Structural and spectroscopic study of 5,7-dihydroxy-flavone and its complex with aluminum

Article abstract of DOI:10.1016/j.saa.2003.11.022

The structure, stability and molar absorptivity of the complex formed between AlCl₃ and 5,7-dihydroxy-flavone in methanol were investigated using UV-Vis spectroscopy and the AM1 method. The molar ratio method and Job's method of continuous variation were applied to ascertain the stoichiometric composition of the complex in methanol at constant ionic strength. A 1:2 complex was indicated by both methods. The molar absorptivity and stability constant of the complex were determined using a simple and accurate procedure that requires solutions having the ligand and metal ion in the stoichiometric proportion. The high stability constant demonstrates that the complexation reaction is total. The structure of this complex, obtained by the quantum semi-empirical AM1 method, indicates that two classes of metal-ligand interactions are involved in the formation of the metal complex: (a) two simple covalent bonds between the aluminum atom and the oxygen atoms of o-hydroxyl groups of 5,7-dihydroxy- flavone; (b) two stronger Coulombic interactions between the aluminum atom and the carbonyl oxygen atoms of the ligand.

Full text of DOI:10.1016/j.saa.2003.11.022

Spectrochimica Acta Part A 60 (2004) 2235–2241
Structural and spectroscopic study of 5,7-dihydroxy-flavone
and its complex with aluminum
G.T. Castro, S.E. Blanco∗
Area de Qu´ımica-F´ısica, Departamento de Qu´ımica, Facultad de Qu´ımica, Bioqu´ımica y Farmacia,
Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700 San Luis, Argentina
Received 29 September 2003; accepted 25 November 2003
Abstract
The structure, stability and molar absorptivity of the complex formed between AlCl3 and 5,7-dihydroxy-flavone in methanol were inves-
tigated using UV-Vis spectroscopy and the AM1 method. The molar ratio method and Job’s method of continuous variation were applied to
ascertain the stoichiometric composition of the complex in methanol at constant ionic strength. A 1:2 complex was indicated by both methods.
The molar absorptivity and stability constant of the complex were determined using a simple and accurate procedure that requires solutions
having the ligand and metal ion in the stoichiometric proportion. The high stability constant demonstrates that the complexation reaction
is total. The structure of this complex, obtained by the quantum semi-empirical AM1 method, indicates that two classes of metal–ligand
interactions are involved in the formation of the metal complex: (a) two simple covalent bonds between the aluminum atom and the oxygen
atoms of o-hydroxyl groups of 5,7-dihydroxy-flavone; (b) two stronger Coulombic interactions between the aluminum atom and the carbonyl
oxygen atoms of the ligand.
© 2003 Elsevier B.V. All rights reserved.
Keywords: 5,7-Dihydroxy-flavone; Al(III) complex; Stability; Complex structures; UV-Vis spectroscopy; AM1 calculations
1. Introduction
with AlCl3 [10–12]. In this work, we determine the sto-
ichiometry of the complex formed between AlCl3 and
5,7-dihydroxy-flavone in methanol, its equilibrium constant
and molar absorptivity. On the other hand, the structures of
free and complexed molecules of the ligand are determined
using the semiempirical AM1 method.
Flavonoids are natural products widely distributed in the
vegetable kingdom and currently consumed in large amounts
in the daily diet [1,2]. They are known to have a broad
spectrum of biological and pharmaceutical activities and an
elevated chemical reactivity [3–6]. Their complexating ca-
pacity, widely used for elucidating the structure of natural
flavonoids, can also contribute to the bioactivity of these
compounds, by acting as carriers and regulators of metal
concentration. Some flavonoids possess a strong antioxi-
dant effect by their radical-scavenging activity and metal ion
chelation [7–9].
2. Experimental
2.1. Reagents
Fig. 1 shows the structure and chemical numbering sys-
tem of 5,7-dihydroxy-flavone (1). Compound 1 from Sigma
was purified by repeated crystallization from ethanol–water.
The purity control was performed determining its melting
point [13] (mp = 285 ◦C) and its TLC chromatographic
properties [14]. Crystallized anhydrous aluminum chloride,
crystallized sodium chloride, and methanol (MeOH) of
spectroscopic grade from Fluka, were used without further
purification.
In order to determine the chelating capacity of metallic
ions of simple flavonoids and benzophenones of biological
interest, we recently studied the complexating reaction of
2-hydroxy-benzophenone and 2,4-dihydroxy-benzophenone
∗
Corresponding author. Tel.: +54-2652-423789;
fax: +54-2652-430224.
