Separation and Purication Technology 120 (2013) 310318

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Separation and Purication Technology

journal homepage: www.elsevier.com/locate/seppur

Simple and efcient preparation of biochanin A and genistein from

Dalbergia odorifera T. Chen leaves using macroporous resin followed by
ash chromatography
Fei-Yue Ma, Meng Luo, Chun-Jian Zhao, Chun-Ying Li, Wei Wang, Cheng-Bo Gu, Zuo-Fu Wei, Yuan-Gang Zu,
Yu-Jie Fu
State Engineering Laboratory for Bio-Resource Eco-Utilization, Northeast Forestry University, Harbin 150040, PR China
Engineering Research Center of Forest Bio-Preparation, Ministry of Education, Northeast Forestry University, Harbin 150040, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a simple rand efcient protocol is developed for preparation of biochanin A and genistein
Received 28 January 2013 from Dalbergia odorifera T. Chen leaves using macroporous resin followed by ash chromatography. The
Received in revised form 3 August 2013 adsorption and desorption capacity of 14 macroporous adsorption resins for biochanin A and genistein
Accepted 29 September 2013
were evaluated, and AL-2 resin showed better properties for biochanin A and genistein. After treatment
Available online 9 October 2013
with AL-2 resin, the contents of biochanin A and genistein in the enriched product were 6.60-fold and
6.41-fold increased with recovery yields of 87.13% and 84.60%, respectively. Furthermore, the operating
Keywords:
parameters of ash chromatography were optimized. The optimal conditions were as follows: stationary
Flash chromatography
Macroporous resin
phase: silica gel, elution system: n-hexane/ethyl acetate, sample/silica gel ratio: 1.3:40 and ow rate:
Separation 50 mL/min. After one ash chromatography run, purities of biochanin A and genistein effectively reached
Dalbergia odorifera T. Chen leaves over 95%, their recovery yields were 80.13% and 73.11%, respectively. The developed protocol was simple,
efcient, scalable and economical, which represented an excellent alternative for the separation and puri-
cation of bioactive compounds from plants.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Flavonoids, a large category of plant polyphenol secondary
metabolites, are widely distributed in medicinal herbs, fruits, teas,
etc. [1]. They possessed particular interest with regard to human
health effects [2]. As a kind of avonoids, isoavonoids can be
potentially used as clinical therapeutic agents, food additive and
health care products [38]. D. odorifera leaves, a natural renewable
resource, usually were discarded as useless material. Our previous
studies found that biochanin A and genistein are the main active
constituents present in D. odorifera leaves [9]. Biochanin A and gen-
istein are naturally occurring plant-derived phytoestrogen, and
possess anticancer, antioxidant and antiosteoporosis effect. In view
of these benecial effects, it is necessary to obtain high purity of
biochanin A and genistein for further medical studies and
applications.
Preparation of isoavonoids from plant extract is a challenging
task because plant extract was a multi-component system. Over
Corresponding author at: State Engineering Laboratory for Bio-Resource Eco-
Utilization, Northeast Forestry University, Harbin 150040, PR China. Tel./fax: +86
451 82190535.
E-mail address: yujie_fu2002@yahoo.com (Y.-J. Fu).
the past decade, various physical and chemical techniques for the
enrichment of bioactive compounds from plant extract have been
investigated, such as liquidliquid extraction [10,11], membrane
ltration [12], ion exchange [13], solid phase microextraction
[14] and adsorptiondesorption [15]. Among them, adsorption
desorption is considered as an economical and efcient method
for preparative enrichment of bioactive components from plant ex-
tracts. Macroporous resins adsorption technology has been
increasingly viewed as a representative adsorptiondesorption
for enriching bioactive components [1618]. Macroporous resins
have unique superiorities, such as high mechanical strength, high
selectivity, good acid and alkali resistance, low cost, convenience
and easy regeneration [19,20]. Open column chromatography is
traditionally used for separating bioactive components from plant
extracts. However, this method is time-consuming, laborious and
requires large volumes of solvents [1]. These limitations warranted
to explore a fast and efcient method for preparation of natural
products from plant extract. Flash chromatography, also known
as medium pressure chromatography, can efciently overcome
these limitations. It is considered as a fast, inexpensive and ef-
cient separation technique for the separation of natural active
components from extracts, and is easy to handle. This technique
is an excellent alternative for slow and often inefcient gravity-
fed chromatography. Compared to traditional gravity-fed

