Acidic hydrolysis of astilbin and its application for the preparation of taxifolin from Rhizoma Smilacis Glabrae

Article abstract of DOI:10.1177/1747519820948357

The acidic hydrolysis of astilbin to produce its aglycone, taxifolin, was investigated in this study. The effects of aq. HCl concentration and temperature on the reaction were studied, and the kinetic parameters were calculated. The results showed that with higher aq. HCl concentration and temperature, the hydrolysis of astilbin became faster. The activation energy of the hydrolysis reaction under 1 mol L^?1 aq. HCl was calculated with a value of 148.6 kJ mol^?1. The reaction was successfully applied to produce taxifolin from a sample of Rhizoma Smilacis Glabrae. A simple method for the purification of taxifolin from Rhizoma Smilacis Glabrae was developed with purity of 97.5%.

Full text of DOI:10.1177/1747519820948357

9
research-arti
4cle2020 8357CHL0010.1177/1747519820948357Journal of Chemical ResearchQiu and Zhang
Research Paper
Journal of Chemical Research
–5
1
Acidic hydrolysis of astilbin and its application
for the preparation of taxifolin from
Rhizoma Smilacis Glabrae
© The Author(s) 2020
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1
1,2
Xiao-Lin Qiu and Qing-Feng Zhang
Abstract
The acidic hydrolysis of astilbin to produce its aglycone, taxifolin, was investigated in this study. The effects of aq. HCl
concentration and temperature on the reaction were studied, and the kinetic parameters were calculated. The results
showed that with higher aq. HCl concentration and temperature, the hydrolysis of astilbin became faster. The activation
energy of the hydrolysis reaction under 1 molL− aq. HCl was calculated with a value of 148.6 kJmol . The reaction was
successfully applied to produce taxifolin from a sample of Rhizoma Smilacis Glabrae. A simple method for the purification
of taxifolin from Rhizoma Smilacis Glabrae was developed with purity of 97.5%.
1
−1
Keywords
acidic hydrolysis, astilbin, kinetics, Rhizoma Smilacis Glabrae, taxifolin
Date received: 20 May 2020; accepted: 18 July 2020
OH
OH
OH
OH
OH
O
OH
O
O
H+
O
OH
H
+
H
2
O
+
Rhamnose
O
OH
OH
O
OH
OH
OH
Astilbin
Taxifolin
8
Taxifolin, also called dihydroquercetin, is a flavanonol confectionery. Due to the safety and health promoting
1
commonly found in many plants, such as onions, milk activities of taxifolin, it is the active component of many
2
thistle, and particularly, with high content, in the genus of health products and drugs in America and Europe, such as
Larix, for example, Dahurian Larch (Larix gmelinii Rupr).3 silymarin (Legalon™), Pycnogenol , and Ascovertin (a
4
® 4
Modern pharmacological studies have shown that taxifolin complex of taxifolin and vitamin C).9
possesses many bioactivities and may have therapeutic
Dahurian Larch is usually the resource for taxifolin prep-
promise for major disease states such as cancer, cardiovas- aration at present. However, this tree grows in the most
4
5,6
cular disease, and liver disease. Theriault et al. found northerly area in the world with extremely stressful
that taxifolin could significantly suppress the syntheses of
cholesterol, triacylglycerol, and phospholipid in HepG2
cells by inhibition of the activity of HMG-CoA reductase,
1College of New Energy and Environmental Engineering, Nanchang
diacylglycerol acyltransferase, and triglyceride transfer
7
Institute of Technology, Nanchang, P.R. China
protein. Sato et al. found that taxifolin has the ability to
2
Jiangxi Key Laboratory of Natural Product and Functional Food,
College of Food Science and Engineering, Jiangxi Agricultural University,
Nanchang, P.R. China
prevent the aggregation and β-sheet formation of amyloid
β-proteins, and may be a worthy candidate for Alzheimer
disease therapeutics. More recently, the European Food
Safety Authority confirmed the safety of Dahurian Larch
Corresponding author:
Qing-Feng Zhang, Jiangxi Key Laboratory of Natural Product and
Functional Food, College of Food Science and Engineering, Jiangxi
Agricultural University, Nanchang 330045, P.R. China.
8
extracts with taxifolin content > 90%. The extract is
intended to be added as an ingredient to many foods, for
example, non-alcoholic beverages, yogurt, and chocolate Email: zhqf619@126.com
Creative Commons Non Commercial CC BY-NC: This article is distributed under the terms of the Creative Commons
Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use,
reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and
Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