E-mail address: sblanco@unsl.edu.ar (S.E. Blanco).
1386-1425/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2003.11.022

2236
G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
ing different volumes of equimolar stock solutions of 1
and AlCl3 (5.10 × 10−3 M). In these solutions, the sum of
the total concentration of ligand and metal was constant
(2.48 × 10−4 M). Solutions containing only compound 1
in the same concentration as in the mixture were used for
corrections of Job’s plot.
In the determination of the εC and KC of the complex, a
spectrophotometric method designed by Ferretti et al. [17]
was used. This procedure requires the preparation of solu-
tions maintaining the molar ratio of ligand to metal ion, in
the stoichiometric proportion. With this purpose, adequate
aliquots of stock solutions of 1 and AlCl3 were added to
volumetric flasks containing approximately 4 g of MeOH to
obtain a progressively increased concentration of the com-
plex. The analytic concentrations of 1 and AlCl3 in the re-
action mixture were from 3.61×10−5 to 1.58×10−4 M and
from 1.80 × 10−5 to 7.84 × 10−5 M, respectively. The ionic
strength was adjusted to the value 2.80×10−2 with NaCl. Af-
ter maintaining the reacting solutions for 1 h at 25 0.1 ◦C,
the respective equilibrium absorbances (Ae) were read at
382 nm, which is the maximum absorption wavelength of
the complex in MeOH. Fig. 2 shows the UV-Vis spectra
of 1, both free and complexed with AlCl3 in MeOH. At
382 nm the absorption of the free ligand is lower than in the
complex.
Fig. 1. Structure and chemical numbering system of 5,7-dihydroxy-flavone.
2.2. Procedures
All solutions were prepared by weighing with an accuracy
of 0.0001 g. The experiments comprised: (a) the determi-
nation of the stoichiometric composition of complex formed
by AlCl3 and 1 in MeOH; (b) the determination of the molar
absorptivity (εC) and stability constant (KC) of this complex.
The molar ratio method, introduced by Yoe and Jones
[15], was used for the spectrophotometric determination
of the complex composition. For this method, solutions
containing a constant concentration of 1 (1.124 × 10−4 M)
and variable concentrations of AlCl3 (from 1.068 × 10−5
to 2.926 × 10−4 M) were prepared in MeOH containing
NaCl (2.80 × 10−2 M) for constant ionic strength. Under
these operative conditions, about 1 h was necessary to reach
the complexation equilibrium at 25.0 0.1 ◦C. After this,
the UV-Vis spectra were recorded on a Shimadzu UV 160
A double beam spectrophotometer with 1 cm thermostati-
cally controlled cells, between 215 and 450 nm at the same
temperature.
3. Calculations
The molecular formula of the ligand chrysin or 5,7-
dihydroxy-flavone is C15H10O4, while that of the obtained
For the application of the continual variation method
(Job’s method) [16], the solutions were prepared by mix-
Fig. 2. UV-Vis spectra of 5,7-dihydroxy-flavone free and complexed with AlCl3 in methanol.

G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
2237
ions. The calculations were performed on an Intel Pentium
IV 1.4 GHz CPU with 512 MB of RAM. Fig. 3 shows the
practical numbering system adopted for carrying out the
calculations.
4. Results and discussion
The UV-Vis spectrum of 1 in MeOH (Fig. 2) is character-
ized by two major absorption bands with maxima at 313 nm
(band I) and 268 nm (band II). Band I is considered to be
associated with the absorption due to the B-ring cinnamoyl
system, and band II with the absorption involving the A-ring
system [23]. In particular, this compound, which is oxy-
genated in the A-ring but not in the B-ring, shows a spectrum
with a pronounced band II and a weak band I. With AlCl3,
flavones containing hydroxyl groups at C-3 or C-5 form sta-
ble complexes between C-4 keto function and either the 3-
or 5-hydroxyl group, producing a large bathochromic shift
of band I. In Fig. 2, a bathochromic shift of 69 nm can be ob-
served for band I, greater than that reported for 5(OH)flavone
(60–61 nm) [23,24] and 3(OH)flavone (56–58 nm) [22,23].