1383-5866/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.seppur.2013.09.035
 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318
chromatography, ash chromatography gives a greatly improved
reproducibility, higher resolution and recovery yield, and a re-
duced consumption of organic solvents. Moreover, with a UV
detector and an automatic fraction collector, the sample solvent
could be collected according to chromatograms prole during elu-
tion process.
The aim of this work was to establish a simple and efcient pro-
tocol for preparation of biochanin A and genistein from D. odorifera
leaves using macroporous resin followed by ash chromatography.
Up to now, there has never been the report on separation of bioch-
anin A and genistein by ash chromatography. The operating
parameters of macroporous resin and ash chromatography were
optimized. Hopefully, this work is helpful for the scale-up applica-
tion for the production of biochanin A and genistein from the D.
odorifera leaves and other plants.
2. Materials and methods
2.1. Materials, chemicals and adsorbents
The Dalbergia odorifera T. Chen leaves were collected from Hai-
nan Province China, and authenticated by Professor Shaoquan Nie
from the Key Laboratory of Forest Plant Ecology, Ministry of Educa-
tion, Northeast Forestry University, China. The leaves were dried in
shade at room temperature, powdered by a disintegrator and then
stored in dark.
Reference compounds biochainin A (40 ,5,7-trihydroxyisoav-
one-7-glucoside,P98%) and genistein (40 ,5,7-trihydroxyisoav-
one,P98%) were purchased from Fluka (Buchs, Switzerland).
Reagents of HPLC grade including methanol and formic acid were
purchased from J&K Chemical LTD. (Beijing, China). Deionized
water was produced by a Millipore Direct-Q purication system
(Millipore Corp., Bedford, MA, USA). Ethanol of analytical grade
was obtained from Tianjin Chemical Reagents Co. (Tianjin, China).
The solvents (n-hexane, ethyl acetate, chloroform and petroleum)
used for preparative ash chromatography were of analytical
grade.
Fourteen macroporous resins including AB-8, ADS-5, ADS-11, D-
101, FL-1, FL-2, FL-3, HPD500, HPD826, HPD-D, ME-2, AL-2, SA-3
and NKA-9 were purchased from Nankai Hecheng S&T (Tianjin,
China) and Bonchem (Hebei, China). Their polarities ranged from
non-polar to strong polar. All the resins were pretreated according
to the manufacturers recommendation prior to use in order to re-
move the monomers and porogenic agents trapped inside the
pores during the synthesis process [4,21]. The moisture contents
of the tested resins were determined by drying the beads at
100 C to constant weight in a drying oven for over 24 h. Toyopearl
HW-40S gel resin and silica gel (300400 mesh) were purchased
from Tosoh Corporation (Tokyo, Japan) and Qingdao Meigao Chem-
ical Co. Ltd., (Qingdao, China), respectively. TLC (Kieselgel GF254)
was purchased from Taizhou Luqiao Sijia Biochemical Plastic Fac-
tory (Taizhou, China).
2.2. Preparation of D. odorifera leaves extracts
Pulverized D. odorifera leaves (5 kg) were extracted with 30 L of
80% ethanol at room temperature for 3 days, repeated twice. The
ltered solutions were gathered and concentrated to dryness by
removing the ethanol solvent using a rotary evaporator device
(RE52AA, Shanghai Huxi Instrument Co., China) at 40 C. The dried
extracts were obtained, and dissolved in 30% ethanol to get sample
solution (5 mg extract/mL) at the concentration of 0.2058 mg/mL
for biochanin A and 0.0530 mg/mL for genistein, respectively
(Table 1).
311
2.3. HPLC analysis of biochanin A and genistein
Biochanin A and genistein were analyzed using an Agilent 1200
HPLC system. Chromatographic separation was carried out on a
HIQ Sil C18V reversed-phase column (250 mm  4.6 mm i.d.,
5 lm). All samples were ltered through 0.45 lm nylon mem-
branes prior to HPLC analysis. The mobile phase consisted of aceto-
nitrile (A) and waterformic acid (B). The gradient elution program
was as follows: 05 min, 510% A; 510 min, 1028% A; 10
30 min, 2860% A; 3035 min, 60% A; 3537 min, 6080% A; 37
42 min, 80100% A; 4255 min, 100% A. The injection volume
was 5 lL. The ow rate and column temperature was 1 mL/min
and 30 C, respectively. Biochanin A and genistein were quantied
at wavelength 262 nm. The chromatographic peaks of the analytes
were conrmed by comparing their retention times and UV spec-
trum with those of the reference compounds. Eight experimental
points were employed for establishing a calibration curve. The
regression lines for biochanin A and genistein were Y = 84 698
x + 78.7 (R2 = 0.9949) and Y = 71 148 x + 1.37 (R2 = 0.9927), where
Y is the peak area of analyte, and x is the concentration of reference
compound (mg/mL).
2.4. Enrichment of biochanin A and genistein by macroporous resin
2.4.1. Screening of macroporous resins
Macroporous resins can selectively adsorb and desorb constitu-
ents from sample solutions due to their specic physical and
chemical properties. The adsorption and desorption capacity of dif-
ferent resins towards biochanin A and genistein were investigated.
The adsorption tests were performed as follows: pre-weighed hy-
drated resins (equal to about 1.0 g dry resin) and 100 mL sample
solution were added into 250 mL asks with stopper. The asks
were shaken in an incubation shaker (120 rpm) for 4 h at 25 C.
After adsorption, the solutions were separated from the resins
and analyzed by HPLC. Then, the resins were subsequently des-
orbed with 100 mL 80% ethanol solution. The asks were continu-
ally shaken (120 rpm) for 6 h at 25 C. The contents of biochanin A
and genistein in desorption solutions were determined by HPLC.
The adsorption properties of resins were evaluated based on the
adsorption and desorption capacities and ratio of desorption. The
equations were as follows:
Adsorption evaluation:
C 0  C e V i
Qe
W
where Qe is the adsorption capacity at adsorption equilibrium (mg/g
resin); C0 and Ce are the initial and equilibrium concentrations of
solute in the solutions, respectively (mg/mL); Vi is the volume of
sample solution, and W is the weight of the dry resin.
Desorption evaluation:
Cd V d
D  100%
C 0  C e V i
CdV d
Qd
W
where Qd is the desorption capacity after adsorption equilibrium
(mg/g resin); D is the desorption ratio (%); Cd is the concentration
of solute in the desorption solution (mg/mL); Vd is the volume of
the desorption solution (mL); C0, Ce, W and Vi are the same as de-
scribed above.
2.4.2. Determination of macroporous resins/sample ratio
A series of adsorptions was carried out with different macropo-
rous resins/sample ratios (2:1, 3:1, 4:1, 4.5:1 and 5:1, w/w) to
determine the optimal macroporous resins/sample ratio.
312 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318

