Preparative isolation and purification of six volatile compounds from essential oil of Curcuma wenyujin using high-performance centrifugal partition chromatography doc

Research ArticlePreparative isolation and purification of six volatile compounds from essential oil of Curcuma wenyujin using high-performance centrifugal partition chromatography Six vo

Research Article

Preparative isolation and purification of six volatile compounds from essential oil of Curcuma wenyujin using high-performance centrifugal partition chromatography

Six volatile compounds, curdione (1), curcumol (2), germacrone (3), curzerene (4), 1,8-cineole (5) and b-elemene (6), were successfully isolated from the essential oil of Curcuma wenyujin by high-performance centrifugal partition chromatography using a nonaqueous two-phase solvent system consisting of petroleum ether-acetonitrile-acetone (4:3:1 v/v/v)

A total of 8 mg of curdione (1), 4 mg of curcumol (2), 10 mg of germacrone (3), 18 mg of curzerene (4), 9 mg of 1,8-cineole (5) and 17 mg of b-elemene (6) were isolated from the essential oil (300 mg) in 500 min Their structures were determined by comparison of their retention times and MS data with those of the authentic samples as well as NMR spectroscopic analysis

Keywords: Curcuma wenyujin / High-performance centrifugal partition

chromato-graphy / Preparative separation / Volatile components DOI 10.1002/jssc.200900453

1 Introduction

Essential oils (also called volatile oils) generally extracted by

distillation from plants are hydrophobic liquids containing

volatile aromatic compounds The essential oils are widely

used in perfumes, cosmetics as well as in food and drink as

flavor additives Some essential oils show multiple

pharmaco-logical activities and have been considered as the major active

fractions of herbal medicines [1–3] Preparative separation of

the volatile components is very important not only for the

quality control of the crude herbs and the products containing

the essential oil but also for the bio-evaluation However,

preparative separation of these pure volatile components is a

challenge because of their structural similarity, strongly

hydrophobic properties and poor stability Column

chromato-graphy over silica gel is broadly used to separate the volatile

components, which is time-consuming and arduous work In

most cases, the major components in essential oils cannot be

retained on silica gel even using hexane or heptane as mobile

phase Poor solubility in aqueous solvents prevents them from

being separated in large scale over the reverse stationary phase

GC is the conventional method for analysis of the essential components usually coupled with a mass spectrometer Preparative GC (Prep-GC) has been successfully used in the separation of the volatile components nowadays [4–6] However, thermolabile volatile components could decompose during separation by Prep-GC Samples injected into Prep-GC must be fully vaporized onto column to ensure radial distribution of the sample across the column The compounds

of interest are eluted out at very dilute concentrations from the column; therefore, it is very difficult to extract or condense those compounds from the gas stream Efficient parking of large GC columns is also difficult All these economic and technical difficulties limit wide use of Prep-GC in chemical separation ([4], Scott, R P W., Chrom-Ed Book Series- GC Chromatography, http://www.library4science.com)

Counter-current chromatography (CCC) including high-speed CCC (HSCCC) and high-performance centrifugal partition chromatography (HPCPC) is a liquid–liquid partition chromatographic technique based on partition of compounds between two immiscible liquid phases This method provides

an advantage over the conventional column chromatography

by eliminating the use of a solid support, which may result in irreversible adsorption [7, 8] HSCCC and HPCPC have increasingly been used to isolate and purify a multitude of natural products [7–10] But only a few researches have been reported in the separation of one or two volatile components from essential oils with this technique [11–15]

Eighty-four herbal drugs including Rhizoma Curcumae (Ezhu) are among 472 Chinese herbal materials recorded in China Pharmacopoeia (2005) Their therapeutic effects have been attributed to essential oils [16, 17] The rhizomes of

Yuan-Ye Dang

Xiao-Cen Li

Qing-Wen Zhang

Shao-Ping Li

Yi-Tao Wang

Institute of Chinese Medical

Sciences, University of Macau,

Taipa, Macau SAR, P R China

Received June 29, 2009

Revised January 20, 2010

Accepted February 23, 2010

Abbreviations: CCC, counter-current chromatography;

