Kaempferol Binding to Zinc(II), Efficient Radical Scavenging through Increased Phenol Acidity

Article abstract of DOI:10.1021/acs.jpcb.8b08284

Zinc(II) enhances radical scavenging of the flavonoid kaempferol (Kaem) most significantly for the 1:1 Zn(II)-Kaem complex in equilibrium with the 1:2 Zn(II)-Kaem complex both with high affinity at 3-hydroxyl and 4-carboxyl coordination. In methanol/chloroform (7/3, v/v), 1:1 Zn(II)-Kaem complex reduces β-carotene radical cation, β-Car^?+, with a second-order rate constant, 1.88 × 10⁸ L·mol^-1·s^-1, while both Kaem and 1:2 Zn(II)-Kaem complex are nonreactive, as determined by laser flash photolysis. In ethanol, 1:1 Zn(II)-Kaem complex reduces the 2,2-diphenyl-1-picrylhydrazyl radical, DPPH^?, with a second-order rate constant, 2.48 × 10⁴ L·mol^-1·s^-1, 16 times and 2 times as efficient as Kaem and 1:2 Zn(II)-Kaem complex, respectively, as determined by stopped-flow spectroscopy. Density functional theory calculation results indicate significantly increased acidity of Kaem as ligand in 1:1 Zn(II)-Kaem complex other than in 1:2 Zn(II)-Kaem complex. Kaem in 1:1 Zn(II)-Kaem complex loses two protons (one from 3-hydroxyl and one from phenolic hydroxyl) forming 1:1 Zn(II)-(Kaem-2H) during binding with Zn(II), while Kaem in 1:2 Zn(II)-Kaem complex loses one proton in each ligand forming Zn(II)-(Kaem-H)₂, as confirmed by UV-vis absorption spectroscopy. Zn(II)-(Kaem-2H) is a far stronger reductant than Kaem and Zn(II)-(Kaem-H)₂ as determined by cyclic voltammetry. Significant rate increases for the 1:1 complex in both β-Car^?+ scavenging by electron transfer and DPPH^? scavenging by hydrogen atom transfer were ascribed to decreases of ionization potential and of bond dissociation energy of 4′-OH for deprotonated Zn(II)-(Kaem-2H), respectively. Increased phenol acidity of plant polyphenols by 1:1 coordination with Zn(II) may explain the unique function of Zn(II) as a biological antioxidant and may help to design nontoxic metal-based drugs derived from natural bioactive molecules.

Full text of DOI:10.1021/acs.jpcb.8b08284

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules
Kaempferol Binding to zinc(II). Efficient Radical
Scavenging through Increased Phenol Acidity
Yi Xu, Lingling Qian, Jing Yang, Rui-Min Han, Jian-Ping Zhang, and Leif H. Skibsted
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b08284 • Publication Date (Web): 08 Oct 2018
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Kaempferol Binding to Zinc(II). Efficient Radical
Scavenging Through Increased Phenol Acidity
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Yi Xu†, Ling-Ling Qian†, Jing Yang†, Rui-Min Han†*, Jian-Ping Zhang†, Leif H. Skibsted‡*
†Department of Chemistry, Renmin University of China, Beijing, 100872
‡Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1058 Frederiksberg C,
Denmark
E-mail addresses of authors：
Rui-Min Han: rmhan@ruc.edu.cn
Yi Xu: yixu@ruc.edu.cn
Ling-Ling Qian: qianlingling@ruc.edu.cn
Jing Yang: yangjing2015@ruc.edu.cn
Jian-Ping Zhang: jpzhang@ruc.edu.cn
Leif H. Skibsted: ls@food.ku.dk
*Corresponding author
Ph. D. Rui-Min Han
Tel: +86-10-6251-6604; Fax: +86-10-6251-6444
E-mail: rmhan@ruc.edu.cn
and
Professor Leif H. Skibsted
Tel: +45-3533-3221
E-mail: ls@food.ku.dk
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ABSTRACT: Zinc(II) enhances radical scavenging of the flavonoid kaempferol (Kaem) most
significantly for the 1:1 Zn(II)-kaempferol complex in equilibrium with the 1:2 Zn(II)-kaempferol
complex both with high-affinity at 3-hydroxyl and 4-carboxyl coordination. In methanol/chloroform (7/3,
v/v), 1:1 Zn(II)-kaempferol complex reduces β-carotene radical cation, β-Car•+, with a second-order rate
constant, 1.88×108 L·mol−1·s−1, while both Kaem and 1:2 Zn(II)-kaempferol complex are non-reactive, as
determined by laser flash photolysis. In ethanol, 1:1 Zn(II)-kaempferol complex reduces the 2,2-diphenyl-
1-picrylhydrazyl radical, DPPH•, with a second order rate constant, 2.48×104 L·mol−1·s−1, 16 times and 2
times as efficient as Kaem and 1:2 Zn(II)-kaempferol complex, respectively, as determined by stopped-
flow spectroscopy. Density functional theory (DFT) calculation results indicate significantly increased
acidity of Kaem as ligand in 1:1 Zn(II)-kaempferol complex other than in 1:2 Zn(II)-kaempferol complex.
Kaem in 1:1 Zn(II)-kaempferol complex loses two protons (one from 3-hydroxyl and one from phenolic
hydroxyl) forming 1:1 Zn(II)-(Kaem−2H) during binding with Zn(II), while Kaem in 1:2 Zn(II)-
kaempferol complex loses one proton in each ligand forming Zn(II)-(Kaem‒H)2, as confirmed by UV-vis
absorption spectroscopy. Zn(II)-(Kaem−2H) is a far stronger reductant than Kaem and Zn(II)-(Kaem−H)2
as determined by cyclic voltammetry. Significant rate increases for the 1:1 complex in both β-Car•+
scavenging by electron transfer (ET) and DPPH• scavenging by hydrogen atom transfer (HAT) were
ascribed to decreases of ionization potential (IP) and of bond dissociation energy (BDE) of 4´-OH for
deprotonated Zn(II)-(Kaem−2H), respectively. Increased phenol acidity of plant polyphenols by 1:1
coordination with Zn(II) may explain the unique function of Zn(II) as a biological antioxidant and may
help to design non-toxic metal-based drugs derived from natural bioactive molecules.
