Food Chemistry 126 (2011) 1959–1963

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Analytical Methods

Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography Kunyou Zhan a,b, Kun Xu b,c,⇑, Hongzong Yin d,⇑ a

Office of Academic Affairs, Shandong Agricultural University, Tai’an 271018, China State Key Laboratory of Crop Biology, Tai’an 271018, Shandong, China c College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, Shandong, China d College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018, China b

a r t i c l e

i n f o

Article history: Received 8 January 2009 Received in revised form 22 October 2010 Accepted 8 December 2010 Available online 15 December 2010 Keywords: High speed counter-current chromatography Isolation 6-Gingerol 8-Gingerol 10-Gingerol

a b s t r a c t A novel method for purifying gingerols from ginger was developed using a high-speed counter-current chromatography (HSCCC). The two-phase solvent system such as light petroleum (bp 60–90 °C)–ethyl acetate–methanol–water (5:5:6.5:3.5, v/v/v/v) was applied to the separation and purification of 6-, 8and 10-gingerol from a crude extract of ginger. The experiment yielded 30.2 mg of 6-gingerol, 40.5 mg of 8-gingerol, 50.5 mg of 10-gingerol from 200 mg of crude extract in one-step separation. And the purity of these compounds was 99.9%, 99.9% and 99.2%, respectively, as determined by high-performance liquid chromatography (HPLC). Their structures were identified by gas chromatography–mass spectrometry (GC/MS) and 1H, 13C nuclear magnetic resonance (NMR). Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Ginger (Zingiber officinale Roscoe) had been widely used as a traditional medicine in China and India (McKenna, Jones, & Hughes, 2002). The major bioactive constituents in ginger were the socalled ‘‘pungent principles’’, the gingerols (Fig. 1), which had been reported to possess the following activities: anti-platelet aggregation (Hibino et al., 2008; Nie et al., 2008), anticancer (Bode, Ma, Surh, & Dong, 2001; Katiyar, Agarwal, & Mukhta, 1996; Shukla & Singh, 2007), anti-oxidation and anti-inflammation (Lam, Woo, Leung, & Cheng, 2007; Minghetti et al., 2007), inhibition of COX-2 expression (Kim, Chun, Kundu, & Surh, 2004; Kim, Kim, Na, Surh, & Kim, 2007; Kim et al., 2005), antifungal (Ficker et al., 2003) and so on. Therefore the isolation and purification of the gingerols from ginger are of great interests. To carry out the various in vitro and in vivo studies about their function and metabolism and to evaluate the quality of various ginger and food contained ginger as reference standard, large quantities of pure gingerols are needed. However, conventional separation and purification methods are needed a multi-step protocol based on column chromatography (CC), thin layer chromatography (TLC) and high performance liquid chromatography ⇑ Corresponding authors. Address: College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018, China. Tel.: +86 538 824 1783 (K. Xu), tel.: +86 538 824 2174 (H.Z. Yin). E-mail addresses: [email protected] (K. Xu), [email protected] (H. Yin). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.12.052

(HPLC) (He, Bernart, Lian, & Lin, 1998; Hiserodt, Franzblau, & Rosen, 1998; Zaeoung, Plubrukarn, & Keawpradub, 2005; Sajjad, Salma, Deepak, and Shivananda (2006)) and result a relatively lower preparative capacity due to irreversible adsorptions of separation materials onto the solid support during separation (Lei et al., 2001). Hence, alternative method has gained growing importance. High-speed countercurrent chromatography (HSCCC) is a liquid chromatographic technique in which the stationary phase is a liquid. Due to the absence of any solid stationary phase, adsorption losses are minimised compared to that caused by CC, TLC, HPLC. HSCCC has been successfully applied to the isolation of various natural products, e.g. phenylethanoid glycosides (Li et al., 2008), ursolic acid (Frighetto, Welendorf, Nigro, Frighetto, & Siani, 2008), lignans (Shi, Zhang, Huang, Liu, & Zhao, 2008) in large quantities. In this contribution, a method for the separation of the gingerols from the crude ethanol extract of ginger by HSCCC is presented. The purity and the structures of three gingerols are confirmed by nuclear magnetic resonance (NMR) spectrometry and gas chromatography/mass spectrometry (GC/MS). 2. Materials and methods 2.1. Reagents All organic solvents used for sample preparation or HSCCC separation were of analytical grade. Solvents of chromatographic

