Preparation and properties of rutin-hydrolyzing enzyme from tartary buckwheat seeds

Article abstract of DOI:10.1016/j.foodchem.2011.10.032

A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70 kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37 °C for 30 min in the presence of 20% ethanol, and its K_m value for rutin is 1.04 × 10 ^-3 M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by ¹H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.

Full text of DOI:10.1016/j.foodchem.2011.10.032

Food Chemistry 132 (2012) 60–66
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Preparation and properties of rutin-hydrolyzing enzyme from tartary
buckwheat seeds
⇑
Xiao-dong Cui, Zhuan-hua Wang
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 August 2010
Received in revised form 28 September
A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammo-
nium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an
apparent molecular weight of about 70 kDa determined by SDS–PAGE, with an isoelectric point (pI)
2
011
(determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incu-
Accepted 11 October 2011
Available online 18 October 2011
bated together with rutin at 37 °C for 30 min in the presence of 20% ethanol, and its K value for rutin is
m
ꢁ3
1
.04 ꢀ 10 M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and
1
this is confirmed by H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product
is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydro-
lyzing enzyme, providing a new enzymatic preparation method for quercetin.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Fagopyrum tataricum Moench
Rutin
Quercetin
Rutin-hydrolyzing enzyme
1
. Introduction
frangibility and permeability of blood capillaries caused by various
diseases, for example, vascular-based pathological hemophilia, le-
Flavonoids are a group of polyphenolic compounds, containing
a basic skeleton of diphenylpropane (C –C –C ). More than 4000
sion in the retina in diabetes. Quercetin has a higher antioxidant
activity than rutin, exhibiting antiallergenic activities, anti-carci-
noma activity, blood glucose reducing activity, and antibacterial
activity (Gaberscik et al., 2002). High concentrations of quercetin
are found in onions (300 mg/kg), kale (450 mg/kg), broccoli
(100 mg/kg), beans (50 mg/kg), apples (50 mg/kg), blackcurrants
(40 mg/kg), and tea (30 mg/kg) (Hollman & Arts, 2000). In 1999,
the International Agency for Research on Cancer (IARC) concluded
that quercetin is a potential anti-cancer agent for humans. In the
United States and Europe, supplements of quercetin are commer-
cially available (Okamota, 2005).
6
3
6
different flavonoids exist and are classified into the following sub-
classes: flavones, flavonols, flavan-3-ols, isoflavones, flavanones,
anthocyanins and chalcones (Murakami, Ashida, & Terao, 2008).
Flavonoids, widely found in fruits and vegetables, have attracted
0
0
much attention as potential anti-carcinogens. Quercetin (3,3 ,4 ,
,7-pentahydroxylflavone) is a typical flavonoid present in the
5
plant kingdom as a secondary metabolite. Compared to other flavo-
noids, quercetin has been demonstrated to be very effective in
anti-tumour, antioxidant and anti-inflammatory activities in vitro
systems (Hidalgo, Sánchez-Moreno,
&
Pascual-Teresa, 2010;
Buckwheat, belonging to Polygnaceae Fagopyrum Mill, is a very
important cereal grain that is loaded with nutritional material.
Buckwheat, especially tartary buckwheat, contains biologically
important rutin, quercetin and fagopyrins not found in other cereal
crops, and the content of flavonoids in tartary buckwheat flour is
10 to 100 times higher than that in common buckwheat (Fabjan,
Rode, Kosir, Wang, & Zhang, 2003). Therefore tartary buckwheat
products are regarded as a source of medicine with some unique
components, and much attention has recently been paid to querce-
tin considering its important role in treating and preventing vari-
ous diseases (Gaberscik et al., 2002). The molecular structures of
rutin and quercetin are similar except rutinoside in C-ring is re-
placed with –OH in quercetin (Fig. 1), indicating that they have
isogenous relationship. In order to convert rutin into quercetin,
in this study, we purified a specific rutin hydrolyzing enzyme
(RHE) from tartary buckwheat, analyzed its enzymatic properties,
Hodek, Trefil, & Stiborova, 2002). These activities of quercetin are
mainly due to the o-hydroxy structure in the B ring, the 2, 3 double
bond in conjugation with the 4-oxo function in the C ring and the
3
- and 5-OH groups with the 4-oxo function in the A and C rings
(Lien, Ren, Bui, & Wang, 1999).
