Biotransformation of the flavonoid tiliroside to 7-methylether tiliroside: Bioactivity of this metabolite and of its acetylated derivative

Article abstract of DOI:10.1007/s000180050076

Incubation of kaempferol-3-O-β-D-(6'-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) with Aspergillus nidulans gives the 7-methyl ether of tiliroside (2) which is a new compound. Its structure is determined by spectroscopic methods. Cytotoxic studies of 2

Full text of DOI:10.1007/s000180050076

CMLS, Cell. mol. life sci. 53 (1997) 587–592
1420-682X/97/070587-06 $ 1.50+0.20/0
Biotransformation of the flavonoid tiliroside to 7-methylether tiliroside: bioactivity of this
metabolite and of its acetylated derivative
C. Demetzosa,*, P. Magiatisa, M. A. Typasb, K. Dimasc, R. Sotiriadouc, S. Perezc and D. Kokkinopoulosc
aLaboratory of Pharmacognosy, Department of Pharmacy, Uni6ersity of Athens, GR-157 71 Athens (Greece)
bDi6ision of Genetics and Biotechnology, GR-157 01 Athens (Greece), e-mail: cdemetzo@atlas.uoa.gr
cHellenic Anticancer Institute, Department of Immunology, 171 Alexandras A6e., GR-115 22 Athens (Greece)
Recei6ed 18 February 1997; recei6ed after re6ision 8 April 1997; accepted 6 May 1997
Abstract. Incubation of kaempferol-3-O-i-D-(6¦-E-p-coumaroyl)-glucopyranoside (tiliroside) (1) with Aspergillus
nidulans gives the 7-methyl ether of tiliroside (2) which is a new compound. Its structure is determined by
spectroscopic methods. Cytotoxic studies of 2 and of its acetylated derivative 2a were carried out in vitro against
fourteen human leukemic cell lines. Results clearly show that compound 2 is ineffective against all leukemic cell
lines tested. On the contrary, compound 2a exhibited cytotoxic activity against four of the cell lines (HL60,
DAUDI, HUT78 and MOLT3) and additionally, a dose- and time-dependent effect on DNA synthesis.
Key words. Aspergillus nidulans; tiliroside; kaempferol glycosides; biotransformation; cytotoxicity; DNA synthesis.
Biotransformations are useful tools for the production distilled H2O (1000 ml) served as minimal medium
of medicinal chemicals from natural products. Such a (MM). Complete medium (CM) was prepared by addi-
product is kaempferol-3-O-i-D-(6¦-E-p-coumaroyl)- tion of yeast extract (15 g), malt extract (15 g) and
glucopyranoside (tiliroside) (1), which has been isolated bacteriological peptone (15 g) per 1000 ml of MM. The
from natural sources [1–3]. Flavonoids are known to ability of A. nidulans to metabolize substance 1 was
exhibit a broad spectrum of pharmacological activities, examined in initial screening tests. A series of conical
including antitumor, antimicrobial, antiviral, central flasks containing either CM or MM with the addition of
vascular system and enzyme inhibiting activities [4]. 0.25, 0.5, 0.75, 1, 1.5 and 3 mg/ml of 1, were inoculated
Our knowledge of the microbial transformation of with identical inocula of A. nidulans spores and optimal
flavonoids, especially flavonoid glycosides, is very lim- experimental conditions (MM +1.5 mg/ml of 1) were
ited at present. Aspergillus niger has been used success- established. A time course of the consumption of 1 and
fully to transform in vivo flavone, flavanone and its biotransformation into new substances was followed
isoflavone [5, 6]. Isolates of the genus of Aspergilli are by collecting the content of two independent flasks
capable of producing extracellular enzymes that de- (from a series of flasks), at 3 h intervals, until the
grade flavonols [7]. Compound 1 is known to be an cultures had passed late exponential phase (i.e. several
inactive product against a panel of human cell lines [1, hours in the stationary phase). The samples were cen-
8]. Some metabolites may be more active than their trifuged and the resulting supernatant was condensed by
parent compounds [9], and our work examines the lyophilization. TLC (silica gel) was used as an analytical
biotransformation of 1 by A. nidulans in order to study method. UV-Vis was used as a method for percentage
the cytotoxicity of its metabolites. Thus, we report in determination of the uptake of compound 1.
this paper the structure of the metabolic product 7-
methylether tiliroside (2) as well as its cytotoxic activity
and effect on DNA synthesis together with its acety-
lated derivative 2a.
Materials and methods
Microorganism and culture conditions. A prototrophic
strain of A. nidulans [10] was used throughout this work.
The fungus was grown at 37 °C under continuous shak-
ing conditions (200 rpm). A chemically defined medium
containing : sucrose (2 g) , NaNO3 (2 g), MgSO4 7H2O
(1 g), KH2PO4 (1 g), KCl (0.5 g), FeCl3 (0.01 g) in
* Corresponding author.

