The influence of the carbohydrate anomeric linkage on the free radical scavenging activity of enzymatically-synthesized phenolic glycosides

Article abstract of DOI:10.1039/c6ra06572d

Phenolic glycosides, widely recognized for their nutraceutical properties exist in nature as α- and β-glycosides. Interestingly, the β-phenolic glycosides are the predominant form in natural products. In this work, the pure α- and β-glucosides and galacto

Full text of DOI:10.1039/c6ra06572d

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The influence of the carbohydrate anomeric
linkage on the free radical scavenging activity of
Cite this: RSC Adv., 2016, 6, 45452
enzymatically-synthesized phenolic glycosides†
Jorge A. Gonzalez-Rıos,a Jose Pedraza-Chaverri,b Omar N. Medina-Campos,b
´
´
´
Miguel Reina,c Yanet Romero,c Ana Martınez,c Agustın Lopez-Munguıaa
´
´
´
´
a
*
and Edmundo Castillo
Phenolic glycosides, widely recognized for their nutraceutical properties exist in nature as a- and b-
glycosides. Interestingly, the b-phenolic glycosides are the predominant form in natural products. In this
work, the pure a- and b-glucosides and galactosides of homovanillyl alcohol (HVA) were enzymatically
synthesized. Their free radical scavenger (FRS) activity was evaluated using the DPPHc endpoint assay.
According to the EC50 values, all glycosides are better FRSs than the corresponding aglycone, with lower
EC50 values found for the b-anomers. In contrast, a DPPHc kinetic analysis revealed that HVA-aglycone is
more reactive than the glycosides. The analysis based on both EC50 and kinetic behaviour suggested
that, although the HVA-aglycone is kinetically a better free radical scavenger, glycosidation renders HVA-
aglycone more stable for long-term reactions. The molecular electrostatic potential and conformational
analysis of a- and b-HVA glycosides demonstrated that b-HVA glycosides are better free radical
scavengers than the corresponding a-anomers. In fact, the higher flexibility of b-HVA glycosides allow
the exposure of advantageously negatively charged regions favouring their FRS capacity.
Received 11th March 2016
Accepted 2nd May 2016
DOI: 10.1039/c6ra06572d
www.rsc.org/advances
Phenolic glycosides (PGs) are an important class of glyco-
sides, very common in plants, where their activity has been
Introduction
associated to cellular damage response and protection against
stress.8 The aglycones in PGs are in general phenolic
compounds (PCs) such as phenolic acids and phenolic alcohols,
or polyphenols such as avonoids, tannins, stilbenes, curcu-
minoids, coumarins, lignans and quinones. PCs in the form of
aglycones usually show low solubility and chemical instability.
In fact, in their natural environment they exist mainly in the
glycosylated form, a chemical condition that improves their
function and distribution inside the plant.9,10
A better understanding of the structure–function relation-
ship of PGs, particularly those structural features associated to
their free radical scavenger activity, is required for a rational
synthesis of novel PGs with enhanced properties. Although it
has been widely accepted that PGs functional properties are
inuenced by the nature of the PC, the effect of the number and
nature of the sugar moieties as well as the type of linkage to the
aglycone has received less attention. Indeed, while the presence
of phenolic hydroxyls in aglycones, notably in the form of an
ortho-diphenolic arrangement has been recognized as
a requirement for an efficient free radical scavenger activity of
the PGs, little is known concerning the requirements in the
carbohydrate moiety.11–13 In recent years, we reported the
enzymatic synthesis of a series of phenyl propanoid glycosides
(PPGs) demonstrating that, from a mechanistic point of view,
their radical scavenging activity is associated to the PC when
Glycosides result from the condensation of a carbohydrate and
a non-sugar moiety (aglycone) such as an amino acid, a lipid,
a nucleotide, a phenolic compound, an alkaloid, or a terpenoid.
The carbohydrate moiety in glycosides is signicant in nature
because it modulates the aglycone function, its pharmacoki-
netics, its nutraceutical activity and in some cases serves for
molecular recognition, particularly in secondary metabolites.1
In fact, the most relevant effect of carbohydrates in glycoside
molecules has been related to the modication of physico-
chemical properties such as solubility, stability and volatility.2–4
From a functional point of view, glycoside pharmacological
activities, such as antitumoral, antiviral, anti-inammatory,
antimicrobial, free radical scavenging, or oxidative DNA
damage protection, have been associated to the aglycone
molecule.3–7
aDepartamento Ingenierıa Celular
´
´ ´
y Biocatalisis, Instituto de Biotecnologıa,
´
´
Universidad Nacional Autonoma de Mexico, Apartado Postal 510-3, Cuernavaca,
Morelos, CP 62250, Mexico. E-mail: edmundo@ibt.unam.mx
b
´
´
´
Departamento de Biologıa, Facultad de Quımica, Universidad Nacional Autonoma de
´
´
Mexico, Cd. Universitaria, 04510 Mexico DF, Mexico
cDepartamento de Materiales de Baja Dimensionalidad, Instituto de Investigaciones en
´
´
Materiales, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, CP
´
04510, Mexico DF, Mexico
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ra06572d
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Scheme 1 Glucosides (Glc) and galactosides (Gal) of homovanillyl alcohol (HVA) in a (Glc-a-HVA and Gal-a-HVA) and b (Glc-b-HVA and Gal-b-
HVA) configuration.