In plants, quercetin exists as glycosylated forms such as gluco-
side, galactoside, rhamnoside, arabinoside and rutinoside (Herrera
Luque de Castro, 2004). Rutin is a flavonol glycoside synthesized
&
by higher plants to defend against ultraviolet radiation and dis-
eases (Gaberscik, Voncina, Trost, & Germ, 2002). Rutin and other
rutin derivatives are used for the treatment and curing of increased
⇑
Corresponding author. Tel.: +86 351 7019371; fax: +86 351 7011499.
E-mail address: zhwang@sxu.edu.cn (Z.-h. Wang).
0
308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.10.032

X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
61
Fig. 1. Molecular structure of rutin and quercetin. A is rutin and B is quercetin.
and eventually developed an affordable technique for preparation
of quercetin on a large scale.
10,000g for 1 min. Electrophoresis was conducted under a constant
current of 20 mA for 3 h. The gels were stained with Coomassie
Brilliant Blue R-250 (Pharmacia, Uppsala, Sweden). The molecular
weight of individual protein bands was determined using a Gene-
Genius densitometric image analysis system (Syngene, Cambridge,
UK) with protein standards. In addition, the isoelectric points (pI)
of the proteins were analyzed by isoelectric focusing under reduc-
2
. Materials and methods
2.1. Materials
ing conditions. RHE (0.5 mg) was solubilized in 500
ll of a rehydra-
Tartary buckwheat seeds were harvested from ShouYang
tion buffer containing urea (7 M), thiourea (2 M), CHAPS (2%, w/v),
IPG buffer pH 3–10 (2%, w/v), and a trace of Bromophenol Blue dye.
The mixture was kept at room temperature for 1 h, and then cen-
trifuged at 12,000g for 5 min. The aliquots of the sample solution
were loaded onto IPG strips (pH 3–10, 13 cm; GE Healthcare, Upp-
sala, Sweden). The strips were allowed to rehydrate overnight in a
reswelling tray. The IEF was carried out in a Multiphor II unit (GE
Healthcare, Piscataway, NJ). Focusing was performed at 20 °C un-
der the following conditions: 100 V (gradient over 1 min); 100 V
County, China, in October 2009. Resource™ Q (6.4 ꢀ 30 mm) anion
exchange column and Superdex™ 75 10/300 GL gel filtration col-
umn were purchased from GE Healthcare (Uppsala, Sweden). Quer-
cetin and rutin were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). All solvents for HPLC were of chromatography grade. All
other reagents were of the highest purity available.
2.2. Extraction and purification of RHE
(
fixed for 120 min); 500 V (gradient over 1 min); 3500 V (gradient
Air-dried tartary buckwheat seeds were ground into powder in a
over 90 min); 3500 V (fixed for 6 h). After IEF, the IPG strips were
stained with Coomassie Brilliant Blue R-250. Completely bleaching
IPG strips, the position of band was measured with a ruler. The pI
was determined according to the relative position of protein bands
in the gel.
mini grinder (Huaxin, China), and the powder could pass through 40
mesh sieve. All operations were carried out at 4 °C. Fifty grams of the
powder were mixed in 500 ml of 20 mM acetate buffer (ACE buffer,
pH 5.0) for 24 h at 4 °C. After the insoluble material was removed by
4 2 4
centrifugation at 8000g for 20 min, solid (NH ) SO was slowly
added to the supernatant to 80% saturation and continuously stirred
for 4 h. The precipitate was collected by centrifugationat 12,000g for
2.4. Catalytic activity of RHE
3
0 min at 4 °C, and was then redissolved in 20 mM Tris–HCl buffer
Rutin, at a final concentration of 1 mg/ml, was dissolved in eth-
(
pH 7.0). Then the crude proteins were dialyzed in deionized water
anol and 100
water and 50
l
l
l of which was then mixed with 350
ll of deionized
at 4 °C for 4 h. The dialyzate was finally centrifuged and lyophilized
to obtain crude proteins for subsequent purification.
