Flavonoids from silkworm droppings and their promotional activities on heme oxygenase-1

Article abstract of DOI:10.1080/10286020.2011.555331

A new flavane glucoside, 7,2'-dihydroxy-8-hydroxyethyl-4'-methoxyflavane- 2'-O-β-d-glucopyranoside (3), along with three known flavonoids, 7,2'-dihydroxy-8-prenyl-4'-methoxyflavane (1), euchrenone a7 (2), and 7,2'-dihydroxy-8-prenyl-4'-methoxy-2'-O-β-d-glucopyranosylflavane (4), was isolated from silkworm droppings. The structures of the compounds were elucidated on the basis of 1D and 2D NMR spectroscopic analyses and optical rotational characteristics. The compounds isolated from silkworm droppings were evaluated for their effects on heme oxygenase-1 (HO-1) activity. Compounds 1 and 3 increased the expression of HO-1 in HepG2 cells. HO-1 is an antioxidant enzyme that catabolizes heme to carbon monoxide, free iron, and biliverdin, all of which are involved in the suppression of inflammatory mediators.

Full text of DOI:10.1080/10286020.2011.555331

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Flavonoids from silkworm droppings and
their promotional activities on heme
oxygenase-1
Ji-Hae Park a , Dae-Young Lee a , Pyeong Yun b , Seung-Woo Yeon c
, Jong Hee Ko c , Yong-Soon Kim d & Nam-In Baek a
a College of Life Sciences & Plant Metabolism Research Center,
Kyung Hee University , Yongin, 446-701, South Korea
b Cosmetic R&D Center, Kolmar Korea Co. , Bucheon, 420-801,
South Korea
c Research Laboratories, Ildong Pharmaceutical Co., Ltd ,
Hwaseong, 445-170, South Korea
d Department of Agricultural Biology , National Academy of
Agricultural Science, RDA , Suwon, 441-853, South Korea
Published online: 30 Mar 2011.
To cite this article: Ji-Hae Park , Dae-Young Lee , Pyeong Yun , Seung-Woo Yeon , Jong Hee Ko ,
Yong-Soon Kim & Nam-In Baek (2011) Flavonoids from silkworm droppings and their promotional
activities on heme oxygenase-1, Journal of Asian Natural Products Research, 13:04, 377-382, DOI:
10.1080/10286020.2011.555331
To link to this article: http://dx.doi.org/10.1080/10286020.2011.555331
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Journal of Asian Natural Products Research
Vol. 13, No. 4, April 2011, 377–382
Flavonoids from silkworm droppings and their promotional activities
on heme oxygenase-1
Ji-Hae Parka, Dae-Young Leea, Pyeong Yunb, Seung-Woo Yeonc, Jong Hee Koc,
Yong-Soon Kimd and Nam-In Baeka*
aCollege of Life Sciences & Plant Metabolism Research Center, Kyung Hee University,
Yongin 446-701, South Korea; Cosmetic R&D Center, Kolmar Korea Co., Bucheon 420-801,
b
c
South Korea; Research Laboratories, Ildong Pharmaceutical Co., Ltd, Hwaseong 445-170,
d
South Korea; Department of Agricultural Biology, National Academy of Agricultural Science,
RDA, Suwon 441-853, South Korea
(Received 21 October 2010; final version received 13 January 2011)
A new flavane glucoside, 7,20-dihydroxy-8-hydroxyethyl-40-methoxyflavane-20-O-b-
D-glucopyranoside (3), along with three known flavonoids, 7,20-dihydroxy-8-prenyl-40-
methoxyflavane (1), euchrenone a7 (2), and 7,20-dihydroxy-8-prenyl-40-methoxy-20-O-
b-D-glucopyranosylflavane (4), was isolated from silkworm droppings. The structures
of the compounds were elucidated on the basis of 1D and 2D NMR spectroscopic
analyses and optical rotational characteristics. The compounds isolated from silkworm
droppings were evaluated for their effects on heme oxygenase-1 (HO-1) activity.
Compounds 1 and 3 increased the expression of HO-1 in HepG2 cells. HO-1 is an
antioxidant enzyme that catabolizes heme to carbon monoxide, free iron, and
biliverdin, all of which are involved in the suppression of inflammatory mediators.
