Fluorescence spectroscopy and docking study in two flavonoids, isolated tectoridin and its aglycone tectorigenin, interacting with human serum albumin: A comparison study

Article abstract of DOI:10.1002/bio.2918

Two flavonoids, tectoridin (TD) isolated from rhizomes of Iris tectorum and hydrolyzed aglycone tectorigenin (TG) were prepared and characterized to compare their different interaction ability with human serum albumin (HSA). Based on the results, the affi

Full text of DOI:10.1002/bio.2918

Research article
Received: 19 November 2014,
Revised: 12 March 2015,
Accepted: 12 March 2015
Published online in Wiley Online Library: 28 April 2015
(wileyonlinelibrary.com) DOI 10.1002/bio.2918
Fluorescence spectroscopy and docking study
in two flavonoids, isolated tectoridin and its
aglycone tectorigenin, interacting with human
serum albumin: a comparison study
Yuanzhi Li, Qing Wang, Jiawei He, Jin Yan and Hui Li*
ABSTRACT: Two flavonoids, tectoridin (TD) isolated from rhizomes of Iris tectorum and hydrolyzed aglycone tectorigenin (TG)
were prepared and characterized to compare their different interaction ability with human serum albumin (HSA). Based on the
results, the affinity of TG–HSA was stronger than that of TD–HAS, and TG combined more closely with HSA than did TD. HSA
fluorescence was quenched by TD/TG. The interactions between TD/TG and HSA involved static quenching. The thermodynamic
parameters indicated that both binding processes were spontaneous; hydrogen binding and van der Waals force were the main
forces between TD and HSA, whereas a hydrophobic interaction was the main binding force between TG and HSA. Synchronous
and 3D fluorescence spectra showed that the binding of TD/TG to HSA induced conformational changes. Moreover, a docking
study confirmed the experimental results. Copyright © 2015 John Wiley & Sons, Ltd.
Keywords: human serum albumin; flavonoids; tectoridin; tectorigenin; interaction
regarding binding, including quenching mechanisms, binding
Introduction
parameters, thermodynamic parameters, binding modes and the
Flavonoids are widely distributed in the plant kingdom as
high-affinity binding site of HSA interacting with TD/TG were
secondary metabolites (1), and compounds in this class possess a
systematically investigated using spectroscopic methods. More-
broad spectrum of biological activities (2,3). Almost all flavonoids
over, the competition between TD/TG with HSA was studied in a
exist as β-glycosides in nature (4). In most cases, flavonoid glyco-
sides are hydrolyzed to their aglycones to produce effects in vivo
ternary system. Molecular docking was also used to gain insight
into the binding modes and further identify the key residues in
(5,6). Studies on biologically active flavonoids have grown expo-
binding. We hope this work will clarify the binding mechanism of
nentially in recent years (7). Knowledge of the binding parameters
TD/TG with protein at the molecular level, and also provide a
and binding mechanism are of the uttermost importance (8–10).
theoretical basis for understanding their effect on protein function
Tectoridin (TD) is the 7-glucoside of tectorigenin (TG), and both
during blood transportation.
compounds are widely distributed in plants, such as Iris tectorum
(11), Belamcanda chinensis (12) and Pueraria thunbergiana (13).
TD and TG have been shown to have many pharmacological
Experimental
activities (14–19). TD, which can be metabolized to TG by human
intestinal bacteria in a deglycosylation procedure, is considered
to be a prodrug (20). The metabolic fates of TD and TG are associ-
Materials and reagents
ated with the hydrolysis of 7-O-glucosyl, O-methylation, sulfation,
disulfation and glucuronidation of hydroxyl groups and mono-
and dihydroxylations (21,22). Comparative studies have shown
that TG has more potent biological activity than TD (20,23,24).
Human serum albumin (HSA) is a major protein component of
blood plasma and has important roles in the blood (25).
Compounds that bind to HSA with high affinity usually interact
with one or two specific sites on the protein (26). Studies on the
interaction mode of small molecules with HSA are quite important
(27). The interaction between flavonoids and serum albumin has
been well studied (28). Most reports have focused on the binding
process between flavonoids and serum albumins; only a few
have discussed the effect of flavonoid glycosylation on the
characteristics of binding with protein (29,30).
Commercially available I. tectorum rhizomes were purchased from
Sichuan Limin Chinese Medicine Pieces Co. Ltd (Chengdu, China)
and identified using pharmacognostical methods. HSA (fatty-acid
free) was purchased from Sigma-Aldrich (Milwaukee, WI, USA)
and used without further purification. Warfarin and ibuprofen were
obtained from the National Institute for Control Pharmaceutical
and Products (Beijing, China). Acetonitrile (HPLC grade) was
supplied by Merck (Darmstadt, Germany). All other materials and
reagents were of analytical grade and ultrapure water was used
throughout.
* Correspondence to: H. Li, College of Chemical Engineering, Sichuan University,
Chengdu 610065, People’s Republic of China. E-mail: lihuilab@sina.com
In this study, two flavonoids, TD and TG, were prepared and
their binding properties to HAS were investigated. Information
College of Chemical Engineering, Sichuan University, Chengdu 610065,
People’s Republic of China
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Interaction of human serum albumin with tectoridin and tectorigenin
TD and TG were identified by correlation with 1H NMR spectro-
scopic data (17,31–33), which were recorded on an AV II-400MHz
instrument (Bruker, Switzerland) (data not shown).