analysed against 2,2-diphenyl-1-picrylhydrazyl radical (DPPHc). far as the sugar nature is concerned, glucose and galactose
From a Sequential Proton Loss Electronic Transfer mechanism, (Gal), the two most frequently found sugars in natural glyco-
we came to the conclusion that the position of the hydroxyl sides were selected. Results are reported in terms of both, the
group in these PPGs has an important effect on their scavenging classical 50% inhibitory concentration parameter (EC50) and
activity.14 Nevertheless, not only aglycones may be responsible a rst order rate constant, obtained from a simplied pseudo-
for the PGs free radical scavenger activity but, as recently re- rst order kinetic model. Moreover, theoretical calculations
ported, some carbohydrates such as glucose (Glc), sucrose, and a conformational analysis are reported in order to explain
fructose, 1-kestotriose, inulin and trehalose may also act as the effects of the anomeric linkage on the radical scavenging
good scavengers of the hydroxyl and superoxide radicals.15
In spite of the lack of knowledge concerning the structural
characteristics that explain the contribution of carbohydrates to
the free radicals scavenger activity of glycosides, it is a fact that
activity.
Materials and methods
glucose is frequently found as the rst sugar linked to the Commercially available enzymes were used in glycoside
aglycone in glycosylated PGs.11 An important additional feature synthesis. Cyclodextrin-glucanotransferase (CGTase) from
is the eventual inuence of the anomeric conguration. In fact, Thermoanaerobacter sp. was kindly provided by Novozymes A/C
´
it has been reported that more than 80% of plant PGs exist as b- (Novozymes Mexico, Mexico City); glucoamylase from Asper-
anomers,16–19 a conguration that makes them more stable in gillus niger was purchased from Enzymes Development Corpo-
nature, where a-glucosidase activity is common and easily ration (New York, NY, USA), while b-galactosidase from
assimilated by humans through b-glucosidase activity of the gut Kluyveromyces lactis (3000 LAU mLꢀ1) from Maxilact (DSM Food
microbiota.20,21
Specialties, the Netherlands). Methanol, 2-methyl-2-propanol
The contribution of the anomeric conguration to the bio- and acetonitrile HPLC grade, were purchased to J.T. Baker
logical and physical functions of carbohydrates has been widely (Phillipsburg, NJ, USA); a-galactosidase from green coffee
recognized. For instance, crucial differences in functional beans, homovanillyl alcohol (HVA), 6-hydroxy-2,5,7,8-
properties of disaccharides are observed between maltose (a- tetramethylchroman-2-carboxylic acid and 2,2-diphenyl-1-
glucose disaccharide), cellobiose (b-glucose disaccharide) and picrylhydrazyl (DPPHc) were all from Aldrich (WI, USA); b-
other disaccharides, commonly attributed to the exibility and/ ciclodextrin, uorescein, starch, and cellobiose were from
or rigidity conferred to the molecules by the anomeric bond.22,23 Sigma Chemical Co. (St. Louis, MO, USA). The enzyme b-
However, as far as glycosides is concerned, information glucosidase from Thermotoga maritima was obtained through
explaining how exibility and/or rigidity impacts functionality recombinant strategies using the genome sequence provided by
´
in PGs, in particular, their free radical scavenger properties, is Dr Enrique Morett (IBT-UNAM, Mexico) and cloned into
´
scarce. An exemption to this fact is the recent report by Sanchez plasmid pET-22b(+). E. coli C-43 was transformed with this
et al.,24 describing the close relationship between exibility in construction. IPTG (0.2 mM) was used as inductor of gene
caffeic acid esters and amides, and its free radical scavenging expression during the 6 hours culture carried out at 37 ꢁC in
capacity against DPPHc.
YT2x medium (triptone 16 g Lꢀ1; yeast extract 10 g Lꢀ1; NaCl 5 g
In this work the effect of the sugar nature and the anomeric Lꢀ1) agitated at 250 rpm. Aer cell disruption the recombinant
linkage (a and b anomers) on the free radical scavenging activity enzyme in the protein extract was puried through an affinity
is reported. For this purpose, four different glycosides of chromatography column on a Ni-chelating column (HiTrap®-
homovanillyl alcohol (HVA) as aglycone were enzymatically Chelating HP column from Amersham Pharmacia Biotech). The
synthesized (Scheme 1) and evaluated against DPPHc assay. As proteins were eluted with a linear gradient (0–100%) with
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NaH2PO4 : NaCl (20 mM : 0.5 M) buffer containing 0.5 M conditions described above for b-glycosides but and additional
imidazole as eluent.
purication step was needed. The second purication step
consisted of a C18 GRACE® column (12 g, 0.04 mm) eluted with
a 0 : 100 to 40 : 60 methanol : water gradient. The products in
this step were also identied with UV detector at 280 nm. The
Enzymatic synthesis and purication of glycosides
Glc-b-HVA. Glc-b-HVA (2-[4-hydroxy-3-methoxyphenyl]ethyl- yield of Glc-a-HVA quantied by HPLC was 17% and 8% aer
b-D-glucopyranosyde) was synthesized with b-glucosidase from purication, both on the basis of HVA converted.
