Improving the NQO1-Inducing Activities of Phenolic Acids from Radix Salvia miltiorrhiza: A Methylation Strategy

Article abstract of DOI:10.1111/j.1747-0285.2011.01171.x

NAD(P)H: quinone oxidoreductase1 (NQO1) is an important detoxification enzyme that can protect mammalian cells against toxic quinones and reduce the risk of tumorigenesis. In this study, it was found that salvianolic acid B (SaB), lithospermic acid (LA), and rosmarinic acid (RA), three main hydrophilic constituents in Danshen, conjugated with glutathione (GSH) easily in vitro but exhibited no NQO1-inducing activities in Hepa 1c1c7 cells, which might attribute to their poor absorptions. After a simple methylation strategy that aimed at improving the liposolubility, both the NQO1-inducing activities and the absorptions in cells of the phenolic acids improved obviously, without losing the GSH-conjugating abilities. The concentration to double the specific activity of NQ01 values of methylated products of lithospermic acid and rosmarinic acid were 17.86±2.34μg/mL and 11.97± 0.60μg/mL, respectively. The findings indicated that methylation is an effective strategy to improve the NQO1-inducing activities of phenolic acids in Danshen. In the present study, a methylation strategy was employed to improve the NQO1 inducing activities of phenolic acids in Danshen. After methylation, both the NQO1 inducing activities and the absorptions in cells of the phenolic acids improved obviously without losing their GSH conjugating abilities.

Full text of DOI:10.1111/j.1747-0285.2011.01171.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01171.x
Chem Biol Drug Des 2011; 78: 558–566
Research Article
Improving the NQO1-Inducing Activities of
Phenolic Acids from Radix Salvia miltiorrhiza:
a Methylation Strategy
Xiaoyu Zhang1, Zhihang Song1, Jinzhong
Received 21 June 2010, revised 17 June 2011 and accepted for publica-
tion 27 June 2011
Xu1,2,* and Zhongjun Ma1,2,
*
1
School of Pharmaceutical Sciences, Zhejiang University, No.866
Yuhangtang Road, Hangzhou 310058, China
Department of Ocean Science and Engineering, Zhejiang
The root of Salvia miltiorrhiza ('Danshen' in Chinese) is one of the
most popular traditional Chinese medicines (TCM) and has been
used extensively for the treatment of angina pectoris, myocardial
infarction, cerebrovascular diseases, chronic renal failure, and dys-
menorrheal for a long history (1–3). The lipophilic ingredients (tan-
shinone IIA and miltirone) and the hydrophilic ingredients
[salvianolic acid B (SaB), lithospermic acid (LA), and rosmarinic acid
(RA)] in Danshen possess extensive biological activities such as
anti-tumor, anti-inflammatory, and anti-virus effects (4–6). In China,
Fufang Danshen Dripping Pill (which is a composite of Salvia mil-
tiorrhiza, Panax notoginseng, and Cinnamomum camphora) is the
most widely used products and has been officially listed in the Chi-
nese pharmacopoeia (7). It is also the first TCM product approved
for phase II and III clinical trials by the Food and Drug Administra-
tion (FDA) in 1997 (IND No. 56956).
2
University, No.866 Yuhangtang Road, Hangzhou 310058, China
*Corresponding authors: Jinzhong Xu, xujinzhong@zju.edu.cn;
Zhongjun Ma, mazj@zju.edu.cn
NAD(P)H: quinone oxidoreductase1 (NQO1) is an
important detoxification enzyme that can protect
mammalian cells against toxic quinones and
reduce the risk of tumorigenesis. In this study, it
was found that salvianolic acid B (SaB), lithosper-
mic acid (LA), and rosmarinic acid (RA), three main
hydrophilic constituents in Danshen, conjugated
with glutathione (GSH) easily in vitro but exhibited
no NQO1-inducing activities in Hepa 1c1c7 cells,
which might attribute to their poor absorptions.
