Synthesis of various β-D-glucopyranosyl and β-Dxylopyranosyl hydroxybenzoates and evaluation of their antioxidant activities

Article abstract of DOI:10.1515/hc-2016-0214

Various β-D-glucopyranosyl and β-Dxylopyranosyl hydroxybenzoates were efficiently prepared from 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (TAGB) or 2,3,4-tri-O-acetyl-α-D-xylopyranosyl bromide (TAXB), respectively, by amine-promoted glycosylation.

Full text of DOI:10.1515/hc-2016-0214

Heterocycl. Commun. 2017; aop
Yasutaka Shimotori*, Masayuki Hoshi, Yosuke Osawa and Tetsuo Miyakoshi
Synthesis of various β-D-glucopyranosyl and β-D-
xylopyranosyl hydroxybenzoates and evaluation
of their antioxidant activities
DOI 10.1515/hc-2016-0214
Received November 23, 2016; accepted January 5, 2017
Introduction
Abstract: Various β-D-glucopyranosyl and β-D- Hydroxybenzoic acid derivatives such as protocatechuic,
xylopyranosyl hydroxybenzoates were efficiently prepared vanillic, and syringic acids are widely distributed in
from 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide plants and foods derived from natural products [1–6]. In
(
(
TAGB) or 2,3,4-tri-O-acetyl-α-D-xylopyranosyl bromide recent years, there have been many reports on the bio-
TAXB), respectively, by amine-promoted glycosylation. logical activities of hydroxybenzoic acid derivatives. It has
Regioselective deacetylation of the resulting acetylated been reported that vanillic acid shows anticoagulant [7],
β-D-gluco- and β-D-xylopyranosyl hydroxybenzoates was anti-inflammatory [8], and nephroprotective effects [9],
investigated using Novozym 435 as a lipase catalyst. In the as well as estrogen-like [10] and antinociceptive activi-
case of β-D-glucopyranosyl hydroxybenzoates, Novozym ties [11]. Syringic acid has an antihyperglycemic effect
4
35-catalyzed deacetylation is regioselective at C-4 and C-6 [12] and antiangiogenic and antimicrobial activities [13,
positions. On the other hand, β-D-xylopyranosyl hydroxy- 14]. In many cases, these hydroxybenzoic acids exist
benzoates are deacetylated only at the C-4 position. Anti- predominantly as ester and glycoside forms with only
oxidant activities of free hydroxybenzoic acids and the lesser amounts of them being present in free carboxylic
respective β-D-gluco- and β-D-xylopyranosyl hydroxyben- acid forms [15]. Many plants containing these hydroxy-
zoates were evaluated by DPPH˙ radical scavenging as well benzoic acid derivatives have been used as traditional
as their inhibitory effect on autoxidation of bulk methyl medicines. Conyza sumatrensis leaves have been used
linoleate. The β-D-xylopyranosyl protocatechoate, as well for traditional treatment of malaria in Cameroon [16].
as quercetin and α-tochopherol, show high antioxidant The stems of Berchemia racemosa are used in Japan for
activity for the radical scavenging activity by 1,1-diphenyl- treatment of gall stones, liver diseases, neuralgia and
2
-picrylhydrazyl (DPPH˙). In bulk methyl linoleate, the stomach cramp [17]. Conyza sumatrensis contains various
antioxidant activities of β-D-gluco- and β-D-xylopyranosyl flavonoids as phenolic components [18]. The occurrence
protocatechoates are higher than that of α-tocopherol.
of β-D-glucopyranosyl syringoate in Berchemia racemosa
has been confirmed by Inoshiri and coworkers [19]. Addi-
tionally, it is known that various Spanish Cistus species
include β-D-glucopyranosyl vanilloate [20]. In traditional
folk medicine, Cistus is used as anti-inflammatory, anti-
ulcerogenic, wound healing, antimicrobial, cytotoxic and
vasodilator remedies [21]. These activities of the plants
containing hydroxybenzoic acid derivatives are related to
their ability to act as antioxidants [22], metal ion chelators
Keywords: antioxidant activity; β-D-glycopyranosyl
hydroxybenzoate; hydroxybenzoic acid; lipase-catalyzed
regioselective deacetylation; methyl linoleate.
[
23], radical scavengers [24] and inhibitors of prooxidant
enzymes [25]. Many researchers have studied the antioxi-
dant effects of hydroxybenzoic acid derivatives [22, 26–28].
We have previously reported preparation of β-D-gluco- and
β-D-xylopyranosyl hydroxycinnamoates using amine-pro-
moted glycosylation and Novozym 435-catalyzed regiose-
lective deacetylation, and evaluation of their antioxidant
activities [29, 30]. β-D-Glucopyranosyl feruloyl and synap-
inoates and β-D-xylopyranosyl cafferoate show similar or
higher antioxidant activities compared with α-tocopherol.
*
Corresponding author: Yasutaka Shimotori, Department of
Biotechnology and Environmental Chemistry, Kitami Institute of
Technology, 165 Koen-cho, Kitami, Hokkaido 090-8507, Japan,
e-mail: yasu@mail.kitami-it.ac.jp
Masayuki Hoshi: Department of Biotechnology and Environmental
Chemistry, Kitami Institute of Technology, 165 Koen-cho, Kitami,
Hokkaido 090-8507, Japan
Yosuke Osawa and Tetsuo Miyakoshi: Department of Applied
Chemistry, School of Science of Technology, Meiji University, 1-1-1
Higashi-mita, Tama-ku, Kawasaki 214-8571, Japan
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ꢀY. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoates
R2
R2
R3
R3
R1
R1
R1
RCOOH
amine
MeOH
Novozym 435
O
O
O
AcO
AcO
AcO
AcO
HO
AcO
O
O
R4
R4
MeCN, MS 4 Å
rt, 24 h, under N2
Solvent
OAc
OAc
OAc
Br
O
O
50 °C, 24 h
1
a–e
3a–e
4a–e
1
TAGB (R = CH OAc)
2a–e
2
TAXB (R1 = H)
1
1
a: R2 = R3 = R4 = H
(Benzoicacid)
(p-hydroxybenzoic acid)
(Protocatechuicacid)
1
2
: R = CH OAc 3: R = CH OH
2
2
b: R = R = H; R3 = OH
2
4
1
: 4: R = H
c: R = H; R3 = R = OH
2
4
d: R = H; R = OH; R4 = OMe (Vanillicacid)
2
3
2
4
3
e: R = R = OMe; R = OH
(Syringicacid)
Scheme 1ꢁGlycosylation of hydroxybenzoic acid derivatives and deacetylation using Novozym 435.
In this study, we investigated the synthesis of various β-D-glucopyranosyl p-hydroxybenzoyl and protocatecho-
β-D-gluco- and β-D-xylopyranosyl hydroxybenzoates and ates, whichhavephenolichydroxylgroups, werelowerthan
described the antioxidant activities of hydroxybenzoic that of β-D-glucopyranosyl benzoate. Jover and co-workers
acids and their gluco- and xylopyranosates as well as have reported the relationship between chemical structures
some vanillic acid-related compounds such as p-hydroxy- and acidity for benzoic acid derivatives [35]. The acidity
benzoic, protocatechuic and syringic acids and their gly- of benzoic acid derivatives used in this work decreases
cosates. Their antioxidant activities were evaluated based in the order benzoic acidꢀ>ꢀsyringic acidꢀ>ꢀprotocatechuic
on their inhibitory effects on the autoxidation of methyl acidꢀ>ꢀvanillic acidꢀ>ꢀp-hydroxybenzoic acid. On the other
linoleate in a bulk system and the radical scavenging hand, the yields of β-D-glucopyranosyl hydroxybenzoates
activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH˙).
decrease in the order benzoic acidꢀ>ꢀvanillic acidꢀ>ꢀsyringic
acidꢀ>ꢀp-hydroxybenzoic acidꢀ>ꢀprotocatechuic acid. It was
expected that the yield of β-D-glucopyranosyl benzoate 1a
would be highest because the stability of benzoate anion
is highest among these benzoic acid derivatives. Galland
and co-workers performed the glycosylation of hydroxy-
cinnamic acids such as ferulic and caffeic acids after
methateification at carboxylic group [36]. It seemed that
the decrease in yield is not proportional to the acidity of
benzoic acids themselves because glycosylation of com-
Results and discussion
Amine-promoted glycosylation of hydroxy-
benzoic acids and Novozym 435-catalyzed
regioselective deacetylation
We have previously reported synthesis of β-D- peting phenoxy anion and carboxylate anion takes place.
glucopyranosyl hydroxycinnamoates by amine-promoted Additionally, it can be assumed that the rate of glycosyla-
glycosylation and Novozym 435-catalyzed regioselec- tion of the phenolic hydroxyl groups for protocatechuic
tive deacetylation [29]. β-D-Glucopyranosyl feruloyl and acid is higher than that of p-hydroxybenzoic acid because
sinapinoates show high antioxidant activities compared protocatechuic acid has two phenolic hydroxyl groups.
with α-tocopherol and quercetin. It is known that glyco-
β-D-Xylopyranosyl hydroxybenzoates were syn-
syl benzoate analogues have various biological activities thesized using tri-O-acetyl-α-D-xylopyranosyl bromide
such as antioxidant [31], cytotoxic [32, 33] and enzyme (TAXB) as a saccharide donor (Scheme 1). Glycosylation
inhibitory properties [34]. Therefore, we synthesized β-D- was performed under previously described conditions
glucopyranosyl hydroxybenzoates by previous methods that were used for the synthesis of β-D-xylopyranosyl
using various hydroxybenzoic acids such as vanillic acid hydroxycinnamoates [30]. As in the case of the synthesis
and protocatechuic acid as an aglycone (Scheme 1).
