Biotransformation of major flavonoid glycosides in herb epimedii by the fungus Cunninghamella blakesleana

Article abstract of DOI:10.1016/j.molcatb.2015.05.021

Biotransformation of icariin (1), epimedin C (2), epimedoside A (3), epimedin A (4) and epimidin B (5), five major components of E. koreanum, were performed by using Cunninghamella blakesleana. And they could be metabolized efficiently to icariside II (1a), 2″-O-rhamnosylikarisoside II (2a), epimedoside b (3a), baohuoside VII (4a) and sagittatoside B (5a) with high yields of 95.1%, 97.7%, 93.7%, 95.8% and 96.4%, respectively. And these transformed products as major forms of herb epimedii in vivo exhibited the more significant anti-osteoporosis activities. Our method could be applied for enriching these rare flavonoids in herb epimedii, for further development of anti-osteoporosis medicines or functional foods.

Full text of DOI:10.1016/j.molcatb.2015.05.021

Accepted Manuscript
Title: Biotransformation of major flavonoid glycosides in herb
epimedii by the fungus Cunninghamella blakesleana
Author: XiuLan Xin Guang-Jun Fan Zheng Sun Ning Zhang
Ye Li Rong Lan Liang Chen PeiPei Dong
PII:
S1381-1177(15)00159-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.05.021
MOLCAB 3204
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
21-1-2015
18-5-2015
19-5-2015
Please cite this article as: X.L. Xin, G.-J. Fan, Z. Sun, N. Zhang, Y. Li, R. Lan, L.
Chen, P.P. Dong, Biotransformation of major flavonoid glycosides in herb epimedii by
the fungus Cunninghamella blakesleana, Journal of Molecular Catalysis B: Enzymatic
(2015), http://dx.doi.org/10.1016/j.molcatb.2015.05.021
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*Graphical Abstract (for review)
Graphical abstract
R2
R2
HO
O
O
GlcO
O
O
Cunninghamella blakesleana
AS 3.970
OR1
OR1
OH
OH
1
2
a
a
R1= Rha
R2= OMe
1
2
R1= Rha
R2= OMe
R = Rha-Rha R = OMe
R = Rha-Rha R = OMe
1
2
1
2
3
a
R = Rha
R = OH
3
4
R = Rha
R = OH
1
2
1
2
4a R = Rha-Glc R = OMe
1 2
5a R1=Rha-Xyl R2= OMe
R = Rha-Glc R = OMe
1
2
5
R1=Rha-Xyl R2= OMe
Page 1 of 22

*Highlights (for review)
Highlight:
(
(
(
1) Icariin (1), epimedin C (2), epimedoside A (3), epimedin A (4) and epimidin B (5)
could be transformed to rare and bioactive products with the yield of above 95%.
2) Some transformed products showed exhibited the more significant anti-
osteoporosis activity.
3) Our investigation provided an efficient biotransformation approach to enrich the
rare and more biological active flavonoids in herb epimedi.
Page 2 of 22

*Manuscript
Click here to view linked References
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Biotransformation of major flavonoid glycosides in herb
epimedii by the fungus Cunninghamella blakesleana
a,+
b,*
c
b,c
a
a
3
XiuLan Xin , Guang-Jun Fan , Zheng Sun , Ning Zhang , Ye Li , Rong Lan ,
a
c,*
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5
Liang Chen , PeiPei Dong
a
College of Bioengineering, Beijing Polytechnic, Beijing 100029, China;
The Second Affiliated Hospital of Dalian Medical University, Dalian 116044,
China;
b
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7
8
c
College of Pharmacy, Dalian Medical University, Dalian 116044, P. R.China.
