JSSC 201901174

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Optimization Extraction and Purification of Biological Activity Curcumin from Curcuma longa L by
High Performance Counter-Current Chromatography
Yan pan1, Ronghui Ju1, Xueli Cao2*, Hairun Pei2*, Tianhao Zheng1, Wei Wang3
From 1Beijing Vocational College of Agriculture，Beijing, 102442, China
2
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Technology & Business University, Beijing 100048, China.
3
Beijing Center for Physical and Chemical Analysis, 100094, China.
To whom correspondence should be addressed: Xueli Cao: Beijing Advanced Innovation
Center for Food Nutrition and Human Health, Beijing Technology & Business University,
Beijing 100048, China. Phone: +86-10-68984898, Email: caoxl@th.btbu.edu.cn; Hairun Pei:
Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing
Technology & Business University, Beijing 100048, China. Phone: +86-10-68984485, Email:
peihairun@th.btbu.edu.cn;
Abbreviations: HPCCC, high performance counter-current chromatography; K, partition
coefficient; HPLC, high performance liquid chromatography; DAD, diode-array detector; CC,
curcumin; DMCC, demethoxycurcumin; BDMCC, bisdemethoxycurcumin; NMR, nuclear
Received: 18/11/2019; Revised: 08/01/2020; Accepted: 03/02/2020
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process, which may lead to differences
between this version and the Version of Record. Please cite this article as doi: 10.1002/jssc.201901174.
This article is protected by copyright. All rights reserved.

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magnetic resonance; L-I-H, lower phase as mobile phase elute in head-to-tail mode; DPPH,
1, 1-Diphenyl-2-picrylhydrazyl.
Abstract： The extraction condition of curcumin from Curcuma longa L was optimized
through four factors and three levels orthogonal experiment based on the results of single
factor tests. Under the optimal conditions：the concentration of ethanol 80 %, extraction
temperature 70 °C, the ratio of liquid to material 20, and extraction time 3 h, a crude
extract with the yield of curcumin 56.8 mg/g could be obtained. The isolation and
purification of curcuminoids from the crude extract was performed on high performance
counter current chromatography employing an optimized solvent system n-hexane-ethyl
acetate-methanol-water (2/3/3/1, v/v). From 97 mg crude sample (in which the purity of
curmumin was 68.56 %), 67 mg curmumin, 18 mg demethoxycurcumin and 9.7 mg
bisdemethoxycurcumin with HPLC purity of 98.26 %, 97.39 % and 98.67 % were respectively
obtained within 70 min. The antioxidant activities and cytotoxicity of purified curcumin was
comparable to that of the commercial product, indicating that the biological activity of
curcumin could be maintained by this method.
Key words：High performance counter-current chromatography; curcuminoids; curcumin;
demethoxycurcumin; bisdemethoxycurcumin.

1. Introduction
Turmeric, Curcuma longa L., is a well-known herb of the Zingiberacea family and a natural
colorant. As the main component of Curcuma longa L, curcuminoids have been explored in
antiprotozoal[1], antioxidant[2], anticancer[3-5], preventing diabetic nephropathy[6] and
preventing Alzheimer’s disease[7]. Curcumin (CC), demethoxycurcumin (DMCC) and
bisdemethoxycurcumin (BDMCC) are the three main curcuminoids (Fig. 1A), and CC is the
most important active component[8]. Due to the suppression effect of CC on a crucial event
in tumor metastasis-epithelial mesenchymal transition[4], it has been evaluated in clinical
trials for the treatment of various cancers and liver diseases, rheumatoid arthritis, infectious
diseases, and Alzheimer's disease[7]. Although DMCC and BDMCC have similar structure with
curcumin, the slight differences in structure make them great different in anti-tumor,
anti-oxidation and other aspects[9]. However, curcumin extract on the market is usually a
mixture of CC, DMCC and BDMCC. Therefore, it is necessary to further isolate and purify
curcumin compounds, which is of great significance for the identification of curcuma, the
quality control of curcuma preparations and the modification of the three curcumins results.
Counter-current chromatography (CCC)[10, 11] is a wildly applied method for purification of
natural components. Each component could be separated according its distribution
coefficients during the liquid-liquid extraction and centrifugal distribution prcoess[12].
Compare to conventional column chromatography, CCC eliminates irreversible adsorptive
loss of samples onto solid support matrix and avoids devitalization of active compounds [13,