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1. INTRODUCTION
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Zinc, as an important micronutrient, is a cofactor for more than 300 metalloenzymes essential for
growth and development of nearly all organisms.1-4 Zn(II) is crucial for the function of superoxide
dismutase through structural support for the Cu(I)/Cu(II) cycling.5 Zinc has also been found to improve
resistance to oxidative stress as an antioxidant despite that Zn(II) is the only oxidation state of importance
for zinc in biological systems.6-10 Zinc biofortification recently was also found improving nutritional
quality and antioxidant system of green bean grown under greenhouse conditions.11 Otherwise the
antioxidative activity of Zn(II) based on molecular level is poorly understood.
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Flavonoids, as secondary plant metabolites of polyphenols family, have important biological activities
such as antioxidant, anti-inflammatory, and anticancer.12,13 Kaempferol (Kaem, Scheme 1a) found in a
variety of plants and plant-derived common foods, contributes approximately ~4 mg/day in the total
average intake of flavonoid in a normal diet.14 Kaem has been reported to have important biological
functions as metal chelators and radical scavengers.15-16 Notably, most metal chelates of flavonoids have
often been found to be better antioxidants and to have more significant biological effects than their parent
flavonoids.17-28 Such enhancement in antioxidative activity of flavonoid complexes may be understood
for transition metal ions like copper(II) and iron(III) as a result of the reduction of central metal ions.17,
29-31
Zn(II) has, however, been found to coordinate flavonoids and other biomolecules, which may affect
the transport and uptake of zinc,32 and also alter the radical scavenging properties of such plant
polyphenols.9,33-36 Zn(II) complexes of Kaem, have recently been found to have anticancer activity.10For
the redox-inert metal zinc, different from copper and iron containing different oxidation states, other
mechanisms must be operating, but have not been identified or studied in any details despite a growing
interest in medical application of Zn(II) coordination compounds. Zinc, as a non-toxic metal, often
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becomes the first choice as a carrier or modifier of biological active secondary plant metabolites like the
flavonoids in the development of new metal-based drugs.33
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3'
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OH
2'
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O
C
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HO
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A
B
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OH
4
5
O
OH
(a)
(b)
(c)
Scheme 1. Molecular structures of (a) kaempferol (Kaem), and complexes of Kaem with Zn(II) ion, (b) 1:2 Zn(II)-
(Kaem‒H)2 and (c) 1:1 Zn(II)-(Kaem‒H)+ and deprotonated Zn(II)-(Kaem‒2H), with and without 7-phenolic proton, as
the most acidic group based on the following DFT calculations.
Reported are results from a study of Zn(II) complexes of Kaem combining spectroscopy for structural
characterization with results from investigations of reaction dynamics of the Zn(II) complexes with
radicals. Reactions with the very reactive β-carotene radical cation, β-Car•+, and with the semi-stable 1,1-
diphenyl-2-picrylhydrazyl radical, DPPH•, show common features for Kaem complexes despite the
different time regime for their reactions. The results point toward a more general mechanism for zinc as
an antioxidant entailing coupling with proton dissociation of plant polyphenols.
2. EXPERIMNENTAL SECTION
2.1 Chemicals. Kaempferol (Kaem, >98%) and apigenin (Api, >98%) from Huike Plant Exploitation Inc,
(Shanxi, China), zinc acetate dihydrate (Zn(CH3COO)2·2H2O, >99%), from Beijing Chemical Reagents
Company, China, acetic acid (CH3COOH, >99.5%) from Titan Scientific,(Shanghai, China), 1,1-
diphenyl-2-picrylhydrazyl radical (DPPH•) from ZhongShengRuiTai Technology Inc (>97%, Beijing,
China) were used as received. All-trans-β-carotene (β-Car, >93%), ferrocene (>98%), NaClO4 (>98%),
methanol-d4 (>99.8%) and chloroform-d (>99.8%) from Sigma-Aldrich (St. Louis, MO, USA) were used
as received. Spectrophotometric grade methanol and ethanol (99.9%, Fine Chemical Industry Research
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Institute, Tianjin, China) were used as received. Chloroform (>99.0%, Beijing Chemical Works, Beijing,
China) was purified before use by passing through an alumina column (AR, Tianjin Fuchen Chemical
Plant, Tianjin, China).
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2.2 Reaction of Zn(II) with Kaempferol. All UV-vis absorption spectra were measured on a Cary50
spectrophotometer (Varian, Inc., Palo Alto, CA, USA) using 1.0 cm quartz cells in a thermostated room
at 25°C. The solutions were prepared by mixing solutions of Kaem and Zn(CH3COO)2 with total molar
concentrations of 5.0×10−5 M in Kaem/Zn(II) molar ratios varying from 19:1 to 1:19.
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2.3 IR, NMR and Mass Spectra. IR spectra were obtained on Bruker Tensor 27 FT-IR spectrometer
(Karlsruke, Germany). Solid samples of Zn(II)-kaempferol complexes for IR tableting were prepared
from a solutions of Kaem and Zn(CH3COO)2 in ratios of 1:1 and 1:2 by evaporation of ethanol with a
nitrogen gas flow.
1H-NMR spectra were obtained on a Bruker AM 400 MHz spectrometer (Karlsruhe, Germany). Sample
of Zn(II)-kaempferol complexes for 1H-NMR determination were prepared by dissolving the solid sample
as obtained for IR spectra in deuterated methanol and chloroform.
MS spectra were obtained on a Thermo Scientific™ Q Exactive™ HF (Waltham, MA, USA) in the
positive ion mode. Sample of Zn(II)-kaempferol complexes were prepared by filtering solutions of Kaem
and Zn(CH3COO)2 after mixing through a nylon membrane with 220 nm sieve pores. The samples were
analyzed by direct infusion ESI by means of a syringe pump (Thermo UltiMate 3000,Waltham, MA, USA)
at a flow rate of 5 μL/min. Capillary temperature was 320°C and spray voltage was 3.50 kV.
2.4 β-Carotene Radical Cation and DPPH• Scavenging. The laser flash photolysis apparatus for the
study of the kinetics of β-Car•+ scavenging has previously been described in detail.37,38 The laser pulses
at 532 nm (4 mJ/pulse, 7 ns, and 10 Hz) for the bleaching assay were supplied by a Nd3+: YAG laser
(Quanta-Ray PRO-230, Spectra Physics Lasers, Inc., Mountain View, CA, USA). 940 and 510 nm probe
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lights were provided by a laser-driven white light source (LDLS-EQ-99 LAMP MODULE, Energetiq
Technology, Inc., Woburn, MA). The absorbance was detected with a photodiode (model S3071,
Hamamatsu Photonics, Hamamatsu, Japan). The optical path length of the flow cuvette was 1 cm. All of
the measurements were carried out in a thermostated room of 25 °C. Methanol/chloroform (7/3, v/v) was
used as the solvent for the equilibrated solution of β-Car and Zn(CH3COO)2 and Kaem.
