of DOI:10.1135/cccc20070334

3
34
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
LIGNAN GLUCOSIDES AND SEROTONIN PHENYLPROPANOIDS FROM
+
THE SEEDS OF Leu zea ca rtha m oides
1
,
2
3
Juraj HARMATHA *, Miloš BUDĚŠÍNSKÝ , Karel VOKÁČ , Milan PAVLÍK,
Karel GRÜNER and Věra LAUDOVÁ
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic,
1
Flemingovo nám. 2, 166 10 Prague 6, Czech Republic; e-mail: harmatha@uochb.cas.cz,
2
3
budesinsky@uochb.cas.cz, vokac@uochb.cas.cz
Received Decem ber 30, 2006
Accepted March 5, 2007
Two lign an glucosides, th e kn own trach eloside an d th e n ew carth am oside, togeth er with
th eir two kn own aglycon es, trach elogen in an d (7E,8′S)-7,8-dideh ydroarctigen in (cf. carth a-
m ogen in ), were isolated from th e seeds of Leuzea carthamoides. Addition al four ph en yl-
propan oid derivatives of tryptam in e, th e E an d Z isom ers of N-feruloylseroton in an d N-iso-
feruloylseroton in , were also isolated an d iden tified. Th eir structure determ in ation an d
iden tification were perform ed by spectroscopic, m ain ly NMR an alysis.
Keyw ord s: Leuzea carthamoides; Asteraceae; Lign an s; Ph en ylpropan oids; Trach eloside;
Carth am oside; N-Feruloylseroton in ; N-Isoferuloylseroton in ; Glycosides; Natural com poun ds.
Leuzea carthamoides (Willd.) DC., syn . Rhaponticum carthamoides (Willd.)
Iljin (Asteraceae) is an en dem ic species of south ern Siberia, but curren tly
also cultivated an d widely produced as a m edicin al plan t in Eastern an d
Cen tral Europe. Th is plan t is kn own for th e con ten t of various ecdy-
1
steroids , in sect m oultin g h orm on e an alogues. It h as been used as a rich
2
,3
4
source of ecdysteroids for our ph ytoch em ical , ch em otran sform ation
an d ph ototran sform ation 5 studies. Th e com poun ds were m ain ly utilised
,6
in specific bioassays reflectin g th eir affin ity to th e in sect ecdysteroid re-
7
,8
ceptor . Th e ph arm acological poten cy an d utilisation of L. carthamoides
h as been so far attributed to a h igh con ten t an d rich structural variabil-
9
,10
ity of ecdysteroids
. Th e ecdysteroid con ten t prom oted utilisation of
1
1
12
13
L. carthamoides for ph ytoth erapeutic , dietary or cosm etic purposes.
Biological effects of various preparation s produced from th is species
1
,11
+
Part 60 in th e series Plan t Substan ces; Part 59 see ref.3
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346
2007 Institute of Organic Chemistry and Biochemistry
doi:10.1135/cccc20070334
©

Lignan Glucosides and Serotonin Phenylpropanoids
335
m ay be also in fluen ced by oth er biologically active con stituen ts, wh ich
h ave been so far less in ten sively studied com pared with ph ytoecdysteroids.
Few years ago, th e presen ce of a series of guaian olides in th e gen us Leuzea
1
4
was reported . Th ese bioactive sesquiterpen e lacton es are widely distrib-
1
5
uted, an d th us ch aracteristic in th e wh ole subtribus Cen taurein ae . Iden ti-
fication of various flavon oids an d flavon oid glycosides was also occasion al-
1
6
ly publish ed by several auth ors, as sum m arised in ref. an d addition ally in
1
7
18
ref. . Th e ran ge of Leuzea ph en ylalkan oids was en larged by reportin g th e
presen ce of a stilben e derivative19 an d four seroton in ph en ylpropan oids20.
Our ph ytoch em ical in terest in biologically active ecdysteroid agon ists
2
1,22
an d in in sect growth regulatin g ph en ylpropan oids23
an d an tagon ists
led us to an alyse an d com pare th e con ten t an d com position of th e above
m en tion ed com poun ds in differen t organ s of th is plan t. Seeds were selected
for in vestigation because of th eir h igh con ten t an d structural variability of
2
4
16–20
ecdysteroids an d prom isin g con ten t of ph en ylpropan oids
. Seeds were
in our case extracted an d separated in a sim ilar procedure as described
2
,3
earlier for roots . Besides ecdysteroids, we isolated an d iden tified from
polar ecdysteroid-con tain in g fraction s also lign an glycosides an d rath er
rare seroton in ph en ylpropan oids. Iden tification of th ese com poun ds is h ere
described.
