Cytotoxicity evaluation of natural coptisine and synthesis of coptisine from berberine

Article abstract of DOI:10.1016/S0014-827X(01)01121-1

The crude extract (80% MeOH in water) of Chelidonii herba exhibited very interesting cytotoxicity against brine shrimp (Artemia salina Leach) nauplii and cultured human tumour cell in vitro, the colon carcinoma HT 29 (144 h treatment). Fractionation of the crude extract and bioassay-guided procedures showed that the cytotoxic and the antitumour activities were concentrated in the basic extract. On the basis of IR, MS and ¹H NMR the compound responsible of the cytotoxic activity was determined to be coptisine. Cytotoxicity evaluation of coptisine was next extended to a panel of human and murine cell lines in comparison with the established antitumour drugs mitoxantrone, doxorubicin (Dx) and cisplatin (CDDP). Coptisine was cytotoxic on LoVo and HT 29 and less potent on L-1210, and it was partially crossresistant on the human tumour colon cell line resistant to Dx, LoVo/Dx, whereas it was not significantly crossresistant on the murine leukaemia cell line resistant to CDDP, L-1210/CDDP. Coptisine alkaloid was then synthesised in gram amount from commercial berberine. A four-step synthetic route was elaborated. The overall yield was about 8-10%. The structural identity of synthetic coptisine was verified by IR and NMR methods. A comparison of the cytotoxic effects on the human tumour colon cell line LoVo and on the murine leukaemia L1210 showed, for both natural and synthetic coptisines, a comparable cytotoxic activity more evident against HT 29 cell line and LoVo cell line, while the activity was lower against the L1210 cell line. Copyright

Full text of DOI:10.1016/S0014-827X(01)01121-1

www.elsevier.nl/locate/farmac
Il Farmaco 56 (2001) 403–409
Cytotoxicity evaluation of natural coptisine and synthesis of
coptisine from berberineꢀ
Maria Laura Colombo a,*, Carlo Bugatti b, Andrea Mossa, Nicoletta Pescalli b,
Laura Piazzoni b, Gabriella Pezzoni b, Ernesto Menta b, Silvano Spinelli b,
Francis Johnson c, Ramesh C. Gupta c, Lakkaraju Dasaradhi c
a Department of Plant Biology, Faculty of Pharmacy, Uni6ersity of Turin, Via Mattioli 25, 10125 Turin, Italy
b No6usPharma, Research and De6elopment Center, Monza, Milan, Italy
c Chem-Master International Inc., East Setauket, NY, USA
Received 31 October 2000; accepted 10 January 2001
Abstract
The crude extract (80% MeOH in water) of Chelidonii herba exhibited very interesting cytotoxicity against brine shrimp
(Artemia salina Leach) nauplii and cultured human tumour cell in vitro, the colon carcinoma HT 29 (144 h treatment).
Fractionation of the crude extract and bioassay-guided procedures showed that the cytotoxic and the antitumour activities were
1
concentrated in the basic extract. On the basis of IR, MS and H NMR the compound responsible of the cytotoxic activity was
determined to be coptisine. Cytotoxicity evaluation of coptisine was next extended to a panel of human and murine cell lines in
comparison with the established antitumour drugs mitoxantrone, doxorubicin (Dx) and cisplatin (CDDP). Coptisine was cytotoxic
on LoVo and HT 29 and less potent on L-1210, and it was partially crossresistant on the human tumour colon cell line resistant
to Dx, LoVo/Dx, whereas it was not significantly crossresistant on the murine leukaemia cell line resistant to CDDP,
L-1210/CDDP. Coptisine alkaloid was then synthesised in gram amount from commercial berberine. A four-step synthetic route
was elaborated. The overall yield was about 8–10%. The structural identity of synthetic coptisine was verified by IR and NMR
methods. A comparison of the cytotoxic effects on the human tumour colon cell line LoVo and on the murine leukaemia L1210
showed, for both natural and synthetic coptisines, a comparable cytotoxic activity more evident against HT 29 cell line and LoVo
´
cell line, while the activity was lower against the L1210 cell line. © 2001 Editions scientifiques et me´dicales Elsevier SAS
Keywords: Chelidonium majus; Protoberberine alkaloids; Coptisine; Synthesis; Cytotoxicity
1. Introduction
C. majus, Greater Celandine, a traditional medicinal
herb widespread in Europe, represents a rich source of
isoquinoline alkaloids and flavonoids [1], employed lo-
cally to remove warts, papillomas and condylomas [2].