Methods
Preparation and characterization of TD and TG
Iris tectorum rhizomes (500 g) were reflux extracted for 2 h with
500 mL ethanol: water (75: 25, v/v) at a temperature of 353 K.
After evaporating the extraction solvent, the residue was diluted
with H2O and successively extracted with petroleum ether and
EtOAc. The EtOAc-soluble fraction (4.2g) was chromatographed
over silica gel using CHCl3: MeOH (7: 3, v/v) to give TD (4 g,
colorless needles) (31).
Fluorescence measurements
An HSA stock solution was prepared at a concentration of
2.0 × 10-5 mol/L in 0.1mol/L Tris/HCl buffer (pH 7.4) containing
0.1 mol/L NaCl. TD and TG were prepared at a concentration
of 1.0 × 10-3 mol/L in ethanol. All the stock solutions were
prepared weekly and stored in the dark at 278 K.
In this study, TG was synthesized by hydrolysis of TD (Fig. 1)
rather than separated from I. tectorum (32). Purified TD (1.5 g)
was mixed with 3% HCl in MeOH: H2O (1: 1, v/v) at 353 K and
refluxed for 4 h. The reaction mixture was then partitioned with
ethyl ether. The ethyl ether fraction was neutralized, dehydrated
and concentrated to obtain the resulting fraction. The resulting
fraction was recrystallized to obtain pure TG (563 mg, pale yellow
needles).
The purities of TD and TG were confirmed using the HPLC area
normalization method. HPLC analysis of the crude extract, TD
and TG were performed on a Shimadzu LC-2010AHT HPLC system
fitted with a Kromasil C18 column (250 mm × 4.6mm, 5 μm). A
gradient elution system acetonitrile (A) and water (B) was
used: 17% A at 0–10 min, 17–46% A at 10–25 min and 46% A at
25–33min (33). All solvents were passed through a 0.22μm filter
prior to use. The flow rate was kept at 1.0 mL/min, and the effluents
were monitored at 265nm using a UV detector. HPLC chromato-
grams of TD, TG and I. tectorum crude extract are shown in Fig. 2.
The relative content of the two components shows that both have
a purity of> 97%. Under the specific HPLC conditions, HPLC
analysis showed that TD is eluted at 16.825 min and TG is eluted
at 26.942 min. This shows the difference in polarity between
the two compounds and that the polarity of TD is reduced after
hydrolysis to TG.
Ethanol was used to dissolve TD and TG, and had almost no effect
on the conformation change and fluorescence quenching of HSA at
the experimental concentration used in the following tests: 2.0 mL
of 1.0 × 10-6 mol/L HSA was titrated with 0.1–1.0% (v/v) ethanol
while its UV–Vis spectra and fluorescence spectra were monitored.
The spectral profiles (data not shown) showed no changes.
All fluorescence spectra were recorded with a Cary Eclipse fluo-
rescence spectrophotometer (Varian, Inc., Palo Alto, CA, USA)
equipped with 1.0 cm quartz cells. A stabilized HSA concentration
(2.0× 10-6 mol/L) and various TD and TG concentrations (from 0.0
to 12.0 × 10-6 mol/L) were added to each volumetric flask and then
diluted to 10 mL with Tris/HCl buffer (pH 7.4). Prior to measuring
the fluorescence, the solutions were vortex-mixed and held for
1 h in a thermostat water bath at different temperatures (298,
310 and 315 K). An excitation wavelength of 280 nm was
employed and emission spectra were recorded from 300 to
500 nm; excitation and emission slit widths were set at 5 and
10nm, respectively.
Six independent binding experiments were conducted per
group. The maximum experimental error in the measurements
was 5%. In this study, the inner filter effect was corrected for using
the equation (34):
Fc ¼ Fm ꢀ eðA1þA2Þ=2
(1)
where Fc and Fm are the corrected and observed fluorescence
intensities, and A1 and A2 are the absorption of the systems at
the excitation and emission wavelengths.
Synchronous fluorescence spectra were recorded at Δλ =15nm
and Δλ = 60 nm. The HSA concentration was fixed at 2.0 × 10-6 mol/L
in a quartz cell and the concentrations of TD and TG were varied
from 0 to 12.0 × 10-6 mol/L by successive additions.
3D fluorescence spectra were measured under the following
conditions: the emission wavelength was recorded between 200
and 500 nm, the initial excitation wavelength was set at 200nm
with increments of 5 nm, and the other scanning parameters were
the same as those for the fluorescence emission spectra.
Figure 1. Molecular structure of TD (A) and TG (B), and the acid hydrolysis process.
Site marker competitive experiments
Competitive binding experiments were conducted using warfarin
and ibuprofen. The ratio of site markers to HSA in the
warfarin/ibuprofen–HSA mixture solution was maintained at 1: 1
(2.0× 10-6 mol/L each). After 1 h incubation, various concentrations
of TD and TG were gradually added to the warfarin/ibuprofen–HSA
systems and then incubated for a further 1 h. The fluorescence
spectra were recorded at 298 K under the experimental conditions
described above.
The effect of TD on the binding constant of TG with HSA (or vice
versa) was studied. When the concentration of one (interferent) in
a series of test tubes containing HSA was kept constant, the
concentration of the other (target) was successively changed.
