Purification of six lignans from the stems of Schisandra chinensis by using high-speed counter-current chromatography combined with preparative high-performance liquid chromatography.

Food Chemistry xxx (2014) xxx–xxx

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Purification of six lignans from the stems of Schisandra chinensis by using high-speed counter-current chromatography combined with preparative high-performance liquid chromatography Lijie Zhu a,b,1, Bin Li a,1, Xiuying Liu a,b, Guohui Huang c,⇑, Xianjun Meng a,⇑ a b c

College of Food, Shenyang Agricultural University, Shenyang 110866, People’s Republic of China College of Chemistry, Chemical Engineering and Food Safety, Food Science Research Institute, Bohai University, Jinzhou 121013, People’s Republic of China Department of Horticulture, Eastern Liaoning University, Dandong 118003, People’s Republic of China

a r t i c l e

i n f o

Article history: Available online xxxx Keywords: Schisandra chinensis Lignans High-speed counter-current chromatography Preparative HPLC

a b s t r a c t A method for the preparative purification of lignans from Schisandra chinensis was established using a combination of high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (HPLC). The crude extracts obtained from S. chinensis by using 70% ethanol were separated on a macroporous resin column and then eluted with a graded ethanol series. A two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v) was used for HSCCC, and a mobile phase of acetonitrile–water (50:50, v/v) was used for preparative HPLC. The results obtained using HSCCC were compared with those obtained using preparative HPLC, and their advantages were further integrated to improve the separation efficiency. Six known lignans were identified by electrospray ionisation mass spectrometry and 1H nuclear magnetic resonance (NMR) and 13C NMR analyses; the purities of all the compounds were more than 91%. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Schisandra chinensis (Turcz.) Baill (Wuweizi in Chinese) is a member of the Magnoliaceae family and is found in northeast Asia (Hancke, Burgos, & Ahumada, 1999). It is used in China, Korea, and Japan for the treatment of various pathological conditions resulting from liver injury induced by alcohol or some other toxic chemical reagents. The fruits and stems of S. chinensis are listed officially as a food and herbal medicine in the Chinese Pharmacopoeia Commission, and they have thus been widely added to functional foods and used to treat various diseases (Chinese Pharmacopoeia Commission, 2010). S. chinensis contains various bioactive constituents, including lignans, triterpenoids, polysaccharides, and sterols (Xiao et al., 2008). Since it was increasingly used in 2002, S. chinensis was enrolled in the list of health foods by the Ministry of Healthy of the People’s Republic of China. Lignans are considered as the main functional constituents of some plants of the Schisandra genus (Huang, Song, Liu, & Liu, 2008). They are known to possess some beneficial pharmacological effects, including anti-hepatotoxic and anti-proliferative effects and penile erection ⇑ Corresponding authors. Tel./fax: +86 24 88488277. 1

E-mail addresses: [email protected] (G. Huang), [email protected] (X. Meng). L. Zhu and B. Li contribute equally to this article.

(Gnabre et al., 2010; Ip, Mak, Li, Poon, & Ko, 1996; Kim et al., 2011). Lignans are generally isolated and purified using a multistep protocol based on repeated column chromatography, which is then often followed by a final purification on preparative thin layer chromatography (TLC; Opletal, Sovová, & Bártlová, 2004). However, the drawbacks of these methods are that they are expensive, time-consuming, and unsuitable for large-scale isolation. High-speed counter-current chromatography (HSCCC) is a liquid–liquid partition chromatography technique developed by Ito (Oka, Harada, & Ito, 1998). Unlike traditional separation methods, HSCCC offers various advantages such as low solvent consumption, high recovery, and rapid separation (Ha, Kang, Na, Park, & Kim, 2011; Wu et al., 2012). Furthermore, the unique feature of HSCCC is that it can be predictably scaled up from analytical to preparative scales. However, HSCCC cannot always obtain a better separation for some structural analogues. Since numerous types of lignans are known to be found in S. chinensis, Peng and Huang could separate 2 types of lignans from this species by using HSCCC (Huang, Shen, & Shen, 2005; Peng, Fan, Qu, Zhou, & Wu, 2005). Preparative high-performance liquid chromatography (HPLC), a classical chromatographic method extensively used in the separation and purification of natural products, was also considered for the isolation of lignans (Aravind, Arimboor, Rangan, Madhavan, & Arumughan, 2008; Jin, Qian, & Du, 2013; Qu, Fan, Peng, & Mi,

http://dx.doi.org/10.1016/j.foodchem.2014.09.008 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Zhu, L., et al. Purification of six lignans from the stems of Schisandra chinensis by using high-speed counter-current chromatography combined with preparative high-performance liquid chromatography. Food Chemistry (2014), http://dx.doi.org/10.1016/ j.foodchem.2014.09.008

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L. Zhu et al. / Food Chemistry xxx (2014) xxx–xxx

Fig. 1. Chemical structures of the 6 lignans isolated from the stems of Schisandra chinensis.

