YU SUN ISOLATION AND PURIFICATION OF LIGNANS FROM … › CICEQ › 2103 › No3 › CICEQ_Vol19_ No3_p435-440... · 2017-02-03 · Baillon., lignans, high-speed counter-current chromatography.

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Chemical Industry & Chemical Engineering Quarterly 19 (3) 435−440 (2013) CI&CEQ

435

YU SUN1,2

SHUANGSHUANG XU1

YANLING GENG1

XIAO WANG1

TIANYOU ZHANG2 1Shandong Analysis and Test

Center, Shandong Academy of Sciences, Jinan, China

2Shandong MingRen Freda Pharmaceutical co., LTD, Jinan,

Shandong, China

SCIENTIFIC PAPER

UDC 582.678.2:543.544:615.89

DOI 10.2298/CICEQ120504078S

ISOLATION AND PURIFICATION OF LIGNANS FROM Schisandra chinensis BY COMBINATION OF SILICA GEL COLUMN AND HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY

Silica gel column combined with high-speed counter-current chromatography separation was successfully applied to the separation of schizandrin (I), ange-loylgomisin H (II), gomisin A (III), schisantherin C (IV), deoxyschizandrin (V),γ-schisandrin (VI) and schisandrin C (VII) from the fruits of Schisandra chinen-sis (Turcz.) Baillon. The petroleum ether extracts of the fruits of S. chinensis were pre-separated first on a silica gel column and divided into two fractions as sample 1 and sample 2. 260 mg of sample 1 was separated by HSCCC using petroleum ether–ethyl acetate–methanol–water (10:8:10:8, v/v) as the two-phase solvent system and 18.2 mg of schizandrin, 15.7 mg of angeloylgomisin H, 16.5 mg of gomisin A and 16.7 mg of schisantherin C were obtained. 230 mg of sample 2 was separated using petroleum ether–ethyl acetate–methanol–water (10:0.5:10:1, v/v) as the two-phase solvent system and 19.7 mg of deoxyschi-zandrin, 23.4 mg of γ-schisandrin and 18.2 mg of schisandrin C were obtained. The purities of the separated compounds were all over 94% as determined by HPLC. The chemical structures of these compounds were confirmed by ESI-MS and 1H-NMR.

Keywords: Schisandra chinensis (Turcz.) Baillon., lignans, high-speed counter-current chromatography.

Schisandra chinensis fructus (Wuweizi in Chinese), the dried fruits of Schisandra chinensis (Turcz.) Baillon, is officially listed in the Chinese Phar-macopoeia and one of the most famous traditional Chinese medicine [1]. It is distributed in northeastern China, Russia, Japan and Korea [2]. Traditionally, the fruits of S. chinensis are used for the treatment of chronic cough, nocturnal emission, spermatorrhea, enuresis, frequent urination, protracted diarrhea, night sweating, spontaneous sweating, palpitation and insomnia [1]. It is also widely used as a functional ingredient and nutritional in foods, such as beer, wine, beverages, jam and other products [3]. Additionally, it is known to be a rich source of lignans with a diben-zo[a,c]cyclooctadiene skeleton [4,5], which have attracted considerable interest because of their biphe-

Correspondence: X. Wang, Shandong Analysis and Test Cen-ter, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, 250014, China. E-mail: [email protected] Paper received: 4 May, 2012 Paper revised: 13 August, 2012 Paper accepted: 19 August, 2012

nyl-type structures and multiple pharmacological acti-vities. In particular, pharmacological research indi-cated that these lignans can inhibit LTB4 production [6], afford protection against hepatic damage induced by CCl4 [7] and protect the liver from injury after administration of acetaminophen [8].

