Biotransformation of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production and DPPH free radical

Article abstract of DOI:10.1080/10286020.2012.702756

prim-O-Glucosylcimifugin (PGCN), a highest content chromone in the roots of Saposhnikovia divaricata, was incubated with human intestinal flora (HIF), and two biotransformation products were obtained from the incubated solution by chromatographic methods. The chemical structures of the two biotransformation products were elucidated as cimifugin (CN) and 5-O-methylvisamminol (MVL), respectively, on the basis of NMR and MS data. The biotransformation product CN was formed through a deglucosylation of PGCN by -glucosidase secreted from the HIF, and then the hydroxymethyl group of CN was reduced to lead to occurrence of MVL. All of these compounds were evaluated for their effect on the inhibition of nitric oxide production induced by lipopolysaccharide in macrophage cell line RAW 264.7 and for 1,1-diphenyl-2-picrylhydrazyl free-radical scavenging activity in cell-free bioassay system.

Full text of DOI:10.1080/10286020.2012.702756

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Biotransformation of prim-O-
glucosylcimifugin by human intestinal
flora and its inhibition on NO
production and DPPH free radical
Bo Zhao a , Xin-Bao Yang b c , Xiu-Wei Yang a & Jian-Xun Liu b c
a State Key Laboratory of Natural and Biomimetic Drugs,
Department of Natural Medicines , School of Pharmaceutical
Sciences, Peking University , Beijing , 100191 , China
b Beijing University of Chinese Medicine , Beijing , 100029 , China
c Research Center, Xiyuan Hospital, China Academy of Chinese
Medical Sciences , Beijing , 100091 , China
Published online: 23 Aug 2012.
To cite this article: Bo Zhao , Xin-Bao Yang , Xiu-Wei Yang & Jian-Xun Liu (2012) Biotransformation
of prim-O-glucosylcimifugin by human intestinal flora and its inhibition on NO production
and DPPH free radical, Journal of Asian Natural Products Research, 14:9, 886-896, DOI:
10.1080/10286020.2012.702756
To link to this article: http://dx.doi.org/10.1080/10286020.2012.702756
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Journal of Asian Natural Products Research
Vol. 14, No. 9, September 2012, 886–896
Biotransformation of prim-O-glucosylcimifugin by human intestinal
flora and its inhibition on NO production and DPPH free radical
Bo Zhaoa1, Xin-Bao Yangbc1, Xiu-Wei Yanga* and Jian-Xun Liubc*
aState Key Laboratory of Natural and Biomimetic Drugs, Department of Natural Medicines,
School of Pharmaceutical Sciences, Peking University, Beijing 100191, China; bBeijing University
of Chinese Medicine, Beijing 100029, China; cResearch Center, Xiyuan Hospital, China Academy
of Chinese Medical Sciences, Beijing 100091, China
(Received 19 January 2012; final version received 10 June 2012)
prim-O-Glucosylcimifugin (PGCN), a highest content chromone in the roots of
Saposhnikovia divaricata, was incubated with human intestinal flora (HIF), and two
biotransformation products were obtained from the incubated solution by chromato-
graphic methods. The chemical structures of the two biotransformation products were
elucidated as cimifugin (CN) and 5-O-methylvisamminol (MVL), respectively, on the
basis of NMR and MS data. The biotransformation product CN was formed through a
deglucosylation of PGCN by b-glucosidase secreted from the HIF, and then the
hydroxymethyl group of CN was reduced to lead to occurrence of MVL. All of these
compounds were evaluated for their effect on the inhibition of nitric oxide production
induced by lipopolysaccharide in macrophage cell line RAW 264.7 and for 1,1-
diphenyl-2-picrylhydrazyl free-radical scavenging activity in cell-free bioassay system.
Keywords: Saposhnikovia divaricata; chromones; prim-O-glucosylcimifugin; nitric
oxide; DPPH free radical
1. Introduction
Figure 1) has been defined as index
component in many editions of Chinese
Pharmacopoeia. It has been reported that
PGCN inhibited the proliferation of vas-
cular smooth muscle cells stimulated by
tumor necrosis factor-a in the primary cell
culture from the isolated rat thoracic aorta
[3] and exhibited significant analgesic,
antipyretic, anti-inflammatory, and anti-
platelet aggregation action [4]. These
reports suggest that PGCN may be valuable
anti-inflammatory and chemopreventive
agents. Recently, absorption, distribution,
metabolism, excretion, and toxicity
(ADMET) have been introduced into the
early processes of drug development
instead of serial development because of
some failure in clinical trials due to
Saposhnikovia
divaricata
(Turcz.)