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The kinetics of DPPH• scavenging by Zn(II)-kaempferol complexes was investigated by using a rapid
mixing, stopped-flow technique using the same method as previously, performed on RX2000 Rapid-
Mixing Stopped-Flow Unit (Applied Photophysics Ltd, Surrey, United Kingdom). For one syringe
solution, DPPH• was dissolved in ethanol to obtain an absorbance of 0.92±0.02 (extinction coefficient ɛ
= 9660 L·mol‒1·cm‒1) at 516 nm, the final concentration of the DPPH• was calculated to be 100 μM. The
other syringe solution was an equilibrated solution of Zn(CH3COO)2 and Kaem.
2.5 Determination of Oxidation Potentials. Cyclic voltammetry (CV) was performed on a three-
electrode CHI 760D electrochemical analyzer (ChenHua Instruments Inc., Shanghai, China). The working
electrode was a glassy carbon piece (diameter=4 mm), the reference electrode was a silver wire, and the
auxiliary electrode was a platinum wire. 0.10 M NaClO4 was used as supporting electrolyte. 5.0×10-5 M
ferrocene was used as internal standard, and CVs were obtained in the potential range of ‒0.4 V to 0.6 V
at 0.1 V/s scan rate.
2.6 Quantum Chemical Calculations. Structural optimizations, calculation of electronic density and
bond lengths of Kaem and three Zn(II)-kaempferol complexes were performed using the Gaussian 09
package with the Becke3 and Lee Yang Parr (B3LYP) hybrid functional method.39 The 6-31+G(d) basis
set and the LANL2DZ pseudopotential, were chosen for C, O, and H atoms, and for the Zn(II) cation,
respectively.
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The proton dissociation enthalpy (PDE), ionization potential (IP) and bond dissociation enthalpy (BDE)
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of both Kaem and Zn(II)-kaempferol complexes were calculated as gas phase enthalpy difference:
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Ar-OH → Ar-O• +H•
Ar-OH → Ar-O− + H+
Ar-OH →Ar-OH•+ + e
Ar-O− →Ar-O• + e
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Ar represents an unsaturated group linked to hydroxyl in Kaem or Zn(II)-kaempferol complexes.
Enthalpies of H• (−312.64 kcal·mol−1) and H+ (1.48 kcal·mol−1) were also calculated and the enthalpy of
electron (0.75 kcal·mol−1) was obtained from literature.40
3. RESULTS
3.1 Formation and Characterization of Two Zn(II)-Kaempferol Complexes. Zn(CH3COO)2 red-
shifts the UV-visible absorption spectrum of kaempferol (Kaem) as seen in Figure 1a for Kaem dissolved
in ethanol together with Zn(CH3COO)2 in a Kaem/Zn(II) ratio varying from 19:1 to 1:19. The spectral
changes indicate binding of Zn(II) to Kaem in a stepwise manner with an initial broadening of the Kaem
spectrum for increasing Zn(II) addition followed by appearance of a new absorption band at 440 nm. The
spectral changes are indicative of formation of two species, Zn(II)-Kaemp complex dominating for Kaem
in excess as equilibrium of equation (1) and Zn(II)-Kaemq complex dominating for increasing Zn(II) in
excess as equilibrium of equation (2):
(1)
(2)
in which p > q > 0, where both p and q may be integers or decimal numbers.
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a
Kaem : Zn(II) (a)
1.0
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19 : 1
0.8
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0.4
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(b)
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425 nm
Zn-Kaemp
Kaem
Zn-Kaem
q
1.2
1.0
0.8
0.6
0.4
0.2
0.0
(c)
(
p=2)
(q=1
)
368 nm
425 nm
440 nm
350
400
450
500
Wavelength / nm
0.4
0.3
0.2
0.1
0.0
440 nm
(d)
0.06
0.04
0.02
0.00
485 nm
Zn-Kaemp
p=2
440 nm
(
)
Zn-Kaemq
(q=1
)
0.0
0.2
0.4
0.6
0.8
1.0
FZn(II)
Figure 1. (a) Absorption spectra of Kaem and Zn(CH3COO)2 in a total concentration 50 µM in different ratios varying
from 19:1 to 1:19 in ethanol. (b) Absorption difference spectra of all spectra in Figure 1a subtracting spectra of Zn-
Kaemq(q=1). Detailed description was presented in text. (c) Normalized absorption spectra of Kaem and deconvoluted
spectra of Zn(II)-Kaemp (p=2) and Zn(II)-Kaemq (q=1). (d) Job’s-Plots of spectra in Figure 1a at 440 nm and
deconvoluted absorbances of Zn-Kaemp (p=2) at 440 nm and Zn-Kaemq (q=1) at 485 nm against molar fractions of zinc
ion, FZn(II)
.
The absorption spectrum of Zn(II)-Kaemq obtained from normalized spectra (for condition where
changes were not seen for further addition of Zn(II) ) with zinc (II) in excess as seen in Figure 1a. A series
of difference spectra excluding Zn(II)-Kaemq as component but with contribution from Zn(II)-Kaemp and
Kaem are shown in Figure 1b after subtraction the calculated Zn(II)-Kaemq spectrum from the original
spectra using equation:
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A
0_485nm
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4
S
=
S
-
S
×
Zn(II)- Kaemq
0
(3)
A
Zn(II)- Kaemq _ 485nm
S0
in which,
S
,
and
represent spectra after (Figure 1b) and before (Figure 1a) subtracting the
q
S
Zn ( II)- Kaem
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7
8
Zn(II)-Kaemq component, and spectrum of Zn(II)-Kaemq (Figure 1c), respectively.
A
and
0 _485nm
9
represent absorbances at 485 nm for original spectra before subtracting the spectrum of
A
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Zn(II)-Kaem _485nm
q
Zn(II)-Kaemq and the spectrum of Zn(II)-Kaemq, respectively.
The absorption spectrum of Zn(II)-Kaemp obtained using the same method as for Zn(II)-Kaemq is
shown in Figure 1c together with spectra of Zn(II)-Kaemq and Kaem. The Job’s-Plots41 for 440 nm based
on spectra in Figure 1a and the independent contribution to absorbance for Zn(II)-Kaemp at 440 nm and
Zn(II)-Kaemq at 485 nm as a function of the molar fraction of Zn(II) are shown in Figure 1d.
