Preparative isolation and purification of psoralen and isopsoralen from Psoralea corylifolia by high-speed counter-current chromatography.
ABSTRACT Psoralen and isopsoralen were separated from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). A two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:4.5:5.5, v/v) was used for HSCCC separation, and yielded, from 100 mg of crude extract, 39.6 mg of psoralen and 50.8 mg of isopsoralen each at over 99% purity as determined by high performance liquid chromatography (HPLC). The identification of psoralen and isopsoralen were performed with 1H NMR and 13C NMR.
- SourceAvailable from: academicjournals.org
- [Show abstract] [Hide abstract]
ABSTRACT: Psoralea corylifolia (Bakuchi), a weed, which possesses a highly potent and medicinally important compound psoralen. P. corylifolia has been widely exploited since ages for its biological potential. Fifteen root nodulating bacteria as pure culture collection (PCC) were isolated from P. corylifolia in India. Further, these strains were evaluated for their effect on the psoralen content in P. corylifolia. High performance liquid chromatography (HPLC) method was used for the estimation of psoralen in P. corylifolia seed extracts. The effectiveness of these rhizobial strains was assessed on the basis of screening of various plant growth promoting attributes. The 16S ribosomal RNA sequencing analysis revealed the identity of two most effective rhizobial isolates PCC2 and PCC7 as Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. The R. leguminosarum PCC2 (JN546144) and Ensifer meliloti PCC7 (JN546145) strains showed solubilization of insoluble inorganic phosphate, secreted indole acetic acid (IAA), produced siderophore, showed ACC deaminase activity, and were positive for nodulation and nitrogen fixing genes. Seeds of P. corylifolia were bacterized with combination of R. leguminosarum PCC2 and Ensifer meliloti PCC7 along with their individual application that resulted in enhancement of various early vegetative and late reproduction parameters of plants in two consecutive field trials in the year 2009 and 2010. The psoralen content in the seeds of P. corylifolia was observed to be increased in the field trials where the combination of rhizobial strains PCC2 and PCC7 was used (2.79%) compared to control (1.91%). These findings indicate that rhizobial strains PCC2 and PCC7 showing good plant growth promoting attributes can be effective for increasing the psoralen content in the seeds of P. corylifolia to a certain level.Pharmacognosy Magazine 10/2013; 9(Suppl 1):S57-65. · 1.11 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: High-speed countercurrent chromatography (HSCCC) is a liquid-liquid separation chromatographic technique which uses no solid supporting medium. During its rapid development in the last 30 years, great progress has been made in the instrumentation, the basic study and application of HSCCC. It has significant advantages over other instrumental separation techniques, in its high efficiency and continuous processing capability. In recent years, HSCCC has been widely used in research and development of natural medicines and functional foods, including preparative separation, fingerprint analysis and screening of bioactive constituents. A review of the technique is presented in this paper.Central European Journal of Chemistry 06/2011; 10(3). · 1.33 Impact Factor
Journal of Chromatography A, 1057 (2004) 225–228
Preparative isolation and purification of psoralen and isopsoralen from
Psoralea corylifolia by high-speed counter-current chromatography
Renmin Liua,∗, Aifeng Lia, Ailing Suna, Lingyi Kongb
aCollege of Chemistry and Chemical Engineering, Liaocheng University, Liaocheng 252059, China
bDepartment of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
Received 11 June 2004; received in revised form 5 September 2004; accepted 16 September 2004
Psoralen and isopsoralen were separated from Psoralea corylifolia by high-speed counter-current chromatography (HSCCC). A two-phase
solvent system composed of n-hexane–ethyl acetate–methanol–water (5:5:4.5:5.5, v/v) was used for HSCCC separation, and yielded, from
100mg of crude extract, 39.6mg of psoralen and 50.8mg of isopsoralen each at over 99% purity as determined by high performance liquid
chromatography (HPLC). The identification of psoralen and isopsoralen were performed with1H NMR and13C NMR.
© 2004 Elsevier B.V. All rights reserved.
Keywords: High-speed counter-current chromatography; Psoralea corylifolia; Psoralen; Isopsoralen
Psoralea corylifolia (Chinese traditional medicinal herb,
Buguzhi in Chinese) has been widely used in traditional Chi-
nese medicine for the treatment of various kinds of disor-
ders such as asthma, cough, nephritis, vitiligo, and calvities
. The effective components of this herb are coumarins.
Psoralen and isopsoralen are the major components. Phar-
macological test revealed that they have antitumor , an-
tibacterial and antivirus activities and can affect metabolism
of some remedy . Psoralen and isopsoralen are used as
reference standards in the quality control of Buguzhi and its
products. So the isolation and purification of psoralen and
isopsoralen are of great interest.
