American Journal of Anal yt ical Chemistry, 2011, 2, 665-674
doi:10.4236/ajac.2011.26076 Published Online October 2011 (http://www.SciRP.org/journal/ajac)
Copyright © 2011 SciRes. AJAC
Chemical Profiling and Quantification of Isoflavone
Phytoestrogens in Kudzu Using LC/UV/MSD
Qing-Li Wu1*, Yong-Hong Yang2, Jim Simon1
1New Use Agriculture and Natural Plant Products Program Department of Plant Biology and Pathology,
Rutgers University, New Brunswick, USA
2Key Laboratory, Department of Plant Pathology, Yunnan Agricultural University, Kunmin, China
E-mail: *qlwu@aesop.rutgers.edu
Received February 21, 2011; revised May 3, 2011; accepted May 20, 2011
Abstract
Method development for determination of isoflavones in kudzu was achieved by HPLC/UV/ESI-MSD. Us-
ing three kudzu species of Pueraria lobata, P. thomsonii and P. edulis, and analyzing the isoflavones sepa-
rately by species and from different plant tissues (roots, stems, leaves, flowers and fruits) in each species, a
total of 25 isoflavones were identified by their molecular ions and characteristic fragment ion peaks using
LC/MSD under MS and MS/MS mode, and in comparison with standard isoflavones. Two main chemical
groups were identified: 1) 8-C-glycosyl isoflavone of puerarin and the analogues of 5-OH puerarin, 3’-OH
puerarin, 3’-OMe puerarin, and their glycosides; and 2) daidzein, genistein, glycitein and their glycosyl and
malonyl derivatives, which are similar to those known in soy. To accurately quantitate total isoflavones,
acidic hydrolysis during extraction of kudzu samples was applied to convert the oxygen glycosides into their
respective isoflavone aglycones of daidzein, genistein and glycitein, or non-hydrolyzed carbon glycosides of
puerarin, 5-OH puerarin, 3’-OH puerarin and 3’-OMe puerarin. Under the multiple optimized conditions, all
seven isoflavones in acidic hydrolyzed kudzu extracts were successfully separated within 30 min and quanti-
fied individually with calycosin used as internal standard by both UV and MS detectors. For the quantitative
study, several standards e.g. 5-OH puerarin, 3’-OH puerarin and 3’-OMe puerarin are not commercially
available. Using polyamide, sephdex-LH20 chromatography and Prep-HPLC, we purified these three stan-
dards from kudzu extracts and then elucidated their structures by UV, MS and NMR spectrometric methods.
This is the first method to simultaneously quantitate all the isoflavones in kudzu.
Keywords: Kudzu, Pueraria, Isoflavones, LC/UV/MSD
1. Introduction
The kudzu root, called “Gegen” in Chinese medicine
which is obtained mainly from Pueraria lobata (lobed
kudzuvine), P. thomsonii (Thomson kudzuvine) and P.
edulis (edible kudzuvine), has been primarily used for
the treatment of common cold, influenza, and wrist and
shoulder stiffness, or as antidipsotropic agent [1]. Kudzu
was reported to contain high amounts of phytoestrogenic
isoflavones, such as daidzein, genistein, puerarin and
their derivatives [2-4]. These compounds based on the
structural similarity to internal estrogen have received
much interest for the prevention of menopausal symp-
toms, osteoporosis, high cholesterol, heart disease and
cancer [5-10]. Several laboratories have provided evi-
dence that the major isoflavones isolated from kudzu are
effective in reducing alcohol intake [11-15]. Therefore, it
is important to better understand the biology of the plant,
and the tissues and sites of isoflavone accumulation.
Several methods for determining isoflavones in kudzu
and the derived products using high performance liquid
chromatography combined with ultraviolet and/or mass
spectrometric detector have been reported [14-21]. Qua-
litative studies on kudzu have only led to the identifyca-
tion of numerous isoflavones. The quantitative studies
focused on the original or acidic hydrolyzed fractions
kudzu extracts. However, only part of major components,
puerarin, and soy-like isoflavones daidzein, genistein and
the glycosides were quantified, and the researchers were
not able to determine accurate levels of the total isofla-
vones in kudzu and the derived products. The purpose of
this research was to chemically profile the isoflavones,
Q.-L. WU ET AL.