2
Journal of Chemical Research 00(0)
OH
OH
OH
OH
250
OH
O
OH
O
O
H+
O
OH
H
+
H
2
O
+
Rhamnose
200
O
OH
OH
O
OH
OH
Astilbin after hydrolysis for 45 min
OH
Astilbin
Taxifolin
150
Astilbin after hydrolysis for 10 min
100
Scheme 1. The acidic hydrolysis equation of astilbin.
5
0
3
environmental conditions. Thus, the tree is slow growing.
Hence, to develop modern medicaments using taxifolin, a
search for more plant sources and the development of sim-
ple and convenient methods for taxifolin purification is
needed. Rhizoma Smilacis Glabrae (RSG) is a herbal mate-
rial commonly used in China. Phytochemical analysis
showed that astilbin is the dominant flavonoid in RSG with
Astilbin
0
0
5
10
15
20
Minute (min)
(
a)
2
000
10,11
content ranging from 0.15% to 4.7%.
Astilbin is the
rhamnoside of taxifolin. Considering that the glucosidic
bond could be hydrolyzed by acid treatment, RSG is a poten-
tial resource for taxifolin production. Acidic hydrolysis is a
common chemical method in cleaving glucosidic bond,
which has been extensively used to produce the aglycones
1
500
1000
Astilbin
Taxifolin
Area sum of Astilbin and Taxifolin
12
13,14
of natural glycosides, such as saponin and flavonoids.
However, to the best of our knowledge, the specific acidic
hydrolysis of astilbin to produce its aglycone has not been
studied yet. Hence, in this study, the kinetics of acidic
hydrolysis of astilbin was studied, and a method for the pre-
parative purification of taxifolin from RSG was developed.
5
00
0
0
20
40
60
80
100
120
Time (min)
(
b)
Results and discussion
Astilbin is the rhamnoside of taxifolin. Under acidic condi- Figure 1. (a) The chromatograms of astilbin before and
tion, it can be hydrolyzed to produce its aglycone, taxifolin after hydrolysis; (b) peak area change of astilbin and taxifolin
versus time during hydrolysis. Hydrolysis was conducted with
(
Scheme 1). Figure 1(a) shows the high-performance liquid
−1
temperature of 80 °C and aq. HCl concentration of 1 molL .
Peaks: 1—astilbin; 2—taxifolin.
chromatography (HPLC) chromatogram of astilbin after
−
1
incubation with 1 molL aq. HCl at 80 °C for different
times. As shown, after 10-min incubation, the peak area of
astilbin decreased significantly, and a new peak, which is
taxifolin, was found in the chromatogram. The retention
time of astilbin was 15.4 min, and taxifolin was 0.6 min
behind. After 45 min, the peak of astilbin disappeared, and
only taxifolin was found in the chromatogram. Figure 1(b)
shows the change of peak areas due to astilbin and taxifolin
with time during the incubation. The peak area of astilbin
quickly decreased with time, while that of taxifolin steady
increased. After 20 min, only 12.5% of the initial astilbin
where C and C are the concentrations of astilbin at 0 and t
0
t
min under the given hydrolysis conditions (in this study, the
concentrations were represented by the peak areas), respec-
−1
tively. Parameter k is the hydrolysis rate constant (s ). The
time needed for 50% astilbin hydrolysis is called the half-
life time (t ), and is calculated by the following equation
1/2
t1/2 = −ln 0.5×k−1
The major factors influencing the acidic hydrolysis of
was left. After 45 min, all astilbin had been hydrolyzed. flavonoid glycosides are acidity (reflected by aq. HCl con-
1
3,14
The total peak area of astilbin and taxifolin slightly centration) and temperature.
decreased with the time, which meant that a small part of lated k and t1/2 versus HCl concentration. All linear fitting
these flavanonols had decomposed.
coefficients for the k calculation were bigger than 0.99,
Figure 2 plotted the calcu-
As shown in Scheme 1, the hydrolysis of astilbin is a sec- which implied the accuracy of pseudo-first-order esti-
ond-order reaction. However, because the concentration of mates. As shown, the values of k rapidly increased, while
water remains almost constant in the solution, the reaction t1/2 quickly decreased, with the rise of aq. HCl concentra-
−
1
could be considered as pseudo-first-order. Thus, a first-order tion. With 0.25molL of aq. HCl, the values of k and t1/2
−
4
−1
reaction model was applied for kinetic analysis of astilbin were 1.4×10
s
and 4968s, respectively. The k value
−
3 −1
hydrolysis under various conditions. The model is expressed as increased to 2.1×10 s , while t decreased to 324s
1
/2
with 2molL− of aq. HCl. These results showed that the
1
ln(C / C ) = −k ×t
hydrolysis of the glucosidic bond in astilbin increased with
t
0