Schischk. (Umbelliferae family) is distrib-
uted widely in northeast China. The dried
roots of S. divaricata (Saposhnikoviae
Radix), called ‘Fang-Feng’ in Chinese, are
well known as a basic traditional Chinese
medicine for thousands of years. Even
today they are widely used in many Chinese
medicine preparations. Saposhnikoviae
Radix has anti-inflammatory and anti-
spasm activities and is used for treating
pyrexia, rheumatism, headache, and con-
vulsion [1]. The Saposhnikoviae Radix
contains about 1% total chromones [2],
among which prim-O-glucosylcimifugin
(PGCN, chemical structure shown in
*Corresponding authors. Email: xwyang@bjmu.edu.cn; liujx0324@sina.com
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2012 Taylor & Francis
http://dx.doi.org/10.1080/10286020.2012.702756
http://www.tandfonline.com

Journal of Asian Natural Products Research
887
8
9
O
O
O
O
O
4
Me
O
OH
7
OH
O
O
OH
2'
O
1"
HO
8a
4a
2
3
6
4'
HO
HO
3'
O
5
OMe
MVL
O
6"
OMe
OMe
CN
OH
PGCN
OH
Figure 1. Chemical structures of PGCN, CN, and MVL and possible biotransformation pathways
of PGCN.
ADMET and pharmacokinetics. It is well
known that human intestinal flora (HIF)
plays an important role in the biotrans-
formation of drugs and/or compounds
administered orally or excreted into bile.
Some drugs and/or compounds are trans-
formed by intestinal flora before absorption
from the gastrointestinal tract. Thus,
intestinal bacteria have been used exten-
sively for the metabolic study in vitro of
natural products, such as flavonoids [5,6],
coumarins [7], triterpenoids [8], and
steroids [9], and numerous important
results have been reported [10]. What is
more interesting is that some drugs and/or
compounds are biotransformed to more
active compounds. For example, rhein
anthrone, the metabolite of rhein produced
by human intestinal bacteria, was shown to
be responsible for the diarrheogenic effect
of rhubarb [11]. Therefore, our research
group has carried out investigations on
the biotransformation of PGCN by HIF.
This paper will describe the isolation and
structural elucidation of two biotransform-
ation products of PGCN by HIF and the
inhibition on nitric oxide (NO) production
in lipopolysaccharide (LPS)-activated
macrophage cell line RAW264.7 and 1,1-
diphenyl-2-picrylhydrazyl (DPPH) free-
radical scavenging activity in cell-free
bioassay system of all compounds.
normal butanol (n-BuOH) extract obtained
at 12h after anaerobic incubation with the
HIF. Two new peaks, together with the peak
of the added PGCN, could be detected in the
HPLC-diode array detector (DAD) chro-
matograms and the amount of the two
biotransformation products increased with
increasing time. For the purpose of obtain-
ing enough of these biotransformation
products, 48h was selected as the incu-
bation time by preliminary experiment,
from which the parent compound PGCN
was undetectable. In addition, HPLC
analysis also showed that when 2.0 mg of
PGCN was added to 10 ml of HIF
incubation solution, the transformation
rate was highest. Based on the above-
mentioned results, optimal culture con-
ditions for the enlarged incubation were
determined. Furthermore, PGCN was also
incubated in general anaerobic medium
(GAM) broth without HIF under the same
conditions, and the results showed that only
the peak of PGCN was detected in the
extracts of the incubation solutions, con-
firming that the two peaks were biotrans-
formation products produced by the HIF. As
described in Section 3.4, the biotransform-
ation products cimifugin (CN) and 5-O-
methylvisamminol (MVL) were yielded.
CN (Figure 1) was obtained as color-
28
less needles with ½aꢀD þ 87.0 (c ¼ 0.3,
CHCl3). The EI-MS showed a molecular
ion peak at m/z 306 [M]þ (base peak).
Comparing the NMR signals of CN with
those of parent PGCN, CN showed the
absence of a glucosyl group but being
attached to C-9 in PGCN; and the signals
of Ha-9 at dH 4.79 (1H, d, J ¼ 14.4 Hz) and
Hb-9 at dH 4.64 (1H, d, J ¼ 14.4 Hz) were
2. Results and discussion
2.1 Isolation and identification of
biotransformation products
HPLC analysis of the samples obtained at
different incubation time intervals showed
that the obvious peaks were detected in the

888
B. Zhao et al.
shifted upfield to dH 4.49 (2H, br s); and
the signal of C-9 at dC 67.3 was shifted
upfield to dC 61.0, corresponding to the
loss of one glucosyl group of PGCN. This
was also supported by the good agreement
of its NMR data with those of CN [12–14].
The 1-hydroxy-1-methylethyl group was
a-orientated according to the study about
The standard calibration curves were
described by the following equations:
y ¼ 14.359x þ 76.811 (r 2 ¼ 0.9999) for
PGCN, y ¼ 37.885x þ 13.426 (r 2
¼
0.9994) for CN, and y ¼ 30.972x þ
134.06 (r 2 ¼ 0.9999) for MVL.