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The resulting mycelial pellets were washed three times line or PBML (1×106 cells/ml final cell density). An
equivalent amount of DMSO was added to control
cultures. After the addition of the flavonoids the cells
were cultured in microplates for 48 hours in a moist
atmosphere of 5% CO2 in air. Cytotoxicity was deter-
mined by the MTT method [28, 29]. Four hours before
the end of the 48 h incubation period, MTT (3-(4,5-
dimethylthiazol-2-yl)-2-5 diphenyl tetrazolium bromide,
Sigma) dissolved in PBS (phosphate buffered saline)
at 5 mg/ml, was added in the cell cultures at a final
concentration of 50 mg/ml. At the end of the 48 h
incubation period, acid isopropanol was added to the
wells and the optical density was measured with an
ANTHOS HT II Microelisa reader, using a test wave-
length of 550 nm. Using this method the lethal dose 50
(LD50) (i.e. the concentration that provoked a 50%
death rate at the cell population compared to the con-
trol) was determined. Data were analysed using a two-
tailed Student’s t-test.
Measurements of DNA synthesis. DNA synthesis was
assayed at 1, 4, 24 and 48 h after the addition of the
compound to be tested. The cells were incubated for
1 h, with 10 mCi of [3H]thymidine (Amersham, U.K.),
before the end of each interval under the same condi-
tions as for the MTT assay. At the end of the incuba-
tion period the cells were harvested in an automatic cell
harvester and the amount of radioactivity incorporated
into macromolecules was measured in a liquid scintilla-
tion counter (Packard IL) and expressed as counts per
minute (CPM). The compound 2a was tested at three
concentrations 2, 10 and 20 mg/ml. Doses higher than
20 mg/ml were found to be significantly cytotoxic for
resting PBML. Therefore 20 mg/ml of 2a was the highest
concentration examined in this kind of experiment.
in Ringer’s solution, broken by sonication and their
cytosolic content including 1 and new substances was
extracted with MeOH. The purity of the compounds
were examined by TLC on silica gel (Kieselgel 60 F254
Merck) with CH2Cl2-MeOH (99:1–90:10). Stock solu-
tions of 1 were made up in DMF. To determine the
nature of new substances prepared from 1, larger vol-
ume cultures were employed in flasks containing a total
of 800 mg of 1.
Isolation of 2. The mycelial pellets of A. nidulans were
extracted with MeOH. The residue (600 mg) was chro-
matographed over 20 g silica gel, using CH2Cl2 and
CH2Cl2-MeOH mixtures of increasing polarity followed
by pure MeOH. A total of 200 fractions were obtained.
Fractions 160–185 (×20 ml) were examined by HPLC
with a gradient system consisting of two LKB2248
pumps. Compound 2 was detected with a single wave-
length absorbance detector at 311 nm and was injected
on a Techsil 10C18 column (25 cm×7.8 mm). Mixtures
of MeOH-AcOH 5% from 40% to 50% in 20 min were
used as eluent at a flow rate of 4 ml/min. 7-methylether
tiliroside (2) was obtained as yellow powder (24 mg).
Compound 2 was dissolved in Ac2O-Py and left for
48 h at room temperature to yield 2a.
1H- and 13C-NMR spectra were recorded on a Brucker
HX 200 spectrometer operating at 200 and 50 MHz
respectively; UV spectra were taken on UV-160A
Shimadzu spectrometer; MS were recorded with a
Nermag R 1010C spectrometer. Thioglycerol was used as
a matrix.
Cytotoxic activity. Compound 1 is known to be an
inactive product against a panel of human and murine
cell lines [8]. Thus for reasons of comparison we exam-
ined the metabolic compound 2 and its acetylated
derivative 2a for cytotoxic activity on leukemic cells.
The following cell lines have been used: CCRF-CEM
[11, 12], MOLT3 [13], JURKAT [14], HUT78 [15], H9
[16] , (T cells) KM3 [17], NAMALWA [18], DAUDI
[19, 20], SDK [21], JIYOYE [22] , CCRF-SB [23] (B cell
lines), HL60 [24] (promyelocytic cell line), K562 [25]
(proerythrocytes), U937 [26] (monocytes). All cell lines
were grown as exponentially proliferating suspension
cultures in RPMI-1640 medium (Gibco Europe Ltd,
Scotland), supplemented with 10% heat inactivated fetal
calf serum (Myoclone Gibco), 2 mM L-glutamine
(Gibco), and 50 mg/ml gentamycin and incubated at
37 °C, in a humidified atmosphere and 5% CO2. Viabil-
ity was assessed by trypan blue dye exclusion and was
always greater than 98%. Peripheral blood mononuclear
leukocytes (PBML) were obtained from the peripheral
blood of normal volunteers after Ficoll-Hypaque cen-
trifugation [27]. The compounds to be tested were dis-
solved in DMSO at the appropriate concentrations and
stored at −40 °C. To determine their cytotoxic activity,
Results and discussion
Identification of compound 2. Compound 2 had
a
molecular formula C31H28O13. The UV (MeOH) spec-
trum showed absorption maxima at 268 and 315 nm.
On addition of base (NaOAc), no bathochromic shift
was observed in contrast to 1, suggesting that it is
indeed a 7-hydroxyl derivative of flavonoid. The 1H-
and 13C-NMR chemical shifts of 2 (tables 1 and 2) were
assigned on the basis of H-H and C-H shift correlation
NMR spectral analysis. The spectral data of 2 are very
similar to those of 1 [2, 3] and related compounds [30].
The presence of an additional methoxy signal (l 3.80)
showed that compound 2 is a methylether of 1. This
was further substantiated by long range H-H shift cor-
relation between the OMe (l 3.80) and the H-6 (d 6.20)
and H-8 (l 6.42) of 2.
Compound 1, which has been isolated previously by us
[1], was dissolved in Ac2O-Py and left for 48 h at room
temperature to yield 1a.
The 1H-NMR signals H-6 and H-8 of 2a showed upfield
the compounds were added at the same time to each cell shifts in comparison with those of hepta-acetyltiliroside