Using the numerical methods in reference,42 p = 2 and q = 1 were resolved using equations (S12) and
(S13) in Supporting Information. Equations (1) and (2) may accordingly be written as new forms:
(4)
(5)
Formation constants in equations (4) and (5) are calculated to have values K1 = 4.8×105 L∙mol−1 for
Zn(II)-Kaem and K2 = 2.5×105 L∙mol−1 for Zn-Kaem2, respectively (see Supporting Information). A
similar analyses based on Jobs method for equilibrium spectra of Kaem and Zn(CH3COO)2 in
methanol:chloroform (7/3, v/v) gave the stability constants K1=1.0×105 L∙mol−1 for Zn(II)-Kaem and
K2=2.0×105 L∙mol−1 for Zn-Kaem2. Equilibrium fractions of Kaem and two complexes for the
concentration ratios used for the structural characterization and for radical scavenging dynamics in ethanol
and methanol:chloroform (7/3, v/v) are shown in Table 1.
The spectral changes for Zinc(II) reaction with Kaem are different from Zn(II)-apigenin complex with
chelation occurring at 4-carbonyl and 5-hydroxyl (Figure S2), but similar ~50 nm spectral red shift
compared to parent flavonoids was observed for 1:2 Zn(II)-Kaem2 and Zn(II)-3-hydroxyflavone
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complex.43 Chelating site in Zn(II)-kaempferol complexes is accordingly ascribed to 3-hydroxyl and 4-
carbonyl groups. The remaining isolated 5-, 7- or 4´-hydroxyls can not associated with metal chelation10
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Table 1. Fractions (F, %) of Kaem, Zn(II)-(Kaem‒H)2 and Zn(II)-(Kaem‒2H) in ethanol and in methanol:chloroform
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(7/3) at indicated varying ratios.
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ethanol / methanol:chloroform(7:3)
Kaem: Zn(II)
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15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
FKaem(%)
FZn(II)-(Kaem‒H)2(%)
FZn(II)-(Kaem‒2H)(%)
1:0
100/100
61.3/61.9
13.8/19.2
0/0
0/0
1:0.2
1:0.5
37.6/36.6
75.7/71.4
1.1 / 1.5
10.5/9.4
1:1
2.7/6.5
1.1/3.3
0.4/1.5
0.2/0.7
0.1/0.4
57.1/67.4
36.3/54.4
18.0/36.4
9.9/24.4
5.2/15.0
40.2/26.1
62.6/42.3
81.6/62.1
89.9/74.9
94.7/84.6
1:2
1:5
1:10
1:20
Solutions: 100 µM Kaem with 0, 20, 50, 100, 200, 500, 1000, 2000 µM Zn(CH3COO)2, respectively.Zn(II)-(Kaem‒H)2
and Zn(II)-(Kaem‒2H) are the specific forms of 1:2 Zn-Kaem2 and 1:1 Zn-Kaem, respectively, and will be further
explained in the following..
The 15 nm red-shift of the absorption of Zn(II)-Kaem compared to Zn(II)-Kaem2 in Figure 1c is
indicative of phenolic deprotonation in Zn(II)-Kaem as confirmed by acid sensitivity of the visible
spectrum of Zn(II)-Kaem. Zn(II)-Kaem was found to transform into Zn(II)-Kaem2 with the spectral red-
shift reversed, by addition of acetic acid see Figure 2.
1.0
Zn(II)-Kaem2
Zn(II)-Kaem
440nm
[CH3COOH]
425 nm
0.8
0.6
0.4
0.2
0.0
0 µΜ
250 µΜ
500 µΜ
750 µΜ
1000 µΜ
1250 µΜ
300
350
400
450
500
Wavelength / nm
Figure 2. Absorption spectra changes of 50 μM Kaem in the presence of 500 μM Zn(CH3COO)2 with addition of 250,
500, 750, 1000 and 1250 μM CH3COOH.
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Formation of 1:2 complex involves loss of a proton from 3-hydroxyl of each Kaem ligand when binding
1
2
3
with zinc thus written as Zn(II)-(Kaem‒H)2 (Scheme 1b), similar to reported 1:2 Cu(II)-quercetin and
4
5
6
7
Zn(II)/Mg(II)-primuletin complexes:44,45
2H+
8
Zn(II) + 2Kaem
Zn(II)-(Kaem H)2
(6)
9
While, formation of 1:1 complex involves loss of one proton from 3-hydroxyl of Kaem generates a
cationic form, Zn(II)-(Kaem‒H)+ (with 7-phenolic proton in Scheme 1c).10,46 A second proton from 7-
phenolic hydroxyl (the most acidic group among three unchelated phenolic hydroxyls, will be further
explained based on the following DFT calculations) is lost from the same Kaem ligand eventually forming
Zn(II)-(Kaem‒2H) (without 7-phenolic proton in Scheme 1c):
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H+
H+
Zn(II)-(Kaem H)+
Zn(II)-(Kaem 2H)
Zn(II) + Kaem
(7)
The equilibrium transformation between two complexes, 1:1 Zn(II)-(Kaem‒2H) and 1:2 Zn(II)-(Kaem‒
H)2, depends on concentrations of hydrogen ion and Zn(II) ion (Scheme 2):
2Zn(II)-(Kaem 2H)+2H+
Zn(II)-(Kaem H)2+Zn(II)
(8)
Accordingly, Zn(II)-(Kaem‒H)+ and Zn(II)-(Kaem‒2H) are used for representing 1:1 Zn(II)-Kaem
before and after second deprotonation, respectively, and Zn(II)-(Kaem‒H)2 for 1:2 Zn(II)-Kaem2 in the
following texts.
IR spectrum data of Zn(II)-kaempferol complexes in Table SI further support above assignment of
1
Kaem binding to Zn(II) with 3-hydroxyl and 4-carbonyl groups. Mass spectrometry and H-NMR
spectroscopy (Figures S3, S4 and Tables S2, S3) confirmed the composition of Zn(II)-(Kaem−H)2 and
Zn(II)-(Kaem−2H) as obtained by Jobs method of continuous variation using Uv-visible spectra. (see
Supporting Information)
3.2 Determinations of Oxidation Potentials. Kaem was found to be oxidized by a quasi-reversible
process using cyclic voltammetry and has an oxidation potential of 0.179 V versus ferrocene from the CV
scan of 100 μM Kaem and Zn(II) for ratios varying from 5:1 to 1:20 as shown in Figure 3a and Table2.
