Efficient preparation of rare Sagittatoside A from epimedin A, by recyclable aqueous organic two-phase enzymatic hydrolysis

Article abstract of DOI:10.1080/14786419.2018.1519820

The rare secondary flavonol glycoside Sagittatoside A has much better in vivo bioactivities than epimedin A in Epimedii Folium. However, its current preparation methods are of low efficiency, with byproducts generated. The aim of this study was to establi

Full text of DOI:10.1080/14786419.2018.1519820

Natural Product Research
Formerly Natural Product Letters
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Efficient preparation of rare Sagittatoside A from
epimedin A, by recyclable aqueous organic two-
phase enzymatic hydrolysis
Yuping Shen, Yi Lu, Jing Gao, Yeting Zhu, Man Wang, Shunli Jing, Lili Xu, Huan
Yang & Xiaobin Jia
To cite this article: Yuping Shen, Yi Lu, Jing Gao, Yeting Zhu, Man Wang, Shunli Jing, Lili Xu,
Huan Yang & Xiaobin Jia (2018): Efficient preparation of rare Sagittatoside A from epimedin A,
by recyclable aqueous organic two-phase enzymatic hydrolysis, Natural Product Research, DOI:
10.1080/14786419.2018.1519820
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1519820
Efficient preparation of rare Sagittatoside A from
epimedin A, by recyclable aqueous organic two-phase
enzymatic hydrolysis
Yuping Shena, Yi Lua, Jing Gaoa, Yeting Zhua, Man Wanga, Shunli Jinga, Lili Xua,
Huan Yanga
and Xiaobin Jiaa,b
b
aSchool of Pharmacy, Jiangsu University, Zhenjiang, China; School of Traditional Chinese Medicine,
China Pharmaceutical University, Nanjing, China
ABSTRACT
ARTICLE HISTORY
Received 17 July 2018
Accepted 2 September 2018
The rare secondary flavonol glycoside Sagittatoside A has much bet-
ter in vivo bioactivities than epimedin A in Epimedii Folium.
However, its current preparation methods are of low efficiency, with
byproducts generated. The aim of this study was to establish a
novel catalysis system for effective and convenient preparation of
Sagittatoside A from epimedin A based on recyclable and integrated
aqueous organic two-phase enzymatic hydrolysis. The system was
consisted of propyl acetate and HAc-NaAc buffer (pH4.5) containing
b-dextranase/epimedin A, and the hydrolysis was performed at
60 ꢀC for 1 h. Consequently, epimedin A was completely hydrolyzed
to sagittatoside A, and 95.02% of the product was transferred into
the organic phase. Moreover, 90% of its initial activity was retained
after seven cycles of hydrolysis. Additionally, the procedure was
simpler than conventional enzymatic hydrolysis. Collectively, the
newly proposed strategy is an efficient and promising approach
for the preparation of sagittatoside A in industrial application.
KEYWORDS
Epimedin A; Sagittatoside A;
two-phase enzymatic
hydrolysis; reusability;
Epimedii Folium
1. Introduction
Epimedii Folium is the dried leaves of Epimedium brevicomu, Epimedium sagittatum,
Epimedium pubescens and Epimedium koreanum. The herb is one of the most
CONTACT Huan Yang
yanghuan1980@ujs.edu.cn; Xiaobin Jia
jxiaobin2005@hotmail.com
School of
Pharmacy, Jiangsu University, Zhenjiang, Jiangsu Province 212013, P.R. China
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1519820.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
Y. SHEN ET AL.
recognized herbal medicines for the treatment of cardiovascular diseases, bone loss
and impotence. Previously, it has demonstrated lots of bioactivities, such as anti-osteo-
porosis (Xue et al. 2016), anti-Alzheimer’s disease (Lan et al. 2017), anti-inflammatory,
promotion of sexual function (Chen et al. 2015) and anti-aging (Ma et al. 2011). The
major bioactive constituents of Epimedii Folium are 8-prenyl-flavonoids, such as epi-
medin A, epimedin B and epimedin C (Qian et al. 2012; Sun et al. 2014; Hou et al.
2016). The contents of these compounds vary from species to species as well as from
one part to another part of a particular species (Zhang et al. 2008). It is reported that
their contents (mg/g) were 7.8, 3.7 and 4.8 respectively in Epimedium pubescens (Liu
et al. 2006). Epimedin A (Figure S1) can promote the proliferation of osteoblast-like
cells and largely represented the corresponding clinical efficacy of this herb (Zhang
et al. 2008). However, only a very small amount of epimedin A could pass through
small intestine due to its great hydrophilicity and they were hardly absorbed into
blood (Zhou et al. 2015), which has restricted its potential medicinal applications.
Sagittatoside A is of very low content (ꢁ1.41 mg/g) in Epimedium (Chen et al. 2008),
and it is also a secondary flavonol glycoside by specific hydrolysis of -OR2 on epimedin
A (Zhou et al. 2015), and could be readily transported into plasma and play an import-
ant bioactive role in vivo (Xin et al. 2015).
In recent years, many efforts have been made to obtain sagittatoside A; however, the
traditional chemical hydrolysis generates many unwanted byproducts due to the uncon-
trollable catalysis. In addition, biotransformation was also not preferred because of its
time-consuming and inconvenient procedures (Xin et al. 2015). Alternatively, enzymatic
hydrolysis proved to be effective and predominant to produce bioactive ingredients
(Benhabiles et al. 2012; Yang et al. 2013; Dziril et al. 2015; Abdi et al. 2018).
In this study, we aimed to establish a novel recyclable and integrated catalysis sys-
tem for efficient preparation of sagittatoside A. The enzyme of the best performance
was selected among five commercial enzymes, and enzymatic hydrolysis was per-
formed in an aqueous organic two-phase system. Then, the key technique was devel-
oped by comprehensive optimization of conditions to achieve higher efficiency and
more convenience. Furthermore, the two-phase system was recycled and continuously
used to investigate its reusability.
2. Results and discussion
2.1. High performance liquid chromatography - ultraviolet (HPLC-UV)
chromatograms and standard curves
HPLC chromatograms of epimedin A, sagittatoside A and sample solution are shown
in Figure S3. Standard curves of two analytes were Y ¼ 6940.4782X ꢂ 11.4558
(r2 ¼ 0.9997) and Y ¼ 11555.8117X ꢂ 10.5461 (r2 ¼ 0.9998), indicating good linear rela-
tionships in concentration ranging from 1.563 to 200.0 lg/mL (Figure S4).
2.2. Screening of enzymes for integrated two-phase hydrolysis
As shown in Figure 1A, 1B, epimedin A has been completely hydrolyzed by b-glucosi-
dase, b-dextranase or cellulase (400 mg/mL), but cellulase showed a weaker ability than