HPCPC, high-performance centrifugal partition

chromato-graphy; HSCCC, high-speed CCC; Prep-GC, preparative GC

Correspondence: Dr Qing-Wen Zhang, Institute of Chinese

Medical Sciences, University of Macau, Taipa, Macau SAR,

P R China

E-mail: qwzhang@umac.mo

Fax: 1853-2884-1358

three species of Curcuma including C wenyujin, C

phaeo-caulis and C kwangsiensis are used as Ezhu, which displays

wide and diverse medicinal value [16] The essential oil of C

wenyujin is considered as an effective part of Rhizoma

Curcumae, which is reported to possess anti-tumor [18, 19]

and anti-viral activities [20, 21] Several components

including b-elemene, curcumol, germacrone and curdione

have been reported to be the biologically active ingredients

in the essential oil [22, 23] Germacrone and curdione were

isolated and purified using two-phase solvent system

composed of petroleum ether/ethanol/diethyl ether/water

(5:4:0.5:1 v/v/v/v) by HSCCC by Yan et al [15] The

ther-mally labile compounds in ezhu oil, germacrone, curdione,

furanodienone and furanodiene, were found to degrade to

b-elemenone, curcumol, curzerenone and curzerene,

respectively, under heat while zurzerene was transformed

into callitrin and callitrisin in aqueous media in previous

studies [24–26] Therefore, it is preferable to use a

non-aqueous solvent system at low temperature to separate those

unstable volatile components An HPCPC method using a

nonaqueous two-phase solvent system was successfully

developed for the separation of six components including

curdione (1), curcumol (2), germacrone (3), curzerene (4),

1,8-cineole (5) and b-elemene (6) (Fig 1) from the essential

oil of C wenyujin GC-MS analysis showed that the six

isolated compounds except curcumol (2) were the major

components in the essential oil of C wenyujin

2 Materials and methods 2.1 Apparatus

The separation was performed on an SIC CPC240 HPCPC (Ever Seiko Corporation, Tokyo, Japan) at room tempera-ture Total rotor volume of this model is 240 mL and total number of cells is 3136 in which cell length is 15 mm and the distance to the cell center is 82.5 mm Descending and ascending modes can be interconverted by a four-way switching valve The rotation speed is up to 2000 rpm (increase and decrease by 100 steps) The two-phase solvent system was pumped with a Syknm S 1021 pump using a dual piston solvent delivery system (Syknm, Germany), equipped with HPCPC to pump Maximum sample injection volume was 5 mL and the flow rate of the pump was set between 0 and 30 mL/min Maximum pressure of the HPCPC equipment is 6.0 MPa An ultra-violet multiple wavelength detector of Agilent Technologies 1200 series (Agilent Technologies, USA) performed the preliminary analysis and detection for all the effluent from CPC240 constantly The chromatogram was recorded with Agilent ChemStation Fractions were collected by a CF-1 fraction collector (Spectrum, USA)

GC-MS analysis was performed with an Agilent 6890 gas chromatography instrument coupled to an Agilent 5973 mass spectrometer and an Agilent ChemStation software (Agilent Technologies)

2.2 Reagents and materials

All organic solvents used for HPCPC were of analytical or chromatographic grade Petroleum ether (b.p 60–901C) of analytical grade was purchased from Uni-Chem (Guang-zhou, China) Acetonitrile and acetone of chromatographic grade were purchased from Merck (Darmstadt, Germany) The essential oil of Curcuma wenyujin was purchased from Zhejiang Ruian Pharmaceutical (Ruian, China) The refer-ence compounds of curdione (1), curcumol (2), germacrone (3), curzerene (4) and b-elemene (6) were previously isolated from the above commercial oil [25]

2.3 Preparation of two-phase solvent system and sample solutions

The two-phase solvent systems were selected mainly according to the partition coefficient K of the target compounds The K values were determined by GC-MS analysis as follows: One drop (about 5 mg) of the essential oil was added to the mixture of equal volume of the upper phase and the lower phase of the two-phase solvent system The solutions were then mixed thoroughly by shaking The upper phase and the lower phase were analyzed by GC-MS, respectively, after equilibration was established

O

O

O

OH O

O

O

2 1

4 3

Figure 1 Chemical structures of curdione (1), curcumol (2),

germacrone (3), curzerene (4), 1,8-cineole (5), b-elemene (6).