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Oxidation potentials are obtained from initial oxidation peak corresponding to oxidation of phenolic
group.47,48 The significant decrease in oxidation potential from 0.179 V to −0.097 V by increasing addition
of Zn(II) ion agrees well with the increase of the Zn(II)-(Kaem−2H) percentage as shown in Figure 3b.
Kaem and 1:2 Zn(II)-(Kaem−H)2 are found to have very similar oxidation potential, ~0.170 V, while
Zn(II)-(Kaem−2H) is far more reducing with an oxidative potentials, approximately −0.097 V, obtained
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
from sample of Kaem and Zn(II) in ratio of 1:20. Such remarkable decreases in oxidation potentials were
also observed for complexes of Zn(II), Al(III) and Eu(III) with quercetin, rutin and galangin.35,49
6.0
(a)
Kaem : Zn(II)
1 : 0
1 : 0.5
1 : 1
4.0
1 : 5
1 : 10
2.0
0.0
-0.4 -0.2 0.0
0.2
0.4
0.6
Potential / V
(b)
Figure 3. (a) Cyclic voltammograms of 100 μM Kaem and solutions of 100 μM Kaem with 20, 50, 100, 200, 500 and
1000 μM Zn(CH3COO)2 in relative to 50 μM ferrocene and 0.1 M NaClO4. (b) Oxidation potentials obtained from
Figure 3a (blue circle), observed first order rate constants obtained from single exponential fitting the decay of β-Car•+
at 940 nm in Figure 4a (red square), and molar fractions (solid lines) of Kaem and deconvoluted Zn(II)-(Kaem−H)2 and
Zn(II)-(Kaem−2H) against concentration of Zn(CH3COO)2 calculated from formation constants, K1=1.0×105 L∙mol−1
and K2=2.0×105 L∙mol−1. Solvent is methanol / chloroform (7/3, v/v).
3.3 Radical Scavenging Kinetics. Antioxidant activities of Kaem and complexes of Zn(II) with Kaem
were evaluated using two radical scavenging methods: (i) scavenging of the photo-induced carotenoid
radical cations β-Car•+ in methanol/chloroform (7/3, v/v), and (ii) scavenging of the semi-stable radical
DPPH• in ethanol.
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3.3.1 β-Car•+ Radical Scavenging Kinetics. Bleaching of β-Car in methanol/chloroform to generate β-
Car•+ using laser flash photolysis and time resolved absorption spectroscopy were previously established
for comparison of the antioxidant activity of polyphenols.37,38 The decay of β-Car•+ absorbing at 940 nm
is not affected by the presence of Zn(II) or Kaem alone, or even Kaem and Zn(II) with ratio at 1:0.2, but
is increasingly accelerated by addition of Zn(II) to Kaem solutions at varying Zn(II):Kaem ratios from
1:0.5 to 1:20 as seen in Figure 4a.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0.10
0.05
0.00
(a)
940 nm
Kaem:Zn(II)
0 : 0
0 : 10
1 : 0
1 : 0.2
1 : 0.5
1 : 1
1 : 2
1 : 5
1 : 10
1 : 20
-0.01
-0.02
-0.03
510 nm
0
500 1000 1500 2000
Delay time / µs
20
15
10
5
(b)
β-Car•+ scavenging
k2 = 1.88×108 L⋅mol-1⋅s-1
0
0
20 40 60 80
[Zn-(Kaem-2H)] / µM
Figure 4. (a) Decay of β-Car•+ as monitored at 940 nm and bleaching recovery of β-carotene ground state as absorbance
at 520 nm in the absence and presence of 100 μM Kaem, of 100 μM Zn(CH3COO)2, and of 100 μM Kaem with 20, 50,
100, 200, 500, 1000 and 2000 μM Zn(CH3COO)2, respectively. (b) Observed first order rate constants obtained from
single exponential fitting the decay of β-Car•+ in Figure 4a against concentration of Zn(CH3COO)2. Second-order rate
constant is calculated to be k2=1.88±0.05×108 L·mol−1·s−1. All in methanol/chloroform (7/3) at 25°C.
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The recovery of β-Car bleaching at 510 nm is similarly as kinetics at 940 nm, not affected by presence
of Zn(II) or Kaem alone, or solution of Kaem:Zn(II)=1:0.2, but is significantly accelerated by increasing
addition of Zn(II) to Kaem.
1
2
3
4
5
6
7
8
9
Absorbance decay at 940 nm could be accommodated by a single exponential function, and the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
k
observed rate constants,
listed in Table 2, are found in good agreement with the increase of the
·+
Car
fraction of Zn(II)-(Kaem−2H) present and with the decrease in the oxidation potential of the equilibrium
k
mixture as shown in Figure 3b. Linear dependence of
on the concentration of Zn(II)-(Kaem−2H)
·+
Car
(Figure 4b), implicates that Zn(II)-(Kaem-2H) complex is the only β-Car•+ radical scavenger with very
low oxidation potential −0.097 V. Kaem and 1:2 Zn(II)-(Kaem-H)2 are non-reactive in β-Car•+ scavenging
due to their higher oxidation potential ~0.170 V than −0.097 V for Zn(II)-(Kaem-2H).
Simultaneous increase in rates of β-Car•+decayat 940 nm and of bleaching recovery at 510 nm confirm
electron transfer from Zn(II)-(Kaem-2H) to β-Car•+, i.e. β-Car•+ was reduced and regenerated to β-Car by
Zn(II)-(Kaem-2H):
Zn(II)-(Kaem 2H)
k 2
+
(9)
¡¤
¦Â-Car + Zn(II)-
¦Â-Car + Zn(II)-(Kaem 2H)
The second-order rate constant k2 for Zn(II)-(Kaem-2H) reducing β-Car•+ is obtained to be
(1.88±0.05)×108 L·mol−1·s−1 based on the linear relationship in Figure 4b.