666
accurately quantify the total isoflavones in different parts
(root, stem, leaf, flower and fruit) of kudzu and to estab-
lish a more robust and inclusive analytical method pro-
viding both a qualitative and quantitative method for the
isoflavones in kudzu. In this study, we conducted field
investigations, collected and authenticated various kudzu
samples of P. lobata, P. thomsonii and P. edulis from
China. Using HPLC/UV/MSD, different parts (root, stem,
leaf, flower and fruit) of the three species were chemi-
cally profiled, and led to the identification of 25 isofla-
vones. The isoflavones can be categorized into two
chemical groups: 1) puerarin (8-C-glucoside daidzein)
and analogues, e.g. 3’-OMe-puerarin and 5/3’-OH-puer-
arin (Figure 1), and 2) daidzein, genistein, glycitein and
their glycosyl and malonyl derivatives, which are similar
to those found in soy. Under optimized conditions, the
total content of isoflavones in acidic hydrolyzed kudzu
was accurately quantified with calycosin used as an in-
ternal standard by both UV and MS detectors.
2. Experimental
2.1. Materials
Standard compounds, genistein and glycitein were pur-
chased from Indofine Chemical Company, Inc. (Somer-
ville, NJ) and daidzein from Sigma Chemical Co. (St.
Louis, MO). The internal standard calycosin was purified
from hydrolyzed extracts of the above ground biomass
from red clover and the standards puerarin, 5-OH puer-
arin, 3’-OH puerarin and 3’-OMe puerarin were purified
from kudzu extract in this laboratory. HPLC-grade
methanol (MeOH), acetonitrile (ACN), ethanol (EtOH),
aqueous ammonia, and concentrated hydrochloric acid
(HCl) were procured from Fisher Scientific Co. (Fair
Lawn, NJ); formic acid was purchased from Acros Or-
ganics (NJ); and polyamide 6 and sephdex-LH20 were
Puerarin: R1 = R2 =H
3’-OH-puerarin: R1 = H, R2 = OH
5-OH-
p
uerarin: R1 = OH, R2 = H
Figure 1. The structures of puerarin and its analogues.
purchased from Sigma-Aldrich Co. (St. Louis, MO).
HPLC-grade water (18 m) was prepared using a Milli-
pore Milli-Q purification system (Millipore Corp., Bed-
ford, MA), and was used to prepare all solutions. All the
kudzu samples including each of the different plant parts
from the roots, stems, leaves, flowers and fruits from P.
lobata, P. thomsonii and P. edulis were collected in
Yunnan Province, China. All botanical samples were
identified and authenticated by species in Key laboratory,
Department of Plant Pathology, Yunnan Agricultural
University, Kunmin, Yunnan, China.
2.2. Apparatus
HPLC separation was performed on a Phenomenex Pro-
disyl ODS (3) column, 5 µm, 150 × 3.2 mm I.D. (Phe-
nomenex Inc., Torrace, CA). For LC/ESI-MS and LC/
MS/MS experiments, an Agilent 1100 Series LC/MSD
trap (Agilent Technologies, Waldbronn, Germany) equi-
pped with quaternary pump, photodiode array and multi-
ple wavelength detector, thermostated column compart-
ment, degasser, MSD trap with an electrospray ion
source (ESI) and software of HP ChemStation, Bruker
Daltonics 4.1 and DataAnalysis 4.1 was used. Waters
Prep-HPLC with a Phenonex Luna Phenyl-Hexyl column,
10 µm, 250 × 30 mm I.D. (Phenomenex Inc., Torrace,
CA), Delta 600 pump, 2487 Dual λ absorbance detector,
600 controller, 717 autosampler, fraction collector II,
in-line degasser AF and software of Millennium 32 was
used for standard purification.