RESULTS AND DISCUSSION
Lign an glycosides 1 an d 2 were isolated from th e m eth an ol extract, in itially
2
,3
prepared for recovery of a series of expected an d also n ew ecdysteroids
Com poun d 1 was th e m ajor n on -steroid con stituen t of th is fraction . In its
.
–
1
IR spectrum , th e presen ce of a five-m em bered lacton e rin g (1765 cm ),
–1
–1
arom atic ring (1515, 1591 and 1606 cm ) and OH groups (3532 and 3431 cm )
were detected. HR-MS con firm ed th e com position C H O with [M + 1]+
2
7
34 12
5
51 an d m ain fragm en ts at m/z 388 [M – Glc] (for th e lign an aglycon e),
m/z 151 (for th e dim eth oxyben zyl fragm en t) an d m/z 136 (for th e h ydroxy-
1
(
m eth oxy)ben zyl fragm en t). Th e H NMR spectrum con firm ed th e presen ce
of th ree arom atic m eth oxy groups (sin glets at δ 3.802, 3.800 an d 3.797)
an d th e β-D-glucose m oiety (an om eric H-1 as doublet at δ 4.86 with J(1,2) =
1
13
7
.6 Hz). Com plete an alysis of H an d C NMR spectra (Table I) an d th e op-
2
0
tical rotation [α]
–50.2 proved th e iden tity of com poun d 1 with th e
trach eloside .
Th e secon d m ajor n on -steroid con stituen t, com poun d 2, sh owed in th e
IR spectrum th e presen ce of OH groups (3412 cm ), arom atic rin gs (1515,
597 cm ) an d a five-m em bered lacton e rin g (1745 cm –1) con jugated with
D
2
5
–
1
–
1
1
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

3
36
double bon d (1644 cm –1). HR-MS con firm ed th e com position C H O
32 11
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
2
7
+
+
with [M + 1] 533 an d m ain fragm en ts at m/z 370 [M – Glc] (for lign an
aglycon e) an d m/z 151 (for dim eth oxyben zyl fragm en t). Th e H NMR spec-
1
trum con firm ed th e presen ce of th ree arom atic m eth oxy groups (sin glets at
δ 3.884, 3.777 an d 3.773), β-D-glucose m oiety (an om eric H-1 as doublet at
δ 4.99 with J(1,2) = 7.3 Hz) an d on e vin yl proton (doublet at δ 7.49 with
1
13
allylic couplin g J = 2.0 Hz). Com plete an alysis of H an d C NMR spectra
Table I) an d th e optical rotation [α]D20 –7.6 in dicated th at th e structure
(
2
6
could be iden tical with th e h em islin B glucoside . Even th e con sideration
th at com poun ds 1 an d 2 m ay h ave a close biogen etic relation , docum en ted
by th e α-position s of th e tertiary OH group in 1 an d th e double bon d in 2
(
possibly form ed by a sim ple deh ydration of 1), in dicated structural rela-
tion con siderin g R con figuration at C-8′. However, th e iden tity h as been
rejected by h ydrolysis of 2 providin g aglycon e 4 with a distin ct positive op-
tical rotation (see below), eviden cin g en an tiom eric structure 2 for our n ew
lign an glycoside carth am oside.
Trach eloside (1) an d carth am oside (2) were h ydrolysed to obtain th e re-
spective aglycon es. From trach eloside (1) was obtain ed aglycon e iden tical
2
5
20
with trach elogen in (3) proven also by th e optical rotation [α]
–31.6 (in
D
2
7
con trary to +29.6 for en an tiom eric (+)-wikstrom ol ). From carth am oside
2) was obtain ed aglycon e carth am ogen in
cal with th e (+)-7,8-dideh ydroarctigen in ([α]D20 +16.8). Th e positive opti-
2
2,28
20
(
(4) (with [α]D +62.1) iden ti-
29
cal rotation of carth am ogen in (4) reflects th e (7E,8′S) con figuration , wh ich
2
0
30
is supported also by positive optical rotation s [α]D +86 of gadain an d
65.7 of an oth er diben zylbutyrolacton e an alogue from Peperonia duclouxii31
both with 8′S con figuration ). Th e so far kn own carth am oside isom ers with
+
(
2
0
32
th e 8′R con figuration h ave n egative optical rotation s ([α]
–
–25, ref. an d
D
3
3
34
16.4, ref. ), sim ilarly as oth er (7E,8′R) an alogues . Th e presen ce of two
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

Lignan Glucosides and Serotonin Phenylpropanoids
337
OH groups in 3 an d on e OH group in 4 togeth er with th eir position s in th e
m olecule were con firm ed by 1D- an d 2D-ROESY spectra after in situ tri-
3
5
ch loroacetyl isocyan ate acylation (TAI m eth od) . Th eir structures were
1
13
con firm ed by com plete an alysis of th eir H an d C NMR spectra (Table II)
an d by optical resolution as above referred. Th e obtain ed results fall well in
th e relation between optical rotation s an d con figuration s at C-8 an d C-8′ in
3
6
diben zylbutyrolacton e lign an s as presen ted earlier . Th e revealed en an tio-
m eric diversity of m ajor lign an con stituen ts in th e sam e plan t species as
observed in th is case, well illustrates a con siderable diversity in lign an bio-
3
7
syn th esis .