Table 1 Pre-weighed resins were added to the sample solution. The mix-
The purities and yields of the compounds in proposed separation procedure. ture was shaken in an incubation shaker (120 rpm) for 4 h at
Compound Procedure Purity Yield Total 25 C. After adsorption, the solutions were separated from the res-
(%) (%) yield ins and analyzed by HPLC. The adsorption ratios of biochanin A and
(%) genistein were calculated according to the following equation:
Biochanin A Crude extract 4.12 69.82
Macroporous resin enrichment 27.20 87.13 C0  Ce
E  100%
Separation by silica gel ash >95 80.13 C0
chromatography
Genistein Crude extract 1.06 61.85 where E is the adsorption ratio (%), which is the percent of the ad-
Macroporous resin enrichment 6.79 84.60
sorbed quantity to the initial quantity under equilibrium; C0 and Ce
Separation by silica gel ash >95 73.11
chromatography
are the initial and equilibrium concentrations of solute in the solu-
tions, respectively (mg/mL).
After determining the adsorption resin and resin/sample ratio
based on the above experiments, scaled-up enrichment of

Fig. 1. The actual ash chromatography system (A), schematic representation of the ash chromatography system (B), and schematic diagram of purication process for
biochanin A and genistein from D. odorifera leaves (C). a mobile phase; b pump; c chromatographic column; d UV detector; e automatic collector; f data
acquisition system.
 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318 313

Fig. 2. Adsorption capacities and desorption ratios of biochanin A (A) and genistein (B) on different macroporous resins.

biochanin A and genistein was carried out to obtain enriched prod-
uct for ash chromatography experiment.
2.5. Separation and purication of biochanin A and genistein by ash
chromatography
TLC, the eluate was divided into three fractions: fraction A
(Fr.A) containing biochanin A; fraction B (Fr.B) containing biocha-
nin A and genistein, and fraction C (Fr.C) containing genistein.