Due to these particular pharmacological and clinical effects of lignans separated from S. chinensis, it is necessary to establish an efficient method for the preparative separation and purification of these com-pounds from this plant. Recently, several extraction, isolation and purification methods of S. chinensis lig-nans have been reported, such as ionic liquid-based ultrasonic-assisted, ionic liquid based microwave simultaneous, macroporous resins and ion exchange resin [9–12]. High-speed counter-current chromato-graphy (HSCCC) is a liquid-liquid partition chromato-graphic technique that can eliminate irreversible adsorption of sample onto the solid support [13]. It has been widely used in preparative separation and purification of various natural products [14–17]. In the previous studies, Peng et al. [18] obtained schizan-drin and gomisin A from S. chinensis by HSCCC and

Y. SUN et al.: ISOLATION AND PURIFICATION OF LIGNANS FROM S. chinensis… CI&CEQ 19 (3) 435−440 (2013)

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Huang et al. [19] separated deoxyschizandrin and γ-schisandrin from S. chinensis using this method too. In order to get more pure compounds, a simple and feasible method needs to be established. In this paper, an efficient method, combination of silica gel column and HSCCC, was reported. Seven lignans were successfully isolated and purified from S. chi-nensis by HSCCC.

EXPERIMENTAL

Reagents and materials

Chromatographic grade methanol (Tedia Com-pany Inc, Fairfield, USA) was used for HPLC analysis. Organic solvents including petroleum ether (60–90 °C), ethyl acetate, ethanol and methanol were all of analytical grade (Damao Chemical Factory, Tianjin, China). The water used in solutions and dilutions was treated with a Milli–Q water purification system (Milli-pore, USA).

The fruits of S. chinensis were purchased from a local drug store. The botanical identification was made by Dr. Zongyuan Yu, Shandong Academy of Chinese Medicine, China. Silica gel (200–300 mesh, Haiyang Chemical Factory, Qingdao, China) was used for sample preparation.

Apparatus

A model GS10A–2 Preparative HSCCC (Beijing Emilion Science & Technology Co., Beijing, China) equipped with a PTFE multilayer coil (1.6 mmI.D.×110 m, with a total capacity of 230 mL). The β values of this preparative column range from 0.5 at internal to 0.8 at the external (β = r/R, where r is the distance or the rotation radius from the coil to the holder shaft, and R (R = 8 cm), the revolution radius or the dis-tances between the holder axis and central axis of the centrifuge). The rotation speed is adjustable from 0 to 1000 rpm, and 800 rpm was used in this experiment. The two-phase solvent was pumped into the column with a model NS–1007 constant-flow pump. Conti-nuous monitoring of the effluent was achieved with a model 8823A–UV monitor at 254 nm. A model 3057–11 portable recorder was employed to record the chromatogram.

The HPLC equipment used was a Waters Em-power system (Milford, MA, USA) including a model 600 system controller, a model 600 pump, a model 600 multisolvent delivery system, a model 996 photo-diode array detector.

Preparation of crude extract

About 500 g of the dried fruits of S. chinensis were milled to powder (about 40 mesh) and extracted

with 3 L 95% ethanol for three times (2 h each time) at the temperature of 70 °C. The extracts were com-bined and evaporated to dryness with a rotary evapo-rator at 50 °C. Then the ethanol extracts were dis-solved in water and extracted with petroleum ether for 3 times. The petroleum ether extraction solutions were concentrated to dryness, which yielded 36.8 g of crude extract. Then the petroleum ether extract was further subjected to the silica gel column (200 g of silica gel H, 200–300 mesh) eluted stepwise with pet-roleum ether–ethyl acetate (5:1 and 2:1, v/v) to obtain two fractions. The petroleum ether–ethyl acetate (2:1, v/v) effluent was collected and evaporated to dryness with a rotary evaporator at 50 °C and about 28.6 g of powder was obtained (sample 1). The petroleum ether–ethyl acetate (5:1, v/v) effluent was also col-lected and evaporated to dryness with a rotary eva-porator at 50 °C and about 4.2 g of powder was obtained (sample 2). All these samples were stored in a refrigerator until subsequent HSCCC separation.