High-speed counter-current chromatography (HSCCC) is
a form of liquid–liquid partition chromatography, which was
The liquid stationary phase is immobilized in the column
by centrifugal force. When the mobile phase is pumped
through the column, sample components are partitioned be-
∗Corresponding author. Tel.: +86 6358239840
E-mail address: firstname.lastname@example.org (R. Liu).
tween the two phases and they are separated on the basis
of difference in partition coefficients. Comparing with tradi-
tional liquid–solid column chromatography, HSCCC elimi-
recovery and efficiency . So it is very suitable for separa-
herbs and other natural products. Many successful applica-
tions of HSCCC have been reported for the separation of
various components such as alkaloids [6,7], flavonoids [8,9],
polyphenols [10,11], terpenoids , antibiotics , dyes
, and coumarin .
extract of P. corylifolia. The optimum conditions were ob-
isopsoralen with the purity of each at over 99%.
The HSCCC instrument employed in the present study
is TBE-300A high-speed counter-current chromatography
0021-9673/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
R. Liu et al. / J. Chromatogr. A 1057 (2004) 225–228
(Tauto Biotechnique Company, Shanghai, China) with three
multilayer coil separation column connected in series (i.d.
of the tubing=1.6mm, total volume=260mL) and a 20mL
sample loop. The revolution radius was 5cm, and the β
values of the multilayer coil varied from 0.5 at internal
terminal to 0.8 at the external terminal. The loop volume
of the sample injection is 20mL. The revolution speed
of the apparatus can be regulated with a speed controller
in the range between 0 and 1000rpm. The experimental
temperature was adjusted by HX 1050 constant tempera-
ture circulating implement (Beijing Boyikang Lab Imple-
ment, Beijing, China). An¨AKTA prime (Amersham Phar-
macia Biotechnique Group, Sweden) was used to pump
the two-phase solvent system and perform the UV ab-
sorbance measurement. It contains a switch valve and a
mixer, which can be used for gradient formation. The data
were collected with Sepu 3000 chromatography workstation
(Hangzhou Puhui Science Apparatus Co. Ltd., Hangzhou,
The HPLC equipment used was Agilent1100 system in-
Nuclear magnetic resonance (NMR) spectrometer used
here was Mercury Plus 400 NMR (Varian Inc., USA).
All solvents used for preparation of crude sample and
HSCCC separation were of analytical grade (Jinan Reagent
Factory, Jinan, China). Methanol used for HPLC was chro-
matographic grade (Yucheng Chemical plant, Yucheng,
China), and water used was distilled water.
The dried seeds of P. corylifolia were purchased from a
local drug store and identified by Professor Yongqing Zhang
(Shandong University of Traditional Chinese Medicine, Ji-
2.3. Preparation of crude sample 
The dried seeds of P. corylifolia were ground to powder
(about 30 mesh). The powder (1000g) was marinated with
2000mL of 50% ethanol for 24h and then filled in a glass
column and eluted with 8000mL of 50% ethanol for 8 days
at room temperature. The extract was evaporated under re-
duced pressure to about 3500mL, and the concentrate was
generated in the solution was extracted with 400mL of light
petroleum (boiling range 60–90◦C) at 85◦C for 2h. The ex-
traction was repeated five times. Then the light petroleum
extracts were combined and evaporated under reduced pres-
sure. 10.3g of crude extract powder was obtained. It was
stored in a refrigerator (4◦C) for the subsequent HSCCC
2.4. Selection of two-phase solvent system
Selecting the solvent system for HSCCC means simulta-
neously choosing the stationary phase and the mobile phase.
The solvent system was selected according to the partition
coefficients (K) of the target compounds. The K-values were
determined by HPLC as following: suitable amount of crude
extract powder was dissolved in the upper phase of the sol-
vent system and analyzed by HPLC. The peak areas of the
peaks were recorded as A1. Then equal volume of the lower
phase was added to the solution and mixed thoroughly to
reach partition equilibrium. The upper phase was then ana-
lyzed by HPLC again. The latter peak areas were recorded as
A2. The K-values were calculated according to the following
2.5. Preparation of two-phase solvent system and
In the present study, the two-phase solvent system com-
posed of n-hexane–ethyl acetate–methanol–water was used
for HSCCC separation. Each solvent was added to a separa-
The upper phase and the lower phase were separated and
degassed by sonication for 30min shortly before use.