The method was fully validated for its
specificity, linearity, lower limits of detec-
tion (LLODs), lower limits of quantifi-
cation (LLOQs), accuracy, precision,
and extraction recovery. The LLODs of
PGCN, CN, and MVL were 0.21, 0.14, and
0.13 mg/ml, respectively. And their
LLOQs were 0.62, 0.43, and 0.40 mg/ml,
respectively. The results of precision and
accuracy are listed in Table 1 which
indicated that the proposed methods are
sufficient for determination of the analytes
PGCN, CN, and MVL in the HIF
incubation system.
·
½aꢀD value of CN [14].
MVL (Figure 1) was also obtained as
20
colorless needles with ½aꢀD þ 89.7 (c 0.3,
CHCl3). The EI-MS showed a molecular
ion peak at m/z 290 [M]þ (base peak). The
NMR data of MVL were very similar
to those of CN except for the signals of
C2-Me at dH 2.28 (3H, s) and dC 19.8 in
MVL instead of C2-CH2OH group at dH
4.49 (2H, br s) and dC 61.0 in CN. The data
of NMR and ½aꢀD of MVL were in good
agreement with those of MVL [14].
Specificity was established by the lack
of interference peaks at the retention time
for PGCN, CN, and MVL. The retention
times of the three compounds were 4.9 min
for PGCN, 7.5 min for CN, and 13.9 min
for MVL (Figure 2).
2.2 Performance of the tested method
The standard calibration curves were linear
at the concentration range of 20–
1000 mg/ml for PGCN, CN, and MVL in
the heat-inactivated incubation solution.
Table 1. Precision and accuracy of analytes PGCN, CN, and MVL determination in the heat-
inactivated HIF incubation solution (n ¼ 3).
Concentration (mg/ml)
Analyte
PGCN
Added Measured (mean ^ SD) Precision (RSD, %) Accuracy (%)
Intra-day
Inter-day
Intra-day
Inter-day
Intra-day
Inter-day
50
200
500
50
51.62 ^ 0.35
200.38 ^ 1.80
479.45 ^ 6.62
48.16 ^ 0.47
199.46 ^ 2.93
487.45 ^ 9.55
51.74 ^ 1.26
216.84 ^ 3.99
484.35 ^ 5.96
54.43 ^ 4.84
193.04 ^ 3.57
464.10 ^ 17.17
46.41 ^ 4.32
203.54 ^ 3.95
517.25 ^ 13.55
52.47 ^ 1.23
190.78 ^ 3.51
471.7 ^ 9.25
0.68
0.90
1.38
0.97
1.47
1.96
2.35
1.84
1.23
0.89
1.85
3.70
0.93
1.94
2.62
2.34
1.84
1.96
103.24
100.19
95.89
96.32
99.73
97.49
200
500
50
200
500
50
200
500
50
CN
103.48
108.42
96.87
108.86
96.52
102.75
92.82
MVL
200
500
50
200
500
101.77
103.45
104.94
95.39
94.34

Journal of Asian Natural Products Research
889
Figure 2. Typical chromatograms of PGCN, CN, and MVL: (A) blank incubation solution;
(B) standard solutions of PGCN (1524.14 mg/ml), CN (1279.55 mg/ml), and MVL (675.12mg/ml). The
incubation solution obtained at 1 h (C), 12 h (D), and 48 h (E), respectively, after biotransformation of
PGCN by HIF. The retention time of PGCN, CN, and MVL was 4.9, 7.5, and 13.9 min, respectively.

890
B. Zhao et al.
Table 2. Extraction recovery of PGCN, CN, and MVL from the heat-inactivated HIF incubation
solution (n ¼ 3).
Analyte
Concentration (mg/ml)
RSD (%)
Recovery (%) (mean ^ SD)
PGCN
50
200
500
50
200
500
50
3.47
3.84
0.96
4.17
2.67
2.86
3.42
2.94
3.92
82.70 ^ 4.20
82.96 ^ 4.63
81.24 ^ 1.18
85.25 ^ 4.89
86.35 ^ 3.09
84.00 ^ 3.41
84.05 ^ 4.07
85.09 ^ 3.45
85.77 ^ 4.56
CN
MVL
200
500
Extraction recovery experiments were
carried out. The extraction recoveries from
the heat-inactivated HIF incubation sol-
ution were 81.24–86.35%. The results of
recovery studies are shown in Table 2.
after prolonged incubation, suggesting that
it was further converted to other product(s)
and was a biotransformation intermediate.
The product MVL was produced gradually
after 24 h of incubation, suggesting that
MVL was a biotransformation product
derived from CN. Based on the character-
istics of chemical structures of PGCN, CN,
MVL, and the above-mentioned time-
course curve, the biotransformation course
of PGCN by HIF could be primarily
speculated.
2.3 Time course and ability of the
biotransformation of PGCN by HIF
Figure 3 shows the time course of
biotransformation of PGCN by a HIF. An
anaerobic incubation of PGCN with the
HIF indicated that PGCN was almost
completely consumed 8 h after the start of
incubation. At the early stage (0.25 h) of
incubation, product CN can be detected
which reached a maximum concentration
at 12 h, whereas it decreased gradually
As shown in Figure 1, PGCN was first
deglucosylated by b-glucosidase secreted
from HIF, leading to the occurrence of CN.