2.3. Purification of Internal Standard of
Calycosin from the Aerial Part of Red
Clover and Standards of Puerarin, 5-OH
Puerarin, 3’-OH Puerarin and 3’-OMe
Puerarin from Kudzu Roots
The internal standard of calycosin was purified from the
above ground biomass of red clover as described in our
previous paper [22]. For purification of the standards
puerarin and it’s analogues, approximately 100 g of dried
roots of P. lobata was used as the starting material. The
kudzu material was first refluxed in 500 mL 80%
methanol for 2 hours for 2 times. The filtrations were
combined and the solvent was evaporated under reduced
pressure to obtained methanol extract (~12 g). The ex-
tract was then chromatographed on polyamide column
using a step-gradient MeOH-H2O (10% - 50% MeOH)
and 250 ml fraction was collected. Those fractions con-
taining the four target components as judged by LC/MS
were collected and then re-chromatographied on seph-
dex-LH20 prior to further purification using Prep-HPLC.
Prep-HPLC was performed using the mobile phase of
Copyright © 2011 SciRes. AJAC
Q.-L. WU ET AL.667
MeCN-H2O (7% MeCN) at a flow rate of 7 mL/min to
get puerarin (40 mg), 5-OH puerarin(12 mg), 3’-OH pu-
erarin (14 mg) and 3’-OMe puerarin (8 mg). The struc-
tures of these four compounds were then determined and
verified by UV, MS and NMR spectrometric methods.
2.4. Preparation of Stock Solutions and
Calibration Standards
Individual stock solutions of 7 standards were prepared
by dissolving the appropriate amounts of ~5.0 mg in 15.0
mL of diluent (water and MeOH, 3:7). The final volume
of each solution was then diluted to 25 mL with diluent.
Calibration standards were prepared by diluting the stock
solutions with diluent and spiked with same amount of
internal standard of calycosin. The calibration curve
ranges for UV and MS methods show excellent linearity
(Table 1). In the calibration plots, 8 and 6 different con-
centration levels were used for UV and MS detection,
respectively.
2.5. Plant Sample Preparation
For qualitative study, ~200 mg of finely ground material
was extracted with 10 mL 80% methanol using sonica-
tion for 1 hour at room temperature. The extracts were
filtered through 0.45 µm filter and 20 μL extract was
injected for each analysis. The extraction procedure for
quantitative analysis was adopted from our prior studies
[22,23]. Approximately 1000 mg of powdered kudzu
material was placed into a 250 mL flask along with 50
mL of ethanol, 20 mL of DI water, and 8 mL of concen-
trated HCl. The mixture was refluxed for 2 hours pro-
tected by N2. The solution was filtered and diluted to
volume of 100 mL. Each hydrolyzed sample of 5 μL
filtered over 0.45 µm filter was analyzed by triplicate
injections.
2.6. Liquid Chromatographic and Mass
Spectrometric Conditions for Identification
of Isoflavones
HPLC separation was performed with the mobile phase
consisting of solvent A and B in gradient, where A was
0.1% formic acid (v/v) in water and B was 0.1% formic
acid (v/v) in acetonitrile. The linear gradient profile was
from 10% to 40% B in 40min. The wavelength of UV
detection was 254 nm. Column compartment was set at
25˚C. The flow rate was 1.0 mL/min. The electrospray
ion mass spectrometer (ESI-MS) was operated under
positive ion and auto MS/MS mode (Threshold, 30,000)
and optimized collision energy level of 80%, scanned
from m/z 100 to 700. ESI was conducted using a needle
voltage of 3.5 kV. High-purity nitrogen (99.999%) was
used as dry gas and nebulizer at a flow rate of 12 L/Min,
and capillary temperature at 350˚C. Helium was used as
collision at 60 psi. The ESI interface and mass spec-
trometer parameters were optimized to obtain maximum
sensitivity. The auto MS/MS total ion chromatogram was
processed by extracting the molecular ions of each
isoflavone for identification.