Keywords: Bombyx mori; 7,20-dihydroxy-8-hydroxyethyl-40-methoxyflavane-20-O-b-
D-glucopyranoside; HO-1; silkworm droppings
1. Introduction
were shown to lower cholesterol levels
and blood pressure, and to exhibit anti-
diabetic characteristics and anticancer
activity [3,4].
Silkworm droppings, also known in Korea
as Jambun, Jamsa, Wonjamsa, and Won-
jamsi, refer to the excrement of the
silkworm (Bombyx mori L.). When the
silkworm ingests the leaves of the mulberry
tree, more than half of the leaves are
excreted without being digested [1]. There-
fore, silkworm droppings are likely com-
posed of both mulberry leaf and various
constituents biotransformed by microbes or
enzymes in the intestine of the silkworm.
Studies have demonstrated that silkworm
droppings are nontoxic and can be used to
strengthen the internal organs of humans,
treat diabetes, and protect against diplegia
[2]. Additionally, silkworm droppings
We have also found that alcohol
extracts of silkworm droppings exhibited
higher immunosuppressive activities than
those of cyclosporine, a well-known
immune inhibitor, against IL-6, MCP-1,
and MIP-1a cytokines. We searched for
immune inhibition compounds in silkworm
droppings using SiO2 and octadecyl silica
gel (ODS) column chromatography (CC)
and isolated a new flavane, 7,20-dihydroxy-
8-hydroxyethyl-40-methoxyflavane-20-O-b-
D-glucopyranoside (3), together with three
known flavonoids 1, 2, and 4 (Figure 1).
*Corresponding author. Email: nibaek@khu.ac.kr
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2011 Taylor & Francis
DOI: 10.1080/10286020.2011.555331
http://www.informaworld.com

378
J.-H. Park et al.
OH
5'
OCH3
2"
1"
8
6'
1'
4'
3'
OCH3
OH
O
HO
8a
4a
7
2
3
2'
HO
O
HO
O
6
4
H5O
OR1
OH
O
O
O
HO
HO
OH
1 R1 = H
4 R1 = β-D-glc
2
3
Figure 1. Chemical structures of flavonoids isolated from silkworm droppings. b-D-glc is b-D-
glucopyranoside.
This paper describes the procedures for the
isolation and structural determination of the
four flavonoids from silkworm droppings in
addition to evaluating the flavonoids for
their effects on the expression of heme
oxygenase-1 (HO-1).
comparison of the physical and spectro-
scopic data with those reported in the
literature [5,6].
Compound 3 was isolated as a pale
yellow amorphous powder and showed IR
absorbance bands of hydroxyl (3364 cm21
)
and aromatic (1609 cm21) groups. The
molecular weight was determined to be
478 from the molecular ion peak m/z 477
[M 2 H]2 in the negative FAB-MS
spectrum, and a molecular formula of
C24H30O10 was determined by the pseudo-
molecular ion ([M 2 H]2, m/z 477.1772)
in the HR-FAB-MS. The 1H NMR
spectrum exhibited three olefin methine
signals of a 1,2,4-trisubstituted benzene
ring at dH 7.34 (1H, d, J ¼ 8.4 Hz), 6.85
(1H, d, J ¼ 2.4 Hz), and 6.63 (1H, dd,
J ¼ 8.4, 2.4 Hz), and two olefin methine
signals of a 1,2,3,4-tetrasubstituted ben-
zene ring were seen at dH 6.73 (1H, d,
J ¼ 8.0 Hz) and 6.33 (1H, d, J ¼ 8.0 Hz).
An oxygenated methine signal at dH 5.50
(1H, dd, J ¼ 9.6, 2.0 Hz), an oxygenated
methylene signal at dH 3.65 (2H, m, H-200),
a methoxy signal at dH 3.78 (3H, s, 40-
OMe), and three methylene signals at dH
2.90 (2H, m, H-100), 2.85 (1H, m, H-4a),
2.61 (1H, m, H-4b), 2.19 (1H, m, H-3a), and
1.82 (1H, m, H-3b) were also observed.
In addition, a hemiacetal proton signal at
dH 4.91 (1H, d, J ¼ 7.2 Hz), oxygenated
methylene signals at dH 3.92 (1H, dd,
J ¼ 12.0, 2.0 Hz) and 3.70 (1H, dd,
J ¼ 12.0, 6.4 Hz), and methine signals at
dH 3.45 (1H, m), 3.44 (1H, m), and 3.38
(1H, m) resulted from the hexose moieties.