After that, biotransformation involved
reduction of hydroxymethyl group in CN
backbone to produce MVL.
1000
800
600
400
200
0
PGCN
MVL
CN
0
4
8
12
16
20
24
Time (h)
28
32
36
40
44
48
Figure 3. Time course of biotransformation of PGCN by HIF (n ¼ 3, mean ^ SD).

Journal of Asian Natural Products Research
891
2.4 DPPH free-radical scavenging
activity of PGCN, CN, and MVL
showing the level of nitrite production was
measured by the Griess method [19]. The
cell viability in 3-(4,5-dimethyl-2-thiazo-
lyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT) assay was found to exceed 95% at
the treated concentrations (6.25–200 mM)
of all the assay compounds, suggesting no
cytotoxicity due to PGCN, CN, and MVL
exposure. The parent compound PGCN
and its biotransformation products CN and
MVL slightly inhibited the NO production
in LPS-activated RAW264.7 cells,
whereas the inhibitory rates of both CN
and MVL were always higher than those of
PGCN (Table 3).
DPPH is a stable free radical and accepts an
electron to become a stable molecule [15]
and has been widely used to evaluate the
free-radical scavenging effects of various
antioxidant substances [16–18]. In this
assay, the free-radical scavenging activity
of PGCN, CN, and MVL, compared
with the positive control such as Vc, was
determined using the DPPH method. Vc
showed DPPH radical scavenging activity
in a concentration-dependent manner.
PGCN, CN, and MVL exhibited DPPH
radical scavenging activity at different
degrees in a concentration-dependent man-
ner (Figure 4). Among assay compounds,
CN showed significant activity with IC50
value of 178.20 mM. On the other hand, the
scavenging activity of the biotransform-
ation products CN and MVL was always
stronger than that of the parent compound
PGCN, but weaker than that of Vc.
In conclusion, PGCN can be converted
to CN and MVL by HIF, and the inhibition
on the NO production in LPS-activated
RAW264.7 cells and DPPH radical
scavenging activity in cell-free bioassay
system of biotransformation products were
always stronger than those of the parent
compound PGCN. A high abundance
of the biotransformation products of
PGCN in the human intestine shows that
its bioavailability depends largely on its
biotransformation by the gut microflora.
On the other hand, the biotransformation
product CN was a well-absorbed com-
pound in the human Caco-2 cell mono-
layer model [20]. The potential importance
of the gut microflora in explaining the
biological effects of PGCN is emphasized.
2.5 Inhibitory effects of PGCN, CN,
and MVL on the production of NO
The inhibition of PGCN, CN, and MVL on
NO production was evaluated by LPS-
stimulated murine macrophage cell line,
RAW264.7 cell. L-N 6-(1-Iminoethyl)-
lysine (L-NIL) was used as a positive
control in this assay. The total activity
100
80
60
40
20
0
PGCN
CN
MVL
700
Vc
800
0
100
200
300
400
500
600
Concentration (µM)
Figure 4. Inhibition of PGCN and its two biotransformation products (n ¼ 3, mean ^ SD). Vc was
as a positive control.

892
B. Zhao et al.
Oxoid BR0055 anaerobic indicator was
used. MCO-15 AC carbon dioxide (CO2)
incubator (Sanyo Electric Co. Ltd, Osaka,
Japan), DL-CJ-2NDII clean bench (Beijing
Donglian Electronic & Technology Devel-
opment Co. Ltd, Beijing, China), and
Automated Microplate Reader (Multiskan
MK3, Thermo Labsystems, Vantaa, Fin-
land) were used. An MD-200-2 sample
concentrator (Hangzhou Allsheng Instru-
ments Co. Ltd, Hangzhou, China) was used
for biosample concentration. Silica gel
200–300 mesh (Qingdao Marine Chemical
Factory, Qingdao, China) was used for
column chromatography (CC). HPLC
analysis was done on a Agilent 1200
infinity series instrument equipped with a
1260 quaternary gradient system, a 1260
injector, a 1260 DAD, a 1260 thermostated
column oven, and ChemStation for LC 3D
system (Revision B.04.03) for data collec-
tion. Semi-preparative HPLC was done on
a CXTH HPLC system (Beijing Chuang
Xin Tong Heng Sci. Technol. Co. Ltd,
Beijing, China) with two P3050 pumps,
UV3000 UV–vis detector, and a A1359
liquid handler. HPLC-grade acetonitrile
(MeCN) and methanol (MeOH) were
purchased from Fisher Chemicals (Fair
Lawn, NJ, USA). Deionized water (H2O)
was purchased from Wahaha Co. Ltd
(Hangzhou, China). Milli-Q grade H2O
was used for assay. Other reagents were of
analytical grade and were purchased from
Beijing Fine Chemicals (Beijing, China).