Selection of the two-phase solvent systems

Approximately 2 mg of the test sample was weighed in a 10 ml test tube to which 2 ml of each phase of the equilibrated two-phase solvent system was added. The tube was capped and shaken vigo-rously for 1 min to equilibrate the sample thoroughly with the two phases. Equal volumes of each phase were then analyzed by HPLC to obtain the partition coefficients (KD). The KD value was expressed as the peak area of compound in the upper phase divided by the peak area of compound in the lower phase.

HSCCC Separation

In each separation process, the multilayer coiled column was first entirely filled with the upper phase (stationary phase) of the solvent. The apparatus was then rotated at 800 rpm, while the lower phase (mobile phase) was pumped into the column at a flow rate of 2 mL/min. After hydrodynamic equilibrium was reached, as indicated by a clear mobile phase eluting at the tail outlet, the sample solution was injected through the sample port. The effluent from the outlet of the column was continuously monitored with a UV detector at 254 nm. The chromatogram was recorded for 50 min after sample injection. Each peak fraction was manually collected according to the UV absor-bance profile and analyzed by HPLC.

Analysis and characterisation of HSCCC fractions

The two samples and each peak fraction from HSCCC were analyzed by HPLC. The analyses were accomplished by a Shim–Pack VP–ODS column (250 mm×4.6 mm I.D., 5 μm) at a column temperature of


437

25 °C. Mobile phase was performed with methanol–water (75:25, v/v). The flow rate was 1.0 mL/min. Detection wave was 254 nm.

The HSCCC fractions were all analyzed by ESI–MS on an Agilent 1100/MS–G1946 (Agilent, Santa Clara, CA, USA) and NMR spectra on a Varian–600 NMR spectrometer (Varian, Palo Alto, CA, USA) with chloroform (CDCl3) as solvent.

RESULTS AND DISCUSSION

In HSCCC separation, the choice of a suitable two-phase solvent system, which can provide an ideal range of the KD for the targeted compounds, is the first and critical step. In general, the most suitable range of the KD value is close to 1 [13]. Too large KD values tend to produce excessive sample band broadening, while too small KD values usually result in poor peak resolution. Several two-phase solvent systems were tested and the KD values were mea-sured, and summarized in Table 1.

It was found that no two-phase solvent system was suitable for separation of the target compounds by one-step HSCCC separation according to the KD values shown in Table 1. Thus, the petroleum ether extracts of the fruits of S. chinensis were pre-sepa-rated first on a silica gel column. Different kinds of solvent systems such as petroleum ether–ethyl ace-tate, petroleum ether–diethyl ether, trichlormethane–methanol were tested for the separation. Different elu-tion gradients were also investigated. It was found that when petroleum ether–ethyl acetate (5:1 and 2:1, v/v) was used for the separation, the crude extract was separated into two fractions. Sample 1 (2:1 fraction) mainly contained compounds I–IV, and sample 2 (5:1 fraction) mainly contained compounds V–VII. Meanwhile, these compounds were largely enriched after the separation of silica gel column. Thus, sample 1 was used for HSCCC separation of compounds I–IV,

and sample 2 for compounds V–VII. The HPLC chro-matograms of sample 1 and sample 2 are shown in Figure 1.

In accordance with the KD values of compounds I–IV shown in Table 1, it can be seen that both petro-leum ether–ethyl acetate–methanol–water with volume ratios of 10:8:10:8 and 10:8:9:8 were suitable for separation of compounds I–IV. So these solvent sys-tems were tested for HSCCC separation. When petroleum ether–ethyl acetate–methanol–water (10:8:10:8, v/v) was used as the two-phase solvent system, the separation result was better than that of petroleum ether–ethyl acetate–methanol–water (10:8:9:8, v/v) was used. So 260 mg of sample 1 was separated by HSCCC with the solvent system of petroleum ether–ethyl acetate–methanol–water (10:8:10:8, v/v). The HSCCC chromatogram of sample 1 is shown in Figure 2A. The fractions of HSCCC were collected according to HPLC analysis. 18.2 mg of schizandrin (I), 15.7 mg of angeloylgomisin H (II), 16.5 mg of gomisin A (III) and 16.7 mg of schisan-therin C (IV) were obtained with the purities of 98.5, 94.4, 97.7 and 95.6%, respectively. The HPLC chro-matograms of compounds I–IV are shown in Figure 1a–d.

