Phytomedicine 10: 300–308, 2003

© Urban & Fischer Verlag

http://www.urbanfischer.de/journals/phytomed
Phytomedicine

In vitro antiproliferative effects on human tumor cell

lines of extracts from the Bangladeshi medicinal plant
Aegle marmelos Correa
I. Lampronti1, D. Martello1, N. Bianchi1, M Borgatti1, E. Lambertini1, R. Piva1, S. Jabbar2,
M. Shahabuddin Kabir Choudhuri2, M. Tareq Hassan Khan2,*, and R. Gambari1,3
1
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy
2
Pharmacology Research Laboratory, Faculty of Pharmaceutical Sciences, University of Science and Technology Chittagong,
Chittagong, Bangladesh
3
Laboratory for the Development of Pharmacological and Pharmacogenomic Therapy of Thalassaemia, Biotechnology Cen-
tre, University of Ferrara, Ferrara, Italy
*
Present address: International Center for Chemical Sciences, H.E.J. Research Institute of Chemistry, University of Karachi,
Karachi, Pakistan

Summary
In the present paper we show that extracts from Aegle marmelos Correa are able to inhibit the in
vitro proliferation of human tumor cell lines, including the leukemic K562, T-lymphoid Jurkat, B-
lymphoid Raji, erythroleukemic HEL, melanoma Colo38, and breast cancer MCF7 and MDA-
MB-231 cell lines. Molecules present within the studied Aegle marmelos C. extracts were identified
by gas-chromatography/mass-spectrometry analysis; three derivatives (butyl p-tolyl sulfide, 6-
methyl-4-chromanone and butylated hydroxyanisole) were found to exhibit strong activity in in-
hibiting in vitro cell growth of human K562 cells. The antiproliferative activity of these compounds
was found to be comparable to that of known antitumor agents, including cisplatin, chromomycin,
cytosine arabinoside and 5-fluorouracil. In addition, the antiproliferative activity of butyl-p-tolyl
sulfide, 6-methyl-4-chromanone and 5-methoxypsolaren was associated to activation of the differ-
entiation pattern of K562 cells.

Key words: Aegle marmelos, antiproliferative activity, anticancer agents, medicinal plants

j Introduction
An increasing number of research papers and reviews
clearly indicate that medicinal plants exhibit a variety
of therapeutic properties (Ahmad et al. 1998; Datta
et al. 1998; Abo et al. 2000; Graf, 2000; Ankli et al.
2002; Neto et al. 2002) and could provide health secu-
rity to rural people in primary health care.
Among medicinal plants, Aegle marmelos Correa ap-
pears to be relevant (Rana et al. 1997; Shoba and
Thomas, 2001). This plant is available in India,
Bangladesh, Burma and Sri Lanka. Its distribution is
mainly within the sub-Himalayan forests, in dry hilly
places ascending to 4,000 ft. It is called “Shivadume”,
the tree of Shiva. Since ancient time, its leaves are of-
fered to Shiva and Parvathi. A. marmelos has an impor-
tant place in indigenous systems of medicine. With re-
spect to pharmacology, alcoholic and aqueous extracts
of the leaves had similar effects as digoxin in amplitude
and contractions of the frog heart and methanolic ex-