Thermotoga maritima at a concentration of 5 U mLꢀ1. b-Gluco-
Gal-a-HVA. Gal-a-HVA (2-[4-hydroxy-3-methoxyphenyl]ethyl-
sidase of Thermotoga maritima with cellobiose (0.8 M) as glu- a-D-galactopyranoside) was synthesized by a-galactosidase from
cosyl donor and HVA (0.8 M) as acceptor in pH 7, 100 mM green coffee beans (20 U mLꢀ1) with 2-nitrophenyl a-D-gal-
Na2HPO4 buffer. Reactions were incubated at 70 ꢁC for 24 hour actopyranoside (0.2 M) as galactosyl donor and HVA (0.1 M) as
with samples taken from the reaction mixture at relevant time acceptor in 50 mM, pH 6.5 malic acid buffer containing 20% v/v
ꢁ
intervals, heated for 10 min at 100 C to stop the reaction and acetonitrile (20%) as the reaction medium. Reactions were
ꢁ
centrifuged for 10 min at 1150 ꢂ g at 25 C to eliminate sus- incubated at 27 ꢁC with stirring for 24 hours. At the end of the
pended solids. The Glc-b-HVA was puried using a Flash reaction the crude of reaction was heated for 5 min at 100 ꢁC to
Chromatography method on a Reveleris® equipment (GRACE®, stop the reaction and centrifuged for 10 min at 1150 ꢂ g at 25 ꢁC
Columbia, MD, USA). First, the samples were extracted from the to eliminate suspended solids. The purication of Gal-a-HVA
crude reaction twice with chloroform in order to eliminate was carried with the two step purication strategy described
residual HVA. Aerwards, the products in the residual aqueous above for a-glucoside. The yield of Gal-a-HVA quantied by
phases were extracted with 2-methyl-2-butanol (2M2B), which HPLC was 5% and 3% aer purication, both on the basis of
was then adsorbed onto silica gel (230–400 mesh, 0.04–0.063 HVA converted.
mm) and evaporated. An Amino GRACE® column (12 g, 0.04
mm) was used as the stationary phase using an ethyl aceta- HPLC analysis of HVA glycosides
te : methanol gradient for elution, starting with ethyl acetate
The glycosides were analysed by HPLC using a Waters 600E
system controller (Waters Corp., Milford, MA) at 35 C, equip-
and increasing the methanol content to reach 45% (v/v) in 8
minutes, followed by 3 min of isocratic ow with the 45%
methanol solvent at a ow rate of 20 mL minꢀ1. The eluted
products were identied with an UV detector at 280 nm. The
yield of Glc-b-HVA quantied by HPLC was 24% and 10% aer
purication, both on the basis of HVA converted.
ꢁ
ped with both a Waters 996 photodiode array detector and
a refraction index detector (Waters Corp., Milford, MA). A Kro-
masil NH2 column (4.6 mm ꢂ 250 mm, 5 mm; Sigma-Aldrich,
USA) was used for separation, using an isocratic phase of
acetonitrile/water (80 : 20) as a mobile phase at a ow rate of 1
Gal-b-HVA. Gal-b-HVA (2-[4-hydroxy-3-methoxyphenyl]ethyl-
mL minꢀ1
.
b-D-galactopyranosyde) was synthesized as previously reported
14
´
´
by Lopez-Munguıa et al., using b-galactosidase from Kluver-
omices lactis (12 U mLꢀ1) as biocatalyst with lactose as galactosyl
donor (1 M), HVA (250 mM) as acceptor in 50 mM, pH 6.5
phosphate buffer at 35 ꢁC. The products of both galactosylation
reactions were analysed either by TLC using ethyl acetate/
metanol/water (24 : 4 : 3 v/v/v) as a solvent or by HPLC (see
later). Samples were stored at ꢀ18 ꢁC until purication. The
purication of Gal-b-HVA was carried out under the same
conditions described above for b-glucoside of HVA. The yield of
Gal-b-HVA quantied by HPLC was 47% and 30% aer puri-
cation, both on the basis of HVA converted.
NMR and HRMS analysis of HVA glycosides
HRMS FAB + spectra in a matrix of m-nitrobenzyl alcohol were
recorded on a JEOL JMX-AX 505 HA mass spectrometer. The
NMR spectra of Gal-b-HVA were recorded on a Varian Unity
spectrometer at 400 MHz. While the Glc-a-HVA, Glc-b-HVA and
Gal-a-HVA were recorded in a Brucker Advance III at 500 Mhz.