of β-D-glucopyranosates 1b and 1c, yields of products 2b
β-D-Xylopyranosyl hydroxybenzoates were also syn- and 2c are lower than that of β-D-xylopyranosyl benzoate
thesized because β-D-xylopyranosyl hydroxycinnamoates 2a. By contrast, reactions of vanillic and syringic acids give
show higher antioxidant activities compared to the corre- almost the same yields as β-D-xylopyranosyl benzoate 2a.
sponding β-D-glycopyranosyl hydroxycinnamoates. Glyco- The decrease of the yields occurrs for the same reason as
sylation of each hydroxybenzoic acid was performed under in the case of synthesis of β-D-glucopyranosyl benzoate.
the previously described conditions [29, 30]. As in the case
Subsequently, deacetylation of glucopyrano-
of glycosylation of hydroxycinnamic acids, the yields of sates 1 was investigated (Scheme 1). We have reported
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Y. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoatesꢀꢂꢁꢁꢁꢀ3
Novozym 435-catalyzed regioselective deacetylation of under mildly acidic conditions of pH 6, a citrate buffer solu-
2
,3,4,6-tetraacetyl-β-D-glucopyranosyl hydroxycinnamo- tion of 1a without lipase was stirred at 40°C for 24 h; the
ates [29]. Novozym 435 catalyzed deacetylation at C-4 and hydrolysis was not observed. In addition to Novozym 435,
C-6 positions takes place regioselectively without metha- lipase AS Amano (from Aspergillus niger), lipase AK Amano
nolysis of a hydroxycinnamoyl group at the anomeric (from Pseudomonas fluorescens), lipase AYS (from Candia
position. Since antioxidant activities of deacetylated rugosa), lipase PS (from Burkholderia cepacia), and PPL
β-D-glucopyranosyl cinnamoates were improved, dea- (from porcine pancreas) were used. All these lipases show
cetylation of β-D-glucopyranosyl hydroxybenzoates 1a–e no reactivity for deacetylation of 1a. Deacetylation of β-D-
was also investigated using Novozym 435 as a catalyst xylopyranosyl benzoate 2a at C-2 and C-3 positions was also
(
Scheme 1). Methanolysis of 1 was performed in methyl investigated using the same conditions described above.
tert-butyl ether (MTBE) for 24 h at 50°C. A mixture of All lipases except lipase AYS Amano showed little reactiv-
MTBE/benzene, 3:1, was used as a solvent because β-D- ity. Hydrolysis of a benzoate group at the C-1 position was
glucopyranosyl p-hydroxybenzoyl, protocatechoyl and confirmed by analysis of NMR spectra. By contrast, deacet-
vanilloates 1b, 1c and 1d are sparingly soluble in MTBE. ylated compound 4a was obtained by regioselective hydrol-
In the case of all substrates, methanolysis progressed to ysis at the C-4 position for Novozym 435, lipase AK Amano
yields in a range of 84–90%. As with β-D-glucopyranosyl and lipase PS Amano. Lipase can hydrolyze non-water
hydroxycinnamoates, methanolysis of the hydroxyben- soluble lipids and esterase catalyzes hydrolysis of water-
zoate moiety at the anomeric position was not observed. soluble lipids [37]. We have previously reported PPL-cata-
By using 2D NMR analysis, it was confirmed that acetyl lyzed hydrolysis of N-methyl-5-acetoxyalkanamides [38].
groups at C-4 and C-6 positions were deacetylated regiose- Although the reaction mixture is a suspension because the
lectively regardless of the difference in the benzoyl part. As water solubility of N-methyl-5-acetoxualkanamides is poor,
with β-D-glucopyranosyl hydroxycinnamoates, Novozym lipase-catalyzed hydrolysis progresses with high enantiose-
4
35-catalyzed methanolysis of β-D-glucopyranosates lectivity. The reaction mixtures of β-D-glucopyranosyl and
1
exhibits high regio- and functional group selectivity. β-D-xylopyranosyl benzoates 1a and 2a are also heteroge-
Additionally, Novozym 435 shows high affinity toward all neous but the water solubility of xylose is higher than that
substrates because all deacetylated β-D-glucopyranosates of glucose [39]. It can be assumed that β-D-xylopyranosyl
2
were obtained in high yield after 24 h.
Methanolysis of β-D-xylopyranosyl hydroxybenzoates pared with β-D-glucopyranosate 1a.
a–e was also performed using Novozym 435. The solubil-
benzoate 2a shows slightly higher affinity for lipase com-
2
ity of compounds 2b,c in MTBE is low, and a mixture of
MTBE/benzene, 3/1, was used as a solvent. Novozym 435 DPPH˙ radical scavenging activities
catalyzed deacetylation of β-D-xylopyranosyl hydroxycin-
namoates proceeds regioselectively at the C-4 position The antioxidant properties of hydroxybenzoyl acids and
[
30]. Deacetylation of β-D-xylopyranosyl hydroxybenzo- β-D-glucopyranosates 1b–e were evaluated by the dem-
ates 2 was also investigated using Novozym 435 in the onstration of DPPH˙ radical scavenging activity (Figure 1).
expectation that it would show high regio- and functional
group selectivity. Methanolysis progressed to about 80%
yields for all reactions. The hydroxybenzoate moiety
did not react, and deacetylation at the C-4 position was
only observed. Novozym 435 also shows high regio- and
functional group selectivity toward β-D-xylopyranosyl
hydroxybenzoates 2 and β-D-glucopyranosates 1.
0.1 µmol/mL
0.5 µmol/mL
1.0 µmol/mL
100.0
90.0
80.0
70.0
6
5
4
3
0.0
0.0
0.0
0.0
Novozym 435 catalyzes methanolysis of acetyl groups
at C-4 and C-6 positions for β-D-glucopyranosyl hydroxy-
benzoates 1 in MTBE. We have previously reported that
deacetylation of β-D-gluco- and xylopyranosyl hydroxy-
cinnamoates increases antioxidant properties [29, 30].
For deacetylation at C-2 and C-3 positions, regioselective
hydrolysis of β-D-glucopyranosyl benzoate 1a was inves-
tigated using various lipases in pH 6 citrate buffer with
20.0
Figure 1ꢁDPPH˙ radical scavenging activities of free hydroxylbenzoic
MTBE as a co-solvent. To confirm that 1a is not hydrolyzed acids and their β-D-glucopyranosates.
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4ꢀ ꢀY. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoates
The activity decreases in the order of protocatechuic p-hydroxybenzoate 3b exhibit lower activities than the
acidꢀ>ꢀsyringic acid≈glucopyranosyl protocatechoates corresponding free hydroxybenzoic acids. Fukumoto and
1
c and 3cꢀ>ꢀvanillic acidꢀ>ꢀglucopyranosyl syringoate co-workers have suggested that the size of the steric hin-
3
eꢀ>ꢀdeacetylated glucopyranosyl vanilloate 3dꢀ>ꢀdea- drance of the sugar moiety reduces the antioxidant prop-
cetylated glucopyranosyl p-hydroxybenzoate 3bꢀ>ꢀglu- erties, and our results are in good agreement with their
copyranosyl syringoate 1eꢀ>ꢀglucopyranosyl vanilloate suggestion [22].
1
d≈p-hydroxybenzoic acidꢀ>ꢀglucopyranosyl p-hydroxy-
benzoate 1b.
On the other hand, the DPPH˙ radical scavenging
activities of xylopyranosates 2 and 4 decrease in the
There are many reports of DPPH˙ radical scaveng- order of xylopyranosyl protocatechoate 2cꢀ>ꢀdeacety-
ing activities of hydroxybenzoic acids. Karamać and lated xylopyranosyl protocatechoate 4c≈protocatechuic
co-workers have reported that the activities decrease in acidꢀ>ꢀsyringic acidꢀ>ꢀvanillic acidꢀ>ꢀxylopyranosyl syrin-
the order syringic acidꢀ>ꢀprotocatechuic acidꢀ>ꢀ>ꢀvanil- goates 2e and 4e≈xyropyranosyl vanilloates 2d and
lic acidꢀ>ꢀ>ꢀp-hydroxybenzoic acid [40]. Fujimoto and 4dꢀ>ꢀxylopyranosyl p-hydroxybenzoates 2b and 4b≈p-
co-workers have reported different results: vanillic hydroxybenzoic acid (Figure 2). Xylopyranosyl protocat-
acidꢀ>ꢀ>ꢀprotocatechuic acidꢀ>ꢀsyringic acid [22]. Further- echuate 2c shows slightly higher activity compared with
more, Noipa and co-workers have reported the order of free protocatechuic acid. Deacetylated xylopyranosyl pro-
syringic acidꢀ>ꢀprotocatechuic acidꢀ>ꢀvanillic acid [28]. tocatechoate 4c also shows comparatively high activity
Still, different results were obtained in this work. It can with free protocatechuic acid.
be suggested that the differences between our and other
These activities are almost of the same strength as
results are due to the measurement conditions. On the those of quercetin and α-tocopherol. In the case of glu-
other hand, our results are in good agreement with copyranosyl protocatechoates 1c and 3c, the activities
Gadow’s results [41]. Free protocatechuic acid shows decrease compared with that of free protocatechuic acid.
