Food Chemistry 135 (2012) 2253–2259

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Food Chemistry
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Enzymatic bioconversion of citrus hesperidin by Aspergillus sojae naringinase:

Enhanced solubility of hesperetin-7-O-glucoside with in vitro inhibition of
human intestinal maltase, HMG-CoA reductase, and growth of Helicobacter pylori
Young-Su Lee a, Ji-Young Huh a, So-Hyun Nam a, Sung-Kwon Moon b, Soo-Bok Lee a,⇑
a
Department of Food and Nutrition, Brain Korea 21 Project, Yonsei University, Seoul 120-749, Republic of Korea
b
Department of Food Biotechnology, Chungju National University, Chungju 380-702, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Hesperetin-7-O-glucoside (Hes-7-G) was produced by the enzymatic conversion of hesperidin by Asper-
Received 20 March 2012 gillus sojae naringinase due to the removal of the terminal rhamnose. Extracts from orange juice and peel
Received in revised form 31 May 2012 containing the hesperidin were so treated by this enzyme that the hesperidin could also be converted to
Accepted 2 July 2012
Hes-7-G. The solubility of Hes-7-G in 10% ethanol was enhanced 55- and 88-fold over those of hesperidin
Available online 14 July 2012
and hesperetin, respectively, which may make Hes-7-G more bioavailable. Hes-7-G was 1.7- and 2.4-fold
better than hesperidin and hesperetin, respectively, in the inhibition of human intestinal maltase. Hes-7-
Keywords:
G was more potent by 2- and 4-fold than hesperidin in the inhibition of human HMG-CoA reductase.
Hesperidin
Hesperetin-7-O-glucoside
Additionally, Hes-7-G exhibited more effective inhibition of the growth of Helicobacter pylori than hes-
Enzymatic bioconversion peretin, while its effectiveness was similar to that of hesperidin. Therefore, the results suggest that bio-
Naringinase converted Hes-7-G is more effective and bioavailable than hesperidin, as it has enhanced inhibitory and
Solubility solubility properties.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction hesperidin helps reduce abnormal capillary leakiness or excess

Flavonoids are the most abundant polyphenolic compounds,
characterised by a C6–C3–C6 carbon skeleton, natural occurrence
in fruits, vegetables, and other plant-derived products, and their
health-promoting activities (Garg, Garg, Zaneveld, & Singla, 2001;
González-Barrio et al., 2004). The health beneficial properties of
flavonoids include biological activities such as anticarcinogenic,
anti-diabetic, anti-inflammatory, antibacterial, immune-stimulat-
ing, antiviral, bone-loss preventive and estrogenic effects, based
on the free radical scavenging and antioxidant activities of these
compounds (Chiba et al., 2003; Havsteen, 2002; Liu, 2004; Tapiero,
Tew, Nguyen Ba, & Mathé, 2002; Tripoli, La Guardia, Giammanco,
Di Majo, & Giammanco, 2007). Among flavonoids, the flavanone
hesperidin is the major flavonoid present in citrus fruits such as
sweet oranges and lemons (Brand et al., 2008; Bredsdorff et al.,
2010; González-Barrio et al., 2004). In immature oranges, the
amount of hesperidin may make up to 14% of the fresh weight of
the fruit (Barthe, Jourdan, McIntosh, & Mansell, 1988). Hesperidin
is structurally a flavanone b-glycosides at C-7 of hesperetin agly-
cone, which is 30 ,5,7-trihydroxy-40 -methoxy flavanone. Its sugar
moiety is a disaccharide rutinose composed of rhamnose and
glucose, that is, O-a-L-rhamnosyl-(1 ? 6)-glucose. Supplemental
⇑ Corresponding author. Tel.: +82 2 2123 3124; fax: +82 2 312 5229.
E-mail address: soobok@yonsei.ac.kr (S.-B. Lee).
swelling in the legs due to fluid accumulation (Garg et al., 2001).
Both hesperidin and hesperetin provide health benefits such as
antioxidant, anti-inflammatory, and antimicrobial effects and pre-
vent bone loss (Chiba et al., 2003; Garg et al., 2001).
Hesperidin can be deglycosylated by a-rhamnosidase to yield
rhamnose and hesperetin 7-O-glucoside (Hes-7-G), which is
known to remove the bitterness from citrus juices and to improve
the aroma in grape juices and wines (Kamiya, Esaki, & Tanaka,
1985; Manzanares et al., 2003). The enzymatic bioconversion of
hesperidin to Hes-7-G can change the absorption site and enhance
its bioavailability in juices and tea (Arts, Sesink, Faassen-Peters, &
Hollman, 2004; Bredsdorff et al., 2010; González-Barrio et al.,
2004; Nielsen et al., 2006). The sugar moiety is proposed to be a
major determinant of the absorption site and bioavailability of
the flavonoid. Hes-7-G and hesperetin aglycone, produced by the
hydrolysis of enzymatic treatments, are better absorbed from the
small intestine compared to hesperidin glycoside with rhamnose
(Nielsen et al., 2006). The bioavailability of the flavonoid has been
demonstrated to increase when hesperidin is enzymatically trans-
formed into Hes-7-G (Bredsdorff et al., 2010; Day et al., 2000; Hab-
auzit et al., 2009; Manach, Morand, Gil-Izquierdo, Bouteloup-
Demange, & Rémésy, 2003; Németh et al., 2003). However, it is
not clearly explained why Hes-7-G is more bioavailable than native
hesperidin. After it is absorbed, the intestinal intracellular hespere-
tin aglycone is subsequently conjugated into glucuronidated and

