Isolation of two new C-glucofuranosyl isoflavones from Pueraria lobata (Wild.) Ohwi with HPLC-MS guiding analysis

Article abstract of DOI:10.1080/10286021003745445

A profiling analysis of the total isoflavone extract of the root of Pueraria lobata (Wild.) Ohwi was performed using HPLC coupled with ESI-ion trap mass spectrometry. A total of seven isoflavones were identified according to their retention times, UV, MS data, and comparing with the literature data. Among them, two proposed new compounds were isolated and their structures were determined to be 8-C-α-glucofuranosyl-7,4'-dihydroxyisoflavone and 8-C-β-glucofuranosyl-7,4'-dihydroxyisoflavone, named as neopuerarin A (7) and neopuerarin B (6), on the basis of chemical and spectral analyses.

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Isolation of two new C-glucofuranosyl
isoflavones from Pueraria lobata (Wild.)
Ohwi with HPLC–MS guiding analysis
Hai-Jiang Zhang a , Xiao-Ping Yang b & Kui-Wu Wang c
a College of Life Science and Chemical Engineering, Huaiyin
Institute of Technology , Huai'an, 200023, China
b College of Pharmaceutical Science and Technology, Tianjin
University , Tianjin, 300072, China
c College of Food Science Biotechnology and Environmental
Engineering, Zhejiang Gongshang University , Hangzhou, 310035,
China
Published online: 21 Apr 2010.
To cite this article: Hai-Jiang Zhang , Xiao-Ping Yang & Kui-Wu Wang (2010) Isolation of two new C-
glucofuranosyl isoflavones from Pueraria lobata (Wild.) Ohwi with HPLC–MS guiding analysis, Journal
of Asian Natural Products Research, 12:4, 293-299, DOI: 10.1080/10286021003745445
To link to this article: http://dx.doi.org/10.1080/10286021003745445
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Journal of Asian Natural Products Research
Vol. 12, No. 4, April 2010, 293–299
ORIGINAL ARTICLE
Isolation of two new C-glucofuranosyl isoflavones from Pueraria lobata
(Wild.) Ohwi with HPLC–MS guiding analysis
Hai-Jiang Zhanga*, Xiao-Ping Yangb and Kui-Wu Wangc
aCollege of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huai’an
200023, China; bCollege of Pharmaceutical Science and Technology, Tianjin University, Tianjin
300072, China; cCollege of Food Science Biotechnology and Environmental Engineering, Zhejiang
Gongshang University, Hangzhou 310035, China
(Received 8 December 2009; final version received 2 March 2010)
A profiling analysis of the total isoflavone extract of the root of Pueraria lobata (Wild.)
Ohwi was performed using HPLC coupled with ESI-ion trap mass spectrometry. A total
of seven isoflavones were identified according to their retention times, UV, MS data, and
comparing with the literature data. Among them, two proposed new compounds were
isolated and their structures were determined to be 8-C-a-glucofuranosyl-7,40-
dihydroxyisoflavone and 8-C-b-glucofuranosyl-7,40-dihydroxyisoflavone, named as
neopuerarin A (7) and neopuerarin B (6), on the basis of chemical and spectral analyses.
Keywords: Pueraria lobata; kudzu; isoflavone; HPLC–MS; neopuerarin A;
neopuerarin B
1. Introduction
(Figure 1) [1,2]. In this paper, HPLC–MS
analysis of the commercial total isoflavone
extract of P. lobata was performed and
two proposed new C-glucofuranosyl iso-
flavones were found. They were then
isolated and their structures were eluci-
dated by chemical and spectral analysis.
Mass spectrometry (MS) is one of the most
sensitive techniques for the rapid identifi-
cation of compounds. HPLC–MS analysis
for the natural product extract could
provide very useful structural information
on the constituents and be suitable for
guiding the subsequent isolation for the
target compounds. It is especially helpful
for the isolation of the new constituents,
particularly at minor or trace level in the
plants so that much time-consuming and
tedious useless work could be avoided.