Reference compound PGCN was iso-
lated and purified from the Saposhnikoviae
Radix by chromatographic methods
described in a previous report [12] and
was identified on the basis of spectral data,
including optical rotation, IR, UV, 1H
NMR, 13C NMR, and MS. The purity by
HPLC–UV analysis was above 99%.
Table 3. Inhibition (%) of PGCN, CN, and
MVL at concentrations of 50–200 mM on NO
production in LPS-activated RAW264.7 cells
(n ¼ 3).
Compound
50 mM
100 mM
200 mM
PGCN
CN
MVL
L-NILa
3.54
10.03
10.35
64.67
5.52
16.95
21.89
81.69
7.22
18.38
38.64
98.71
a L-NIL was used as a positive control.
The results provided a firm basis for
evaluating the anti-inflammatory proper-
ties of PGCN and their potential value as
therapeutic agents.
3. Experimental
3.1 General experimental procedures
Melting point was measured on a X-4
micro-melting point apparatus (Beijing
Taike Instrument Co. Ltd, Beijing, China)
and is uncorrected. Optical rotation was
measured on an Autopol III polarimeter
(Rudolph Research Analytical, Flanders,
NJ, USA) with MeOH or CHCl3 as solvent.
UV spectra were acquired on a Cary 300
UV–vis spectrophotometer (Varian, Inc.,
Palo Alto, CA, USA) in EtOH solution. IR
spectra were recorded on a Nexus-470 FT-
IR spectrometer (Thermo Nicolet, Inc.,
Madison, WI, USA) as KBr disks. EI-MS
data were obtained on a TRACE 2000 GC-
MS spectrometer (Thermo Finnigan, San
Jose, CA, USA), and ESI-MS data were
acquired using an MDS SCIEX QSTAR
mass spectrometer (Applied Biosystems/
MDS Sciex., Foster City, CA, USA)
equipped with an electrospray ionization
(ESI) source. NMR spectra were obtained
on a Bruker AV-400 spectrometer (Bruker
¨
BioSpin AG Facilities, Fallanden, Switzer-
land; 400 MHz for 1H NMR and 100 MHz
for 13C NMR) with trimethylsilane as an
internal standard. A Thermo Scientific
1029 Forma Anaerobic System (Forma
Scientific, Inc., Marietta, OH, USA) was
used to create anaerobic conditions, and
Sodium thioglycolate, L-cysteine
hydrochloride, and glucose were pur-
chased from Sinopharm Chemical Reagent
Co. Ltd (Beijing, China). Tryptone, beef
extract, beef liver extract powder, digest-
ibility serum powder, soya peptone, yeast

Journal of Asian Natural Products Research
893
extract, and proteose peptone were pur-
chased from Beijing Shuangxuan Micro-
organism Medium Product Factory
(Beijing, China). Dulbecco’s modified
Eagle’s medium (DMEM) and fetal bovine
serum (FBS) were purchased from
GibcoTM (Grand Island, NY, USA). Peni-
cillin and streptomycin were purchased
from Huabei Pharmaceutical Group Co.
Ltd (Shijiazhuang, China). L-Ascorbic acid
(Vc), LPS, DPPH, L-NIL, MTT, and Griess
reagent were purchased from Sigma-
Aldrich Co. (St Louis, MO, USA).
previously [5,6,21]. The obtained GAM
was dispensed in tubes, autoclaved at
0.07 MPa at 1158C for 30 min, and stored
at 48C or was immediately used. HIF was
prepared as described in our report [5,21].
3.4 Incubation and isolation of
biotransformation products
PGCN (1 g) was dissolved in 5 ml of MeOH
and added to 1000ml of HIF solution. The
mixture was then anaerobically incubated
for 48h at 378C. After terminating the
incubation, the mixture was extracted with
1000mlwater-saturatedn-BuOHfivetimes.
The combined n-BuOH solution was
evaporated under reduced pressure to give
a residue (5.4g). The residue was subjected
to silica gel CC eluting with gradient
chloroform–MeOH (19:1 and 9:1) and
purified by semi-preparative HPLC using
MeOH–H2O (1:1) as the eluent to yield
products CN (280mg) and MVL (150mg).
The murine macrophage cell line,
RAW264.7, was obtained from the Cell
Resource Center, IBMS, CAMS/PUMC
(Beijing, China). 96-Well plate (Corning
Costar #3599) was purchased from
Corning Inc. (Cambridge, MA, USA).