2. Results and discussion
When 80% methanol extracts of silkworm
droppings were developed on silica gel thin
layer chromatography (TLC), some spots
exhibited UV absorption and yellow and
dark brown coloration when sprayed with
10% H2SO4 solution and heated, indicating
the presence of flavonoids in the extracts.
The methanol extracts were fractionated
into EtOAc, n-BuOH, and H2O layers
through solvent fractionation. Repeated
silica gel and ODS CC of the EtOAc
fractions produced four flavonoids. The
chemical structures of the compounds were
determined through the analysis of data
from 1H, 13C NMR, DEPT, gradient
correlation spectroscopy, gradient hetero-
nuclear
single-quantum
coherence
(gHSQC), gradient heteronuclear multiple-
bond connectivity (gHMBC), electron ion-
ization mass spectrometry (EI-MS), fast
atom bombardment mass spectrometry
(FAB-MS), and infrared spectroscopy (IR).
The three compounds 1, 2, and 4 were
identified to be 7,20-dihydroxy-8-prenyl-40-
methoxyflavane (1), euchrenone a7 (2), and
7,20-dihydroxy-8-prenyl-40-methoxy-20-O-
b-D-glucopyranosylflavane (4) on the basis
of spectroscopic data and chemical evi-
dence. These findings were confirmed by

Journal of Asian Natural Products Research
379
The side chain was deduced to be an ethyl
alcohol moiety based on the signals of an
oxygenated methylene proton at dH 3.65
and a methylene proton at dH 2.90. As a
result, compound 3 was assumed to be a
flavane monoglycoside combined with an
ethyl alcohol group.
(C-20). Based on these results, the ethyl
alcohol and the b-D-glucose moieties were
determined to be located at C-8 and C-20,
respectively. As a result, compound 3 was
determined to be a 7,20-dihydroxy-8-
hydroxyethyl-40-methoxyflavane-20-O-b-
D-glucopyranoside. The stereostructure of
a chiral carbon, C-2, was determined as
S by the comparison of not only the
chemical shifts (dC 73.5, dH 5.50) of the
carbon and proton signals, but also through
the comparison of the specific rotation
value of 248.1 with those of similar
compounds in the literature [7]. The
chemical shifts of C-2 and H-2 in flavane
moiety with 2R structure were usually
observed at dC 78.5 and dH 4.85, whereas
those with 2S structure were at dC 73.5 and
dH 5.50 [8]. The sugar of compound 3 was
determined to be D-glucopyranose from the
The 13C NMR spectrum showed 24
carbon signals, including a hexose carbon
signal at d 102.9 (C-1000), 78.2 (C-3000), 78.1
(C-5000), 74.9 (C-2000), 71.4 (C-4000), and 62.5
(C-6000), which were identified as those of a
b-glucopyranose. The multiplicity of each
carbon was determined using a DEPT
experiment. Two benzene rings showed 12
carbon signals: four oxygenated olefin
quaternary signals at d 161.3 (C-40), 156.1
(C-20), 155.5 (C-7), and 155.2 (C-8a), three
olefin quaternary signals at d 125.4 (C-10),
114.4 (C-4a), and 113.4 (C-8), and five
olefin methine signals at d 128.2 (C-5),
127.9 (C-60), 108.8 (C-50), 108.4 (C-6), and
103.1 (C-30). There were also one oxyge-
nated methine signal at d 73.5 (C-2), one
methoxy signal at d 55.8, and two
methylene signals at d 30.4 (C-3) and 25.8
(C-4). Consequently, compound 3 was
identified as a flavane glucoside. The
stereostructure of the anomer carbon was
confirmed to be b from the coupling
constant of the hemiacetal proton signal
(J ¼ 7.2 Hz) in the 1H NMR spectral data.
The side chain was determined to be an
ethyl alcohol group from an oxygenated
methylene at d 62.6 (C-200) and a methylene
at the d 27.7 (C-100) carbon signals. To
determine the locations of the glucose and
the ethyl alcohol group in compound 3, 2D
NMR, such as gHSQC and gHMBC
experiments, was conducted. In the
gHMBC spectrum, the methylene proton
signal at dH 2.90 (H-100) showed cross peaks
with two oxygenated olefin quaternary
carbon signals at d 155.5 (C-7) and 155.2
(C-8a), and with one olefin quaternary
carbon signal at d 113.4 (C-8). Also, the
anomeric proton signal at dH 5.50 (H-1000)
showed a correlation with the oxygenated
olefin quaternary carbon signal at d 156.1
25
specific rotation value (½aꢀD þ47.4,
c ¼ 1.2, H2O)ofthesugar obtainedthrough
acid hydrolysis of the glycoside 3. As a
result, compound 3 was determined to be a
new compound.
