SUPPLEMENTARY MATERIAL
Preparative Isolation of Paclitaxel and Related Taxanes from Cell
Cultures of Taxus Chinensis Using Reversed Phase Flash
Chromatography
Zhi-kun Lianga,b, Ruo-gu Huanga,b, Zhi-sheng Xiea,b and Xin-jun Xua,b
a
School of Pharmaceutical Sciences, Sun Yat-Sen University, 510006 Guangzhou, P.
R. China
b
Guangdong Technology Research Centre for Advanced Chinese Medicine, 510006
Guangzhou, P. R. China
Correspondence: Xinjun Xu, School of Pharmaceutical Sciences, Sun Yat-Sen University. 132
Waihuan Rd. East, Guangzhou Higher Education Mega Center, Guangzhou, 510006, P. R. China.
Tel: +86 13924061779, +86 20 39943041. E-mail address: xxj2702@sina.com
In this study, paclitaxel, baccatin III, taxuyunnanine C and sinenxane C were
successfully separated by reversed phase flash chromatography on a
manually-packed C18 column from Taxus chinensis cell culture extract. The
crude cell culture extract was firstly treated with Al2O3 column
chromatography and then divided into two parts: fraction 1 and fraction 2. 10
mg of baccatin III and 19 mg of paclitaxel were obtained from 100 mg dried
fraction 1. 52 mg of taxuyunnanine C and 11 mg sinenxane C were obtained
from 100 mg dried fraction 2. The purities of the four compounds were 98.02%,
98.53%, 98.93% and 98.76%, respectively. Their structures were characterized
by UV, MS, and NMR. These results indicate that paclitaxel and related
taxanes including baccatin III can be obtained from cell culture, in a highly
pure state, using reversed phase flash chromatography.
Keywords:Taxus chinensis cell culture; baccatin III; paclitaxel; taxuyunnanine
C; sinenxane C; reversed phase flash chromatography
Experimental
Apparatus
The separation was performed on a BiotageIsolera Rapid preparation liquid
chromatograph with an automatic fraction collector, and a PeakTrak software system
(AB company, Sweden). HPLC analysis was performed on a Lab alliance HPLC with
1500 pump, an AS1000 autosampler and a UV6000 detector (SSI, USA). An
SB25-12DTD ultrasonic machine (Ningbo Scientz Bio-technology Co., Ltd, China), a
KERN ABT 220-5DM electronic balance (KERN, Germany) and a Yarong RE-300
rotational vacuum concentrator (Shanghai, China) were employed in preparing
samples. Characterization was performed on a Finnigan LCQ DECA XP Liquid
Chromatography Mass Spectrometer (Thermo, USA) and a BrukerAvance III 400
Nuclear Magnetic Resonance Spectrometer (Bruker, Germany).
Reagents and materials
Suspension cells of Taxus chinensis (Pilger)Rehd (Batch Number: 20130201) and
paclitaxel reference substance were supplied by Guangdong Kelun Pharmaceutical
Co., Ltd (Meizhou, China). Alkaline Al2O3 chromatographic supports (70-150 µm)
were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Preparative C18 reversed phase silica gel was purchased from Tianjin BonnaAgela
Technologies Co., Ltd (Lot: BL0001L2201, size: 20~45 µm, Tianjin, China).
Methanol and acetonitrile were HPLC grade from SK Chemicals (Korea). Ultrapure
water was obtained from a Milli-Q RG purification unit (Millipore, Bedford, MA,
USA). Other chemicals were of analytical-reagent grade and purchased from Tianjin
Damao Chemical Reagent Factory (Tianjin, China).
Preparation of crude sample
Suspension cells originated from the callus of Taxus chinensis (Pilger) Rehd were
maintained in darkness at 24 °C with shaking at 150 rpm. Suspension cells were
cultured in B5 medium supplemented with 30 g/L sucrose, 10 µM naphthalene acetic
acid, 0.2 µM 6-benzylamino purine, 1 g/L casein hydrolysate and 1 g/L
2-(N-morpholino) ethanesulfonic acid (MES). Cell cultures were transferred to fresh
medium every 2 weeks. In prolonged culture for production, 2 and 3% (w/v) maltose
were added to culture medium at day 9 and 21, respectively, and 10 µM AgNO3 was
added on the initiation of culture as an elicitor (Choi, et al. 1999).
After culture, 10 g of the biomass was weighed and put into a beaker with 150
mL of methanol. The beaker was placed in an ultrasonic apparatus at 40 °C, setting
frequency at 40 KHz (duration 6 s, interval 1.5 s, power 1000 W) for 30 minutes.
Then, the filtered solution was collected and another 150 mL of methanol was added
into the beaker for another 30 minutes. After the process was repeated 4 times, the
overall extract was pooled and evaporated to dryness in a rotary evaporator under
reduced pressure at 40 °C.