3.2 HPLC conditions
HPLC analysis was carried out on an
analytical DiamonsilTM ODS C18 column
(250 £ 4.6 mm, 5 mm) using an Agilent
1200 infinity series instrument equipped
withathermostatedcolumnovenoperatedat
308C. The gradient solvent system consisted
of MeOH (A) and H2O (B). The elution
condition was 50% A (v/v) from 0 to 8 min,
and then linearly changed to 90% A (v/v) at
12min, isocratic elution with 90% A (v/v)
from 12 to 15min, and linearly changed to
50% (v/v) from 15 to 18min. The flow rate
was 1.0 ml/min, the detection wavelength
was at 254 nm, and the injection volume was
10ml. The purification of biotransformation
products by preparative HPLC was carried
out by a CXTH HPLC system using a
Daisogel C18 column (250 £ 30 mm,
10mm) with MeOH–H2O (1:1, v/v) as the
eluent system. The flow rate was 15 ml/min,
the detectionwavelength was at254 nm, and
the injection volume was 1 ml.
3.4.1 prim-O-Glucosylcimifugin
28
A white amorphous powder; ½aꢀD þ 7.0
(c ¼ 0.3, MeOH); UV lEtOH (nm) (log 1):
229 (3.81), 244 (3.82), 250 (3.62), and 293
(4.18); IR (KBr) nmax (cm21): 3384, 1659,
1623, 1607, 1471, 1385, 1094, and 857;
1H NMR (400 MHz, CD3OD) d: 6.66
(1H, s, H-8), 6.41 (1H, s, H-3), 4.80 (1H,
t, J ¼ 8.4 Hz, H-20), 4.79 (1H, d, J ¼
14.4 Hz, Ha-9), 4.64 (1H, d, J ¼ 14.4 Hz,
Hb-9), 4.45 (1H, d, J ¼ 7.6 Hz, H-100), 3.97
(3H, s, 5-OMe), 3.91 (1H, d, J ¼ 11.6 Hz,
Ha-600), 3.70 (1H, dd, J ¼ 11.6 and 3.8 Hz,
Hb-600), 3.38 (2H, br d, J ¼ 8.4 Hz, Ha-30,
Hb-30), 3.28–3.41 (4H, m, H-200, H-300, H-
400, H-500, overlapped), 1.33 (3H, s, 40-Me),
and 1.27 (3H, s, 40-Me); 13C NMR
(100 MHz, CD3OD) d: 165.1 (C-2), 111.0
(C-3), 179.6 (C-4), 112.4 (C-4a), 167.1 (C-
5), 118.5 (C-6), 161.3 (C-7), 94.6 (C-8),
157.0 (C-8a), 67.3 (C-9), 92.7 (C-20), 28.8
(C-30), 72.3 (C-40), 25.4 (C40-Me), 25.5
(C40-Me), 61.1 (5-OMe), 104.1 (C-10),
75.0 (C-20), 78.2 (C-30), 71.6 (C-40), 78.0
3.3 Preparation of the GAM broth
and HIF
The composition and preparation of
GAM broth were reported as described

894
B. Zhao et al.
(C-50), and 62.8 (C-60); ESI-MS m/z 469
[M þ H]þ, 959 [2M þ Na]þ.
the solvent, respectively. Calibration
samples were prepared in heat-inactivated
nutrient medium of HIF at concentrations
of 20–1000 mg/ml for PGCN, CN, and
MVL, respectively. Calibration curves
were obtained by plotting peak area versus
PGCN, CN, and MVL concentrations
in the standard sample. Quality control
samples in heat-inactivated nutrient med-
ium of HIF were prepared at low, medium,
and high concentration levels of 50, 200,
and 500 mg/ml for PGCN, CN, and MVL,
respectively. The nutrient medium sol-
ution standards were prepared using
PGCN-, CN-, and MVL-free nutrient
medium solution.
3.4.2 Cimifugin
Colorless needles (E ¼ tOH), mp 110–
28
1128C; ½aꢀD þ 87.0 (c ¼ 0.3, CHCl3); UV
lEtOH (nm) (log 1): 229 (4.28), 244 (4.12),
251 (4.10), and 292 (4.00); IR (KBr) nmax
(cm21): 3376, 1657, 1620, 1581, 1470,
1381, 1097, and 855; 1H NMR (400 MHz,
CDCl3) d 6.47 (1H, s, H-8), 6.26 (1H, s,
H-3), 4.72 (1H, t, J ¼ 8.8 Hz, H-20), 4.49
(2H, br s, H2-9), 3.93 (3H, s, 5-OMe), 3.26
(2H, br d, J ¼ 8.8 Hz, H-30), 1.37 (3H, s,
40-Me), and 1.25 (3H, s, 40-Me); 13C NMR
(100 MHz, CDCl3) d: 164.6 (C-2), 109.5
(C-3), 177.5 (C-4), 112.2 (C-4a), 165.3 (C-
5), 117.2 (C-6), 159.5 (C-7), 93.8 (C-8),
155.9 (C-8a), 61.0 (C-9), 91.4 (C-20), 27.8
(C-30), 71.6 (C-40), 24.3 (C40-Me), 26.2
(C40-Me), and 61.2 (5-OMe); EI-MS m/z
306 [M]þ (base peak), 247, 229.