Figure 2. HPLC chromatograms of TD (A), TG (B) and the crude extract of I. tectorum (C).
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Y. Li et al.
Molecular modeling
Table 1. KSV and Kq values for the complex of TD/TG–HSA at
different temperatures.
Docking calculations were performed using the CDOCKER
protocol in Discovery Studio 3.1 (State Key Laboratory of
Biotherapy, Sichuan University, China), which is available from
Accelrys Software Inc (San Diego, CA, USA). The crystal structure
of HSA used in the docking studies was obtained from the RCSB
Protein Data Bank (PDB ID: 1H9Z).
Compound
TD
T (K)
KSV (L/mol)
Kq (L/mol/S)
R*
298
310
315
298
310
315
44 794
39 729
38 810
60 752
66 748
73 486
4.479 × 1012
3.973 × 1012
3.881 × 1012
6.075 × 1012
6.675 × 1012
7.349 × 1012
0.9968
0.9961
0.9980
0.9992
0.9993
0.9971
TG
Results and discussion
The fluorescence quenching spectra and quenching
mechanism
* The correlation coefficient for the regression equations.
The emission spectra of HSA in the absence and presence of TD
and TG are shown in Fig. 3. When the excitation wavelength is
set at 280 nm, HSA shows a strong emission band around
338 nm, whereas TD and TG do not show any detectable emission
under this condition. With the continuous addition of TD and TG,
the emission intensity of HSA decreased gradually, which reveals
that both TD and TG quenched the intrinsic fluorescence of HSA.
Fluorescence quenching is mainly caused by dynamic and static
quenching. To explore the quenching mechanism qualitatively,
the Stern–Volmer equation is utilized (35):
usually described by deriving the binding constant (Ka) and the
number of binging sites (n) from the following double-logarithm
regression curve (38):
log ½ðF0 - FÞ=Fꢁ ¼ logKa þ n log½Qꢁ
(3)
where Ka is the binding constant of the interaction between
quenchers and HSA and n is the number of binding sites per has
molecule. The values of Ka and n can be obtained from the plot
of log(F0 – F)/F versus log[Q]. The values obtained are shown in
Table 2.
F0=F ¼ 1 þ KSV ½Qꢁ ¼ 1 þ Kq τ0 ½Qꢁ
(2)
where F0 and F are the fluorescence intensities in the absence and
presence of quencher, respectively; KSV is Stern–Volmer quenching
constant; [Q] is the concentration of the quencher, TD/TG here;
Kq = KSV/τ0, is the apparent bimolecular quenching rate constant;
τ0 is the average lifetime of the molecule without quencher,
which is ~ 10-8 s. KSV can be calculated from the Stern–Volmer plots
of F0/F versus [Q] at three temperatures (inset to Fig. 3A,B), then Kq
can be calculated. Values of KSV and Kq are listed in Table 1. In
general, the collisional quenching constant for various types of
quencher with biopolymer is 2.0× 1010 L/mol/s (36). In this study,
the bimolecular quenching constants are both much great than
this value in the interaction between TD/TG and HSA, which
implies that there is non-fluorescent complex formation between
TD/TG and HSA. The static quenching may be the main
mechanism of the fluorescence quenching of HSA by TD/TG under
our experiment conditions (37).
The values of n are ~ 1, suggesting that there is one major
binding site in HSA for both TD and TG during their interactions.
For TD, the values of Ka decrease with increases in temperature,
and indicate that the stability of the TD–HSA complex decreases
at increasing temperature (39). The increase in Ka values with
increasing temperature suggested that the binding between TG
and HSA is endothermic (40). More importantly, the value of Ka
(KaTG > KaTD) confirms that the affinity of TG–HSA is stronger than
that of TD–HAS, and TG binds more closely with HSA than TD (41).
The decreasing affinity of TD for HSA after glycosylation may be
caused by the increase in molecular size and polarity, and transfer
to a non-planar structure (42). When the hydroxyl group is
replaced by a glycoside in the TD molecule, steric hindrance may
take place, which may weaken the affinity to HSA. Another
possible explanation is that glycosylation decreases the hydropho-
bicity of flavonoids (43).
Our results partly support the hypothesis that flavonoid
aglycones are more easily absorbed than flavonoid glycosides
(6). The fairly large and highly polar flavonoid glycosides cannot
be absorbed after oral ingestion, but are hydrolyzed to their
aglycones by bacterial enzymes in vivo (5,6). This is supported by
Binding parameters
For a static quenching interaction, the relationship between the
fluorescence intensity and the concentration of quencher can be
Figure 3. Emission spectra of HSA (2.0 × 10-6 mol/L) in various TD (A)/TG (B) concentrations at 298 K. [TD or TG]: (a → g) 0, 2.0 × 10-6, 4.0 × 10-6, 6.0 × 10-6, 8.0 × 10-6, 1.0 × 10-5,
1.2 × 10-5 mol/L. [h] = 1.2 × 10-5 mol/L for TD (A) /TG (B) only. (Inset) Stern–Volmer plot.
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Interaction of human serum albumin with tectoridin and tectorigenin
Table 2. Values of Ka, n and thermodynamic parameters of the TD/TG–HSA system at different temperatures.