Under the presumption that certain alkaloid con-
stituents contained in C. majus exhibit inhibitory prop-
erties on cancer cell growth, our attention was focused
on the toxicity of the basic extract. The natural com-
pound coptisine was isolated from the basic extract and
identified as the cytotoxic component of C. majus aerial
parts. The alkaloid coptisine was then synthesised in
gram amount from berberine.
In a program of evaluation of the antiproliferative
activity of plants, a large number of extracts from
higher plants, mainly belonging to the mediterranean
flora, were tested. A preliminary screening was per-
formed against brine shrimp (Artemia salina Leach.)
nauplii and a cultured human tumour cell in vitro, the
colon carcinoma HT 29.
Among the tested plants, Chelidonium majus basic
extract showed interesting cytotoxicity in replicate tests.
Natural and synthetic coptisine were tested in an
enlarged tumour cell line panel: carcinoma cell line
LoVo and its subline resistant to doxorubicin (Dx),
murine leukaemia cell line L1210 and its subline resis-
tant to cisplatin (CDDP).
ꢀ Giornata di Studio SIPHAR (Italian Society for Pharmacog-
nosy), Assisi, (Italy), 19 September 2000.
* Corresponding author.
E-mail address: marialaura.colombo@unimi.it (M.L. Colombo).
´
0014-827X/01/$ - see front matter © 2001 Editions scientifiques et me´dicales Elsevier SAS
PII: S0014-827X(01)01121-1

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M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
2. Materials and methods
2.1. Extraction scheme
coptisine, on the basis of MS and IR data. MS and
NMR spectra and biological activity of recovered cop-
tisine were performed in comparison to berberine chlo-
ride (Sigma Chemical Co., UK), a protoberberine
alkaloid structurally related to the coptisine.
Plant material was extracted according to the Har-
borne procedure [3]. Briefly, the vegetable tissue was
reduced into small particles of uniform size in a ho-
mogeniser: 35 g of fresh material was treated with 200
ml MeOH–H2O (2:1 v/v) at 4°C for 5 min. The hy-
droalcoholic extract was recovered by filtration. The
vegetable material was extracted again with 100 ml of
the above solution and filtered. The two hydroalcoholic
extracts were mixed and reduced in volume (1/10) to
obtain the crude extract (CE). The CE was then aci-
dified with 2 N H2SO4 (pH 2) and then extracted in a
separatory funnel with CHCl3 (50 ml×3). The mixed
organic phases were treated with anhydrous sodium
sulfate, filtered and reduced in volume (1/10). This step
gave the moderately polar extract (MPE). The residual
aqueous phase was alkalinised with 30% NH4OH (pH
9) and then extracted a first time with 50 ml of CHCl3
and then with CHCl3–MeOH (50 ml×2). The three
organic phases were mixed, treated with anhydrous
sodium sulfate and reduced in volume (1/10). The or-
ganic extract was defined as the basic extract (BE).
The CE, MPE and BE extracts were first monitored
by TLC (SiO2 F254, Merck, Darmstadt, Germany). The
mobile phase was CHCl3–MeOH–H2O–CH3COOH
(85:15:1:2 v/v).
2.3. Cytotoxicity assay procedure
The CE, MPE and BE crude extracts were first tested
against A. salina Leach (brine shrimp) nauplii, and
cultured human tumour cell, the colon carcinoma HT
29.