1
The experiments realized to each molecule included H NMR,
1H-1H COSY, HSQC and HMBC and 100 MHz for 13C NMR using
CD3OD as solvent. Chemical shis are reported in parts per
million (ppm) and referenced to the proton resonance of the
solvent used for each sample.
Glc-a-HVA. Glc-a-HVA (2-[4-hydroxy-3-methoxyphenyl]ethyl-
a-D-glucopyranoside) was synthesized with CGTase of Ther-
moanaerobacter (20 U mLꢀ1) using starch 10% w/v (0.1 M) as
Evaluation of radical scavenging activity in glycosides
glucosyl donor and 1 M of HVA as acceptor in a pH 6.0, 50 mM The diphenyl-picrylhydrazine (DPPHc) was performed as re-
Na2HPO4 buffer. Reactions were performed at 50 ꢁC with ported by Yamaguchi et al.25 The reaction mixture containing
continuous stirring. Aer 24 hours the reaction was heated for 5 2.95 mL of a 100 mM ethanolic DPPHc solution and 0.050 mL of
min at 100 ꢁC to stop the reaction. In order to transform all the the sample dissolved in methanol at an adequate concentration
glucosylated products to the monoglucosylated form, the reac- was shaken vigorously on a vortex mixer and incubated at room
tion mixture was treated with 258 U mLꢀꢁ 1 of glucoamylase from temperature until no changes in absorbance were observed.
Aspergillus niger during 48 hours at 50 C. Aliquots of the reac- Ascorbic acid, a well-known and common free radical scavenger
tion mixture were withdrawn at relevant time intervals, heated molecule, was used as reference. Unless otherwise stated, for all
ꢁ
for 5 min at 100 C to stop the reaction and centrifuged for 10 DPPHc EC50 determinations, samples were taken considering
min at 1150 ꢂ g at 25 ꢁC to eliminate suspended solids. The that all reactions reached steady state aer 90 min. The non-
purication of Glc-a-HVA was carried out under the same reacted DPPHc was determined colorimetrically at 517 nm
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using a Beckman DU 650 spectrophotometer. Test compounds dissociated one by one. The corresponding dehydrogenated
and changes in the absorbance of the samples were expressed in molecules were fully optimized. Molecular electrostatic poten-
terms of EC50, indicating the concentration of a compound tial is the force acting on a positive test charge (a proton) and
required for a 50% decrease in the DPPHc absorbance. The EC50 indicates the molecular charge distribution. It is visualized
parameter was calculated from a concentration–response curve. through mapping its values onto a surface. In this case,
For kinetic determinations, the decay prole on DPPHc absor- a surface with an isovalue of 0.0004 e auꢀ3 was used.
bance was evaluated during short times (<4 min of reaction)
considering an initial DPPHc concentration in excess with
respect to glycosides in order to analyse the data as a pseudo-
Results and discussion
rst order kinetic model. All analyses were carried out in trip-
licates. The standard error of the mean (SEM) and EC50 values
were obtained with Prism (GraphPad Soware, Inc.®, La Jolla,
CA, USA).
Synthesis of glycosides and free radical scavenger
measurements
In a previous work we reported the enzymatic synthesis of PPGs
using vanillyl and homovainillyl b-galactosides as precursors.
We found that both PPGs and b-galactosides showed free
radical scavenging activity.14 In the course of those syntheses,
some important questions arose regarding the effect of the
anomeric linkage (a and b anomers) and the sugar nature of the
glycosides in this property. We therefore decided to further
explore the structure–function relationship based on the
already described HVA b-galactoside and extending the enzy-
matic synthesis to the corresponding a-galactoside, including
the a- and b-glucosides of HVA to explore the effect of the
chemical nature of the sugar. For this purpose, synthesis reac-
tions employing K. lactis b-galactosidase, green coffee beans a-
galactosidase, Toruzyme® CGTase and T. maritima b-glucosi-
dase were performed. The HPLC analyses of these reactions
showed yields from low (5% for Gal-a-HVA) to moderate (47%
for Gal-b-HVA) on the basis of HVA converted (see ESI†). The
products from these reactions were puried and structurally
characterized. Under these reaction conditions, high regio-,
chemo- and enantioselective glycosylated molecules were ob-
tained, as all products showed 100% anomeric purity, with
glycosylation performed exclusively on the primary hydroxyl
group of HVA with no alternative glycosylation in the phenolic
hydroxyl.
Statistical analysis
Data of DPPHc tests were expressed as mean ꢃ SEM. Scavenging
activity was expressed as EC50 value, which denotes the
concentration required to give a 50% reduction in scavenging
capacity of the radical. The data was compared using one way
analysis of variance (ANOVA) followed by the Dunnett compar-
ison test. Values with p < 0.05 were considered signicant.
Computational details
Conformers were generated using the Marvin Sketch soware
(ChemAxon version 6.1) and their geometry-optimized using the
MMFF94 force eld method.26–30 The search was limited to 20
confomers, with a diversity limit of one and 10 000 seconds of
optimization time. The visualization of conformers and
measurement of distances and dihedral angles (uHVA-gly) were
¨
performed using the PyMOL soware (Schroedinger version
1.74).