•+
−1
•
• , s−1), and oxidation
k
k
DPPH
Table 2. Observed first order rate constants for the decay of β-Car ( •+ , s ) and of DPPH (
Car
potentials determined by CV at indicated varying ratios.
k
k
•+
Kaem:Zn(II)
•
Potential (V)
(s−1)
(s−1)
Car
DPPH
1:0
2.2±0.1×102
2.7±0.2×102
1.7±0.2×103
3.9±1.0×103
6.9±1.1×103
1.1±0.1×104
1.4±0.1×104
1.6±0.1×104
0.15±0.01
---
0.67±0.01
1.19±0.03
1.79±0.08
---
0.179
0.165
0.156
0.064
−0.006
−0.058
−0.091
−0.097
1:0.2
1:0.5
1:1
1:2
1:5
1:10
1:20
1.99±0.07
---
For Kaem:Zn(II) with varying ratios, the concentration of Kaem is fixed to be 100 μM.Oxidation potentials were relative
to ferrocene for the actual solution used for β-Car•+ scavenging.
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3.3.2 DPPH• Radical Scavenging Kinetics. The reaction between 2,2-diphenyl-1-picrylhydrazyl radical
(DPPH•) and Kaem as well as the Zn(II)-kaempferol complexes were followed as changes in absorbance
in ethanol by stopped-flow spectroscopy as shown in Figure 5. Upon addition of 100 μM Kaem to 100
μM DPPH• solution, the decay of absorbance at 516 nm is accelerated, while no effect is seen for addition
of up to 1000 μM Zn(II). The rate of DPPH• scavenging for Kaem is strongly accelerated by addition of
Zn(II).
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
1.0
Kaem : Zn(II)
0.8
•
DPPH
0 : 0
0 : 10
1 : 0
1 : 0.5
1 : 1
1 : 2
1 : 10
0.6
0.4
0.2
0.0
A
0
5
10
15
Time / s
Figure 5. Absorbances at 516 nm for DPPH• scavenged by 100 μM Kaem, by 100 μM Zn(CH3COO)2, and by 100 μM
Kaem with 50, 100, 200 and 1000 μM Zn(CH3COO)2 in ethanol at 25°C, respectively.
DPPH• radical scavenging is generally accepted as hydrogen atom transfer (HAT) mechanism.50 Kaem,
is in equilibrium with the 1:1 and 1:2 Zn(II)-kaempferol complexes in the ethanol solutions, and the
reaction rate depends on three parallel reactions:
Kaem
k2
(10)
DPPH + Kaem
DPPH-H + (Kaem H)
Zn(II)-(Kaem H)
2
k2
(11)
(12)
DPPH-H + Zn(II)-[(Kaem H)2 H]
DPPH + Zn(II)-(Kaem H)2
Zn(II)-(Kaem 2H)
k2
DPPH-H + Zn(II)-[(Kaem 2H) H]
DPPH + Zn(II)-(Kaem 2H)
k2Kaem k Zn(II)-(Kaem- H)
k Zn(II)-(Kaem- 2H)
2
in which,
,
and
are second order rate constants for reactions of DPPH• with
2
2
Kaem, Zn(II)-(Kaem−H)2, and Zn(II)-(Kaem−2H), respectively. The changes in absorbance including the
decay of DPPH• and formation of reaction product DPPH-H, could for all conditions be described as an
exponential decay with an observed rate constant,
, within a 2 s time period:
k
•
DPPH
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•t
DPPH
A
+ A
= B×e- k
(13)
(14)
•
DPPH-H
DPPH
1
2
3
•t
e
• c • l + e
c
l = B×e- k
DPPH
DPPH-H DPPH-H
DPPH
DPPH
4
5
6
‒1
‒1 51
A
e
c
and
in which,
A
and
,
e
(9660 L·mol ·cm ) and
,
c
are absorbances, the
•
•
•
DPPH-H
DPPH-H
DPPH-H
DPPH
DPPH
DPPH
7
8
extinction coefficients and concentrations of DPPH• and the stable reaction product DPPH-H ，
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
e
respectively. B is pre-exponential factor. The extinction coefficient of DPPH-H is calculated to be
DPPH-H
4
=644 L·mol‒1·cm‒1, based on complete transformation of initial 100 μM (10− M) DPPH• to the stable
4
reaction product DPPH-H ( DPPH-H =10− −
c
• ). Reaction rate equation can be written as:
c
DPPH
•t
c （e • - eDPPH-H）=B×e- k
-10- ×e
4
DPPH
•
DPPH-H
DPPH
DPPH
(15)
The initial reaction rates are calculated as:
dc
B×k
DPPH
•
•
(16)
DPPH
ratet= 0 = -
=
t= 0
dt
e • - e
DPPH
DPPH-H
Considering the reactions of equations (10-12), the initial reaction rate may also be written as:
dc
•
t= 0
Kaem
Zn(II)-(Kaem-H)
k2
t= 0
Zn(II)-(Kaem- H)
Zn(II)-(Kaem- 2H)
k2
DPPH
Kaem
2
2
-
t= 0 = ct= 0 ×k ×c
+
×c
+
2
×ct= 0
(17)
(
)
•
Zn(II)-(Kaem- 2H)
DPPH
dt
k2Kaem
k2Zn(II)-(Kaem- H)
2
Individual second order rate constants,
=(1.59±0.01)×103 L·mol−1·s−1,
k2Zn(II)-(Kaem- 2H)
=(1.30±0.07)×104 L·mol−1·s−1 and
=(2.48±0.03)×104 L·mol−1·s−1 are calculated from the
distributions of Kaem, Zn(II)-(Kaem−H)2 and Zn(II)-(Kaem−2H) (Table 1) at initial time (t=0 s) and the
k
observed rate constant
at varying Zn(II):Kaem ratios (Table 2). Zn(II)-(Kaem−H)2 is found to be
•
DPPH
~8 times as fast as the parent Kaem in DPPH• scavenging, while Zn(II)-(Kaem−2H) is found to be ~16
times as fast as Kaem.
3.4 Structural and Thermodynamic Analyses. Molecular structures of Zn-kaempferol complexes and
of Kaem for comparison were optimized using DFT method and are shown in Figure S5. H exa-coordinate
structures of all Zn-kaempferol complexes are more stable than tetra-coordinated as indicated by
imaginary frequencies found in frequency calculation for tetra-coordination.52 In the optimized structures
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of both 1:1 and 1:2 Zn(II)-kaempferol complexes, Zn(II) ion coordinate to oxygen atoms of Kaem. The
other coordination sites in octahedron are occupied by solvent ethanol, not shown in Figure S5 for clarity.