1. The bioassay against A. salina Leach, brine shrimp
nauplii, was performed as described previously [5] and
developed with minor modifications. Cysts were from
Aquafauna Bio-Marine Inc., Hawthorne, CA. To
obtain nauplii the dry cysts (ca 50 mg) were hatched
in artificial sea water prepared from sea salt (Sigma)
40 g/l in double-distilled water, in a rectangular box
(20×30 cm) in a warm room (25–27°C) in the
darkness. After 48 h incubation, the nauplii were
collected by pipette after attracting them with a light
source and after separation from their shells. Plant
extract cytotoxicity was evaluated in a range of 1000,
100 and 10 mg/ml, each test was made on five groups
of 10 animals for each dose of toxin, with a final test
mixture of 5 ml. The plant extracts were dissolved in
DMSO (stock solution) prior to dilution. The maxi-
mum DMSO concentration was 0.5%. Negative con-
trols using 0.5% DMSO were used. Each assay was
performed on nauplii after 48 h. Food (yeast) was
added during the hatching not during test procedure.
Survival data were evaluated as ‘immobility’. In the
cases where control deaths occurred the data were
corrected using Abbott’s formula:% death=[(test−
control)/control]×100. LD50 was determined from
the 24 h counts using the probit analysis method.
2. Cultured human tumour cell (colon carcinoma HT
29 line) established cultures have been propagated
in McCoy’s 5a medium with 15% foetal bovine
serum.
The CE, MPE and BE extracts were subsequently
monitored by HPLC under isocratic conditions.
Column: Alltech silica gel (250×4 mm i.d., 10 mm
particle size). The mobile phase was 0.015 M sodium
acetate in acetic acid–methanol–1,4 dioxane–iso-
propanol (2:68:10:20 v/v). Detection was at 280 nm,
with a flow rate of 1.5 ml/min [4].
2.2. Fractionation of the BE extract
The BE extract (270 mg) adsorbed on celite was
fractionated on SiO2 packed column. SiO2 was imbibed
with
CHCl3–MeOH–H2O–CH3COOH
conc.
(90:10:1:1 v/v) with slow addition of methanol until the
solvent solution was clear. We collected 60 fractions
(8–10 ml each) before coptisine isolation.
2.4. Enlarged cancer cell line panel
Then the column chromatography mobile phase was
changed in order to elute other more polar components.
The new mobile phase CHCl3–MeOH (50:50 v/v) al-
lowed the collection of 10 fractions (8–10 ml) from 61
to 70. Eventually, the column chromatogram was
washed with 100% methanol, thus obtaining 10 frac-
tions from 71 to 80.
The TLC system showed that fractions 64–66 had a
yellow–green spot as the main component; these frac-
tions were then pooled together. The main component
was isolated, crystallised as a chloride and identified as
1. Human tumour colon carcinoma cell line LoVo
and its subline resistant to Dx were cultured in Ham’s
F12 medium supplemented with 10% foetal calf
serum+1% l-glutamine (200 mM)+1% antibiotic
solution+2% HEPES buffer.
2. Murine leukaemia cell line L1210 and its subline
resistant to CDDP L1210/Pt were cultured in RPMI
medium supplemented with 20% foetal calf serum+
1% l-glutamine (200 mM)+0.5% b-mercaptoethanol.
All the cell lines were maintained at 37°C in an
atmosphere of 5% CO2 and 90% relative humidity.

M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
405
2.5. MTT assay
Adam’s catalyst (200 mg) in Parr hydrogenator at 3–4
atm. The absorption of 2 mol of hydrogen took 10–12
h. At the end of the reduction the solution was diluted
with water (150 ml) and the catalyst filtered off. The
filtrate was basified using aq. ammonia (100 ml) and the
almost white tetrahydroberberine chloride was collected
by filtration. Recrystallisation with hot ethanol gave
tetrahydroberberine (2) as colourless crystals (7 g) in
85% yield. The TLC solvent system was hexane–ethyl
acetate, 8:2, Rf=0.8 (see Scheme 1).