Th e aglycon es 3 an d 4 were used as referen ce com poun ds for search in g
th ese two lign an lacton es also in th e n ative extract. In th is way, both
lign an s, trach elogen in (3) an d carth am ogen in (4) were detected, isolated
an d structurally iden tified in low polarity fraction s of th e extract. Both
lign an s 3 an d 4 togeth er with th eir glycosides 1 an d 2 were in cluded in our
curren t bioactivity in vestigation , an d som e results were already pub-
2
2,23,38
lish ed
. Un fortun ately, th e C-8′con figuration s of carth am oside an d
carth am ogen in were in th e paper22 im properly presen ted.
From th e lign an glycosides 1 an d 2 con tain in g polar fraction , an addi-
tion al com poun d 5 was obtain ed. Its IR spectrum in dicated th e presen ce
of arom atic rin g (1513 an d 1594 cm –1), C=C double bon d (1653 cm ), C–O
bon d (1273, 1207, 1183, 1124 an d 1029 cm –1) an d OH group (3385 cm –1).
–1
1
Th e H NMR spectrum con firm ed th e presen ce of 20 proton s, in cludin g on e
arom atic m eth oxy group (δ 3.88) an d four labile exch an geable proton s (two
1
3
2
OH an d two NH). C NMR sh owed 20 carbon s: 17 sp -carbon atom s (n in e
of th e –CH= type, seven of th e >C= type an d on e C=O), two m eth ylen e an d
on e m eth oxy group. HR-MS con firm ed th e com position C H N O with
2
0
30
2
4
+
[
M + 1] 353 an d m ain fragm en ts at m/z 177 (for th e feruloyl m oiety) an d
m/z 159 (for th e h ydroxy(eth yl)in dolyl fragm en t). Com plete an alysis of H
an d C NMR spectra (Table III) proved th e iden tity of com poun d 5 with
N-(E)-feruloylseroton in .
1
1
3
In order to in vestigate biological activities of th ese com poun ds we scaled
up separation s by a m odified procedure20 usin g com poun d 5 as an in tern al
stan dard. Th e N-(E)-feruloylseroton in -con tain in g fraction obtain ed by col-
um n ch rom atograph y was an alysed an d purified by HPLC in several solven t
system s, resultin g in on e sin gle-peak product. Th e NMR spectra, h owever,
sh owed th at th e obtain ed sam ple was a m ixture of four isom eric com -
poun ds, on e m ajor an d th ree m in ors. Th e previously described HPLC con -
2
0
20
dition s (reviewed in ref. ) an d also th ose used in our experim en ts , were
foun d in sufficien t for separation of th is fam ily of com poun ds. A m ajority
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

3
38
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
TABLE I