l of purified RHE (50 g/ml). The mixture was incu-
l
bated for 15 min at 37 °C, and the enzyme reaction was terminated
by addition of 500 l of methanol. After centrifugation, the mixture
was subjected to HPLC using a C18 column (250 ꢀ 4.6 mm, i.d.
m, Waters, Milford, MA, USA). Specific activity of RHE was cal-
culated from the amount of quercetin released from the rutin as a
substrate. In order to simply monitor the activity of RHE, enzyme
reactions were kept at room temperature for 30 min until yellow
precipitation completely took place. After 12,000g centrifugation,
supernatant was removed and precipitation was dissolved in etha-
nol. The supernatant and precipitation were identified with HPLC.
Crude proteins (10 mg) obtained above was dissolved in 10 ml
of 20 mM Tris–HCl buffer (pH 7.0) and centrifuged at 10,000g for
l
2
0 min. The supernatant (1 ml) obtained was loaded onto a Re-
source™ Q column equilibrated with 20 mM Tris–HCl buffer (pH
.0) and then eluted with the same buffer containing 1 M NaCl. Un-
5
l
7
bound protein and eluted proteins were collected in Eppendorf
tubes. The proteins in different tubes were measured for RHE activ-
ity according to the method described in the section below. The
fraction containing RHE activity was further separated on a Super-
dex™ 75 10/300 GL gel filtration column (GE Healthcare, Uppsala,
Sweden). The purified RHE was lyophilized for subsequent re-
search. Protein concentration was determined according to Brad-
ford (1976) with bovine serum albumin as a standard.
2.5. Product analysis by HPLC
HPLC analysis was carried out at room temperature on a Waters
2
.3. SDS–PAGE and IEF analysis
1525 system using a Waters 2487 photodiode array detector set at
60 nm. The products which were prepared as described above
were dissolved in methanol. After centrifugation, 20 l of the
supernatant were injected onto a 250 ꢀ 4.6 mm Hypersil ODS-2
column, 5 m particle size (Thermo Fisher Scientific, Walthan,
3
SDS–PAGE was performed to estimate the molecular weight of
l
RHE using the method of Laemmli (1970). Samples were dissolved
in 6ꢀ loading buffer, heated at 100 °C for 3 min, and centrifuged at
l

6
2
X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
MA, USA). Mobile phase was methanol–0.4% phosphoric acid
50:50, v/v), flow rate 1 ml/min. At the same time, standards rutin
(without NaCl) when pH is 5.0. Based on this property, we specu-
late that RHE belongs to albumins.
(
and quercetin were used as controls. Prepared products were iden-
tified on the basis of their retention times in HPLC by direct chro-
matographic comparison with quercetin. Purity of the product was
calculated according to the peak areas.
After centrifugation, the supernatant was subjected to HPLC.
Mobile phase was methanol–0.4% phosphoric acid (50:50, v/v),
The crude proteins, following dialysis in 20 mM Tris–HCl buffer
(pH 7.0), were loaded onto a Resource™ Q anion exchange column
and eluted as described in the Section 2. As shown in Fig. 2A, peak
II, corresponding to RHE, was eluted between 34.0 and 43.0 ml by
1 M NaCl. Peak II has two major protein bands on SDS–PAGE with
approximate molecular weights of 70 and 20 kDa (data not shown).