The cytotoxic activity of the compounds was deter-
mined by the MTT colorimetric assay. The MTT col-
orimetric assay [6] is based on the mitochondrial
reduction of tetrazolium salt by living cells. The viable
cell number is proportional to the production of forma-
zan salts. The crystals of formazan were dissolved in
DMSO and the optical density was measured spec-
trophotometrically (Microplates reader, Series 750,
Cambridge Technology Inc.).
2.6. Experimental procedures in the synthesis of coptisine
2.6.2. Second step
For the preparation of tetrahydroxytetrahydrober-
berine chloride (3) to a stirred solution of tetrahy-
droberberine (2) (3.7 g, 9.8 mmol) in dry DCM (120 ml)
under nitrogen at −40°C, a solution of 1 M borontri-
bromide in DCM (60 ml, 59 mmol) was added over a
2.6.1. First step
For the preparation of tetrahydroberberine (2)
berberine chloride (1) (10 g) suspended in 50% aq.
acetic acid (150 ml) was hydrogenated in presence of
Scheme 1. Four-step procedure in the synthesis of coptisine.

406
M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
Table 1
and the filtrate was concentrated at reduced pressure.
The dark-coloured residue was diluted with water (50
ml) and extracted with ether (3×100 ml). The com-
bined organic layers were washed with water, dried
over anhydrous MgSO4, filtered and concentrated to
furnish a yellow solid. Recrystallisation with ethyl ace-
tate gave the tetrahydrocoptisine (4) (1.5 g, 20%). The
TLC solvent system was hexane–ethyl acetate, 8:2,
Rf=0.6.
Chemical and physical data of the synthesised coptisine
1%
1 cm
UV
umax
E
m
Solvent HCl 0.1 N in
MeOH–H2O
266
494
−22 115
357
458
506
113
22 630
5039
FT-IR
KBr pellets
(cm−1
3037
2899
1508
1478
1215
1057
1039
)
2.6.4. Fourth step
For the preparation of coptisine iodide (5) a mixture
of tetrahydrocoptisine (4) (0.65 g, 1.8 mmol) and
sodium acetate (2.08 g, 2.5 mmol) was dissolved in a 2:1
mixture of boiling ethanol and acetic acid (30 ml); into
this warm solution at 50°C a solution of iodine (1.04 g,
4.05 mmol) in ethanol (20 ml) was added slowly over a
period of 20 min. The mixture was stirred overnight at
r.t. and the precipitated coptisine iodide (5) was re-
moved by filtration and washed with ethanol. It was
further purified by heating in aq. acetone to give cop-
tisine iodide (5) as yellow crystal (0.55 g, 67%) decom-
posing at 285°C.
1H NMR
Solvent: DMSO-d6
(ppm)
9.954
8.958
8.032
7.838
7.793
7.088
6.541
6.176
4.875
3.200
s
s
d
s
1H
1H
1H
1H
1H
1H
2H
2H
2H
2H
m
s
s
s
m
m
Chemical and physical data of synthetic coptisine are
summarised in Table 1.
Elemental analysis
% Theor.
51.03
3.16
3.13
28.38
% Found
50.35
3.21
3.07
27.50
C
H
N
I
3. Results
HPLC
r.t.: 13.8°
Plant crude extracts obtained from plant material
following a standard procedure [3] were tested in a
preliminary step using a rapid cytotoxic-like assay: A.
salina Leach, brine shrimp nauplii, and the antiprolifer-
ative test against HT 29 human colon carcinoma cul-
tured cells. The biological activity of the methanolic
extract of papaveraceous C. majus was significantly
interesting against brine shrimp (A. salina Leach) nau-
plii and colon carcinoma HT 29 cells. Fractionation of
the crude extract and bioassay-guided procedures
showed that the cytotoxic activity was concentrated in
the basic extract LD50=250 mg/ml in A. salina and
IC50=1.14 mg/ml in colon carcinoma HT 29 cell
proliferation.