Compound
TD
T (K)
Ka (mol/L)
n
R*
ΔH (kJ/mol)
–25.177
ΔG (kJ/mol)
ΔS ( J/mol/K)
298
310
315
298
310
315
102 235
71 040
58 492
130 858
138 548
140 832
1.0772
1.0544
1.0418
1.0535
1.0693
1.0783
0.9977
0.9993
0.9968
0.9990
0.9994
0.9996
–28.58
–28.79
–28.75
–29.19
–30.51
–31.05
11.48
TG
3.43
109.47
* R is the correlation coefficient for the regression equations.
the metabolic process of TD shown previously (26–30), which
proposes that TD is first hydrolyzed to TG, and TG shows more
potent biological activity than TD.
quantum yield of the donor. In this case, K2 = 2/3, N = 1.33 and
Φ = 0.13 (44). J can be obtained from equation (6) (35):
J
¼ ∑ FðλÞ εðλÞ λ4 Δλ =∑ FðλÞ Δλ
(6)
FRET measurements analysis
Where F(λ) is the fluorescence intensity of donor without
acceptor at wavelength λ and ε(λ) is the UV molar absorption
coefficient of the acceptor molecule when the wavelength is λ.
J, R0, E and r were obtained from the above equations and the
experimental data, and are listed in Table 3. It can be seen from
Table 3 that the distance between the donor and acceptor
molecules (r) was < 8 nm, and 0.5R0 < r < 1.5R0, which indicates
that energy transfer occurs from HSA to TD/TG with high
probability and the presence of a static type of quenching
mechanism (44). This result correlates nicely with the binding
parameters and quenching mechanism discussed previously.
According to Förster’s theory, the efficiency of fluorescence
resonance energy transfer (FRET) depends on the following factors
(35): (1) the relative orientation of donor and acceptor dipoles; (2)
the degree of overlap between the fluorescence emission
spectrum of the donor and the absorption spectrum of the
acceptor; and (3) the distance between the donor and acceptor.
The overlap between the fluorescence emission spectrum of HSA
and the UV–vis absorption spectra of TD and TG, which was
recorded in UV and visible absorption spectroscopy (TU1900,
Persee, Beijing, China), is shown in Fig. 4. The binding distance, r,
between TD/TG and HSA, and the energy transfer efficiency E
between the donor and acceptor could be calculated using
equation (4) (35):
Thermodynamic parameters and nature of the binding forces
The thermodynamic parameters of TD/TG–HSA complex are
calculated from the following van’t Hoff equation (38):
À
Á
6
E ¼ 1 ꢂ F=F0 ¼ R06= R0 þ r6
(4)
InKa
¼
ꢂ ΔH=ðRTÞ þ ΔS=R
(7)
(8)
Where F and F0 are the fluorescence intensities of HSA in the
presence and absence of TD/TG, r is the distance between the
acceptor and donor and R0 is the critical distance when the transfer
efficiency is 50%. R0 is calculated from equation (5) (35):
ΔG ¼ ꢂ RT InKa
where Ka is the binding constant at the corresponding tempera-
ture, R is the gas constant (8.3145 J/mol/K), and T is the experimen-
tal temperature.
According to equation (7), ΔH and ΔS can be calculated from the
slope and intercept of the plot of lnKa versus 1/T. ΔG is calculated
using lnKa values for different temperatures. The thermodynamic
parameters are summarized in Table 2.
R0 ¼ 8:79 ꢀ 10ꢂ25 K2 Nꢂ4 Φ J
(5)
6
Where K2 is the spatial orientation factor of the dipole, N is the
refractive index of the medium, and Φ is the fluorescence
For these two HSA binding systems, negative values of ΔG
indicate that the binding is spontaneous. The values of ΔH and
ΔS indicate that hydrogen binding and van der Waal’s force are
the main forces between TD and HSA (38), whereas a hydrophobic
interaction is considered to be the main binding force between
TG and HSA (38). This can be explained by the differences in
molecular polarity, molecule planarity and molecular size between
TD and TG.
Table 3. Energy transfer parameters for TD/TG–HSA system
System
J (cm3/L/mol)
R0 (nm)
E
r (nm)
TD–HSA
TG–HSA
2.740 × 10–14
3.034 × 10–14
2.96
3.01
0.108
0.219
4.21
3.72
Figure 4. The overlap of fluorescence spectrum of (a) HSA and the absorption of (b)
TD and (c) TG. [HSA] = [TD] = [TG] = 2.0 × 10-6 mol/L.
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Y. Li et al.
However, in the TG–HSA system, the positive values of ΔH
indicate that the binding is endothermic, and the binding capacity
of TG to HSA is enhanced by increasing temperature. The basis of
hydrophobic forces can explain on (40).
Identification of the sites of action of TD and TG on HSA
To identify the binding sites of TD and TG on HSA, warfarin and
ibuprofen were used as probes to label sites І and II, respectively,
in the competition experiments (45). In the warfarin competition
experiments, on regular addition of TD or TG, the fluorescence
intensity of HSA gradually decreased, indicating that the binding
of both TD and TG to HSA is affected by the addition of warfarin.