2007). Although multiple sample treatments are always required before preparation, very efficient purification of high-purity compounds is enabled in a single step (Yao, Luo, Huang, & Kong, 2008). Occasionally, HSCCC alone could not be used to obtain a satisfying result; therefore, a combination of two chromatographic methods might be required to achieve the separation of target compounds. Thus, we speculated that high-purity lignans could be isolated from S. chinensis by using HSCCC coupled with preparative HPLC. In this study, this method was successfully used to separate 6 lignans from S. chinensis, and their chemical structures were elucidated (Fig. 1). Of the isolated lignans, gomisin J and angeloylgomisin H showed good prospects for application as a potential hypoglycemic drug and liver cancer prevention agent, respectively (Lee, Kim, Lee, Yun, & Nho, 2009; Zhang, Shi, & Zheng, 2010). Our findings suggest that our method can be efficiently used for the large-scale preparation of lignans and evaluating their overall activities. In addition, to our knowledge, pregomisin, angeloylgomisin H, and angeloylgomisin Q were isolated for the first time by using this method.

Organic solvents used for sample preparation and HSCCC separation were of analytical grade and purchased from Shanghai Chemical Reagent Corporation (Shanghai, China). Acetonitrile used for analytical and preparative HPLC was of chromatography grade (Merck, Germany). Water was purified using an NW-10VF Water Purification System (Heal Force, China). AB-8 macroporous adsorption resin was purchased from Cangzhou Bon Adsorber Technology Co., Ltd. (Cangzhou, China). Stems of S. chinensis were purchased from the College of Horticulture, Shenyang Agricultural University (Shenyang, China).

2. Materials and methods

2.3. Preparation of resin-purified sample

2.1. Apparatus

Stems of S. chinensis were dried at a constant temperature of 60 °C and pulverized to 30 mesh size by using a disintegrator. The powder (1 kg) was extracted using 2500 mL ethanol (70%) at 60 °C for 3 h. The extraction procedure was then repeated to reextract the residue twice, and the extracts were combined together. After the extracts were filtered using a ceramic filter, the filtrate was concentrated to remove ethanol in vacuum to obtain an aqueous fluid. This fluid was then subjected to separation by using a glass column (5 100 cm) packed with 500 mL of macroporous resin AB-8 and eluted with 0%, 30%, and 70% ethanol. The eluent of 70% ethanol was evaporated to dryness in vacuum, and 16.2 g of dried powder was obtained as resin-purified sample. It was then stored in a refrigerator (4 °C) for further use.

The HSCCC instrument used in this study was a TBE-300B highspeed counter-current chromatograph (Tauto Biotechnique, China) with 3 polytetrafluoroethylene multi-layer coil separation columns connected in series (internal diameter of the tubing: 1.8 mm, total volume: 300 mL) and a 25-mL sample loop. The revolution radius was 5 cm, and the b of the multilayer coil varied from 0.6 at the internal terminal to 0.8 at the external terminal (b = r/R, where r is the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge). An HX 1050 constant-temperature circulating implement (Beijing Boyikang Lab Instrument, China) was used to control the separation temperature. An S-1007 constant flow pump (Shenyitong Tech & Exploitation, China) and a C-635 UV photometer (Buchi, Switzerland) were also included in this system. Model N2000 chromatography workstation (Zhejiang University, China) was used for data collection. The analytical HPLC equipment had a 1525 pump and a 2487 detector (Waters, USA) and was

controlled using ‘‘Breeze’’ software. The preparative HPLC equipment was an LC-6AD system and Shimadzu HPLC workstation (Shimadzu, Japan). The nuclear magnetic resonance (NMR) spectrometer was the Agilent 400/54 Premium Shielded NMR Magnet System (Agilent, USA). Electrospray ionisation-mass spectrometry (ESI-MS) was performed using Agilent 1100 Series LC–MS Trap SL (Agilent, USA). 2.2. Reagents and materials