14]
which can achieve identical purities and yields producing matching chromatograms [15, 16].
Notably, CCC has been already successfully used in the purification of phytosterol[17], fatty
acids[18], anthocyans[14], polyphenols[19], flavones[20] and many other natural and synthetic
products[21]. It has been reported that CC could be separated by counter-current
chromatography[22] or centrifugal partition chromatography[5], but the solvent system
contains chloroform which is forbidden in China. Recently, with the development of
counter-current chromatography technology, a new high-performance counter-current
chromatography (HPCCC) equipment with higher separation efficiency has been
developed[16]. HPCCC is a recently developed countercurrent chromatography which
accelerates the separations complete in minutes rather than hours that generally
experienced with HSCCC. Solvent systems, that was difficult to be applied before, can also
achieve the separation with improved retention of the stationary phase, and solvent
systems used in counter-current chromatography are gradually becoming less toxic. In
addition, it needs to point that CC is chemical instability and is sensitive to pH, temperature
and light[23]. In this way, the bioactivity of the extracted CC is an important indicator to
evaluate the purification method.
Herein, we developed a more friendly solvent system composed of n-hexane/ethyl
acetate/methanol/water to directly separate and purify curcumin from crude extract.
Complete separation of curcumin, demethoxycurcumin and bisdemethoxycurcumin with
high purity could be achieved efficiently in about one hour through the optimized solvent

system. Moreover, the bioactivity (antioxidant activities and tumor cell cytotoxicity) of
extracted CC shows no significant difference with commercial product.
2. Materials and methods
2.1 Reagents and materials
Curcuma longa L was bought from Tongrentang Chinese Medicine. The curcumin,
demethoxycurcumin, and bisdemethoxycurcumin standard products were obtained from
Sigma-Aldrich (Sigma-Aldrich company, China). Ethyl acetate, n-hexane and methanol were
purchased from Macklin (AR, Shanghai Macklin Biochemical Co. Ltd, China). Ethanol was
bought from National Pharmaceutical (AR, China National Pharmaceutical Group
Corporation, China) and distilled water were used. Chromatographic grade methanol used
for HPLC analysis were purchased from Fisher (HPLC, Fisher scientific, USA) and HPLC water
was supplied by Milli-Q Plus system (Millipore, Bedford, USA). HeLa (human cervical cancer
cells) cells were obtained from the Cell Resource Center (Peking Union Medical College
Headquarters of National Infrastructure of Cell Line Resource, NSTT). Cells were cultured in
Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine
serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37◦C and 5% CO2.
2.2 Apparatus
The CCC apparatus used in this study was Spectrum HPCCC (Dynamic Extraction, UK).
Spectrum HPCCC contains two columns, an analytical (0.8 mm i.d. tubing, 22.5 mL) and a
semi-preparative (1.6 mm i.d., 133.5 mL), and the rotation speed was adjustable from 0 to