The sample solution for HSCCC separation was prepared
by dissolving suitable amount of crude extract in the upper
phase of the solvent system.
2.6. HSCCC separation procedure
HSCCC was performed with a TBE-300A HSCCC in-
strument. The upper phase (stationary phase) and the lower
phase (mobile phase) of the two-phase solvent system were
pumped into the column simultaneously by using¨AKTA
prime system, with the volume ratio of 60:40. When the col-
umn was totally filled with the two phases, the lower phase
was pumped at a flow rate of 2.0mLmin−1, and at the same
time, the HSCCC apparatus was run at a revolution speed
of 900rmp. After hydrodynamic equilibrium was reached
ing 100mg of the crude extract was injected into the separa-
recorded 100min after sample injection. All through the ex-
monitored with a UV absorbance detector at 254nm. Each
peak fraction was collected according to the chromatogram
solved in methanol for subsequent study.
2.7. HPLC analysis and identification of HSCCC peak
The crude extract of P. corylifolia and each peak frac-
tion from HSCCC separation were analyzed by HPLC.
R. Liu et al. / J. Chromatogr. A 1057 (2004) 225–228
Fig. 1. HPLC chromatograms of crude extract from P. corylifolia. Con-
ditions: column, reversed-phase YWG C18column (200mm×4.6mm i.d.,
detection wavelength, 254nm.
The column used for HPLC analysis was YWG C18 col-
umn (200mm×4.6mm i.d. 10?m). The mobile phase
was methanol–water (40:60, v/v) and the flow rate was
1.0mlmin−1. The effluent was monitored at 254nm.
The identification of HSCCC peak fractions was carried
out by1H NMR and13C NMR.1H NMR and13C NMR
spectra were recorded on a Mercury Plus 400 NMR with
TMS (for1H NMR) and C2HCl3(for13C NMR) as internal
3. Results and discussion
3.1. Optimization of HPLC method
The partition coefficient of each component in crude ex-
tract was determined by HPLC analysis. So in the first place,
a good HPLC method should be developed for analysis
of crude extract. Different mobile phases (methanol–water,
acetonitrile–water) were used in HPLC to separate psoralen
and isopsoralen from the crude extract of P. corylifolia. The
results indicated that when methanol–water (40:60, v/v) was
used as the mobile phase, psoralen and isopsoralen could ob-
tain baseline separation. Analyzed by Agilent 1100 worksta-
tion, the purity factor of each peak was within the calculated
threshold limit. The HPLC chromatogram of crude extract
from P. corylifolia was given in Fig. 1.
3.2. Optimization of HSCCC conditions
According to the HPLC analysis results (Fig. 1), there
are two major compounds present in the crude extract from
compounds in HSCCC separation, the K-values of these two
compounds in different solvent systems were determined by
HPLC as the procedure shown in Section 2.4. The results are
The K-values of psoralen and isopsoralen
Ethyl acetate–water (5:5, v/v)
Ethyl acetate–methanol–water (5:1:5, v/v)
Ethyl acetate–methanol–water (5:2:5, v/v)
Ethyl acetate–methanol–water (5:3:5, v/v)
Ethyl acetate–methanol–water (5:4:5, v/v)
No phase separation
aK is expressed as the solute concentration in the upper stationary phase
divided by that in the lower mobile phase.
very big. By adding methanol to ethyl acetate–water, the K-
values could be reduced. But the phase separation could not
of n-hexane to ethyl acetate–methanol–water system could
improve the phase separation. According to the K-values, n-
hexane–ethyl acetate–methanol–water solvent systems with
used as the solvent system, the separation time was too long.
was used, the two compounds could not get satisfactory
separation. When n-hexane–ethyl acetate–methanol–water
(5:5:4.5:5.5, v/v) was used as the solvent system, good sep-
aration results and acceptable separation time could be ob-
ture, the retention percentage of the stationary phase and the
revolution speed of the separation coil were also optimized.
The HSCCC chromatogram of crude extract from P. coryli-
folia obtained under the optimum conditions was shown in
Fig. 2. 39.6mg of psoralen (peak I) and 50.8mg of isopso-
in one-step separation.
3.3. HPLC analysis and identification of HSCCC peak
The purity of each peak fraction of HSCCC was deter-
mined by HPLC. The HPLC chromatogram and the UV
spectra of each fraction were shown in Fig. 2. The purity of
psoralen (peak I in Fig. 2) and isopsoralen (peak II in Fig. 2)
was 99.4 and 99.6%, respectively.