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.007
2254 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259
sulfated metabolites, which are detected in human blood and urine
(Brand et al., 2008; Gardana, Guarnieri, Riso, Simonetti, & Porrini,
2007).
Naringinase (EC 3.2.1.40) is commercially useful in the pharma-
ceutical and food industries, because of the hydrolysis bioconver-
sion of sugar moieties in antibiotics, flavonoids, or glycolipids
(Ribeiro & Ribeiro, 2008). Naringinase is also used in debittering
citrus juices and wine (Puri & Banerjee, 2000; Vila-Real et al.,
2007, 2010). In fact, naringinase is known to catalyse the deglyco-
sylation of many natural glycosides, including flavonoids (Ribeiro
& Ribeiro, 2008). The enzyme has both a-L-rhamnosidase and b-
D-glucosidase activities. Recently, it has been shown that the ruti-
noside, O-a-L-rhamnosyl-(1 ? 2)-glucose of naringin is hydrolysed
by the naringinase produced by an Aspergillus sojae strain isolated
from a traditional Korean fermented soybean, to preferably yield a
rhamnose and naringenin-7-O-glucoside, prunin (Chang et al.,
2011). Prunin is considered to have a potential use as a sweetening
agent in diabetes therapy without concurrent undesirable bitter-
ness, and it possesses many biological activities, such as antiviral,
anti-inflammatory, hypoglycemic, and hypocholesterolemic effects
(Choi, Yokozawa, & Oura, 1991; Jeon, Park, & Choi, 2004; Kaul, Mid-
dleton, & Ogra, 1985). The naringinase from A. sojae has generally
recognised as safe (GRAS) status (Chang et al., 2011) and thus
can be used in food applications. This particular form has high a-
L-rhamnosidase activity and relatively very low b-D-glucosidase
activity, thus acting like a-L-rhamnosidase for the hydrolysis of
rhamnose of naringin in grapefruit.
In this study, the hydrolysis of O-a-L-rhamnosyl-(1 ? 6)-glucose
of hesperidin by the naringinase from A. sojae was examined. The
bioconversion of hesperidin to the intermediate Hes-7-G was mon-
itored and the intermediate was produced by batch-type reactions.
The biological properties of the flavonoid, including solubility and
in vitro inhibition of enzymes and Helicobacter pylori growth, were
investigated and compared based on the sugar moiety.
2. Materials and methods
2.1. Materials, enzymes, and microorganisms
Hesperidin, hesperetin, naringin, naringenin, L-rhamnose,
p-nitrophenyl a-L-rhamnopyranoside (pNPaR), p-nitrophenyl b-D-
glucopyranoside (pNPbG), b-nicotinamide adenine dinucleotide
20 -phosphate tetrasodium salt (NADPH), DL-3-hydroxy-3-methyl-
glutalyl coenzyme A sodium salt (HMG-CoA), and dithiothreitol
(DTT) were purchased from Sigma–Aldrich (St. Louis, MO, USA).
The juice and peel of oranges, which were purchased at a local
supermarket, were prepared by slight modifications of the previ-
ous method (Gil-Izquierdo, Gil, & Ferreres, 2002). The juice of or-
anges was obtained by squeezing and centrifuging fruit to collect
the supernatant. The peel of the oranges was blended, extracted
with 80% ethanol in an ultrasonic bath for 2 h at room temperature
and concentrated to remove ethanol by evaporisation. The juice
supernatant and peel extract of oranges were lyophilised for fur-
ther use. Naringinase from Penicillium decumbens was also ob-
tained from Sigma–Aldrich and dialysed against 50 mM Tris–HCl
buffer (pH 7.0), followed by concentration for use. Human intesti-
nal maltase, which is an N-terminal catalytic domain of intestinal
maltase-glucoamylase, was kindly provided by Professor Doman
Kim at Chonnam National University after expression in Pichia pas-
toris and subsequent purification (Ryu, Seo, Kang, Kim, & Kim,
2011; Sim, Quezada-Calvillo, Sterchi, Nichols, & Rose, 2008). Hu-
man HMG-CoA reductase (the catalytic domain) was also obtained