2.4. Selection of the two-phase solvent systems n-Hexane–methanol–water, ethyl acetate–n-butanol–water, and n-hexane–ethyl acetate–methanol–water were used as the systems to select according to the partition coefficients (K) of the


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target compounds in the resin-purified sample. The K values were determined using HPLC analysis as follows: 1 mg of resin-purified sample was added to a 10-mL test tube, and 4 mL of each phase of the two-phase solvent system was added. The test tube was shaken vigorously for 3 min to equilibrate the sample thoroughly. The peak areas of the upper phase analysed using HPLC were recorded as A, whereas the lower phase was recorded as B. K values of target components were obtained according to the peak areas by using the following equation: K = A/B. 2.5. Preparation of the two-phase solvent system and sample solution In the present study, the two-phase solvent system used for HSCCC separation consisted of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v). Each component of the solvent system was added to a separatory funnel and thoroughly equilibrated at room temperature for 12 h. The upper and lower phases were separated and degassed by sonication for 30 min shortly before use. For HSCCC separation of the resin-purified sample, the sample solution was prepared by dissolving 200 mg of enriched lignans in 3 mL of the lower phase. 2.6. Preliminary fractionation by using HSCCC HSCCC separation was performed as follows: the multilayer coil column was first filled with the upper phase as a stationary phase. The lower aqueous mobile phase was then pumped into the head end of the column inlet at a flow rate of 2.0 mL/min while the apparatus was rotated at 800 rpm. The sample solution was loaded via the injection valve after the system reached hydrodynamic equilibrium. The entire separation experiment was conducted at room temperature (25 °C). The effluent was continuously monitored using a UV detector at 254 nm and collected using a fraction collector set at 5 min for each tube. The HSCCC chromatogram is shown in Fig. 2. Peak fractions I and II were manually collected according to the chromatogram. When the general separation was completed, the residual solution in the column was blown out by nitrogen gas and collected as well. The above 2 fractions were concentrated under reduced pressure, and their residuals were dissolved in methanol for subsequent HPLC analysis. 2.7. HPLC analysis and identification of HSCCC peak fractions HPLC analyses of the resin-purified sample and HSCCC peak fractions were performed using a Hypersil ODS-2 C18 column (150 4.6 mm, 5 lm) at 25 °C. The mobile phase was acetonitrile–water (50:50, v/v) in isocratic mode, with a run time of

Table 1 Purity and recovery of the lignans from Schisandra chinensis after enrichment by macroporous resin. Compound

Schizandrol A Gomisin J Schizandrol B Pregomisin Angeloylgomisin H Angeloylgomisin Q

Purity (%)

Recovery (%)

70% Ethanol extract

Resin-purified sample

1.90 0.43 1.10 0.39 0.68 0.74

10.8 2.81 6.18 2.87 4.31 4.53

72.4 77.9 71.7 81.2 79.3 70.5

20 min. The flow rate was maintained at 1.0 mL/min, and UV detection was set at 205 nm. The injection volume was 15 lL. 2.8. Subsequent purification and analysis by using preparative HPLC After 4 times separation with HSCCC, 150 mg of fraction II was further purified using preparative HPLC on a Shim-pack PREP-ODS (H) (250 mm 20 mm, 5 lm) column at 25 °C. The mobile phase was acetonitrile–water (50:50, v/v) in isocratic mode, and the sample was injected through a 900-lL loop. The effluent was monitored at 205 nm, and the flow rate was constantly maintained at 8.0 mL/min. The purified fractions were analysed using a Shim-pack PREP-ODS (H) Kit (250 mm 4.6 mm, 5 lm), and the flow rate was constantly maintained at 1.0 mL/min. 3. Results and discussion 3.1. Selection of the pretreatment process The stems of S. chinensis are rich sources of lignans, and these lignans can be efficiently extracted using 50–95% ethanol, as reported in a previous study (Opletal et al., 2004). Hence, we selected 70% ethanol to ensure the presence of less water-soluble impurities in the extracts. Several sample pretreatment methods, such as ethyl acetate and reversed-phase C18 open column chromatography, have been used to remove other fat-soluble components from the crude extract in previous studies (Shi, Zhang, Huang, Liu, & Zhao, 2008; Zhu, Liu, Xu, Lin, & Wang, 2012). Unlike those methods, the AB-8 macroporous adsorption resin showed unique advantages and was successfully used in the enrichment of lignans (Caichompoo et al., 2009; Wang et al., 2005). Thus, 6 major lignans, with a considerable yield, were enriched from the total ethanol extract in our study. HPLC conditions reported

Fig. 2. High-speed counter-current chromatography (HSCCC) chromatograms of samples obtained from Schisandra chinensis. Two-phase solvent system: n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v); mobile phase: the lower phase; flow rate: 2.0 mL/min; revolution speed: 800 rpm; detection wavelength: 254 nm; sample size: 200 mg of the resin-purified sample dissolved in a mixture of 3 mL of the lower phase of the two-phase solvent system; separation temperature: 25 °C; retention of the stationary phase: 55%.


