Efficient
separation
bisdemethoxycurcumin
of
from
curcumin,
turmeric
demethoxycurcumin
using
supercritical
and
fluid
chromatography: from analytical to preparative scale
Wei Song a, Xue Qiao a, Wen-fei Liang a, Shuai Ji a, Lu Yang a, Yuan Wang a, Yong-wei Xu c,
Ying Yang c, De-an Guo a,b, Min Ye a,b,*
Affiliations:
a
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, 38 Xueyuan Road, Beijing 100191, China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China
c
Waters Corporation, Building 13, No. 1000 Jinhai Road, Pudong New District, Shanghai
201206, China
*
Corresponding author. Tel.: +86 10 82801516. Fax: +86 10 82802024. Email address:
[email protected] (M. Ye).
1
List of Contents
Experimental Section
Quantitative analysis of curcuminoids using UPC2 (Tables S1-S3 included)
Figure S1. Optimization of different types of columns.
Figure S2. Optimization of different additives in mobile phase.
Figure S3. Optimization of different modifiers in mobile phase.
Figure S4. Optimization of different sample solvents.
Figure S5. Purity test of curcumin by HPLC.
Figure S6. Purity test of demethoxycurcumin (DMC) by HPLC.
Figure S7. Purity test of bisdemethoxycurcumin (BDMC) by HPLC.
Figure S8. 1H NMR (400 MHz, acetone-d6) spectrum of curcumin.
Figure S9. 13C NMR (100 MHz, acetone-d6) spectrum of curcumin.
Figure S10. 1H NMR (400 MHz, acetone-d6) spectrum of demethoxycurcumin (DMC).
Figure S11. 13C NMR (100 MHz, acetone-d6) spectrum of demethoxycurcumin (DMC).
Figure S12. 1H NMR (400 MHz, acetone-d6) spectrum of bisdemethoxycurcumin (BDMC).
Figure S13. 13C NMR (100 MHz, acetone-d6) spectrum of bisdemethoxycurcumin (BDMC).
Table S4. NMR spectroscopic data for curcumin, DMC, and BDMC (400 MHz, acetone-d6).
2
Experimental Section
Reagents
Methanol, acetonitrile and isopropanol (Fisher Chemical, New Jersey, USA); ethanol (Duksan
Pure Chemicals, Ansan-City, Korea); n-hexane (Dikma, Beijing, China); and oxalic acid
(Sigma-Aldrich, St. Louis, USA) were of HPLC grade. High-purity carbon dioxide (CO2,
99.995%) was purchased from Beijing Chengweifeng Gases Co. Ltd. (Beijing, China).
Acetone-d6 for NMR analysis was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Plant materials
Dried rhizomes of Curcuma longa L. were purchased from Pengzhou City, Sichuan Province,
China, in December 2009. The plant was identified by Professor Min Ye at Peking University.
A voucher specimen (No. JH200912) was deposited at the School of Pharmaceutical Sciences,
Peking University, Beijing, China.
Preparation of turmeric extract
The rhizomes of Curcuma longa L. (15.0 g) was pulverized into fine powder and extracted
twice in an ultrasonic water bath, with 150 ml of methanol for each time. The methanol
extracts were combined and filtered. The filtrate was evaporate to dryness in vacuo to obtain
the turmeric extract, and weighed as 0.9649 g. The dry extract was finally dissolved in 15 ml
of methanol and filtered through 0.22-μm membrane before use.
Analytical supercritical fluid chromatography (UPC2)
A Waters ACQUITY Ultra Performance Convergence ChromatographyTM (UPC2) system was
used for analytical scale separation. The UPC2 system consists of a convergence manager, a
3
sample manager, a binary solvent manager, a column manager, and a photodiode array
detector (PDA). An ACQUITY UPC2 BEH column (Waters, 100 mm × 3 mm, 1.7 μm) was
selected for sample separation. The column temperature was 40°C. The mobile phase was
consisted of CO2 as solvent A and methanol containing 10 mM oxalic acid as solvent B. The
flow rate was set at 0.9 mL/min with the back pressure maintained at 1800 psi. A gradient
program was used as follows: 0-3 min, 5-30% B; 3-4.5 min, 30-40% B; 4.5-4.8 min, 40-5% B;
4.8-6.0 min, 5% B. An aliquot of 1 μL sample was injected for analysis. The PDA detector
was monitored at a wavelength of 410 nm. All data acquisition and analysis were controlled
by Waters Empower TM 3 software.