By contrast, there was almost no change on addition of ibuprofen,
indicating that ibuprofen does not prevent the binding of these
two flavonoids to their usual binding location. The binding
constants (Ka) for the competitive experiments were calculated
and the results are given in Table 4. In the presence of warfarin,
the binding constant (apparent binding constant) changed
significantly, whereas in the presence of ibuprofen, the change in
the binding parameters was relatively small. The experimental
Table 4. Binding constants of competitive experiments of
TD/TG–HSA system (T = 298 K)
Compounds
TD
Site probe
Ka (L/mol)
None
102 235
457
94 762
130 858
6 457
Warfarin
Ibuprofen
None
Warfarin
Ibuprofen
TG
113 586
Figure 5. Synchronous fluorescence spectra of the TD–HSA system in Δλ = 15 nm (A) and Δλ = 60 nm (B); TG–HSA system in Δλ = 15 nm (C) and Δλ = 60 nm (D). The synchronous
fluorescence quenching degree of HSA by TD and TG (E). [HSA] = 2.0 × 10-6 mol/L; [TD or TG]: (a → g) 0, 2.0 × 10-6, 4.0 × 10-6, 6.0 × 10-6, 8.0 × 10-6, 1.0 × 10-5, 1.2 × 10-5 mol/L (T = 298 K).
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Interaction of human serum albumin with tectoridin and tectorigenin
results and analysis demonstrate that the binding site for TD and
TG to HSA is mainly located within site I (subdomain IIA) of HSA.
(40.06% for TG), and that of Tyr is decreased to 70.12%
(54.81% for TG), suggesting that both flavonoids reached
subdomain IIA of HSA, where only one Trp residue (Trp214) is
located (47). Moreover, there is a large hydrophobic cavity in
subdomain IIA where many drugs can bind (48). Therefore, the
quenching of the fluorescence intensity of Trp residues is
stronger than that of Tyr residues, suggesting that Trp residues
contribute more to the quenching of the intrinsic fluorescence.
These results suggest that both flavonoids are closer to Trp
residues than Tyr residues, or the binding site is mainly focused
on a tryptophan moiety (49).
Also, as shown in Fig. 5(E), the slopes of the curves for [TG–HSA]
are steeper than for [TD–HSA] when Δλ is either 15 or 60 nm,
indicating that TG has more micro-environmental influence than
TD on the amino acid residues of HAS. This conclusion agrees well
with the binding parameters results.
Synchronous fluorescence spectra
When the intervals (Δλ) between the excitation and emission
wavelengths are 15 and 60nm, the synchronous fluorescence
spectra show characteristics of the tyrosine (Tyr) and tryptophan
(Trp) residues of HSA, respectively (46). The effect of these two
flavonoids on the synchronous fluorescence spectra is shown in
Fig. 5. It can be seen from Fig. 5(B,D) that, when the concentrations
of TD or TG increase, the maximum emission wavelength of the
Trp residue becomes slightly red-shifted (from 281 to 283.5nm
for TD, and from 281 to 284 nm for TG), although the Tyr residues
do not show any significant shift in TD or TG (Fig. 5A,C). This
indicates that the conformation of the Trp micro-environment is
changed and the polarity is decreased with increased hydrophobic
binding of TD or TG (46).
Moreover, as the concentration of the two flavonoids is
gradually increases, the synchronous fluorescence intensities
decrease. To explore the conformational changes in HSA induced
by the addition of the two flavonoids (46–48), F/F0 is plotted
against [Q] for the complexes at Δλ = 15 nm and Δλ =60nm
(Fig. 5E).
TD and TG competitive binding experiment
To determine whether the interaction between one flavonoid
and HSA is altered in the presence of another, we explored
the competitive binding between TD and TG in a ternary system
(45,49). When TD or TG was added to a solution containing
TG–HSA or TD–HSA, the fluorescence intensity of the solution
decreased. So TD or TG can interact with TG–HSA or TG–HSA
Comparing Δλ = 15 nm with Δλ = 60 nm, on addition of TD (TG),
the fluorescence intensity of Trp residues is decreased to 67.91%
binary systems to form
a
three-component complex,
TG–(HSA–TD) or TD–(HSA–TG), via apparently hydrophobic
hydrogen binding or van der Waal’s forces, which are the main
interaction forces discussed previously.
The curves of F/F0 versus [Q] for the complexes are plotted
in Fig. 6. Comparison of the quenching curves in the binary
and ternary systems provides general information about the
change in affinity of one to serum albumin in the presence of the
other (50).
The quenching curves of the TG–HSA and TG–(HSA–TD) sys-
tems overlapped (Fig. 6a,b). This means that TD does not
affect, or contributes only weakly to the quenching of HSA
fluorescence in the presence of TG in the ternary system
TG–(HSA–TD). By contrast, it can be observed from Fig. 6 that
the quenching curves of TD–HSA and TD–(HSA–TG) systems
are quite different. It can be concluded that the presence of
TG probably inhibits formation of the TD–HSA complex or
makes the TD–HSA interaction more difficult. This phenomenon
may be caused by TG, which reduces the affinity of TD to HSA,
displaces the TD molecule and stabilizes the complex due to
the hydrophobic interactions.
Figure 6. Quenching curves in the binary system (a: TG effect HSA; c: TD effect HSA)
and ternary system (b: TG effect HSA when TD fixed; d: TD effect HSA when TG fixed).
[HSA] = 2.0 × 10-6 mol/L.
Figure 7. 3D fluorescence spectra of HSA (A), TD-HSA (B) and TG-HSA (C). [HSA] = [TD] = [TG] = 2 × 10-6 mol/L.
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Y. Li et al.