The identification of peak fractions in Fig. 2 was per-
data of each fraction were given as follows:
R. Liu et al. / J. Chromatogr. A 1057 (2004) 225–228
v/v); mobile phase, the lower phase; flow rate, 2.0mlmin−1; revolution speed, 900rpm; detection wavelength, 254nm; separation temperature, 20◦C; sample
size, 100mg of crude sample dissolved in 5ml of the upper phase; retention percentage of the stationary phase, 60%. I: psoralen (collected during 78–112min);
II: isopsoralen (collected during 150–202min).
HSCCC peak I in Fig. 2:1H NMR (400MHz, C2HCl3):
C12–H), 7.69 (1H, s, C5–H), 7.49 (1H, s, C8–H), 6.84 (1H,
d, J=2.4Hz, C11–H), 6.39 (1H, d, J=10Hz, C3–H);13C
NMR (400MHz, C2HCl3): δppm: 161.10 (C-2), 156.37 (C-
7), 151.96 (C-9), 146.91 (C-12), 144.12 (C-4), 124.86 (C-6),
119.82 (C-5), 115.37 (C-10), 114.60 (C-3), 106.35 (C-11),
HSCCC peak II in Fig. 2:1H NMR (400MHz, C2HCl3):
C12–H), 7.45 (1H, d, J=8.8Hz, C6–H), 7.40 (1H, d,
J=8.8Hz, C5–H), 7.15 (1H, d, J=2.4Hz, C11–H), 6.41 (1H,
d, J=9.6Hz, C3–H);13C NMR (400MHz, C2HCl3): δppm:
160.66 (C-2), 157.33 (C-7), 148.93 (C-9), 145.90 (C-12),
144.59 (C-4), 141.25 (C-3), 123.82 (C-5), 117.04 (C-8),
114.10 (C-10), 108.84 (C-6), 104.11 (C-11).
According to the literature , peaks I and II in Fig. 2
were identified as psoralen and isopsoralen, respectively.
In conclusion, HSCCC was successfully used for the
isolation and purification of psoralen and isopsoralen from
P. corylifolia. 39.6mg of psoralen and 50.8mg of isopso-
ralen each at over 98% purity can be obtained from 100mg
of the crude extract in one-step separation.
 Jiangsu New Medicinal College, Dictionary of Traditional Chinese
Herbs, Shanghai Science and Technology Publishing House, Shang-
hai, 1985, p. 1178.
 S.N. Wu, Z.H. Zhang, J.B. Zhao, Chin. J. Chin. Mater. Med. 23
 S.Q. Mi, X. Hong, T.L. Huang, S.M. Ye, W.P. Ou, M.R. Feng, Q.W.
Xu, Q.D. Liu, Tradit. Chin. Drug Res. Clin. Pharmacol. 9 (1998)
 Y. Ito, J. Chromatogr. 214 (1981) 122.
 X. Han, T.Y. Zhang, Y. Wei, X.L. Cao, Y. Ito, J. Chromatogr. A 971
 F.Q. Yang, Y. Ito, J. Chromatogr. A 923 (2001) 281.
 L.M. Yuan, M. Zi, X.X. Chen, Z.Y. Li, R.N. Fu, T.Y. Zhang, J.
Chromatogr. A 927 (2001) 91.
 X.F. Ma, P.F. Tu, Y.J. Chen, T.Y. Zhang, Y. Wei, Y. Ito, J. Chro-
matogr. A 992 (2003) 193.
 X.L. Cao, Y. Tian, T.Y. Zhang, X. Li, Y. Ito, J. Chromatogr. A 855
 L. Chen, Y.S. Han, F.Q. Yang, T.Y. Zhang, J. Chromatogr. A 907
 F.Q. Yang, T.Y. Zhang, Y. Ito, J. Chromatogr. A 919 (2001) 443.
 H.B. Li, F. Chen, J. Chromatogr. A 925 (2001) 109.
 W. Wang-Fan, E. K¨ usters, C. Mak, Y. Wang, J. Chromatogr. A 925
 A. Weisz, E.P. Mazzola, J.E. Matusik, Y. Ito, J. Chromatogr. A 923
 F.Q. Yang, T.Y. Zhang, Q.H. Liu, G.Q. Xu, Y.B. Zhang, S. Zhang,
Y. Ito, J. Chromatogr. A 883 (2000) 67.
 D.P. Yu, Lishizhen Med. Mater. Med. Res. 11 (2000) 592.
 D.C. Chen, Handbook of Reference Substance for Traditional Chi-
nese Herbs, China Pharmaceutical Technology Publishing House,
Beijing, 2000, p. 91, 107.