Hydrolysis of flavonoid glycosides by propolis β-glycosidase

Article abstract of DOI:10.1080/14786419.2010.541877

Flavonoids generally occur as O-glycosides with sugars bound in nature, while aglycones and their derivatives are the main flavonoids in propolis. The objective of this work was to study the propolis β-glycosidase activities toward flavonoid β-glycosides and their conjugated forms. β-Glycosidase was extracted from propolis, incubated with avonoid glycosides, and analysed for aglycone formation by HPLC. The results demonstrated that glucose conjugates were rapidly hydrolysed, but not conjugates with other sugars, i.e. rutin and naringin. The rate and extent of deglycosylation depends on the structure of the avonoid and the position of the sugar substituitions. Quercetin 3-O-glucoside had the highest percent of hydrolysis, while quercetin 7-O-glucoside was the least hydrolysed. The Km values for hydrolysis of apigenin 7-glucoside and luteolin-7-O-glucoside were 13M and 20M, respectively.

Full text of DOI:10.1080/14786419.2010.541877

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Hydrolysis of flavonoid glycosides by
propolis β-glycosidase
a
a
a
Cui-Ping Zhang , Gang Liu & Fu-Liang Hu
a
College of Animal Sciences, Zhejiang University, Hangzhou
10029, China
3
Version of record first published: 19 Aug 2011.
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glycosides by propolis β-glycosidase, Natural Product Research: Formerly Natural Product Letters,
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Natural Product Research
Vol. 26, No. 3, February 2012, 270–273
SHORT COMMUNICATION
Hydrolysis of flavonoid glycosides by propolis b-glycosidase
Cui-Ping Zhang, Gang Liu and Fu-Liang Hu*
College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
(
Received 19 July 2010; final version received 11 November 2010)
Flavonoids generally occur as O-glycosides with sugars bound in nature,
while aglycones and their derivatives are the main flavonoids in propolis.
The objective of this work was to study the propolis ꢀ-glycosidase activities
toward flavonoid ꢀ-glycosides and their conjugated forms. ꢀ-Glycosidase
was extracted from propolis, incubated with Favonoid glycosides, and
analysed for aglycone formation by HPLC. The results demonstrated that
glucose conjugates were rapidly hydrolysed, but not conjugates with other
sugars, i.e. rutin and naringin. The rate and extent of deglycosylation
depends on the structure of the Favonoid and the position of the sugar
substituitions. Quercetin 3-O-glucoside had the highest percent of hydro-
lysis, while quercetin 7-O-glucoside was the least hydrolysed. The Km
values for hydrolysis of apigenin 7-glucoside and luteolin-7-O-glucoside
were 13 mM and 20 mM, respectively.
Keywords: propolis; ꢀ-glycosidase; flavonoid glucosides; hydrolysis
1
. Introduction
Propolis, a resinous hive product collected by bees, is known to have been used in
folk medicine since as early as 300 BC. It was reported that this product possesses
such versatile biological activities as antibacterial, antiviral, fungicidal, local
anaesthetic, antiulcer, immunostimulating etc. (Bankova, Popov, & Marekov,
1
983). The main propolis substance group are the flavonoid aglycones, 136
flavonoid aglycones or derivatives have been identified in propolis (Zhang & Hu,
009). However, flavonoids mainly consist of the sugar conjugates of anthocyani-
2
dines, flavones, flavonols and flavanones in nature (Nuutila, Kammiovirta, &
Oksman-Caldentey, 2002). Therefore, some people presumed that flavonoid glyco-
sides were hydrolysed by ꢀ-glycosidase during propolis collection and processing
(Banskota, Tezuka, & Kadota, 2001; Bonvehi, Coil, & Jorda, 1994; Lu, Wu, &
Yuan, 2004; P.G. Pietta, Gardana, & A.M. Pietta, 2002). It does not conform to the
fact that isorhamnetin-3-O-rutinoside was isolated from propolis (sample from
Crete; Popova, Chinou, Marekov, & Bankova, 2009), and rutin (quercetin
3-O-rhamnoglucoside) has been identiEed by HPLC in European, Asian and
South American propolis (Bonvehi & Call, 1994).
*Corresponding author. Email: flhu@zju.edu.cn
ISSN 1478–6419 print/ISSN 1478–6427 online
ß 2012 Taylor & Francis
http://dx.doi.org/10.1080/14786419.2010.541877
http://www.tandfonline.com