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funnel at room temperature, separated and degassed by ultrasonic bath for 30 min before use. The sample solution for HSCCC separation was prepared by dissolving 200 mg of crude extract in 10 ml lower phase of the solvent system. 2.6. Separation procedure

Fig. 1. Chemical structures of 6-gingerol (I), 8-gingerol (II) and10-gingerol (III) in extract of Zingiber officinale Roscoe.

grade used for HPLC and MS analysis were purchased from Basf (Tianjin, China). 2.2. HSCCC apparatus The preparative HSCCC separation was performed on a CCC TBE300A instrument (Tauto Biotechnique Company, Shanghai, China), equipped with three PTFE (Polytetrafluoroethylene) multilayer coils separation column connected in series (the tubing I.D. 1.6 mm and a total volume of 260 ml) and A manual sample injection valve with a 20 ml sample loop. The revolution radius was 5 cm, and the b values of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. The revolution speed of the apparatus can be regulated with a speed controller in the range between 0 and 1000 rpm. An HX 1050 constant-temperature circulating implement (Beijing Boyikang Lab Instrument Co. Ltd., Beijing, China) was employed to control the separation temperature. An ÄKTA prime (Amersham Pharmacia Biotechnique Group, Sweden) was employed to pump the two-phase solvent system and perform the UV absorbance measurement. It contains a switch valve and a mixer, which can be used for gradient formation. The data were collected with Sepu 2000 chromatography workstation (Hangzhou Puhui Science Apparatus Co. Ltd., Hangzhou, China). 2.3. Preparation of crude extract The fresh ginger was sliced into thin slices, dried at 40 °C and ground into powders (4 meshes). The powders were filtered after being soaked in ethanol for 72 h. The filtrates were combined and the solvent was evaporated under reduced pressure at 40 °C. The crude extraction yield of ginger (2.0 kg dry weight) was (3.5%) 70 g. Then the extract was stored at 4 °C for HSCCC use. 2.4. Selection of the two-phase solvent system and sample solution An aqueous biphasic solvent system was selected according to the partition coefficient (K) of each target component. The K-values were determined by HPLC as follows: about 2 mg of crude extract was added to a test tube, to which 3 ml of each phase of the biphasic solvent system was added. The test tube was shaken violently for several minutes to get equilibrium. Then the upper and lower phases were analysed by HPLC. The partition coefficients (K) of all components in sample were obtained by peak area obtained from the upper phase to that of the lower phase. 2.5. Preparation of two-phase solvent system and sample solution In present study, the two-phase solvent system composed of light petroleum (bp 60–90 °C)–ethyl acetate–methanol–water with different volume ratios was applied for HSCCC separation. The solvent mixture was thoroughly equilibrated in a separation

The HSCCC system was operated in head to tail mode using the upper phase as stationary phase. After loading the tube with stationary phase, the partition was performed at a revolution speed of 800 rpm, while the mobile phase was pumped through the system at a flow rate of 2 ml/min. The hydrodynamic equilibrium was reached after an hour, and then the dissolved samples were immediately injected. The temperature was controlled at 25 °C throughout the whole separation process. The run was monitored at 280 nm. Each separated fraction was collected according to the chromatogram peak and evaporated to dryness under reduced pressure at 30 °C. The collected samples were dissolved in chromatographic grade n-hexane at 30 °C and crystallised at 4 °C. 2.7. HPLC analysis and identification of HSCCC peak fractions The crude extract of ginger and each peak fraction from HSCCC separation were analysed by HPLC. The HPLC system used throughout this study was comprised of a Waters 1525 binary HPLC pumps fitted with a 20 ll sample loop, a Waters 2487 dual absorbance detector, a ODS RP-C18 column (250  4.6 mm I.D. 5 lm) and a Waters Breeze software package for data collection (Milford, MA, USA). The identification of HSCCC peak fractions was carried out by GC–MS, 1H and 13C NMR. A GC–MS instrument (GCMS-QP2010 Plus, Shimadzu, Japan) with a fused-silica capillary column Rtx-5ms (30 m length, 0.25 mm diameter, 0.25 lm film thickness) was used for analysis. The GC–MS conditions were as follows: carrier gas, helium (1.2 ml/min); injection mode, split ratio (10:1); The temperature program was 80 °C hold for 3 min, then ramped at 20 °C/min to 140 °C and hold for 2 min, then ramped at 6 °C/min to 230 °C and hold 3 min, then ramped at 15 °C/min to 260 °C and hold for 5 min. Electron ionisation mass spectra in the full-scan mode were recorded at 0.8 kV electron energy in the 45–550 amu range. Ion source temperature and interface temperature was 230 and 250 °C, respectively. The data were identified by comparison with standard mass spectra and mass data with those found in the literature (Jolad et al., 2004) . The NMR data were obtained on a Mercury Plus 400 NMR (Varian Inc., USA) with CDCl3 as solvent and TMS as internal standard.