almost the same high DPPH˙ radical scavenging activity as The water solubility of xylose is higher than that of glucose
quercetin and α-tocopherol. Antioxidants that have many [39]. It can be assumed that the activities of xylopyrano-
phenolic hydroxyl groups show high antioxidant activities syl protocatechoates 2c and 4c increase compared with
[
42, 43]. Free syringic and vanillic acids show high radical glucopyranosyl protocatechoates 1c and 3c because the
scavenging activities compared with free p-hydroxyben- affinities of xylopyranosates 2c and 4c with DPPH˙ radical
zoic acid. Ortho-methoxy groups as electron-donating are higher than those of glucopyranosates 1c and 3c.
groups at the phenolic hydroxyl group improve antioxi- Whereas xylopyranosyl p-hydroxybenzoyl, vanilloyl and
dant activity and stabilize phenoxy radicals [42, 43]. The syringoates 2b, 2d and 2e show about 30% activities, dea-
radical scavenging activity of free vanillic acid is higher cetylation increases the activities about 10%. In contrast,
than that of free p-hydroxybenzoic acid. Syringic acid has there are no significant differences between activities of
two methoxy groups at the ortho positions of phenolic xylopyranosyl vanilloyl and syringoates 2d and 2e and
hydroxyl groups. The phenoxy radical of syringic acid is deacetylated xylopyranosates 4d and 4e. The activities
more stable than that of vanillic acid. It can be assumed of xylopyranosyl p-hydoxybenzoates 2b and 4b and free
that syringic acid shows almost the same activity as vanil-
lic acid because the steric hindrance of syringic phenoxy
0
.1 µmol/mL
0.5 µmol/mL
1.0 µmol/mL
radicals is larger than that of vanillic acid. Only vanillic
acid among free hydroxybenzoic acids shows concentra-
tion dependency. These results are in good agreement
with those of Shyamala [44] and Sang [45]. DPPH˙ radical
scavenging activities of glucopyranosyl protocatechoates
1
00.0
90.0
80.0
7
6
5
4
0.0
0.0
0.0
0.0
1
c and 3c are highest in glucopyranosyl hydroxybenzo-
ates 1 and 3. In the case of glucosyl p-hydroxybenzoyl,
vanilloyl and syringoates, the corresponding deacety-
lated glucopyranosates 3b, 3d and 3e show about 10%
higher activities than acetylated glucopyranosates 1b,
30.0
0.0
2
1
d and 1e. Regioselective deacetylation at C-4 and C-6
positions increases water solubility and the increase of
affinity to DPPH˙ free radicals increases the activity. All
Figure 2ꢁDPPH˙ radical scavenging activities of free hydroxybenzoic
glucopyranosyl hydroxybenzoates except glucopyranosyl acids and their β-D-xylopyranosates.
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Y. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoatesꢀꢂꢁꢁꢁꢀ5
p-hydroxybenzoic acid are similar. p-Hydroxybenzoic acid protocatechoate 3c shows only the activity like free syrin-
and its xylopyranosates show low activities compared gic acid. Comparing free hydroxybenzoic acids for DPPH˙
with other hydroxybenzoic acid derivatives, and the sugar radical scavenging activity, syringic and vanillic acids
moiety does not affect activities. Xylopyranosyl proto- have relatively high activities. In contrast, these hydroxy-
catechoates 2c and 4c have the highest activities in this benzoic acids have very low activities on the inhibition
investigation.
of autoxidation of methyl linoleate. Porter has advanced
the so-called ‘polar paradox’ by the observation that
polar antioxidants are more effective in nonpolar lipids,
whereas nonpolar antioxidants are more effective in polar
lipid emulsions [47]. The water solubility of hydroxyben-
zoic acid derivatives used in this investigation decreases
Inhibitory effect on autoxidation of bulk
methyl linoleate
Formation of hydroperoxides in bulk methyl linoleate at in the order of protocatechuic acidꢀ>ꢀp-hydroxybenzoic
0°C was estimated based on the absorbance at 234 nm acidꢀ>ꢀsyringic acidꢀ>ꢀvanillic acid [48, 49]. The antioxi-
generated during the initial oxidation stage (Figure 3). dant activity of p-hydroxybenzoic acid is one of the lowest
4
Modifications were made to the original method [46].
because there is only one phenolic hydroxyl group and no
The antioxidant activity decreases in the order of methoxy group to stabilize the phenoxy radical. Glyco-
protocatechuic acidꢀ>ꢀglucopyranosyl protocatechoate sylation causes the decrease in antioxidant activity com-
1
cꢀ>ꢀα-tocopherolꢀ>ꢀglucopyranosyl syringoate 1eꢀ>ꢀdea- pared with free hydroxybenzoic acids in almost all cases.
cetylated glucopyranosyl protocatechoate 3c≈syringic A possible explanation for the decrease of antioxidant
acidꢀ>ꢀdeacetylated glucopyranosyl syringoate 3eꢀ>ꢀdea- activities might be a decrease of polarity by glycosylation
cetylated
glucopyranosyl
vanilloate
3dꢀ>ꢀvanillic compared with free hydroxybenzoic acids.
As with glucopyranosyl hydroxybenzoates 1 and 3,
acid≈glucopyranosyl vanilloate 1d≈p-hydroxybenzoic
acid≈glucopyranosyl p-hycroxybenzoates 1b and 3b. Free antioxidant activities of xylopyranosyl hydroxybenzo-
protocatechuic acid and glucopyranosyl protocatechoate ates 2 and 4 were also investigated using methyl linoleate
1
c) show higher antioxidant activities than α-tocopherol. (Figure 4). The antioxidant activity decreases in the
The antioxidant activity of protocatechuic acid is higher order of protocatechuic acidꢀ>ꢀxylopyranosyl protocat-
than that of other hydroxybenzoic acids. Glycopyra- echoate 2cꢀ>ꢀdeacetylated xylopyranosyl protocatecho-
nosyl protocatechoate 1c has high antioxidant activity ate 4cꢀ>ꢀα-tocopherolꢀ>ꢀxylopyranosyl syringoates 2e
among the glucosyl hydroxybenzoate synthesized in this and 4eꢀ>ꢀsyringic acidꢀ>ꢀp-hydroxybenzoic acid≈vanillic
investigation. Surprisingly, deacetylated glucopyranosyl acidꢀ>ꢀxyropyranosylvanilloates2dand4d≈xylopyranosyl
3.00
2.50
2.00
1.50
1.00
0.50
0.00
Control
α-Tocopherol
Quercetin
p-Hydroxybenzoic acid
Glucosyl p-hydroxybenzoate 1b
Glucosyl p-hydroxybenzoate 3b
Protocatechuic acid
Glucosyl protocatechoate 1c
Glucosyl protocatechoate 3c
Vanillic acid
Glucosyl vanilloate 1d
Glucosyl vanilloate 3d
Syringic acid
Glucosyl syringoate 1e
Glucosyl syringoate 3e
0
5
10
15
20
25
30
Oxidation time (days)
Figure 3ꢁInhibition of formation of hydroperoxide in bulk methyl linoleate by free hydroxybenzoic acids and their β-D-glucopyranosates.
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ꢀY. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoates
4
3
3
2
2
1
1
0
0
.00
.50
.00
.50
.00
.50
.00
.50
.00
Control
α-Tocopherol
Quercetin
p-Hydroxybenzoic acid
Xylosyl p-hydroxybenzoate 2b
Xylosyl p-hydroxybenzoate 4b
Protocatechuic acid
Xylosyl protocatechoate 2c
Xylosyl protocatechoate 4c
Vanillic acid
Xylosyl vanilloate 2d
Xylosyl vanilloate 4d
Syringic acid
Xylosyl syringoate 2e
Xylosyl syringoate 4e
0
5
10
15
20
25
30
Oxidation time (days)
Figure 4ꢁInhibition of formation of hydroperoxide in bulk methyl linoleate by free hydroxybenzoic acids and their β-D-xylopyranosates.
p-hydroxybenzoates 2b and 4b. In addition to free proto- amine-promoted glycosylation. Deacetylation of products
catechuic acid, xylopyranosyl protocatechoates 2c and 4c 1 and 2 was investigated using Novozym 435 as a lipase
have higher antioxidant activities than α-tocopherol.
catalyst until the reaction progressed with over 80%
Xylopyranosyl syringoates 2e and 4e show activi- yields in all cases. β-D-Glucopyranosyl hydroxybenzoates
ties that are similar to that of α-tocopherol for as long as 1 are regioselectively deacetylated at C-4 and C-6 posi-
5
days. Although glycosylation decreases the antioxidant tions in the presence of Novozym 435. Regioselective dea-
activity of protocatechuic acid, deacetylated xylopyrano- cetylation at the C-4 position exclusively was confirmed
syl protocatechoate 4c is more active than the correspond- for β-D-xylopyranosyl hydroxybenzoates 2. Novozym 435
ing glucopyranosate 3c. Moreover, antioxidant activities of is most effective for regioselective deacetylation among
xylopyranosyl syringoates 2e and 4e are better compared various lipase catalysts used in this investigation. In the
with free syringic acid. Xylose exhibits 1.5 times higher case of DPPH˙ radical scavenging activities, the activities
water solubility than glucose [39]. It can be suggested that of β-D-glucopyranosyl hydroxybenzoates 1 and 3 decrease
the water solubility of xylopyranosates is higher than that compared with the corresponding free hydroxybenzoic
of glucopyranosates. For this reason, some xylopyrano- acids. In contrast, β-D-xylopyranosyl protocatechoates 2c
syl hydroxybenzoates show higher antioxidant activities and 4c show almost the same high antioxidant activities
than the corresponding free hydroxyl benzoic acids and as quercetin and α-tocopherol. Moreover, the antioxidant
glucopyranosates. We previously reported a similar inves- activities of β-D-glucopyranosyl and β-D-xylopyranosyl
tigation of hydroxycinnamic acids derivatives [29, 30]. In protocatechoates 1c, 2c and 4c in inhibition of autoxi-
almost all cases, hydroxybenzoic acid derivatives show dation of bulk methyl linoleate are higher than that of
lower antioxidant activities than hydroxycinnamic acid α-tocopherol.
derivatives. The phenoxy radical of cinnamic acids is
stabilized because these acids have an α,β-unsaturated
C-C double bond in the phenylpropane structure. These
Experimental
results are in good agreement with related literature
reports [46, 50].