1H an d C NMR data of com poun ds 1 an d 2 in CD OD
13
3
1
2
Position
δ(C)
δ(H)(JH,H
)
δ(C)
δ(H)(JH,H)
1
2
3
4
5
6
7
133.34
115.92
147.17
150.53
117.78
124.15
41.81
–
128.29
117.40
150.40
149.68
113.44
125.17
138.40
–
6.79 d (2.0)
7.23 d (2.1)
–
–
–
–
7.08 d (8.3)
6.69 dd (8.3;2.0)
3.16 d (13.6)
6.82 d (7.7)
7.22 dd (7.7;2.1)
7.49 d (2.0)
2
–
–
–
.90 d (13.6)
8
9
1
2
3
4
5
6
7
77.30
180.44
131.70
113.93
149.16
150.59
113.22
122.26
32.16
130.04
174.94
131.93
114.17
149.44
150.80
115.02
122.05
38.36
–
–
′
′
′
′
′
′
′
–
~6.72 m
6.73 d (2.1)
–
–
–
–
6.87 d (7.8)
~6.72 m
7.23 d (8.0)
6.74 dd (7.7;2.1)
2.94 dd (14.2;5.3)
2.76 dd (14.2;8.6)
4.06 m
2.80 dd (13.7;5.1)
2
.52 dd (13.7;9.5)
8
9
′
′
44.59
71.80
56.78
2.44 m
40.42
71.70
(
8.6;6.8;5.3;2.0;1.9)
4.36 dd (9.2;6.8)
.30 dd (9.2;1.9)
~3.99 m
4
OCH3
3.802 s
3.800 s
3.797 s
56.87
56.52
56.40
3.884 s
3.777 s
3.773 s
5
5
6.56
6.54
Glc
1
102.83
74.86
77.80
71.28
78.17
64.22
4.86 d (7.6)
102.25
74.94
77.85
71.28
78.31
62.49
4.99 d (7.3)
2
3.48 dd (9.6;7.6)
3.45 t (9.6;9.6)
~3.38 m
3.53 dd (9.2;7.3)
3.49 dd (9.2;8.7)
3.40 dd (9.6;8.7)
3.47 ddd (9.6;5.6;1.8)
3.90 dd (12.1;1.8)
3.70 dd (12.1;5.6)
3
4
5
~3.38 m
6
3.85 dd (12.1;1.8)
3
.68 dd (12.1;5.4)
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

Lignan Glucosides and Serotonin Phenylpropanoids
339
TABLE II
1H an d 13C NMR data of com poun ds 3 an d 4 in CDCl
3
a
a
3
3 + TAI
δ(H)(JH,H
–
4
4 + TAI
Position
δ(C)
δ(H)(JH,H
)
)
δ(C)
δ(H)(JH,H
)
δ(H)(JH,H)
1
2
125.94
112.12
–
125.63
112.26
–
–
6.72 d
(
7.12 d
(1.9)
7.04 d
(2.0)
7.13 d
(1.5)
2.0)
3
4
5
145.08
147.82
114.29
–
–
–
147.60
148.09
114.96
–
–
–
–
–
6.84 d
7.15 d
(8.2)
6.99 d
(8.2)
7.23 d
(7.6)
(
8.0)
6.63 dd
8.0;2.0)
3.09 d
13.7)
.93 d
13.7)
6
7
123.17
42.19
6.86 dd
(8.2;1.9)
123.92
137.42
7.22 dd
(8.2;2.0)
7.22 dd
(7.6;1.5)
(
3.39 d
(14.2)
7.53 d
(1.9)
7.57 d
(2.0)
(
2
(
3.20 d
(14.2)
8
9
76.41
178.29
130.94
111.37
–
–
–
–
126.53
172.74
130.41
111.49
–
–
–
–
–
–
–
1
2
′
′
–
6.63 d
6.46 d
(2.0)
6.68 d
(2.0)
6.64 d
(2.1)
(
2.1)
3
4
5
′
′
′
146.59
149.10
112.60
–
–
–
146.71
149.13
112.68
–
–
–
–
–
6.80 d
6.77 d
(8.2)
6.81 d
(8.1)
6.79 d
(8.2)
(
8.2)
6.68 dd
8.2;2.1)
2.96 m
6
7
′
′
120.82
31.57
6.52 dd
(8.2;2.0)
120.68
37.26
6.75 dd
(8.1;2.0)
6.70 dd
(8.2;2.1)
(
2.49 dd
3.08 dd
3.03 dd
(
13.5;4.6)
2.35 dd
13.5;10.5)
2.93 m
10.5;4.6;8.2;7.9)
4.29 dd
8.8;8.2)
.19 dd
8.8;7.9)
(14.4;4.2)
(13.5;4.6)
~
2.53 m
2.65 dd
(14.4;10.2)
2.69 dd
(13.5;10.0)
(
8
9
′
′
43.85
70.04
~2.53 m
~4.03 m
39.68
69.69
3.83 m