For the geometry optimization with density functional
theory, the initial geometries were set up analysing different
conformers generated previously with a force eld MMFF94.26–30
Conformers with the lowest energy were fully optimized.
Geometry optimizations and frequency calculations were
carried put using the B3LYP31 functional in conjunction with
the 6-311G(d) basis set with water as solvent (smd). Local
minima were identied by the absence of imaginary frequen-
cies. All electronic calculations were performed with Gaussian
09 program series.32 Two reaction mechanisms were analysed in
order to study the free radical scavenger capacity: single elec-
tron transfer (SET) and hydrogen atom transfer (HAT). For the
SET mechanism it is necessary to calculate the vertical ioniza-
tion energy (IE) and the vertical electron affinity (EA). In order to
compute IE and EA single-point calculations using the opti-
mized structures of the neutral molecule were performed with
a different basis set (6-311+G(d)). IE was calculated as the
difference between the energy of the cation and the neutral
molecule, assuming that both have the ground-state nuclear
conguration of the neutral molecule. EA was also calculated as
vertical, and represents the energy difference between the
neutral and the anion, calculated with the ground-state nuclear
conguration of the neutral molecule. For the HAT mechanism,
we analysed the bond dissociation energy (D0) of one hydrogen
atom. To calculate the D0 values, the hydrogen atoms were
Indeed, HMBC experiments and/or d 13C conrmed that HVA
substrate was linked to sugars through the C-8 carbon atom
(–CH2–OH). In one hand, considering the long-range correla-
tions between C-10 of sugars and the H-8 pair of aglycone or the
long-range correlation between C-8 of aglycone and the H-10 of
Table 1 Free radical scavenging activity of HVA-glycosides evaluated
by DPPHc assay. Activity is reported as the half maximal effective
concentration (EC50). Ascorbic acid is included for comparison. The
relative increment of the free radical scavenging activity is also
reported
Molecule
EC50-DPPHc (mM)
Relative increment DPPHca (%)
HVA
56.10 ꢃ 2.91
49.64 ꢃ 1.07
48.31 ꢃ 0.36
44.03 ꢃ 1.11
44.47 ꢃ 0.67
22.60 ꢃ 0.13
—
12
14
21
22
60
Glc-a-HVA
Gal-a-HVA
Glc-b-HVA
Gal-b-HVA
Ascorbic acid
a
Relative increment of free radical scavenging activity of the EC50
compared to EC50 of HVA aglycone. Data are mean ꢃ SEM. p < 0.1 vs.
Glc-b-HVA.
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sugars. On the other hand, the downeld chemical shi of the product research, the single EC50 value determination is
C-8 carbon in glycosides from around d 61.77 for the free C-8 of a limited view of the free radical scavenging capacity of the
HVA to around d 68.75–72.05 observed in HVA glycosides (see evaluated compound. Actually, as the DPPHc assay is nothing
ESI for details†).
but a chemical reaction, it has been suggested that this evalu-
The DPPHc assay has been commonly used to evaluate and ation should include the reaction kinetic parameters avoiding
compare free radical scavenging activity of several isolated the comparison made with xed endpoint measurements.33 In
molecules or natural extracts. Despite some limitations in the fact, EC50 endpoint assays employing DPPHc have recently been
application of this assay,33,34 the assay was here selected due to strongly criticised as not reliable.34 Foti (2015), emphasized that
the stability of the radical, the easiness of application and its EC50 is not a kinetic parameter and its purported correlation
generalized adoption.
with the antioxidant properties of chemical substances is not
The free radical scavenger activity of the four synthesized always justied. One of the eventual problems associated with
glycosides (Gal-a and -b-HVA, Glc-a and -b-HVA) was evaluated EC50 data, is that for large reaction times the secondary product
using the radical DPPHc and reported as their corresponding formed from phenolic antioxidants can also scavenge free
EC50 values (EC50-DPPHc). These results are shown in Table 1, radicals. Under this consideration, we evaluated the effect of
using ascorbic acid as reference. As described, all glycosylated glycoside concentration in the initial rate of the DPPHc radical
HVAs have lower EC50 values than the corresponding aglycone consumption (VO). For this purpose, the DPPHc radical
(HVA). It is clear that glycosylated HVAs are not better free consumption was followed during the rst four minutes of
radical scavengers than ascorbic acid, but nevertheless, they are reaction, from an initial DPPHc concentration in excess with
better scavengers than non-glycosylated HVA. In quantitative respect to the initial concentration of glycosides, as reported in
terms, considering the EC50-DPPHc values, HVA glycosides Fig. 1. From these results, it may be observed that a rst order
show an increment of the free radical scavenging activity of 12– kinetic behaviour is established for the rate dependence for
14% for the a-glycosides and up to 21–22% for the b-glycosides, HVA and each HVA glycoside. Considering that this is actually
as compared to the HVA-glycone. Although it may not be a second order reaction in which the initial DPPHc radical
surprising to nd that glycosylation improves the free radical concentration was the same for all the reaction assays, and well
scavenging capacity of the aglycone, it is interesting to point out in excess with respect of the glycoside (AGly) we can state that:
that the improvement is higher for the b-glycosides. We may
VO ¼ k00[DPPHc][AGLy] ¼ k0[AGLy]
conclude that, under the perspective of the EC50 values, glyco-
sylation alone is not enough to explain the improvement of the
free radical scavenging activity of HVA, as the nature of
anomeric linkage between the sugar and the aglycone seems to
The k0 values obtained for the evaluated glycosides, are
shown in Fig. 2 and Table 2, where an interesting feature may be
observed, supporting the need for kinetic evaluation of the free
radical scavenging data instead of a single EC50-DPPHc
contribute also to this effect.