To obtain pure protein and analyze its properties and function, peak
II, collected from the anion exchange columns was further separated
by size exclusion chromatography (SEC) on a Superdex™ 75 10/300
GL column with 20 mM Tris–HCl buffer at pH 7.0 (Fig. 2B). The pro-
tein eluted in the first peak (elution volume 11.0 ml) displayed cat-
alytic activity to hydrolyze rutin, and SDS–PAGE revealed a highly
purified single protein band (Fig. 3, lane 3) with an approximate
molecular weight of 70 kDa. RHE was successfullypurified 5.25 folds
from the powder prepared from dried tartary buckwheat seeds, with
a total recovery of 60% (Table 1). Isoelectric focusing was used to
determine the pI of RHE. According to relative mobility of RHE in
IPG strip, the pI of RHE was calculated to be 6.7. This is different than
glycosidases from common buckwheat seeds (Bourbouze, Pratviel-
Sosa, & Percheron, 1974), which indicates that these enzymes are
not identical to RHE described here.
flow rate 1 ml/min, injection volume 20 ll. At the same time, rutin
and quercetin standards were used as positive controls. Products
were identified on the basis of their retention times in HPLC by di-
rect chromatographic comparison with standards. Purity of the
product was estimated according to the peak areas.
2.6. Nuclear Magnetic Resonance spectroscopy (NMR)
Prepared product compounds were analyzed by 1H NMR on a
DRX–300 instrument (Bruker, Fallanden, Switzerland). Sample
10.0 mg) and quercetin standard (10.0 mg) were dissolved in
.5 ml deuterated methanol in dimethyl sulphoxide (DMSO) for
(
0
NMR analysis, and residual signals (2.30 ppm) used for internal
standard. Chemical shifts and coupling constants were expressed
in d (ppm) and Hz, respectively.
3.2. Activity of RHE
2.7. LC-ESI-MS/MS analyses
Rutin was used as a substrate for the activity analysis of RHE.
Since the solubility of rutin and quercetin in water were very
Liquid chromatography electrospray ionization mass spectrom-
etry (LC-ESI-MS) is often used to identify phenolic compounds
Seeram, Lee, Scheuller, & Heber, 2006). The analysis was carried
(
0
10
20
30
40
50
60
out using a Waters ACQUITY™ UPLC system. An ACQUITY UPLC™
BEH C18 column (50 ꢀ 2.1 mm, 1.7 mm, Waters Corp., Milford,
MA, USA) was employed for separation, with the column tempera-
ture maintained at 35 °C. The gradient elution for UPLC analysis
consisted of two solvent compositions: Solvent A: 1% acetonitrile
in water (pH 3.0) and solvent B: 0.1% formic acid in acetonitrile.
The constant gradient began with 20.0% eluent A. After establishing
the final conditions for the chromatographic analysis of samples,
the detector interface and mass spectrometer were systematically
1
00
8
00
A
Peak II
80
600
Peak I
6
4
0
0
4
00
ꢁ
200
0
optimized to maximize the response for [M–H] ion with detection
20
0
in the multiple reaction monitoring (MRM) mode. Throughout the
UPLC process, the flow rate was set at 0.3 ml/min and the run time
was 5 min while the volume of injection was 10
l
l.
0
10
20
30
40
50
60
A Waters TQDTM tandem quadropole mass spectrometer
E lution volum e(m l)
(
Waters Corp., Milford, MA, USA) equipped with an electrospray ion-
ization (ESI) interface was used for analytical detection. The ESI
source was set in negative ionization mode. Quantification was per-
formed using MRM of the transitions of m/z 301.187 ? 151.109 for
quercetin and the samples, with scan time of 0.10 s per transition.
The optimal MS parameters were as follows: capillary voltage
0
5
10
15
20
2
1
1
0 0
1 0 0
Peak II
B
8
6
0
0
5 0
Peak I
3
.5 kV, cone voltage 30 V, source temperature 120 °C and desolva-
0 0
tion temperature 350 °C. Nitrogen was used as the desolvation gas
with a flow rate of 60 L/h. All data collected in multi-channel analy-
sis (MCA) mode were acquired and processed using MassLynx™ V
4 0
2 0
0
5
0
0
4
.1 software with QuanLynx™ V 4.1 program (Waters Corp., Milford,
MA, USA).