After a simple methylation strategy that aimed at
improving the liposolubility, both the NQO1-induc-
ing activities and the absorptions in cells of the
phenolic acids improved obviously, without losing
the GSH-conjugating abilities. The concentration
to double the specific activity of NQ01 values of
methylated products of lithospermic acid and ros-
marinic acid were 17.86 2.34 lg ⁄ mL and 11.97
0.60 lg ⁄ mL, respectively. The findings indicated
that methylation is an effective strategy to
improve the NQO1-inducing activities of phenolic
acids in Danshen.
NAD(P)H: quinone oxidoreductase1 (NQO1) is one of the phase II
detoxifying enzymes and could protect mammalian cells by convert-
ing toxic quinones to hydroquinones. Thus, reduction in quinones by
NQO1 is an important detoxification pathway in the life processes
(8). The induction of NQO1 is regulated via Keap1-Nrf2 signaling
pathway. Keap1 maintains steady-state levels of nuclear factor ery-
throid-2-related factor 2 (Nrf2) by proteasomal degradation of Nrf2
through a Keap1-Cul3-Rbx-dependent mechanism in which Keap1
serves as the substrate adaptor subunit in the E3 ubiquitin ligase
(9,10). Upon oxidizing stress, Nrf2 accumulates and transports into
the nucleus, then binds to the 5¢-upstream regulatory antioxidant
response element (ARE) regions of detoxification genes, and accel-
erates the expression of NQO1 (11–13).
Key words: glutathione, methylation, NAD(P)H: quinone oxidoreduc-
tase1, phenolic acids, Radix Salvia miltiorrhiza
Abbreviations: ARE, antioxidant response element; CID, collision-
induced dissociation; ECL, enhanced chemiluminescence; FDA, Food and
Drug Administration; GSH, glutathione; HRP, horseradish peroxidase; LA,
lithospermic acid; LA-Me, methylation of lithospermic acid; LC–MS,
liquid chromatography–tandem mass spectrometry; NQO1, NAD(P)H: qui-
none oxidoreductase1; Nrf2, nuclear factor erythroid-2-related factor 2;
PMSF, phenylmethylsulphonyl fluoride; RA, rosmarinic acid; RA-Me,
methylation of rosmarinic acid; SaB, salvianolic acid B; SaB-Me, methyl-
ation of salvianolic acid B; SDS–PAGE, sodium dodecyl sulfate–poly-
acrylamide gel electrophoresis; SIC, selected ion chromatograms; TCM,
traditional Chinese medicines.
Depleting the cellular level of glutathione (GSH) and feedback acti-
vating Keap1-Nrf2 signaling pathway is one of the important mech-
anisms involved in the induction of phase II detoxifying enzymes
(14). In previous study, it has been reported that electrophilic com-
pounds that could alkylate cellular GSH had potent NQO1-inducing
activities (15,16). So, GSH can be employed as a thiol group-con-
taining probe to screen the electrophilic compounds (17). The
screening method is suitable for the rapid discovery of phase II
detoxifying enzymes inducers from plant, food, and other natural
sources.
