Fumaria densiflora DC. Alkaloids

Article abstract of DOI:10.1135/cccc19961064

Twenty isoquinoline alkaloids were identified in the aerial part of Fumaria densiflora DC. In addition to bicuculline, densiflorine, fumaritine, fumariline, scoulerine, coptisine, cryptopine, parfumine, protopine, and sinactine which have been found previously, the following alkaloids were isolated for the first time from this species: adlumine, fumaricine, fumaritridine, fumarophycine, fumarofine, corytuberine, cis-N-methylstylopinium iodide, stylopine, and two new 1-benzylisoquinoline alkaloids, viz. fumaflorine and fumaflorine methyl ester. The cytoprotectivity of extracts of F. densiflora and F. officinalis, and that of protopine, cryptopine, and parfumine was tested on primary cultures of rat hepatocytes intoxicated with carbon tetrachloride. The cytoprotective effect of the extracts is associated with the alkaloids.

Full text of DOI:10.1135/cccc19961064

1064
Taborska et al.:
Fumaria densiflora DC. ALKALOIDS
a1
a
b1
c
Eva TABORSKA , Hana BOCHORAKOVA , Jaromir SOUSEK , Petr SEDMERA ,
Ctirada VAVRECKOVAb2 and Vilim SIMANEK
a Institute of Biochemistry, Masaryk University, 662 43 Brno, Czech Republic; e-mail:
1 taborska@med.muni.cz
*
b3,
b Institute of Medical Chemistry, Palacky University, 775 15 Olomouc, Czech Republic;
e-mail: 1 jaromir@tunw.upol.cz, 2 ctirada@tunw.upol.cz, 3 vilim@tunw.upol.cz
c Institute of Microbiology, Academy of Sciences of the Czech Republic, 142 20 Prague 4,
Czech Republic; e-mail: sedmera@biomed.cas.cz
Received January 31, 1996
Accepted March 10, 1996
Twenty isoquinoline alkaloids were identified in the aerial part of Fumaria densiflora DC. In addition
to bicuculline, densiflorine, fumaritine, fumariline, scoulerine, coptisine, cryptopine, parfumine,
protopine, and sinactine which have been found previously, the following alkaloids were isolated for
the first time from this species: adlumine, fumaricine, fumaritridine, fumarophycine, fumarofine, co-
rytuberine, cis-N-methylstylopinium iodide, stylopine, and two new 1-benzylisoquinoline alkaloids,
viz. fumaflorine and fumaflorine methyl ester. The cytoprotectivity of extracts of F. densiflora and
F. officinalis, and that of protopine, cryptopine, and parfumine was tested on primary cultures of rat
hepatocytes intoxicated with carbon tetrachloride. The cytoprotective effect of the extracts is associated
with the alkaloids.
Key words: Fumaria densiflora DC.; Isoquinoline alkaloids; Cytoprotective effect.
Fumaria densiflora DC. (F. micrantha) is an annual plant wildly growing in southeastern
Europe and in the Middle East. It is one of the 40 species of the genus Fumaria (Fuma-
riaceae), which have been employed for centuries in folk medicine and some of which
are components of phytotherapeutic preparations exerting choleretic and spasmolytic
effects1,2. The following isoquinoline alkaloids have been identified in F. densiflora:
fumaramine3,4, adlumidiceine, densiflorine, coptisine, cryptopine, palmatine, protopine,
sinactine5, adlumidine, bicuculline fumadensine, fumaridine, fumariline, fumaritine,
hydrastine, isosalutaridine, N-methylhydrasteine, parfumidine, parfumine, and scou-
lerine6. In the context of a phytochemical investigation of the genus Fumaria we re-
examined F. densiflora of Bulgarian origin. The plant was collected at the same site as
in 1983 (ref.5). The current paper deals with the isolation and characterization of alka-
* The author to whom correspondence should be addressed.
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

Fumaria densiflora DC. Alkaloids
1065
loids, with the cytoprotective activity of Fumaria extracts and of three major alkaloids
of this plant, using a model of rat hepatocytes.