measurement. In effect, contrary to the conclusions driven by
Kinetic description of the DPPHc measurements
the EC50-DPPHc data for HVA and its glycosides, the k0 values
Although the determination of the free radical scavenging
indicate that HVA, the aglycone, is a faster free radical scavenger
activity of chemicals based on EC50 values of the DPPHc assay is
of DPPHc than its glycosides. Moreover, in terms of the nature
an extensively applied method in food science and natural
of the glycosylation, the kinetic results presented in Fig. 1 and 2,
Fig. 1 Initial rates of DPPHc free radical scavenging as a function of
glycoside concentration (mM). The initial rates were obtained from Fig. 2 Pseudo-first order rate constant (k0) of the radical scavenging of
lineal adjustment on the residual DPPHc concentration determined DPPHc reaction with the corresponding glycosides. Data are mean ꢃ
during the initial reaction time. Data are mean ꢃ SEM.
SEM. * ¼ p < 0.05 vs. Glc-b-HVA. ** ¼ p < 0.05 vs. Gal-b-HVA.
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Table 2 Pseudo first-order rate constant (k0vel) of the radical scav- Table 3 Vertical Ionization Energy (IE), Electron Affinities (EA) and
enging of DPPHc reaction with the corresponding glycosidea
dissociation energies (D0) for the molecules under study. Experimental
values for the antiradical activity is also included
Molecule
k0vel (mol DPPHc per min mol glycoside)
EC50 (mM)
HVA
47.57 ꢃ 5.6
12.67 ꢃ 1.84
15.79 ꢃ 1.04
25.41 ꢃ 2.58
32.36 ꢃ 0.57
Molecule
IE (eV)
AE (eV)
D0 (eV)
(DPPHc)
Glc-a-HVA
Gal-a-HVA
Gal-b-HVA
Glc-b-HVA
Glc-a-HVA
Glc-b-HVA
5.95
5.97
0.79
0.79
3.51
3.46
49.64
44.03
a
Data are mean ꢃ SEM.
Experimental information concerning the antiradical capacity
is also included. IE and EA are practically the same for both
molecules, indicating that the SET mechanism does not explain
the experimental results. For the HAT mechanism, the D0 value
for Glc-b-HVA is slightly lower than for Glc-a-HVA. This indi-
cating that Glc-b-HVA is better antiradical than Glc-a-HVA, in
agreement with the experimental results.
The experiments of the antiradical capacity using the DPPHc
as free radical are not fully explained with the bond dissociation
energy because DPPHc is a big molecule and usually free radical
scavengers transferred one electron to DPPHc and do not
transfer and H atom. As explained in the methodology section,
the SET mechanism is associated with IE and EA and in this
case, these two parameters do not explain the experimental
differences between Glc-a-HVA and Glc-b-HVA with the DPPHc
assay. In summary, the hydrogen bond dissociation energy give
us an indication that Glc-b-HVA is a better free radical scavenger
than Glc-a-HVA, but does not explain the reaction with DPPHc.
as well as in Table 2, show that independently of the sugar
incorporated to the HVA glycoside, the compounds bearing the
b anomeric linkage are faster free radical scavengers than those
bearing a anomeric linkage. These results conrm the obser-
vations drawn from results shown in Table 1, where b glycosides
also showed the best free radical scavenging properties.
From the results of both EC50 values and the kinetic behav-
iour of HVA and its glycosides, it is worth mentioning that in
one hand, HVA is the best free radical scavenger, as this phenyl
alcohol reacts faster with DPPHc than all the glycosides evalu-
ated. Conversely, the conclusions derived from the EC50-DPPHc
determinations suggest that HVA glycosides have a higher free
radical scavenger capacity. Certainly, for all cases, lower
concentration of glycosides was required to achieve a decrease
in 50% of the initial DPPHc concentration. From both analysis it
seems that glycosidation decrease the reactivity of the aglycone
but enhances its long-term reaction stability against free radical
scavengers. In agreement with previous reports, it is important
to stress the fact that the sole determination of EC50 in the
DPPHc assay provides limited information concerning the free
radical scavenging activity of a given compound, particularly for
a comparative purpose. Hence, and as recently reviewed, the
evaluation of both, the EC50 and the kinetic parameters allow
a better evaluation of the free radical scavenging activity of any
given compound.33,34
Up to this point, these results allow us to conclude that HVA
glycosylation results in stable long-term free radical scavengers.