from Sigma–Aldrich. H. pylori ATCC 43526 was purchased from the
Korean Culture Center of Microorganisms (KCCM). All other chem-
icals used were of analytical grade.
2.2. Preparation and assay of naringinase from A. sojae
The naringinase enzyme from the liquid culture of A. sojae was
purified according to a previous method (Chang et al., 2011). The
crude enzyme in the culture solution was precipitated with 80%
(w/v) (NH4)2SO4 (7000g for 30 min at 4 °C), dissolved in a 50 mM
sodium citrate buffer (pH 6.0), and dialysed. The protein sample
was purified by the rechromatography of a Q-Sepharose column
in a 50 mM Tris–HCl buffer (pH 7.0) at a flow rate of 1 ml/min. Ac-
tive fractions that exhibited a-rhamnosidase activity were pooled,
dialysed, and concentrated with an ultrafiltration membrane (Mil-
lipore Co., Bedford, MA, USA) for further investigation.
a-L-Ramnosidase activity of the purified naringinase was spec-
trophotometrically assayed under standard conditions at 25 °C in
a 50 mM sodium citrate buffer (pH 6.0) using pNPaR as the sub-
strate for hydrolysis (Chang et al., 2011). b-D-Glucosidase activity
of the enzyme was also spectrophotometrically determined at
400 nm using pNPbG in the same buffer at 25 °C. The concentration
of the liberated p-nitrophenol was determined at 400 nm with a
diode array spectrophotometer (DU 730; Beckman Inc., Fullerton,
CA, USA). One unit (1 IU) of the enzyme activity was defined as
the amount of enzyme that releases 1 lmol of p-nitrophenol per
min under standard conditions. The protein concentration was
determined according to the Bradford method with bovine serum
albumin (BSA) as a standard (Bradford, 1976).
2.3. Enzymatic bioconversion of hesperidin by naringinase
A substrate solution (0.1–1.0 mM) of hesperidin in a 50 mM so-
dium citrate buffer (pH 6.0) containing 10% dimethylsulfoxide
(DMSO) or 10–20% ethanol was incubated with naringinase at
37 °C for 12–24 h. After the reaction was terminated with 0.1 M
HCl solution, the reaction mixture was centrifuged at 12,000g for
15 min and the resulting supernatant was filtered through a
0.22-lm pore-size membrane (Millipore Co., Bedford, MA, USA).
The bioconversion of hesperidin by A. sojae naringinase was then
investigated by both thin-layer chromatography (TLC) and high
performance liquid chromatography (HPLC). TLC analysis of the
hesperidin hydrolysis was performed on Whatman K5F silica gel
plates (Whatman, Kent, UK) with isopropyl alcohol–ethyl ace-
tate–water (3:1:0.3, v/v/v) as the solvent system (Chang et al.,
2011). The TLC plate was developed, dried, and visualised by dip-
ping it in a solution containing 0.3% (w/v) N-(1-naphthyl)-ethy-
lenediamine and 5% (v/v) H2SO4 in methanol and heating it for
10 min at 110 °C. HPLC analysis was carried out qualitatively and
quantitatively to determine the hesperidin and the hydrolysed
products, such as Hes-7-G and hesperetin, on a Waters symmetry
C18 column (4.6  250 mm; Waters Co., Milford, MA, USA) con-
nected to a Waters model 510 system with a 996 photodiode array
detector at 254 nm (Ribeiro & Ribeiro, 2008). The linear HPLC gra-
dient was composed of two solvents, solvent A and solvent B, 10%
and 90% (v/v) acetonitrile, respectively, in water. After a 20 ll
injection of each sample, solvent B was increased from 10% to
30% in 20 min, increased to 100% in 5 min, held at 100% for
10 min, and then reduced to 10% in 10 min. The time-dependent
change in the composition of the reaction mixture that was period-
ically sampled was also analysed by HPLC. The hesperidin
(1.3 mM) was incubated with naringinase (0.07 mg/ml) in a
50 mM sodium citrate buffer (pH 6.0) containing 10% DMSO at
37 °C for 12 h.
The lyophilised extracts of the juice and peel of oranges were
treated with 10% DMSO and 10% ethanol, respectively, to solve
the hesperidin flavanone in orange. Hesperidin (0.17 mM dissolved