1H NMR (500 MHz) and 13C NMR (126 MHz) spectra were recorded on
a JNM-ECA-500 spectrometer with tetramethylsilane as the internal
standard using CDCl and DMSO-d as solvents. Structural determina-
3
6
tion of all compounds was performed using COSY, HMQC and HMBC
NMR techniques. ESI-MS spectra were taken on an AccuTof GCv 4G
Conclusions
(
JEOL, Tokyo, Japan) mass spectrometer. IR spectra were obtained in
Various β-D-gluco- and β-D-xylopyranosyl hydroxy- KBr pellets on an FT-IR JASCO 460 plus spectrometer. Melting points
benzoates 1 and 2 were efficiently synthesized by were determined on an MP-500D instrument and are uncorrected.
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Y. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoatesꢀꢂꢁꢁꢁꢀ7
Optical rotations were determined with a JASCO P-1010 polarimeter. (ddd, Jꢀ=ꢀ9.2 Hz, 4.6 Hz and 2.3 Hz, 1H, H-5), 4.17 (dd, Jꢀ=ꢀ12.6 Hz and
Lipase AS Amano, lipase AK amano, lipase AYS amano and lipase 2.3 Hz, 1H, H-6a), 4.33 (dd, Jꢀ=ꢀ12.6 Hz and 4.6 Hz, 1H, H-6b), 5.20 (t,
PS Amano were purchased from Wako Pure Chemical Industries, Jꢀ=ꢀ9.2 Hz, 1H, H-4), 5.35 (m, 2H, H-2 and H-3), 5.88 (d, Jꢀ=ꢀ9.2 Hz, 1H,
1
3
Ltd. Japan. Porcine pancreas lipase (PPL) was obtained from Nacalai H-1), 6.92 (d, Jꢀ=ꢀ8.0, 1H, Ph), 7.56 (m, 2H, Ph); C NMR (CDCl ): δ 20.6
3
Tesque Inc., Japan.
(CH , OAc at C-2 and C-4), 20.7 (CH , OAc at C-3 and C-6), 61.6 (C-6),
3
3
6
8.0 (C-4), 70.3 (C-2), 72.6 (C-3, C-5), 92.2 (C-1), 115.1 (Ph), 116.7 (Ph),
1
24.6 (Ph), 164.3 (O-C(=O)-Ph), 169.7, 170.1, 170.2, 171.0 (C=O, OAc at
+
C-2, C-3, C-4 and C-6). HRMS. Calcd for C H O , [M] : m/z 484.1216;
General procedure for glycosylation of hydroxybenzoic
acids
21 24 13
found: m/z 484.1215.
2
,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl vaniloate (1d)ꢁYield
25
A
mixture of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide 73%; colorless solid; mp 111–112°C; R 0.33 (hexane-AcOEt, 1:1); [α]
f
D
(
TAGB, 411 mg, 1.0 mmol) or 2,3,4-tri-O-acetyl-α-D-xylopyranosyl −30.1 (c 1.0, THF); IR: 3427 (O-H), 2944 (C-H), 1752 (C=C-C=O), 1595
−1 1
bromide (TAXB, 339 mg, 1.0 mmol), a hydroxybenzoic acid (2.0 mmol (Ar, C=C), 1515(Ar, C=C), 1218 (C-O-C=O), 1035 (C-O-C=O) cm ; H NMR
for 1, 3.0 mmol for 2) and i-Pr NEt (388 mg, 3.0 mmol for 1, 129 mg, (CDCl ): δ 2.06 (m, 12H, CH , OAc at C-2, C-3, C-4 and C-6), 3.95 (m,
2
3
3
1
.0 mmol for 2) and 4 Å molecular sieves (1 g) in CH CN (5 mL) was 2H, H-5 and OCH at Ph), 4.14 (dd, Jꢀ=ꢀ12.6 Hz and 2.3 Hz, 1H, H-6a),
3
3
stirred for 24 h at room temperature under argon. Progress of the 4.34 (dd, Jꢀ=ꢀ12.6 Hz and 4.2 Hz, 1H, H-6b), 5.20 (m, 1H, H-4), 5.35 (m,
reaction was monitored by thin layer chromatography. Upon the 2H, H-2 and H-3), 5.88 (m, 1H, H-1), 6.95 (d, Jꢀ=ꢀ8.0 Hz, 1H, Ph), 7.53
1
3
completion of the reaction, the mixture was concentrated under (d, Jꢀ=ꢀ1.7 Hz, 1H, Ph), 7.65 (dd, Jꢀ=ꢀ8.0 Hz and 1.7 Hz, 1H, Ph); C NMR
reduced pressure, the residue was neutralized with aqueous NaHCO3 (CDCl ): δ 20.6 (CH , OAc at C-2, C-3, C-4 and C-6), 61.5 (C-6), 67.9 (C-4),
3
3
and extracted with AcOEt. The extract was washed with brine, dried 70.2 (C-2), 72.6 (C-3, C-5), 92.2 (C-1), 125.1 (Ph), 114.4 (Ph), 112.1 (Ph),
over anhydrous MgSO , and concentrated under reduced pressure. 164.1 (O-C(=O)-Ph), 169.3, 169.4, 170.1, 170.6 (C=O, OAc at C-2, C-3, C-4
4
+
The crude product was purified by silica gel chromatography eluting and C-6). HRMS. Calcd for C H O , [M] : m/z 498.1373; found: m/z
2
2
26 13
with hexane/AcOEt.
498.1398.
2
,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl benzoate (1a)ꢁYield 2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl syringoate (1e)ꢁYield
25
25
8
4%; colorless solid; mp 140–141°C; R 0.55 (hexane-AcOEt, 1:1); [α]
71%; colorless solid; mp 67–68°C; R 0.28 (hexane-AcOEt, 1:1); [α]
f
D
f
D
−
24.71 (c 1.0, THF); IR: 2951 (C-H), 1737 (C=C-C=O), 1452 (Ar, C=C), 1251 −39.1 (c 1.0, THF); IR: 3439 (O-H), 2944 (C-H), 1755 (C=C-C=O), 1515 (Ar,
−1 1
−1
1
(
C-O-C=O), 1066 (C-O-C=O) cm ; H NMR (CDCl ): δ 2.02 (m, 12H, CH , C=C), 1223 (C-O-C=O), 1076 (C-O-C=O) cm ; H NMR (CDCl ): δ 2.06
3
3
3
OAc at C-2, C-3, C-4 and C-6), 3.95 (m, 1H, H-5), 4.14 (d, Jꢀ=ꢀ12.6 Hz, 1H, (m, 12H, CH , OAc at C-2, C-3, C-4 and C-6), 3.91 (m, 7H, H-5 and 2OCH3
H-6a), 4.33 (dd, Jꢀ=ꢀ12.6 Hz and 4.6 Hz, 1H, H-6b), 5.21 (m, 1H, H-4), at Ph), 4.14 (dd, Jꢀ=ꢀ12.6 Hz and 2.3 Hz, 1H, H-6a), 4.35 (dd, Jꢀ=ꢀ12.6 Hz
3
5
.36 (m, 2H, H-2, H-3), 5.94 (m, 1H, H-1), 7.46 (t, Jꢀ=ꢀ7.4 Hz, 2H, Ph), 7.61 and 4.6 Hz, 1H, H-6b), 5.21 (m, 1H, H-4), 5.36 (m, 2H, H-2 and H-3), 5.87
t, Jꢀ=ꢀ7.4 Hz, 1H, Ph), 8.05 (d, Jꢀ=ꢀ17.4 Hz, 2H, Ph); 1 C NMR (CDCl ): δ (d, Jꢀ=ꢀ8.0 Hz, 1H, H-1), 7.33 (s, 2H, Ph); C NMR (CDCl ): δ 20.6 (CH ,
3
13
(
3
3
3
2
0.5 (2CH , OAc at C-2 and C-4), 20.6 (CH , OAc at C-3 and C-6), 61.4 OAc at C-2, C-3, C-4 and C-6), 56.5 (CH , OCH at Ph), 61.5 (C-6), 67.8
3
3
3 3
(
C-6), 67.9 (C-4), 70.1 (C-2), 72.7 (C-3, C-5), 92.3 (C-1), 128.3 (Ph), 128.5 (C-4), 70.3 (C-2), 72.4 (C-3, C-5), 92.2 (C-1), 107.1 (Ph), 164.1 (-O-(C=O)-
(
Ph), 130.1 (Ph), 134.0 (Ph), 164.5 (O-(C=O)-Ph), 169.3 (C=O, OAc at Ph), 169.3 (C=O in OAc at C-2), 169.4 (C=O, OAc at C-3), 170.0 (C=O, OAc
+
C-2), 169.4 (C=O, OAc at C-3), 170.0 (C=O, OAc at C-4), 170.6 (C=O, OAc at C-4), 170.6 (C=O, OAc at C-6). HRMS. Calcd for C H O , [M] : m/z
2
3
28 14
+
at C-6). HRMS. Calcd for C H O , [M] : m/z 452.1319; found: m/z 528.1479; found: m/z 528.1451.