Pueraria lobata (Wild.) Ohwi (kudzu)
is a very important Chinese traditional
medicine and has been used as antipyretic,
diaphoretic, and antiemetic agents for
many centuries in China. The main active
compounds in P. lobata are believed to be
the isoflavones. Up to now, many iso-
flavones have been isolated from this plant
2. Results and discussion
2.1 HPLC–MS analysis
The HPLC–MS conditions for the total
isoflavone extract of P. lobata were
optimized as described in Section 3.2 and
the HPLC–MS chromatograms are shown
in Figure 2. Based on mass spectral and
UV spectral data (see Table 1), and
comparing with the reference samples
and the literature data [1–6], seven main
compounds were identified as shown in
Table 1.
*Corresponding author. Email: zhanghj3@hyit.edu.cn
ISSN 1028-6020 print/ISSN 1477-2213 online
q 2010 Taylor & Francis
DOI: 10.1080/10286021003745445
http://www.informaworld.com

294
H.-J. Zhang et al.
R1
H
R2
H
R3
H
H
H
H
H
R4
H
H
H
H
H
R5
H
R3
Daidzein
Daidzin
O
O
H
Glc
H
H
R2O
Formononetin
H
CH3
H
Genistein
Genistin
OH
H
R4
OH Glc
H
Genistein
8-C-glucoside
R1
OH
H
Glc
Glc
H
H
H
OR5
Puerarin
PG-1
H
H
H
H
H
H
H
H
H
Glc OH
Glc: β-pyranoglucose
PG-3
Glc OCH3
Figure 1. Structures of some isoflavones in the root of P. lobata.
As literatures reported [3–5], the
neutral loss of 120 amu from the deproto-
nated ion in negative mode is believed to
be the characteristic of C-glycoside and
could be used for the differentiation from
O-glycoside, the typically neutral loss of
which is 162 amu. Therefore, all the peaks
in Figure 2 were identified as the
C-glycosides except for peak 4, which
showed a neutral loss of 162 amu.
UV spectra. Comparing with the literature
data [1,2], peak 5 was identified as
genistein 8-C-glycoside, while peak 1
was identified as PG-1.
From the MSn spectra of peak 3, the
fragmentation of [M2H]2 ion at m/z 445
gave product ions at m/z 415 [M2H2
CH2O]2, 325 [M2H2120]2, 310
[M2H21202CH3]2, 297 [M2H
21202CO]2, and 282 [M2 H2120
2CH32CO]2. It indicated a ZOCH3
group in the aglycone skeleton.
By comparing with the literature data
[1,2], peak 3 was identified as PG-3.
Peaks 6 and 7 were the puerarin isomers
of C-glycosyl isoflavone, which were
identified as new compounds. The isolation
and structural elucidation of them were then
carried out, as reported in brief [7].
Based on the retention time, MS and
UV spectral data, and comparing with the
reference samples, peaks 2 and 4 were
unambiguously identified as puerarin and
daidzin, respectively.
Peaks 1 and 5 were the isomeric
compounds with the similar MSn spectra,
in which the consecutive neutral losses of
120 and 28 amu from [M2H]2 of m/z 431
were observed. But they showed different
(a)
(b)
Intens.
×106
8
1
2
mAU
2
1000
800
600
400
200
0
6
4
2
0
4
1
3
4
6
3
5
7
6
5
7
5
10
15
min
0
2
4
6
8
10
12
14
16 Time [min]
TIC -All MS
Figure 2. HPLC–MS analysis for the total isoflavone extract of P. lobata. (a) HPLC–UV
chromatogram and (b) total ion current (TIC) chromatogram.

Journal of Asian Natural Products Research
295
Table 1. The HPLC–MSn results of the total isoflavone extract of P. lobata.