3D fluorescence spectra analysis
The 3D fluorescence spectra of HSA in the absence and presence
of TD or TG are shown in Fig. 7, and the related characteristics
are presented in Table 5. As shown in Fig. 7, peak a is the Rayleigh
scattering peak (λex = λem), and peak b is the second-order
scattering peak (λex = 2λem). Peak 1 mainly reveals the spectral
characteristics of tryptophan and tyrosine residues (51), because
their intrinsic fluorescence is primarily exhibited when HSA is
excited at 280 nm, whereas the fluorescence of the phenylalanine
residue can be negligible. In addition to peak 1, there is another
visible peak (peak 2), which is mainly caused by the transition of
π → π* of characteristic polypeptide backbone structure C = O of
HSA (51).
The intensities of peaks 1 and 2 are obviously changed,
although to different extents, in the absence and presence of TD
or TG (Table 5). The decrease in the fluorescence intensity of the
two peaks results from a conformational change in HSA, increasing
the exposure of some previously buried hydrophobic regions to
TD and TG.
As shown in Fig. 7 and Table 5, the addition of TD/TG to HSA led
to a significant reduction in fluorescence intensities with a
slightly blue shift in the maximum emission wavelength for peak
1 and a red shift for peak 2. The above phenomena reveal that
the interactions between TG and HSA induced some micro-
environmental and conformational changes in HSA.
Figure 8. The schematic diagram generated using the 2D diagram feature of
Accelrys Discovery Studio 3.1 shows the interactions between TD (A)/TG (B) and its
neighbouring residues.
Docking studies
To confirm the experimental data, molecular docking studies were
carried out. The optimum poses of the lowest CDOCKER interac-
tion energy in 2D docking of HSA–TD/TG are shown in Fig. 8. Based
on the docking results, both TD and TG have more affinity to site I
than site II in HSA, which is in agreement with the site marker
experimental results.
transitions, suggesting that the stability of the TG–HSA system
is enhanced, which is in accordance with the binding mode
proposed above.
TD is mainly surrounded by Leu260, Arg257, Leu238, Tyr150,
Ser287, Ile264, Ile290, Leu219, Ala291, Arg222, Arg218, Glu292,
Lys195, Trp214 and Lys199 (site I). A hydrogen bond is found
between ligand oxygen and Arg257. In addition, residues
Arg222, Arg218, Lys195 and Lys199 in the proximity of the ligand
are conjugated with TD (Fig. 8A). These forces stabilize the docking
conformation and are consistent with the binding mode obtained
from the thermodynamic parameters, which confirms the
experimental results.
Figure 8(B) shows that TG was mainly surrounded by Trp214,
Lys199, Arg218, Arg222, Leu260, Ser287, Ala261, Arg257, Val241
and Leu238 (site I). Residues Lys199, Arg222 and Arg257 in the
proximity of the ligand are conjugated with TD as π → π*
Conclusion
In this study, TD was isolated from I. tectorum rhizomes and
hydrolyzed to obtain TG. We then studied the interaction of
TD/TG with HSA using spectroscopic fluorescence and molecular
docking techniques. The results revealed that the HSA fluores-
cence was quenched by TD/TG. Static quenching could be the
main mechanism of the fluorescence quenching of HSA by
TD/TG under our experiment conditions. The affinity of TG–HSA
was stronger than that of TD–HAS, and TG combined more closely
with HSA than TD. The negative values of ΔG indicated that the
binding processes were both spontaneous, and the values of ΔH
and ΔS indicated that hydrogen binding and van der Waal’s force
were the main forces between TD and HSA, whereas hydrophobic
interactions were considered the main binding force between TG
and HSA. Synchronous and 3D fluorescence spectral analysis
showed that the binding of TD/TG led to conformational changes
in the protein. In addition, a docking study suggested that both TD
and TG could enter into the large hydrophobic cavity of site I,
consistent with the binding mode obtained from the thermody-
namic parameters. The competitive binding characters of TD and
TG in a ternary system were explored, and the result showed that
TG reduced the affinity of TD for HSA, displaced the TD molecule
and stabilized the complex.
Table 5. 3D fluorescence spectral parameters of HSA alone
and in the presence of TD/TG
System
Peak
Peak position
Intensity (a.u.)
[λex/λem] (nm)
HSA
1
2
1
2
1
2
280/340
225/333
280/337
225/335
280/332
225/338
582.28
688.96
446.01
560.10
385.65
548.59
TD–HSA
TG–HSA
The drug delivery system is important in the efficacy of a
drug. Studies of the interactions between transporters like HSA
and drugs are thus valuable. In this case, the interaction
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Luminescence 2016; 31: 38–46

Interaction of human serum albumin with tectoridin and tectorigenin
22. Chen Y, Song W, Peng ZH, Ge BY, Han FM. Identification of metabolites
of tectoridin in vivo and in vitro by liquid chromatography-tandem
mass spectrometry. J Pharm Pharmacol 2008;60:709–16.
23. Park EK, Shin YW, Lee HU, Lee CS, Kim DH. Passive cutaneous
anaphylaxis-inhibitory action of tectorigenin, a metabolite of tectoridin
by intestinal microflora. Biol Pharm Bull 2004;27:1099–102.
24. Zhang WD, Qi LW, Yang XL, Huang WZ, Li P, Yang ZL. Identification of
the major metabolites of tectorigenin in rat bile by liquid chromatogra-
phy combined with time-of-flight and ion trap tandem mass spectrom-
etry. Rapid Commun Mass Spectrom 2008;22:2677–84.