The separation and purication of biochanin A and genistein

were performed on a commercial ash chromatography system
(Leisure, Shanghai, China) equipped with a binary liquid pump, a
UV detector and an automatic collector. The actual ash chroma-
tography system and schematic representation of the ash chro-
matography system were shown in Fig. 1A and B.
The enriched product of biochanin A and genistein was dis-
solved in methanol. The same amount of silica gel (300
400 mesh) was added to the sample solutions, and then removed
solvent by rotary evaporator device at 40 C. The mixture of en-
riched product and silica gel was loaded on a column
(26 mm  125 mm I.D.) which was pre-packed with silica gel
(300400 mesh). Separation of biochanin A and genistein was
achieved using gradient elution system. The eluate was fraction-
ated and collected by automatic collector according to the chro- Fig. 3. Effect of macroporous resins/sample ratio on the adsorption ratios of
matograms at 262 nm. Based on the chromatograms prole and biochanin A and genistein.
314 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318

Fig. 4. HPLC chromatograms of crude extract (A), enriched product with AL-2 resin (B) and puried biochanin A (C) and genistein (D).

Then, the fraction A, B and C were evaporated by rotary evapora-
tion, respectively. The fraction A and C were analyzed by HPLC to
determine the purities and recovery yields of obtained biochanin
A and genistein, respectively. In order to screen optimum elution
system, three elution systems, n-hexaneethyl acetate, n-hexane
chloroform, n-hexaneacetone, were used as gradient elution sol-
vent. The optimum sample/silica gel ratio and ow rate were also
studied systematically in this work.
Another chromatogram column (26 mm  125 mm I.D.) was
packed with Toyopeal HW-40S gel resin. The enriched product of
biochanin A and genistein (1.0 g) was dissolved in 6 mL methanol,
and then loaded on the Toyopeal HW-40 gel resin column. The
biochanin A and genistein were eluted with pure methanol. The
ow rate was kept at 30 mL/min. The schematic diagram of puri-
cation process for biochanin A and genistein from D. odorifera
leaves was shown in Fig. 1C.
3. Results and discussion
3.1. Enrichment by macroporous resin
3.1.1. Screening of macroporous resins
To enrich effectively biochanin A and genistein from D. odorifera
leaves extracts by macroporous resins, the optimum type of mac-
roporous resin was screened rstly. The properties of these resins
were compared in terms of their adsorption and desorption capac-
ities as well as desorption ratios. As can be seen from Fig. 2, the
adsorption capacities of FL-1, HPD-500 and SA-3 resins towards
genistein were higher, and the adsorption capacities of FL-1, ME-
2 and HPD-500 resins towards biochanin A were better. The
adsorption capacity not only correlates with the physical and
chemical properties of adsorbent, but also with the size and chem-
ical features of the adsorbed substance. However, their lower
 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318 315

desorption capacities resulted in a lower desorption ratio. Desorp- resin which is based on hydroxylated methacrylic polymer [22].
tion ratios of AL-2 resin for genistein and biochanin A were obvi- Recently, due to high mechanical strength, good chemical stability
ously higher than those of others, and AL-2 resin possessed and much better regeneration efciency, it has been more and
moderate adsorption capacity towards genistein and biochanin A. more widely applied in the separation and purication of natural
Therefore, AL-2 resin was chosen as the optimum resin for the products [2225]. Especially, it exhibited potential capacity for
enrichment. separation and purication of avonoids [22,23]. Fig. 5A showed
the chromatogram proles of separating biochanin A and genistein
3.1.2. Effect of macroporous resin /sample ratio on adsorption ratio by silica gel ash chromatography. Separation of biochanin A and
The effect of macroporous resins/sample ratio on the adsorption genistein was achieved by the following elution program: 0
ratio was investigated at macroporous resins/sample ratio of 2:1 13.5 min, n-hexaneethyl acetate (10:1, v/v); 13.545 min, n-hex-
5:1. The results are shown in Fig. 3. It can be seen that the adsorp- aneethyl acetate (6:1, v/v). The ow rate was kept at 30 mL/min.
tion ratios increased with the macroporous resins/sample ratio. The sample/silica gel ratio was 1:40 (1.0 g enrichment product:
When the macroporous resins/sample ratio is 3:1, the adsorption 40 g silica gel). Based on the chromatograms prole and TLC, we
ratio of bochanin A reached 90%. But in this case, the adsorption ra- collected about 2128 min and 32.538 min as Fr. A and C to yield
tio of genistein was till lower. When the macroporous resins/sam- biochanin A and genistein, respectively. After HPLC analysis, the
ple ratio is 5:1, the adsorption ratios of bochanin A and genistein
reached 97.45% and 90.24%. Considering simultaneous enrichment
of biochanin A and genistein, a macroporous resins/sample ratio of
5:1 was chosen.
In order to obtain enriched product for the following ash chro-
matography experiment, scaled-up enrichment of biochanin A and
genistein was carried out. The chromatograms of the samples be-
fore and after treatment with AL-2 resin were shown in Fig. 4A
and B. The contents of biochanin A and genistein in crude extracts
were 4.12% and 1.06%. After the enrichment on AL-2 resin, the con-
tents of biochanin A and genistein in the enriched product reached
27.20% and 6.79%, which were 6.60-fold and 6.41-fold to those in
crude extract, respectively, and the recovery yields were 87.13%
and 84.60%, respectively (Table 1). This process achieved easy
and effective enrichment of biochanin A and genistein by using
AL-2 resin.