Fig. 3. Practical numbering system adopted in the calculations.
complex is C30H18O8–Al–Cl. It is evident that this metallic
complex possesses a high number of heavy atoms, which
implies that the study of its structure by ab initio or DFT
methods would require a high CPU time. Therefore, the
structure of the complex was studied by the semiempirical
MO AM1 method [18] included in the Gaussian 98 [19]
program packages. It must be noted that the AM1 method
does not allow analyzing the hexacoordinated aluminum
atom in the obtained complexes; however, it is useful for
description of the structural changes undergone by sev-
eral compounds. This method has been previously used to
explain the conformation, hydrogen bonding and UV sol-
vatochromic shifts of benzophenones in primary alcohols
[20]. Furthermore, the AM1 method has proved to be use-
ful for satisfactorily describing the structural differences
between free and chelated benzophenone molecules [10]
and flavonoids [21,22] used as ligands of various metallic
4.1. Complex stoichiometry in methanol
The stoichiometry of the complex was determined apply-
ing the molar ratio method. The absorbance of band I of 1
decreases at 313 nm and a new band, which increases with
the amount of added AlCl3, appear at 382 nm. The data ob-
tained from these experiments at 25.0 0.1 ◦C are sum-
marized in Fig. 4. In this figure, we plotted Ae against the
[AlCl3]/[1] ratio. The straight lines built with the data of Ae
obtained at 313 and 382 nm intersect at [AlCl3]/[1] = 0.5,
revealing the formation of an Al(III)/1 = 1:2 complex. It
Fig. 4. Determination of the stoichiometry of the complex formed between AlCl3 and 5,7-dihydroxy-flavone in methanol, by applying the molar ratio
method.

2238
G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
can also be observed that the molecule of 1 is completely
complexed for a ratio [AlCl3]/[1] = 1.
equilibrium (t = ∞). Considering that L and C, but not M,
absorb some radiation at the selected wavelength (as fre-
quently occurs), the total absorbance of the reacting solution
in the equilibrium will be:
Analyzing the complexation reactions between aluminum
and flavonoids in MeOH, it can be stated that the chelating
power of 1 is similar to that reported for 3(OH)flavone
but greater than that determined for 5(OH)flavone [24]
and 3ꢀ,4ꢀ(OH)2flavone [25]. Boudet et al. [22] have ob-
served a complex Al(III)/3(OH)flavone = 1:2, and a ratio
[AlCl3]/[ligand] of 1 is necessary to obtain a full com-
plexation of 3(OH)flavone. Cornard et al. have studied the
complexation of 5(OH)flavone [24] and 3ꢀ,4ꢀ(OH)2flavone
[25] and have determined the formation of complexes of 1:1
stoichiometry. These molecules are completely complexed
for a ratio [AlCl3]/[ligand] of 1.5 and 2, respectively.
Job’s method (continual variation method) was applied
to validate the stoichiometric composition of the chelate.
The absorbance plots at 382 nm (Fig. 5) against the molar
fraction of 1 (X1) have a maximum at X1 = 0.64 (close to
0.66), confirming that the stoichiometric ratio of AlCl3 to 1
in the complex is 1:2.
Ae = εL(L0 − 2x)ꢀ + εCxꢀ
(2)
where εL is the molar absorptivity of the ligand and ꢀ the op-
tical path length. Under the experimental conditions adopted
in this work, L0 = 2M0, the stability constant of the com-
plex is expressed as,
x
2x
KC =
=
(3)
(M0 − x)(L0 − 2x)2
(L0 − 2x)3
Combining Eqs. (2) and (3) and solving the obtained cubic
equation, the following expression is obtained [17]:
Ae
2εL − εC
1
= εC +
·
(4K)1/3 (M0)2/3
(4)
ꢀM0
Considering ꢀ = 1 cm, the experimental data obtained at
ionic strength 2.80 × 10−2 and temperature 25.0 0.1 ◦C
were plotted according to Eq. (4) as shown in Fig. 6. The
values obtained for εC and KC from the intercept and slope
of the straight line were:
4.2. Stability constant and molar
absorptivity of the complex
εC (382 nm) = 12, 092 l mol−1 cm−1
KC (MeOH) = 4.55 × 1011
Considering that the stoichiometry of complex is 1:2 and
omitting the charges for the sake of simplicity, the complex-
ation reaction can be written as,
+
2L
ꢀ
(1)
M
C
It is interesting to highlight that Eq. (4), besides permit-
ting the determination of the εC and KC values, would con-
stitute an alternative for confirming the 1:2 stoichiometry.