Conditions — Column: TOSOHAAS ODS; 25 cm×4.6 mm; p.s.:
5 mm; Eluent: water–acetonitrile–dioxan, 65:30:5 with 20 mM
KH2PO4+5 mM octansulfonic acid sodium salt; pH 2.7 (H3PO4).
Flow eluent: 1 ml/min. Detector UV at 240 nm
m.p. (DSC)\270°C
period of 20 min so that a constant temperature of
−40°C was maintained throughout the addition. The
reaction was allowed to come to room temperature
(r.t.) and stirred for a further 12 h. After cooling the
reaction mixture to 0°C the excess of borontribromide
was quenched by slow addition of methanol (50 ml).
The reaction mixture was concentrated to half its vol-
ume and the insoluble tetrahydroxy compound (3) was
removed by filtration and washed with DCM (20 ml).
The crude tetrahydroxytetrahydroberberine (3) (3.2 g,
96%) was used as such in the next step without any
further purification.
Solvent partitioning of the basic extract concentrated
the cancer cell growth inhibitory activity into a
methanol-soluble fraction that was further separated by
column chromatography (see Section 2). The chromato-
graphic separation over a large silica gel column of the
BE yielded an active fraction in which the cytotoxicity
was concentrated: LD50 98 mg/ml (A. salina) and IC50
0.49 mg/ml (HT 29 cell line). After purification and
crystallisation of the active fraction, yellow fine needles
of an alkaloid compound were obtained. The com-
pound was identified as coptisine (1) on the basis of
2.6.3. Third step
A solution of 3 (7.05 g, 19 mmol) in dry DMF (100
ml) under nitrogen was heated to 60°C and a solution
of bromochloromethane (2.5 ml, 39 mmol) in DMF (5
ml) was added over a period of 10 min. The tempera-
ture of the reaction was raised to 95°C and left for 24
h. After cooling to r.t., the inorganics were filtered off
1
TLC, IR, MS and H NMR data, by comparison with
berberine (2), a protoberberine alkaloid structurally
related to coptisine (Fig. 1).

M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
407
The isolated coptisine was tested against an enlarged
panel of cultured human cancer cells carcinoma cell line
LoVo and its subline resistant to Dx and against
murine leukaemia cell line L1210 and its subline resis-
tant to CDDP. The results of activities were sum-
marised in Fig. 2. Coptisine showed
a similar
cytotoxicity against the LoVo cell line (IC50=0.87 mg/
ml) and L1210 (IC50=0.87 mg/ml), its cytotoxicity nev-
ertheless was twice higher on HT 29 cell line
(IC50=0.49 mg/ml). The alkaloid berberine, tested in
comparison with coptisine, was more powerful on the
leukaemic line (IC50=0.33 mg/ml) while it had a lower
activity on HT 29 and LoVo cells (IC50=1.89 and 1.90
mg/ml, respectively). As regards the crossresistance with
CDDP on L12010/CDDP cells or with Dx on LoVo/Dx
cells, the isolated coptisine is partially crossresistant.
To confirm the identification of the activity and
provide more material for biological testing the synthe-
sis seemed more practical than re-isolation. The alka-
loid coptisine was then synthesised from commercial
berberine. To our knowledge, there are no early reports
on coptisine synthesis. The only mention is in a foot-
note of a Japanese paper where the authors stated that
the conversion of berberine to coptisine will be possible
and that it would be published in a separate paper,
which has never appeared [7].
Fig. 1. (a) Coptisine chemical structure; and (b) berberine chemical
structure.
Fig. 2. IC50 values (mg/ml) of natural coptisine and berberine evaluated on HT 29, LoVo and LoVo/Dx, L1210 and L1210/CDDP tumour cell
lines.