3.84 m
(10.0;5.8;4.6;2.7;2.0)
(
4.28 d
(4.2)
4.32 dd
(8.8;5.8)
(
4
(
4.29 dd
(8.8;2.7)
OCH3
55.95
3.860 s
3.800 s
3.797 s
3.860 s
3.851 s
3.846 s
55.97
55.92
55.88
3.921 s
3.886 s
5
5
5.92
5.85
3.864 s (2)
3.857 s (2)
Th e sign als of NH proton s in OTAC groups: a 8.73 s an d 8.68 s; b 8.74 s.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

3
40
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
TABLE III
13
1H an d C NMR data of com poun ds 5–8 in CD OD
3
5
6
7
8
Position
δ(C)
δ(H)(JH,H
)
δ(C)
δ(H)(JH,H
)
δ(H)(JH,H
)
δ(H)(JH,H)
2
3
4
124.32
112.55
103.56
7.03 bs
–
124.27
112.51
103.54
7.03 bs
–
6.95 bs
–
6.95 bs
–
6.96 dd
6.96 dd
(2.4;0.6)
–6.95 dd
(2.3;0.6)
6.94 dd
(2.4;0.5)
(
2.3;0.6)
5
6
151.20
111.60
–
151.15
112.66
–
–
–
6.66 dd
6.66 dd
6.66 dd
6.66 dd
(
8.6;2.3)
(8.6;2.4)
(8.6;2.3)
(8.7;2.4)
7
112.42
7.16 dd
112.40
7.16 dd
7.15 dd
7.15 dd
(
8.6;0.6)
(8.6;0.8)
(8.6;0.6)
(8.7;0.5)
8
9
133.19
129.49
26.46
–
133.14
129.45
26.45
–
–
–
–
–
–
–
1
0
1
2.94 bt
2.93 bt
(7.2)
2.88 bt
(7.2)
2.88 bt
(7.2)
(
7.2)
1
41.49
3.58 bt
41.48
3.57 bt
(7.2)
3.51 bt
(7.2)
3.52 bt
(7.2)
(
7.2)
1
2
′
′
128.39
112.69
–
129.55
112.51
–
–
–
7.12 d
7.04 d
(2.1)
7.35 d
(2.0)
7.04 d
(2.2)
(
2.0)
3
4
5
′
′
′
149.33
149.86
116.48
–
150.86
148.03
114.55
–
–
–
–
–
–
–
6.79 d
6.91 d
(8.3)
6.72 d
(8.3)
6.75 d
(8.4)
(
8.2)
6
7
8
9
′
′
′
′
123.25
142.00
118.95
7.03 dd
122.05
141.73
119.51
7.00 dd
(8.3;2.1)
6.93 dd
(8.3;2.0)
6.90 dd
(8.4;2.2)
(
8.2;2.0)
7.74 d
7.41 d
(15.7)
6.61 d
(12.7)
6.58 d
(12.6)
(
15.6)
6.42 d
6.40 d
(15.7)
5.84 d
(12.7)
5.85 d
(12.6)
(
15.6)
169.29
56.40
–
169.12
56.37
–
–
–
OCH3
3.88 s
3.88 s
3.80 s
3.83 s
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

Lignan Glucosides and Serotonin Phenylpropanoids
341
of n egative effects of separation discussed in th at paper was rem oved by us-
in g a colum n packed with Separon SGX Ph en yl ph ase. Four com poun ds
were detected in th is ch rom atogram , wh ich were, after separation , iden ti-
fied by NMR spectroscopy (Table III) as N-(E)-feruloylseroton in (5), N-(E)-
isoferuloylseroton in (6), N-(Z)-feruloylseroton in (7), an d N-(Z)-isoferuloyl-
seroton in (8). Th e isom ers were eluted from th e ph en yl ph ase colum n in
two groups. In th e first group were presen t Z isom ers (feruloyl followed by
th e isoferuloyl isom er), in th e secon d group were E isom ers (again feruloyl
followed by th e isoferuloyl isom er) with th e predom in an t N-(E)-isoferuloyl-
seroton in (6).