3. Results and discussion 3.1. Optimisation of HPLC method The partition coefficient (K) of each component in crude extract was determined by HPLC analysis, so an optimal HPLC method should be developed for analysis of crude extract at first. Different mobile phases (methanol–water, acetonitrile–water) were used in HPLC to separate target components from the crude extract. The results indicated that when the mobile phase was acetonitrile and nanopure water (65:35, v/v), these targets could obtain good separation. The HPLC chromatogram of crude extract from Zingiber officinal was given in Fig. 2, the relative area of peaks I, II, III, IV-1, IV-2 in Fig. 2A was 28.07%, 4.75%, 9.78%, 2.37% and 5.87% on the peak area percent, respectively.

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Fig. 2. HPLC chromatograms of crude extract of Zingiber officinale (A) and purified peak fraction I (B), II (C), III (D). Column: column ODS RP-C18 (250  4.6 mm I.D. 5 lm); mobile phase: acetonitrile/nanopure water (65:35, v/v); flow rate: 1.2 ml/min; detection wavelength: 280 nm.

3.2. Optimisation of HSCCC conditions The selection of the two-phase solvent system for the target compounds was the most important step in HSCCC. For effective isolation of principle compounds in HSCCC separation, the K-values of these compounds in different solvent systems were determined

Table 1 The K of target compounds in light petroleum (bp 60–90 °C)–ethyl acetate– methanol–water solvent systems at different volume ratio. Volume ratio

KI

KII

KIII

KIV

5:5:6:4 5:5:6.5:3.5 5:5:7:3

0.44 0.69 0.90

0.65 1.15 1.61

1.20 2.11 3.85

1.40 2.52 5.01

by HPLC as the procedure shown in Section 2.4. The results were shown in Table 1. For effective separations with respect to resolution and short elution time, one of the most important factors for a successful CCC separation is the choice of a suitable solvent system (Ito, 2005). When light petroleum (bp 60–90 °C)–ethyl acetate–methanol–water (5:5:6.5:3.5, v/v/v/v) was used as the solvent system, good separation results and acceptable separation time could be obtained. At room temperature the separation time of the two phases was 17 s. The separation time correlating to the retention of the stationary phase should not be more than 20 s. For efficient separations the retention of the stationary phase should be 50% or higher (Ito, 2005) .With about 53% in the present study, the recommended level was achieved. The flow rate of the mobile phase, the separation temperature, the retention percentage of the stationary phase, the revolution

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Fig. 3. HSCCC separation chromatogram. two-phase solvent system combined light petroleum (bp 60–90 °C)–ethyl acetate–methanol–water (5:5:6.5:3.5, v/v/v/v). The retention percentage of the stationary phase was 53%. The 200 mg of crude sample dissolved in 10 ml of the mobile phase: flow rate 2.0 ml/min, revolution speed 800 rpm, detection wavelength 280 nm, separation temperature 25 °C. I: collected during 90–101 min; II: collected during 111–125 min; III: collected during 152–164 min.