2.7. Liquid Chromatographic and Mass
Spectrometric Conditions for Quantification
of Isoflavones
MS detection was conducted under collision energy level
of 80% and scanned from m/z 100 to 600. Other MS pa-
rameter and LC conditions were the same as described
above. Under SIM mode (selected ion monitoring), pro-
tonated [M++H] ions were isolated for each isoflavone.
The mass spectrometer was set into two time segments: 1)
from 0 to 16.5 min for 3’-OH puerarin; puerarin; 3’-OMe
puerarin and 5-OH puerarin with isolation of m/z 417,
433 and 447; 2) from 16.5 to 30 min for daidzein, gly-
citein, genistein and calycosin of m/z 255, 271 and 285.
The isolation width was set as 1.0 m/z. The calibration
curves were plotted using a 1/x-weighted quadratic mo-
del for the regression of peak area acquired from UV and
MS detector versus analyte concentration.
3. Results and Discussion
3.1. Characterization of Isoflavones
Simultaneous UV and processed auto MS/MS chroma-
tograms of 80% methanol extract of P. lobata root are
illustrated in Figure 2. The identities, retention time,
protonated [M+H]+ and characteristic fragment ions for
individual peaks are listed in Table 2. In kudzu, the car-
bon glucoside of daidzein, puerarin (7, peak 6 in Figure
2) is well known to dominate the isoflavone constituents.
Therefore, some minor analogues of puerarin e.g. 5-OH
puerarin, 3’-OH puerarin, 3’-OMe puerarin and their
glycosides are also detected on the basis of MS and
MS/MS spectral interpretation and some of them by
comparison to the authenticated standards. The MS spec-
tra of puerarin and its analogues showing the protonated
molecular ions [M+H]+ are illustrated in Figure 3. Based
on the above analysis, a total of 10 puerarin analogues
were determined in kudzu (Compounds 1-9 and 12 in
Table 2), with some of the structures of puerarin and its
analogues shown in Figure 1. Daidzein, genistein, gly-
citein and their derivatives of glycoside and glycoside
malonate, which are well-known to be present in soy,
were also detected in kudzu (Compounds 10 , 11 and
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Copyright © 2011 SciRes. AJAC
668
3.2. Quantification of Isoflavones
13-25 in Table 2). Their structures were identified by
analysis of MS spectral data, and by comparison to those
in soy as well. Some of them e.g. daidzein, genistein,
glycitein and the glucosides were also verified by com-
parison to the authenticated standards. Comparative eva-
luation of the different plant tissues indicated that the
chemical profile of kudzu root and stem is very similar,
among which the puerarin is the major peak. In contrast,
the content of puerarin in kudzu leaf is relatively lower
than daidzein and its malonyl derivative. In the flower,
the major isoflovones are daidzein, genistein, glycitein
and their glycoside derivatives, while only trace levels of
isoflavones were detected in kudzu fruit. The chemical
profile of kudzu root from different plant sources of P.
lobata, P. thomsonii and P. edulis are very similar (Fig-
ure 4).
A qualitative study revealed that kudzu contains large
mount of isoflavones, and most of these are available as
glycoside and malonate conjugates, e.g. glycoside of
daidzein, genistein and puerarin; and glycoside malonate
of daidzein and genistein. Therefore, in this study to fa-
cilitate the quantification and accurately evaluate the
total isoflavones in kudzu, the kudzu samples were hy-
drolyzed during extraction. Under optimized conditions,
all three isoflavone aglycones of daidzein, genistein and
glycitein, and four non-hydrolyzed carbon glycosides of
puerarin, 5-OH puerarin, 3’-OH puerarin and 3’-OMe
puerarin were successfully quantified by using HPLC/
UV/ESI-MSD. This is the first method to quantify all the
isoflavones in kudzu, and therefore to provide an accu-
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Time ( mi n)
1
2
3
4 5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23 24
1 2
3
4 5
6
9
7
8
10
11
12 14
17 13
15
16
18
19
20
21
22
23 24
(a)
(b)
Figure 2. Simultaneous LC-UV (a) and pr ocessed auto MS/MS (b) chromatograms of P. lobata root extract. The identities, tR
value and MS of each peak are listed in Table 2.