The high value of KC shows that equilibrium is totally dis-
placed towards the formation of the complex. The value of
KC for this complexation reaction in MeOH is comparable to
(L0−2x)
x
(M0−x)
where M represents AlCl3; L represents ligand 1, and C rep-
resents the metal complex. From now on, t is the time; M0
and L0 the molar concentrations of AlCl3 and 1 for t = 0,
respectively; and x the molar concentration of complex at
Fig. 5. Method of continual variations. Absorbance plot at 382 nm (λmax of chelate) vs. molar fraction of ligand.

G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
2239
Fig. 6. Determination of stability constants and molar absorptivity of the complex formed between AlCl3 and 5,7-dihydroxy-flavone in methanol. Error
bars = 0.2%.
that reported for the chelation of aluminum by 3(OH)flavone
(2×1012) [25], also with 1:2 stoichiometry. Other flavonoids
and benzophenones that complexate aluminum in a 1:1 stoi-
Table 1
chiometric relationship have lower equilibrium constant val-
Calculated structural magnitudes with the AM1 method and using at the
COSMO model for the ligand and metal complex methanol, at 298 K
ues (approximately 106) [10,12,25].
Magnitude
Methanol
Ligand
4.3. Structural properties of the complex
Metal complex
It is evident that the ligand suffers some structural changes
when it reacts with AlCl3 to form the metal complex (Figs. 3
and 7). Recently, Lau et al. [26] carried out a very exhaustive
conformational study on 1 by means of ab initio methods at
the RHF/6-31G(d) level of theory. These authors determined
(in vacuum) that by rotation of the plane that involves the
aromatic B-ring with respect to the plane containing the rest
DM
4.61
27.5
7.11
26.7
26.9
D (C17C12C2C3)
D (C47C42
q (O5)
q (O19
q (O35
q (O49
q (Al21
q (Cl20
r (Cl20–Al21
r (C4–O5)
r (C9–O19
r (C34–O35
r (C39–O49
r (O19–Al21
r (O49–Al21
C
37C32
)
–
−0.476
−0.401
−0.410
−0.398
−0.408
0.900
−0.385
2.219
1.268
1.347
1.268
1.345
1.745
1.744
2.347
2.331
124.2
121.8
124.4
122.4
118.7
121.0
)
)
)
)
)
−0.292
–
–
–
–
ꢀ
of the molecule around the single bond C2 − C1 (Fig. 1),
)
–
the dihedral angle D (C17C12C2C3) (Fig. 3) varies between
26.40◦–27.43◦. Table 1 reports the optimized values of sev-
eral structural properties calculated for both ligand and com-
plex with the AM1 method. The data indicate that 1 has a
non-planar structure.
1.255
1.364
)
)
)
)
)
)
)
)
–
–
–
–
–
–
Our calculations performed in MeOH at 298 K for ligand
and complex show that the dihedral angles D (C17C12C2C3)
are 27.5◦ and 26.7◦, respectively. This implies that the fold-
ing of phenyl B-ring of both species with respect to the plane
that comprises the rest of the molecule practically remains
constant. This is to say, the above mentioned dihedral angle
of the free molecule of 1 is not substantially modified when
it is complexed. On the other hand, and as it should be ex-
pected, other molecular magnitudes are very different. As an
example, the dipole moment (DM) of the complex is higher
than 1. This fact is closely linked to the changes in the bond
lengths and net charges of the atoms involved in the forma-
d (O5–Al21
d (O35–Al21
A (C6C4O5)
A (C6C9O19
A (C36C34O35
A (C36C39O49
A (C9O19Al21
123.2
124.0
)
)
)
–
–
–
–
A (C39O49Al21
)
DM: dipolar moment (Debye); D: dihedral angle between the indicated
atoms (◦) (Figs. 3 and 7); q: total atomic charge of the indicated atom
(a.u.); r: bond length between the indicated atoms (Å); d: Coulombic
interaction distance between the indicated atoms (Å); A: bond angle
between the indicated atoms (◦).

2240
G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
Fig. 7. Structure of the complex formed between AlCl3 and 5,7-dihydroxy-flavone in methanol.
tion of the complex, namely: O5, O19, O35, O49 and Al21.