Preparative supercritical fluid chromatography
A Waters Prep 100q SFC system was used to perform preparative scale separation of
curcuminoids. The system consisted of a Thar SFC high pressure pump, a Waters 2545
quaternary gradient module, a Thar SFC injection module, a Waters 2767 sample manager, a
Waters column oven, a Waters back pressure regulator, three Waters heat exchanger, a Waters
2998 photodiode array detector, and a series III pump. A Viridis BEH OBD column (Waters,
250 mm × 19 mm, 5 μm) was used for sample separation. The composition of the mobile
phase was consistent with that used in UPC2 system, and a brief gradient elution was applied
(0-10.0 min, 8-15% B; 10.0-15.0 min, 15-8% B). The flow rate was 80 ml/min. The back
pressure was maintained at 120 bar. The UV detection wavelength was set at 410 nm. The
injection volume was 100 μL. MassLynx V4.1 software was used for system control and data
processing.
High-performance liquid chromatography (HPLC)
For HPLC purity test, an Agilent 1100 HPLC system was applied, which consisted of a
4
quaternary pump, an auto-sampler, a column temperature controller, and a PDA detector.
Samples were separated on an Agilent SB-C18 column (250 mm × 4.6 mm, 5 μm). The flow
rate was 1.0 mL/min and the column temperature was maintained at 30 °C. The mobile phase
consisted of acetonitrile (A) and water containing 0.1 % formic acid (v/v, B). The following
elution gradient was used: 0 min, 19% A; 8 min, 55% A; 20 min, 70% A; 25-30 min, 95% A.
The detection wavelength was set at 410 nm.
Post-purification and structural identification
To remove the residues of oxalic acid, the three compounds isolated by preparative SFC
system were suspended in 15 ml of deionized water, respectively, and centrifuged at a speed
of 9000 rpm at 4C for 10 min. The precipitates were then dissolved in 2 mL of methanol,
respectively, and suspended in 20 mL of water for freeze drying. The dried powder of each
compound was weighed for recovery calculation. The NMR spectra of the purified
compounds were obtained at 400 MHz for 1H and 100 MHz for 13C, respectively, on a Bruker
AVANCE III-400 spectrometer in acetone-d6 with TMS as reference.
5
Quantitative analysis of curcuminoids by UPC2
Preparation of standard solutions
Curcumin (0.58 mg), DMC (0.83 mg), and BDMC (0.44 mg) were accurately weighed and
dissolved in methanol to prepare their individual stock solutions (1 mg/mL, respectively). The
three stock solutions were equally mixed and then serially diluted (dilution factor = 1.67, 3.33,
5.00, 10.00, 16.67, 33.33, and 100.00) to produce calibration standard solutions. An aliquot of
1 μL was injected for UPC2 analysis.
Preparation of sample solutions
The methanol solution of turmeric extract used for preparative SFC experiment was diluted to
1/300, and filtered through 0.22 μm membranes before use. A 1-μL aliquot was injected for
UPC2 analysis.
Calibration curves of curcumin, DMC and BDMC
The standard solution samples were analyzed to establish the calibration curves. The curves
were constructed by plotting the peak areas of the samples against the concentration of the
compound. The calibration curves, linear ranges, and correlation coefficients (r2), and original
data were summarized as follows：
Table S1. Calibration curves, linear ranges, and correlation coefficients (r2)
compound
calibration curve
linear range
r2
curcumin
y = 9313.6x + 2607.7
10.00-100.00
0.9995
DMC
y = 7927.8x - 9848.9
3.33-66.67
0.9996
BDMC
y = 10264x - 9039.3
3.33-66.67
0.9999
Table S2. Original data for the standard samples
6
concentration
curcumin
DMC
BDMC
(μg/mL)
Area
relative error
Area
relative error
Area
relative error
3.33
/
/
21164
4.26%
28195
2.32%
10.00
90333
4.56%
70292
3.19%
93236
2.58%
20.00
185576
1.36%
144384
2.66%
195599
0.25%
33.33
319212
-2.05%
250207
-0.34%
328744
0.32%
66.67
633730
-1.47%
521725
-3.97%
677482
-2.00%
100.00
926327
1.06%
/
/
/
/
Contents and recoveries of curcumin, DMC, and BDMC
A total of 2.2 mL of samples (equivalent to 2.2 g of turmeric) was injected for preparative
SFC separation (22 injections, 100 μL for each time). Thus, there contents of curcumin, DMC,
and BDMC in 2.2 mL turmeric sample were calculated to be 29.7 mg, 9.8 mg, and 5.2 mg,
respectively. Compared to the amounts obtained after SFC separation and post-purification
(20.8 mg, 7.0 mg, and 4.6 mg), the recoveries of curcumin, DMC and BDMC were 70.03 %,
71.43 % and 88.46 %, respectively.
Table S3. The contents and recoveries of curcumin, DMC and BDMC.
compound
peak area
curcumin
DMC
BDMC
421364
108686
73956
concentration
(μg/mL)
44.96
14.95
8.08
content
(mg, in 2.2mL)
29.7
9.8
5.2
7
prepared amount
(mg)
20.8
7.0
4.6
recovery
70.03%
71.43%
88.46%
HSS C18
BEH 2EP
BEH
Figure S1. Optimization of different types of columns: ACQUITY UPC2 BEH 2-EP (1.7 μm),
ACQUITY UPC2 HSS C18 SB (1.8 μm), and ACQUITY UPC2 BEH (1.7 μm). All provided
by Waters (Milford, MA, USA) with column dimensions of 100 mm ×3.0 mm I.D.