speed of the separation coil and loading concentration were also optimised. The HSCCC chromatogram of crude extract obtained under the optimal conditions was shown in Fig. 3. The whole separation procedure yielded 30.2 mg 6-gingerol (peak I) at 99.9% purity, 40.5 mg 8-gingerol (peak II) at 99.9% purity, and 50.5 mg 10-gingerol (peak III) at 92.4% purity, respectively, in 170 min only in one CCC run from 200 mg crude extract. While HSCCC peak fraction IV contained two compounds with HPLC retention time at 9.44 min and 11.65 min. And the HPLC chromatograms of each of the three major compounds were shown in Fig. 2 (B–D).

2004); 1H NMR (400 MHz, CDCl3, TMS): d 0.88 (s, 3H, C12), 1.25– 1.28 (m, 12H, C6-C11, 2.72 (m, 1H, C5), 2.76(d, 2H, C4), 2.81(t, 2H, C2), 2.84(t, 2H, C1), 3.87(s, 3H, AOMe), 4.03 (s, 1H, C5AOH), 6.65–6.84 (m, 3H, C20 ,C50 ,C60 ), 7.27 (s, 1H, C40 AOH); 13C NMR (100 MHz, CDCl3, TMS): d 14.28 (C-12), 22.90 (C-11) , 25.68 (C10), 29.51 (C-9), 29.92 (C-8), 31.81(C-7), 32.02 (C-6), 36.68 (C-1), 45.66 (C-2), 49.57 (C-4), 56.09 (AOCH3), 67.86 (C-5), 111.18 (C10 ), 114.57 (C-60 ), 120.95 (C-50 ), 132.86 (C-20 ), 144.18 (C-30 ), 146.63 (C-40 ), 211.80 (C@O). We can conclude that the obtained product was as 8-gingerol.

3.3. Structure identification of HSCCC peak fractions

3.3.3. Data of HSCCC peak III MS m/z (rel. int.): 350 ([M]+, 13%), 332 (6), 205 (9), 194 (14),179 (7), 177 (7), 151 (43), 150 (12) and 137 (100), and the data were consistent with the mass spectra of 10-gingerol (Jolad et al., 2004); 1H NMR (400 MHz, CDCl3, TMS): d 0.88 (s, 3H, C14), 1.26– 1.47 (m, 16H, C5-C13), 2.46 (m, 1H, C5), 2.72 (d, 2H, C4), 2.75 (t, 2H, C2), 2.84 (t, 2H, C1), 3.87 (s, 3H, OMe), 4.03 (s, 1H, C5-OH), 6.65–6.84 (m, 3H, C20 ,C50 ,C60 ), 7.26 (s, 1H, C40 AOH); 13C NMR (100 MHz, CDCl3, TMS): d 14.34 (C-14), 22.90 (C-13), 25.67 (C12), 29.51 (C-11), 29.53 (C-10), 29.76 (C-9), 29.80 (C-8), 31.81(C7), 32.11 (C-6), 36.68 (C-1), 45.66 (C-2), 49.57 (C-4), 56.09 (30 OCH3), 67.86 (C-5), 111.18 (C-10 ), 114.57 (C-60 ), 120.95 (C-50 ), 132.86 (C-20 ), 144.18 (C-30 ), 146.63 (C-40 ), 211.69 (C@O). On the basis of the above data, we concluded that the fraction III was 10-gingerol.