2
1
24 11
4
68.1331.
2
,3,4-Tri-O-acetyl-β-D-xylopyranosyl benzoate (2a)ꢁYield 70%;
25
2
,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl p-hydroxybenzoate colorless solid; mp 119–120°C; R 0.63 (hexane-AcOEt, 1:1); [α]
f
D
(
1b)ꢁYield 49%; colorless solid; mp 131–132°C; R 0.45 (hexane- −52.9 (c 1.0, THF); IR: 2949 (C-H), 1755 (C=C-C=O), 1602 (Ar, C=C),1244
f
2
5
−1 1
AcOEt, 1:1); [α] −79.45 (c 1.0, THF); IR: 3270 (O-H), 2935 (C-H), 1754 (C-O-C=O), 1088 (C-O-C=O) cm ; H NMR (CDCl ): δ 2.06 (m, 9H, CH ,
D
3
3
−1
1
(
C=C-C=O), 1519 (Ar, C=C), 1237 (C-O-C=O), 1087 (C-O-C=O) cm ; H OAc at C-2, C-3 and C-6), 3.64 (dd, Jꢀ=ꢀ12.6 Hz and 8.2 Hz, 1H, H-5a),
NMR (CDCl ): δ 2.06 (m, 12H, CH , OAc at C-2, C-3, C-4 and C-6), 4.03 4.24 (dd, Jꢀ=ꢀ12.6 Hz and 4.6 Hz, 1H, H-5b), 5.18 (ddd, Jꢀ=ꢀ8.2 Hz, 7.8 Hz
3
3
(
1
dd, Jꢀ=ꢀ12.6 Hz and 2.3 Hz, 1H, H-6a), 4.20 (dd, Jꢀ=ꢀ12.6 Hz and 4.6 Hz, and 4.6 Hz, 1H, H-4), 5.22 (dd, Jꢀ=ꢀ8.2 Hz and 6.4 Hz, 1H, H-2), 5.29
H, H-6b), 4.27 (ddd, Jꢀ=ꢀ9.7 Hz, 4.6Hz and 2.3 Hz, 1H, H-5), 5.03 (t, (t, Jꢀ=ꢀ8.2 Hz, 1H, H-3), 5.97 (d, Jꢀ=ꢀ6.4 Hz, 1H, H-1), 7.46 (m, 2H, Ph),
1
3
Jꢀ=ꢀ9.7 Hz, 1H, H-4), 5.11 (dd, Jꢀ=ꢀ8.6 Hz and 9.7 Hz, 1H, H-2), 5.51 (t, 7.60 (m, 1H, Ph), 8.05 (m, Ph); C NMR (CDCl ): δ 20.6 (CH , OAc at
3
3
Jꢀ=ꢀ9.7 Hz, 1H, H-3), 6.12 (d, Jꢀ=ꢀ8.6 Hz, 1H, H-1), 6.88 (m, 3H, Ph), 7.78 C-2, C-3 and C-4), 62.5 (C-5), 68.0 (C-4), 69.1 (C-2), 70.3 (C-3), 92.5 (C-1),
1
3
(
(
m, 2H, Ph); C NMR (CDCl3): δ 20.6 (2CH , OAc at C-2 and C-4), 20.7 128.5 (Ph), 129.6 (Ph), 133.5 (Ph), 164.5 (O-(C=O)-Ph), 169.3, 169.6, 169.8
3
+
CH , OAc at C-3 and C-6), 61.2 (C-6), 67.9 (C-4), 70.1 (C-2), 71.3 (C-5), (C=O, OAc at C-2, C-3 and C-4). HRMS. Calcd for C H O , [M] : m/z
3
18 20
9
7
1.6 (C-3), 91.3 (C-1), 131.4 (Ph), 115.6 (Ph), 163.4 (C(=O)-Ph), 169.3, 380.1107; found: m/z 380.1096.
1
69.5, 169.8, 169.9 (C=O, OAc at C-2, C-3, C-4 and C-6). HRMS. Calcd for
+
C H O , [M] : m/z 468.1268; found: m/z 468.1281.
2
1
24 12
2,3,4-Tri-O-acetyl-β-D-xylopyranosyl p-hydroxybenzoate (2b)ꢁ
Yield 51%; colorless solid; mp 205–206°C; Rf 0.43 (hexane-AcOEt,
2
5
2
,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl protocatechoate (1c)ꢁ 1:1); [α] −68.3 (c 1.0, THF); IR: 3372 (O-H), 3000 (Ar, C-H), 1755 (C=C-
D
Yield 29%; colorless solid; mp 65–66°C; R 0.33 (hexane-AcOEt, 1:1); C=O), 1619 (Ar, C=C), 1518 (Ar, C=C), 1241 (C-O-C=O), 1152 (C-O-C=O)
f
2
5
−1
1
[
α] −10.7 (c 1.0, THF); IR: 3423 (O-H), 2964 (C-H), 1750 (C=C-C=O), cm ; H NMR (CDCl ): δ 2.06 (m, 9H, CH , OAc at C-2, C-3 and C-4),
D
3
3
−1
1
604 (Ar, C=C), 1521 (Ar, C=C), 1222 (C-O-C=O), 1070 (C-O-C=O) cm ; 3.95 (ddd, Jꢀ=ꢀ9.6 Hz, 4.6 Hz and 2.3 Hz, 1H, H-4), 4.13 (dd, Jꢀ=ꢀ12.6 Hz
1
H NMR (CDCl ): δ 2.06 (m, 12H, CH , OAc at C-2, C-3, C-4 and C-6), 3.97 and 2.3 Hz, 1H, H-5a), 4.32 (dd, Jꢀ=ꢀ12.6 Hz and 4.6 Hz, 1H, H-5b), 5.35
3
3
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ꢁꢁꢁꢂ
8ꢀ ꢀY. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoates
(
m, 2H, H-2 and H-3), 5.90 (d, Jꢀ=ꢀ9.2 Hz, 1H, H-1), 6.74 (s, 1H, OH at Ph) 435 was filtered off the mixture and MTBE was then removed under
.87 (m, 2H, Ph), 7.93 (m, 2H, Ph); 1 C NMR (CDCl ): δ 20.6 (CH , OAc reduced pressure. The residue was subjected to flash chromatog-
3
6
3
3
at C-2, C-3 and C-4), 61.6 (C-5), 69.9 (C-2), 72.6 (C-3 and C-4), 92.5 (C-1), raphy (CHCl /MeOH, 9:1) to give the partially deacetylated product
3
1
15.5 (Ph) 132.6 (Ph), 164.2 (O-(C=O)-Ph), 169.6, 169.8, 170.2, (-C=O in 3a–e or 4a–e.
+
OAc at C-2, C-3 and C-4). HRMS. Calcd for C H O , [M] : m/z 396.1057;
18
20 10
found: m/z 396.1062.
2,3-Di-O-acetyl-β-D-glucopyranosyl benzoate (3a)ꢁYield 85%;
25
colorless solid; mp 136–137°C; R 0.48 (CHCl /MeOH, 10:1); [α] −54.7
f
3
D
2
,3,4-Tri-O-acetyl-β-D-xylopyranosyl protocatechuate (2c)ꢁYield (c 1.0, THF); IR: 3437 (O-H), 3069 (Ar, C-H), 1750 (C=C-C=O), 1601 (Ar,
25
−1 1
4
4%; colorless solid; mp 124–125°C; R 0.3 (hexane-AcOEt, 1:1); [α]
C=C), 1236 (C-O-C=O), 1028 (C-O-C=O) cm ; H NMR (DMSO-d ): δ 2.06
6
f
D
−
55.0 (c 1.0, THF); IR: 3367 (O-H), 2964 (C-H), 1728 (C=C-C=O), 1605 (m, 6H, CH , OAc at C-2 and C-3), 3.68 (m, Jꢀ=ꢀ9.2, 4.6, 2.3 Hz, 1H, H-4),
3
−1
1
(
Ar, C=C), 1533 (Ar, C=C), 1213 (C-O-C=O), 1052 (C-O-C=O) cm ; H 3.87 (m, 2H, H-6a, H-6b), 3.96 (m, 1H, H-3), 5.21 (t, Jꢀ=ꢀ9.2 Hz, 1H, H-5),
NMR (CDCl ): δꢀ=ꢀ2.06 (m, 9H, CH , OAc at C-2, C-3 and C-4), 3.62 (dd, 5.26 (dd, Jꢀ=ꢀ10.3 Hz and 9.2 Hz, 1H, H-2), 5.93 (d, Jꢀ=ꢀ9.2 Hz, 1H, H-1),
3
3
1
3
Jꢀ=ꢀ12.4 Hz and 8.2 Hz, 1H, H-5a), 4.22 (dd, Jꢀ=ꢀ12.4 Hz and 5.0 Hz, 1H, 7.46 (t, Jꢀ=ꢀ8.0 Hz, 2H, Ph), 7.60 (m, 1H, Ph), 8.02 (m, 2H, Ph); C NMR
H-5b), 5.04 (ddd, Jꢀ=ꢀ8.7 Hz, 8.2 Hz and 5.0 Hz, 1H, H-4), 5.21 (dd, (DMSO-d ): δ 20.6 (CH , OAc at C-2 and C-3), 61.5 (C-6), 68.9 (C-4), 70.2
6
3
Jꢀ=ꢀ8.7 Hz and 6.9 Hz, 1H, H-2), 5.30 (dd, Jꢀ=ꢀ8.7 Hz and 8.2 Hz, 1H, H-3), (C-2), 75.5 (C-3), 76.5 (C-5), 92.7 (C-1), 128.5 (Ph), 130.1 (Ph), 134.0 (Ph),
1
3
5
.89 (d, Jꢀ=ꢀ6.9 Hz, 1H, H-1), 6.91 (m, 1H, Ph), 7.56 (m, 2H, Ph); C NMR 147.6 (-C(=O)C-Ph), 164.5 (C(=O)CH=CH-), 169.5 (C=O, OAc at C-2), 171.2
+
(
CDCl ): δ 20.6 (CH , OAc at C-2, C-3 and C-4), 62.5 (C-5), 68.3 (C-4), (C=O, OAc at C-3). HRMS. Calcd for C H O , [M] : m/z 368.1107; found:
3
3
17 20 9
6
9.6 (C-2), 70.7 (C-3), 92.5 (C-1), 115.0 (Ph), 116.7 (Ph), 124.4 (Ph), 164.4 m/z 368.1125.