Zn(II) ion and 4 oxygen atoms in 1:2 Zn(II)-(Kaem−H)2 are coplanar with Ci point group symmetry. 4-
O-Zn bonds in all Zn(II)-kaempferol complexes are longer than 3-O-Zn bonds due to stronger electron
withdrawal effect of 4-carboxyl than 2,3-carbon-carbon double bond. Both 3- and 4-O-Zn bonds in 1:2
Zn(II)-(Kaem−H)2 are longer than those in 1:1 Zn(II)-(Kaem−H)+ and Zn(II)-(Kaem−2H) implicating
stronger binding between oxygen and Zn(II) in 1:1 complexes. Two Kaem ligands simultaneously
coordinated to Zn(II) together disperse the electronic density and weaken bond strength.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Thermodynamic parameters, including proton dissociation enthalpy (PDE), ionization potential (IP),
bond dissociation energy (BDE) of Zn(II)-kaempferol complexes and of Kaem for comparison, were
calculated and are listed in Table 3. Significant effects are seen for phenol acidity increase for 5, 7 and
4´-hydroxyls of Kaem in 1:1 Zn(II)-(Kaem−H)+ through coordination to Zn(II) with the lowest PDE value
(276.32~278.07 kcal∙mol‒1), ~50 kcal∙mol‒1 less than Zn(II)-(Kaem−H)2 and Kaem with similar PDE
values (326~328 kcal∙mol‒1). 1:1 Zn(II)-(Kaem−H)+ with much lower PDE values than Kaem is thus easy
to lose the second proton (the first from 3-hydroxyl by coordination with Zn(II)) forming Zn(II)-
(Kaem−2H), which is in good agreement with above experimental observations of 15 nm red-shift of
Zn(II)-(Kaem−2H) relative to Zn(II)-(Kaem−H)2 as seen in Figure 1a, 1c and 2. While the acidity of three
phenols in Zn(II)-(Kaem−H)2 remains almost unaffected by coordination with similar PDE values to
phenols in Kaem. PDE values of three phenols (5, 7 and 4´) in Zn(II)-(Kaem−H)+ are very close. 7-
Hydroxyl has slightly lower PDE value than the other two, and 7-phenolate is accordingly used as the
most dominating deprotonated forms of Zn(II)-(Kaem−2H) in Scheme 1, and in the following descriptions
of BDE, IP as well as radical scavenging mechanism as shown in Scheme 2.
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Table 3. Calculated bond lengths of LO-Zn (Å), proton dissociation energy(PDE), ionization potential (IP), and bond
1
2
dissociation energy (BDE) of Kaem and Zn-kaempferol complexes in kcal·mol−1.
3
4
5
6
7
8
9
Compound
Kaem
Zn(II)-(Kaem‒H)2
Zn(II)-(Kaem‒H)+
Zn(II)-(Kaem‒2H)
LO-Zn
(Å)
3-O-Zn
4-O-Zn
3-OH
---
---
2.06
2.15
2.00
2.08
1.98
2.02
331.97
PDE
(kcal·mol−1)
5-OH
337.27
333.63
278.07
7-OH
4´-OH
322.58
322.54
321.79
324.04
276.32
277.21
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-O−
7-O−
4´-O−
---
7-O−
4´-O−
5-O−
4´-O−
5-O−
7-O−
122.26
122.10
121.43
---
IP
167.02
140.32
199.62
(kcal·mol−1)
3-OH
5-OH
78.12
92.34
79.95
70.48
76.72
66.46
87.08
78.45
73.91
83.67
83.54
78.99
BDE
(kcal·mol−1)
7-OH
83.46
77.76
68.15
67.35
4´-OH
IP values in Table 3 show that, compared to parent Kaem, coordination of Kaem decreases IP of both
Zn(II)-(Kaem−2H) and Zn(II)-(Kaem−H)2, but not for Zn(II)-(Kaem−H)+. Due to electron withdrawal by
Zn(II) ion, coordination of the first Kaem to Zn(II) decreases PDE in Zn(II)-(Kaem−H)+ and further
decreases IP in Zn(II)-(Kaem−2H), but coordination of the second Kaem in Zn(II)-(Kaem−H)2 more than
outbalance this electron withdrawal effect, resulting in Zn(II)-(Kaem−H)2 having a higher IP than Zn(II)-
(Kaem−2H) but lower than Kaem. The IP of Zn(II)-(Kaem−2H) (~122 kcal∙mol‒1), is far lower than that
of Zn(II)-(Kaem−H)2 (140.32 kcal∙mol‒1), Kaem (167.02 kcal∙mol‒1) and Zn(II)-(Kaem−H)+ (199.62
kcal∙mol‒1), in agreement with far lower oxidation potential ‒0.097 V than the potentials of ~0.170 V for
both Zn(II)-(Kaem−H)2 and Kaem. The lowest oxidation potential for Zn(II)-(Kaem−2H) agrees well
with this complex being the only electron donor observed in β-Car•+ radical scavenging.
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Scheme 2. Proposed mechanism of Kaem binding to Zn(II) and radical scavenging of Zn(II)-kaempferol complexes.
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Zn(II)-(Kaem−2H) is also found having the lowest BDE among all Zn(II)-kaempferol complexes and
Kaem. Although 7-hydroxyl for 4-deprotonation in Zn(II)-(Kaem−2H) has the lowest BDE, 66.46
kcal·mol‒1, 4´-hydroxyl for 7-deprotonation in Zn(II)-(Kaem−2H) with the second lowest BDE, 67.35
kcal·mol‒1, is believed to be the most dominating DPPH• scavenging site due to 7-phenolate form being
the dominating deprotonated form of Zn(II)-(Kaem−2H), as seen in Scheme 2. Similarly, the 4´-hydroxyl
in 1:2 Zn(II)-(Kaem−H)2 also has relatively lower BDE, 73.91 kcal·mol‒1, lower than parent Kaem, 77.76
kcal·mol‒1.53 These results may explain 16 times for Zn(II)-(Kaem−2H) and 8 times for Zn(II)-
(Kaem−H)2, as fast as Kaem for scavenging rates in DPPH• radical scavenging.