25. Yang F, Zhang Y, Liang H. Interactive association of drugs binding to
human serum albumin. Int J Mol Sci 2014;15:3580–95.
26. Yamasaki K, Chuang VT, Maruyama T, Otagiri M. Albumin–drug
interaction and its clinical implication. Biochim Biophys Acta
1830;2013:5435–43.
27. Anand U, Mukherjee S. Binding, unfolding and refolding dynamics of
serum albumins. Biochim Biophys Acta 1830;2013:5394–404.
between HSA and TD/TG will be important factor in our
understanding of the pharmacokinetics and pharmacological
effects of TD/TG.
References
1. Falcone Ferreyra ML, Rius SP, Casati P. Flavonoids: biosynthesis,
biological functions, and biotechnological applications. Front Plant
Sci 2012;3:222.
2. Singh M, Kaur M, Silakari O. Flavones: an important scaffold for
medicinal chemistry. Eur J Med Chem 2014;84:206–39.
3. Thilakarathna SH, Rupasinghe HP. Flavonoid bioavailability and attempts
for bioavailability enhancement. Nutrients 2013;5:3367–87.
4. Chen Z, Zheng S, Li L, Jiang H. Metabolism of flavonoids in human: a
comprehensive review. Curr Drug Metab 2014;15:48–61.
5. Walle T. Absorption and metabolism of flavonoids. Free Radical Biol
Med 2004;36:829–37.
6. Walle T, Browning AM, Steed LL, Reed SG, Walle UK. Flavonoid
glucosides are hydrolyzed and thus activated in the oral cavity in
humans. J Nutr 2005;135:48–52.
7. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant polyphenols:
chemical properties, biological activities, and synthesis. Angew Chem
Int Ed Engl 2011;50:586–621.
8. Ionescu S, Matei I, Tablet C, Hillebrand M. New insights on flavonoid–
serum albumin interactions from concerted spectroscopic methods
and molecular modeling. Curr Drug Metab 2013;14:474–90.
9. Chaudhuri S, Sengupta B, Taylor J, Pahari BP, Sengupta PK. Interactions
of dietary flavonoids with proteins: insights from fluorescence
spectroscopy and other related biophysical studies. Curr Drug Metab
2013;14:491–503.
28. Pal S, Saha C.
A review on structure–affinity relationship of
dietary flavonoids with serum albumins. J Biomol Struct Dyn 2014;32:
1132–47.
29. Cao H, Wu D, Wang H, Xu M. Effect of the glycosylation of flavonoids on
interaction with protein. Spectrochim Acta A 2009;73:972–5.
30. Xiao J, Cao H, Wang Y, Zhao J, Wei X. Glycosylation of dietary flavonoids
decreases the affinities for plasma protein. J Agric Food Chem
2009;57:6642–8.
31. Farag SF, Backheet EY, El-Emary NA, Niwa M. Isoflavonoids and flavone
glycosides from rhizomes of Iris carthaliniae. Phytochemistry
1999;50:1407–10.
32. Han T, Cheng G, Liu Y, Yang H, Hu YT, Huang W. In vitro evaluation of
tectoridin, tectorigenin and tectorigenin sodium sulfonate on antioxi-
dant properties. Food Chem Toxicol 2012;50:409–14.
33. Sun Y, Liu Z, Wang J. Ultrasound-assisted extraction of five isoflavones
from Iris tectorum. Maxim Sep Purif Technol 2011;78:49–54.
34. Huang Y, Cui LJ, Wang JM, Huo K, Chen C, Zhan WH, et al. Interaction of
aconitine with bovine serum albumin and effect of atropine sulphate
and glycyrrhizic acid on the binding. J Lumin 2012;132:357–61.
35. Lakowicz JR. Principles of fluorescence spectroscopy. Plenum Press:
New York, 2006.
36. Matei I, Hillebrand M. Interaction of kaempferol with human serum
albumin: a fluorescence and circular dichroism study. J Pharm Biomed
Anal 2010;51:768–73.
37. Naik KM, Nandibewoor ST. Spectroscopic studies on the interaction
between chalcone and bovine serum albumin. J Lumin 2013;
143:484–91.
38. Tang L, Zuo H, Li S. Comparison of the interaction between three
anthocyanins and human serum albumins by spectroscopy. J Lumin
2014;153:54–63.
39. Pu H, Jiang H, Chen R. Combined multispectroscopic and molecular
docking investigation on the interaction between strictosamide and
human serum albumin. Luminescence 2013;28:482–9.
10. Jaldappagari S, Balakrishnan S, Hegde AH, Teradal NL, Narayan PS.
Interactions of polyphenols with plasma proteins: insights from
analytical techniques. Curr Drug Metab 2013;14:456–73.
11. Sun Y, Li W, Wang J. Ionic liquid based ultrasonic assisted extraction of
isoflavones from Iris tectorum Maxim and subsequently separation and
purification by high-speed counter-current chromatography.
J
Chromatogr B Analyt Technol Biomed Life Sci 2011;879:975–80.
12. Jiang Y, Zhao W, Feng C, Zhou T, Fan G, Wu Y. Isolation and purification
of isoflavonoids from Rhizoma belamcandae by two-dimensional
preparative high-performance liquid chromatography with column
switch technology. Biomed Chromatogr 2009;23:1064–72.