Th e con ten t of Z isom ers (cis-cum aroyl-, feruloyl- or isoferuloylseroton in s)
in plan ts is usually m uch lower th an th e con ten t of E isom ers (trans-
an alogues). Th erefore, it is possible th at E isom ers as m in or com pon en ts
were n ot detected in earlier an alyses, an d th us n ot reported in th e respec-
tive papers (as surveyed in refs2
0,39,40
). Th e iden tification of Z an d E isom ers
of N-(p-cum aroyl)seroton in s, presen t in Amorphophallus konjac in alm ost
4
1
equivalen t ratio , as well as our results, support in directly such assum p-
tion . Ch rom atograph ic an alysis of N-feruloylseroton in isom ers 5–8 h as
sh own th at sim ple colum n ch rom atograph y, as well as com m on reverse or
n orm al ph ase HPLC separation s allowed on ly isolation of a m ixture of iso-
2
0
m ers . Fin al separation of in dividual isom ers required special con dition s,
in th is case use of a colum n filled with Ph en yl ph ase.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

3
42
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
Biological activities of th e isolated an d iden tified Leuzea lign an s an d
feruloylseroton in con jugates were assessed in various test m odels available
to our research partn ers. Th e in sect feedin g preferen ces were tested again st
selected in sect stored-product pests23 an d com pared with a series of oth er
structurally related n atural or ch em ically m odified lign an s. Activities of th e
Leuzea lign an glucosides 1 an d 2 varied from stron g an tifeedan t (for
Tribolium confusum) up to weak attractan t (for Sitophilus granarius) extrem es,
2
3
in con trast to th e oth er, m ostly stron g feedin g deterren t lign an s . Th e
effect on th e ecdysteroid receptor assessed in th e specific Drosophila
melanogaster B cell bioassay for ecdysteroid agon ist an d an tagon ist activi-
II
9
,22
ties
was sim ilar to oth er tested lign an s: carth am oside (2) sh owed a low
2
2
agon ist activity, trach eloside (1) rem ain ed in active . Trach eloside (1) was
in cluded in a series of selected lign an s tested for th eir im m un om odulatory
3
8
properties . Its activity was rem arkably weak com pared with th e structur-
ally related lign an s possessin g n on -polar m eth oxy or m eth ylen edioxy sub-
1
8
stituen ts . An algesic an d also an xiolytic effects of th e n atural crystallin e
N-feruloylseroton in fraction con tain in g isom ers 5–8 h as been tested on
4
2
n ociception an d an xiety in Wistar rats . Th e N-feruloylseroton in fraction
h ad selective effects especially in th e h igh -pain th resh old rats. It decreased
an xiety by in creasin g exploratory activity, an d reduced stress-in duced an al-
4
3
gesia after acute stress . Our in vestigation in dicates th at L. carthamoides
m ay com e to be a n ew rich n atural source of seroton in derivatives for vari-
ous bioactive prepretion s.
Our reported an d also un publish ed prelim in ary results in dicate th at
1
–3
Leuzea carthamoides con tain s, besides bioactive ecdysteroids , also oth er
valuable secon dary m etabolites with a poten cy to be effective in assum ed
th erapeutic exploitation .
EXPERIMENTAL
In frared spectra (in cm –1) were recorded on a Bruker IPS-88 in strum en t usin g KBr pellets.
Optical rotation s were m easured at 20 °C on a Rudolph research an alytical Autopol IV
polarim eter in m eth an ol. NMR spectra (δ, ppm ; J, Hz) were m easured on a Varian Un ity-500
1
13
1
spectrom eter ( H at 500 MHz, C at 125.7 MHz) in CD OD an d/or CDCl . H NMR ch em i-
3
3
cal sh ifts were referen ced to TMS (in CDCl ) an d/or residual solven t peak (δ 3.31 in
3
1
3
CD OD). C NMR ch em ical sh ifts were referen ced to th e solven t sign al (δ(CDCl ) 77.0 an d
3
3
δ(CD OD) 49.00). Hom on uclear 2D-COSY an d 2D-ROESY spectra were used for structure as-
3
1
3
sign m en t of proton s. C APT spectra an d h eterocorrelated 2D-HMQC or 2D-HMBC spectra
were com bin ed to assign all carbon sign als. Mass spectra were recorded on a ZAB-EQ spec-
trom eter with fast atom bom bardm en t (FAB) ion isation usin g a glycerol–th ioglycerol m ix-
ture as a m atrix.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

Lignan Glucosides and Serotonin Phenylpropanoids
343
Plan t Material
Seeds of Leuzea carthamoides (W illd.) DC., syn . Rhaponticum carthamoides (W illd.) Iljin
Asteraceae) were collected at th e h orticulture station Velký Osek (Czech Republic) from cul-
(
tivated four-years-old plan ts.
Extraction an d Isolation
Seeds (700 g) of Leuzea carthamoides were first processed by CO2 supercritical fluid extraction
in order to exploit th e obtain ed seed oil as a side product in ten ded for special application .
Th e defatted residue was extracted with MeOH (yieldin g 137 g of extract) to recover ch arac-
3
teristic Leuzea ph ytoecdysteroids . Two portion s of th is extract were separated in two differ-
en t ways: by colum n ch rom atograph y on silica gel (as described below), or by low-pressure
2
0
liquid ch rom atograph y on Seph adex LH-20 (as described earlier ).