0
5
10
15
20
3
. Results
E lu tio n v o lu m e(m l)
3
.1. Isolation of rutin hydrolysis enzyme (RHE)
Fig. 2. Chromatographic characterization of RHE. (A) Anion exchange chromatog-
raphy. Crude extracts were loaded onto a Resource Q anion exchange column
(6.4 ꢀ 30 mm) pre-equilibrated with 20 mM Tris–HCl buffer (pH 7.0) and were
eluted using 1 M NaCl in the same buffer. Rutin as substrate is used to monitor RHE
activity. (B) Size exclusion chromatography. Peak II recovered from the anion
exchange column, which contained RHE activity, was loaded onto a Superdex™ 75
Seed storage proteins are a set of proteins that accumulate to
high levels in seeds during the late stages of seed development.
They are usually classified into four groups: globulins, albumins,
prolamins, and glutelins. We used different solutions to extract
RHE, and found that RHE has the highest solubility in ACE buffer
10/300 GL gel filtration column equilibrated with 20 mM Tris–HCl buffer (pH 7.0).
The column was eluted with the same buffer at a constant flow rate of 0.5 ml/min.

X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
63
kDa Mr
4.0
1
2
3
In order to further determine the structure of hydrolysates, LC-
ESI-MS/MS was employed to determine the molecular structure of
the RHE-hydrolyzed products. UPLC–MS/MS chromatograms of
standard quercetin and the hydrolysis product are shown in
Fig. 5A and B. The retention times for standard quercetin and the
hydrolysis product were similar, approximately 3.70 and
9
RHE (70kDa)
67.0
43.0
3
.82 min, respectively. Fig. 5C and D are characteristic ion scan
spectra for quercetin and the hydrolysate. We found that some
characteristic groups are present in both quercetin and the hydro-
lysate, such as 78.56 m/z, 97.29 m/z, 194.45 m/z. Combining these
30.0
1
results with the H NMR and HPLC data, it can be conclude that
the hydrolysate by RHE is indeed quercetin.
2
1
0.1
4.4
4
. Discussion
The importance of endogenous enzymes on the stability of ac-
tive ingredients has rarely been investigated in the past. Endoge-
nous enzymes may influence the quality and the nutritional
value of foods. In the food industry, the appropriate processing
technology is essential for food preservation.
Fig. 3. SDS–PAGE analysis of RHE purified by gel filtration. A 12.5% solution of
polyacrylamide gel (containing 1% sodium dodecyl sulphate) was used to separate
the samples. Lane1 is molecular weight marker; lane 2 is peak I protein fraction
purified by gel filtration (target protein); lane 3 is peak II from gel filtration.
We started our investigations by using different methods to
analyze the contents of flavonoids in tartary buckwheat seeds
and found a dramatic difference in the proportion of rutin and
quercetin. Assuming that quercetin may be a hydrolyzing product
of rutin, we aimed at the isolation of specific rutin hydrolysis en-
zyme (RHE). Using a 2-step purification method described here,
we succeeded in isolating a RHE with a molecular weight of
70 kDa, which is responsible for rutin hydrolysis in tartary buck-
wheat seeds. The isoelectric points for glycosidases from common
buckwheat seeds were around 3.7 (Bourbouze et al., 1974), which
indicates that these enzymes are not identical to RHE described
here. On the other hand, isoelectric point determined here for
RHE is in the range of those reported for other plant b-glucosidases
(Schliemann, 1987).
different, we adopted a low-alcohol solution as a reaction system.
In this system, we held the ethanol concentration at 20% because
high concentrations of ethanol (>30%) would inhibit the activity
of RHE. K
m
value of RHE for rutin was calculated to be 1.04 ꢀ
M according to the peak areas obtained from HPLC.