EXPERIMENTAL
The melting points were measured on a Mettler FP51 instrument and are not corrected. Thin layer
chromatography (TLC) was performed on TLC plates coated with 60F254 silica gel (Merck) using the
following systems: cyclohexane–diethylamine 9 : 1 (S1), cyclohexane–chloroform–diethylamine 7 : 2 : 1
(S2), methanol–25% ammonia 200 : 1 (S3), and methanol–water–25% ammonia 15 : 3 : 1 (S4). The
alkaloids were detected either with UV radiation at 235 or 326 nm or by spraying with potassium
iodoplatinate(IV). The UV and IR spectra were scanned on Perkin–Elmer PE-552 and ATI MATT-
SON Genesis FT IR spectrometers, respectively. NMR spectra were measured on a Varian VXR-400
spectrometer (400 MHz for 1H NMR, 100 MHz for 13C NMR) at 25 °C. The chemical shifts are
expressed in ppm with respect to tetramethylsilane, the spin–spin interaction constants are in Hz. The
carbon signal multiplicity was measured by APT and DEBT procedures. The mass spectra were ob-
tained on a Finnigan MAT 90 instrument. Alkaloid standards used for identification were obtained
from collections of the Institute of Medical Chemistry in Olomouc and Institute of Biochemistry in
Brno. Hepabene is a preparation of Merckle GmbH.
4
4
5
5
3
2
MeO
MeO
MeO
MeO
6
6
N
N
Me
Me
14
1
12
HO
H
O
16
11
9
O
10
9
O
10
O
O
O
1
2
5
5
4
4
6
6
3
MeO
HO
MeO
HO
N
Me
N
Me
OH
8
OH
O
9
MeO
HO
O
9
13
13
10
O
12
12
O
O
3
4
6
MeO
MeO
3
N
7
COOR
1
8
6’
O
O
1’
O
4’
5,
R = Me
R = H
6,
Collect. Czech. Chem. Commun. (Vol. 61) (1996)

1066
Taborska et al.:
Plant Material
The flowering aerial parts of F. densiflora DC. were collected in the surroundings of Galata (near
Varna, Bulgaria) in May 1994 and identified by Dr M. E. Popova (Institute of Pharmacognosy, Varna
University). A herbal specimen is deposited with the Institute of Medical Chemistry, Faculty of
Medicine, Palacky University in Olomouc.
Extraction and Isolation of Alkaloids
The plant was dried at room temperature. The amount of 2.7 kg of the dry drug was then ground and
extracted with methanol at 60 °C. The volume of the extract was reduced by evaporation in a vacuum
and dissolved in 1% aqueous sulfuric acid. The lipoid fraction was removed by extraction with pe-
troleum ether, and the L-fraction was separated off by extraction with ether7. The aqueous layer was
made alkaline with 40% aqueous NaOH to a pH > 12, and this fraction B was extracted. The remain-
ing aqueous fraction was made acidic to pH 4 with 1 M aqueous H2SO4, and a saturated solution of
potassium iodide was added. The strongly polar alkaloids (fraction I) were extracted into chloroform.
Fraction A was dissolved in 1% H2SO4 and made alkaline with NaOH, and the majority of non-
phenolic tertiary bases (A1) was extracted into ether. The aqueous phase was adjusted to pH 6 and
alkalinized with ammonia. The phenolic bases (A2) were separated by extraction into ether.
Fraction A1
Protopine. Obtained as a crystalline substance (1.46 g); m.p. 219–221 °C (MeOH) (ref.8: 222–225 °C);
1
the UV, H NMR, and 13C NMR spectra were identical with those of the authentic sample.
Cryptopine. Obtained as a crystalline substance (0.37 g), m.p. 187 °C (Me2CO) (ref.8: 185–186 °C);
1
the UV, H NMR, and 13C NMR spectra were identical with those of the authentic sample.
Stylopine. Obtained as a crystalline substance (7.2 mg), m.p. 203–205 °C (EtOH) (ref.8: 204 °C);
1
the UV, H NMR, and 13C NMR spectra were identical with those of the authentic sample.
Sinactine. Obtained as a crystalline substance (12.1 mg), m.p. 168–170 °C (EtOH) (ref.8: 169–170 °C);
1
the UV, H NMR, and 13C NMR spectra were identical with those of the authentic sample.
Adlumine (1). Obtained as a crystalline substance (8.5 mg), m.p. 181–182 °C (MeOH) (ref.8: 178–180 °C),
[αD] +42° (CHCl3). UV spectrum (MeOH), λmax, nm (log ε): 288 (3.97), 324 (3.99). FAB-MS, m/z
1
384 (M + H)+. EI-MS, m/z (%): 206 (100), 190 (10), 177 (3). H NMR spectrum (CDCl3): 2.46 ddd,
1 H, J = 15.4, 3.2, 3.0 (H-5a); 2.56 ddd, 1 H, J = 11.0, 10.9, 3.0 (H-6a); 2.65 s, 3 H (NMe); 2.78 ddd,
1 H, J = 15.4, 11.0, 4.2 (H-5b); 3.08 ddd, 1 H, J = 10.9, 4.2, 3.2 (H-6b); 3.76 s, 3 H (3-OMe); 3.82 s,
3 H (2-OMe); 4.08 d, 1 H, J = 3.4 (H-14); 5.69 dd, 1 H, J = 3.4, 0.9 (H-13); 6.08 s, 2 H (–OCH2O–);
6.38 s, 1 H (H-4); 6.70 s, 1 H (H-1); 6.88 d, 1 H, J = 7.9 (H-11); 7.17 dd, 1 H, J = 7.9, 0.9 (H-12).