To determine whether the isolated
compounds induced the expression of
HO-1, we exposed HepG2 cells to 20mM
concentration of compounds 1–4.
The results showed that compounds 1
and 3 increased the expression of HO-1
in a concentration-dependent manner
(Figure 2). The inductions of HO-1
Figure 2. HO-1 expressions in HepG2 cells
exposed to compounds 1 and 3 isolated from
silkworm droppings. Cells were incubated with
20mM concentrations of compounds 1–4 for
16h. HO-1 protein expression was evaluated
using antiHO-1 antibodies as described in
the experimental methods. One representative
Westernblotfromthreeindependentexperiments
is shown.

380
J.-H. Park et al.
expression by compounds 1 and 3 were
almost the same as those of a chalcone,
(E)-1-[2-methoxy-4,6-bis(methoxymethox-
y)phenyl]-3-(3-nitrophenyl)prop-2-en-1-
one [9] and a triterpenoid, 23-hydroxyur-
solic acid [10], which were reported to
significantly increase HO-1 expression.
HO-1 is considered to be an antioxidant
enzyme that catabolizes heme to carbon
monoxide, free iron, and biliverdin, all
of which are involved in the suppression
of inflammatory mediators or factors
that may be used to improve atopy-related
symptoms.
3.2 Materials
Silkworm droppings were supplied in
January 2008 from the Department of
Agricultural Biology, National Academy
of Agricultural Science, RDA, Suwon,
Korea. A voucher specimen (KHU08-
0102) has been reserved at the Laboratory
of Natural Products Chemistry, Kyung Hee
University, Yongin, Korea.
3.3 Extraction and isolation
Dried silkworm droppings (10 kg) were
extracted at room temperature using 80%
aqueous MeOH (36 L £3) for 24 h,
followed by filtration. The filtrates were
evaporated in vacuum, and the obtained
MeOH extracts were suspended in H2O
(3 L) and then extracted successively with
ethyl acetate (EtOAc, 3 L £5) and n-buta-
nol (n-BuOH, 2.8 L £3). The organic and
aqueous layers were concentrated to
produce the residues of the EtOAc fraction
(SDE, 61 g), the n-BuOH fraction (SDB,
30 g), and the H2O fraction (SDW, 136 g).
The EtOAc extract (SDE, 60 g) was
applied to SiO2 CC (w 8.0 £ 15.0 cm) and
eluted with n-hexane–EtOAc (5:1 !
3:1 ! 1:1 ! 1:3, 30 L of each) and
CHCl3 –MeOH (10:1 ! 7:1 ! 5:1 !
3:1, 25 L of each). The eluting solutions
were monitored using TLC and ultimately
produced 23 fractions (SDE-1 to SDE-23).
Fraction SDE-5 [2.9 g, Ve/Vt (elution
volume/total volume) 0.07–0.11] was
subjected to SiO2 CC (w 5.0 £ 11.0 cm)
and was eluted with n-hexane–EtOAc
(5:1 ! 1:1, 2.5 L of each) and CHCl3–
MeOH (10:1 ! 7:1, 2.5 L of each), yield-
ing 22 fractions (SDE-5-1 to SDE-5-22).