Moreover, it has been also established that in all the assays the
compounds bearing the b anomeric linkage are better free
radical scavengers (DPPHc assay) than the corresponding a-
anomers.
In order to contribute to a better understanding of the struc-
tural HVA-glycosides determinants of these enhanced properties,
particularly those between the a and b anomers, two structural
analysis were applied: a quantum chemistry study in order to
dene the electrostatic potential surface of the glycosides and to
explore how the negatively charged part of the molecules is
exposed, and a conformational analysis, in order to establish
geometrical differences in the glycosides structure, describing
their exibility and their ability to expose of their negative charge.
Theoretical study
In order to analyse the antiradical capacity of Glc-a-HVA and
Fig. 3 Molecular electrostatic potential of (A) Glc-a-HVA; (B) Glc-b-
Glc-b-HVA, Table 3 reports IE, EA, and D0 of both compounds. HVA. Negative values in red, positive values in blue.
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Fig. 4 View of the most closed conformers generated for Glc-b-HVA (A) and Glc-a-HVA (B). The shortest IRD observed for Glc-b-HVA
(conformer 3, see ESI†) and Glc-a-HVA (conformer 40, see ESI†) conformers are 3.91 A˚ and 6.59 A˚ respectively.
The main difference between Glc-a-HVA and Glc-b-HVA is between these compounds is their spatial geometry, we
the geometry. For this reason, the explanation concerning the searched for 20 conformers of each glycoside under study using
differences in reactivity against DPPHc should be rather related the MMFF94 force eld (see Materials and methods section).
with the structure and with the molecular electrostatic poten- The aim of the conformational analysis was to explore the effect
tial. Since glycosides transferred electrons to DPPHc, it is of rotation of the dihedral angle around the glycosidic linkage
important to analyse the region of the molecules that is nega- on the free radical scavenger properties of the glycosides.
tively charged. Electrons would be preferentially transferred Indeed, although structural properties of disaccharides have
from this section of the molecule to the DPPHc. Fig. 3 presents been well described using f, j and U dihedral angles around
the molecular electrostatic potentials of Glc-a-HVA (Fig. 3A) and the O–C–O–C arrangement, it seems that the torsion angle
Glc-b-HVA (Fig. 3B). In red is the negatively charged zone and should have different parameters when O–C–C–C arrangement
blue indicates the region with less electrons. For Glc-a-HVA, is present like in glycosides (blue line in Fig. 5A).
electrons are mainly in the middle of the molecule, whilst for
Visual analysis of the generated conformers of HVA glyco-
Glc-b-HVA the external part of the molecule is negatively sides showed different free rotation around HVA. Accordingly,
charged. Electrons are more available in Glc-b-HVA than in Glc- there are structures with a more closed conformation, in which
a-HVA, and this may explain the differences as electron donors. the distant residues may become closer due to the higher ex-
In summary, Glc-b-HVA may be a better antiradical because the ibility of the molecule (Fig. 4). Actually, in Fig. 4 it may be
negatively charged part of the molecule is more exposed than in observed that Glc-b-HVA may adopt a more closed conforma-
˚
Glc-a-HVA.
tion (Fig. 4A; 3.91 A between the two most distant atoms, shown
˚
in red in Fig. 5A) than Glc-a-HVA (Fig. 4B; 6.59 A between the
two most distant atoms, also shown in red in Fig. 5A), sug-
gesting that Glc-b-HVA, is a better antiradical molecule, due to
its higher exibility with respect to Glc-a-HVA.
Conformational analysis
The theoretical study suggests that the more exposed the
negatively charged region of Glc-b-HVA the better its antiradical
properties. The ability of a molecule to expose a specic struc-
tural region is dependent on its exibility and some molecular
properties depend on this structural feature. Indeed, Castro
et al. suggest that the anomeric conguration of alkyl glycosides
determines the exibility and spatial orientation of their alkyl
moiety and therefore the three dimensional arrangement of the
polar and non-polar moieties of the surfactant.35 In disaccha-
rides it has been reported that this exibility is dependent on
the accessible conformational space of the molecules.36
It is worth mentioning that in terms of free radical scav-
enging activity assayed against DPPHc it has been also reported
that the molecular exibility seems to play a very important role
in these properties, where the more exible molecules showed
higher inhibition of the DPPHc radical than those with higher
rigidity.24
In order to visualize the exibility of conformers as a func-
tion of the distances between the more distant atoms in the
conformers, we established the Intramolecular Relative
Distance (IRD) between the two most distant atoms in the
planar molecules (CH2OH oxygen in the sugar molecule and
–OH phenolic hydroxyl in HVA, atoms shown in red in Fig. 5A).