0944-7113/03/10/04-300 $ 15.00/0
 In vitro antiproliferative effects on human tumor cell lines
tracts of roots inhibited the beating rate by approxi-
mately 50% of cultured mouse myocardial cells (Kaki-
uchi et al. 1991). Alcoholic extracts of the roots and
fruits showed hypoglycaemic activity (Karunanayake
et al. 1984). With respect to clinical applications, it
should be noted that the roots are astringent, bitter and
febrifuge. They are useful in diarrhoea, dysentery, dys-
pepsia, stomachalgia (Shoba and Thomas, 2001), car-
diopalmus, seminal weakness, vomiting, intermittent
fever and swellings. The leaves of A. marmelos are use-
ful as laxative, febrifuge and expectorant, also in oph-
thalmia, deafness, inflammations, catarrah, diabetes,
asthmatic and antifungal complaints (Rana et al. 1997).
Finally, the effect of these extracts was examined in the
regulation of hyperthyroidism (Kar et al. 2002).
Despite these interesting clinical applications, no re-
ports in the literature are available on the possible ef-
fects of extracts from A. marmelos as antitumor agents.
It should be underlined that several medicinal
plants can give rise to antitumor extracts (Popov et al.
2001; Richardson, 2001; Steenkamp et al. 2001;
Mukherjee et al. 2001; Wargovich et al. 2001; Yu
et al. 2001; Chang et al. 2002; Ruffa et al. 2002; Tat-
man and Mo, 2002). For instance, in a recent study an-
titumor activity of Emblica officinalis has been report-
ed (Jose et al. 2001; Khan et al. 2002). Aqueous ex-
tracts of E. officinalis were found to display cytotoxic
effects on L929 cells in culture in a dose-dependent
manner. Interestingly, E. officinalis extracts were
found to reduce ascites and solid tumors in tumor-
bearing mice (Jose et al. 2001; Sharma et al. 2000;
Jeena et al. 1999).
In order to determine whether A. marmelos Correa
exhibits antiproliferative activity, we analysed
petroleum ether, ethyl acetate and carbontetrachloride
fractions of A. marmelos extracts on chronic myeloge-
nous leukemia K562 (Bianchi et al. 2001), T-lym-
phoid Jurkat (Siddiqui et al. 2001), B-lymphoid Raji
(Pompeia et al. 2001), erythroleukemic HEL (Zhang
et al. 2001), melanoma Colo38 (Corti et al. 1998),
breast cancer MCF7 (Chauvier et al. 2002) and MDA-
MB-231 (Nomoto et al. 2002) human cell lines. The
activity of Aegle marmelos extracts was compared to
that of the already characterized antitumor extracts
from Emblica officinalis (Sharma et al. 2000; Jose et
al. 2001) and to those of Saraca asoka (Varghese et al.
1993), Paederia foetida (De et al. 1994), Cassia so-
phera (Tiwari and Misra, 1975) and Hemidesmus in-
dicus (Qureshi et al. 1997; Prabakan et al. 2000; Rav-
ishankara et al. 2002), all of them well known to ex-
hibit biological activity (either anti-tumor, anti-in-
flammatory or anti-microbial). Bioactive derivatives
were identified by gas-chromatography coupled with
mass-spectrometry (GC/MS) analysis of the fractions
of A. marmelos C.
301
j Materials and Methods
Plant extracts, tumor cell lines and chemical
compounds
The plant extracts were produced as follows. The dried
fruits of Emblica officinalis were extracted with abso-
lute ethanol and the yield was 9.33%. The stem barks of
Aegle marmelos (voucher # EPL-106), Saraca asoka
(voucher # EPL-624), Paederia foetida (voucher #
BNH-25372), and Cassia sophera (voucher # BC-654)
were also extracted with absolute ethanol and the ob-
tained yields were 15.31%, 15.13%, 11.92%, and
12.03%, respectively and the Hemidesmus indicus
(voucher # BC-1083) was extracted with 80% ethanol-
water (8.9% yield). Then the ethanolic extract of the A.
marmelos was further fractionated with petroleum ether
(9.89% yield), carbontetrachloride (8.98%), ethyl ac-
etate (11.04%), methanol (12.08%) and remain aqueous
portion (13.08%). Butylated hydroxyanisole, butyl p-
tolyl sulfide, 6-methyl-4-chromanone, 5,6-dimethoxy-
1-indanone, palmitic acid, methyl linoleate,
5-methoxypsoralen were obtained from Aldrich (Mil-
waukee, WI, USA). Cisplatin, cytosine arabinoside,
chromomycin, 5-fluorouracil were obtained from
Sigma Chemicals Co. (St. Louis, MO, USA).
Assays of in vitro antiproliferative activity
In vitro antiproliferative activity was assayed as fol-
lows. Cell number/ml was determined by using a
model ZBI Coulter Counter (Coulter Electronics,
Hialeah, FL). Usually, cells were seeded at the initial
cell concentration of 3 × 104 cells/ml and the cell num-
ber/ml determined after 2, 3, 4, 5 days of cell culture.
IC50 was determined usually after 4 days, when untreat-
ed cells are in the log phase of cell growth.
Benzidine-staining procedure
Differentiation of K562 cells was analyzed by benzi-
dine-staining procedure using a benzidine/hydrogen
peroxide solution (0.2% benzidine in 5 mol/l glacial
acetic acid, 10% H202), as reported elsewhere (Gambari
et al. 1984).
Gas-chromatography/Mass-spectrometry (GC/MS)
analysis of the fractions of Aegle marmelos extracts
A Fisons (Thermo Finnigan, San Jose, CA) model GC
8000 gas chromatograph interfaced to a Fisons model
MD 800 quadrupole mass spectrometer was used for
all measurements. The fused-silica gas chromatograph-
ic capillary column was a MEGA SE 54 (methyl
phenyl polysiloxanes), 25 m × 0.25 mm I. D. and
0.25 µm film thickness. The head pressure of the carri-
er gas (helium, 99.99% purity) was 50 kPa
(7.2 p.s.i.). One µl of sample dissolved into appropriate
solvents was injected into the gas chromatograph. The
302 I. Lampronti et al.