13. Kim C, Shin S, Ha H, Kim JM. Study of substance changes in flowers
of Pueraria thunbergiana Benth. during storage. Arch Pharm Res
2003;26:210–3.
14. Akther N, Andrabi K, Nissar A, Ganaie S, Chandan BK, Gupta AP, et al.
Hepatoprotective activity of LC-ESI-MS standardized Iris spuria rhizome
extract on its main bioactive constituents. Phytomedicine
2014;21:1202–7.
15. Shim M, Bae JY, Lee YJ, Ahn MJ. Tectoridin from Maackia amurensis
modulates both estrogen and thyroid receptors. Phytomedicine
2014;21:602–6.
16. Chen Y, Wu CM, Dai RJ, Li L, Yu YH, Li Y, et al. Combination of HPLC
chromatogram and hypoglycemic effect identifies isoflavones as the
principal active fraction of Belamcanda chinensis leaf extract in diabetes
40. Wang YQ, Zhang H-M, Zhang GC, Tao WH, Tang SH. Binding of brucine
to human serum albumin. J Mol Struct 2007;830:40–5.
41. Cao H, Wu D, Wang H, Xu M. Effect of the glycosylation of flavonoids on
interaction with protein. Spectrochimica Acta A 2009;73:972–5.
42. Xiao J, Cao H, Wang Y, Zhao J, Wei X. Glycosylation of dietary flavonoids
decreases the affinities for plasma protein. J Agric Food Chem
2009;57:6642–8.
43. Tavel L, Andriot I, Moreau C, Guichard E. Interactions between
beta-lactoglobulin and aroma compounds: different binding
behaviors as a function of ligand structure. J Agric Food Chem
2008;56:10208–17.
treatment.
J Chromatogr B Analyt Technol Biomed Life Sci
2011;879:371–8.
17. Jung SH, Lee YS, Lim SS, Lee S, Shin KH, Kim YS. Antioxidant activities of
isoflavones from the rhizomes of Belamcanda chinensis on carbon
tetrachloride-induced hepatic injury in rats. Arch Pharm Res
2004;27:184–8.
44. Gökoğlu E, Kıpçak F, Seferoğlu Z. Studies on the interactions of
3,6-diaminoacridine derivatives with human serum albumin by
fluorescence spectroscopy. Luminescence 2014;29:872–7.
45. Bozoğlan BK, Tunç S, Duman O. Investigation of neohesperidin
dihydrochalcone binding to human serum albumin by spectroscopic
methods. J Lumin 2014;155:198–204.
18. Pan CH, Kim ES, Jung SH, Nho CW, Lee JK. Tectorigenin inhibits
IFN-gamma/LPS-induced inflammatory responses in murine macro-
phage RAW 264.7 cells. Arch Pharm Res 2008;31:1447–56.
19. Liu M, Yang S, Jin L, Hu D, Wu Z, Yang S. Chemical constituents of the
ethyl acetate extract of Belamcanda chinensis (L.) DC roots and their
antitumor activities. Molecules 2012;17:6156–69.
46. Bi S, Yan L, Pang B, Wang Y. Investigation of three flavonoids binding to
bovine serum albumin using molecular fluorescence technique. J
Lumin 2012;132:132–40.
47. Dangkoob F, Housaindokht MR, Asoodeh A, Rajabi O, Zaeri ZR, Doghaei
AV. Spectroscopic and molecular modeling study on the separate and
simultaneous bindings of alprazolam and fluoxetine hydrochloride to
human serum albumin (HSA): with the aim of the drug interactions
probing. Spectrochim Acta A 2015;137:1106–19.
20. Bae EA, Han MJ, Lee KT, Choi JW, Park HJ, Kim DH. Metabolism of 6′′-O-
xylosyltectoridin and tectoridin by human intestinal bacteria and their
hypoglycemic and in vitro cytotoxic activities. Biol Pharm Bull
1999;22:1314–8.
21. Kang KA, Lee KH, Chae S, Zhang R, Jung MS, Kim SY, et al.
Cytoprotective effect of tectorigenin, a metabolite formed by transfor-
mation of tectoridin by intestinal microflora, on oxidative stress
induced by hydrogen peroxide. Eur J Pharmacol 2005;519:16–23.
Luminescence 2016; 31: 38–46
Copyright © 2015 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/luminescence

Y. Li et al.
48. Cheng Z, Liu R, Jiang X, Xu Q. The interaction between cepharanthine
and two serum albumins: multiple spectroscopic and chemometric
investigations. Luminescence 2014;29:504–15.
49. Cheng ZJ. Studies on the interaction between scopoletin and two
serum albumins by spectroscopic methods. J Lumin 2012;132:2719–29.
50. Chamani J, Asoodeh A, Homayoni-Tabrizi M, Tehranizadeh ZA,
Baratian A, Saberi MR, et al. Spectroscopic and nano-molecular
modeling investigation on the binary and ternary bindings of colchi-
cine and lomefloxacin to human serum albumin with the viewpoint
of multi-drug therapy. J Lumin 2010;130:2476–86.
51. Pu H, Jiang H, Chen R, Wang H. Studies on the interaction between
vincamine and human serum albumin: a spectroscopic approach.
Luminescence 2014;29:471–9.
wileyonlinelibrary.com/journal/luminescence
Copyright © 2015 John Wiley & Sons, Ltd.
Luminescence 2016; 31: 38–46