1600 rpm. The column temperature was controlled by a SH150-1500 constant temperature
regulator (Lab Tec, Beijing, China). A KNAUER Smartline HPLC system (Berlin, Germany)
containing two p-1000 pumps, a UV-2500 detector and a EuroChrom workstation was
equipped with Spectrum HPCCC. An Agilent 1260 HPLC system (Agilent, USA) with
diode-array detector DAD-10A UV (Agilent, USA) was used in this study. The NMR data were
obtained on an Aglient DD2 600 MHz NMR spectrometer (Agilent, USA) with
tetramethylsilane as an internal standard. SpectraMax 190 (Molecular Devices, USA) ELISA
was used in this study.
2.3 The orthogonal test
The extraction of Curcuma longa L. was carried out through a four-factor, three-level
orthogonal test based on the single-test results. The optimization of the extraction
conditions was listed in Table 1 with the concentration of ethanol (X1), extraction
temperature (X2), the ratio of liquid to material (X3) and the extraction time (X4). Table 2
represented the experiment variables and 9 experiment points. The content of curcumin
was determined by external standard method (y = 117675x – 654802, R2 = 0.9978).
2.4 Counter-Current Chromatography Separation and identification
The selection of two-phase solvent system was the key point for CCC where the separation
is based on the partition coefficient (K) and separation factors (α) of each target component.
The K value of each component in crude Curcuma longa L extract was measured by HPLC.
Generally, 10 mg of crude sample was dissolved in the selected two-phase solvents (Table 3)
with 1 mL of upper phase and 1 mL of lower phase. After violent shaking and equilibrating
6

the two phases completely, 0.5 mL solution of each phase was taken and analyzed by HPLC.
The K values of each target component were calculated from the peak area obtained of the
upper phase divided by that of the lower phase. Separation factors (α) represented the ratio
of the K values of two adjacent target components.
The multilayer column was filled thoroughly with the upper phase (stationary phase), and
the mobile phase was then pumped into the Spectrum-CCC column at the flow rate of 5
mL/min while the apparatus was rotated at speed of 1600 rpm. After the equilibration, the
crude extract (97 mg) dissolved in 2 mL upper phase was loaded into the 133.5 mL column.
Chromatograms were recorded at 425 nm and the peak fractions were collected.
The purity of the three components was analyzed by HPLC, and their chemical structures
were identified by comparing the retention times with that of standard samples in HPLC and
1
H-NMR analysis. The HPLC conditions were as follows: column: Agilent ZORBAX SB-C18 (4.6
× 250 mm, 5 μm) used at the temperature of 35 ºC; mobile phase: acetonitrile : H2O (0.5 %
acetic acid) = 44 : 56 (v/v) at the flow rate of 1 mL/min; injection volume : 10 μL; detection
wavelength: 425 nm.
2.5 Antioxidant activitie
The antioxidant activity of the purified curcumin was determined using the 1,
1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay[22]. Generally, the
purified curcumin solutions at concentrations of 25, 50, and 100 μg/mL were prepared in
tubes, 4 mL DPPH (0.1 mM, dissolved in methanol) were added into each tube and mixed

thoroughly. The tubes were then incubated without light at room temperature for 20 min. A
control sample was prepared without any curcuminoids and used for baseline corrections.
The absorbance of sample solutions at 517 nm was recorded. The radical scavenging activity
was expressed as the inhibition percentage according to formula:
Radical scavenging activity (RSA %) = [(Acontrol – Aanalyte)/Acontrol] × 100% (1)
where A were the absorbance values measured for the test and control sample respectively.
All analyses were repeated three times, and the results were expressed as mean ± SD.
2.6 Cell viability
HeLa (human cervical cancer cells) cells were obtained from the Cell Resource Center
(Peking Union Medical College Headquarters of National Infrastructure of Cell Line
Resource, NSTT). Cells were cultured in Dulbecco's Modified Eagle's medium (DMEM)
(Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin
(Gibco) at 37 ºC and 5 % CO2[24].
To evaluate the cytotoxicity of extracted curcumin and standard sample, CCK-8 cell viability
was carried out. Basically, 5-8 × 103 cells were seeded in 96-well plates and incubated
overnight for further adherence. Cells were treated with extracted curcumin and standard
curcumin with concentrations ranging from 0 to 20 μg/mL for 24 h. Next, CCK-8 was added
to the culture medium at a tenth of the volume and incubated at 37 ºC for 1 h. Cells without
curcumin treatment were used as control. The absorbance at 450 nm was measured and cell
viability was calculated as formula:

Cell viability (%) = [(Acontrol – Aanalyte)/Acontrol] × 100% (2)
Where A was the absorbance of cells treated with or without CCK-8 at 450 nm. Each
concentration of curcumin was repeated five times, and the results were expressed as mean
± SD.
3. Results and discussion
3.1 HPLC analysis of the standards and crude samples
Fig.1 peresents the HPLC analysis chromatograms of standards (B) and the crude sample (C)
under optimized condition. By comparing the retention times with that of standard samples,
peak 1, 2 and 3 in crude sample were identified respectively as bisdemethoxycurcumin,
demethoxycurcumin and curcumin tentatively, which accounted for 10.32 %, 19.01 % and
68.56 % of the crude extract of Curcuma longa L sample.
3.2 Optimization for extraction of curcumin
In order to optimize the extraction conditions, an orthogonal L9 (3)4 test was designed
involved four factors with three variation levels: X1 (extraction time, 1, 2, 3 h), X2 (extraction
temperature, 60, 70, 80 °C), X3 (the concentration of ethanol, 70, 80 and 90 %) and X4 (the
ratio of liquid to material, 15, 20 and 25 mL/g) which listed in Table 1. From the results
(Table 2), the maximum extraction yield of curcumin was 54.2 mg/g. However, it could not
be considered as the best extraction conditions. Because the coefficients of the three
products are R3 > R4 > R2 > R1, the influence of extraction parameters decreased in the order
of X3 > X4 > X2 > X1. Therefore, it seems that the concentration of ethanol was found to be
9

the most important parameter in the curcumin extraction process. In this case, the
conditions used to achieve maximum yield of curcumin were optimized as the
concentration of ethanol 80%, temperature 70 °C, time 3 h, and liquid to material ratio 20
mL/g, respectively. Through confirmatory test, the highest yield of curcumin was 56.8 ± 1.2
mg/g.
3.3 Selection of solvent system and Counter-Current Chromatography separation
Several prerequisites should be considered. According to the golden rules in selecting
optimum separation conditions, partition coefficients of different solvent systems in the
range of 0.5 - 2 for the target component are important for successful separation by CCC
method[11]. Moreover, the separation factor between two constituents (α = K1 / K2, K1 > K2)
is at least 1.5. In addition, solubility and stability of the analytes in solvent should be taken
into consideration at the same time, as is the same case to the rapid and clear separation of
solvent system into phases. It has been reported that the curcumin had been separated by
n-hexane/chloroform/methanol/water (5/10/7.5/2.5, v/v) [22] solvent system. However, due
to the toxicity concern, chloroform was not easily available as a controlled product in china.
In this article, we try to use ethyl acetate to replace chloroform to achieve the separation.
According to another previous research, the curcumin couldn’t be separated by solvent
system, n-hexane/ethyl acetate/methanol/water (1/1/1/1, v/v)[25]. However, our
optimization based on the determination of the K and α values of the target components, as
shown in Table 3, revealed that n-hexane/ethyl acetate/methanol/water (2/3/3/1, v/v)
could be a potential solvent system to produce a satisfactory separation.
10