558

Methylation to Improving NQO1-Inducing Activity
Extraction, methylation, and isolation
Methods and Materials
The dried roots of S. miltiorrhiza (1 kg) were extracted with 95%
ethanol (8 L · 3) to give a crude ethanol extract. This residue
was dissolved in H2O (1 L) and then extracted successively with
petroleum ether (1 L · 3), dichloromethane (1 L · 3), and n-buta-
nol (1 L · 3). Each solvent was evaporated off under reduced
pressure to yield extracts of petroleum ether, dichloromethane,
and n-butanol. The n-butanol extract of S. miltiorrhiza (6 g) and
K2CO3 (132 g) in 120 mL of dry acetone was added 50 mL of
CH3I. The reaction mixture was heated to reflux for 24 h. After
filtration of the inorganic salts, the solvent was dissolved in
500 mL of H2O and then extracted with dichloromethane
(500 mL · 3). The solvent was washed with isovolumic H2O three
times and evaporated under reduced pressure. The crude methy-
lated product (3.8 g) was subjected to silica gel column chroma-
tography (200–300 mesh, 12 · 120 cm), eluted with petroleum
ether ⁄ ethyl acetate (100:0, 100:1, 100:2.5, 100:5, 100:10, 100:20,
100:50 (v ⁄ v)) to afford seven fractions (Fr. A-G). The Fr. D was
further separated by preparative HPLC using methanol ⁄ H2O as
the mobile phase (95% methanol, flow rate 10 mL ⁄ min) to yield
RA-Me (80.3 mg). The Fr. E was separated by preparative HPLC
using methanol ⁄ H2O as the mobile phase (78% methanol, flow
rate 10 mL ⁄ min) to yield LA-Me (52.5 mg). The Fr. G was sepa-
Materials and chemical
The roots of S. miltiorrhiza were collected at Shanxi Province,
People's Republic of China, in December 2007. The plant material
was identified by the authors, and a voucher specimen (No.
SM071211) had been deposited in the herbarium of School of
Pharmaceutical Sciences, Zhejiang University. GSH was purchased
from Sigma-Aldrich (St. Louis, MO, USA). HPLC-grade acetonitrile
(Merck, Darmstadt, Germany) and formic acid (Tedia, Fairfield, OH,
USA) were utilized for the HPLC analysis. Deionized water was pre-
pared using a Milli-Q system (Millipore, Bedford, MA, USA). All
other chemicals and solvents were of analytical-reagent grade. Sal-
vianolic acid B (SaB), lithospermic acid (LA), and rosmarinic acid
(RA) were purchased from National Institute for the Control of
Pharmaceutical and Biological Products (Beijing, China). Methyla-
tions of salvianolic acid B (SaB-Me), lithospermic acid (LA-Me), and
rosmarinic acid (RA-Me) were isolated from methylated products of
the n-butanol extract of S. miltiorrhiza. The structures of three
methylated compounds were elucidated by NMR spectroscopic
analysis (Bruker Ultrashield Plus 500 MHz spectrometer). Anti-Nrf2
antibody was purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
A
B
C
Figure 1: HPLC chromatograms before (A) and after (B) incubation of the n-butanol extract of S. miltiorrhiza with GSH and the correspond-
ing mass spectrum of each GSH conjugate (C).
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Zhang et al.
A
B
C
Figure 2: HPLC chromatograms before and SIC chromatograms after incubation of SaB (A), LA (B) and RA (C) with GSH and the corre-
sponding mass spectrum of each GSH conjugate.
560
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Methylation to Improving NQO1-Inducing Activity
rated by preparative HPLC using methanol ⁄ H2O as the mobile
phase (68% methanol, flow rate 10 mL ⁄ min) to yield SaB-Me
(120.3 mg).
separated by sodium dodecyl sulfate–polyacrylamide gel electropho-
resis (SDS–PAGE) on a 12% polyacrylamide gel. Proteins were
transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA)
and probed with anti-Nrf2 antibody and then incubated with a
horseradish peroxidase (HRP)–conjugated secondary antibody. Pro-
teins were visualized using ECL western blotting detection reagents
(Millipore, Billerica, MA, USA).
Cell culture
Hepa 1c1c7 murine hepatoma cells were obtained from the Ameri-
can Type Culture Collection (ATCC) and maintained in a-minimum
essential medium supplemented with 0.1% penicillin–streptomycin
and 10% fetal bovine serum (GIBCO, Grand Island, NY, USA). The
cells were incubated in 5% CO2 at 37 ꢀC.