Alkaline Al2O3 adsorption chromatography
The powders of chromatographic supports alkaline Al2O3 were activated 4-6 h at
120 °C. The column (25 mm×150 mm i.d.) was packed with the activated alkaline
Al2O3 by dry column packing and equilibrated with adequate dichloromethane. A
sample of crude cell culture extract was dissolved in appropriate amount of
dichloromethane and loaded into the column, followed by dichloromethane-methanol
(98: 2, v/v) elution to remove low-polar compounds at 3.0 mL/min for 50 min. Then,
isocratic elution was performed with dichloromethane-methanol (95: 5, v/v) at 5
mL/min for 30 min. The eluent was detected at 227 nm with a UV monitor. Two
elution fractions (fraction 1: 5% methanol eluent; fraction 2: 2% methanol eluent)
were collected for about 150 mL. The above two fractions were evaporated to dryness
by rotary vaporization under reduced pressure at 40 °C and analyzed by HPLC. The
residues were stored at 4 °C for further reversed phase flash chromatography
separation. Fraction 1 was used for separation of paclitaxel, and fraction 2 for other
taxanes.
HPLC conditions
The crude extract and isolated fractions were analyzed by HPLC under the following
conditions. A Dikma Diamonsil C18 column (150 mm×4.6 mm, 5 µm) was used. The
mobile phase was composed of water (A) and acetonitrile (B). The flow rate was 1.0
mL/min and elution gradient was: 0~15min: 20%B→40%B; 15~30 min: 40%B;
30~50 min: 40%B→90%B; 50~70 min: 90%B. Detection wavelength was set at 227
nm and column temperature was 35 °C.
Isolation and purification of paclitaxel and related taxanes by flash
chromatography
A reversed phase C18 column manually packed (30 g, 30 cm×1.5 cm, average particle
size: 51 µm) was used. Flash chromatography conditions of fraction 1 and fraction 2
were as follows: the mobile phase was composed of water (A) and methanol (B). The
flow rate was 8 mL/min. The elution gradient procedure for fraction 1 was: 0~60 min:
55% B; 60~70 min: 55%→70% B; 80~90 min: 70% B. The elution gradient
procedure for fraction 2 was: 0~60 min: 80% B; 60~65 min: 80%→95% B; 65~85
min: 95% B. Monitoring wavelength was set at 227 nm and column temperature was
30 °C. Both tested samples were prepared by mixing about 100 mg dried extract with
2 g C18 packing material.
HPLC Purity determination of isolated compounds
In separation process, the effluent from the column was collected into the test tubes
with a fraction collector set at 8 mL for corresponding tube. Fractions of peak 1, peak
2, peak 3 and peak 4 (corresponding to compound 1, compound 2, compound 3 and
compound 4, respectively) were collected, and subsequently, concentrated to dryness
under reduced pressure. Compound 1, compound 2, compound 3 and compound 4
were dissolved in 80% aqueous methanol solution respectively, and then analyzed by
HPLC-DAD.
Figures
Figure S1. The chemical structures of 1: baccatin III (C31H38O11); 2: paclitaxel
(C47H51NO14); 3: taxuyunnanine C (C28H40O8); 4: sinenxane C (C31H46O8).
Figure S2. HPLC chromatogram of Taxus chinensis cell culture extracts (a), fraction
1 (b) and fraction 2 (c).
Figure S3. Flash chromatogram of fraction 1 (a) and fraction 2 (b) from Taxus
chinensis cell culture extract. Peak fractions 1, 2, 3 and 4 correspond to compounds 1,
2, 3 and 4.
Figure S4. HPLC chromatograms of compound 1 (a), compound 2 (b), compound 3
(c) and compound 4 (d).
TABLE S1 Repeatability of the separation procedure of baccatin III, paclitaxel,
taxuyunnanine C and sinenxane C from Taxus chinensis cell culture extracts using
reversed phase flash chromatography.
Fraction1
Compound1
Compound2
Fraction2
Compound3
Compoun4
Extract
Yield
Purity
Yield
Purity
Extract
Yield
Purity
(mg)
(mg)
(%)
(mg)
(%)
(mg)
(mg)
(%)
(mg)
(%)
102
10.4
97.53
19.1
98.01
103
52.4
98.56
10.5
98.24
107
11.5
98.02
20.4
98.09
101
50.3
98.93
11.4
98.76
101
9.8
97.87
18.2
98.53
103
51.6
98.75
Yield
10.7
TABLE S2 1H NMR (400 MHz) and 13C NMR (400 MHz) spectroscopic data of
baccatin III and paclitaxel (Chloroform-d, δ in ppm).