The union bond lengths and net charges of the chlorine and
aluminum atoms in a normal AlCl3 molecule are:
charges of the oxygen atoms of carbonyl (q (O5), q(O35))
and hydroxyl (q (O19), q (O49)) groups. While the q (O5),
q (O35) of the complex are lower (in absolute value) than
the q (O5) values of free molecules, the opposite occurs in
the case of the q (O19) charges. These observations permit
to conclude that the formation of the complex implies an
r (Al–Cl) = 1.874 Å;
q (Cl) = −0.153 a.u.
q (Al) = 0.460 a.u.;
=
increase of length bond C O together with a decrease of q
(O5), and a decrease of length bond o-C–OH accompanied
by an increase of q (O19). Overall, the structural changes
suffered by 1 upon forming the complex bear a certain
similarity with those previously observed in the complex-
ation reaction of AlCl3 with o-hydroxy-benzophenones
[10].
From Table 1, it can be seen that the three above mag-
nitudes are markedly higher in the complex, as is also the
case of the q (O19), q (O49) charges and the r (C4–O5), r
(C34–O35) bond lengths. It is obvious that the simultaneous
increase of a bond’s length and the charges of the implied
atoms will markedly increase the polarization of the bond,
and therefore the DM of the complex molecule. This ex-
plains why the DM of the complex is much higher that the
DM of the free ligand.
5. Conclusions
It was proposed that two classes of metal–ligand interac-
tions are involved in the formation of the metal complex: (a)
two simple covalent bonds between the aluminum atom and
the oxygen atoms of o-hydroxyl groups of 1 (r (O19–Al21)
and r (O49–Al21)); (b) two stronger Coulombic interactions
between the central atom and the carbonyl oxygen atoms of
the ligand (d (O5–Al21) and d (O35–Al21)). From Table 1,
it can be observed that although the d (O35–Al21) distance
is lower than d (O5–Al21), the difference between these
two magnitudes is very small (under 1%). Furthermore, it
must be pointed out that the values for r (O19–Al21) and r
(O49–Al21) are practically identical. It is also observed that
the bond lengths o-C–OH (r (C9–O19), r (C39–O49)) in the
free molecules of 1 suffer a slight decrease of 0.017 Å when
they are complexed. The opposite occurs in the case of the
The UV-Vis spectra of 1 in MeOH exhibit two main
absorption bands at 313 and 268 nm. This compound, with
AlCl3 in pure MeOH at 25 ◦C maintaining the ionic strength
constant, reacts easily reaching the complexation equilib-
rium at about 1 h. Therefore, band I suffers bathochromic
shifts of 69 nm. Applying the molar ratio method and Job’s
method, it was determined that the stoichiometric composi-
tion of the complex formed is 1:2. Furthermore, additional
experiments allowed obtaining the value of the constant of
stability. Considering the elevated value of the equilibrium
constant of the investigated complexation reaction, it can
also be inferred that 5,7-dihydroxy-flavone constitutes an
analytical reagent of great interest due to its potential use in
the quantitative determination of metallic ions. The struc-
ture of the complex was satisfactorily explained by means
of the theoretical study performed using the AM1 method.
The calculated length bonds, energy and reactivity indices
permitted to conclude that the formation of the complex
=
double bond C O (r (C4–O5), r (C34–O35)). The carbonylic
bond lengths in the complex are approximately 0.013 Å
=
above the length bond C O that characterizes the free lig-
and. Opposite behaviors are also observed in the total atomic

G.T. Castro, S.E. Blanco / Spectrochimica Acta Part A 60 (2004) 2235–2241
2241
[10] S.E. Blanco, M.C. Almandoz, G.T. Castro, F.H. Ferretti, J. Mol.
Struct. (Theochem) 586 (2002) 177–192.
[11] M.I. Sancho, M.C. Almandoz, S.E. Blanco, F.H. Ferretti, J. Mol.
Struct. (Theochem) 634 (2003) 107–120.
[12] G.T. Castro, S.E. Blanco, S.L. Arce, F.H. Ferretti, Spectrochim. Acta.
Part A 59 (2003) 2685–2696.
[13] D.R. Lide, Handbook of Chemistry and Physics, 78th ed., CRC
Press, NY, 1997–1998, pp. 3–78.
[14] B. Fried, J. Sherma, Thin-Layer Chromatography (Chromatographic
Science, V. 81), 4th ed., Marcel Dekker, USA, 1999.
[15] J.H. Yoe, A.L. Jones, Ind. Eng. Chem. Anal. Ed. 16 (1944) 111.
[16] P. Job, Anal. Chim. 9 (1928) 113.