A two-phase solvent system composed of petroleum

ether/acetonitrile/acetone (4:3:1 v/v/v) was selected for the

HPCPC separation after a partition experiment of the

essential oil in a series of solvent systems (Table 1) by

GC-MS analysis The solvent mixture was equilibrated

comple-tely in a separatory funnel at room temperature Both of the

upper and the lower phases were degassed for 30 min by

ultrasonication before use

Aliquots of 300 mg of the essential oil were diluted by

2.4 mL of the mixed solution of lower phase and upper

phase (1:1 v/v) of the optimized solvent system used for

HPCPC separation

2.4 HPCPC separation procedure

The upper phase of the two-phase solvent system was

performed as the stationary phase and the lower phase as

the mobile phase in the present HPCPC separation The

upper stationary phase was first pumped into HPCPC at a

flow-rate of 5.0 mL/min and the apparatus was run at

300 rpm under ascending separation mode After about

50 min when the column was fully filled with stationary

phase, the lower mobile phase was pumped into HPCPC at

a flow rate of 1.5 mL/min and the rotation speed was set at

1700 rpm under descending separation mode The sample

was injected through the sample loop after hydrodynamic

equilibrium was reached, as indicated by a clear mobile

phase eluting at the outlet The HPCPC separation was

running for 600 min and effluent from the outlet of HPCPC

was continuously monitored by an ultra-violet multiple

wavelength detector at UV 210 nm Per 3 mL fractions of

effluent were collected and analyzed by GC-MS The

fractions were pooled according to GC-MS results and

evaporated at 401C under reduced pressure to avoid thermal degrading At the end of the separation, all the upper stationary phase was collected to calculate the retention of the stationary phase

2.5 GC-MS analysis

GC-MS was performed on a capillary column (30 m  0.25

mm, id) coated with 0.25 mm film 5% phenyl methyl siloxane was used for the separation The carrier gas was high-purity helium and its flow rate was at 1.0 mL/min The optimized GC-MS conditions were as follows: the column temperature was set at 601C for injection, then programmed

at 51C/min to 1201C and held for 13 min, then at 251C/min

to 1451C and held for 20 min, finally, at 301C/min to 2801C Split injection (2 mL) was conducted with a split ratio of 10:1 The mass spectrometer was set in electron-impact (EI) mode in which the scan range was between 40 and 550 amu, the ionization energy was 70 eV and the scan rate was 0.34 s per scan The inlet, ionization source temperatures were 250 and 2801C [27]

3 Results and discussions

The selection of the two-phase solvent system is the first and critical step for an HPCPC or an HSCCC separation Successful separation by HSCCC or HPCPC depends on the suitable selection of a two-phase solvent system, which should have good solubility and stability for target compounds, an ideal partition coefficient (K), short settling time (o30 s) and satisfactory stationary phase retention (>50%) [9, 28]

Partition coefficient is a very important parameter for solvent system selection A compound with a small K value may come out with the solvent front and result in a loss of peak resolution while a large K value produces excessive running time and band broadening Ideally, the partition coefficient K has to be close to 1, usually in a range of 0.5–2 [8, 9] However, all partition coefficients are difficult to maintain in the range of 0.5–2 when separating more than four compounds in a one-step HSCCC or HPCPC run In practice, a K value between 0.2 and 5 can be used without the excessive elution time associated with band broadening [7, 29]

In this study, K 5 Aup(peak area of compound in upper phase)/Alow(peak area of compound in lower phase) Previous study indicated some components degraded in aqueous solution [26] Hence, the non-aqueous solvent system was given priority in the selection of solvent system Eight kinds of two-phase nonaqueous solvent systems were studied, such as petroleum ether/acetonitrile/acetone (2:1:1, 5:3:2, 4:3:1 and 7:6:1 v/v/v) and n-heptane/acetonitrile/ethyl acetate (7:6:1, 4:3:1, 5:3:2 and 2:1:1 v/v/v) With the GC-MS results and the calculation of K 5 Aup/Alow, the different