Qiu and Zhang
3
-3
3
2.5x10
6x10
1
400
3
-3
5x10
2.0x10
3
4
x10
-3
300
1.5x10
3
3
3
3
x10
a
-
3
1
.0x10
2
00
2
2
k
t1/2
2x10
-4
5
.0x10
1x10
b
c
100
0
.0
0
2.25
0
.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
HCl Concentration (mol/L)
0
0
5
10
15
20
25
Figure 2. The calculated k and t1/2 versus aq. HCl
Minutes
concentration.
Figure 3. The chromatograms of RSG extract before (a) and
after hydrolysis (b), and the purified taxifolin product (c). Peaks:
1—astilbin; 2—taxifolin.
Table 1. The calculated k, t , sand E of astilbin hydrolysis
with 1molL of aq. HCl at different temperatures.
1
/2
a
−
1
T (°C)
k×10−4 (s−1)
R2
t1/2 (s)
E (kJmol )
a
−1
hydrolysis, the peak of astilbin disappeared from the chro-
matogram (line b). However, the peak of taxifolin was
found (peak 2 in chromatogram b). The retention time dif-
ference between astilbin and taxifolin was 0.6min, which
was in accordance with Figure 1(a). Besides RSG, astilbin
was also found in many other Smilacaceae genus plants, for
6
6
7
7
8
0
5
0
5
0
0.89±0.06
1.44±0.14
2.41±0.21
7.78±0.25
17.33±0.52
0.996
0.996
0.999
0.998
0.999
7800±550 148.6±16.1
4812±519
2874±276
894±36
396±20
1
6
17
example, Smilax corbularia and Smilax china. Sakurai
et al. identified two new taxifolin-O-glucosides in Taxillus
increasing acidity. It was also found that with 0.25molL−1
of HCl, the total area of astilbin and taxifolin was equal to
the initial area of astilbin, and was almost unchanged with
incubation time (data not shown). The results implied that
the molar absorption coefficient of taxifolin was equal to
that of astilbin, and they were relatively stable under such
acidic conditions. However, their total area decreased
more quickly with 2molL of aq. HCl than that with
1
astilbin and taxifolin was faster. The decomposition of fla-
vonoids under strong acidic condition was found in many
18
kaempferi (DC.) Danser. As revealed in this study, the
glucosidic bond of taxifolin glucosides could be success-
fully hydrolyzed by acid treatment. Hence, these plants
may also be the potential resource for taxifolin production.
A simple method for the preparative separation of taxi-
folin from RSG was developed. The total flavonoids in RSG
was first purified by a macroporous resin adsorption-des-
−
1
orption process. Then, the flavonoids were hydrolyzed with
−
1
molL of aq. HCl, which meant the decomposition of
−1
1
molL aq. HCl at 80°C for 1h followed by ethyl acetate
extraction and recrystallization from water to give taxifolin
with purity of 97.5%. Using 1000g of a sample of RSG,
1
3,14
other studies.
3
.96±0.21g of taxifolin was obtained. The taxifolin was
The hydrolysis rate constants of astilbin with 1molL−1
of HCl at different temperatures are listed in Table 1. As
shown, the k values quickly increased with the rise of tem-
perature (T). According to the Arrhenius equation, the acti-
further characterized by MS analysis (Figure 4). The
molecular weight ([M–H] , 303.0602m/z) and fragmenta-
−
tion pattern (285.0463, 177.0246, 125.0285m/z) were the
19
same as those of taxifolin in the literature, which con-
vation energy (E ) of the reaction can be calculated from
the intercept by plotting lnk versus 1/T to give a straight
line. In the present, the linear coefficient (R ) of lnk versus
a
firmed its chemical identity.
2
1
1
/T was 0.954. The E of the hydrolysis of astilbin using Conclusion
a
−1 −1
molL of aq. HCl was 148.6kJmol . The relatively high
Astilbin is successfully converted into taxifolin through
acidic hydrolysis. The reaction is acidity- and temperature-
dependent. By the reaction, RSG is a potential resource for
taxifolin production. A simple method for the purification
of taxifolin from RSG through acidic hydrolysis of astilbin
was developed with purity of 97.5%.
activation energy implied that the hydrolysis could hardly
occur at room temperature. Even at 60°C, the k value was
as small as 0.89×10 s and t1/2 was as long as 7800s.
However, acidic hydrolysis at high temperatures (e.g.
−
4 −1
1
4
>
90°C) may also cause the degradation of flavonoids.
Hence, based on the optimization study, hydrolysis condi-
tions with temperature of 80°C, aq. HCl concentration of
−
1
1
molL , and duration of 1h were chosen for RSG extract Experimental
hydrolysis in this study.
Chemicals and materials
In Figure 3, line a is the chromatogram of RSG extract
before hydrolysis. The biggest peak in the chromatogram is The sample of RSG was gift from Guandong Shixin
1
5
astilbin, which is the dominant flavanonol in RSG. After Pharmaceutical Co., Ltd (Jieyang, China) and was