Liquid chromatography–tandem mass
spectrometry (LC–MS) analysis of GSH
conjugates
The n-butanol extract of S. miltiorrhiza and each test compound
were incubated with 1 mM GSH at 37 ꢀC for 2 h in a total volume
of 200 lL of 25 mM Tris–HCl buffer (pH 8.0) (the final concentration
of the n-butanol extract of S. miltiorrhiza and each test compound
were 1.0 mg ⁄ mL and 400 lM, respectively). The reaction products
were analyzed using Thermo Finnigan LCQ Deca XPplus ESI ion trap
mass spectrometer (San Jose, CA, USA) in negative ion mode
equipped with a Agilent 1100 HPLC system (Waldbronn, Germany)
and a Zorbax SB-C18 column (4.6 mm · 250 mm, 5 lm, Agilent
Technologies, USA). The mobile phase consisted of A (HCOOH:
H2O = 0.2:100) and B (HCOOH: CH3CN = 0.2:100). For the n-butanol
extract of S. miltiorrhiza, a gradient program was used according to
the following profile: 0–15 min, 95% A ⁄ 5% B – 70% A ⁄ 30% B;
15–55 min, 70% A ⁄ 30% B – 50% A ⁄ 50% B; 55–65 min, 50%
A ⁄ 50% B – 45% A ⁄ 55% B; 65–70 min, 45% A ⁄ 55% B -100% B.
For SaB, LA, and RA, a 30-min gradient was used from 90%
A ⁄ 10% B to 50% A ⁄ 50% B and maintained 50% A ⁄ 50% B during
the next 10 min. For SaB-Me, LA-Me, and RA-Me, a 30-min gradi-
NQO1 induction assay
NQO1-inducing activity was assessed using Hepa 1c1c7 cells as
described previously (18). Induction of NQO1 activity was deter-
mined by the CD value, which was the concentration required to
double the specific activity of NQO1 in vehicle-treated cells. A ser-
ies of concentrations ranging from 1 to 30 lg ⁄ mL of test sample
were tested for their NQO1-inducing activities and then the CD
value could be calculated. Experiments were carried out three times
on separate occasions.
Western blot analysis
After treatment, Hepa 1c1c7 cells were lysed in 50 mM Tris–HCl
(pH 7.4), 150 mM NaCl, 1% Triton X-100 containing 1 mM phenylm-
ethylsulphonyl fluoride (PMSF) at 4 ꢀC for 30 min and then centri-
fuged at 13 200 g for 10 min to obtain total protein. Fifty
micrograms of protein from total protein lysates were used and
Figure 3: The methylation reaction route.
Chem Biol Drug Des 2011; 78: 558–566
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Zhang et al.
A
B
C
Figure 4: HPLC chromatograms before and SIC chromatograms after incubation of SaB-Me (A), LA-Me (B) and RA-Me (C) with GSH and
the corresponding mass spectrum of each GSH conjugate.
562
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Methylation to Improving NQO1-Inducing Activity
ent was used from 90% A ⁄ 10% B to 100% B and maintained
100% B during the next 10 min. The flow rate was 0.5 mL ⁄ min,
and the column temperature was set at 30 ꢀC. The UV spectra
were recorded at 280 nm. The MS operating parameters were as
follows: collision gas, ultra-high-purity helium (He); nebulizing gas,
high-purity nitrogen (N2); ion spray voltage, )4.5 kV; sheath gas
(N2), five arbitrary units; capillary temperature, 275 ꢀC; capillary
voltage, )15 V; tube lens offset voltage, )30 V. The collision
energy for collision-induced dissociation (CID) was between 30%
and 45%, and the isolation width of precursor ions was 3.0 Th. For
cell lysates, Hepa 1c1c7 cells were treated with 20 lg ⁄ mL of test
compound for 2 h. The cells were lysed and mixed with isovolumic
methanol to denature and precipitate the proteins. After centrifuga-
tion at 14 000 rpm for 10 min, 20 lL supernatant was analyzed by
LC–MS. The mobile phase, gradient program, and MS operating
parameters were the same as described earlier.