(
-C(=O)C-), 170.0, 170.1, 170.2 (-C=O in OAc at C-2, C-3 and C-4); HRMS:
+
m/z [M] Calcd for C H O : 412.1006; found: 412.1030.
2,3-Di-O-acetyl-β-D-glucopyranosyl
p-hydroxybenzoate
18
20 11
(
3b)ꢁYield 88%; colorless solid; mp 131–132°C; R 0.33 (CHCl /MeOH,
f
3
2
5
2
,3,4-Tri-O-acetyl-β-D-xylopyranosyl vaniloate (2d)ꢁYield 73%; 10:1); [α] −31.0 (c 1.0, THF); IR: 3379 (O-H), 3014 (Ar, C-H), 1732 (C=C-
D
2
D
5
colorless solid; mp 91–92°C; R ꢀ=ꢀ0.48 (hexane-AcOEt, 1:1); [α] −14.2 C=O), 1610 (Ar, C=C), 1517 (Ar, C=C), 1241 (C-O-C=O), 1086 (C-O-C=O)
f
−1
1
(
c 1.0, THF); IR: 3353 (O-H), 3000 (Ar, C-H), 1732 (C=C-C=O), 1595 (Ar, cm ; H NMR (DMSO-d ): δ 2.06 (m, 6H, CH , OAc at C-2 and C-3),
6 3
−1 1
C=C), 1519 (Ar, C=C), 1249 (C-O-C=O), 1054 (C-O-C=O) cm ; H NMR 3.58 (m, 3H, H-4, H-6a and H-6b), 3.70 (d, Jꢀ=ꢀ12.0 Hz, 1H, H-5), 4.99
(
CDCl ): δ 2.06 (m, 9H, CH , OAc at C-2, C-3 and C-4), 3.62 (dd, Jꢀ=ꢀ12.6 Hz (t, Jꢀ=ꢀ9.2 Hz, 1H, H-2), 5.18 (t, Jꢀ=ꢀ9.2 Hz, 1H, H-3), 5.88 (d, Jꢀ=ꢀ8.0 Hz,
3
3
13
and 8.6 Hz, 1H, H-5a), 3.95 (s, 3H, OCH at Ph), 4.22 (dd, Jꢀ=ꢀ12.6 Hz and 1H, H-1), 6.91 (d, Jꢀ=ꢀ7.4 Hz, 2H, Ph), 7.19 (d, Jꢀ=ꢀ7.4 Hz, 2H, Ph); C NMR
3
5
.2 Hz, 1H, H-5b), 5.04 (dd, Jꢀ=ꢀ8.6 Hz and 5.2 Hz, 1H, H-4), 5.23 (dd, (DMSO-d ): δ 20.6 (CH in OAc at C-2 and C-3), 55.9 (OCH at Ph), 59.8
6
3
3
Jꢀ=ꢀ8.6 Hz and 6.9 Hz, 1H, H-2), 5.30 (t, Jꢀ=ꢀ8.6 Hz, 1H, H-3), 5.90 (d, (C-5), 67.1 (C-6), 70.5 (C-2), 74.3 (C-3), 77.2 (C-4), 92.0 (C-1), 115.5 (Ph),
Jꢀ=ꢀ6.9 Hz, 1H, H-1), 6.17 (s, 1H, OH at Ph) 6.95 (d, Jꢀ=ꢀ8.0 Hz, 1H, Ph), 131.7 (Ph), 163.6 (-C=O in OAc at C-2), 169.5 (-C=O in OAc at C-3). HRMS.
1
3
+
7
.54 (d, Jꢀ=ꢀ1.7 Hz, 1H, Ph), 7.65 (dd, Jꢀ=ꢀ8.6 Hz and1.7 Hz, 1H, Ph);
C
Calcd for C H O , [M] : m/z 384.1057; found: m/z 384.1072.
17
20 10
NMR (CDCl ): δ 20.6 (CH , OAc at C-2, C-3 and C-4), 62.8 (C-5), 68.4
3
3
(
C-4), 69.5 (C-2), 70.8 (C-3), 92.6 (C-1), 112.0 (Ph), 114.3 (Ph), 124.9 (Ph),
2
,3-Di-O-acetyl-β-D-glucopyranosyl protocatechuate (3c)ꢁYield
1
64.2 (O-(C=O)-Ph), 169.4, 169.7, 169.8, (C=O, OAc at C-2, C-3 and C-4).
25
8
4%; colorless solid; mp 95–96°C; R 0.10 (CHCl /MeOH, 20:1); [α]
f
3
D
+
HRMS. Calcd for C H O , [M] : m/z 426.1162; found: m/z 426.1177.
1
9
22 11
−57.9 (c 1.0, THF); IR: 3423 (O-H), 2941 (C-H), 1733 (C=C-C=O), 1604
−
1 1
(
Ar, C=C), 1521 (Ar, C=C), 1221 (C-O-C=O), 1033 (C-O-C=O) cm ; H NMR
2
7
,3,4-Tri-O-acetyl-β-D-xylopyranosyl
syringoate
(2e)ꢁYield
(
DMSO-d ): δ 1.91 (s, 3H, CH , OAc at C-3), 2.06 (s, 6H, CH , OAc at
6
3
3
1%; colorless solid; mp 103–104°C; R ꢀ=ꢀ0.33 (hexane-AcOEt, 1:1);
f
C-2), 3.54 (m, 3H, H-4, H-6a and H-6b), 3.68 (d, Jꢀ=ꢀ12.0 Hz, 1H, H-5),
4.93 (dd, Jꢀ=ꢀ10.3 Hz and 8.6 Hz, 1H, H-2), 5.13 (m, 1H, H-3), 5.89 (d,
Jꢀ=ꢀ8.6 Hz, 1H, H-1), 6.82 (d, Jꢀ=ꢀ8.6 Hz, 1H, Ph), 7.27 (dd, Jꢀ=ꢀ2.3 Hz and
2
5
[
α] −51.8 (c 1.0, THF); IR: 3439 (O-H), 2948 (C-H), 1752 (C=C-C=O),
D
−
1
1
610 (Ar, C=C), 1515 (Ar, C=C), 1217 (C-O-C=O), 1039 (C-O-C=O) cm ;
1
H NMR (CDCl ): δ 2.06 (m, 9H, CH , OAc at C-2, C-3 and C-4), 3.60
13
3
3
8.6 Hz, 1H, Ph), 7.31 (d, Jꢀ=ꢀ2.3 Hz, 1H, Ph); C NMR (DMSO-d ): δ 20.6
6
(
(
1
dd, Jꢀ=ꢀ12.0 Hz and 8.6, Hz, 1H, H-5a), 3.94 (s, 6H, OCH at Ph), 4.21
3
(CH , OAc at C-2 and C-3), 59.9 (C-5), 67.2 (C-6), 70.6 (C-2), 74.7 (C-3),
3
dd, Jꢀ=ꢀ12.0 Hz and 5.2 Hz, 1H, H-5b), 5.06 (dd, Jꢀ=ꢀ8.6 Hz and 5.2 Hz,
7
7.3 (C-4), 91.6 (C-1), 116.4 (Ph), 118.9 (Ph), 122.2 (Ph), 145.1 (C(=O)
H, H-4), 5.27 (dd, Jꢀ=ꢀ8.6, 6.9 Hz, 1H, H-2), 5.33 (t, Jꢀ=ꢀ8.6 Hz, 1H, H-3),
C-Ph), 163.7 (C=O, OAc at C-2), 169.4 (C=O, OAc at C-3). HRMS. Calcd
1
3
5
.85 (d, Jꢀ=ꢀ6.9 Hz, 1H, H-1), 6.04 (s, 1H, OH at Ph), 7.33 (s, 2H, Ph);
C
+
for C H O , [M] : m/z 400.1005; found: m/z 400.1008.