Natural Product Research
271
Quercetin 3-O-glucoside: R
Quercetin 7-O-glucoside: R
Apigenin 7-O-glucoside: R
Rutin: R = O-Glucose(6 1)rhamnose; R
Luteolin 7-O-glucoside: R =R =H; R = OH; R
4
1
= O-glucose; R
=OH; R =OH; R
=R =R =H; R = glucose
=OH; R =R
= glucose
2
= OH; R
3 4
= R =H
1
2
3
=H; R =glucose
4
1
2
3
4
1
←
2
3
4
=H
1
3
2
1
Naringin: R = Glucose(2←1)rhamnose
Figure 1. Structure of flavonoid glycosides.
Table 1. Initial rates and extent of hydrolysis of flavonoid glycosides with ꢀ-glycosidase from
propolis.
Initial rate of
hydrolysis
(m mol min
Hydrolysis after different time (%)
ꢀ
1
ꢀ1
g )
Substrate
90 min
120 min
180 min
Quercetin 3-O-glucoside
Quercetin 7-O-glucoside
Rutin
3.80 ꢁ 0.45
1.60 ꢁ 0.26
0
28.64 ꢁ 0.72
20.59 ꢁ 0.56
0
35.77 ꢁ 0.81
27.63 ꢁ 0.67
0
49.55 ꢁ 0.65
41.96 ꢁ 0.60
0
Naringin
Apigenin 7-O-glucoside
Luteolin 7-O-glucoside
0
0
0
0
2.10 ꢁ 0.20
22.19 ꢁ 0.46
28.65 ꢁ 0.52
43.20 ꢁ 0.83
3.24 ꢁ 0.33
25.59 ꢁ 0.35
32.72 ꢁ 0.25
46.47 ꢁ 0.42
In nature, the flavonols are found mainly as the 3-O-ꢀ-glycoside, the flavones,
flavanones and isoflavones, are found mainly glycosylated in the 7 position (Merken &
Beecher, 2000). Aryl ꢀ-glycosidase activity was detected in propolis in our previous
experiments (Zhang, Zheng, & Hu, 2011). The aim of this study is to use propolis crude
extracts to show whether there is ꢀ-glycosidase activity towards six different glyco-
sides, i.e. quercetin 3-O-glucoside, quercetin 7-O-glucoside, apigenin 7-O-glucoside,
luteolin 7-O-glucoside, rutin and naringin (naringenin 7-O-rhamnoglucoside).
Figure 1 shows the structures of flavonoid glycosides used in this article. All their
aglycones were found in propolis.
2
. Results and discussion
Hydrolysis of flavonoid glycosides by ꢀ-glycosidase extracts from propolis was
investigated. Supplementary Figure S1 (online only) shows representative chro-
matograms from 0, 90, 120 and 180 min incubations of luteolin-7-O-glucoside with
propolis ꢀ-glycosidase extract, demonstrating the conversion of luteolin-7-O-
glucoside to luteolin by hydrolysis of the ꢀ-glucoside bond. Table 1 shows the
initial rates and the rate of deglycosylation of various flavonoid glycosides.
Quercetin 3-O-glucoside, quercetin 7-O-glucoside, apigenin 7-glucoside and luteolin

2
72
C.-P. Zhang et al.
Table 2.
ꢀ
m
K and Vmax of Apigenin 7-glucoside and luteolin-7-O-glucoside by
-glycosidase from propolis.
ꢀ1
ꢀ1
g
V
max (m mol min
Substrate
K
m
(n ¼ 3)
propolis) (n ¼ 3)
Apigenin 7-glucoside
Luteolin 7-O-glucoside
12.93 ꢁ 1.73
19.49 ꢁ 2.76
1.91–3.80
3.54–5.66
Notes: One unit of activity is defined as one m mol of product formed per min at
ꢂ
37 C. K
of values measured in different propolis samples.
m
values are expressed as mean ꢁ SD, whereas Vmax is shown as the range
7
-O-glucoside are all hydrolysed by propolis ꢀ-glycosidase extract. However, with
rutin and naringin no aglycone peak was detectable under our standard HPLC
conditions. Even when incubating for 6 h, no aglycone was detected. When treated
with propolis ꢀ-glycosidase extract, quercetin 3-O-glucoside had the highest percent
of hydrolysis, quercetin 7-O-glucoside was the least hydrolysed.
Despite the fact that we cannot exclude the possibility that the enzyme
preparations may contain other glycosidases as impurities, it is obvious that the
raw enzyme extraction of propolis is inactive with rutin and naringin. These results
supported that flavonoid mono-glucosides in plant resins were hydrolysed by
propolis ꢀ-glycosidase during propolis collection and processing, and the results
agreed closely with the present results that isorhamnetin-3-O-rutinoside and rutin
were found in propolis. Whether this reaction is of physiological importance or not,
these results are of significance for the understanding of the constituents of propolis.
ꢀ
-Glycosidase activity at various concentrations of apigenin 7-glucoside and
luteolin-7-O-glucoside was measured in enzyme extract. The results suggested that
the same enzyme extract is responsible for catalysing the hydrolysis reaction, with a
Km of 13 mM for apigenin 7-O-glucoside and 20 mM for luteolin-7-O-glucoside
(Table 2). The total amount of the ꢀ-glycosidase activity varies in different propolis
samples.
3
. Conclusion
Studies of additional flavonoid glycosides demonstrated that glucose conjugates were
rapidly hydrolysed by propolis ꢀ-glycosidase, but not conjugates with other sugars,
i.e. rutin, and naringin. The rate and extent of deglycosylation depends on the
structure of the Favonoid and the position of the sugar substituitions. This supported
the contention that flavonoid glycosides were hydrolysed by ꢀ-glycosidase during
propolis collection and processing, it may also explain the presence of isorhamnetin-
3
-O-rutinoside and rutin in propolis.
Supplementary material
Experimental details relating to this paper are available online, alongside Figure S1.

Natural Product Research
273
Acknowledgements
This research was financially supported by the grant from Chinese Ministry of Agriculture
[
[
Project no. NYCYTX-43] and Zhejiang Provincial Natural Science Foundation of China
Project no. R3090332].
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