according to their mass spectra (Figure 1C). The results were further
validated by the subsequent analysis of GSH conjugations with
three main phenolic acids in the n-butanol extract, SaB, RA, and LA
(with the retention time 26.71, 25.41, and 23.60 min, respectively,
in Figure 1A). After incubation of GSH with SaB, LA, and RA,
respectively, SaB–GSH conjugate, LA–GSH conjugate, and RA–GSH
conjugate could be easily found in their selected ion current (SIC)
chromatograms (Figure 2A–C). We found that all three molecular
weights of GSH conjugates were the molecular weight of test com-
pound plus that of GSH and loss of two protons, which could be
explained that in basic condition, ring A of the compound (take SaB
as an example) would interconvert to a quinone formation and thus
could be attacked by nucleophilic GSH (Figure S1, supporting infor-
mation). Although there were a, b-unsaturated ketone moieties in
the chemical structures of SaB, LA, and RA, they did not react with
GSH through Michael addition because the alkene was not reduced
according to MS analysis, which was confirmed by NMR study. SaB
(4 mg), GSH (40 mg), and NaHCO3 (40 mg) reacted in D2O at 37 ꢀC
1
Results and Discussion
for 2 h and then H NMR spectrum was recorded. As shown in Fig-
ure S2 (supporting information), the alkene proton signals did not
disappear after the reaction.
Reaction of GSH with the n-butanol extract of
S. miltiorrhiza and three main phenolic acids in
it
To screen the electrophilic compounds in Danshen, GSH was
employed to incubate with the n-butanol extract of S. miltiorrhiza
and then the incubation solution was analyzed by LC–MS. In HPLC
chromatogram after the incubation, three extra peaks formed (Fig-
ure 1B), which were identified as SaB–GSH conjugate (peak 1),
RA–GSH conjugate (peak 2), and LA–GSH conjugate (peak 3)
Determination of NQO1-inducing activities and
absorption in cells of the n-butanol extract of
S. miltiorrhiza, SaB, LA, and RA
Next, we tested whether SaB, LA, and RA, three main constituents in
the n-butanol extract of S. miltiorrhiza which could form GSH conju-
gates, had NQO1-inducing activities. In contrast to our presumption,
Figure 5: NQO1-inducing activities before and after methylation of the n-butanol extract of S. miltiorrhiza, SaB, LA and RA.
Chem Biol Drug Des 2011; 78: 558–566
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Zhang et al.
neither the extract nor the three compounds possessed NQO1-induc-
ing activities (the CD values were all larger than 20 lg ⁄ mL), which
might attribute to the hydrophilic property of these compounds and
thus they could not be absorbed in cells. Subsequently, LC–MS analy-
sis of the cell lysates verified our hypothesis. After Hepa 1c1c7 cells
were treated with 20 lg ⁄ mL of SaB, LA, and RA for 2 h, respectively,
we could find neither the compounds nor their GSH conjugates in cell
lysates in the SIC chromatograms (Figure S3, supporting information).
From this point of view, although the compounds conjugated with
GSH potently in vitro, they could not react with the cellular GSH
because of their poor absorption.
large number of carbon signals between d 50.0 and d 60.0, which
represented methoxyl carbons (Figure S4, supporting information),
indicating the methylation carried out to a large extent. Then, SaB-
Me, LA-Me, and RA-Me were isolated from the reaction product.
The reaction route was shown in Figure 3.
Reaction of GSH with SaB-Me, LA-Me, and
RA-Me
Subsequently, we analyzed the GSH conjugations with SaB-Me, LA-
Me, and RA-Me by LC–MS. In HPLC chromatograms after incuba-
tion of LA-Me and RA-Me with GSH, RA-Me–GSH conjugate and
LA-Me–GSH conjugate could easily be found in their SIC chromato-
grams (Figure 4B,C). However, the conjugation of SaB-Me with GSH
was much weaker (Figure 4A). We noticed that all three molecular
weights of GSH conjugates were the molecular weight of test com-
pound plus that of GSH, which meant the alkene was reduced.