=
implies an increase of bond C O length together with a
decrease of q (O5), and a decrease of length bond o-C–OH
accompanied by an increase of q (O19). This is in agree-
ment with the observed MD. Two classes of metal–ligand
interactions are involved in the formation of the metal
complex: (a) two simple covalent bonds between the alu-
minum atom and the oxygen atoms of o-hydroxyl groups
of 1; (b) two stronger Coulombic interactions between
the aluminum atom and the carbonyl oxygen atoms of the
ligand.
[17] F.H. Ferretti, R.A. Olsina, C.B. Marone, Anales Asoc. Qu´ım. Ar-
gentina 70 (1982) 501–508.
[18] M.J.S. Dewar, E.G. Zoebisch, E.F. Healey, J.J.P. Stewart, J. Am.
Chem. Soc. 107 (1985) 3902–3909.
Acknowledgements
[19] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb,
J.R. Cheeseman, V.G. Zakrzewski, J.A. Montgomery, Jr., R.E. Strat-
mann, J.C. Burant, S. Dapprich, J.M. Millam, A.D. Daniels, K.N.
Kudin, M.C. Strain, O. Farkas, J. Tomasi, V. Barone, M. Cossi, R.
Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochter-
ski, G.A. Petersson, P.Y. Ayala, Q. Cui, K. Morokuma, D.K. Malick,
A.D. Rabuck, K. Raghavachari, J.B. Foresman, J. Cioslowski, J.V.
Ortiz, A.G. Baboul, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz,
I. Komaromi, R. Gomperts, R.L. Martin, D.J. Fox, T. Keith, M.A.
Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W.
Gill, B. Johnson, W. Chen, M.W. Wong, J.L. Andres, C. Gonzalez,
M. Head-Gordon, E.S. Replogle, J.A. Pople, Gaussian 98, Revision
A.9, Gaussian Inc., Pittsburgh, PA, 1998.
[20] S.E. Blanco, J.J. Silber, F.H. Ferretti, J. Mol. Struct. (Theochem)
582 (2002) 91–105.
[21] J.P. Cornard, A.C. Boudet, J.C. Merlin, J. Mol. Struct. 508 (1999)
37–49.
[22] A.C. Boudet, J.P. Cornard, J.C. Merlin, Spectrochim. Acta. Part A
56 (2000) 829–839.
This work was supported by grants from National Uni-
versity of San Luis (Argentine Republic).
References
[1] O. Benavente-Garc´ıa, J. Castillo, F.R. Marin, A. Ortuño, J.A. Del
R´ıo, J. Agric. Food Chem. 45 (1997) 4505–4515.
[2] G. Di Carlo, N. Mascolo, A.A. Izzo, F. Capasso, Life Sci. 65 (1999)
337–353.
[3] A. Nagao, M. Seki, H. Kobayashi, Biosci. Biotechnol. Biochem. 63
(1999) 1787–1790.
[4] A. Jain, M.C. Martin, N. Parveen, N.U. Khan, J.H. Parish, S.M.
Hadi, Phytother. Res. 13 (1999) 609–612.
[5] S.E. Blanco, J.J. Silber, G.E. Narda, L.J. Yamin, F.H. Ferretti, J.
Colloid Interface Sci. 180 (1996) 144–148.
[6] S.A. Andujar, M.A. Filippa, S.E. Blanco, F.H. Ferretti, J. Mol. Struct.
(Theochem) 636 (2003) 157–166.
[7] C.G.M. Heijnen, G.R.M.M. Haenen, J.A.J.M. Vekemans, A. Bast,
Environ. Toxicol. Pharmacol. 10 (2001) 199–206.
[8] T. Koide, H. Noda, W.W. Liu, T. Ogata, H. Kamada, Anal. Sci. 16
(2000) 1029–1032.
[23] T.J Mabry, K.R. Markham, M.B. Thomas, The Systematic Identifi-
cation of Flavonoids, Springer–Verlag, NY, 1970, pp. 41–56.
[24] J.P. Cornard, J.C. Merlin, J. Mol. Struct. 569 (2001) 129–138.
[25] J.P. Cornard, A.C. Boudet, J.C. Merlin, Spectrochim. Acta. Part A
57 (2001) 591–602.
[9] M.E. Bodini, M.A. del Valle, R. Tapia, F. Leighton, P. Berrios,
Polyhedron 20 (2001) 1005–1009.
[26] K.S. Lau, A. Mantas, G.A. Chass, F.H. Ferretti, M.R. Estrada, G.
Zamarbide, I.G. Csizmadia, Can. J. Chem. 80 (2002) 845–855.