In addition, we also determined the dihedral angle of glycosides
(uHVA-gly) formed between the more exible atoms in the
structure (blue line in Fig. 5A). For the analysis of relative
mobility, the relationship between uHVA-gly and IRD was dened
and is shown in Fig. 5B. It may be observed in this gure that for
all Glc-b-HVA and Gal-b-HVA conformers the shortest IRD are
found at uHVA-gly of 80 and 280 degrees. On the contrary, for all
Glc-a-HVA and Gal-a-HVA conformers the shortest IRD were
observed at uHVA-gly near to 180 degrees. Nevertheless, in all
cases the shortest IRD in the a-glycosides were higher than
those observed for b-glycosides. It is interesting to note that Glc-
b-HVA and Gal-b-HVA conformers showed a wider dispersion of
the mean IRD than the dispersion of the mean IRD for Glc-a-
HVA and Gal-a-HVA conformers (Fig. 5C). This indicating that
To explore changes on exibility of HVA glycosides resulting
from the accessible conformational space of these molecules,
we accomplished a conformational analysis of the a- and
b glycosides. Considering that one of the main differences
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Fig. 5 Relationship between dihedral angle uHVA-gly and IRD (B), and mean and dispersion of IRD values (C). The IRD values were determined
from distances between the two most distant atoms in the planar molecules, atoms in red (A). The dihedral angles (uHVA-gly) were defined as the
angle formed between the more flexible atoms in the structure, blue line in structure (A). For the sake of clarity, tendency lines are included in (B).
The molecule shown is Glc-b-HVA (A).
these b-glycosides may adopt a wider conformational disper- assays allow us to support the conclusion that glycosyl-residue
sion between open and closed conformations. In consequence, on glycosides has an important contribution to the free
b-glycosides showing a higher exibility than a-glycosides may radical scavenging activity of the PPG.
expose easily their negatively charged groups and therefore
exhibit better free radical scavenger properties.
The kinetic study of the DPPHc assay demonstrated that the
HVA aglycone is more reactive than its glucose or galactose
glycosides, two of the most frequently found sugars in natural
glycosides. Interestingly, more signicant differences in free
radical scavenger activity were found based on the nature of the
Conclusions
Four different a- and b-glycosides of HVA containing glucose anomeric linkage rather than on the nature of the sugar residue,
and galactose were enzymatically-synthesized using different with b-glycosides displaying better free radical scavenging
glycosidases. Contrary to most chemical methods, this activity than a-glycosides.
straightforward and highly selective enzymatic method allowed
We propose that the increase of the free radical scavenger
us to glycosylate exclusively the primary hydroxyl of HVA to capacity found for b-glycosides is due to the exibility of the
obtain 100% pure anomers without protection-de-protection glycosides and is explained with the aid of the molecular elec-
strategies. According to the EC50 values obtained in DPPHc trostatic potential. This parameter indicates that Glc-b-HVA is
assay, these enzymatically-synthesized glycosides displayed a better free radical scavenger than Glc-a-HVA due to the large
better free radical scavenger properties than the corresponding exposure of the negatively charged region on the b-glycoside
HVA-aglycone. Among all glycosides, the b-glycosides exhibited molecule. Furthermore, b-glycosides show higher exibility
the highest free radical scavenger activity. Thus, these endpoint than a-glycosides exposing advantageously their negatively
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charged groups, something that favours the free radical scav- 11 C. Rice-Evans, N. Miller and G. Paganga, Structure-
enger capacity. These results are very good examples of the
correlation between the structure, the molecular charge distri-
bution and the free radical scavenger capacity.
antioxidant activity relationships of avonoids and
phenolic acids, Free Radical Biol. Med., 1996, 20(7), 933–956.
12 M. Soobrattee, V. Neergheen, A. Luximon-Ramma,
O. Aruoma and T. Bahorun, Phenolics as potential
antioxidant therapeutic agents: Mechanism and actions,
Mutat. Res., Fundam. Mol. Mech. Mutagen., 2005, 579, 200–
213.
Acknowledgements
This research was supported by PAPIIT-UNAM (grant IN209016)
13 T. Vogt, Phenylpropanoid Biosynthesis, Mol. Plant, 2009,
3(1), 2–20.
´
´
and CONACyT (grant 219728). J.A. Gonzalez-Rıos, Y. Romero
and M. Reina thank CONACyT for scholarships 313184, 332509,
3077279 respectively. Authors thank Fernando Gonzalez for
technical assistance and Novozymes-Mexico for their generous
donation of CGTase. We thank DGTIC-UNAM for the assistance.
Mitztli supercomputer of DGTIC to do all the quantum chem-
ical calculations.
´
´
´
14 A. Lopez-Munguıa, Y. Hernandez-Romero, J. Pedraza-
´
Chaverri, A. Miranda-Molina, I. Regla, A. Martınez and
E. Castillo, Phenylpropanoid glycoside analogues:
enzymatic synthesis, antioxidant activity and theoretical
study of their free radical scavenger mechanism, PLoS One,
2011, 6(6), 1–9.
´
15 S. Stoyanova, J. Geuns, E. Hideg and W. Ende, The food
additives inulin and stevioside counteract oxidative stress,
Int. J. Food Sci. Nutr., 2011, 62(3), 207–214.
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