8
10mM oxalic acid in methanol
No oxalic acid in methanol
Figure S2. Optimization of different additives in mobile phase: acidic additive (10 mM oxalic
acid in methanol) and no additive.
9
A
ethanol/acetonitrile (1/1, v/v)
ethanol
methanol
ethanol/acetonitrile (1/1, v/v)
B
ethanol
methanol
Figure S3. Optimization of different modifiers in mobile phase: methanol, ethanol, and an
equal mixture of methanol and acetonitrile (10 mM oxalic acid were added, respectively).
A, full view; B, enlarged profiles.
10
methanol
ethanol
acetonitrile
isopropanol
Figure S4. Optimization of different sample solvents: methanol, ethanol, acetonitrile, and
isopropanol.
11
mAU
150
254 nm
100
50
0
mAU
250
0
5
10
15
20
25
min
5
10
15
20
25
min
5
10
15
20
25
min
365 nm
200
150
100
50
0
mAU
0
410 nm
600
400
200
0
0
Figure S5. Purity test of curcumin by HPLC (99.99%).
Samples were separated on an Agilent SB-C18 column (250 mm × 4.6 mm, 5 μm). The UV
detection wavelength was set at 254 nm, 365 nm, and 410 nm.
mAU
254 nm
40
20
0
mAU
80
0
5
10
15
20
25
min
5
10
15
20
25
min
5
10
15
20
25
min
365 nm
60
40
20
0
mAU
0
410 nm
200
150
100
50
0
0
Figure S6. Purity test of demethoxycurcumin (DMC) by HPLC (92.62%).
12
mAU
254 nm
40
20
0
mAU
0
80
5
10
15
20
25
min
5
10
15
20
25
min
25
min
365 nm
60
40
20
0
mAU
0
250
200
150
100
50
0
410 nm
0
the enol form of BDMC
5
10
15
20
Figure S7. Purity test of bisdemethoxycurcumin (BDMC) by HPLC (94.57%).
13
Figure S8. 1H NMR (400 MHz, acetone-d6) spectrum of curcumin.
14
Figure S9. 13C NMR (100 MHz, acetone-d6) spectrum of curcumin.
15
Figure S10. 1H NMR (400 MHz, acetone-d6) spectrum of demethoxycurcumin (DMC).
16
Figure S11. 13C NMR (100 MHz, acetone-d6) spectrum of demethoxycurcumin (DMC).
17
Figure S12. 1H NMR (400 MHz, acetone-d6) spectrum of bisdemethoxycurcumin (BDMC).
18
Figure S13. 13C NMR (100 MHz, acetone-d6) spectrum of bisdemethoxycurcumin (BDMC).
19
Table S4. NMR spectroscopic data for curcumin, DMC, and BDMC (400 MHz, acetone-d6).
curcumin
demethoxycurcumin (DMC)
bisdemethoxycurcumin (BDMC)
Position
C, type
H (J in Hz)
C, type
H (J in Hz)
C, type
1
101.8, CH2
5.97, s
101.8, CH2
5.98, s
101.9, CH2
2, 2
184.1, C
3, 3
122.5, CH
6.70, d (15.6)
122.3, CH/122.5, CH
6.66, d (16.0) / 6.71, d (16.0)
122.3, CH
6.67 (d, J = 15.6 Hz, 2H)
4, 4
141.5, CH
7.60, d (15.6)
141.5, CH/141.2, CH
7.61, d (16.0) / 7.60, d (16.0)
141.2, CH
7.61 (d, J = 15.6 Hz, 2H)
5, 5
128.4, C
6, 6
116.3, CH
7, 7
184.7, C
5.98 (s, 1H)
184.7, C
127.9, C/128.4, C
7.33, s
H (J in Hz)
127.9, C
131.1, CH/116.4, CH
7.57,d (8.8) / 7.34, d (1.2)
131.1, CH
7.57 (d, J = 8.8 Hz, 2H)
148.9, C
117.0, CH/150.0, C
6.91, d (8.8)
117.0, CH
6.91 (d, J = 8.8 Hz, 2H)
8, 8
150.1, C
160.7, C/150.2, C
9, 9
111.7, CH
6.89, d (8.0)
117.0, CH/111.7, CH
6.91, d (8.8) / 6.88, d (8.0)
117.0, CH
6.91 (d, J = 8.8 Hz, 2H)
10, 10
123.9, CH
7.18, d (8.0)
131.1, CH/124.0, CH
7.57, d (8.8) / 7.18, dd (8.0, 1.2)
131.1, CH
7.57 (d, J = 8.8 Hz, 2H)
OCH3
56.5, CH3
3.92, s
56.5, CH3
3.92, s
160.7, C
20