injector and detector temperatures for the gas chro-
matograph were 250 °C and 300 °C, respectively. The
column oven temperature was increased linearly from
40 °C (held for 4 min.) to 200 °C (held for 10 min.) at
10 °C/min. The mass spectrometer operated at source
and interface temperatures of 250 °C. The ionization
mode was Electron Ionization (E.I.) (70 eV), with an
electron multiplier voltage of 50 V above the “tune”
voltage. The “solvent delay”, the time gap of a given
analysis in which the mass spectrometer is turned off,
was 4 min. The GC/MS system was operated in “full
scan” mode. The software utilized was the Mass Spec-
trometry Data Handling System (version 1.12, Fisons,
Thermo Finnigan, San Jose, CA) with NIST library to
recognize all the derivatives found in plant extracts.
j Results
The petroleum ether fraction of Aegle marmelos ex-
tracts was obtained as a liquid material, while the ethyl
acetate and the carbontetrachloride fractions were ob-
Fig. 1. Effects of increasing amounts of the petroleum ether
fraction of Aegle marmelos extracts on cell proliferation of
K562 cells. K562 cells were seeded at the initial cell concen-
tration of 30.000 cells/ml and then cultured for the indicated
lenght of time in the absence (s) or in the presence of 0.1
(d), 1 (u), 10 (j), 100 (e) µg/ml of the petroleum ether
fraction of Aegle marmelos.
tained as solid materials. The biological activities of
A. marmelos fractions were compared to those of Em-
blica officinalis, which is known to exhibit antitumor
effects (Jose et al. 2001), and with those of other plants,
including Paederia foetida, Saraca asoka, Hemi-
desmus indicus and Cassia sophera.
Effects of the pet-ether fraction of A. marmelos
on in vitro proliferation of human leukemic
K562 cells
Fig. 1 shows the effects of increasing amounts of the
petroleum ether fraction of A. marmelos extracts on
cell proliferation of K562 cells. K562 cells were seed-
ed at the initial cell concentration of 30.000 cells/ml
and then cultured for the indicated length of time in the
absence or in the presence of 0.1–100 µg/ml of the pet-
ether fraction of A. marmelos. As it can be easily noted,
Fig. 2. Effects of increasing concentrations of extracts from
Saraca asoka (s), Paederia foetida (d), Aegle marmelos
(u), Emblica officinalis (j), Cassia sophera (e) and
Hemidesmus indicus (r) on cell growth of K562 cells. Cells
were cultured for 4 days as indicated and the cell number/ml
determined and compared to the value obtained using control
untreated K562 cells.
 In vitro antiproliferative effects on human tumor cell lines
1–10 µg/ml the petroleum ether fraction of A. marme-
los are able to inhibit cell growth of K562 cells, while
100 µg/ml fully suppress cell proliferation. In order to
determine the IC50 (concentrations of extracts leading
to 50% inhibition of K562 cell growth), we determined
the values of cell number/ml after 4 days, when un-
treated cells are in the log phase of cell growth (Khan et
al. 2002).
Fig. 2 shows the results of this experiment. In these
assays, Emblica officinalis, Paederia foetida, Saraca
asoka, Hemidesmus indicus and Cassia sophera ex-
tracts were compared. The data obtained show that the
IC50 of the pet-ether fraction of A. marmelos extracts
was about 25 µg/ml, a value higher than that obtained
when E. officinalis extracts were used (0.4 µg/ml), but
significantly lower than the IC50 values obtained with
Paederia foetida, Hemidesmus indicus and Cassia so-
phera. The extracts from Saraca asoka were unable to
inhibit K562 cell growth at the concentrations used in
this experiment.
Fig. 3. Effects of the petroleum ether fraction (s), ethyl ac-
etate fraction (d) and carbontetrachloride fractions (u) of
Aegle marmelos extracts on K562 cells growth. Comparison
Emblica officinalis extract (j) was performed. Cells were
cultured for 4 days as indicated and the cell number/ml deter-
mined and compared to the value obtained using control un-
treated K562 cells.
303
Effects of the ethyl acetate and the
carbontetrachloride fractions of extracts
of A. marmelos on in vitro proliferation
of human leukemic K562 cells
Fig. 3 shows the IC50 values of the ethyl acetate and the
carbontetrachloride fractions of A. marmelos extracts.
As it can be easily observed, both A. marmelos frac-
tions exhibited antiproliferative activity on K562 cells,
even if lower that E. officinalis extracts. Among the
fractions analysed, the most active was the pet-ether
fraction.
Effects of A. marmelos extracts on in vitro
proliferation of human tumor cell lines
In order to determine the activity of A. marmelos ex-
tracts on different human tumor cell lines, the K562,
Fig. 4. Effects of petroleum ether fraction of the Aegle
marmelos extracts on cell proliferation of different human
tumor cell lines, including myelogenous leukemia K562
(s), B-lymphoid Raji (d), T-lymphoid Jurkat (u), ery-
throleukemia HEL (j), melanoma Colo38 (e), breast cancer
MDA-MB-231 (r) and MCF7 (n) cell lines. Cells were cul-
tured for 4 days as indicated and the cell number/ml deter-
mined and compared to the value obtained using control un-
treated cells.
304 I. Lampronti et al.
B-lymphoid Raji, T-lymphoid Jurkat, ery-
throleukemia HEL, melanoma Colo38,
breast cancer MDA-MB-231 and MCF7
cells were employed. Fig. 4 shows the IC50
values obtained using pet-ether fraction of
A. marmelos and these tumor cell lines. The
IC50 values obtained give evidence for an
activity of A. marmelos extracts on all the
tumor cell lines used.