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Fig. 3. Analysis of high-performance liquid chromatography (HPLC) conditions: column, Hypersil ODS-2 C18 column (150 4.6 mm, 5 lm); mobile phase, acetonitrile–water (50:50, v/v); flow rate, 1.0 mL/min; detection wavelength, 205 nm. Sample was obtained from the resin-purified sample (a) and high-speed counter-current chromatography (HSCCC) peak fraction (fraction I, (b); fraction II, (c)).

Table 2 Partition coefficient (K value) of target components in different solvent systems. Solvent system

Ratio (v/v)

K value Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

n-Hexane–methanol–water

5:4:1 4:4:1 3:4:1

0.23 0.31 0.44

0.27 0.42 0.51

0.32 0.47 0.60

0.38 0.55 0.68

0.41 0.69 0.77

0.47 0.78 0.85

Ethyl acetate–n-butanol–water

5:1:3 5:1:4 5:1:5

5.09 5.55 6.74

5.34 6.01 7.88

5.87 6.32 7.97

6.16 6.68 8.21

6.47 6.97 8.42

6.91 7.25 8.81

n-Hexane–ethyl acetate–methanol–water

6:5:5:5 5:6:5:5 1:1:1:1 5:5:4:5 5:5:5:4

0.77 0.89 1.03 1.29 1.34

0.82 1.12 1.67 1.78 1.86

1.04 1.48 1.89 2.04 2.24

1.19 1.76 1.92 2.36 2.47

1.33 1.91 2.09 2.59 2.68

1.45 2.15 2.31 2.88 2.93

previously and the external standard method were used to purify and recover schizandrol A, gomisin J, schizandrol B, pregomisin, angeloylgomisin H, and angeloylgomisin Q after enrichment by using AB-8 resin; the results are shown in Table 1.

3.2. HPLC analysis and identification of HSCCC peak fractions The resin-purified sample and each peak fraction of HSCCC were analysed using HPLC, and their chromatograms are shown in Fig. 3.



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Fig. 4. Preparative high-performance liquid chromatography (HPLC) conditions: column, Shim-pack PREP-ODS (H) (250 mm 20 mm, 5 lm); mobile phase, acetonitrile– water (50:50, v/v); flow rate, 8.0 mL/min; detection wavelength, 205 nm. Sample obtained from fraction II was collected using high-speed counter-current chromatography (HSCCC) (a). Analytical HPLC conditions: column, Shim-pack PREP-ODS (H) Kit (250 mm 4.6 mm, 5 lm); mobile phase, acetonitrile–water (50:50, v/v); flow rate, 1.0 mL/min; detection wavelength, 205 nm. Sample was obtained from the preparative HPLC peak fraction (b–f).

Because the polarity of major lignans was considerably similar, isocratic elution was used to obtain optimum resolution and shorten the analysis time. During separation by HSCCC, 254 nm was selected because of the longer cut-off wavelength of ethyl acetate (254 nm). However, most lignans showed a maximum absorption wavelength between 200 and 220 nm; hence, 205 nm was selected for UV detection by HPLC. Stable baseline and better resolution were obtained using acetonitrile as the mobile phase, because it afforded lower absorption at a lower wavelength, unlike methanol. According to the adopted conditions and area normalisation

method, the purity of fraction I (compound 1) was 91.3%, and 5 main peaks were detected in fraction II. 3.3. Selection of a two-phase solvent system A suitable two-phase solvent system is crucial for the separation of target compounds by using HSCCC. For solvent selection, K is the most important parameter because HSCCC is a liquid– liquid partition separation method (Ito, 2005; Lopes-Lutz, Mudge, Ippolito, Brown, & Schieber, 2011). For simultaneous partition of