in DMSO for the juice extract and 0.77 mM in ethanol for the peel
extract) was incubated at 37 °C for 24 h with crude enzyme solu-
tion (1.7–2 mg/ml), which was prepared by the ammonium sulfate
 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259
precipitation, dialysis, and concentration of the culture broth for A.
sojae. After the reaction was terminated by 0.1 M HCl, the reaction
solution was filtered through a 0.22 lm membrane filter (Millipore
Co.) and analysed by HPLC.
2.4. Repetitive batch-type production of Hes-7-G
To produce Hes-7-G from hesperidin and increase the enzyme
productivity, the enzymatic reaction was performed by a repetitive
batch reaction that recycled the enzyme according to the previous
method of other reports (Chang et al., 2011; Kragl et al., 1993). For
one cycle of the batch reaction in a total volume of 6 ml, naringin-
ase (0.1 mg/ml) was incubated with 0.12 mM hesperidin in a
50 mM sodium citrate buffer containing 10% ethanol at 37 °C for
12 h. The enzyme was repeatedly used in an ultrafiltration mem-
brane (Amicon Ultra-15 device, molecular weight cut-off of
10 kDa; Millipore Co.). The solution bearing the reaction product
was separated by a centrifugation (5000g) at the end of each batch.
The batch reaction was also repeated by the addition of new sub-
strate solution. The filtrate for each cycle was monitored by HPLC
and freeze-dried. This repetitive batch process was continuously
performed for up to 17 cycles.
The major reaction products, Hes-7-G and rhamnose, were puri-
fied from the reaction mixture using a Prevail Carbohydrate ES col-
umn (300  20 mm; Alltech Associates, Inc., Deerfield, IL, USA) that
was connected to a Waters Delta Prep 4000 preparative chroma-
tography system with a Waters™ 486 absorbance detector at
254 nm (Waters Co.). A linear gradient in the HPLC was employed
using two solvents of 20% (v/v) acetonitrile in water (A) and 80% (v/
v) acetonitrile in water (B) for 60 min at a flow rate of 4 ml/min at
room temperature. The solvent B was increased from 10% to 30% in
25 min, also increased to 100% in 10 min, held at 100% for 10 min,
and then reduced to 10% in 10 min. The fraction containing Hes-7-
G was collected, evaporated to remove the acetonitrile, and freeze-
dried. The homogeneity of the purified products was identified by
the TLC and HPLC analyses described above.
2.5. Solubility
Excess hesperidin or purified reaction product, such as Hes-7-G
and hesperetin, was mixed with 200 ll of distilled deionised water
(DDW) or 10% (v/v) ethanol in water by vortexing at 25 °C, fol-
lowed by incubation for 1 h in an ultrasonic bath (5510-DTH; Bran-
son, Danbury, CT, USA) to maximise the solubility of the compound
(Chang et al., 2011; Li, Park, Park, Park, & Park, 2004). After sonica-
tion, each sample was filtered through a 0.22-lm pore-size mem-
brane (Millipore Co.) and the solution concentration was measured
by HPLC analysis.
2.6. In vitro inhibition of human intestinal maltase and HMG-CoA
reductase
The in vitro inhibition of human intestinal maltase was investi-
gated and compared by hesperidin and naringin flavanones, such
as hesperidin, Hes-7-G, hesperetin, naringin, punin, and naringe-
nin, depending on the flavanone structure and the sugar moiety.
The reaction of human intestinal maltase was assayed using malt-
ose at 0.33–3.5 mM as a substrate, and the various flavanones at 0–
5 mM as inhibitors. Reactions were carried out in 50 mM sodium
phosphate buffer (pH 7.0) at 25 °C. The released glucose in the
reaction was determined by using the modified glucose oxidase/
peroxidase method (Kim et al., 1999). The type of inhibition and
the inhibition constant (Ki) were determined using Dixon plots
by a non-linear regression program, DNRPEASY (Duggleby, 1984).
The in vitro inhibition of human HMG-CoA reductase by hesper-