Gas-chromatography/
Mass-spectrometry analysis
of the fractions of A. marmelos extracts
In order to identify the putative antiprolif-
erative compound present within the
A. marmelos fractions, gas-chromatogra-
phy/mass-spectrometry (GC/MS) (Tom-
kins and Sega, 2001) analysis was per-
formed. Gas chromatography coupled to
mass-spectrometry allows to purify and to
identify single components from biological
extracts. A. marmelos extracts were diluted
to a final volume of 1 ml with methanol
(approximately 1 mg/ml). One µl of each
solution was injected into the gas chro-
matograph with an appropriate microsy-
ringe, chromatographed using a fused-sili-
ca capillary column and analyzed with a
quadrupole mass spectrometric detector.
The resulting chromatograms are shown in
Fig. 5 and indicated that one peak
(r.t. = 19.3 min), corresponding to
6-methyl-4-chromanone, is present in two
fraction (ethyl acetate and carbontetrachlo-
ride). The other identified molecules differ
between the analyzed fractions. The com-
pounds corresponding to the major peaks
identified are shown in Table 1. Several
identified compounds (butylated hydrox-
yanisole, butyl p-tolyl sulfide, 6-methyl-4-
chromanone, 5,6-dimethoxy-1-indanone,
palmitic acid, methyl linoleate, 5-metho-
xypsoralen) were commercially available
and were tested for their antiproliferative
activity.