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Table 3 ESI-MS and 1H NMR data of lignan from Schisandra chinensis. Compound Schizandrol A Gomisin J Schizandrol B Pregomisin Angeloylgomisin H Angeloylgomisin Q

ESI-MS (m/z) 433.2 389.2 399.2 391.3 499.0 548.2

+

[M+H] [M+H]+ [M OH]+ [M+H]+ [M H] [M+NH4]+

1

H NMR (400 MHz, CDCl3)

d d d d d d

6.59, 7.19, 6.60, 7.19, 7.20, 6.75,

6.52, 6.56, 6.46, 6.33, 6.63, 6.52,

3.53, 5.71, 5.95, 6.18, 5.81, 5.93,

3.48, 2.48, 5.94, 5.65, 2.65, 5.68,

1.13, 2.38, 3.92, 3.83, 1.54, 3.80,

Reference 0.83 2.17, 3.82, 2.62, 1.40, 3.58,

2.04, 3.50, 2.17, 1.21, 3.46,

2 similar compounds by HSCCC, selecting a suitable two-phase solvent system is important; this system should provide an ideal K value of around 0.5–2 for each compound, and the ratio of two K values should be around 1.5 or higher (Zhang et al., 2012). Although a series of solvent systems such as n-hexane–methanol–water and ethyl acetate–n-butanol–water were considered as candidates for HSCCC analysis, none of them yielded a satisfactory K value for the separation of the 6 main compounds (see Table 2). When n-hexane–ethyl acetate–methanol–water was used, the ratio of two K values between compounds 1 and 2 was better; however, those for the remaining 4 compounds were poor. Among all the solvent systems tested, n-hexane–ethyl acetate– methanol–water (1:1:1:1, v/v) was chosen for preliminary fractionation by HSCCC, and fraction II, which contained several unseparated compounds, was subsequently purified using preparative HPLC. 3.4. Subsequent purification and analysis of preparative HPLC The 5 peak fractions, i.e., compounds 2 (20.4 mg, 39.3 min), 3 (30.9 mg, 41.6 min), 4 (9.4 mg, 53.1 min), 5 (25.2 mg, 57.4 min), and 6 (32.4 mg, 66.6 min), were separated from 150 mg of fraction II by using preparative HPLC, and their HPLC chromatograms are shown in Fig. 4 (a, preparative HPLC; b–f, analytical HPLC). Compared with the analysis results obtained using HPLC chromatograms of fraction II, preparative HPLC successfully yielded a similar resolution of the 5 peak fractions. According to the adopted conditions and area normalisation method, the purities of the 5 compounds (compounds 2–6) were all above 95%. The HSCCC and preparative HPLC peak fractions were identified on the basis of the retention times, together with the data for ESI-MS, 1H NMR, and 13C NMR. 3.5. Comparison and combination of HSCCC and preparative HPLC HSCCC is not a precise tool for analytical purposes. However, it enables the direct analysis of crude materials. Unlike HSCCC, preparative HPLC requires sample treatment before separation, which enables very efficient purification of high-value compounds in a single step. If the time required for the purification steps before preparative HPLC is considered, the productivity of HSCCC would obviously be higher than that of HPLC. Furthermore, since the separation mechanism of HSCCC is completely different from that of preparative HPLC, it can be used as an alternative prospective when efficient separation cannot be obtained using acetonitrile, methanol, or other solvents in C8 or C18 columns. In addition, preparative HPLC always yields excellent resolution and good reproducibility compared to those obtained using HPLC for a similar separation principle. The solvent system could be selected and adjusted by analysing the sample on HPLC first, and then applying the similar method for preparative HPLC. Isocratic and gradient elution can be used in preparative HPLC to separate different types of compounds. In this study, since the 6 compounds could not be separated well by using HSCCC, preparative HPLC was successfully used for the subsequent purification. Because of the combination of

1.95, 2.66, 2.00, 1.10, 2.31,

1.02, 2.32, 1.65, 0.73 2.18,

0.67 1.90, 1.30, 0.80 1.19, 0.78, 0.68 1.79, 1.62, 1.29, 1.17

Hu et al. (2009) Yang, Huang, Pu, Li, and Zhao (2012) Hu et al. (2009) Yang et al. (2012) Ikeya, Taguchi, Yosioka, and Kobayashi (1979) Ikeya, Taguchi, and Yosioka (1979)