idin, Hes-7-G, hesperetin, and naringin was also investigated and
2255
compared (Chang et al., 2011). The enzyme was stored at 80 °C
until analysis. HMG-CoA-dependent oxidation of NADPH was mon-
itored for 30 min at 340 nm with a DU 730 spectrophotometer
(Beckman Inc., Fullerton, CA, USA) equipped with a cell holder that
was maintained at 25 °C. The standard assay mixture (100 ll) con-
tained 200 lM HMG-CoA, 200 lM NADPH, 100 mM NaCl, 1.0 mM
EDTA, 10 mM DTT, and 100 mM sodium phosphate buffer (pH
6.8). All reaction components, including the enzyme (10 lg/ml)
without HMG-CoA, were first monitored to detect potential
HMG-CoA-independent oxidation of NADPH. The reaction was
then initiated by the addition of HMG-CoA (0.05–2 mM) to the
reaction mixture. One unit of HMG-CoA reductase is defined as
the amount of enzyme that catalyses the oxidation of 1 lmol of
NADPH per min. The HMG-CoA-dependent oxidation reactions
were performed with various concentrations (0–80 lM) of hesper-
idin, Hes-7-G, hesperetin, and naringin as the inhibitors.
2.7. In vitro growth inhibition of H. pylori
The effects of the hesperidin and naringin flavanones (0.05–
0.5 mM), such as hesperidin, Hes-7-G, hesperetin, naringin, prunin,
and naringenin, on the growth of H. pylori were examined. H. pylori
was cultured in a test tube containing 5-ml of sterilised brain–
heart-infusion broth (BHI; Difco Laboratories, Detroit, MI, USA)
supplemented with 5% horse serum (Sigma–Aldrich) with shaking
(150 rpm) at 37 °C for 3 days. The test tube was contained in a Gas-
Pak EZ Pouch System (5–15% CO2; BD Diagnostics, MD, USA) under
microaerophilic conditions. At 3 days, 50 ll of culture solution was
cultivated further on a sterilised 96 well-microplate (Nunclon™D
Surface, Thermo Fisher Scientific, Denmark) containing 180 ll
BHI broth supplemented with 5% horse serum and a 20 ll flava-
none solution (or distilled water in the control) per well. The 96
well-microplate with lid was incubated under 10% CO2 at 37 °C.
The growth of bacteria in the microplate was determined by mea-
suring an optical density (OD) at 620 nm several times from 0 to
80 h.
3. Results and discussion
3.1. Enzymatic bioconversion of hesperidin
Hesperidin is an abundant and inexpensive byproduct of citrus
cultivation, which can be obtained from waste orange peel from
the citrus industry (Garg et al., 2001). The bioavailability of hesper-
idin was shown to increase in human subjects when hesperidin
was converted to Hes-7-G (Bredsdorff et al., 2010). In this study,
the A. sojae naringinase (GRAS) was the enzyme used to treat hes-
peridin. The purified naringinase derived from A. sojae converted
the hesperidin mainly to the intermediate Hes-7-G (Fig. 1). The su-
gar moiety of hesperidin is an O-a-L-rhamnosyl-(1 ? 6)-glucose,
which is attached to the 7-OH group of hesperetin aglycone. The
purified naringinase from A. sojae can hydrolyse naringin that con-
tains an O-a-L-rhamnosyl-(1 ? 2)-glucose as the sugar moiety,
mainly removing the rhamnose and producing the intermediate
prunin (Chang et al., 2011). Irrespective of a-L-rhamnnose linkage,
A. sojae naringinase could hydrolyse the rutinoside disaccharide of
hesperidin to convert it to a glucoside, which were determined by
HPLC (Fig. 2). Thus, hesperidin was efficiently converted to the
intermediate Hes-7-G by the rhamnosidase activity of A. sojae
naringinase with scarcely any hesperetin aglycone produced. A
time-dependent bioconversion of hesperidin by A. sojae naringin-
ase was monitored in 50 mM sodium citrate buffer (pH 6.0) con-
taining 10% DMSO at 37 °C for 12 h, as shown in Fig. 3A. The
results show that the enzymatic bioconversion of hesperidin by
the purified naringinase produces gradually increasing levels of
2256 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259