Colum n ch rom atograph y separation of th e MeOH extract (10 g) was perform ed usin g
silica gel (450 g, ICN SiliTech , particle size 32–63 n m , activated at 120 °C for 15 h ). For elu-
tion was used th e gradien t solven t system CHCl –MeOH 20:1 (1400 m l), 18:1 (900 m l), 16:1
3
(
(
900 m l), 14:1 (1000 m l), 12:1 (1850 m l), 10:1 (1400 m l), 8:1 (700 m l), 6:1 (900 m l), 4:1
450 m l), 2:1 (600 m l), followed by m ixin g th e distilled eluen t from previous fraction s with
MeOH in ratio 4:1 (600 m l), 3:1 (350 m l), 2:1 (600 m l), 1:1 (400 m l) an d MeOH (400 m l).
Th e ch rom atograph ic fraction s were after a TLC m on itorin g (usin g Kieselgel GF254 an d
CHCl –MeOH 20:1) com bin ed in to 24 collective fraction s.
3
Solid part from fraction 15 was filtered, wash ed with CHCl3 an d purified on a silica gel
colum n (20 g) with 5% MeOH in CHCl3 providin g com poun d 1 (250 m g). Fraction s 16–18
(
2
1.1 g) were furth er separated on silica gel colum n (50 g) with a m obile ph ase con tain in g
% MeOH in CHCl3 (400 m l), an d 5% MeOH in CHCl3 (900 m l) givin g accordin g to TLC
two sin gle com poun ds 1 (65 m g) an d 2 (170 m g).
Fraction s 6–10 (1.4 g) were furth er separated on a silica gel colum n (60 g) with a m obile
ph ase con tain in g 2% MeOH in CHCl3 (1000 m l), an d 5% MeOH in CHCl3 (900 m l) givin g
accordin g to TLC on e large N-feruloylseroton in -con tain in g fraction (420 m g). Th is fraction
was furth er an alysed an d subsequen tly also separated by HPLC (usin g Kn auer-m odular HPLC
system ). For separation were used sem ipreparative colum n s (250 × 8 m m ) packed with
Separon SGX C-18 (7 µm ) an d Separon SGX Ph en yl (7 µm ), respectively, produced by Tessek
Prah a. MeOH–water m obile ph ase was used un der various ch rom atograph ic con dition s, as
2
0
reported in detail in our previous paper . Four con stituen ts of th is fraction were isolated:
(4.1 m g), 6 (14.6 m g), 7 (2.6 m g) an d 8 (2.5 m g). Com poun ds 5 an d 6 were isolated in
5
purity over 92%, wh ich did n ot ch an ge durin g solven t evaporation an d NMR spectroscopy.
Purity of com poun ds 7 an d 8 ch an ged rath er rapidly durin g th e processin g. Th e fin al purity
reach ed on ly 65%, due to com m on Z/E isom erisation of th eir feruloyl/isoferuloyl m oiety.
Trach eloside (1)
Com poun d 1 was obtain ed as am orph ous solid from fraction 15 by repeated colum n ch ro-
2
0
m atograph y usin g 5% MeOH in CHCl3 for elution . [α]D –50.2 (c 0.159 in MeOH). IR (KBr):
515, 1591, 1606 (arom atic); 1765 (lacton e); 3532, 3431 (OH). FAB-MS, m/z (rel.%): 573 (20)
1
[
[
[
M + Na], 551 (8) [M + 1], 489 (12), 475 (8), 389 (10), 388 (10) [M – Glc], 185 (26), 151 (59)
dim eth oxyben zyl], 137 (58) [h ydroxy(m eth oxy)ben zyl], 93 (100). HR-MS, m/z: 551.210173
+
1
13
M + H] , for C27H35O12 required 551.212852. For H an d C NMR data, see Table I.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

3
44
Harmatha, Buděšínský, Vokáč, Pavlík, Grüner, Laudová:
Carth am oside (2)
Com poun d 2 was obtain ed as am orph ous solid from fraction s 16–18 by repeated colum n
2
0
ch rom atograph y usin g 5% MeOH in CHCl3 for elution . [α]D –7.6 (c 0.629 in MeOH). IR
(
(
[
KBr): 1515, 1597 (arom atic); 1644 (double bon d); 1745 (lacton e); 3412 (OH). FAB-MS, m/z
rel.%): 555 (6) [M + Na], 533 (4) [M + 1], 371 (18) [M – Glc + 1], 181 (26), 151 (100)
+
dim eth oxyben zyl], 137 (17), 115 (68), 110 (48), 91 (84). HR-MS, m/z: 533.207006 [M + H] ,
1
13
for C27H33O11 required 533.202287. For H an d C NMR data, see Table I.