Therefore, it was employed to separate curcumin, demethoxycurcumin, and
bisdemethoxycurcumin in the crude sample on a high-performance counter-current
chromatography (HPCCC) instrument. As a result, the equipment was more robust efficient
than previous HSCCC instruments. The CCC elution was operated in L-I-H[14] (lower phase
as mobile phase, eluted from inner head) mode for 70 min after 97 mg of the crude extract
was loaded into 133.5 mL column. The whole separation complete in 70 minutes rather than
5 hours that generally experienced with HSCCC[22]. Three peak fractions (67 mg F1, 18 mg F2
and 9.7 mg F3) could be baseline separated (Fig. 2). Compared with the retention time of
standard samples, HPLC analysis proved the F1, F2 and F3 were curcumin,
demethoxycurcumin, and bisdemethoxycurcumin, with the purity 98.26 %, 97.39 % and
98.67 %, respectively (Fig. 3).
3.4 Identification of purified target compounds
F1, F2 and F3 were further confirmed for their structures by 1HNMR (Fig. S1). The identity of
F1, F2 and F3 were assigned as curcumin, demethoxycurcumin, and bisdemethoxycurcumin
based on agreement of NMR data with literatures[22, 26].
1
H NMR (DMSO, 600 MHz) of F1: δ 7.516(d, J = 15.81 Hz, 2H), 7.293(d, J = 1.79 Hz, 2H),
7.123(dd, J = 8.21, 1.81 Hz, 2H), 6.795(d, J = 8.14 Hz, 2H), 6.725(d, J = 15.82 Hz, 2H), 6.031(s,
1H), 3.810(s, 6H). All these data are in agreement with that of curcumin.
1
H NMR (DMSO, 600 MHz) of F2: δ 7.535(t, J = 6.71, 6.71 Hz, 3H), 7.503(d, J = 5.64 Hz, 1H),
7.296(d, J = 1.62 Hz, 1H), 7.116(dd, J = 8.20, 1.65 Hz, 1H), 6.794(dd, J = 8.32, 1.47 Hz, 3H),
11

6.731(d, J = 15.81 Hz, 1H), 6.662(d, J = 15.86 Hz, 1H),6.019(s, 1H), 3.811(s, 3H). All these data
are in agreement with that of demethoxycurcumin.
1
H NMR (DMSO, 600 MHz) of F3: δ 7.544(s, 2H), 7.517(d, J = 15.80 Hz, 4H), 6.793(d, J = 8.56
Hz, 4H), 6.665(d, J = 15.83 Hz, 2H), 6.013(s, 1H). All these data are in agreement with that of
bisdemethoxycurcumin.
3.5 Antioxidant activities and cytotoxicity
As a natural bioactive compound, curcumin is chemically instable to pH, temperature and
light. Therefore, avoiding compound inactivation during the purification process is one of
the important indicators to evaluate the purification method. Here we compared the
antioxidant activities and tumor cell cytotoxicity of the isolated high purity curcumin with
commercial curcumin product (purity >95%) to explore the HPCCC method in maintaining
the bioactivity of curcumin. As shown in Fig. 4A, DPPH assay displayed similar capability of
extracted curcumin and commercial curcumin in scavenging DPPH radicals, which is
consistent with other antioxidant profiles of curcuminoids[22, 27]. Moreover, pharmacology
studies had confirmed the anticancer effect of curcumin through its effect on protein
pathways[28]. As shown in Fig. 4B, as an antitumor drug, Curcumin exhibited cytotoxicity with
an IC50 value of 8.88 μg/ml in HeLa cells. Cell viability results suggested no significant
difference of HPCCC purified curcumin and commercial product, which further confirmed
the bioactivity of curcumin was remained during the HPCCC purification.
12