408
M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
Fig. 3. IC50 values (mg/ml) of natural coptisine, synthetic coptisine and berberine evaluated on HT 29, LoVo and LoVo/Dx, L1210 and
L1210/CDDP tumour cell lines.
The synthesis of coptisine was achieved in four steps
starting from berberine chloride (1). Hydrogenation of
1 in presence of Adam’s catalyst gave the tetrahy-
droberberine chloride (2) in 85% yield. The conversion
of 2 to the tetrahydroxy derivative (3) was brought
about using boron tribromide in 96% yield. An attempt
to bis methylate 3 to obtain the tetrahydrocoptisine (4)
under PTC conditions using adogen as catalyst was
futile. The bismethylation was accomplished in 20%
yield using caesium fluoride and bromochloromethane
in DMF. The oxidation of 4 using iodine in acetic acid
gave the desired target molecule coptisine iodide (5) in
67% yield. Chemical and physical data of the synthetic
material matched those of the product isolated from the
plant, as reported in Scheme 1.
(Fig. 3): coptisine is very toxic on HT 29 cells (natural
IC50=0.66 mg/ml and synthetic IC50=0.88 mg/ml).
Coptisine had an antiproliferative activity on LoVo
cells: natural IC50=2.38 mg/ml and synthetic IC50=5.2
mg/ml. On L1210 cell line berberine is more toxic than
natural and synthetic coptisine: IC50=0.37 mg/ml for
berberine and IC50=1.02 mg/ml for natural coptisine
and IC50=3.82 mg/ml for synthetic coptisine. In all the
considered tests it is seen that natural coptisine seemed
more active than the synthetic one. Perhaps the natural
coptisine, recrystallised as chloride, was more soluble in
water than the synthesised iodide salt.
The cytotoxicity results of natural and synthetic cop-
tisine are summarised below
The cytotoxicity of natural coptisine and synthetic
coptisine iodide was evaluated:
“ versus berberine and Dx on human colon carcinoma
cell line LoVo and its subline resistant to Dx, LoVo/
Dx;
LoVo Mitox\Dx\CDDP\coptisine\berberine
HT 29 Mitox\Dx\CDDP=coptisine\berberine
L1210 Mitox\Dx\berberine\coptisine]CDDP
In conclusion we can affirm that the alkaloid cop-
tisine has been identified as the most significant cancer
cell growth inhibitory substance from the aerial parts of
C. majus and should deserve further studies to assess its
antitumour efficacy.
“ versus berberine and CDDP on murine leukaemia
cell line L1210 and its subline resistant to CDDP,
L1210/Pt.
On the basis of all the developed tests, natural and
synthetic coptisines showed a similar cytotoxicity trend
.

M.L. Colombo et al. / Il Farmaco 56 (2001) 403–409
409
chromatographic separation of quaternary alkaloids of Chelido-
nium majus L. root, J. Chromatogr. 393 (1987) 312–316.
[5] B.N. Meyer, N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, D.E.
Nichols, J.L. McLaughlin, Brine shrimp: a convenient general
bioassay for active plant constituents, Planta Medica 45 (1982)
31–34.
[6] T. Mosmann, Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays, J.
Immunol. Methods 65 (1983) 55–63.
References
[1] M.L. Colombo, E. Bosisio, Pharmacological activities of Che-
lidonium majus L. (Papaveraceae), Pharm. Res. 33 (1996) 127–
134.
[2] J.L. Hartwell, Plants used against cancer. A survey, Lloydia,
1967–1971.
[3] J.B. Harborne, Phytochemical Analysis, Chapman and Hall,
London, 1984.
[4] C. Bugatti, M.L. Colombo, F. Tome`, High performance liquid
[7] M. Hanaoka, Chem. Pharm. Bull. 30 (1982) 3446–3449 (foot-
note c15).
.