K values of curdione (1), curcumol (2), germacrone (3),

Table 1 The partition coefficient values of curdione (K1 ),

curcumol (K2), germacrone (K3), curzerene (K4),

1,8-cineole (K5) and b-elemene (K6) in eight kinds of

solvent systems

No Solvent systems (v/v) K 1 K 2 K 3 K 4 K 5 K 6

1 Petroleum ether/acetonitrile/

acetone (2:1:1)

0.36 0.58 0.92 2.25 2.01 6.03

2 Petroleum ether/acetonitrile/

acetone (5:3:2)

0.39 0.60 0.77 1.49 1.66 3.27

3 Petroleum ether/acetonitrile/

acetone (4:3:1)

0.33 0.56 0.76 1.74 1.94 4.53

4 Petroleum ether/acetonitrile/

acetone (7:6:1)

0.17 0.32 0.50 1.02 1.15 2.22

5 n-Heptane/acetonitrile/ethyl

acetate (7:6:1)

0.28 0.54 0.72 2.08 2.17 6.49

6 n-Heptane/acetonitrile/ethyl

acetate (4:3:1)

0.34 0.63 0.75 1.90 1.94 5.22

7 n-Heptane/acetonitrile/ethyl

acetate (5:3:2)

0.43 0.72 0.80 1.49 1.56 2.99

8 n-Heptane/acetonitrile/ethyl

acetate (2:1:1)

0.58 0.82 0.81 1.25 1.26 1.94

curzerene (4), 1,8-cineole (5) and b-elemene (6) were

obtained for those eight kinds of solvent systems (Table 1)

The K values of nearly all target compounds in petroleum

ether/acetonitrile/acetone systems could be enlarged by

increasing the volume ration of acetone In the petroleum

ether/acetonitrile/ethyl acetate systems, increasing the

volume ration of ethyl acetate result in the increasing of the

K values of curdione (1), curcumol (2) and germacrone (3),

and the decreasing of the K values of curzerene (4),

1,8-cineole (5) and b-elemene (6) The K values of all target

compounds in n-heptane/acetonitrile/ethyl acetate (2:1:1 v/

v/v) system lay well in a range of 0.5–2.0 Theoretically, two

compounds with a similar K value could not be separated

from each other by CCC Since K2and K4were close to K3

and K5, respectively, curcumol (2) and germacrone (3), and

curzerene (4) and 1,8-cineole (5) were hardly separated from

each other with this solvent system (n-heptane/acetonitrile/

ethyl acetate 2:1:1 v/v/v) Other factors beside the range of K

value should also be considered for the selection of the

solvent system

Resolution Rs is a critical parameter to measure the

quality of a separation and is expressed as the Knox equation

composing three variables including the efficiency N, the

selectivity a, and the capacity k0:

Rs ¼ ðNÞ1=2½ða  1Þ=a½k0

2=ð1 þ k0

2Þ=4 or

Rs ¼ ðNÞ1=2ða  1Þ½k01=ð1 þ k01Þ=4

ð1Þ

Since K is more practically used than k0, the above

equation will be given as [7]:

Rs ¼ 2VsðK2 K1Þ=ðWb11Wb2Þ ð2Þ

Herein, Vsis the volume of the stationary phase; K1and

K2are the partition coefficients for the two adjacent peaks,

respectively (where K2>K1); Wb1and Wb2are the peak width

for the two target components

It is obvious that the resolution increases with the

volume Vs of the stationary phase, with the selectivity

(K2/K1) or the difference between K2 and K1, and the

reduction in band width

Within the eight tested solvents systems, the differences

between K2 and K3, and between K4 and K5 in solvent

systems (petroleum ether/acetonitrile/acetone 2:1:1 and

4:3:1 v/v/v) are 0.34 and 0.24, and 0.20 and 0.20,

respec-tively, which are larger than those in the other six systems

(Table 1) These two solvent systems (petroleum ether/ acetonitrile/acetone 4:3:1 and 2:1:1 v/v/v) were selected for further study

The settling times for solvent systems of petroleum ether/acetonitrile/acetone (4:3:1 and 2:1:1 v/v/v) were 7 and