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CMLS 53 (1997), Birkha¨user Verlag, CH-4010 Basel/Switzerland
589
Table 1. 1H-NMR spectra of compounds 1a, 2 and 2a.
7-Methylether tiliroside (2). Yellow powder; [h]2D5+67.8
(c. 0.12, MeOH). UV (MeOH) nm: 268, 315; (AlCl3)
275, 306, 396; (AlCl3+HCl) 275, 304, 393; (MeONa)
267, 367; (NaOAc) 268, 315; (NaOAc+H3BO3) 268,
H
1a
2
2a
6
8
6.77 d (2)
7.21 d (2)
7.96 d (8)
7.11 d (8)
5.53 d (8)
6.20 d (2)
6.42 d (2)
7.98 d (8)
6.75 d (8)
5.20 d (8)
6.59 d (2)
6.79 d (2)
7.96 d (8)
7.11 d (8)
5.53 d (8)
5.17 dd (9, 8)
5.29 t (9)
5.07 t (9)
3.67 m
1
315. H-NMR see table 1. 13C-NMR see table 2. Posi-
2%,6%
3%,5%
1¦
2¦
3¦
4¦
5¦
6¦
tive ion FAB-MS m/z [M +1]+, 609.
Biotransformation of product 1 to product 2. Very small
amounts of substance 1 were taken up by A. nidulans
cells until the early exponential phase of the culture, as
most of the substance (\95%) remained unchanged in
the supernatant. It was well after the mid-exponential
phase that the amounts of uptaken 1 increased to 25 to
30% and could be detected in the cytosolic content. This
increase in the uptake of 1 continued until the beginning
of the stationary phase (65%) and 10 h later, cultures
reached maximum uptake values, i.e. 92%. Until late
exponential phase, the amounts of 1 found in the cyto-
solic contents of A. nidulans mycelial cells and those
remaining in the supernatant were in almost direct
correlation. This possibly indicates the absence or low
activity of an extracellular enzyme capable of degrading
1 in the supernatant. In the beginning of the stationary
phase the first traces of substance 2 were detected in
cells; and this, together with the fact that intracellular
and extracellular amounts of 1 did not add up to the
initial amounts provided, was considered as strong evi-
dence that A. nidulans cells had started the biodegrada-
tion and biotransformation of uptaken 1. Similarly, the
time of maximum uptake of 1 coincided with the maxi-
mal amounts of substance 2 detected by TLC, and
confirmed by UV-Vis quantitative determination. Un-
der the same conditions, small traces of other sub-
stances were also observed, but these were never found
in sufficient amounts to determine their nature. When
the above optimum conditions were applied to large
scale experiments, about 40% of substance 1 was found
to remain in an unchanged form. The remaining 60% of
substance 1 had therefore obviously been converted to
other products. Amongst these, the most prominent was