Following the incubation of substrate rutin and purified RHE at
7 °C for 15 min, yellow precipitation was observed in the reaction
ꢁ3
1
0
3
system. The supernatant was removed by centrifugation at 12,000g
for 10 min at 4 °C. The precipitate was dissolved in methanol and
evaluated by HPLC (Fig. 4). Each sample of the supernatant and
the precipitation was injected into HPLC system. The precipitate
eluted at 21.8 min (Fig. 4D), similar to the quercetin standard
(
Fig. 4B), thereby confirming our hypothesis. On the other hand,
2Rutin glucosidase activities have been reported in plant and
microorganisms (Hay, Westlake, & Simpson, 1961; Kurusawa,
Ikeda, & Egami, 1973; Narikawa, Hirofumi, & Takaaki, 2000; Yasuda
& Nakagawa, 1994); different enzymes may be involved in the deg-
radation or modification of genuine flavonol glycosides. In 1959,
pioneer papers dealing with microbial aerobic degradation of flavo-
noids were published (Hattori & Noguchi 1959; Westlake, Talbot,
Blakley, & Simpson, 1959). However, the enzymes which could lead
to rutinose plus quercetin upon action on rutin are only Aspergillus
enzyme and Penicillium enzyme (Hay et al., 1961; Narikawa et al.,
the supernatant was eluted at 7.9 and 21.8 min, respectively, with
very low absorbance (Fig. 4C); and both retention times in the
supernatant were consistent with rutin and quercetin, as shown
in Fig. 4A and B, respectively. This phenomenon suggests that
RHE may effectively convert rutin to quercetin. The elution peak
at 5.7 min may be the absorption peak of RHE. As shown in
Fig. 4D, the RHE reaction product is of high purity. According to
the HPLC peak areas (data not shown), the purity of the product
is about 98%.
2
000). The purified Aspergillus enzyme was shown to be stable
1
3
.3. H NMR spectroscopy and LC-ESI-MS/MS analysis
with an optimum pH of 5.6 .The substrate specificity of the enzyme
was shown to be quite restricted. The K
m
value for rutin substrate
We employed 1H NMR spectroscopy to elucidate the structure
was found to be 7.3 ꢀ 10 M. During rutin hydrolysis a 1:1 ratio
was found between the two products (quercetin and rutinose). Fur-
thermore, the Penicillium enzyme, a b-rutinosidase in microorgan-
isms, was purified from Penicillium rugulosum IFO 7242 (Narikawa
et al., 2000), which can release the disaccharide rutinose from the
flavonoid glycoside rutin. This enzyme had a molecular weight of
245 kDa, a very low optimum pH of 2.2, and had 3-glucosidase
activity for catalyze rutin and isoquercitrin into quercitrin. The
two kinds of enzymes were called rutinosidase. It is noteworthy
ꢁ5
of the hydrolysis product obtained from RHE reaction. Quercetin
1
1
has a simple H NMR spectrum. H NMR showed five phenolic hy-
1
droxyl groups (d 12.5, 10.8, 9.58, 9.35, 9.29, 1H, s). H NMR analysis
of the hydrolysis product indicated chemical shifts in the region of
d 6.00 to 8.00 ppm, characteristics of flavone nucleus: 7.64 (1H, d,
H-2), 7.53 (1H, dd, J = 8.6 Hz, H-6), 6.87 (1H, d, J = 8.6 Hz, H-5), 6.40
1H, J = 2 Hz, H-8), 6.16 (1H, d, J = 2 Hz, H-6). These data coincide
with those of the standard quercetin.
(
Table 1
Purification of RHE from tartary buckwheat seeds.
Stage
Total protein (mg)
Total activity (nkat)
Specific activity (nkat/mg protein)
Recovery (%)
Purification (fold)
Crude extract
Resource Q
Superdex G75
15.19
5.03
1.83
1035
828
654
68
164
357
100
80
60
1
2.41
5.25
nkat is defined as the amount of enzyme required to raise the rate of reaction by 1 nmol/s under defined assay conditions.