9
ABSTRACT：
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Biotransformation of icariin (1), epimedin C (2), epimedoside A (3), epimedin A (4)
and epimidin B (5), five major components of E. koreanum, were performed by
using Cunninghamella blakesleana. And they could be metabolized efficiently to
icariside II (1a), 2″-O-rhamnosylikarisoside II (2a), epimedoside b (3a), baohuoside
VII (4a) and sagittatoside B (5a) with high yields of 95.1%, 97.7%, 93.7%, 95.8%
and 96.4%, respectively. And these transformed products as major forms of herb
epimedii in vivo exhibited the more significant anti-osteoporosis activities. Our
method could be applied for enriching these rare flavonoids in herb epimedii, for
further development of anti-osteoporosis medicines or functional foods.
Keywords: Biotransformation; Icariin; epimedin C; Cunninghamella blakesleana;
epimedii
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------------------------------------------------------------------------------------------------------
Correspondence: Dr. Guang-Jun Fan, the Second Affiliated Hospital of Dalian
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Medical University, Dalian 116044, China. E-mail: faguangjun2014@163.com (G.J.
Fan); maxc1978@126.com (P. P. Dong). + These authors contributed equally to this
work.
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1. Introduction
Herba Epimedii (Berberidaceae) is an important and frequently used as traditional
Chinese medicine [1]. Its major bioactive constituents are flavonoid glycosides with
the isopentene group at C-8, such as icariin, epimedin C, epimedoside A, epimedin A
and epimidin B [2]. Among them, icariin and epimedin C have the natural abundance
with the contents of 0.5% and 1% in herba epimedii, respectively [3]. However, due to
their hydrophilicity of sugar moieties, these flavonoid glycosides are hardly absorbed
from the small intestine of human, which resulting in the poor oral availabilities [4].
Only their secondary glycosides such as icariside II and 2″-O-rhamnosylikarisoside II,
could be transported into the plasma and played an important bioactive role in vivo
[5-8]. Meantime, these secondary glycosides, exhibited the more significant
bioactivities including differentiation and proliferation of osteoblasts [9];
anti-osteoporosis [10], immunological function modulation [11], and anti-cancer [12].
So these secondary glycosides of epimedii had been paid more and more attention.
Unfortunately, their contents were very low in crude material of herba epimedii, and
limited their wide application in areas of medicines and foods. In previous
investigation, some attempts for obtaining these secondary flavonoids had been
reported. The chemical hydrolysis methods usually formed the cyclization products
such as β-anhydroicaritin. And the enzymatic hydrolysis method could transform
icariin to icariside II with the low yield of 47.5%, however other major epimedii
flavonoid glycosides such as epimedin A, epimidin B and epimedin C, could not be
transformed efficiently [13]. So it is very necessary to establish a systemic and
efficient method to obtain the series of rare and bioactive secondary flavonoids
glycosides of epimedii for development of new drugs or functional foods.
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Biotransformation is a useful technique to modify structures of biologically
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active compounds [14-21]. It has the advantage of high region-selectivity, mild
reactions and avoiding the various steps for protection and deprotection to
complicated structures and chiral centers of natural products [22-23].
In this study, biotransformation of icariin (1), epimedin C (2), epimedoside A (3),
epimedin A (4) and epimidin B (5) by cell suspension of C. blakesleana were
performed. And highly selective hydrolysis of C-7 was observed in the
biotransformation process. Totally, icariside II (1a) and 2″-O-rhamnosylikarisoside II
(2a), epimedoside b (3a), baohuoside VII (4a) and sagittatoside B (5a) were
prepared with high yields of 95.1%, 97.7%, 93.7%, 95.8% and 96.4%, respectively.
All metabolites were isolated and identified accurately by NMR and MS techniques.
In addition, their cell proliferation activity for MC3T3-E1 cell was also investigated,
and some metabolites (1a, 2a and 5a) exhibited the potential anti-osteoporosis
activity.
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2. Materials and methods
2.1. Apparatus and Reagents
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H- and C-NMR (nuclear magnetic resonance) spectra were recorded in
DMSO-d6 using TMS as internal standard on a DRX-600 spectrometer (Bruker,
Karlsruhe). ESIMS data were obtained by API3200-MS (AB SCIEX, New York,
USA). Agilent 1100 series HPLC equipped with Diode array detector at 254 nm
was used (Agilent, New York, USA). Silica gel (200-300 mesh) was purchased from
Qingdao Marine Chemical Group, Qingdao, China. ODS (Chromatorex, size
100-200 mesh) was purchased from Fujian Silysia Chemical Group, Fujian, China.