substance 2, but at only 5% of the total 1 taken up in
the cytosolic content. It should also be noted that
substance 2 was never detected, even in traces, in the
supernatant at any stage of growth and it is concluded
that this substance is not secreted into the medium and
that it remains in the cells. Thus, all the above data
confirm that A. nidulans metabolized tiliroside (1) to
7-methyl tiliroside (2).
5.17 dd (9, 8) 3.50 m
5.29 t (9)
5.07 t (9)
3.67 m
3.50 m
3.50 m
3.50 m
4.08 m
4.30 dd (11.9 and 1.9) 4.08 m
4.15 dd (11.9 and 6.8)
2§,6§
3§,5§
7§
7.50 d (8)
7.16 d (8)
7.51 d (16)
6.24 d (16)
–
7.22 d (8.5)
6.71 d (8.5)
7.35 d (15.9)
6.03 d (15.9)
3.80 s
7.50 d (8)
7.16 d (8)
7.51 d (16)
6.24 d (16)
3.80 s
8§
OCH3
COCH3 2.46–2.02
–
2.46–2.02
Recorded in CDCl3 (2 was recorded in DMSO-d6), J (Hz) in
parentheses.
(1a) [1] (table 1). The C-H HETCOR spectrum of 1 and
2 showed couplings between H-6 and C-6, H-8 and C-8,
as well as H-2%,6% and C-2%,6%, and finally between H-3%,5%
and C-3%,5% which confirmed unambiguously the chemi-
cal shifts of the H-6, H-8, H-2%,6% and H-3%,5% [3, 31].
All the above data allowed us to confirm that com-
pound 2 has never been reported previously and that it
is a 7-methylether tiliroside.
Table 2. 13C-NMR spectra of compounds 1a, 2 and 2a.
C
1a
2
2a
2
3
4
5
6
7
8
9
10
1%
2%,6%
3%,5%
4%
156.7
137.1
172.1
151.1
113.5
158.5
108.9
156.2
115.8
128.2
131.8
122.1
152.3
99.6
156.6
133.1
177.5
164.9
97.8
157.1
137.2
172.3
151.2
108.5
158.5
98.6
156.2
115.8
128.1
130.3
122.1
152.3
99.6
73.4
73.5
65.8
72.7
61.6
132.8
129.3, 129.4
121.4, 121.5
153.4
144.1
117.5
165.6
56.1
168.9–169.4
20.6–21.2
164.9
92.1
159.7
104.7
120.6
130.8
115.1
160.1
100.6
74.1
76.2
70.1
74.1
62.9
124.8
130.1
115.6
159.0
144.4
113.4
166.0
55.9
1¦
2¦
73.4
73.5
68.5
72.7
3¦
4¦
5¦
6¦
61.6
1§
132.9
129.3, 129.4
121.4, 121.5
153.6
144.1
117.5
165.9
–
Cytotoxic activity and effect on DNA synthesis.
Flavonoids are one of the major classes of natural
products, with widespread distribution and a broad
spectrum of biological activities [4]. They have been
tested against cancer cell lines, showing promising ef-
fects in many cases [32, 33]. We examined the cytotoxic
activity of compounds 2 and 2a against fourteen human
leukemic cell lines. It was found that compound 2 was
inactive in all cell lines tested, at doses as high as 50
2§,6§
3§,5§
4§
7§
8§
9§
OCH3
COCH3
COCH3
169.1–169.4
20.6–21.1
–
–
Recorded in CDCl3 (2 was recorded in DMSO-d6).