NATURAL PRODUCT RESEARCH
3
Figure 1. Effects of the (A) enzyme, 400 mg/mL, B: no enzyme, E1: b-glucosidase, E2: b-dextranase,
E3: cellulase, E4: naringinase, E5: glucoamylase, 1: epimedin A, 2: sagittatoside A; (B) enzyme con-
centration; (C) enzyme/epimedin A ratio; (D) hydrolysis duration; (E) reaction temperature; (F) pH of
buffer, on the conversion rate of epimedin A.
b-glucosidase and b-dextranase as only 69.4% of epimedin A was converted in 25.0 mg/
mL enzyme solution. In addition, more than 90% of substrate was converted by 12.5mg/
mL b-dextranase, which is higher than that of b-glucosidase (81.0%). As reported, b-glu-
cosidases, b-dextranase or cellulase can catalyze the hydrolysis of glycosidic linkage (Oh
et al. 2018), but they have exhibited difference in hydrolytic efficiency of epimedin A,
which could be caused by various affinity with this specific substrate. Consequently,
b-dextranase was selected due to the best catalysis performance.
2.3. Optimization of integrated enzymatic hydrolysis conditions
2.3.1. Enzyme/epimedin A ratio
As shown in Figure 1C, conversion rate of epimedin A increased from ca. 61% to
100% when the enzyme/epimedin A ratio raised from 1:8 to 1:2 (mg/mg, w/w) or above.
Therefore, the ratio of 1:2 was chosen for the following enzymatic hydrolysis.