All solvents were AR grade from Tianjin Kermel Company. The crude materials of E.
koreanum were purchased from Hongjiu BioTech Co. Jilin Province of China.
2.2. Microorganisms and Culture medium
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C. blakesleeana was purchased from Chinese General Microbiological Culture
Collection Center in Beijing, China. Biotransformation experiments were performed
in potato medium, which was made by the following composition (L): 200 g potato
and 20 g glucose.
2.3. Culture and biotransformation procedures
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Mycelia of C. blakesleeana from agar slants (2 cm ) were transferred to 100 mL
◦
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of medium and cultured at 30 C with 175 rpm for 36 h to make a stock inoculum.
Then 10 mL volume of the inoculum was added to 1000 mL of liquid medium, and
◦
the flask was placed on rotary shaker operating at 175 rpm and 30 C. After 36h of
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preculture, 100 mg of icariin with 2 mL methanol added into 2L of culture medium
for preparative biotransformation with the final concentration of 50mg/L. The
incubation was continued for 5 additional days. The culture was filtered, and the
filtrate was extracted with same volume of EtOAc for five times. And the suspension
cells of C. blakesleeana (5.2 g) were also extracted by using EtOAc (1000 mL).
Then all extraction of EtOAc were collected and concentrated under the reduced
pressure. The residues (1.6 g) were applied to an ODS column (200g, 10×50cm) and
eluted with MeOH-H O (in a gradient manner from 10% to 90%, at a flow rate of 2.0
2
mL/min). Totally, 90.1 mg of icariside II (96.5% purity) was obtained from fraction
of 60% MeOH (500 mL). Similarly, 100 mg of epimedin C (2), epimedoside a (3),
epimedin A (4) and epimidin B (5), respectively, were added into 2L of culture
medium of C. blakesleeana. Other processes of collection and isolation for the target
compounds were same with that of icariin. Finally, 2″-O-rhamnosyli- karisoside II
(2a, 95mg, 95% purity, Rt=16.5min), epimedoside B (3a, 83.5 mg, 94% purity,
Rt=14.1min), baohuoside VII (4a, 89.5mg, 96% purity, Rt=10.2min) and
sagittatoside B (5a, 85.1mg, 95% purity, Rt=7.5min) were prepared by preparative
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HPLC in an isocratic manner of 60% MeOH with a flow rate of 2.0 mL/min.
2.4. HPLC analysis
The samples were analyzed on a DINOEX Ultimate 3000 instrument HPLC
equipped with a Kromasil C-18 column, 4.6 mm × 250 mm (5μm), and diode array
detector (DAD) at 270 nm, and the mobile phase was composed by solvents A
(methanol) and B (0.3% acetic acid) in gradient elution. The gradient program was
as follows: initial 0-5min, using a constant elution A-B (45: 55, v/v), then 5-10 min,
linear change from A-B (45: 55, v/v) to A-B (80: 20, v/v); and then 10-20 min, using
a constant elution A-B (80: 20, v/v); next 20-30 min, linear change to A-B (80: 20,
v/v) to A-B (90: 10, v/v); then 30-35 min, using a constant elution A-B (90: 10, v/v).
The flow rate was 0.8 mL/min and column temperature was 30℃.
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2.5. Bioassay
Mouse osteoblastic cell line (MC3T3-E1, purchased from the Chinese Academy
of Sciences Cell Bank) was cultured under a humidified atmosphere of 5% CO at
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37 °C with the Dulbecco’s modified Eagle’s medium (DMEM) containing 1%
penicillin and 1% streptomycin and 10% fetal bovine serum. In this study, cell
proliferation of MC3T3-E1 cell was measured by MTT assay. Briefly, cells were
maintained in growth media for 24 h at 5% CO . And then, the cells were treated
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with the three different concentrations (1, 10 and 100μM) of the test compounds for
24 h. Finally, MTT (5 mg/mL) was added to the cell cultures (1:10) and samples
were incubated at 37°C for 4h. In addition, the cells in normal culture medium were
treated with same volume of DMSO as control condition, the values of which was
set as 100%.