4. Conclusion
Overall, here we have developed a more efficient and rapid HPCCC method for the
separation and purification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin
from Curcuma longa L. By using n-hexane/ethyl acetate/methanol/water (2/3/3/1, v/v) as
two-phase solvent system, three compounds can be completely separated with high purity
in about 60 min. The purities of the isolated curcumin, demethoxycurcumin, and
bisdemethoxycurcumin can reach 98.26 %, 97.39 % and 98.67 %, while their contents in the
crude extract were 68.56 %, 19.01 % and 10.32 % respectively. The antioxidant activities and
cytotoxicity of purified curcumin is comparable to the commercial product, indicating the
method maintain the biological activity of curcumin. Our results demonstrate that HPCCC
elution method is a feasible method to separate and purify curcumin, demethoxycurcumin,
and bisdemethoxycurcumin from Curcuma longa L, which is more effective than many other
conventional techniques and potential to be applied for commercial industry.
Acknowledgement
This work was supported by grants from Natural Science Foundation of Beijing (No.
2184100) and the National Natural Science Fund of China (No. 21773014). We would like to
thank Yueheng Qi and Hang Gao from the Key Laboratory of Radio Pharmaceuticals of the
Ministry of Education for help with NMR experiments.
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Conflict of interest
The authors have declared no conflict of interest.
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FIGURE 1. Structural formulas of the main curcuminoids (A) and HPLC analyses of curcumin,
demethoxycurcumin, and bisdemethoxycurcumin standards (B) and the crude sample from
Curcuma longa L (C). Conditions: column: Agilent ZORBAX SB-C18 (4.6 × 250 mm, 5μm) was
used; column temperature, 35 ºC; mobile phase, acetonitrile: H2O (0.5 % acetic acid) = 44:
56 (v/v); flow rate, 1.0 mL/min; injection volume, 10 μL; detection wavelength: 425 nm. (A)
Structural formulas of the main curcuminoids. (B) Three standard samples in which black
dotted line is bisdemethoxycurcumin, red dashed line is demethoxycurcumin and blue solid
line is curcumin. (C) HPLC chromatogram of crude ethanol extract from Curcuma longa L.
Peak identities: 1, bisdemethoxycurcumin (10.32 %); 2, demethoxycurcumin (19.01 %); 3,
curcumin (68.56 %).
19

FIGURE 2. HPCCC chromatograms of the crude sample. Conditions: column, Spectrum-133.5
mL; flow rate, 5 mL/min; rotary speed, 1600 rpm; elution time: 70 min in L-I-H mode;
detection wavelength, 425 nm. F1, F2 and F3 were identified to be curcumin,
demethoxycurcumin, and bisdemethoxycurcumin, respectively.
20

FIGURE 3. HPLC-DAD analysis of F1, F2 and F3. Conditions: column, Agilent ZORBAX SB-C18
(4.6 × 250 mm, 5μm); column temperature, 35 ºC; mobile phase, acetonitrile : H 2O (0.5 %
acetic acid) = 44 : 56 (v/v); flow rate, 1.0 mL/min; injection volume, 10 μL; detection
wavelength, 425 nm. (A) F1, (B) F2, (C) F3.
21

FIGURE 4. The antioxidant activities and the cytotoxicity of curcumin on HeLa cells. (A) DPPH
radical scavenging activity (RSA) of curcumin standard and purified curcumin. (B) CCK-8
analysis of curcumin standard and purified curcumin on HeLa cells after 24 h treatment.
22

Table 1. Factors and levels for orthogonal test of extraction
Levels
Variable
1 2 3
X1 extraction time (h) 1 2 3
X2 extraction temperature (°C) 60 70 80
X3 the concentration of ethanol (%) 70 80 90
X4 the ratio of liquid to material (mL/g) 15 20 25
Table 2. Result analysis of L9 (3)4 test
No. X1 X2 X3 X4 Yields (mg/g)
1 1 1 1 1 23.6
2 1 2 2 2 54.2
3 1 3 3 3 36.9
4 2 1 2 3 45.7
5 2 2 3 1 36.4
6 2 3 1 2 36.7
7 3 1 3 2 38.4
8 3 2 1 3 32.7
9 3 3 2 1 48.8
k1 38.233 35.9 31 36.267
k2 39.600 41.100 49.567 43.100
k3 39.967 40.800 37.233 38.433
R 1.732 5.200 18.567 6.833
23

Table 3. Partition coefficients (K) and separation factors (α) of the three components in four
solvent systems
Solvent System CC DMCC BDMCC α

(K2)
n-hexane/ethyl (K1) (K3) K2/K1 K3/K2
acetate/methanol/water（v/v
1/1/1/1 1.77 1.95 2.11 1.10 1.08
1/3/3/1 0.93 1.16 1.25 1.24 1.07
2/3/3/1 0.63 1.05 1.67 1.67 1.59
3/3/3/1 0.47 0.59 0.75 1.25 1.27
24

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www.jss-journal.

com Page 1 Journal of Separation Science

From 1Beijing Vocational College of Agriculture，Beijing, 102442, China

Technology & Business University, Beijing 100048, China.