compound 1
Position
δC
δH multiplicity(J in
compound 2
Position
δC
Hz)
1
79.65
2
74.85
3
45.09
4
80.13
5
84.20
6-α
Hz)
1
79.08
5.60 d
2
76.29
5.64 d(7.1)
3.79 d(7.0)
3
47.92
3.82 d(6.8)
4
82.32
5.00 m
5
85.90
2.59 m
6-α
7
71.93
8
58.82
4.99 dd(2.0, 9.3)
2.47 m
36.65
35.10
6-β
δH multiplicity(J in
1.88 m
6-β
4.46 dd(11.0, 6.8)
7
72.30
8
59.30
1.99 m
4.33 dd(7.7, 10.2)
Purity
98.48
9
204.76
9
205.16
10
75.92
10
76.84
11
131.23
11
134.62
12
146.78
12
142.10
13
68.12
4.87 m
13
72.37
6.16 s
14
38.13
2.31 m
14
37.53
2.43 m
15
42.02
15
44.63
16
19.08
1.11 s
16
22.39
1.13 s
17
26.14
1.13 s
17
26.95
1.15 s
18
15.89
1.97 d(1.3)
18
14.75
1.93 s
19
9.23
1.66 s
19
10.47
1.66 s
4.31 d(8.3)
20-
20-
6.34 s
4.29 d(8.1)
77.53
76.89
20-β
6.44 s
4.17 d(8.4)
20-β
4.18 d(8.2)
2-Ac
166.78
1'
174.51
170.13
2'
74.92
3'
57.75
C=O
4-Ac
4.74 d(2.5)
C=O
Me
22.23
2.29 s
10-Ac
4-OAc
170.98
171.36
C=O
Me
5.66 dd(2.5, 2.6)
C=O
20.45
2.25 s
Bz2-3'/5' 127.26
7.47 m
Me
23.26
2.32 s
10-OAc
171.91
C=O
Bz2-1' 129.80
Me
20.81
2.16 s
Bz2-2'/6' 129.93
Bz2-4' 133.78
8.08 dd(8.4, 1.4) C=O Ph1 167.69
7.60 m
q-Ph1
131.21
o-Ph1
131.40
8.11 d(1.3, 8.4)
m-Ph1
129.74
7.56 m
p-Ph1
134.99
7.59 t(1.3, 7.3)
q-Ph2
134.62
o-Ph2
128.51
7.47 m
m-Ph2
129.62
7.43 m
p-Ph2
132.89
7.33 t(1.5, 7.1)
C=O Ph3 170.30
q-Ph3
140.00
o-Ph3
128.53
7.72 d(1.1, 8.1)
m-Ph3
129.78
7.41 m
p-Ph3
129.05
7.49 m
TABLE S3 1H NMR (400 MHz) and 13C NMR (400 MHz) spectroscopic data of
taxuyunnanine C and sinenxane C (Chloroform-d, δ in ppm).
compound 3
Position
δC
δH multiplicity(J in
compound 4
Position
δC
Hz)
δH multiplicity(J in
Hz)
1
59.25
1.87 d(2.1)
1
59.26
1.88 d(2.1)
2
70.85
5.36 dd(6.4, 2.1)
2
70.57
5.36 dd(6.3, 2.0)
3
42.39
2.95 d(6.4)
3
42.15
2.94 d(6.3)
4
142.58
5
78.53
6
29.11
7-α
4
142.28
5.29 t(2.4)
5
78.17
5.30 t(2.4)
1.79 m
6
28.90
1.80 m
1.96 m(13.2, 7.0)
7-α
34.07
7-β
8
33.82
1.22 m(15.3, 3.5)
7-β
8
37.57
9-α
1.64 dd(14.7, 5.7)
1.24 m
37.28
9-α
44.17
9-β
1.96 m
2.35 m
44.98
2.37 dd(14.8, 12.2)
9-β
6.07 dd(12.5, 5.7)
10
70.12
1.63 m
10
70.36
11
135.68
11
135.34
12
134.96
12
134.84
13-α
2.83 dd(18.1, 8.7)
13-α
39.71
13-β
6.06 dd(5.3, 11.8)
2.86 dd(18.7, 8.8)
39.67
2.41 m
13-β
4.98 dd(9.1, 4.7)
14
70.12
15
39.64
2.40 m
14
70.81
15
39.91
16
25.66
1.65 s
16
25.40
1.67 s
17
31.97
1.11 s
17
31.70
1.12 s
18
22.01
2.08 s
18
21.43
2.09 d(0.4)
19
22.68
0.83 s
19
21.90
0.85 s
5.28 s
20-α
4.83 s
20-β
20-α
117.16
20-β
1'
170.17
4.99 dd(8.7, 4.3)
5.26 s
116.88
1'
4.82 t(0.8)
175.66
2'
21.65
2.05 s
2'
41.08
2.32 m
3'
26.74
1.46 m
4'
11.59
0.89 t(7.0)
5'
16.58
1.11 d(7.0)
2-OAc
170.44
C=O
Me
21.60
2.07 s
5-OAc
170.26
C=O
2-OAc
Me
21.14
2.16 s
170.25
C=O
10-OAc
Me
169.99
21.36
2.02 s
C=O
5-OAc
Me
21.60
2.05 s
169.96
C=O
Me
20.93
2.06 s
10-OAc
169.83
C=O
Me
21.36
2.19 s