4
Journal of Chemical Research 00(0)
authenticated by the HPLC fingerprint we developed pre- Preparation of taxifolin from RSG through
1
5
viously. The specimens are deposited in Jiangxi Key hydrolysis
Laboratory of Natural Product and Functional Food.
RSG sample was homogenized and filtered through 40
mesh sieve. RSG sample (1000g) was extracted twice by
Astilbin (>98%) was purified from RSG in our labora-
tory and was identified by UV, IR, MS, and nuclear mag-
netic resonance (NMR). HPLC-grade acetonitrile was
purchased from Anhui Tedia High Purity Solvents Co.,
Ltd (Anqing, China). All other reagents used were ana-
lytical grade.
1
3
0L of 50% ethanol at room temperature with stirring for
0min. After filtering, the extract was concentrated to
about 10L. Then, the extract was pumped through a H103
resin fixed bed. After adsorption, the resin was desorbed
with 3L of 80% ethanol. The eluent was concentrated to
about 400mL, and concentrated aq. HCl was added with
−1
HPLC analysis
final concentration of 1molL . The mixture was then incu-
bated at 80°C for 1h for hydrolysis. After cooling to room
temperature, the mixture was extracted three times using
equal volumes of ethyl acetate. The ethyl acetate fraction
was concentrated to dryness by vacuum rotary evaporation
at 60°C. Then, the residue was dissolved by about 200mL
of boiling water. The solution was stored under 4°C for
48h. Taxifolin crystals were dried at 60°C after filtration. A
schematic diagram of the preparation procedures was pre-
sented in Figure 5. The purity of taxifolin in the product
was determined by HPLC with the calibration curve of
Y=28.94X, where Y was peak area and X was the concen-
tration of taxifolin. MS analysis was performed on Q-TOF
5600-plus mass Spectrometer (AB Sciex Corporation,
Foster City, USA). The mass spectrometer was operated in
the negative ion mode. Ultrapure nitrogen was used as the
A Symmetry C18 column (250mm×4.6mm i.d., 5μm;
Waters, USA) and an Agilent 1260 HPLC system equipped
with an autosampler and diode array detector (DAD) detector
were used. The analysis conditions were in according to our
15
previous work. Acetonitrile (A) and 0.1% acetic acid aque-
ous solution (B) were used as the mobile phase with flow rate
−1
of 1mLmin and linear gradient program of 0–15min, 16%–
20% A; 15–40min, 20%–40% A. The injection volume was
10μL, and the detected wavelength was 291nm. The column
temperature was set at 40°C.
Astilbin hydrolysis study
A 0.5mL aliquot of astilbin (2mgmL−1 in 50% methanol)
was mixed with 9.5mL of aq. HCl solution with different
2
2
−
1
ion source gas 1 (50lb/in ), ion source gas 2 (50lb/in ), and
concentrations (0.25, 0.5, 1, and 2molL ). The mixture
was incubated in a water bath at different temperatures (60,
2
curtain gas (40lb/in ). The Turbo Ion Spray voltage and
temperature were set at −4500V and 500°C, respectively.
Declustering potential, collision energy, and collision
energy spread were set at 100, −40, and 10V, respectively.
Data acquisition was performed with Analyst 1.6 software
6
5, 70, 75, and 80°C). The mixture was sampled for HPLC
analysis at different time intervals.
(AB Sciex).
5
2
.5x10
303
5
2.0x10
Statistical analysis
285
Data were expressed as the mean ± standard deviation (SD)
of triplicates. Origin 8.0 software (Origin Lab Co.,
Northampton, USA) was used for data analysis and plotting.
5
5
4
1
.5x10
1.0x10
125
177
Declaration of conflicting interests
5.0x10
The author(s) declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
0.0
100
200
300
400
m/z
Funding
The author(s) disclosed receipt of the following financial support
for the research, authorship, and/or publication of this article: This
Figure 4. The MS spectra of purified taxifolin product.
Figure 5. Schematic diagram for preparation of taxifolin from RSG through acidic hydrolysis.

Qiu and Zhang
5
work was supported by the National Natural Science Foundation
of China (grant number 31760461).
8. EFSAPanelonDieteticProducts,NutritionandAllergies(NDA),
Turck D, Bresson JL, et al. EFSA J 2017; 15(2): e04682.
9
. Plotnikov MB, Plotnikov DM, Alifirova VM, et al. Zh Nevrol
Psikhiatr 2004; 104: 33.
ORCID iD
1
0. Chen L, Yin Y, Yi HW, et al. J Pharmaceut Biomed 2007;
3: 1715.
Qing-Feng Zhang
https://orcid.org/0000-0002-1277-042X
4
1
1
1. Zhang QF, Li SC, Lai WP, et al. Food Chem 2009; 113: 684.
2. Tava A, Biazzi E, Mella M, et al. Phytochemistry 2017;
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2