The identification of peak fractions in Fig. 3 was performed with GC–MS, 1H and 13C NMR. The GC–MS and NMR data of each peak were given as follows. 3.3.1. Data of HSCCC peak I MS m/z (rel. int.): 294 ([M]+, 20%), 276 (4), 205 (7), 194 (6), 179 (8), 177 (4), 151 (13), 150 (49) and 137 (100) , and the data were consistent with the mass spectra of 6-gingerol (Jolad et al., 2004); 1H NMR (400 MHz, CDCl3, TMS): d 0.88 (t, 3H, C10), 1.25– 1.28 (m, 8H, C6-C9), 2.46 (m, 1H, C5), 2.60 (d, 2H, C4), 2.74 (t, 2H, C2), 2.84 (t, 2H, C1), 3.87 (s, 3H, AOMe), 4.03 (s, 1H, C5AOH), 6.65–6.82 (m, 3H, C20 , C50 , C60 ), 7.27 (s, 1H, C40 AOH) ; 13C NMR (100 MHz, CDCl3, TMS): d 14.30 (C-10), 22.85 (C-9), 25.38 (C-8), 29.49 (C-7), 31.97 (C-6), 36.61 (C-1), 45.67 (C-2), 49.54 (C-4), 56.09 (30 -OCH3),67.86 (C-5), 111.15 (C-10 ), 114.58 (C-60 ), 120.93 (C-50 ), 132.85 (C-20 ), 144.14 (C-30 ), 146.62 (C-40 ), 211.80 (C@O). From the above data, the obtained product was identified as 6gingerol with high purity. 3.3.2. Data of HSCCC peak II MS m/z (rel. int.): 322 ([M]+, 16%), 304 (4), 205 (7), 194 (5),179 (8), 177 (5), 151 (12), 150 (50) and 137 (100), and the data were consistent with the mass spectra of 8-gingerol (Jolad et al.,

4. Conclusion The conventional procedure for purifying gingerols from ginger was time-consuming, expensive and low efficient. The results in this study demonstrate that optimised HSCCC methods can be successfully used for the isolation and purification of gingerols from the crude extract of Zingiber. In the present study, 30.2 mg of 6-gingerol, 40.5 mg of 8-gingerol, 50.5 mg 10-gingerol were

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obtained with the purities of 99.9%, 99.9%, 92.4%, respectively, only in one CCC run from 200 mg crude extract of Zingiber in 170 min. Acknowledgement This research was supported by the Ministry Agriculture Foundation of China (No. 2006-G15). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.foodchem.2010.12.052. References Bode, A. M., Ma, W. Y., Surh, Y. J., & Dong, Z. (2001). Inhibition of epidermal growth factor-induced cell transformation and activator protein 1 activation by [6]gingerol. Cancer Research, 61, 850–853. Ficker, C., Smith, M. L., Akpagana, K., Gbeassor, M., Zhang, J., Durst, T., et al. (2003). Bioassay-guided isolation and identification of antifungal compounds from ginger. Phytotherapy Research, 17, 897–902. Frighetto, R. T. S., Welendorf, R. M., Nigro, E. N., Frighetto, N., & Siani, A. C. (2008). Isolation of ursolic acid from apple peels by high speed counter-current chromatography. Food Chemistry, 106, 767–771. He, X. G., Bernart, M. B., Lian, L. Z., & Lin, L. Z. (1998). High-performance liquid chromatography–electrospray mass spectrometric analysis of pungent constituents of ginger. Journal of Chromatography A, 796, 327–334. Hibino, T., Yuzurihara, M., Terawaki, K., Kanno, H., Kase, Y., & Takeda, A. (2008). Goshuyuto, a traditional Japanese medicine for migraine, inhibits platelet aggregation in guinea-pig whole blood. Journal of Pharmacological Sciences, 108, 89–94. Hiserodt, R. D., Franzblau, S. G., & Rosen, R. T. (1998). Isolation of 6-, 8-, and 10gingerol from ginger rhizome by HPLC and preliminary evaluation of inhibition of Mycobacterium avium and Mycobacterium tuberculosis. Journal of Agricultural and Food Chemistry, 46, 2504–2508. Ito, Y. (2005). Golden rules and pitfalls in selecting optimum conditions for highspeed counter-current chromatography. Journal of Chromatography A, 1065, 145–168. Jolad, S. D., Lantz, R. C., Solyom, A. M., Chen, G. J., Bates, R. B., & Timmermann, B. N. (2004). Fresh organically grown ginger (Zingiber officinale): Composition and effects on LPS-induced PGE2 production. Phytochemistry, 65, 1937–1954.

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Preparative separation and purification of gingerols from ginger (Zingiber officinale Roscoe) by high-speed counter-current chromatography.

A novel method for purifying gingerols from ginger was developed using a high-speed counter-current chromatography (HSCCC). The two-phase solvent syst...
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