3.2. Separation and purication of biochanin A and genistein by ash

chromatography

3.2.1. Selection of stationary phase

Stationary phase played a decisive role in separation of biocha-
nin A and genistein. Two stationary phases, silica gel and Toyopeal
HW-40S, were tested in this experiment. Toyopeal HW-40S gel re-
sin, as one of the novel stationary phase, is a molecular sieve gel

Fig. 5. Chromatogram proles of enriched product by silica gel ash chromatog-

raphy at 30 mL/min (A), Toyopeal HW-40S gel resin ash chromatography at 30 mL/ Fig. 6. Effects of elution system (A), sample/silica gel ratio (B) and ow rate (C) on
min (B). the yields of biochanin A and genistein.
316 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318
Fig. 7. Chromatogram prole of enriched product by silica gel ash chromatogra-
phy under optimized conditions.
purities of biochanin A and genistein were more than 95%, the
recovery yields reached 76.02% and 70.52%, respectively.
Biochanin A and genistein with weak-polarity was hardly
eluted on Toyopeal HW-40S column by aqueous methanol. So, pure
methanol was employed as mobile phase. The ow rate was kept at
30 mL/min. As shown in Fig. 5B, biochanin A and genistein were
not effectively separated. Only small amounts of Fr.A and C were
obtained. Although the purities of puried biochanin A and geni-
stein were more than 95%, their recovery yields were less than 15%.
Hence, the silica gel ash chromatography was suitable for
preparation of biochanin A and genistein from D. odorifera T. Chen
leaves. The operating parameters of silica gel ash chromatography
were optimized in the following experiments.
3.2.2. Effects of elution system
Biochanin A and genistein are weak-polar compounds, so low
polar solvents were required to separate on normal phase silica
gel column by ash chromatography. Three elution systems with
weak polarity (n-hexaneethyl acetate, n-hexanechloroform, n-
hexaneacetone) were tested. The results were shown in Fig. 6A.
It was found only a little Fr.A and C were obtained using n-hex-
anechloroform (CHCl3) and n-hexaneacetone systems because
biochanin A and genistein could not be completely separated.
Although the content of biochanin A and geinstein in Fr.A and
Fr.C reached more than 90%, the recovery yields of biochanin A
and genistein were extremely low (less than 45%). For n-hexane
ethyl acetate system, weak-polar impurities were effectively re-
moved when n-hexaneethyl acetate (10:1, v/v) elution system
was employed. Biochanin A and genistein with similar polarity
Fig. 8. Negative ESIMS and MS/MS spectra of puried biochanin A (A and B) and genistein (C and D) from D. odorifera leaves, respectively.
were separated by using n-hexane/ethyl acetate (6:1, v/v). Hence,
n-hexaneethyl acetate was used in silica gel ash chromatography
for the separation and purication of biochanin A and genistein.
3.2.3. Effects of sample/silica gel ratio
High sample load may distort peak shape and cause an overall
decrease in separation efciency due to column overload. So, the
inuence of sample load on separation efciency was also investi-
gated by varying the sample/silica gel ratio from 4:40 to 1:40. The
elution program was the same as Section 3.2.1. The results were
shown in Fig. 6B. The yields of biochanin A and genistein increased
drastically with decreasing sample/silica gel ratio from 4:40 to
2:40, but did not continue to signicantly increase when the sam-
ple/silica gel ratio was below 1.3:40. Although the purity of bioch-
anin A was more than 95% at the high sample/silica gel ratio (4:40,
2:40), the recovery yield of biochanin A was remarkably low. The
same trend appeared in the recovery yield of genistein. As ex-
pected, when the sample/silica gel ratio decreased, separation ef-
ciency gradually increased due to less sample load. So, satisfactory
separation of biochanin A and genistein could be achieved with a
sample/silica gel ratio of 1.3:40.
3.2.4. Effects of ow rate
Flow rate, as a parameter of ash chromatography, effects the
retention times, system pressure and separation efciency. In or-
der to keep the same volume of elution solvent, elution time was
adjusted with different ow rate. Fig. 6C described the effect of
ow rate on separation of biochanin A and genistein by silica gel
ash chromatography. As shown in Fig. 6C, the recovery yields of