Fumariline. Obtained as a crystalline substance (28.7 mg), m.p. 137 °C (MeOH) (ref.8: 138 °C or
1
144 °C); the UV, H NMR and 13C NMR spectra were identical with those of the authentic sample.
Fumaricine (O-methylfumaritine) (2). Obtained as a crystalline substance (13.0 mg), m.p. 173–176 °C
(MeOH) (ref.8: 177 °C); [αD] –31° (CHCl3). UV spectrum, λmax, nm (log ε): 212 (4.56), 290 (3.83).
EI-MS, m/z (%): 369 (C21H23NO5, 36, M+), 354 (73), 338 (16), 336 (6), 322 (12), 206 (100), 190 (9),
153 (4), 136 (5), 89 (6), 77 (10). 1H NMR spectrum (CDCl3): 2.41 s, 3 H (NMe); 2.57 m, 1 H
(H-5a); 2.97–3.06 m, 2 H (H-6a, H-5b); 3.28 dd, 1 H, J = 15.6, 0.9 (H-13a); 3.34 dd, 1 H, J = 15.6,
1.2 (H-13b); 3.51 s, 3 H (2-OMe); 3.78 m, 1 H (H-6b); 3.84 s, 3 H (3-OMe); 4.56 brs, 1 H (H-8);
5.94 d, 1 H, J = 1.4 (–OCH2O–); 5.97 d, 1 H, J = 1.4 (–OCH2O–); 6.41 s, 1 H, (H-1); 6.61 s, 1 H
(H-4); 6.72 ddd, 1 H, J = 7.8, 1.2, 0.9 (H-12); 6.76 dd, 1 H, J = 7.8, 0.8 (H-11). 13C NMR spectrum
(CDCl3): 23.03 t (C-5), 38.15 q (NMe), 43.64 t (C-13), 47.53 t (C-6), 55.52 q (2-OMe), 55.73 q
(3-OMe), 74.51 s (C-7), 82.10 d (C-8), 101.40 t (–OCH2O–), 108.51 d (C-11), 110.03 d (C-1),
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Fumaria densiflora DC. Alkaloids
1067
111.46 d (C-4), 115.48 d (C-12), 124.92 s (C-8a), 125.93 s (C-7a), 128.62 s (C-4a), 134.98 s (C-12a),
144.16 s (C-9), 147.18 s (C-2), 147.31 s (C-10), 148.19 s (C-3).
Bicuculline. Substance identified by TLC, RF 0.47 (S1); 0.63 (S2).
Densiflorine. Substance identified by TLC, RF 0.69 (Sl); 0.86 (S2).
Fraction A2
Parfumine. Obtained as a crystalline substance (52.1 mg), m.p. 110–112 °C (MeOH) (ref.8: 111–112 °C);
1
the UV, H NMR and 13C NMR spectra are identical with those of the authentic sample.
Fumaritridine (3). Obtained as a crystalline substance (18.8 mg), m.p. 202–204 °C (EtOH) (ref.8:
198–200 °C), [αD] +18° (CHCl3). UV spectrum (MeOH), λmax, nm (log ε): 208 (4.65), 230–232
(3.97), 290 (3.66). EI-MS, m/z (%): 369 (75, C21H23NO5, M+), 354 (100), 338 (63), 337 (48), 326
(27), 324 (28), 323 (24), 322 (51), 311 (51), 308 (11), 307 (11), 295 (10), 294 (10), 281 (14), 279
(15), 190 (10), 179 (64), 165 (16), 164 (22), 148 (12). 1H NMR spectrum (CDCl3): 2.26 s, 3 H
(NMe); 2.55 ddd, 1 H, J = 14.3, 5.9, 3.8 (H-6a); 2.88 ddd, 1 H, J = 11.0, 11.0, 5.9 (H-5a); 2.97 ddd,
1 H, J = 11.0, 6.8, 6.8 (H-5b); 3.07 s, 3 H (OMe); 3.21 dddd, 1 H, J = 14.3, 11.0, 6.8, 0.7 (H-6b);
3.33 dd, 1 H, J = 16.4, 1.2 (H-13a); 3.46 d, 1 H, J = 16.4 (H-13b); 3.91 s, 3 H (OMe); 4.56 s, 1 H
(H-8); 5.92 d, 1 H, J = 1.5 (–OCH2O–); 6.02 d, 1 H, J = 1.5 (–OCH2O–); 6.67 s, 1 H (H-4); 6.73 dd,
1 H, J = 7.7, 1.2 (H-12); 6.76 d, 1 H, J = 7.7 (H-11); 7.03 s, 1 H (H-1). 13C NMR spectrum
(CDCl3): 33.19 t, 41.33 q, 42.54 t, 50.81 q, 55.80 t, 55.91 q, 70.72 d, 92.82 s, 100.83 t, 108.39 d,
112.07 d, 115.94 d, 116.85 d, 122.88 s, 130.25 s, 132.22 s, 136.40 s, 143.60 s, 144.63 s, 145.64 s,
146.31 s.