Q.-L. WU ET AL.669
Table 1. Calibration curve ranges and regressions (r2) of 7 analytes by UV and MS detection.
Analyte UV (ng/mL) r2 MS (ng/mL) r2
Daidzein 95.70-12250 1 23.93-765.63 0.9977
Glycitein 105.47-13500 0.9998 26.37-843.75 0.9931
Genistein 105.47-13500 1 26.37-843.75 0.9968
Puerarin 103.52-13250 0.9997 25.88-828.13 0.9959
3’-OMe-puerarin 99.61-12750 0.9994 24.90-796.88 0.9969
3’-OH-puerarin 105.47-13500 0.9999 26.37-843.75 0.9987
5-OH-puerarin 109.38-14000 0.9999 27.34-875.00 0.9923
Eight and six concentration levels were used for calibration plots under UV and MS detection, respectively.
Table 2. Peak assignments and the presence of isoflavones in kudzu from different plant sources.
Peak tR
(min)
[M+H]+
(m/z)
MS
fragment
ion (m/z) Identities
PL-R
PL-S
PL-L
PL-Fl
PL-Fr
PT-R
PT-S
PT-L
PE-R
Compound
Code
1 2.3 579 417 Puerarin-G + - - - - + + - - 1
1a 2.3 579 433 5/3’-OH-puerarin-Rha + - - - - - + - - 2
2 3.4 579 417 Puerarin-G + + - - - + + + + 3
3 4.1 433 3’-OH-puerarin* + + T - - + + + + 4
4 5.2 565 433 5/3’-OH-puerarin-Api/Xyl+ - - - - - + T - 5
5 5.9 595 433 5/3’-OH-puerarin-G + T - - - T + + T 6
6 6.5 417 Puerarin* + + + T T + + + + 7
7 7.1 549 417 Puerarin-Api/Xyl + + - - + + + + 8
8 7.2 447 3’-OMe-puerarin* + + T - - + + + + 9
9 7.8 549 417 Puerarin-Api/Xyl + + + - - + + + + 10
10 9.7 417 255 Daidzein-G* + + T - T + + + + 11
11 10.9 447 285 Glycitein-G* + + + + T + + + + 12
12 11.6 433 5-OH-puerarin* + + T - - + + + + 13
13 14.1 519 271 Genistein-G-M + T T - T + T + 14
14 14.8 565 433, 271 Genistein-G-Api/Xyl + + T - - + + T - 15
15 15.1 433 271 Genistein-G* + + + - T + + + + 16
16 15.7 503 255 Daidzein-G-M + + - T T + + T + 17
17 16.2 503 255 Daidzein-G-M + T T - - + + T + 18
18 17.0 503 255 Daidzein-G-M + + + + - + + + + 19
19 17.6 533 285 Glycitein-G-M + T - T + + - - 20
20 20.1 519 271 Genistein-G-M + T T - - + + T T 21
21 20.5 519 271 Genistein-G-M + + + - - + + + + 22
22 21.5 255 Daidzein* + + + - - + + + + 23
23 22.3 285 Glycitein* + T T + - T + 24
24 27.2 271 Genistein* + + + + - + + + + 25
*Identity based on MS spectral and retention data using authentic standards. G: Glucosyl/Galactosyl; Rha: Rhamnosyl; Api: Apiosyl; Xyl: Xylosyl; M: Malonyl.
+: Present; -: Not detectable; T: Trace. PL, P. lobata; PT, P. thomsonii; PE, P. edulis; L, Leaf; S, Stem; R, Root; Fl, Flower; Fr, Fruit.
Copyright © 2011 SciRes. AJAC
Q.-L. WU ET AL.