Fraction SDE-5-12 (630 mg, Ve/Vt 0.19–
0.24) was purified using ODS CC (w 3.5 £
6.0 cm) and was eluted with acetonitrile–
H2O (3:2, 2.6 L) to ultimately produce
compound 1 [SDE-5-12-6, 7.0 mg, Ve/Vt
0.02–0.03, TLC (ODS F254S) Rf 0.50,
acetonitrile–H2O ¼ 3:1]. Fraction SDE-9
(1.9 g, Ve/Vt 0.35–0.42) was subjected to
SiO2 CC (w 4.5 £ 12.0 cm) and eluted with
3. Experimental
3.1 General experimental procedures
UV spectrum was obtained using UV-vis
spectrophotometer (Simadzu UV-1601,
Tokyo, Japan). Optical rotation was
measured on a JASCO P-1010 digital
polarimeter (Jasco, Tokyo, Japan), and IR
spectra were obtained using a Perkin Elmer
Spectrum One FT-IR spectrometer (Buck-
inghamshire, England). EI-MS was
recorded on a JEOL JMS-700 (JEOL,
Tokyo, Japan) and FAB-MS was con-
ducted on a JEOL JMSAX-700 (Tokyo,
Japan). 1H NMR (400 MHz) and 13C NMR
(100 MHz) spectra were recorded on a
Varian Unity Inova AS-400 FT-NMR
spectrometer (Palo Alto, CA, USA). SiO2
(Kiesel gel 60, Merck, Darmstadt,
Germany) and ODS (LiChroprep RP-18,
Merck) resins were used for CC, and TLC
analysis was carried out using Kiesel gel 60
F254 and RP-18 F254S (Merck) plates.
The compounds were detected using a
UV lamp Spectroline Model ENF-240 C/F
(Spectronics Corporation, New York, NY,
USA) and a 10% H2SO4 solution. AntiHO-
1 antibodies (Stressgen, Victoria, Canada)
were used for Western blotting exper-
iments, and proteins from these gels were
then electrophoretically transferred to a
PVDF membrane (Millipore, Bedford,
MA, USA).

Journal of Asian Natural Products Research
381
20
CHCl3–MeOH (15:1, 5.4 L), yielding eight
fractions (SDE-9-1 to SDE-9-8). Fraction
SDE-9-4 (216 mg, Ve/Vt 0.22–0.31) was
purified using ODS CC (w 3.0 £ 4.5 cm)
and eluted with MeOH– H2O (3:2, 690 ml)
to ultimately produce compound 2 [SDE-9-
4-4, 3.5 mg, Ve/Vt 0.22–0.26, TLC (ODS
(4.02); ½aꢀD 254.6 (c ¼ 0.3, MeOH); IR
(CaF2 window in MeOH, cm21) 3364,
2915, 1609, 1075; 1H NMR (400 MHz,
methanol-d4, dH) 7.34 (1H, d, J ¼ 8.4 Hz,
H-60), 6.85 (1H, d, J ¼ 2.4 Hz, H-30), 6.73
(1H, d, J ¼ 8.0 Hz, H-5), 6.63 (1H, dd,
J ¼ 8.4, 2.4 Hz, H-50), 6.33 (1H, d,
J ¼ 8.0 Hz, H-6), 5.50 (1H, dd, J ¼ 9.6,
2.0 Hz, H-2), 4.91 (1H, d, J ¼ 7.2 Hz,
H-1000), 3.92 (1H, dd, J ¼ 12.0, 2.0 Hz,
H-6000a), 3.78 (3H, s, 40-OMe), 3.70 (1H,
dd, J ¼ 12.0, 6.4 Hz, H-6000b), 3.65 (2H, m,
H-200), 3.45 (1H, m, H-3000), 3.44 (1H, m,
H-2000), 3.44 (1H, m, H-4000), 3.38 (1H, m,
H-5000), 2.90 (2H, m, H-100), 2.85 (1H, m,
H-4a), 2.61 (1H, m, H-4b), 2.19 (1H, m,
H-3a), 1.82 (1H, m, H-3b); 13C NMR
(100 MHz, methanol-d4, dC) 161.3 (C-40),
156.1 (C-20), 155.5 (C-7), 155.2 (C-8a),
128.2 (C-5), 127.9 (C-60), 125.4 (C-10),
114.4 (C-4a), 113.4 (C-8), 108.8 (C-50),
108.4 (C-6), 103.1 (C-30), 102.9 (C-1000),
78.2 (C-3000), 78.1 (C-5000), 74.9 (C-2000), 73.5
(C-2), 71.4 (C-4000), 62.6 (C-200), 62.5
(C-6000), 55.8 (40-OCH3), 30.4 (C-3), 27.7
(C-100), 25.8 (C-4); negative FAB-MS: m/z
477 [M 2 H]2; HR-FAB-MS: m/z
477.1772 (calcd for C24H29O10, 477.1761).