biochanin A and genistein gradually increased with increasing ow
rate from 30 to 50 mL/min. When the ow rate is lower, the time of
contact between stationary phase and target compounds is longer,
leading to difculty in elution of target compounds from the chro-
matography column. When ow rate was increased to 70 mL/min,
the recovery yields of biochanin A and genistein decreased appar-
ently. The separation efciency of chromatogram column is related
to its plate number. Plate number rises rstly and then declines
with increasing the ow rate. In a word, excessively high ow rate
has negative effect on column chromatography. Moreover, higher
ow rate resulted in increase of backpressure, which might require
special facilities in industrial production, leading to increase of
 F.-Y. Ma et al. / Separation and Purication Technology 120 (2013) 310318
instrument and consumable costs [26]. These factors become a
greater concern in large-scale produce. So, a ow rate of 50 mL/
min was considered as optimal one. The corresponding gradient
program was: 08 min, n-hexaneethyl acetate (10:1, v/v); 8
35 min, n-hexaneethyl acetate (6:1, v/v).
3.2.5. Validation of Fash chromatography
The enriched product of biochanin A and genistein (1.3 g) was
dissolved in methanol. The same amount of silica gel (300
400 mesh) was added to the sample solutions, and then removed
solvent by rotary evaporator device at 40 C. The mixture of en-
riched product and silica gel was loaded on a pre-packed 40 g silica
gel (300400 mesh) column (26 mm  125 mm I.D.). Separation of
biochanin A and genistein was achieved using n-hexane and ethyl
acetate as gradient elution solvent. The gradient program was: 0
8 min, n-hexaneethyl acetate (10:1, v/v); 835 min, n-hexane
ethyl acetate (6:1, v/v). The ow rate was kept at 50 mL/min. The
chromatogram prole of enriched product by ash chromatogra-
phy was shown in Fig. 7. Under the optimized conditions,
282.98 mg biochanin A and 64.52 mg genistein were obtained by
one ash chromatography run. The purities of biochanin A and
genistein both reached more than 95%, and their recovery yields
were 80.13% and 73.11%, respectively (Table 1). HPLC chromato-
grams of puried biochanin A and genistein were shown in
Fig. 4C and D, respectively. The proposed silica gel ash chroma-
tography boasts high efciency, low running costs and process
automation, and it is a promising method for efcient preparation
of biochanin A and genistein from D. odorifera T. Chen leaves.
3.3. Conrmation of biochanin A and genistein
The structures of puried compounds were conrmed by MS
spectra. ESIMS spectra of puried biochanin A and genistein in
Fig. 8A and C showed a molecular ion [MH] at m/z 282.9 and
269.0, respectively. The MS/MS spectra of puried biochanin A
and genistein in Fig. 8B and D were also consistent with those from
previous publications [27].
4. Conclusions
In this work, AL-2 was used for effective enrichment of biocha-
nin A and genistein from extract of D. odorifera leaves. After treat-
ment with AL-2 resin, the contents of biochanin A and genistein in
the enriched product reached 27.20% and 6.79% with recovery
yields of 87.13% and 84.60%, respectively. Under the optimal con-
ditions of ash chromatography, biochanin A and genistein were
successfully separated after one ash chromatography run, and
their purities were over 95% with the recovery yields of 80.13%
and 73.11%. The total recovery yields of biochanin A and genistein
reached 69.82% and 61.85%, respectively. These results indicated
that the enrichment of macroporous resin followed by the separa-
tion of ash chromatography is an effective procedure for prepar-
ing high-purity biochanin A and genistein with advantages of high
recovery yield, high efciency, low running costs and process auto-
mation. This work provides a promising alternative for large-scale
preparation of avonoids from D. odorifera leaves or other plants.
Acknowledgements
The authors gratefully acknowledge the nancial supports by
Program for Agricultural Science and Technology Achievements
Transformation Fund Program (2012GB23600641), Importation of
International Advanced Forestry Science and Technology, National
317
Forestry Bureau (2012-4-06) and Heilongjiang Province Science
Foundation for Excellent Youths (JC200704).
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