Hes-7-G
A 0.4
0.35 1
rhamnose
0.3
0.25
hesperetin 0.2
hesperidin 0.15
glucose
0.1
G1 0.05
G2 0
G3 -0.05 0 10 20 30 40 50
G4 Time (min)
G5
G6
G7 B 0.2
2
0.15 1

S 1 2 3 4 5 6 0.1

Fig. 1. TLC analysis of the reaction of A. sojae purified naringinase with hesperidin 0.05
at 37 °C. Lane S, standard maltooligosaccharides (G1–G7); lane 1, a-L-rhamnose;
lane 2, hesperidin; lane 3, reaction of A. sojae naringinase with hesperidin for 12 h;
0
lane 4, reaction of P. decumbens naringinase with hesperidin for 12 h; lane 5, Hes-7-
G; lane 6, hesperetin. The reaction was carried out with naringinase at 0.07 mg/ml 0 10 20 30 40 50
and hesperidin at 1 mM in 50 mM sodium citrate buffer (pH 6.0) containing 10% -0.05
DMSO.
Time (min)

Hes-7-G, reaching a plateau (1.2 mM) within 12 h under the de-

scribed condition. A very small amount of hesperidin and hespere- C 0.3
2
tin were detectable (each 0.07 mM, not more than 5%) at the end of 0.25
the reaction. The molar production yield of Hes-7-G based on the
0.2
molarity of the hesperidin used was found to be approximately
90%. To increase the enzyme productivity, the production of Hes- 0.15
7-G was effectively performed by repetitive batch reactions with 0.1
enzyme recycling. As shown in Fig. 3B, the hesperidin (0.13 mM) 3
was dissolved and reacted in each batch (6 ml) of 50 mM sodium 0.05
citrate buffer containing 10% ethanol. This organic solvent is gener- 0
ally used in the food industry and the hesperidin was easily con-
-0.05 0 10 20 30 40 50
verted mainly to Hes-7-G. The enzyme activity was almost
constantly maintained up to 12th batch cycle, and was then largely Time (min)
decreased by 40% of the original activity at the 13th cycle, a level
that was maintained up to the 17th cycle. The total molar produc- Fig. 2. HPLC analysis on the reaction of purified A. sojae naringinase with hesperidin
tion yield of Hes-7-G based on hesperidin (mM) was, on average, at 37 °C. (A) Hesperidin (B) reaction of A. sojae naringinase with hesperidin for 6 h
86% for 12 batch cycles. The stability of the enzyme might be de- (C) reaction of A. sojae naringinase with hesperidin for 12 h. 1, hesperidin; 2, Hes-7-
G; 3, hesperetin.
creased by the organic solution in the repetitive reaction. The
Hes-7-G produced from the reaction solution (5 ml) was easily
purified from the rhamnose product and unreacted hesperidin in et al., 2004). Our results suggest that the GRAS A. sojae naringinase
a single step by Prep-LC, respectively. The purity and homogeneity can be applied to the flavanones from citrus fruit containing hes-
of the separated Hes-7-G was estimated by HPLC to be above peridin as well as naringin to convert the flavanones (flavonoid
99.9%. The rhamnose of the rutinoside was concomitantly pro- rutinosides) to the corresponding glucosides.
duced in an equimolar amount and separately obtained.
Extracts from the peel and juice of oranges containing the
hesperidin were subjected to the treatment of the crude enzyme 3.2. Properties of bioconverted products
solution from A. sojae. As shown in Fig. 4, the hesperidin detected
in the juice and peel of oranges was efficiently converted to Hes-7- 3.2.1. Solubility
G within 12 h, with negligible hesperetin also produced. The molar The solubility of hesperidin was compared with that of both
production yields of Hes-7-G, based on the molarity of hesperidin Hes-7-G and hesperetin in distilled deionised water (DDW) and
existed in the extracts, was evaluated to be approximately 71% for 10% ethanol solution at 25 °C (Table 1). The solubility of Hes-7-G
orange juice and 78% for orange peel, respectively. In fact, the en- was remarkably higher than that of hesperidin approximately as
zyme solution of A. sojae contains some independent b-glucosidase 36-fold greater in DDW and 55-fold greater in 10% ethanol. The
which can convert Hes-7-G to hesperetin. If the purified naringin- solubility of Hes-7-G was also approximately 77-fold greater than
ase was used, the production yield of Hes-7-G would have hesperetin in DDW and 88-fold greater in 10% ethanol. In fact, a
increased. The enzymatic conversion of the flavanones in orange glycosylation of aglycone is well known to increase the water
juice was reported to be performed with 50% conversion of rutino- solubility of flavonoids and other aglycones. The hydrolysis of the
side to the corresponding flavonoid glucoside (González-Barrio a-L-rhamnosyl moiety in hesperidin likely results in the
 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259 2257

A A 0.2
1.5
Concentration (mM)

0.15

Concentration (mM)
1

0.1
0.5

0.05

0 2 4 6 8 10 12 14 0
0 4 8 12 16 20 24 28
Time (h)
Time (h)
B 0.15
B 1
Concentration (mM)