F
254S) Rf 0.60, MeOH–H2O ¼ 3:1]. Frac-
tion SDE-18 (985 mg, Ve/Vt 0.83–0.84)
was subjected to SiO2 CC (4.5 £ 14.0 cm)
and eluted with CHCl3–MeOH (10:1,
2.6 L) to yield eight fractions (SDE-18-1
to SDE-18-8). Fraction SDE-18-6 (156 mg,
Ve/Vt 0.62–0.74) was purified using ODS
CC (2.5 £ 5.5 cm) and eluted with MeOH–
H2O (3:4, 560 ml) to result in nine fractions
(SDE-18-6-1 to SDE-18-6-9), yielding
compound 3 [SDE-18-6-2, 11 mg, Ve/Vt
0.12–0.17, TLC (ODS F254S) Rf 0.40,
MeOH–H2O ¼ 1:1]. Fraction SDE-19
(1.2 g, Ve/Vt 0.84–0.87) was subjected to
ODS CC and was eluted with MeOH–H2O
(1:1, 2.1 L) to produce six fractions (SDE-
19-1 to SDE-19-6) and also yielded
compound 4 [SDE-19-2, 16 mg, Ve/Vt
0.11–0.19, TLC (ODS F254S) Rf 0.50,
MeOH–H2O ¼ 2:1].
3.3.1 7,20-Dihydroxy-8-prenyl-40-
methoxyflavane (1)
3.3.4 7,20-Dihydroxy-8-prenyl-40-meth-
oxy-20-O-b-D-glucopyranosylflavane (4)
A
pale yellow amorphous powder
20
(MeOH); ½aꢀD 223.7 (c ¼ 0.1, MeOH);
A pale yellow amorphous powder (MeOH);
20
IR (CaF2 window in CHCl3, cm21) 3402,
½aꢀD 224.2 (c ¼ 0.1, MeOH); IR (CaF2
2924, 1715, 1616; H and 13C NMR refer
1
window in MeOH, cm21): 3362, 2927,
to the literature [5]; EI-MS: m/z 340 [M]þ.
1
1713, 1069; H and 13C NMR refer to the
literature [5]; negative FAB-MS m/z 501
[M 2 H]2.
3.3.2 Euchrenone a7 (2)
A pale yellow amorphous powder (MeOH);
20
½aꢀD 234.8 (c ¼ 0.45, MeOH); IR (CaF2
3.4 Hydrolysis of compound, isolation
of the sugar, and measurement of specific
rotation value
window in MeOH, cm21): 3310, 2927,
2853, 1708, 1601; 1H and 13C NMR refer to
the literature [6]; EI-MS: m/z 324 [M]þ.
SDE-18-6-1 (21 mg, Ve/Vt 0.00–0.11) and
SDE-18-6-3 (32 mg, Ve/Vt 0.18–0.29),
which were revealed to contain 95% of
compound 3 from the TLC experiment,
were combined and dissolved in H2O
(3 ml), followed by addition of 10%
hydrochloric acid (0.5ml). The solution
3.3.3 7,20-Dihydroxy-8-hydroxyethyl-40-
methoxyflavane-20-O-b-D-glucopyrano-
side (3)
A
(MeOH); UV l
pale yellow amorphous powder
nm (log 1): 260
CHCl3
max

382
J.-H. Park et al.
Acknowledgements
was hydrolyzed at 1208C for 2 h. The
hydrolysate was neutralized by the addition
of Ag2CO3 in excess and filtered through
filter paper. The filtrate was centrifuged at
5000g for 10min. The supernatant was
concentrated in vacuo to give a white
powder (12mg). The powder was dissolved
in H2O (1 ml) and was used for the
measurement of the specific rotation value.
This study was supported by the Mental Health
Food Development Program from the Korea
Food Research Institute (20100797U0054101
S000100) and the Development Program from
the Agricultural Research & Development
Promotion Center (108056033HD110).
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3.5 HO-1 expression in HepG2 cells
HepG2 cells were plated in 6-well plates
at a density of 1 £ 106 cells/well and
were grown at 378C. After 24 h, the cells
were treated with compounds for 16 h,
washed twice with 1 £ PBS, and har-
vested in 1 ml of PBS through centrifu-
gation at 14,000 rpm for 5 min. The cells
were then lysed in 100 ml of TEN buffer
[50 mM Tris–HCl (pH 7.6), 150 mM
NaCl, 2 mM EDTA], 1% Triton X-100, a
protease inhibitor cocktail, and sodium
vanadate. The cell lysates were loaded
onto a 12% SDS-polyacrylamide gel and
separated using electrophoresis. Proteins
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