. The absorbance was measured in an optical 96-well microplate reader at the
wavelength of 570 nm. All the data was present as the mean of three experiments
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(n=3).
3. Results and discussion
3.1. Preliminary screening for biotransformation
15 strains of filamentous fungi from nine genera were initially screened for their
abilities to transform icariin by TLC and HPLC chromatography. Among the cultures
screened, M. spinosus, F. solani and P. janthinellum, showed the ability of
transforming icariin into more polar metabolites. However, C. blakesleeana was
found to be able to convert substrates into one main non-polar metabolite with high
yield and low yield of by-products. Therefore, it was selected for the preparative
biotransformation of substrates including icariin (1), epimedin C (2), epimedoside A
(3), epimedin A (4) and epimidin B (5) in this study. (Figs 2 and 3)
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3.2. Time course of biotransformation of substrates
Fig.4A showed the biotransformaton kinetic of icariin after administration of 50
mg/L. After incubation for 168h with C. blakesleeana it was completely transformed
to icariside II (1a) with the conversion of 95.06% by HPLC analysis. In order to
investigate the capability of C. blakesleeana to metabolize icariin, the effects of pH,
culture temperature, substrate concentration and reaction specificity in the
biotransformation process were also investigated (Fig. 4). Our results suggested that
the optimum pH value should be at 6–7 with the highest transformation rate. And the
preferred temperature was 28-30℃. However, when the concentration was increased
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to 80 mg/L, the substrates were not completely transformed with the rate of 63.18%.
Similarly, C. blakesleeana also exhibited the good ability to transform epimedin C
to 2″-O-rhamnosylikarisoside II (2a). After incubation for 120 h with C.
blakesleeana, epimedin C was completely transformed with the yield of 97.67% by
HPLC analysis, which suggesting that C. blakesleeana had the high selectivity for
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glucose hydrolysis of epimedium flavonoids at C-7. Finally, the optimum pH value,
culture temperature and substrate concentration were determined at pH=7, 28-30℃
and 100 mg/L (supplement data, Figs 4).
In order to elucidate the relationship of biotransformation ability and substrate
structures of epimedium flavonoids. The ability of C. blakesleeana to transform
epimedoside A (3), epimedin A (4) and epimidin B (5), three major derivatives of
icariin in epimedium, were also investigated. After incubation for 168h, they were
completely transformed to epimedoside B (3a), baohuoside VII (4a) and
sagittatoside B (5a) with the yield rates of 93.7%, 95.8% and 96.4%, respectively.
The results were illustrated in Fig. 3.
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3.3. Identification of biotransformation products
Incubation of icariin (1) and other four flavonoid glycosides epimedin C (2),
epimedoside A (3), epimedin A (4) and epimidin B (5) with C. blakesleeana for 7
days yielded five products and their structures were identified as icariside II (1a),
2″-O- rhamnosylikarisoside II (2a), sagittatoside B (3a) baohuoside VII (4a) and
epimedoside b (5a) (Fig. 1), respectively, and by comparing with the literatures [24].