0.1 0.8

Concentration (mM)
0.6
0.05
0.4

0 0.2
0 2 4 6 8 10 12 14
Batch cycle
Fig. 3. Time progress of A. sojae purified naringinase reaction with hesperidin (A)
and repetitive batch-type production of Hes-7-G (B). The reaction was performed in
50 mM sodium citrate buffer (pH 6.0) containing 10% DMSO (A) and 10% ethanol (B)
with naringinase at 0.07 mg/ml at 37 °C for 12 h. The reaction mixture was analysed
by HPLC. Hesperidin, j; Hes-7-G, d; hesperetin, D.
enhancement of solubility. It is generally accepted that the solubil-
ity of naringin is largely increased by its conversion to prunin due
to the removal of a-L-rhamnose during the hydrolysis (Chang et al.,
2011). The enhanced solubility of Hes-7-G is a desirable character-
istic for practical applications in food or pharmacological indus-
tries, which might be capable of improving its bioavailability
through greater absorption as compared to hesperidin and agly-
cone hesperetin. In addition, the absorption of Hes-7-G was sug-
gested to be actively performed in the small intestine rather than
the colon, which was understood on the absorption model through
deglycosylation of flavonoid glucoside at the epithelial cell mem-
brane of the small intestine (González-Barrio et al., 2004; Németh
et al., 2003). The bioavailability of the hesperidin in orange juice
was increased by the enzymatic removal of the a-L-rhamnosyl
moiety from the rutinosides to the glucosides (Bredsdorff et al.,
2010; González-Barrio et al., 2004). Therefore, the enzymatic bio-
conversion of hesperidin to Hes-7-G using A. sojae naringinase
may be useful for enhancing its potential bioavailability in citrus
food products, as well as the bioconversion of naringin to prunin
(Chang et al., 2011).
3.2.2. In vitro inhibitions of human intestinal maltase and HMG-CoA
reductase
Flavonoids including the flavanones were known to inhibit rat
small intestinal and yeast a-glucosidases (Tadera, Minami,
Takamatsu, & Matsuoka, 2006). The flavanones, hesperidin and
naringin, as well as their intermediate compounds and aglycones,
mildly inhibited human intestinal maltase. The flavanones tested
all exhibited competitive inhibition of human intestinal maltase
16 18
0
0 4 8 12 16 20 24 28
Time (h)
Fig. 4. Time course analyses of A. sojae crude naringinase reactions with hesperidin
in the extracts of orange juice (A) and orange peel (B). Hesperidin, j; Hes-7-G, d.
The reaction was performed in 50 mM sodium citrate buffer (pH 6.0) containing
10% DMSO (juice) or 10% ethanol (peel) with crude naringinase at 37 °C for 24 h.
The reaction mixture was analysed by HPLC.
Table 1
Comparison of solubilities of hesperidin, Hes-7-G, and hesperetin at 25 °C.
Compound Solubility in DDWa (lM) Solubility in 10% ethanol (lM)
Hesperidin 19.2 ± 0.1 37.5 ± 0.1
Hes-7-G 688.4 ± 30.8 2063.3 ± 28.4
Hesperetin 8.9 ± 0.2 23.5 ± 0.1
a
DDW indicates distilled deionised water.
with inhibition constants (Ki) of 1.0–4.4 mM (Table 2). The inhibi-
tion constants of the flavanone glucosides, Hes-7-G and prunin,
were slightly lower than those of the flavanone rutinosides and
aglycones. Therefore, the intermediate compounds, Hes-7-G and
prunin, were more potent than the other flavanones tested. These
results indicate that the flavanones were slightly different at bind-
ing the enzyme active site, depending on the type of sugar moiety
of the flavanone competing with the maltose substrate. It has also
been indicated that the glucose moiety of the flavanones may be
preferred at the binding of the enzyme and thus effective at inhib-
iting the human brush border intestinal maltase and therefore
potentially interfere with intestinal digestive functions.
In addition, the in vitro inhibition of human HMG-CoA reductase
by Hes-7-G was compared to the inhibition by hesperidin, hespere-
tin, and naringin. Ki values for these compounds were in the range
of 10–44 lM (Table 2, Supplementary material). HMG-CoA reduc-
tase is a target enzyme for cholesterol-lowering compounds,
because it is known to be involved in a rate-limiting step in
2258 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259