670
433
417
447
433
415
399
329
417
415
313
297
327
313
Relative intensity (%)
100
50
0
(a)
(b)
(c)
(d)
100 200 300 400 500
m/
z
100
50
0
100
50
0
100
50
0
Figure 3. Representative MS spectra of 8-C-glycosyl isoflavones of 3’-OH puerarin (a), puerarin (b), 3’-OMe puerarin (c) and
5-OH puerarin (d).
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Time
(
mi n
)
1 2
3
4
6
7
8
9 10
11
12
13
14
15
16
17
18
19 20
21
22
23 24
(a)
(b)
(c)
Figure 4. Processed auto MS/MS total ion chromatograms of extracts of root of P. lobata (a), P. thomsonii (b) and P. edulis (c).
Total ion chromatograms are extracted with molecular ions of each isoflavones. Peak assignment in A (P. lobata root) is listed
n Table 2. The presence of chemical profiles for each sample is listed in Table 2. i
Copyright © 2011 SciRes. AJAC
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Copyright © 2011 SciRes. AJAC
671
rate method for its quality control.
3.2.1. U V M ethod
Using the conditions optimized under multiple preli-
minary assays, this system enables separation of 7 tar-
get isoflavones within 30 min. The chromatograms of a
standard mixture with internal standard calycosin, and
hydrolyzed extract of P. lobata under UV detection at
wavelength of 254 nm are illustrated in Figure 5. Peak
assignments were made with single compound injections
and MS spectral data. Baseline separation was success-
fully achieved for all analytes.
3.2.2. MS Me thod
In hydrolyzed kudzu extracts, compared to puerarin,
5-OH puerarin, daidzein and genistein, the other 3 ana-
lytes of 3’-OH puerarin, 3’-OMe puerarin and glycitein
were all found in much lower concentrations, requiring
an MS method with higher sensitivity and selectivity.
Under SIM mode, protonated [M++H] ion was isolated
for individual target compounds of daidzein at m/z 255,
genistein at m/z 271, glycitein and calycosin (IS) at m/z
285, puerarin at m/z 417, 5-OH puerarin and 3’-OH pu-
erarin at m/z 433, and 3’-OMe puerarin at m/z 447. Fig-
ure 6 illustrates the MS chromatogram of hydrolyzed red
P. lobata root extract with selected ion monitoring (SIM)
that demonstrates baseline separation of the 7 compo-
nents in complex plant matrices within 30 min.
The content of total isoflavones detected in root of
lobed kudzuvine (P. lobata) and Thomson kudzuvine (P.
thomsonii), which are both collected as the official me-
dicinal plant species in China pharmacopoeia (The
(a)
(b)
1
2
3
4
5
6
IS
7
1
2
3
4
5
6
IS
7
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Time (min)
Figure 5. HPLC/UV chromatograms of mixture of 7 standards with internal standard calycosin (a) and acidic hydrolyzed P.
lobata extract. 1 = 3’-OH puerarin; 2 = puerarin; 3 = 3’-OMe puerarin; 4 = 5-OH puerarin; 5 = daidzein; 6 = glycitein; 7 =
genistein; IS = internal standard, calycosin.
Q.-L. WU ET AL.
672
SIM 417
SIM 433
SIM 447
SIM 271
SIM 255
SIM 285
0.0 5.0 10.0 15.0 20.0 25.0 30.0
Time (min)
(a)
(b)
(c)
(e)
(d)
(f)
1
2
4
5
7
6
IS
3
Figure 6. Processed MS chromatograms of hydrolyzed root extract of P. lobata. Reconstructe d ion chromatogr am for m/z 417
(a), 433 (b), 447 (c), 255 (d), 271 (e) and 285 (f). 1 = 3’-OH puerarin; 2 = puerarin; 3 = 3’-OMe puerarin; 4 = 5-OH puerarin;
5 = daidzein; 6 = glycitein; 7 = genistein; IS = internal standard, calycosin.
Pharmacopoeia Commision of P. R. China, 2005) was
much higher than that found in the edible kudzuvine (P.
edulis) (Table 3). It is interesting that the total isofla-
vones in P. thomsonii stem was very high (Figure 7).