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3.3.1. Icariside (1a): yellow needle crystal, H-NMR (DMSO-d , 600MHz):
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H-NMR (DMSO-d , 600MHz): 5.16 (1H,t, J=6.0 Hz, H-12), 5.27 (1H, d, J=1.6 Hz,
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H-1´´), 6.32(1H, s, H-6), 7.13 (2H, d, J=9.2 Hz, H-3´, H-5´), 7.87 (2H, d, J=9.2 Hz,
H-2´,H-6´), 10.84(1H, br. s, 7-OH), 12.53 (1H, s, 5-OH), 3.85 (3H,s, OCH ), 1.69
3
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(3H, s, H-15), 1.61 (3H, s, H-14) and 0.78 (3H, d, J = 6.6 Hz, H-6´´). C-NMR
(DMSO-d , 150 MHz): 156.6 (C-2), 134.4 (C-3), 178.0 (C-4), 161.6 (C-5), 98.3
6
(C-6), 161.2 (C-7), 106.0 (C-8), 153.7 (C-9), 104.1 (C-10), 21.1 (C-11), 122.3 (C-12),
131.1 (C-13), 25.4 (C-14), 17.8 (C-15), 122.4 (C-1´), 130.43 (C-2´, C-6´), 114.1
(C-3´ and C-5´), 158.8 (C-4´), 55.5 (OCH ), 101.1 (Rha-1´), 70.7 (Rha-2´), 70.4
3
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(Rha-3´), 71.2 (Rha-4´), 70.4 (Rha-5´), 17.4 (Rha-6´).
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3.3.2. 2″-O-rhamnosylikarisoside II (2a): yellow powder (methanol), H-NMR
(DMSO-d ,600MHz):12.57(1H,s,OH-5), 7.86 (2H, d, J=8.4Hz, H-2’, H-6’), 7.13
6
(2H, d, J = 4.5 Hz, H-3’, H-5’), 6.93 (1H, s, H-6’), 6.34 (1H,s), 5.37 (1H, s), 5.15
(1H, d, J = 7.0 Hz), 3.85 (3H, s, OCH -4’),1.68 (3H, s), 1.62 (3H, s), 0.81 (3H, d, J =
3
13
5.4Hz). C-NMR (DMSO-d , 150 MHz): 156.8 (C-2), 134.5 (C-3), 178.0 (C-4),
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161.4 (C-5), 98.5 (C-6), 161.8 (C-7), 106.0 (C-8), 153.8 (C-9), 104.2 (C-10), 21.3
(C-11), 122.3 (C-12), 131.1 (C-13), 25.5 (C-14), 17.9 (C-15), 122.3(C-1’), 130.5
’
’
(C-2’, C-6’), 114.2 (C-3’ and C-5’), 130.5 (C-4’), 55.6 (OCH ), 101.7 (Rha-1 ), 75.6
3
’’
’’
’’
’’
’’
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(Rha-2 ), 70.3 (Rha-3 ), 72.0 (Rha-4 ), 70.2 (Rha-5 ), 17.7 (Rha-6 ), 100.8
’
’’’
’
’’’
’
’’’
’
’’’
’
’’’
(Rha -1 ), 70.6 (Rha -2 ), 70.7 (Rha -3 ), 71.4 (Rha -4 ), 68.9 (Rha -5 ) and 17.6
’
’’’
(Rha -6 ).
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3.3.3 Epimedoside B (3a): yellow powder (methanol), H-NMR (DMSO-d ,
6
600MHz): 12.56(1H, s, OH-5), 10.90 (1H, s, OH-7), 10.27 (1H, s, OH-4’), 7.76 (1H,
d, J=8.4Hz, H-2’), 7.14(2H, d, J= 4.5Hz, H-3’, H-5’), 6.93(1H,s,H-6’), 6.32 (1H, s),
5.27 (1H, s), 5.14 (1H, t, J = 6.6Hz), 1.67(3H,s), 1.62(3H, s), 0.80 (3H,d, J=6.0 Hz).
1
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C-NMR (DMSO-d , 150 MHz): 157.73 (C-2), 134.2 (C-3), 178.1 (C-4), 160.1
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(C-5), 98.4 (C-6), 161.7 (C-7), 106.1 (C-8), 153.9 (C-9), 104.2 (C-10), 21.3 (C-11),
122.4 (C-12), 130.5 (C-13), 25.6 (C-14), 17.9 (C-15), 120.9(C-1’), 130.7 (C-2’,
’’
’’
C-6’), 115.5 (C-3’ and C-5’), 160.0 (C-4’), 102.0 (Rha-1 ), 70.5 (Rha-2 ), 70.8
’’
’’
’’
’’
(Rha-3 ), 71.2 (Rha-4 ), 70.2 (Rha-5 ) and 17.6(Rha-6 ).