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Y. SHEN ET AL.
2.3.2. Hydrolysis duration
It can be seen from Figure 1D that the conversion rate of epimedin A was dramatically
increased to 73.5% within the initial 20 mins, followed by a moderate increase to
100% in the next 20 mins. Therefore, 40 mins was chosen to ensure the complete
hydrolysis. According to the enzyme kinetics, the fastest speed occurred in the initial
period of hydrolysis; then, the rate decreased gradually because of the reduced
amount of substrate and/or impaired enzyme activity. In addition, feedback inhibition
of the product stimulates the reverse reaction and further reduces the speed of
hydrolysis. The concentration of glucose in buffer continuously increased with the pro-
ceeding of the hydrolysis, which in turn led to an inhibitory effect on the catalysis.
2.3.3. Reaction temperature
As shown in Figure 1E, the most suitable temperature of enzymatic hydrolysis was
60 ꢀC at which epimedin A has been all converted within 40 mins. However, the con-
version rate rapidly decreased when the temperature was <50 ꢀC or >70 ꢀC. To
achieve complete hydrolysis, 60 ꢀC was selected for subsequent experiments.
2.3.4. pH of buffer
It can be seen from Figure 1F that more than 80% of epimedin A was converted to
sagittatoside A in the pH range of 4.0 ꢃ 6.0; however, the conversion rate dramatically
declined to less than 5% at pH 7.0 or above. Considering the greater robustness of
performance in more acidic condition, pH 4.5 was chosen for enzymatic hydrolysis
with the highest conversion rate achieved at 100%.
2.3.5. Metal ions
As shown in Table 1, robust inhibition was observed in the presence of Agþ. The other
cations including Fe2þ, Fe3þ, Cu2þ, Co2þ, Zn2þ, Ba2þ, Mn2þ and Al3þ exerted no
advance effects on enzyme activity. And, Ca2þ, Kþ and Mg2þ didn’t show capability to
enhance activity significantly. Thus, no metal icons were added intentionally into
enzymatic solutions in further experiments.
Table 1. Effects of metal ions on the enzymatic hydrolysis of epimedin A (n ¼ 3).
Metal ionsa
Relative conversion rate (%)b
Metal ionsa
Relative conversion rate (%)b
Control
Ca2þ
Kþ
100
100.6 2.3
101.7
62.7 0.5
102.9 2.4
99.3 0.6
93.7 0.9
Cu2þ
Co2þ
Zn2þ
Ba2þ
Mn2þ
Al3þ
95.4 0.3
99.8 0.4
94.0 0.5
96.1 0.1
96.9 1.1
95.5 0.4
1
Agþ
Mg2þ
Fe2þ
Fe3þ
aThe concentration of metal ions used in hydrolysis was 1 mM.
bThe conversion rate without metal ions was considered as 100%.

NATURAL PRODUCT RESEARCH
5
2.4. Integrated enzymatic catalysis
2.4.1. Construction of the integrated system
In initial stage (Figure 2A), the conversion rate increased gradually and then main-
tained at 100% after 1 hr. Epimedin A could be converted completely if propyl acetate,
vinyl acetate, ethyl propionate or isopropyl ether was employed for hydrolysis at 60 ꢀC
for 40 mins. Meanwhile, the conversion rate was at 96.1% by ethyl acetate, suggesting
that the solvent dissolved in aqueous phase could exert a negative impact on the
activity of b-dextranase.
The transfer rate of sagittatoside A kept increasing within the first one hour and
remained stable afterwards (Figure 2B). The transfer rate in isopropyl ether (<5%) was
much lower than others due to the significant difference in polarity between the solv-
ent and sagittatoside A. Furthermore, the transfer rate in ethyl acetate, propyl acetate
and ethyl propionate were all above 90% and higher than that in methyl tert-butyl
ether and vinyl acetate.
Based on the results of the above investigates, propyl acetate was consequently
selected as the most proper organic solvent for the integrated biphasic system since it
has exhibited best performance to maintain activity of b-dextranase and extraction of
sagittatoside A. After hydrolysis for 40 min, the conversion rate of epimedin A and the
transfer rate of sagittatoside A in propyl acetate were 100% and 84.5%, respectively.
As the reaction lasted for another 20 mins, the transfer rate was further increased to
91.6%. Accordingly, 1 hr was chosen for the integrated process.
2.4.2. Optimization for integrated two-phase enzymatic hydrolysis
As shown in Table 2, 87.80% of sagittatoside A was transferred to propyl acetate
when the volume ratio was 1:2 (Vo/Vb), and 95.02% of resulting sagittatoside A was
extracted by the organic phase as the ratio was increased to 1:1. There was no mark-
edly change in the transfer rate of sagittatoside A while volume ratio being increased
from 1:1 to 2:1 subsequently. To reduce the running cost and reaction volume in
application, the optimal volume ratio was set as 1:1.
Figure 2. Conversion rate of epimedin A (A) and transfer rate of sagittatoside A in organic phase
(B) in biphasic system with different organic solvents. I: methyl tert-butyl ether; II: ethyl acetate; III:
vinyl acetate; IV: ethyl propionate; V: isopropyl ether; VI: propyl acetate.