4. DISCUSSION
Flavonoids are among the most extensively investigated phytochemicals due to their diverse
pharmacological and therapeutic activities. The ability of chelating with metal ions for flavonoids has
brought about emergence of a new category of molecules with a broader spectrum of pharmacological
activities. Flavonoid-based metal complexes are mostly found enhancement in antioxidant, anti-
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inflammatory, anti-cancer activity and DNA binding with respect to their parent flavonoids.54,55 Metal-
flavonoid complexes are found sensitive to experimental conditions, i.e. concentration, pH, solvent, thus
hampering acquirement of crystal structures, characterization of metal-flavonoid compounds and
elucidation of molecular mechanism. S. Selvaraj et al. recently gave two proposed enhanced mechanism
of metal-flavonoid complexes as antioxidants in scavenging reactive radicals. One is hydrogen abstraction
by DPPH• radical from an undissociated proton in phenolic hydroxyl chelating with metal ions. The other
is metal ion as radical attacking site as SOD-mimic activity.20 While, both two suggestions lack
experimental evidences.
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Kaempferol (Kaem), as a common flavonoid found in many vegetables and fruits, has been modified
by coordination to metal ions, such as Zn(II), Eu(III), and Cu(II), etc.10,56-59 Such metal-kaempferol
complexes have been found having anticancer and DNA binding activities. In present study, upon addition
Zn(II) to kaempferol, two zinc-kaempferol complexes with stoichiometries of 1:1 and 1:2 are formed, and
characterized by IR, MS and H1-NMR. Radical scavenging reactivities of two complexes towards the
semi-stable radical DPPH• and the highly reactive β-Car•+ are for the first time compared and determined
in quantity based on molecular level.
Zn(II) was found to coordinate Kaem through the 3-hydroxyl and 4-carbonyl group with high affinity
both for the first and for the second ligands. Spectral analyses and DFT calculations confirmed the first
Kaem ligand coordination to Zn(II) significantly increase acidity of phenol in mono-coordinated complex
Zn-(Kaem-H)+ due to electron withdrawal effect Zn(II), thus easy to deprotonate forming 1:1 Zn-(Kaem-
2H). Coordination of the second Kaem in Zn(II)-(Kaem−H)2 balances this electron withdrawal effect, and
deprotonation in phenolic group not occurred in Zn(II)-(Kaem−H)2. Modification of Kaem in Zn(II)-
(Kaem−2H), two proton leaving from each Kaem ligand, accordingly is more significant than that in
Zn(II)-(Kaem−H)2, one proton leaving from each Kaem ligand. Zn(II)-(Kaem-2H) and Zn(II)-
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(Kaem−H)2 transform into each other depending on the concentrations of Zn(II) and hydrogen ions, see
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equation (5).
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Zn(II) is not redox active and Zn(II) coordination on kaempferol is accordingly not disturbed by change
in the oxidation state of the metal7. The radical scavenging kinetics showed a clear pattern. Radical
scavenging efficiencies for 1:1 and 1:2 complexes depend on types of complexes and reactive radicals,
and reaction type. Zn(II)-(Kaem−2H) is the only scavenger for the very reactive β-Car•+ radical
scavenging through ET mechanism, and also is the most efficient scavenger for the moderately reactive
DPPH• through HAT mechanism. While Kaem and with Zn(II)-(Kaem−H)2 are non-reactive in β-Car•+
radical scavenging. Zn(II)-(Kaem−2H) reacts with DPPH• 16 times and 2 times as fast as Kaem and
Zn(II)-(Kaem−H)2, respectively.
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Radical scavenging results are in good agreement with DFT calculations. The IP of the deprotonated
forms of Zn(II)-(Kaem−2H) is significantly smaller than of the protonated forms of Kaem and Zn(II)-
(Kaem−H)2, which notably have a large energy barrier for deprotonation. Accordingly, Zn(II)-
(Kaem−2H) also has the lowest barrier for electron transfer to β-Car•+ as seen in laser photolysis. The
BDE of 4´-OH hydroxyl were smallest among three unchelated phenolic hydroxyls in Kaem, Zn(II)-
(Kaem−H)2 and Zn(II)-(Kaem−2H) (for 7-phenl deprotonation with the lowest PDE), with the lowest
value for Zn(II)-(Kaem−2H) and the highest value for Kaem. The most active scavenger of DPPH• is
accordingly concluded to be Zn(II)-(Kaem−2H), see Scheme 2.
The limited experimental studies on flavonoids partially are due to their poor water solubility. However,
metal-flavonoid complexes are more hydrophilic and water-soluble than the corresponding ligands19.
Such coordination in present study may occur spontaneously after intake of fruits and vegetables with
flavonoids during digestion or may be in design of various kinds of metal-based drugs containing
bioactive molecules of natural extraction. Structure-reactivity relationship obtained in this study, more
efficient radical scavenging through increased phenol acidity in 1:1 Zn(II)-kaempferol complex than 1:2
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Zn(II)-kaempferol complex and parent Kaem, may point toward a general mechanism for zinc as an
antioxidant1,2 and for increasing interest in the use of Zn(II) coordination complexes with low toxicity and
low side effects in medicinal therapeutic applications33.
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5. CONCLUSIONS
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Kaem binds to Zn(II) with high affinity entailing electron withdrawal from Kaem increasing phenolic
acidity. Deprotonation of Kaem mono-coordinated to Zn(II) has a larger effect on electron donation than
electron withdrawal by coordination to Zn(II). The 1:1 Zn(II)-kaempferol complex becomes more
reducing than the uncoordinated Kaem and 1:2 Zn(II)-kaempferol complex. Phenolic acidity and
oxidation potential are unaffected by coordination in the 1:2 Zn(II)-kaempferol complex compared to
uncoordinated Kaem. Deprotonation, however, in general decrease ionization potential, and higher
tendency of deprotonation for Kaem in Zn(II)-(Kaem−2H) than in Zn(II)-(Kaem−H)2 fully outbalance
the effect of electron withdrawal by coordination to Zn(II). The outbalance make Kaem in the 1:1 Zn(II)
complex the very efficient radical scavenger.
Supporting Information.
S1. Reaction of Kaem and zinc(II), S2. Structural characterization of Zn(II)-kaempferol complexes. This
material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* rmhan@ruc.edu.cn
* ls@food.ku.dk
Author Contributions
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The manuscript was written through contributions of all authors. / All authors have given approval to the
final version of the manuscript.
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENT
This work has been supported by grants from Natural Science Foundation of China (Nos. 21573284 and
21673288).
ABBREVIATIONS
Kaem, kaempferol; DFT, density functional theory; β-Car, β-carotene; β-Car•+, β-carotene radical cation;
CV, cyclic voltammetry; DPPH•, 2,2-diphenyl-1-picrylhydrazyl radical; PDE proton dissociation energy,;
IP, ionization potential; BDE, bond dissociation energy.
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