17
20 11
NMR (CDCl ): δ 20.6 (CH , OAc at C-2, C-3 and C-4), 56.3 (OCH at Ph),
3
3
3
6
3.0 (C-5), 68.7 (C-4), 70.0 (C-2), 71.4 (C-3), 93.2 (C-1), 107.4 (Ph), 164.3
2
,3-Di-O-acetyl-β-D-glucopyranosyl vaniloate (3d)ꢁYield 89%;
25
(
O-(C=O)-Ph), 169.4, 169.8 (C=O, OAc at C-2, C-3 and C-4). HRMS. Calcd
colorless solid; mp 127–128°C; R 0.30 (CHCl /MeOH, 10:1); [α] −71.2
+
f
3
D
for C H O , [M] : m/z 456.1268; found: m/z 456.1291.
2
0
24 12
(
c 1.0, THF); IR: 3295 (O-H), 3021 (Ar, C-H), 2974 (C-H), 1752 (C=C-C=O),
−
1 1
1
608 (Ar, C=C), 1519(Ar, C=C), 1223 (C-O-C=O), 1032 (C-O-C=O) cm ; H
NMR (DMSO-d ): δ 1.93 (s, 3H, CH , OAc at C-3), 2.06 (s, 3H, CH , OAc
6
3
3
General procedure for Novozym 435-catalyzed regiose-
lective deacetylation
at C-2), 3.58 (m, 3H, H-4, H-6a and H-6b), 3.70 (d, Jꢀ=ꢀ11.5 Hz, 1H, H-5),
3
.82 (s, 6H, OCH at Ph), 4.97 (t, Jꢀ=ꢀ8.6 Hz, 1H, H-2), 5.17 (t, Jꢀ=ꢀ8.6 Hz,
3
1
H, H-3), 5.89 (d, Jꢀ=ꢀ8.6 Hz, 1H, H-1), 6.90 (d, Jꢀ=ꢀ8.6 Hz, 1H, Ph), 7.41
1
3
A solution of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl hydroxyben- (d, Jꢀ=ꢀ1.7 Hz, 1H, Ph), 7.43 (dd, Jꢀ=ꢀ1.7 Hz and 8.6 Hz, 1H, Ph); C NMR
zoate 1a-e (1.0 mmol) or 2,3,4-tri-O-acetyl-β-D-xylopyranosyl hydroxy- (DMSO-d
): δ 20.6 (CH , OAc at C-2 and C-3), 55.5 (OCH at Ph), 59.9
3 3
6
benzoates 2a-e (1.0 mmol), MeOH (384 mg, 12.0 mmol), and Novozym (C-5), 67.2 (C-6), 70.7 (C-2), 74.5 (C-3), 77.4 (C-4), 91.9 (C-1), 115.4 (Ph),
4
2
35 (0.8 g) in tert-butyl methyl ether (MTBE, 10 mL) was stirred for 119.0 (Ph), 123.9 (Ph), 147.4 (C(=O)C-Ph), 163.6 (C=O in OAc at C-2),
+
+
4 h at 50°C. The progress of the reaction was monitored with thin 169.3 (C=O in OAc at C-3). HRMS. m/z [M] Calcd for C18
H
O
11, [M] : m/z
22
layer chromatography. Upon completion of the reaction, Novozym 414.1162; found: m/z 414.1183.
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Y. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoatesꢀꢂꢁꢁꢁꢀ9
2
,3-Di-O-acetyl-β-D-glucopyranosyl syringoate (3e)ꢁYield 90%; (DMSO-d ): δ 1.95 (s, 3H, CH , OAc at C-3), 2.03 (s, 3H, CH , OAc at
6
3
3
2
5
colorless solid; mp 159–160°C; R 0.73 (CHCl /MeOH, 10:1); [α] −41.5 C-2), 3.52 (dd, Jꢀ=ꢀ9.7 Hz and 10.3 Hz, 1H, H-5a), 3.75 (m, 1H, H-4), 3.81
f
3
D
(
1
c 1.0, THF); IR: 3312 (O-H), 3009 (Ar, C-H), 2995 (C-H), 1743 (C=C-C=O), (s, 3H, OCH at Ph), 3.91 (dd, Jꢀ=ꢀ5.2 Hz and 10.3 Hz, 1H, H-5b), 4.98
3
−
1 1
604 (Ar, C=C), 1516 (Ar, C=C), 1062 (C-O-C=O) cm ; H NMR (DMSO- (dd, Jꢀ=ꢀ8.0 Hz and 9.7 Hz, 1H, H-2), 5.10 (t, Jꢀ=ꢀ9.7 Hz, 1H, H-3), 5.81 (d,
d ): δ 2.06 (m, 6H, CH , OAc at C-2 and C-3), 3.58 (m, 1H, H-4, H-6a and Jꢀ=ꢀ8.0 Hz, 1H, H-1), 6.89 (d, Jꢀ=ꢀ8.6 Hz, 1H, Ph), 7.40 (d, Jꢀ=ꢀ2.3 Hz, 1H,
6
3
1
3
H-6b), 3.70 (d, Jꢀ=ꢀ12.0 Hz, 1H, H-5), 3.81 (s, 6H, OCH at Ph), 4.99 (t, Ph), 7.43 (dd, Jꢀ=ꢀ2.3 Hz and 8.6 Hz, 1H, Ph); C NMR (DMSO-d ): δ 20.6
3
6
Jꢀ=ꢀ9.2 Hz, 1H, H-2), 5.18 (t, Jꢀ=ꢀ9.2 Hz, 1H, H-3), 5.88 (d, Jꢀ=ꢀ8.0 Hz, 1H, (CH , OAc at C-2 and C-3), 55.5 (OCH at Ph), 65.8 (C-5), 66.9 (C-4), 70.3
3
3
1
3
H-1), 7.19 (s, 2H, Ph); C NMR (DMSO-d ): δ 20.6 (CH in OAc at C-2 and (C-2), 74.1 (C-3), 92.5 (C-1), 112.8 (Ph), 115.3 (Ph), 123.9 (Ph), 163.6 (C(=O)
6
3
C-3), 55.9 (OCH at Ph), 59.8 (C-5), 67.1 (C-6), 70.5 (C-2), 74.3 (C-3), 77.2 C-Ph), 169.3 (C=O, OAc at C-2), 169.4 (C=O, OAc at C-3). HRMS. Calcd for
3
+
(
1
C-4), 92.0 (C-1), 107.0 (Ph), 147.6 (C(=O)C-Ph), 163.6 (C=O, OAc at C-2), C H O , [M] : m/z 384.1057; found: m/z 384.1081.
17
20 10
+
69.4 (C=O, OAc at C-3). HRMS. Calcd for C H O , [M] : m/z 444.1268;
1
9
24 12
found: m/z 444.1243.
2,3-Di-O-acetyl-β-D-xylopyranosyl syringoate (4e)ꢁYield 82%;
25
colorless solid; mp 144–145°C; R ꢀ=ꢀ0.30 (CHCl /MeOH, 20:1); [α]
D
f
3
2
,3-Di-O-acetyl-β-D-xylopyranosyl benzoate (4a)ꢁYield 83%; −9 7.1 ( c 1.0, THF); IR: 3480 (O-H), 2985 (C-H), 1732 (C=C-C=O), 1615 (Ar,
25
D
−1 1
colorless solid; mp 149–150°C; R 0.43 (CHCl /MeOH, 20:1); [α] −70.1 C=C), 1516 (Ar, C=C), 1119 (C-O-C=O) cm ; H NMR (DMSO-d ): δ 1.95
f
3
6
(
1
c 1.0, THF); IR: 3411 (O-H), 2950 (C-H), 1738 (C=C-C=O), 1601 (Ar, C=C), (s, 3H, CH , OAc at C-3), 2.03 (s, 3H, CH , OAc at C-2), 3.53 (t, Jꢀ=ꢀ10.8 Hz,
3
3
−
1 1
492 (Ar, C=C), 1069 (C-O-C=O) cm ; H NMR (DMSO-d ): δ 1.93 (s, 3H, 1H, H-5a), 3.76 (m, 1H, H-4), 3.82 (s, 6H, CH at Ph), 3.92 (dd, Jꢀ=ꢀ5.7 Hz
6
3
CH in OAc at C-3), 2.03 (s, 3H, CH in OAc at C-2), 3.93 (dd, Jꢀ=ꢀ5.7 Hz and 10.8 Hz, 1H, H-5b), 5.00 (dd, Jꢀ=ꢀ8.0 Hz and 9.2 Hz, 1H, H-2), 5.20 (t,
3
3
1
3
and 14.9 Hz, 1H, H-5a), 3.58 (ddd, Jꢀ=ꢀ5.7 Hz, 10.3 Hz and 14.9 Hz, 1H, Jꢀ=ꢀ9.2 Hz, 1H, H-3), 5.80 (d, Jꢀ=ꢀ8.0 Hz, 1H, H-1), 7.12 (s, 2H, Ph); C NMR
H-4), 3.93 (dd, Jꢀ=ꢀ5.7 Hz and 10.3 Hz, 1H, H-5b), 4.99 (dd, Jꢀ=ꢀ8.2 Hz (DMSO-d ): δ 20.6 (CH , OAc at C-2 and C-3), 65.9 (C-5), 67.0 (C-4), 70.3
6
3
and 10.3 Hz, 1H, H-2), 5.11 (t, Jꢀ=ꢀ9.2 Hz, 1H, H-3), 5.90 (d, Jꢀ=ꢀ8.2 Hz, 1H, (C-2), 74.0 (C-3), 92.7 (C-1), 107.2 (Ph), 163.7 (C(=O)C-Ph), 169.3 (C=O,
+
H-1), 7.55 (t, Jꢀ=ꢀ8.0, 10.3 Hz, 2H, Ph), 7.69 (t, Jꢀ=ꢀ8.0 Hz, 1H, Ph), 7.92 (dd, OAc at C-2), 169.4 (C=O, OAc at C-3). HRMS. Calcd for C H O , [M] :
1
8
22 11
13
Jꢀ=ꢀ1.7 Hz and 8.0 Hz, 1H, Ph); C NMR (DMSO-d ): δ 20.6 (CH , OAc at m/z 414.1162; found: m/z 414.1183.