Peak no. tR (min) [M2H]2 Fragment ion m/z
lmax (nm)
Identity
PG-1
1
2
3
3.73
5.71
5.33
431
415
445
MS2[431]: 311, 283
250, 290
MS3[431 ! 311]: 283
MS2[415]: 325, 295, 267
MS3[415 ! 295]: 267
250, 306
Puerarin
MS2[445]: 415, 325, 310, 297, 282 250, 286, 308 PG-3
MS3[445 ! 325]: 310, 297, 282
4
5
7.77
8.99
415
431
MS2[461]: 415, 253
MS2[431]: 311, 283
MS3[431 ! 311]: 283
MS2[415]: 295, 267
MS3[415 ! 295]: 267
MS2[415]: 295, 267
MS3[415 ! 295]: 267
250, 298
260
Daidzin
Genistein
8-C-glycoside
New compound
6
7
12.43
17.62
415
415
250, 306
250, 306
New compound
2.2 Structural characterization
the difference in the sugar moiety region,
which suggested that compound 7 is a
typical isoflavone. The 13C NMR and
DEPT spectral data of the sugar moiety
indicated that compound 7 is a C-glycosyl
compound by the chemical shift of
anomeric carbon at 79.9 ppm and the
sugar moiety is an aldohexose unit which
showed one CH2 and five CH groups.
By 1H–1HCOSY, HSQC, and HMBC
spectra shown in Figure 3, the structure
of the aglycone was elucidated and the site
of the sugar linkage to aglycone in
compound 7 was confirmed at the C-8
Compound 7 (Figure 3) was obtained as a
mild yellow amorphous powder with the
UV absorption maxima at 250 and 306 nm.
The IR spectrum showed the absorption
bands at 3383 and 1633 cm21 attributed to
hydroxyl and carbonyl groups, respect-
ively. The molecular formula was deter-
mined as C21H20O9 by HR-ESI-MS at m/z
415.1033 [M2H]2. Comparing the 1H
and 13C NMR spectral data (see Table 2)
of compound 7 with those of puerarin [1],
they showed the similar pattern except for
H
H
O
HO
HO
H
HO
H
OH
H
HO
O
O
H
7
8
5
H
2
9
6
10
H
3
H
4
H
OH
H
H
Figure 3. Key HMBC correlations of compound 7.

296
Table 2. 1H and 13C NMR spectral data of neopuerarin B (6) and neopuerarin A (7) (in DMSO-d6).
Proton Carbon
H.-J. Zhang et al.
Position
6
7
6
7
2
3
4
5
6
7
8
9
8.33 s
8.32 s
152.9
123.6
175.1
126.7
116.7
160.8
111.9
155.2
116.6
122.7
130.5
115.4
157.5
115.3
130.4
80.4
152.8
123.5
175.1
125.7
115.7
161.8
109.8
154.8
116.8
122.6
130.2
115.2
157.4
115.2
130.2
79.9
–
–
–
–
7.96 (d, J ¼ 8.5 Hz)
7.90 (d, J ¼ 8.5 Hz)
6.92 (d, J ¼ 8.5 Hz)
6.88 (d, J ¼ 8.5 Hz)
–
–
–
–
–
–
–
–
10
10
20
30
40
500
600
100
200
300
4
–
–
7.42 (d, J ¼ 7.5 Hz)
7.38 (d, J ¼ 7.5 Hz)
6.83 (d, J ¼ 7.5 Hz)
6.83 (d, J ¼ 7.5 Hz)
–
6.83
7.40
–
6.83
7.38
5.30
4.18
3.80
4.15
3.80
3.46–3.63
5.72
4.32
4.20
4.24
3.80
3.53–3.64
82.1
80.8
77.1
69.0
77.9
75.2
80.9
68.5
500
600
64.0
64.1
position by the appearance of the corre-
lation between the anomeric proton of the
sugar at dH 5.30 (H-100) and the carbon
atoms at dC 160.8 (C-7), 111.9 (C-8), and
powder with the UV absorption maxima at
250 and 306 nm. The IR spectrum showed
the absorption bands at 3403 and
1628 cm21 attributed to hydroxyl and
carbonyl groups, respectively. The mol-
ecular formula was determined as
C21H20O9 by HR-ESI-MS at m/z
415.1107 [M2H]2. The 1D and 2D
NMR spectral data of compound 6 showed