Thus, the conjugation might take place in the b position, but not in
ring A (Figure S5, supporting information), which was different from
the reaction mechanism of phenolic acids.
Methylation of the n-butanol Extract of
S. miltiorrhiza and isolation of SaB-Me, LA-Me,
and RA-Me
To improve the liposolubility of phenolic acids in the n-butanol
extract of S. miltiorrhiza, we took methylation reaction of the
extract. 13C NMR spectrum was used to monitor whether the meth-
ylation was complete. After 24-h reaction, we found there were a
Figure 6: SIC chromatograms of the molecular weight of SaB-Me–GSH conjugate, LA-Me–GSH conjugate and RA-Me–GSH conjugate after
Hepa 1c1c7 cells were treated with 20 lg ⁄ mL of each compound.
564
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Methylation to Improving NQO1-Inducing Activity
was almost unchanged after methylation, indicating that the methy-
lated compound could be easily absorbed in cells and conjugate
with the cellular GSH instantaneously. In general, methylation strat-
egy might serve as an effective approach for the improvement of
the NQO1-inducing activities of phenolic acids in Danshen.
Acknowledgments
Figure 7: Nrf2 levels in Hepa 1c1c7 cells after treatment with
20 lg ⁄ mL of each compound for 2 h.
The work was financially supported by Zhejiang Key Science &
Technological Program (2009C13028), Administration of Traditional
Chinese Medicine of Zhejiang Province (2010ZA049) and Zhejiang
Innovation Program for Graduates (YK2009015).
Determination of NQO1-inducing activities and
absorption in cells of the methylated product of
n-butanol extract of S. miltiorrhiza, SaB-Me, LA-
Me and RA-Me
It was found that after methylation, the NQO1-inducing activities of
extract, LA-Me, and RA-Me increased obviously (Figure 5), and the
CD values of the methylated product of n-butanol extract of
S. miltiorrhiza, LA-Me, and RA-Me were 17.48 € 0.98 lg ⁄ mL,
17.86 € 2.34 lg ⁄ mL, and 11.97 € 0.60 lg ⁄ mL, respectively. Inter-
estingly, we found that the NQO1-inducing activities of SaB and
SaB-Me were almost the same. Then, we investigated the absorp-
tions of these three methylated products in cells. After Hepa 1c1c7
cells were treated with 20 lg ⁄ mL of each compound for 2 h, their
GSH conjugates in cell lysates could easily be found in the SIC
chromatograms (Figure 6), indicating all three methylated products
were absorbed in cells.
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Figure S1. The proposed reaction between SaB and GSH under
basic conditions.
1
Figure S2. H NMR spectra before (A) and after (B) incubation of
SaB with GSH.
Figure S3. LC-MS analysis of the cell lysates after Hepa 1c1c7
cells were treated with 20 lg ⁄ mL of SaB, LA and RA for 2 h. (A)
SIC chromatograms of the molecular weight of SaB, LA and RA. (B)
SIC chromatograms of the molecular weight of SaB-GSH conjugate,
LA-GSH conjugate and RA-GSH conjugate.
Figure S4. 13C NMR spectra before (A) and after (B) methylation
of the n-butanol extract of S. miltiorrhiza.
Figure S5. The proposed reaction between SaB-Me and GSH
under basic conditions.
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functionality of any supporting materials supplied by the authors.
Any queries (other than missing material) should be directed to the
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Improving the NQO1-Inducing Activities of Phenolic Acids from Radix
Salvia miltiorrhiza: a Methylation StrategyIn the present study, a
methylation strategy was employed to improve the NQO1 inducing
activities of phenolic acids in Danshen. After methylation, both the
NQO1 inducing activities and the absorptions in cells of the pheno-
lic acids improved obviously without losing their GSH conjugating
abilities.
Supporting Information
Additional Supporting Information may be found in the online ver-
sion of this article:
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