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Figure 1. Effect of compound 2a on DNA synthesis in T cell lines, H9 (lymphoblast, CD3+CD4+) (A) and MOLT3 (ALL,
CD4+CD8+) (B). Various concentrations of compound 2a were added and DNA synthesis was assayed as described after 1, 4, 24,
48 h. The values represent means of three independent experiments run in triplicate. SD never exceeded 15% of the mean value.
mg/ml, and for time periods as long as 48 h. It was (45.8 mg/ml) cell lines. The remaining 10 leukemic cell
therefore considered unnecessary to examine further the lines seem to be resistant to the influence of compound
effect of this compound on DNA synthesis. On the 2a, at doses as high as 50 mg/ml.
contrary, its acetylated product (compound 2a) exhib- Compound 2a was tested further for a possible antipro-
ited a better cytotoxic activity. Its major cytotoxic effect liferative activity. In order to have a nonsensitive cell
was observed on the promyelocytic HL60 cell line with line as negative control, we used both sensitive and
an LD50 below 20 mg/ml (18.5 mg/ml), followed by nonsensitive cell lines of the B and T lineage. In the four
DAUDI (25.1 mg/ml), HUT78 (40 mg/ml) and MOLT3 cell lines examined (DAUDI and JIYOYE from the B

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CMLS 53 (1997), Birkha¨user Verlag, CH-4010 Basel/Switzerland
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Figure 2. Effect of compound 2a on DNA synthesis in B cell lines, JIYOYE (Burkitt lymphoma with herpes-like virus) (A) and
DAUDI (Burkitt lymphoma with Epstein-Barr virus) (B). As in T cell lines various concentrations of compound 2a were added and
DNA synthesis was assayed after 1, 4, 24, 48 h. The values represent means of three independent experiments run in triplicate. SD never
exceeded 17% of the mean value.
and MOLT3 and H9 from the T lineage), 2a exhibited mg/ml or remained at very low levels at 10 mg/ml until
a significant antiproliferative activity. This flavonoid the end of the incubation period. At 2 mg/ml suppres-
suppressed DNA synthesis at doses lower than 20 mg/ sion of DNA synthesis was observed 1 h after the
ml, even in those cells where its cytotoxic activity was addition of compound 2a in DAUDI, JIYOYE and H9
estimated to be at concentrations greater than 50 mg/ml. and after 4 h of incubation in MOLT3. In two cell lines
The inhibition effect began as early as 1 h after the (DAUDI and JIYOYE), DNA synthesis recovered after
addition of 2a. DNA synthesis declined more at 20 24 h of incubation to 100% of the control level, but only

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