a similar pattern to compound 7, which
suggested that compound 6 is also an 8-C-
aldohexofuranosyl-7,40-dihydroxyisofla-
vone. Comparing the 13C NMR spectral
data of the sugar moiety of compound 6
with those of compound 7, the chemical
shifts of C-200 and C-300 moved about 5 ppm
downfield while the signal of C-400 shifted
nearly 4 ppm upfield. Acid treatment
of compound 6 using Besson’s isomeriza-
tion method [8] yielded puerarin and
compound 7 (Figure 5). Therefore, com-
pound 6 is determined to be 8-C-b-
glucofuranosyl-7,40-dihydroxyisoflavone,
155.2 (C-9) (Figure 3). Comparing the 13
C
NMR spectral data with those of the
puerarin [1,2], the carbon signals of C-100,
C-200, C-400 in the sugar moiety of
compound 7 shifted 6.5–10.8 ppm to the
downfield while the chemical shift of C-500
moved more than 10 ppm to the upfield.
Moreover, the coupling constant between
the anomeric proton and H-200 was not
observed. It suggested that the sugar is in
the furanose form. The 13C NMR spectrum
of the sugar moiety of compound 7 was in
agreement with the reported data for the a-
glucofuranosyl compound [8]. Therefore,
compound 7 is determined to be 8-C-
a-glucofuranosyl-7,40-dihydroxyisofla-
vone, named neopuerarin A (Figure 4).
Compound 6 (Figure 3) was also
obtained as a mild yellow amorphous

Journal of Asian Natural Products Research
297
R
8
HO
O
9
7
6
2
2'
1'
10
3
6
7
R = β-glucofuranose
R = α-glucofuranose
4
3'
5
6'
O
4'
OH
5'
Figure 4. Structures of 6 (neopuerarin B) and 7 (neopuerarin A).
named neopuerarin B (Figure 4). To our
knowledge, it is the second report of
C-glucofuranosyl compounds in nature.
grade from Yuanli Chemical Plant (Tian-
jin, China). Water for the sample prep-
aration and the HPLC analysis was
purified by a Milli-Q academic water
purification system (Millipore, Bedford,
MA, USA).
3. Experimental
3.1 Chemicals and materials
A commercial product of the total
isoflavone extract of P. lobata was kindly
donated by Zhongjing Modern TCM
Company (Nanyang, China). The com-
mercial reference compounds of puerarin
and daidzin (purity . 98%, measured by
HPLC with the area normalization
method) were kindly gifted by Jinfeng
Pharmaceutics Co. Ltd (Qingdao, China)
and Dr Yang Xuedong, College of
Pharmaceutical, Science and Technology,
Tianjin University (Tianjin, China).
Methanol for the HPLC analysis was of
HPLC grade from Caledon (Ontario,
Canada). Formic acid was of analytical
3.2 Instruments
HPLC–MS analysis for the total isofla-
vone extract of P. lobata was performed
on an Agilent 1200-IT 6310 system
equipped with an Agilent G1311A qua-
ternary pump, a G1315D DAD detector,
and a 6310 ion-trap mass spectrometer.
The LC separation was performed using an
Agela Venusil XBP-C18 column
(4.6 £ 250 mm, 5 mm), eluting with A
(0.1% formic acid):B (MeOH) ¼ 65:35
(v:v) at 1.0 ml/min. The column tempera-
ture was set at 308C and the effluent was
monitored at 250 nm. High-purity nitrogen
(a)
(b)
Norm.