1
3.3.4. Baohuoside VII (4a): yellow powder (methanol), H-NMR (DMSO-d ,
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600MHz): 6.34 (1H,s), 5.54 (1H,s), 5.16(1H, t, J = 7.2Hz), 4.25 (1H, d, J=7.8Hz),
1
3
3.86 (3H, s), 1.68 (3H,s), 1.62 (3H,s), 0.86 (3H, d, J = 6.0Hz). C-NMR (DMSO-d ,
6
150 MHz): 156.7 (C-2), 134.2 (C-3), 178.0 (C-4), 161.5 (C-5), 98.5 (C-6), 161.8
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(C-7), 106.0 (C-8), 153.9 (C-9), 104.9 (C-10), 21.4 (C-11), 122.4 (C-12), 131.1
(C-13), 25.5 (C-14), 17.9 (C-15), 122.4(C-1’), 130.6 (C-2’, C-6’), 114.2 (C-3’ and
’’
’’
’’
C-5’), 158.9 (C-4’), 55.6 (OCH ), 101.8 (Rha-1 ), 81.2 (Rha-2 ), 70.4 (Rha-3 ), 71.3
3
’’
’’
’’
’
’
’
(Rha-4 ), 69.9 (Rha-5 ), 17.6 (Rha-6 ), 104.2 (Glc-1 ), 73.8 (Glc-2 ), 76.9 (Glc-3 ),
’
’
’
69.2 (Glc-4 ), 76.3 (Glc-5 ) and 61.1(Glc-6 ).
1
3.3.5: Sagittatoside B (5a): yellow powder (methanol), H-NMR (DMSO-d ,
6
600MHz): 6.34(1H,s), 5.34(1H,s), 5.16(1H,t, J=6.6Hz),4.17(1H,d, J=1.8Hz), 3.86
1
3
(3H, s), 1.68(3H,s), 1.62(3H,s), 0.86(3H, d, J=6.6Hz). C-NMR (DMSO-d , 150
6
MHz): 156.6 (C-2), 134.6 (C-3), 178.1 (C-4), 161.5 (C-5), 98.5 (C-6), 161.8 (C-7),
106.1 (C-8), 153.8 (C-9), 104.1 (C-10), 21.3 (C-11), 122.3 (C-12), 131.1 (C-13),
25.5 (C-14), 17.8 (C-15), 122.4(C-1’), 130.4 (C-2’, C-6’), 114.2 (C-3’ and C-5’),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
’’
’’
’’
158.9 (C-4’), 55.5 (OCH ), 101.1 (Rha-1 ), 80.7 (Rha-2 ), 70.4 (Rha-3 ), 71.8 (Rha-
3
’’
’’
’’
’
’
’
4 ), 70.4 (Rha-5 ), 17.5 (Rha-6 ), 106.5 (Xyl-1 ), 73.8 (Xyl-2 ), 76.3 (Xyl-3 ), 69.4
’
’
(Xyl-4 ) and 65.9 (Xyl-5 ).
3.4 Bioassay
In this study, we evaluated the effects of compounds (1-5 and 1a-5a) on the
osteoblast function using MC3T3-E1 cells. Our results indicated that the bioactivities
of secondary aglycones were more significant than these of flavonoid glycosides of E.
koreanum (Fig. 5). At the concentration of 100μM, the activity of transformed
products (1a-5a) exhibited the significant anti-osteoporosis activity than these of
substrates (1-5), especially for 1a, 2a and 5a with the mean survival rates of 137%,
143% and 158%, respectively (n=3). According to our results, compounds 1a, 2a and
5a may be used new natural resources to develop the anti-osteoporosis agent.