3.6 Sample preparation
The incubation samples (10 ml) of PGCN
with HIF were extracted five times with
10 ml of n-BuOH. The n-BuOH extracts
were combined and then evaporated to
dryness under a gentle stream of nitrogen
gas at 458C. The residue was dissolved in
2.0 ml of MeOH with vortexing, which
also acted as a deproteinization agent. The
MeOH solution was mixed in vortexer for
1 min and centrifuged at 16,000g for
10 min. The supernatant was collected
and filtered through a 0.45-mm filter. A
10 ml aliquot of the supernatant liquor was
subjected to the HPLC system for analysis.
3.4.3 5-O-Methylvisamminol
Colorless needles (EtOAc–hexane), mp
28
140–1418C; ½aꢀD þ 89.7 (c ¼ 0.3,
CHCl3); UV lEtOH (nm) (log 1): 232
(4.26), 244 (4.24), 252 (4.11), and 289
(3.98); IR (KBr) nmax (cm21): 3383, 1657,
1620, 1610, 1596, 1467, 1388, 1098, and
1
850; H NMR (400 MHz, CDCl3) d: 6.53
(1H, s, H-8), 6.01 (1H, s, H-3), 4.73 (1H, dd,
J ¼ 8.8 and 8.4Hz, H-20), 3.94 (3H, s, 5-
OMe), 3.25 (2H, dd, J ¼ 8.8 and 8.4 Hz, H-
30), 2.28 (3H, s, H3-9), 1.36 (3H, s, 40-Me),
and 1.24 (3H, s, 40-Me); 13C NMR
(100MHz, CDCl3) d: 163.4 (C-2), 111.4
(C-3), 177.1 (C-4), 112.0 (C-4a), 164.2 (C-
5), 117.1 (C-6), 159.8 (C-7), 93.9 (C-8),
156.0 (C-8a), 19.8 (C-9), 91.2 (C-20), 27.8
(C-30), 71.8 (C-40), 24.1 (C40-Me), 26.0
(C40-Me), and 61.1 (5-OMe); EI-MS m/z
290 [M] þ (base peak), 231, 213.
3.7 Validation study
The method was fully validated for its
specificity, linearity, LLODs, LLOQs,
accuracy, precision, and extraction recov-
ery. Specificity was established by the
lack of interference peaks at the retention
time for PGCN, CN, and MVL. Linearity
was tested at six levels of concentrations
covering the range of 20–1000 mg/ml for
PGCN, CN, and MVL, r 2 values of which
were 0.9999, 0.9994, and 0.9999, respect-
ively. The LLOD was defined as the
concentration that produced a signal-to-
noise ratio of 3:1. The LLOQ was
3.5 Preparation of stocks, calibration,
and quality control samples
Stock solutions (2.0 mg/ml) of PGCN, CN,
and MVL were prepared with MeOH as

Journal of Asian Natural Products Research
895
determined as the lowest concentration on
the calibration curve for which the assay
precision relative standard deviation (RSD)
was lower than 10% and the accuracy was
between 80% and 120%. The intra-day
variability was determined by assaying
three replicates on the same day, and the
inter-day variability was determined by
assaying three replicates on three consecu-
tive days. The precision was expressed as
the RSD. The accuracy was determined by
comparing the calculated concentration
(obtained from the calibration curve) with
the theoretical concentration. The extrac-
tion recoveries were determined at three
concentrations by comparing peak areas
extracted from heat-inactivated HIF med-
ium solution with those of the same
amounts of standard solutions in MeOH.
microtiter plate. The reaction mixtures
were incubated for 30 min in a dark room
at 258C. Free-radical scavenging capacity
was read spectrophotometrically by moni-
toring the decrease in absorbance at
540 nm using a UVmax Kinetic Microplate
Reader. Finally, the percentage of
DPPH free-radical scavenging activity
was calculated with the following
equation:
DPPH free–radical scavening effect ð%Þ
ðA0 2 A1Þ
¼
£ 100%;
A0
where A0 is the absorbance of the control
and A1 is the absorbance of the samples
and the standards.
3.10 Cell culture and nitrite
determination
3.8 Time course of the
biotransformation of PGCN by HIF
The RAW264.7 cells were cultured in
DMEM supplemented with 10% FBS in a
378C humidified incubator with 5% CO2
prior to seeding of 96-well plates for the
designated experiments and were seeded at
a density of 1.5 £ 106 cells/ml in 100 ml
aliquots in 96-well culture plates. After 12-
h incubation, the cells were treated with
LPS (1 mg/ml) and various concentrations
of assay compounds (6.25, 12.5, 25, 50,
100, and 200 mM), and the plates were
incubated for 20 h. Then, the 100 ml of cell-
free supernatant was collected, which was
mixed with 100 mL of Griess reagent (1%
sulfanilamide and 0.1% naphthyl ethylene
diamine dihydrochloride in 5% H3PO4)
and incubated at room temperature for
15 min to form a purple azodye [22]. The
absorption at 540 nm was determined by a
Microplate Reader. Cell viability (.95%)
was determined with the MTT assay.