Antiproliferative activity
of pure compounds identified
within the A. marmelos extracts
We analysed the seven commercially pure
compounds identified within A. marmelos
Fig. 5. Chromatograms of the Aegle marmelos extracts (A: petroleum extracts on K562 cell proliferation in order
ether fraction, B: ethyl acetate fraction, C: carbontetrachloride fraction). to identify active components (see Fig. 6 for
 In vitro antiproliferative effects on human tumor cell lines 305
the chemical structures). The results are reported in in the ethyl acetate fraction. The antiproliferative ef-
Fig. 7. Interestingly, all the molecules exhibited an- fects of the most active compounds were comparable to
tiproliferative activity, even if at different concentra- some of the most commonly used antitumor agents such
tions. The IC50 values are reported in Table 2. As clear- as cisplatin (Bianchi et al. 2000), 5-fluorouracil (Roobol
ly evident, the most active compounds were butyl p- et al. 1984; Ozaki, 1996; Nagy et al. 2002), chro-
tolyl sulfide (7 µM), 6-methyl-4-chromanone (15 µM) momycin (Inoue et al. 1983; Ono et al. 1982; Baguley,
and butylated hydroxyanisole (BHA, 35 µM), present 1982; Bianchi et al. 2001) and ara-C (Grant, 1998;
Bianchi et al. 2001; Winter et al. 1985) (see Table 2).