6
3
C-2 and C-3), 65.8 (C-5), 66.8 (C-4), 70.2 (C-2), 74.0 (C-3), 92.7 (C-1), 128.9
Ph), 129.2 (Ph), 134.0 (Ph), 163.9 (-C(=O)C-Ph), 169.2 (C=O, OAc at C-2),
(
1
+
69.4 (C=O, OAc at C-3). HRMS. Calcd for C H O , [M] : m/z 338.1002; Lipase screening for deacetylation of 1a and 2a
1
6
18
8
found: m/z 338.1016.
Lipase (0.1 g) (AS from Aspergillus niger, AK from Pseudomonas fluores-
cens, AYS from Candida rugosa, PS from Burkholderia cepacia and PPL
from porcine pancreas) was added to a mixture of 1a (45 mg, 0.1 mmol)
or 2a (38 mg, 0.1 mmol) in pH 7 citrate buffer (10 mL) and MTBE (2.5 mL),
and the mixture was stirred at 40°C for 24 h. The progress of the reac-
tion was monitored with thin layer chromatography. After stirring,
lipase was filtered off and the organic phase was extracted with AcOEt.
The combined extracts were washed with brine, dried over anhydrous
2
,3-Di-O-acetyl-β-D-xylopyranosyl
p-hydroxybenzoate
(
4b)ꢁYield 85%; colorless solid; mp 202–203°C; R 0.23 (CHCl /
f
3
2
5
MeOH, 20:1); [α] −190.8 (c 0.5, THF); IR: 3411 (O-H), 3000 (Ar,
C-H), 2942 (C-H), 1725 (C=C-C=O), 1618 (Ar, C=C), 1518 (Ar, C=C), 1078
D
−1
1
(
C-O-C=O) cm ; H NMR (DMSO-d ): δ 1.92 (s, 3H, CH , OAc at C-3),
6
3
2
.03 (s, 3H, CH , OAc at C-2), 3.51 (t, Jꢀ=ꢀ11.5 Hz, 1H, H-5a), 3.75 (m, 1H,
3
H-4), 3.91 (dd, Jꢀ=ꢀ5.7 Hz and 11.5 Hz, 1H, H-5b), 4.96 (t, Jꢀ=ꢀ8.6 Hz, 1H,
H-2), 5.09 (t, Jꢀ=ꢀ8.6 Hz, 1H, H-3), 5.84 (d, Jꢀ=ꢀ8.6 Hz, 1H, H-1), 6.87 (d,
MgSO and concentrated under reduced pressure. The crude product
4
Jꢀ=ꢀ8.6 Hz, 2H, Ph), 7.77 (d, Jꢀ=ꢀ8.6 Hz, 2H, Ph); 1 C NMR (DMSO-d ): δ
3
6
was purified by flash chromatography (CHCl /MeOH, 9:1).
3
2
0.6 (CH , OAc at C-2 and C-3), 65.7 (C-5), 66.9 (C-4), 70.2 (C-2), 74.2
3
(
C-3), 92.3 (C-1), 115.5 (Ph), 131.7 (Ph), 163.6 (C(=O)C-Ph), 169.1 (C=O,
+
OAc at C-2), 169.4 (C=O, OAc at C-3). HRMS. Calcd for C H O , [M] :
m/z 354.0951; found: m/z 354.0965.
16
18
9
DPPH˙ radical scavenging activity
The antioxidant activity of β-D-glycopyranosyl hydroxybenzoates was
2
8
−
,3-Di-O-acetyl-β-D-xylopyranosyl protocatechoate (4c)ꢁYield
2
5
estimated by measuring their free radical scavenging activity using
0%; colorless solid; mp 78–79°C; R 0.13 (CHCl -MeOH, 20:1); [α]
f
3
D
2
,2-diphenyl-1-picrylhydrazyl (DPPH˙) as free radical according to
68.5 (c 0.5, THF); IR: 3423 (O-H), 2988 (C-H), 1732 (C=C-C=O), 1605
the literature report [51]. The reaction mixture (10 mL) comprised of
freshly made 0.15 mꢂ DPPH˙ in ethanol (7000 μL), different concentra-
tions of each β-D-glycopyranosyl hydroxybenzoates (1, 5 and 10 μmol)
in 300 μL DMSO and tris-HCl buffer (pH 7.4, 100 mꢂ). The reaction
mixture was kept for 30 min in a water bath at 25°C under dark and
optical density was measured at 517 nm. DPPH˙ has an unpaired elec-
tron, which gave purple color, and when this electron is balanced, the
color is lost. The compounds which can give an electron to the DPPH˙
bleaching the reagent, which is monitored as the decrease in absorb-
ance of the DPPH˙ solution. All analyses were carried out in triplicate.
−1 1
(
Ar, C=C), 1524 (Ar, C=C), 1080 (C-O-C=O) cm ; H NMR (DMSO-d ): δ
6
1
.92 (s, 3H, CH in OAc at C-3), 2.02 (s, 3H, CH in OAc at C-2), 3.49 (t,
3
3
Jꢀ=ꢀ10.8 Hz, 1H, H-5a), 3.73 (dd, Jꢀ=ꢀ5.7 Hz and 10.8 Hz, 1H, H-4), 3.89
(
dd, Jꢀ=ꢀ4.6 Hz and 10.8 Hz, 1H, H-5b), 4.93 (dd, Jꢀ=ꢀ8.6 Hz and 9.2 Hz,
H, H-2), 5.06 (t, Jꢀ=ꢀ9.2 Hz, 1H, H-3), 5.81 (d, Jꢀ=ꢀ8.6 Hz, 1H, H-1), 6.82
d, Jꢀ=ꢀ8.6 Hz, 1H, Ph), 7.27 (dd, Jꢀ=ꢀ1.7 Hz and 8.6 Hz, 1H, Ph), 7.31 (d,
1
(
1
3
Jꢀ=ꢀ1.7 Hz, 1H, Ph); C NMR (DMSO-d ): δ 20.6 (CH , OAc at C-2 and C-3),
6
3
6
5.8 (C-5), 66.9 (C-4), 70.3 (C-2), 74.3 (C-3), 92.3 (C-1), 115.4 (Ph), 116.4
(
Ph), 122.3 (Ph), 163.8 (C(=O)C-Ph), 169.1 (C=O, OAc at C-2), 169.5 (C=O,
+
OAc at C-3). HRMS. Calcd for C H O , [M] : m/z 370.0900; found: m/z
16
18 10
370.0921.
Inhibitory effect on autoxidation of bulk methyl linoleate
2
,3-Di-O-acetyl-β-D-xylopyranosyl vaniloate (4d)ꢁYield 88%;
25
colorless solid; mp 40–41°C; R 0.33 (CHCl /MeOH, 20:1); [α] −75.9
f
3
D
(
c 1.0, THF); IR: 3437 (O-H), 3018 (Ar, C-H), 2941 (C-H), 1734 (C=C- To 1 g of methyl linoleate, 25 μL of the acetone solution of the test
−
1
1
compound was mixed in a 50-mL vial, and the mixture was agitated
C=O), 1595 (Ar, C=C), 1515 (Ar, C=C), 1082 (C-O-C=O) cm ; H NMR
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1
0ꢀꢁꢁꢁꢂꢀY. Shimotori et al.: Synthesis of antioxidant glycosyl hydroxybenzoates
under ultrasonic wave for 30 s. A 25 μL aliquot of acetone without [12] Srinicasan, S.; Muthukumaran, J.; Muruganathan, U.; Ven-
sample was added for control. After purging the acetone with nitro-
gen, the mixture was placed in an oven at 40°C in the dark. Final con-
centration of each sample was 0.05 μmol/g. An aliquot was dissolved
in ethanol, and the conjugated diene absorbance was measured at
katesan, R. S.; Jalaludeen, A. M. Antihyperglycemic effect of
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metabolism in experimental diabetic rats. Biomed. Prevent.
Nut. 2014, 4, 595–602.
2
2
34 nm with an UVmini-2400 UV-vis spectrophotometer every 24 h at [13] Karthik, G.; Angappan, M.; VijayaKumar, A.; Natarajapillai, S.
0°C. All tests were run in triplicate.
Syringic acid exerts antiangiogenic activity by downregulation
of VEGF in zebrafish embryos. Biomed. Prevent. Nut. 2014, 4,
2
03–208.
[
14] Shi, C.; Sun, Y.; Zheng, Z.; Zhang, X.; Song, K.; Jia, Z.; Chen,
Y.; Yang, M.; Liu, X.; Dong, R.; et al. Antimicrobial activity of
syringic acid against Cronobacter sakazakii and its effect on
cell membrane. Food Chem. 2016, 197, 100–106.
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