A biotransformation system consists of a
HIF (1 ml) and GAM solution (9 ml)
containing 2 mg of PGCN, and was
anaerobically incubated at 378C. The
biotransformation reaction was stopped at
0.25, 0.5, 1, 2, 4, 8, 12, 24, 36, and 48 h
after the incubation and the system was
mixed with 10 ml of EtOAc. The incu-
bation samples were treated and analyzed
as described in Section 3.6. All the
incubation experiments were carried out
in three replicates at each time point.
3.9 DPPH free-radical scavenging
activity
The free-radical scavenging activity of
PGCN, CN, and MVL was determined
using DPPH as a reagent with Vc used as
standards as described in the previous
report [16–18]. In brief, 100 ml of 160 mM
DPPH free radical in anhydrous ethanol
and 100 ml of each anhydrous ethanol
solution of PGCN, CN, MVL, and Vc at
concentrations of 12.5, 25, 50, 100, 200,
400, and 800 mM were mixed in a 96-well
Acknowledgments
This project was financially supported in part by
the National Sciences and Technology Program
(2011BAI07B08) of China, ‘Major New

896
B. Zhao et al.
Medicine Project’ in Megaprojects of Science
Research of China (2009ZX09301-010), and
Beijing Municipal Special-Purpose Science
Foundation of China (Z0004105040311).
[9] X.W. Yang, Z.T. Xing, J.R. Cui, Y.L. Fan,
R. Zhao, and X.G. Li, J. Peking Univ.
(Health Sci.) 33, 199 (2001).
[10] X.W. Yang, M.R. Hao, and M. Hottori,
Metabolite Analysis for Chemical Con-
stituents of Traditional Chinese Medicine
(China Medical-Pharmaceutical Science
and Technology Publishion House,
Beijing, 2003), p. 265.
Note
1. These authors contributed equally to this
work.
[11] G. Nijis, P. de Witte, and K. Geboes, Eur.
J. Pharmacol. 239, 241 (1993).
[12] B. Zhao, X.B. Yang, X.W. Yang, and
L.X. Zhang, China J. Chin. Mater. Med.
35, 1569 (2010).
[13] Y.Y. Jiang, B. Liu, R.B. Shi, and G.Z. Tu,
Acta Pharm. Sin. 42, 505 (2007).
References
[1] Chinese Pharmacopoeia Commission,
Pharmacopoeia of the People’s Republic
of China (China Medical Science and
Technology Press, Beijing, 2010), Vol. I,
p. 140.
[2] Z.M. Han, R.Y. Yang, Y.H. Wang,
M. Han, W.W. Kong, and L.M. Yang,
Lishizhen Med. Mater. Med. Res. 19, 3035
(2008).
[14] H. Sasaki, H. Taguchi, T. Endo, and
I. Yosioka, Chem. Pharm. Bull. 30, 3555
(1982).
¨
[15] A. Kabouche, Z. Kabouche, M. Oztu¨rk,
U. Kolak, and G. Topcu, Food Chem. 102,
1281 (2007).
[3] L. Wang, R.X. Liang, Y. Cao, and
J.X. Ye, China J. Chin. Mater. Med. 33,
2157 (2008).
[4] B.Y. Xue, W. Li, L. Li, and Y.Q. Xiao,
China J. Chin. Mater. Med. 25, 297
(2000).
[5] T.T. Xu, X.W. Yang, B. Wang, W. Xu,
Y.Y. Zhao, and Q.Y. Zhang, Planta Med.
75, 483 (2009).
[6] X.W. Yang, J.Y. Zhang, W. Xu, J. Li, and
W.Q. Zhang, Acta Pharm. Sin. 40, 717
(2005).
[7] P. Zhang and X.W. Yang, J. Asian Nat.
Prod. Res. 11, 371 (2009).
[8] X.W. Yang, J. Zhao, J.R. Cui, and
W. Guo, J. Peking Univ. (Health Sci.)
36, 31 (2004).
[16] F.R. Wang, X.W. Yang, Y. Zhang,
J.X. Liu, X.B. Yang, Y. Liu, and R. Shi,
J. Asian Nat. Prod. Res. 13, 319 (2011).
[17] N. Wang and X.W. Yang, J. Asian Nat.
Prod. Res. 12, 1044 (2010).
[18] O.P. Sharma and T.K. Bhat, Food Chem.
113, 1202 (2009).
[19] L.C. Green, D.A. Wagner, J. Glogowski,
P.L. Skipper, and J.S. Wishnok, Anal.
Biochem. 126, 131 (1982).
[20] B. Zhao, X.B. Yang, X.W. Yang, Q. Wu,
Y. Wang, L.X. Zhang, and W. Xu, Planta
Med. 77, 1531 (2011).
[21] X.W. Yang and W. Xu, China J. Chin.
Mater. Med. 36, 19 (2011).
[22] M.S. Kyung, H.K. Yang, and S.P. Wan,
Biol. Pharm. Bull. 27, 538 (2004).