4. Conclusions
C. blakesleana exhibited the high selectivity of hydrolysis glucose of C-7 such as
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1
2
3
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icariin (1), epimedin C (2), epimedoside A (3), epimedin A (4) and epimidin B (5), to
produce some rare flavonoid glycosides of epimedii including icariside II (1a), 2″-O-
rhamnosylikarisoside II (2a), sagittatoside B (3a) baohuoside VII (4a) and
epimedoside b (5a), with the yield rates of above 95%. And these transformed
products showed more anti-osteoporosis than these of substrates (1-5). Our
investigation provided an efficient biotransformation approach to enrich the rare and
better bioactive flavonoids in herb epimedii, for further development of anti-
osteoporosis medicines and foods.
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References
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Acknowledgements: We thank the authors thank Microbial transformation and
metabolism of triterpenoids (YZK2014020); the National Natural Science
Foundation of China (21306002); the Application of Chromatography in natural
products research, Program for Research Team in Beijing Polytechnic for financial
support.
1
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Figure captions
Figure 1. The biotransoformation pathway of major epimedii flavonoids by C.
blakesleeana
Figure 2. HPLC chromatograms of the blank of M. spinosus AS 3.3450 (A) and
icariin (B), administrating 1 for 5 days (C), epimedin C (D), administrating 1 for 5
days (E).The DAD spectra of compounds1-2 and 1a-2a.
1
1
1
1
1
1
1
1
1
1
Figure 3. HPLC chromatograms of compound 3 (A), administrating 3 for 120h (B),
compound 4 (C), administrating 4 for 120h (D), Compound 5 (E), administrating 5
for 120h (F).
Figure 4. The time course and effects of pH and temperature for icariin (1) and
epimedin C (2) by Cunninghamella blakesleana. (A) Time course of
biotransformation of icariin; (B) Time course of biotransformation of epimedin C;
(C) Effects of pH value on the biotransformation of 1 and 2; (D) Effects of
temperature values.
Figure 5. Survival rate of compounds1-5 and 1a-5a on MC3T3-E1 cells by MTT
method. Comparison test, *p < 0.05; **p < 0.01
2
2
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0
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Page 15 of 22

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R2
R2
1
R = Rha
1
R = OMe
2
1a R = Rha
R = OMe
2
1
GlcO
O
O
Cunninghamella blakesleana
2
R = Rha-Rha R = OMe
1
2
HO
O
O
2a
R = Rha-Rha R = OMe
1 2
3
4
R = Rha
R = OH
3a R = Rha
R = OH
2
1
2
1
AS 3.970
R = Rha-Glc R = OMe
4a R = Rha-Glc R = OMe
1 2
1
2
OR1
OR1
5
R =Rha-Xyl R = OMe
1 2
5a R =Rha-Xyl R = OMe
1 2
OH
OH
7
8
Figure 1. The biotransoformation pathway of major epimedii flavonoids by C. blakesleeana
9
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Figure. 2. HPLC chromatograms of the blank of M. spinosus AS 3.3450 (A) and
icariin (B), administrating 1 for 5 days (C), epimedin C (D), administrating 1 for 5
days (E).The DAD spectra of compounds1-2 and 1a-2a.
1
1
1
1
1
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Page 17 of 22

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Figure 3. HPLC chromatograms of compound 3 (A), administrating 3 for 120h (B),
compound 4 (C), administrating 4 for 120h (D), Compound 5 (E), administrating 5
for 120h (F).
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Page 18 of 22

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Figure 4. The time course and effects of pH and temperature for icariin (1) and
epimedin C (2) by Cunninghamella blakesleana. (A) Time course of
biotransformation of icariin; (B) Time course of biotransformation of epimedin C;
(C) Effects of pH value on the biotransformation of 1 and 2; (D) Effects of
temperature values.
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Figure 5. Survival rate of compounds1-5 and 1a-5a on MC3T3-E1 cells by MTT
method. Comparison test, *p < 0.05; **p < 0.01
6
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Page 20 of 22

Supplementary Material
Supplementary data
1
Compound 4a H-NMR spectrum
13
Compound 4a C-NMR spectrum
Page 21 of 22

1
Compound 5a H-NMR spectrum
13
Compound 5a C-NMR spectrum
Page 22 of 22