6
4
X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
Fig. 4. HPLC chromatograms for quercetin and the hydrolyzate by RHE. (A) HPLC for rutin; (B) HPLC for quercetin; (C) HPLC for supernatant from reaction system after
centrifugation; (D) HPLC for the hydrolyzate by RHE.
that rutinosidase is a quite specific enzyme discriminating both the
sugar part and the flavonol aglycone part of its substrate (Sylvain,
Pierre, & Gilles, 2010).
Rutin glucosidase in plant has been purified only from buck-
wheat seeds (Yasuda & Nakagawa, 1994; Baumgertel, Grimm,
Eisenbeiß, & Kreis, 2003; Narikawa et al., 2000). By comparison,

X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
65
1
00
3.70 min
MRM of 2 channels ES-
01.187>151.109
A
3
9.11e4
Area
5
0
0
0
1.0
2.0
3.0
4.0
5.0
Retention Time (min)
1
00
3.82 min
MRM of 2 channels ES-
01.187>151.109
B
3
2.37e4
Area
5
0
0
0
1.0
2.0
3.0
4.0
5.0
Retention Time (min)
D
C
Fig. 5. LC-ESI-MS/MS chromatograms analysis of quercetin and the hydrolyzate by RHE in negative mode (301.187 > 151.109 m/z). (A) MRM chromatograms of quercetin. (B)
MRM chromatograms of hydrolyzate by RHE. (C) Characteristic ions scan spectra for quercetin. (D) Characteristic ions scan spectra for hydrolysate by RHE.
we found that these glycosides described so far including rutin-
degrading enzymes (RDE I and RDE II), flavonol-3-O-b-heterodisac-
charide glycosidase (FHG I) and RHE reported here from tartary
buckwheat share some characteristics. They have similar masses
was further degraded and yielded phloroglucinol carboxylic acid
and protocatechuic acid at various stages of a food processing pro-
cess. However glycosidases responsible for the hydrolysis of sec-
ondary plant products have been shown to possess a high degree
of specificity. Our research indicated that there are no other com-
pounds present after RHE incubated with quercetin for a long time.
This phenomenon suggests that RHE is a specific enzyme which
hydrolyzes rutin to quercetin. RHE could be used in the preparation
of quercetin from rutin, a process that was first explored by us and
reported here.
(
(
about 70 kDa), and FHG I and RHE have similar isoelectric points
about 6.5) (Baumgertel et al., 2003; Yasuda & Nakagawa, 1994),
which indicates that these enzymes are not identical to glycosi-
dases from common buckwheat seeds (Bourbouze et al., 1974).
The cleavage of rutin into quercetin and rutinose is the first step
in enzymatic rutin degradation (Surholt & Hosel, 1978). The agly-
cone released by this reaction may further be degraded by the
so-called ‘flavonol oxidases’ (Barz & Koster, 1981) yielding prod-
ucts such as 3,4-dihydroxybenzoic acid and 2,4,5-trihydroxyben-
zoic acid. This process is harmful in the foods, because quercetin
To the best of our knowledge, so for, use of rutin hydrolysis en-
zyme in plants to prepare quercetin has not been reported. Here
we explored the possibility of using rutin hydrolase in the prepara-
tion process of quercetin. Therefore, it can be concluded that this

6
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X.-d. Cui, Z.-h. Wang / Food Chemistry 132 (2012) 60–66
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Acknowledgements
The authors would like to appreciate Dr. Jiao J.A., Vice President
of Hematech. Inc., USA, for comments and revision on the manu-
script. We also would like to thank Dr. Song H. from Shanxi En-
try-exit Inspection and Quarantine Bureau for the analysis of LC-
ESI-MS/MS. This project was supported by the Natural Sciences
Foundation of China (Grant Nos. 30671084, 30870525 and
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