Table 2 would be a more effective mild anti-diabetic and cholesterol-low-

Comparison of inhibition constants (Ki) of flavanones against human intestinal ering flavanone with higher solubility than hesperidin. The greater
maltase and human HMG-CoA reductase.
amount of hesperidin in citrus food may be used by the enzymatic
Compound Human intestinal maltase Human HMG-CoA reductase conversion to Hes-7-G.
Inhibition patterna Ki (mM)b Inhibition pattern Ki (lM)
Hesperidin Competitive 3.1 ± 0.7 Non-competitive 19.0 ± 0.1 3.2.3. In vitro inhibitory effects of flavanones on the growth of H. pylori
Hes-7-G Competitive 1.8 ± 0.1 Non-competitive 9.8 ± 1.2 As shown in Fig. 5, the flavanones, hesperidin and naringin, as
Hesperetin Competitive 4.4 ± 0.6 Non-competitive 9.6 ± 0.4
well as their intermediate compounds and aglycones, showed
Naringin Competitive 2.8 ± 0.1 Non-competitive 43.7 ± 2.3
Prunin Competitive 1.0 ± 0.1 –c –
some inhibitory effects on the growth of H. pylori. The antibacterial
Naringenin Competitive 3.8 ± 0.9 – – activity of flavonoids is being increasingly investigated with
a,b
medicinal significance (Cushnie & Lamb, 2005). The flavonoids
Inhibition pattern and constants (Ki values) were determined by Dixon plots for
the activity of human intestinal maltase (HIM) or human HMG-CoA reductase (HCR)
have inhibitory effects on the growth of H. pylori, which is a path-
in the presence of flavanones (0–5 mM for HIM and 0–80 lM for HCR) as an ogenic bacterium that causes chronic gastric inflammation and
inhibitor. promotes carcinogenesis (Lee, Shin, & Hahm, 2008). The growth-
c
Not determined. inhibiting effect of Hes-7-G was significant and similar to that of
hesperidin, but better than hesperetin at 0.5 mM. Due to the lim-
ited solubility, hesperetin existed in an undissolved state, probably
resulting in relatively lower effects than Hes-7-G and hesperidin at
A 120 this concentration. In addition, the growth-inhibiting effect of
prunin was better than those of naringin and naringenin at
100 0.5 mM. Even though naringenin was restricted in solubility and
Relative growth (%)

undissolved, the growth-inhibiting effect of naringenin was

80 slightly better than that of naringin at 0.5 mM. The enhancement
of the intermediate compounds, Hes-7-G and prunin, at inhibiting
60 the growth of H. pylori might be mainly attributed to the increased
solubilities of the intermediate compounds. The more soluble form
40 of the flavanone might yield greater effects on the inhibition of
bacterial growth, even at the same concentration. The inhibition
20 mechanism of the flavanones against the growth of H. pylori is un-
clear. However, it has been suggested that the antibacterial mech-
0 anism of flavonoids, including some flavanones, is through their
0 12 24 36 48 60 72 84
inhibition of nucleic acid synthesis or cytoplasmic membrane
function (Cushnie & Lamb, 2005). For instance, quercetin exhibits
Time (h)
an inhibition of bacterial DNA gyrase (Ohemeng, Schwender, Fu,
& Barrett, 1993) and an increase in the permeability of the inner
B 120
bacterial membrane (Mirzoeva, Grishanin, & Calder, 1997), and
naringenin exhibits bacterial membrane interference and an
inhibition of bacterial motility (Mirzoeva et al., 1997; Tsuchiya &
100
Iinuma, 2000).
Relative growth (%)

In conclusion, Hes-7-G could be easily produced from hesperi-

80
din by the enzymatic reaction of A. sojae naringinase. Hesperidin
in the extracts of orange juice and peel could also be enzymatically
60
converted to the intermediate Hes-7-G compound. The solubility of
Hes-7-G was much higher than that of either hesperidin or hes-
40
peretin, which may make it more bioavailable. Hes-7-G was also
20
approximately 2-fold better than hesperidin in the inhibition of
human intestinal maltase and HMG-CoA reductase. In addition,
0 Hes-7-G showed superior inhibition of the growth of H. pylori com-
0 20 40 60 80 100 pared to hesperetin. Thus, the intermediate compound, Hes-7-G, is
suggested to be a more bioavailable and functional flavanone with
Time (h) enhanced solubility.

Fig. 5. Inhibitory effects of flavanones on the growth of H. pylori. (A) Relative

growth in the presence (0.5 mM) of hesperidin (j), Hes-7-G (d), and hesperetin (N)
(B) relative growth in the presence (0.5 mM) of naringin (j), prunin (d), and
naringenin (N). Relative growth of the control () indicates 100% at 72-h (A) and 80-
h (B) in the absence of flavanones.
cholesterol biosynthesis (Hilleman, Wurdeman, & Lenz, 2001). The
decrease in the inhibition constant of Hes-7-G in comparison with
that of hesperidin might imply that the glucose sugar moiety of the
intermediate flavanone more suitably bound to the enzyme than
the rutinose disaccharide moiety of hesperidin. The inhibitory ef-
fect of hesperidin was better than naringin, indicating that the
aglycone part might be partly preferable for the inhibition of
HMG-CoA reductase. Finally, our results suggest that Hes-7-G
Acknowledgments
This research was supported by Basic Science Research Program
through the National Research Foundation of Korea (NRF) Grant
funded by the Korean Government (MEST) (2009-0077358), and
partly by the Marine and Extreme Genome Research Center Pro-
gram, Ministry of Maritime Affairs and Fisheries, Republic of Korea.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
07.007.
 Y.-S. Lee et al. / Food Chemistry 135 (2012) 2253–2259 2259

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