14 s, respectively, which indicated that the solvent system of petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) may yield

a larger retention of the stationary phase than the solvent system of petroleum ether/acetonitrile/acetone (2:1:1 v/v/v) The influences of flow rate and revolution speed were also investigated A lower flow rate usually gives a higher retention level of the stationary phase, improves the peak resolution and also requires a longer separation time Using

a lower revolution speed reduces the volume of the stationary phase retained in the column leading to lower peak resolution On the other hand, higher revolution speeds may produce excessive sample band broadening by violent pulsation of the column because of elevated pressure [9] When revolution speed was 1200 rpm and flow rate of the mobile phase was 2.5 mL/min, more than 50% of the stationary phase (upper layer of petroleum ether/acetoni-trile/acetone 4:3:1 v/v/v) was extruded when the mobile phase (lower layer of petroleum ether/acetonitrile/acetone 4:3:1 v/v/v) was pumped into HPCPC When the revolution speed was increased to 1400 rpm and the flow rate of the mobile phase was decreased to 2.0 mL/min, the outflow volume of the stationary phase was about 70 mL before sample injection, which means that the retention of stationary phase was nearly 70% (total rotor volume of this model is 240 mL) The stationary phase could not be held well in the rotor and it came out with the effluent all the time during the whole process of separation Then, the revolution speed was increased to 1600 rpm and the flow rate of the mobile phase was reduced to 1.5 mL/min, yielding nearly 50 mL of the stationary phase eluted after hydrodynamic equilibrium was attained, and the main-tenance of the stationary phase was also improved The purity of the target components from this separation could reach 80% At last, when revolution speed was increased to

1700 rpm and the flow rate of the mobile phase was main-tained at 1.5 mL/min, stationary phase retention was over 85% and rarely any stationary phase eluted after the sample injection The purity of each target component increased to 85% or above in this last experiment For the solvent system

min

mAU

0

500

1000

1500

2000

Time (Min)

6 5

4

Figure 2 HPCPC chromatogram of the

essential oil of C wenyujin Experimental

conditions: solvent system: petroleum ether/ acetonitrile/acetone (4:3:1 v/v/v); mobile phase: lower phase, revolution speed:

1700 rpm; flow rate: 1.5 mL/min; wavelength:

210 nm Fractions 1–6 correspond to curdione, curcumol, germacrone, curzerene, 1,8-cineole and b-elemene, respectively.

of petroleum ether/acetonitrile/acetone (2:1:1 v/v/v), the

retention of stationary phase was less than 50% when

carrying out the same procedures for optimizing

the flow rate and revolution speed as for solvent system of

petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) Thus,

petroleum ether/acetonitrile/acetone (4:3:1 v/v/v) was

selected as the solvent system for the HPCPC separation

and other conditions were optimized as: revolution speed

was 1700 rpm while flow rate of mobile phase was 1.5

mL/min

The HPCPC separation with the optimum condition described above yielded a total of 8 mg of curdione (1), 4 mg

of curcumol (2), 10 mg of germacrone (3), 18 mg of curzerene (4), 9 mg of 1,8-cineole (5) and 17 mg

of b-elemene (6) from the essential oil (300 mg) in 2.4 mL of the selected optimum solvent system (Fig 2) The amount

of each isolated compound from one run was enough for structural analysis by MS and NMR

Repeatability test was chosen to evaluate the system suitability of the developed assay The HPCPC separation

0

200000

1000000

1800000

2600000

Time >

Abundance

A

B

C

D

E

F

G

1 5

0

600000

1400000

2200000

3000000

Time >

Abundance

1 2

0

80000

16000

240000

320000

Time >

0

80000

160000

240000

Time >

0

200000

1000000

1800000

Time >

0

40000

80000

120000

160000

Time >

Abundance 5

0

50000

150000

350000

Figure 3 GC-MS chromato-grams of (A) the essential oil

of Curcuma wenyujin and (B)

Fraction 1, (C) Fraction 2, (D) Fraction 3, (E) Fraction 4, (F) Fraction 5 and (G) Fraction 6 from HPCPC separation 1, curdione; 2, curcumol; 3, germacrone; 4, curzerene; 5, 1,8-cineole; 6, b-elemene.