their advantages, compound 1 was obtained through the initial separation by HSCCC, and compounds 2–6 were further rapidly purified by preparative HPLC. 3.6. Structural identification The peak fractions were structurally identified using ESI-MS, 1H NMR, and 13C NMR. Data for each compound are shown in Table 3. Details for NMR are presented in Supplementary Data. 4. Conclusion In the present study, preparative purification of lignans from the stems of S. chinensis by using HSCCC combined with preparative HPLC was investigated. A complementary action between the 2 methods afforded a new and superior separation mode for lignans. Initial preparation performed using HSCCC enriched certain polarity bands containing the chosen targets, followed by further purification by using preparative HPLC with a 5-lm ODS column; this led to the isolation of high-purity lignans from the stems of S. chinensis. The most significant advantage of this method was that an almost quantitative mass balance of components and better resolution were achieved simultaneously in a short time. The relationship between the elution solvents of HSCCC and preparative HPLC showed that the lignans had partly suitable K values (0.2–5) when the solvent system of n-hexane–ethyl acetate–methanol–water (1:1:1:1, v/v) was used; the lignans could be separated efficiently with 50% acetonitrile by using preparative HPLC. 5 lignans did not separate immediately when this solvent system was used; therefore, some other systems need to be tested in the future. Selection of appropriate solvents might lead to the establishment of optimum separation conditions for most lignans by using HSCCC or preparative HPLC. We intend to use this method for the preparative separation and purification of other natural lignans with biological activities and for improving the efficiency of monomeric compound preparation. Acknowledgement This work was supported by a National Natural Science Foundation of China (No. 31201325). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 09.008. References Aravind, S. G., Arimboor, R., Rangan, M., Madhavan, S. N., & Arumughan, C. (2008). Semi-preparative HPLC preparation and HPTLC quantification of tetrahydroamentoflavone as marker in Semecarpus anacardium and its polyherbal formulations. Journal of Pharmaceutical and Biomedical Analysis, 48, 808–813.


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Purification of two triterpenoids from Schisandra chinensis by macroporous resin combined with high-speed counter-current chromatography.

Preparative isolation and purification of iridoid glycosides from Fructus Corni by high-speed countercurrent chromatography.

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high-performance liquid chromatography and high-speed countercurrent chromatography.

Two-dimensional countercurrent chromatography × high performance liquid chromatography for preparative isolation of toad venom.

Quantitative analysis of six lignans in fruits with different colours of Schisandra chinensis by HPLC.

An efficient strategy for the extraction and purification of lignans from Schisandra chinensis by a combination of supercritical fluid extraction and high-speed counter-current chromatography.

Preparative high-performance liquid chromatography purification of polyunsaturated phospholipids and characterization using ultraviolet derivative spectroscopy.

Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step.

Preparative separation and purification of rosmarinic acid from perilla seed meal via combined column chromatography.

Purification of erucic acid by preparative high-performance liquid chromatography and crystallization.

Two-dimensional countercurrent chromatography×high performance liquid chromatography with heart-cutting and stop-and-go techniques for preparative isolation of coumarin derivatives from Peucedanum praeruptorum Dunn.

Gradient elution in preparative liquid chromatography.

Isolation and purification of schisandrol A from the stems of Schisandra chinensis and cytotoxicity against human hepatocarcinoma cell lines.

Preparative purification of plasmin activity stimulating phenolic derivatives from Gastrodia elata using centrifugal partition chromatography.

Rapid isolation and purification of phorbol esters from Jatropha curcas by high-speed countercurrent chromatography.

Isolation and anti-hepatitis B virus activity of dibenzocyclooctadiene lignans from the fruits of Schisandra chinensis.

Aqueous two-phase extraction combined with chromatography: new strategies for preparative separation and purification of capsaicin from capsicum oleoresin.

Angel rotor countercurrent chromatography.

Preparative purification of adenosine deaminase from human erythrocytes by affinity chromatography.

Preparation of Highly Purified Stearidonic Acid from Echium Oil via an Enzymatic Method Combined with Preparative High Performance Liquid Chromatography.

thermospray mass spectrometry for the identification of bioactive lignans from plant natural products.

Application and comparison of high-speed countercurrent chromatography and high performance liquid chromatography in preparative enantioseparation of α-substitution mandelic acids.

Ultrafiltration liquid chromatography combined with high-speed countercurrent chromatography for screening and isolating potential α-glucosidase and xanthine oxidase inhibitors from Cortex Phellodendri.

Two-steps extraction of essential oil, polysaccharides and biphenyl cyclooctene lignans from Schisandra chinensis Baill fruits.