Scoulerine. Obtained as a crystalline substance (5.2 mg), m.p. 197 °C (MeOH) (ref.8: 198–200 °C);
1
the UV, H NMR and 13C NMR spectra are identical with those of the authentic substance.
Fumarofine (4). Obtained as a crystalline substance (1.7 mg), m.p. 244–246 °C (MeOH) (ref.8:
240–245 °C). UV spectrum (MeOH), λmax, nm (log ε): 212 (4.40). 236 (4.54), 245–258 sh (4.06–4.05),
266–275 sh (3.58–3.54), 339 (3.52). EI-MS, m/z (%): 369 (100, C20H19NO6, M+), 354 (55), 326 (21).
298 (11), 284 (28), 283 (13), 204 (13), 192 (24), 190 (12), 179 (17), 177 (13), 164 (14), 162 (15),
1
151 (13), 77 (10). H NMR spectrum (CD3SOCD3): 1.97 m, 1 H; 2.28 m, 1 H; 2.36 m, 1 H; 2.47 m,
1 H; 2.49 s, 3 H (OMe); 3.79 s, 3 H (OMe); 4.22 d, 1 H, J = 1.0 (H-8); 6.21 d, 1 H, J = 1.0
(–CH2O–); 6.22 d, 1 H, J = 1.0 (–OCH2O–); 6.29 s, 1 H (OH); 6.62 s, 1 H (H-4); 7.13 dd, 1 H, J =
1
7.9, 1.0 (H-9); 7.16 s, 1 H (H-1); 7.31 d, 1 H, J = 7.9 (H-10); 8.68 s, 1 H (OH). H NMR spectrum
(CD3OD): 2.25 m, 1 H; 2.43 m, 1 H; 2.54 m, 1 H; 2.58 s, 3 H (NMe); 2.66 m, 1 H; 3.86 s, 3 H
(OMe); 4.61 s, 1 H; 6.71 s, 1 H; 6.92 s, 2 H (–OCH2O–); 7.24 dd, 1 H, J = 7.9, 0.8; 7.26 d, 1 H, J = 7.9;
7.13 s, 1 H. 13C NMR spectrum (CD3SOCD3): 31.92 t, 45.64 q, 48.94 t, 55.80 q, 72.36 d, 83.03 s,
103.13 t, 113.34 d, 114.66 d, 114.91 d, 119.04 s, 120.06 d, 126.18 s, 132.71 s, 141.61 s, 143.94 s,
144.27 s, 146.21 s, 148.24 s. The sample was too dilute for the C=O signal to be observable.
Fumaritine. Obtained as a crystalline substance (18.7 mg), m.p. 155–157 °C (Et2O) (ref.8: 198–200 °C);
1
the UV, H NMR and 13C NMR spectra were identical with those of the authentic sample.
Fumarophycine. Identified by TLC, RF 0.25 (S1); 0.40 (S2).
Fraction B
Coptisine. Obtained in the chloride form (9.1 mg). The tetrahydro derivative, m.p. 204–205 °C,
was obtained by reduction with zinc in hydrochloric acid; the UV, 1H NMR, and 13C NMR spectra
were identical with those of stylopine.
Fumaflorine methyl ester (5). Obtained as a crystalline substance (10.6 mg), m.p. 184–186 °C
(MeOH). UV spectrum (MeOH), λmax, nm (log ε): 213 (4.62), 236 (4.54), 282–290 sh (4.03–4.01),
334 (4.07). EI-MS, m/z (%): 395 (1, C21H17NO7, M+), 364 (5, C20H14NO6), 353 (8), 352 (37,
Collect. Czech. Chem. Commun. (Vol. 61) (1996)