Effects on K562 differentiation

Fig. 6. Chemical structures of palmitic acid, methyl
linoleate, butylated hydroxyanisole, butyl p-tolyl sulfide, 6-
methyl-4-chromanone, 5,6-dimethoxy-1-indanone and 5-
methoxypsoralen.
Many antitumor compounds are able to exert their an-
tiproliferative activity, by the activation of the terminal
program of differentiation. Therefore, we tested the
ability of both A. marmelos extracts and identified
compounds in inducing differentiation of K562 cells.
After treatment with the analyzed compounds, K562
differentiation was analyzed as reported elsewhere by
benzidine-staining procedure (Gambari et al. 1984).
Two compounds (butyl p-tolyl sulfide and 6-methyl-4-
chromanone) induced erythroid differentiation at con-
centrations higher that those able to give a 50% inhibi-
tion of K562 cell growth; one compound (5-methoxyp-
solaren) was able to induce K562 differentiation when
used at concentrations lower than that causing 50% in-
hibition of K562 cell growth. The other compounds
were unable to induce differentiation. A differential ef-
fect on erythroid induction of K562 cell differentiation

Fig. 7. Effects of commercially pure compounds identified within Aegle marmelos fractions on K562 cell proliferation
(A: petroleum ether fraction, B: ethyl acetate fraction, C: carbontetrachloride fraction). A: palmitic acid (s) and methyl
linoleate (d). B: butylated hydroxyanisole (s), butyl p-tolyl sulfide (d), 6-methyl-4-chromanone (u) and 5,6-dimethoxy-1-
indanone (j). C: 6-methyl-4-chromanone (s) and 5-methoxypsoralen (d). K562 cells were cultured for 4 days as indicated
and the cell number/ml determined and compared to the value obtained using control untreated cells.
306 I. Lampronti et al.
Table 1. Compounds present within Aegle marmelos ex-
tracts and identified by GC-MS.
Fractions of Aegle Identified compounds
marmelos
petroleum ether • palmitic acid
fraction • methyl linoleate
• 7-Isopropyl-4α-methyl-octahydro-
naphthalen-2-one
ethyl acetate • 3-(2-hydroxyphenyl)(E)-2-propenoic
fraction acid
• 2-tert-butylphenol
• butylated hydroxyanisole
• butyl p-tolyl sulfide
• 6-methyl-4-chromanone
• 5,6-dimethoxy-1-indanone
carbontetrachloride • hexachloro ethane
fraction • ethyl phosphonic acid diethyl ester
• 3-(acethoxy)-5,6-epoxy-6-methyl-
(1,2-ethandiyl acetal)-pregnan-20-one
• 6-methyl-4-chromanone
• 3-Methyl-2,3,4,4α,9,9a-hexahydro-
pyrano[2,3-b]indole
• 5-methoxypsoralen
was also noted among known antitumor compounds,
some of which are able to induce differentiation (such
as chromomycin, cytosine arabinoside and cisplatin),
while others (such as 5-fluorouracil) do not exhibit this
property (Table 2 and data not shown).
These results suggest that the antiproliferative activi-
ty of butylated hydroxyanisole, 5,6-dimethoxy-1-in-
danone, palmitic acid and methyl linoleate is not asso-
ciated to induction of differentiation; on the contrary,
the antiproliferative activity of butyl-p-tolyl sulfide,
6-methyl-4-chromanone and 5-methoxypsolaren is as-
sociated to activation of the differentiation pattern of
K562 cells.
j Discussion
A consistent proportion of people in developing coun-
tries depend on traditional medicines for their primary
health needs (Neto et al. 2002; Ankli et al. 2002; Datta
et al. 1998; Ahmad et al. 1998). Accordingly, medicinal
plants are used in prenatal care, in obstetrics, in gynae-
cology, in respiratory disorders, in skin disorders, in
cardiac diseases, in nervous and muscular disorders, in
mental health (Abo et al. 2000; Velazco, 1980; Graf,
2000).
From the data obtained, it is apparent that extracts
from Aegle marmelos Correa are able to inhibit the in
vitro proliferation of K562 (Bianchi et al. 2001),
Table 2. Effects of purified compounds of Aegle marmelos
on K562 cell growth (IC50) and differentiation (% of benzi-
dine-positive cells after 6 days culture at the indicated con-
centrations)
Compounds IC50 (µM) Differentiation
butylated hydroxyanisole 35 6% (1 µM)
butyl p-tolyl sulfide 7 25% (50 µM)
6-methyl-4-chromanone 15 35% (50 µM)
5,6-dimethoxy-1-indanone 70 10% (100 µM)
palmitic acid 85 4% (100 µM)
methyl linoleate 250 4% (200 µM)
5-methoxypsoralen 100 60% (50 µM)
cisplatin 5 75% (8 µM)
5-fluorouracil 50 3% (60 µM)
chromomycin 5 80% (0.2 µM)
cytosine arabinoside 0.25 92% (0.5 µM)
T-lymphoid Jurkat (Siddiqui et al. 2001), B-lymphoid
Raji (Pompeia et al. 2001), erythroleukemic HEL
(Zhang et al. 2001), melanoma Colo38 (Corti et al.
1998), breast cancer MCF7 (Chauvier et al. 2002) and
MDA-MB-231 (Nomoto et al. 2002) human cell lines.
Molecules present within the studied A. marmelos
extracts were identified by gas-chromatography/mass-
spectrometry analysis; three derivatives (butyl p-tolyl
sulfide, 6-methyl-4-chromanone and butylated hydrox-
yanisole) were found to exhibit strong activity in in-
hibiting in vitro cell growth of human K562 cells.
We like to underline that these data demonstrate a
novel property of extracts from A. marmelos, a medici-
nal plant of great relevance in indigenous systems of
medicine for the possible use of both roots, leaves and
fruits in a variety of medical interventions (Rana et al.
1997; Shoba et al. 2001). Despite the interesting clini-
cal applications of A. marmelos extracts, no reports in
the literature are available on their possible effects as
antitumor agents.
Furthermore, our paper allows to identify putative
active compounds presents within A. marmelos ex-
tracts. Among these, butyl p-tolyl sulfide, 6-methyl-4-
chromanone and butylated hydroxyanisole appear to be
the most interesting and exhibit an antiproliferative ef-
fect comparable to that of other antitumor compounds,
including cisplatin, chromomycin, cytosine arabi-
noside and 5-fluorouracil.
Finally, we like to underline that the antiproliferative
activity of butyl-p-tolyl sulfide, 6-methyl-4-chro-
manone and 5-methoxypsolaren is associated to activa-
tion of the differentiation pattern of K562 cells; there-
fore, these compounds could be proposed for further
investigations, in order to develop suitable drugs for
antitumor differentiation therapy.
 In vitro antiproliferative effects on human tumor cell lines
As far as the mechanism of action of A. marmelos
plant extracts and identified bioactive compounds, fur-
ther experiments are needed, both in vitro and in vivo,
including “gene expression profiling” using macro- or
microarray technologies (Nees and Woodworth, 2001;
Chicurel and Dalma-Weiszhausz, 2002; Blower et al.
2002), analysis of possible activation of the apoptotic
pathway (Lee et al. 2001; Giridharan et al. 2002), in-
hibition of angiogenesis and metastasis-associated
genes (Menon et al. 1997; Popov et al. 2001), im-
munostimulating effects (Popov et al. 2001; Sai Ram
et al. 2002).
Acknowledgements
R.G. is granted by CNR-P.F. Biotecnologie, CNR Agenzia
2000 and Finalized Research funds (year 2001) from the Ital-
ian Ministry of Health. M.T.H.K. is supported by the “Third
World Academy of Sciences (TWAS)” for his visit to Pakistan.
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j Address
R. Gambari, Dipartimento di Biochimica e Biologia
Molecolare, University of Ferrara, Via L.Borsari n. 46,
44100 Ferrara, Italy
Tel: ++39-532-291448; Fax: ++39-532-202723;
e-mail: gam@unife.it