Trach elogen in (3)
Com poun d 1 (50 m g) was refluxed in a m ixture of MeOH (5 m l) an d 10% H SO (10 m l) for
2
4
6
0 m in , th en 30 m l H O was added an d th e solven t partly evaporated in order to rem ove
2
MeOH. After coolin g th e m ixture was extracted with AcOEt, dried with an h ydrous MgSO4
an d evaporated. Th e residue was purified on a sh ort silica gel colum n with 10% AcOEt in
CHCl3 yieldin g aglycon e 3 (30 m g). Th e obtain ed com poun d was used as a stan dard for TLC
m on itorin g of seed extract ch rom atograph y fraction s. Com poun d 3 (5 m g) was isolated
from fraction s 3–4 by NP-HPLC usin g a sem ipreparative SGX colum (250 × 8 m m ) with
2
0
m obile ph ase 10% MeOH in h exan e (isocratic con dition s). [α]D –31.6 (c 0.111 in MeOH).
IR (KBr): 1516, 1603 (arom atic); 1769 (lacton e); 3430 (OH). FAB-MS, m/z (rel.%): 411 (4)
[
(
M + Na], 389 (12) [M + 1], 388 (18) [M], 151 (68) [dim eth oxyben zyl], 137 (100) [h ydroxy-
+
m eth oxy)ben zyl], 93 (18). HR-MS, m/z: 388.156096 [M] , for C21H24O7 required 388.152203.
1
13
For H an d C NMR data, see Table II.
Carth am ogen in (4)
Com poun d 2 (50 m g) was processed in th e sam e way as com poun d 1 yieldin g aglycon e 4
(
35 m g) used as a stan dard for m on itorin g fraction s 2–4 of th e seed extract. Com poun d 4
(
12 m g) was later isolated also from fraction s 3–4 by th e sam e HPLC process as described for
2
0
com poun d 3. [α]D
+62.1 (c 0.307 in MeOH). IR (KBr): 1515 (arom atic); 1644 (double
+
bon d); 1745 (lacton e); 3422 (OH). EI-MS, m/z (rel.%): 370 (18) [M] , 219 (24), 151 (100)
+
[
1
dim eth oxyben zyl]. HR-MS, m/z: 370.140908 [M] , for C21H22O6 required 370.141639. For
13
H an d C NMR data, see Table II.
N-(E)-Feruloylseroton in (5)
Com poun d 5 (4.1 m g) was isolated in 92% purity. IR (KBr): 1265, 1513, 1648 (am ide); 3380
+
(
(
[
NH, OH). EI-MS, m/z (rel.%): 352 (43) [M] , 177 (81) [C10H O ], 159 (69) [C H NO], 146
9 3 10 9
10) [C H NO], 133 (70), 116 (10), 113 (18), 106 (41), 89 (100). HR-MS, m/z: 352.138500
M] , for C20H20N O required 352.142307. For H an d C NMR data, see Table III.
9
8
+
1
13
2
4
N-(E)-Isoferuloylseroton in (6)
Com poun d 6 (14.6 m g) was isolated in purity over 95%. IR (KBr): 1267, 1510, 1653 (am ide);
+
3
395 (NH, OH). EI-MS, m/z (rel.%): 352 (7) [M] , 177 (14), 160 (19), 147 (15), 146 (51), 117
+
1
(
5), 91 (7). HR-MS, m/z: 352.138659 [M] , for C20H20N O required 352.142307. For H an d
2 4
1
3
C NMR data, see Table III.
Collect. Czech. Chem. Commun. 2007, Vol. 72, No. 3, pp. 334–346

Lignan Glucosides and Serotonin Phenylpropanoids
345
N-(Z)-Feruloylseroton in (7)
Com poun d 7 (2.6 m g) was isolated in 68% purity. Relevan t 1H NMR data (Table III) were ex-
tracted from th e obtain ed spectra.
N-(Z)-Isoferuloylseroton in (8)
Com poun d 8 (2.5 m g) was isolated in 65% purity. Relevan t 1H NMR data (Table III) were ex-
tracted from th e obtain ed spectra.
The research was supported by the Grant Agency of the Czech Republic (Grants No. 203/04/0298
and No. 203/01/0183) and in part from the research project Z40550506. We thank Dr J. Kohoutová
for MS spectra, Dr S. Vašíčková for IR spectra and Ms M. Kojecká for technical assistance.
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