To whom correspondence should be addressed: Xueli Cao: Beijing Advanced Innovation

Beijing 100048, China. Phone: +86-10-68984898, Email: caoxl@th.btbu.edu.cn; Hairun Pei:

Abbreviations: HPCCC, high performance counter-current chromatography; K, partition

curcumin; DMCC, demethoxycurcumin; BDMCC, bisdemethoxycurcumin; NMR, nuclear

Received: 18/11/2019; Revised: 08/01/2020; Accepted: 03/02/2020

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factor tests. Under the optimal conditions：the concentration of ethanol 80 %, extraction

counter current chromatography employing an optimized solvent system n-hexane-ethyl

curmumin was 68.56 %), 67 mg curmumin, 18 mg demethoxycurcumin and 9.7 mg

curcumin could be maintained by this method.

Key words：High performance counter-current chromatography; curcuminoids; curcumin;

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antiprotozoal[1], antioxidant[2], anticancer[3-5], preventing diabetic nephropathy[6] and

preventing Alzheimer’s disease[7]. Curcumin (CC), demethoxycurcumin (DMCC) and

in tumor metastasis-epithelial mesenchymal transition[4], it has been evaluated in clinical

Counter-current chromatography (CCC)[10, 11] is a wildly applied method for purification of

natural components. Each component could be separated according its distribution

coefficients during the liquid-liquid extraction and centrifugal distribution prcoess[12].

Compare to conventional column chromatography, CCC eliminates irreversible adsorptive

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products[21]. It has been reported that CC could be separated by counter-current

chromatography[22] or centrifugal partition chromatography[5], but the solvent system

contains chloroform which is forbidden in China. Recently, with the development of

counter-current chromatography technology, a new high-performance counter-current

chromatography (HPCCC) equipment with higher separation efficiency has been

developed[16]. HPCCC is a recently developed countercurrent chromatography which

systems used in counter-current chromatography are gradually becoming less toxic. In

evaluate the purification method.

Herein, we developed a more friendly solvent system composed of n-hexane/ethyl

acetate/methanol/water to directly separate and purify curcumin from crude extract.

Complete separation of curcumin, demethoxycurcumin and bisdemethoxycurcumin with

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extracted CC shows no significant difference with commercial product.

2. Materials and methods

2.1 Reagents and materials

demethoxycurcumin, and bisdemethoxycurcumin standard products were obtained from

bought from National Pharmaceutical (AR, China National Pharmaceutical Group

serum (Gibco) and 1% penicillin/streptomycin (Gibco) at 37◦C and 5% CO2.

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tetramethylsilane as an internal standard. SpectraMax 190 (Molecular Devices, USA) ELISA

was used in this study.

2.3 The orthogonal test

was determined by external standard method (y = 117675x – 654802, R2 = 0.9978).

2.4 Counter-Current Chromatography Separation and identification

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of the K values of two adjacent target components.

wavelength: 425 nm.

2.5 Antioxidant activitie

1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity assay[22]. Generally, the

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was expressed as the inhibition percentage according to formula:

Radical scavenging activity (RSA %) = [(Acontrol – Aanalyte)/Acontrol] × 100% (1)

2.6 Cell viability

(Peking Union Medical College Headquarters of National Infrastructure of Cell Line

(Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin

(Gibco) at 37 ºC and 5 % CO2[24].

viability was calculated as formula:

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Cell viability (%) = [(Acontrol – Aanalyte)/Acontrol] × 100% (2)