was repeated three times and the purity of the isolated

components determined by GC-MS (Fig 3, Table 2)

Variation of purity was expressed by the RSDs, which were

less than 2.1% (Table 2)

The structures of compounds 1–4 and 6 were identified

as curdione (1), curcumol (2), germacrone (3), curzerene (4)

and b-elemene (6), respectively, in comparison of their

retention times and MS data (Table 3) with those of the

authentic samples isolated from this oil previously [25]

Compound 5 was identified as 1,8-cineole (5) by

spectro-scopic analysis including MS (Table 3) and NMR as follows:

The1H NMR spectrum of compound 5 showed signals

at d: 1.01 (3H, s, H3-7), 1.21 (6H, s, H3-9 and H3-10 ), 1.36

(1H, m, H-4), 1.45 (2H, m, Ha-3 and Ha-5), 1.47 (2H, m,

Ha-2 and Ha-6 ), 1.61 (2H, m, Hb-2 and Hb-6 ) and 2.00 (2H,

m, Hb-3 and Hb-5 ) The13C NMR spectrum of compound 5

displayed signals at d: 22.8 (C-3 and C-5), 27.5 (C-7), 28.8

(C-9 and C-10), 31.5 (C-2 and C-6), 32.9 (C-4), 69.7 (C-1) and

73.5 (C-8) The NMR data above were in agreement with

those of 1,8-cineole [30]

4 Concluding remarks

After only a one-step separation by HPCPC using a

non-aqueous two-phase solvent system, six components,

namely, curdione (1), curcumol (2), germacrone (3),

curzerene (4), 1,8-cineole (5) and b-elemene (6), were

successfully separated from the essential oil of C wenyujin,

which are pure enough for structural elucidation,

qualitative analysis as a chemical reference The isolated

components (2, 3, 5 and 6) with purity above 90% might be

suitable for biological research The result suggested that

HPCPC might be an effective and efficient method for the isolation and purification of bioactive compounds from volatile oil The method provides an alternative approach to separate the strong hydrophobic components with similar structures

The authors would like to thank X H Gao and F Q Yang for their technical assistance in GC-MS in our laboratory The research was supported by grants from Macao Science and Technology Development Fund (013/2008/A1) and Research committee of University of Macau (RG075/06-07S/WYT/ ICMS)

The authors have declared no conflict of interest

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Table 2 Purity of six components separated by HPCPC

Purity Curdione (1) Curcumol (2) Germacrone (3) Curzerene (4) 1,8-Cineole (5) b-Elemene (6) First run (%) 85.0 a) 91.7 94.0 88.5 94.4 97.8 Second run (%) 86.9 92.8 94.5 89.7 93.2 96.7 Third run (%) 88.6 93.4 92.8 86.8 95.8 96.2 Average (%) 86.8 92.6 93.8 88.3 94.5 96.9

a) Purity (%) 5 100  (peak area of target compound)/(total peak area of all peaks).

Table 3 The retention times (RT) and MS data of six isolated components

Peak Compound RT (min) MS dataa)

1 Curdione 35.60 236(M1,8), 180(96), 167(75), 109(80), 95(34), 82(36), 69(100), 68(64), 67(61), 55(73), 41(67)

2 Curcumol 29.48 236(M 1 ,26), 135(28), 121(100), 119(29), 107(41), 93(40), 91(29), 69(33), 55(44), 43(28), 41(46)

3 Germacrone 33.82 218(M 1 ,8), 136(49), 135(71), 121(24), 107(100), 105(20), 91(33), 79(20), 67(52), 53(22), 41(28)

4 Curzerene 23.59 216(M 1 ,13), 148(30), 133(10), 109(10), 108(100), 105(12), 93(11), 91(16), 79(17), 77(15)

5 1,8-Cineole 6.16 154(M 1 ,36), 111(47), 108(50), 93(36), 84(41), 81(61), 71(48), 69(38), 41(36), 43(100)

6 b-Elemene 16.98 204(M 1 ,2), 147(46), 121(46), 107(67), 93(100), 91(50), 81(93), 79(66), 68(67), 67(75), 41(51)

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