6
Y. SHEN ET AL.
Table 2. Effects of volume ratio on the transfer rate of sagittatoside A in propyl acetate.
Propyl acetate/buffer (mL/mL)
Conversion rate (%)
Transfer rate (%)
1:2
1:1
3:2
2:1
100
100
100
100
87.80
91.55
92.57
95.02
2.4.3. Reusability of b-dextranase
The conversion rate of epimedin A was kept nearly 100% even after the buffer has
been used for four cycles, and the b-dextranase showed an excellent stability against
the reaction and remained almost 90% of its initial activity after seven cycles (Figure
S5). The decrease could be caused by the deactivation on enzyme by the organic solv-
ent, inhibition effect on enzymatic hydrolysis by the increasing amount of glucose in
buffer and the enzyme’s denaturation under heating for a long time.
2.5. Identification of sagittatoside A
Electrospray ionization (positive mode) - mass spectrometry (ESIþ-MS) (Figure S6): m/z
699 [M þ Na]þ, 677 [M þ H]þ, 515 [M þ H-Glc]þ, 369 [M þ H-Glc-Rha]þ; 1H-NMR
(400 MHz, DMSO-d6) d: 12.45 (1 H, s, 5-OH), 7.88 (2 H, d, J ¼ 8.9 Hz, 2’, 6’-H), 7.14 (2 H, d,
J ¼ 9.0 Hz, 3’, 5’-H), 6.32 (1 H, s, 6-H), 5.54 (1 H, s, rha-1-H), 5.16 (1 H, t, 2’’-H), 4.25 (1 H,
d, glc-1-H), 3.85 (3 H, s, 4’-OCH3), 1.68 (3 H, s, 5’’-H), 1.62 (3 H, s, 4’’-H), 0.86 (3 H, d,
J ¼ 6.2 Hz, rha-6-H). 13C-NMR (100 MHz, DMSO-d6) d: 178.41 (C-4), 162.25 (C-7), 161.79
(C-5), 159.31 (C-4’), 156.92 (C-2), 154.23 (C-9), 135.21 (C-3), 131.45 (C-3’’), 130.83 (C-2’,
6’), 122.77 (C-1’), 122.75 (C-2’’), 114.54 (C-3’, 5’), 106.55 (C-8), 104.56 (C-10), 101.50 (rha-
C-1), 98.86 (C-6), 77.10 (rha-C-2), 76.72 (glc-C-3), 74.29 (glc-C-2), 72.16 (glc-C-4), 70.85
(rha-C-4), 70.63 (rha-C-3), 69.77 (rha-C-5), 60.98 (glc-C-6), 55.92 (C-4’-OCH3), 25.86 (C-
4’’), 21.63 (C-1’’), 18.22 (C-5’’), 17.82 (rha-C-6). These data are consistent as reported
(Xin et al. 2015).
3. Conclusions
In this study, a novel recyclable and integrated catalysis system based on two-phase
enzymatic hydrolysis was established for efficient and convenient preparation of a rare
secondary flavonol glycoside sagittatoside A from its original form epimedin A.
Compared with conventional enzymatic hydrolysis, this newly constructed system had
an apparent advantage in terms of the favorable reusability of enzymes and organic
solvent due to the convenience of their recycling. In addition, the conversion of epi-
medin A and extraction of sagittatoside A was realized in an integrated step, largely
facilitating the holistic reaction and eliminating further tedious chromatography separ-
ation. In summary, this novel system was demonstrated to have high efficiency and
convenience, suggesting the feasibility and potential to produce other bioactive com-
pounds such as secondary glycosides or aglycones from natural products.

NATURAL PRODUCT RESEARCH
7
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This study was supported by National Natural Science Foundation of China (81873196), National
Science & Technology Project for ‘New Drugs Innovation & Development’ (2017ZX09301051), Six
Talent Peaks (YY-010) and Qinglan Project (2016).
ORCID
Huan Yang
http://orcid.org/0000-0003-1808-4735
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