Additionally, in the kudzu flower, the soy like isofla-
vones of daizein, genistein and glycitein are the major
isoflavones.
4. Conclusions
High performance liquid chromatography combined with
ultraviolet and electrospray ionization mass spectromet-
ric detector (HPLC/UV/ESI-MSD) has been applied to
the study of isoflavones in plant matrices of kudzu from
various Pueraria species of P. lobata (lobed kudzuvine),
P. thomsonii (thomson kudzuvine) and P. edulis (edible
kudzuvine). Under the multiple optimized HPLC and
MSD conditions, 25 isoflavones including 10 puerarin
analogues (isoflavone C-glycosides) and 15 soy-like
isoflavones (daidzein, genistein, glycitein and the gly-
cosides and glycoside malonates) have been identified
based on analysis of the UV and MS data and by com-
parison to the authenticated standards. A simple method
was developed for quantification of total isoflavones in
hydrolyzed kudzu extracts by HPLC with UV and MS
detectors. Within 30 min, all the 7 isoflavones were to-
tally separated and eluted individually. This is the first
method to quantify all the 7 isoflavones in kudzu organs.
For this study, some standards e.g. puerarin, 5-OH puer-
arin, 3’-OH puerarin and 3’-OMe puerarin are not com-
Copyright © 2011 SciRes. AJAC
Q.-L. WU ET AL.673
Table 3. Isoflavone content in kudzu from different plant sources.
Conten t (%)
Sample Code Daidzein Glycitein Genistein Puerarin3’-OMe-puerarin3’-OH-puerarin 5-OH-puerarinTotal
PL-R 0.2436 0.0166 0.0739 1.9670 0.0240 0.0365 0.1619 2.5235
PL-S 0.0373 0.0016 0.0231 0.0664 0.0028 T 0.0273 0.1586
PL-L 0.0127 0.0011 0.0246 0.0162 T T T 0.0546
PL-Fl 0.0067 0.0963 0.0285 T T T T 0.1315
PL-Fr T T T T T T T T
PT-R 0.1522 0.0034 0.0201 1.4572 0.0112 0.0009 0.1382 1.7831
PT-S 0.2298 0.0157 0.0500 0.7831 0.0117 0.0016 0.0421 1.1340
PT-L 0.0185 0.0024 0.0503 0.0236 T T 0.0192 0.1141
PE-S 0.0810 0.0059 0.0160 0.1625 0.0011 0.0016 0.0196 0.2876
PL, P. lobata; PT, P. thomsonii; PE, P. edulis; L, Leaf; S, Stem; R, Root; Fl, Flower; Fr, Fruit. T, trace level. The content of isoflavones of daidzein, genistein,
puerarin and 5-OH-puerarin was calculated using UV detection and the content of minor isoflavone of Glycitein, 3’-OMe-puerarin and 3’-OH-puerarin calculated
using MS detection.
0
0.5
1
1.5
2
2.5
3
Conten t (%)
PL-R PL-SPL-L PL-FlPL-FrPT-R PT-SPT-LPE-R
Sample Code
Figure 7. Content of total isoflavones in kudzu from differ-
ent plant sources PL, P. lobata; PT, P. thomsonii; PE, P.
edulis; L, Leaf; S, Stem; R, Root; Fl, Flower; Fr, Fruit.
mercial available. Using polyamide, sephdex-LH20 chro-
matography and Prep-HPLC, we purified these four
standards from kudzu extracts and elucidated their struc-
tures by UV, MS and NMR spectrometric methods.
5. Acknowledgements
This work was supported in part by NIH grant OD-
00-004, and the New Use Agriculture and Natural Plant
Products Program and conducted as part of the National
Botanical Center for Age-Related Diseases. Authors also
acknowledge the support by the New Jersey Agricultural
Station, School of Environmental and Biological Sciences,
at Rutgers University. We thank Connie Weaver, Stephen
Barnes, Michael Wyss and Jeevan Prasain for their en-
couragement and discussions.
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