80
Norm.
80
Puerarin
60
Neopuerarin B
60
40
20
0
40
Neopuerarin B
20
Neopuerarin A
0
5
10
15
min
5
10
15
min
Figure 5. HPLC chromatogram of (a) neopuerarin B (6) and (b) acid-hydrolyzed neopuerarin B.

298
H.-J. Zhang et al.
(N2) was used as both drying gas with a
flow rate of 12 l/min and as nebulizing gas
with a pressure of 60 psi. Ultra-high pure
helium (He) was used as the collision gas.
The dry temperature was set at 3508C and
the capillary voltage was set at 3500 V.
The mass spectra were recorded in the
scale from m/z 100 to 1000 in the negative
mode. MSn data were acquired in the Auto
MSn mode.
compound 6 (26.33 mg) and compound 7
(14.88 mg).
3.4.1 Compound 6
A mild yellow amorphous powder; mp
157–1608C. UV (CH3OH) lmax (log 1):
250 (3.33), 306 (2.87) nm; IR (KBr) nmax
:
3403, 1628, 1515, 1257, 1028 cm21; H
NMR (DMSO-d6, 500 MHz) and 13C
NMR (DMSO-d6, 125 MHz) spectral data
are given in Table 2; HR-ESI-MS m/z
415.1029 [M2H]2 (calcd for C21H19O9,
415.1028).
1
Melting points were measured on a
YRT-3 micro-melting point apparatus
(Tianjin, China). The UV spectra were
recorded on a Shimadzu UV-2450 UV/Vis
spectrophotometer. The IR spectra were
obtained on a Bruker Tensor 27 FT-IR
spectrophotometer, with KBr pellets.
The NMR spectra were measured with a
Bruker DRX-500 spectrometer, at
500 MHz (1H) and 125 MHz (13C),
respectively. The HR-ESI-MS spectra
were measured with a Varian IonSpec
7.0T high-resolution ESI-FT-ICR mass
spectrometer.
3.4.2 Compound 7
A mild yellow amorphous powder; mp
149–1528C. UV (CH3OH) lmax (log 1):
250 (3.32), 306 (2.83) nm; IR (KBr) nmax
:
3383, 1633, 1515, 1256, 1037 cm21; H
NMR (DMSO-d6, 500 MHz) and 13C
NMR (DMSO-d6, 125 MHz) spectral data
are given in Table 2; HR-ESI-MS m/z
415.1033 [M2H]2 (calcd for C21H19O9,
415.1028).
1
3.3 Sample preparation
The extract (51.8 mg) was dissolved with
10 ml mobile phase and filtered through
0.45 mm nylon membrane. The filtered
solution was subjected to HPLC–MS
analysis with an injection volume of 40 ml.
3.5 Acid hydrolysis of compound 6
Compound 6 (1 mg) was dissolved in 4 ml
of MeOH–4 M HCl (1:1) and was refluxed
for 1 h. The solution was neutralized with
2 M NaOH and then extracted with 2 ml of
n-BuOH three times. The n-BuOH layer
was combined and concentrated in vacuo.
The residue was dissolved with 2 ml of
MeOH and then subjected to HPLC–MS
analysis (Figure 5).
3.4 Isolation
The extract of total isoflavone (30 g) was
chromatographed on RP-C18 silica gel
(40–60 mm, self-synthesized), eluting
with 25% MeOH, 40% MeOH, and 60%
MeOH, successively. The 40% elutes were
concentrated by Rota-vapor and then
purified by semi-preparative HPLC
(Agela Venusil XBP-C18 column,
10 £ 250 mm, 5 mm), eluting with H2O–
MeOH (60:40 v/v) at 2.0 ml/min with a
Shimadzu LC-10AT pump and the efflu-
ents were monitored at 250 nm using a
Shimadzu SPD-10A UV detector, to afford
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