From Table 1, it can be seen that petroleum ether–ethyl acetate–methanol–water with volume ratios of 10:1:10:1, 10:0.5:10:1 and 10:0.5:10:0.5 were all suitable for separation of compounds V–VII. When petroleum ether–ethyl acetate–methanol–water (10:1:10:1 and 10:0.5:10:0.5, v/v) were used as the two-phase solvent system, compounds VI and VII were successfully separated, however, compound V could not be obtained. While when petroleum ether–ethyl acetate–methanol–water (10:0.5:10:1, v/v) was chosen as the two-phase solvent system, compounds V–VII could all be obtained. So petroleum ether–ethyl acetate–methanol–water (10:0.5:10:1, v/v) was chosen to be the two-phase solvent system for the separation

Table 1. Partition coefficient (KD) values of target compounds in different two-phase solvent systems

Solvent system composition (petroleum ether–ethyl acetate–methanol–water), v/v

Compound

I II III IV V VI VII

1:1:1:1

10:8:10:10

10:8:12:8

10:8:10:8

10:8:9:8

10:5:10:5

10:2:10:2

10:1:10:1

10:0.5:10:1

10:0.5:10:0.5

1.38

1.14

0.25

0.65

0.92

0.24

0.13

0.06

0.06

0.02

2.73

2.31

0.62

1.11

1.65

0.35

0.17

0.07

0.07

0.02

3.72

3.10

0.97

1.48

2.02

0.57

0.29

0.10

0.13

0.06

5.23

4.15

1.28

1.96

2.43

0.96

0.62

0.11

0.15

0.07

15.40

14.63

5.74

11.62

10.01

3.37

1.77

0.43

0.74

0.36

—

—

13.14

—

17.21

8.73

3.02

1.55

1.98

1.03

—

—

10.84

13.76

14.66

6.68

2.17

1.00

1.28

0.70


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Figure 1. HPLC Chromatograms of the ethyl acetate fraction of Magnolia sprengeri and HSCCC peak fractions (I–VII). Experimental conditions: column, Shim–pack VP–ODS column (250 mm×4.6 mm i.d., 5μm); column temperature, 25 °C; mobile phase, methanol–water

(25:75, v/v); flow rate, 1 mL/min; detection, 254 nm; injection volume, 20 µL.


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Figure 2. HSCCC Chromatograms. A) Sample 1. HSCCC Conditions: Two-phase solvent system: petroleum ether–ethyl acetate–

methanol–water (10:8:10:8, v/v); mobile phase: lower phase; flow rate: 2 mL/min; detection, 254 nm; sample size: 260 mg dissolved in 5 mL of the upper phase and 5 mL of the lower phase. B) Sample 2. HSCCC Conditions: Two-phase solvent system: petroleum ether–ethyl acetate–methanol–water (10:0.5:10:1, v/v); mobile phase: the lower phase; flow rate: 2 mL/min; detection, 254 nm; sample size:

230 mg dissolved in 5 mL of the upper phase and 5 mL of the lower phase.

and purification of compound V–VII. The HSCCC chromatogram of sample 2 was shown in Figre 2B. 19.7 mg of deoxyschizandrin (V), 23.4 mg of γ-schi-sandrin (VI) and 18.2 mg of schisandrin C (VII) were obtained from 230 mg of sample 2 with the purities of 94.3, 95.6 and 98.2%, respectively. The HPLC chro-matograms of compounds V–VII are shown in Figure 1e–g.

The chemical structure of each peak fraction of HSCCC was identified according to its ESI-MS and 1H-NMR data. Compared with the data given in [20– -27], peaks I–VII in Figure 2 were indentified as schi-zandrin, angeloylgomisin H, gomisin A, schisantherin C, deoxyschizandrin, γ-schisandrin and schisandrin C.

CONCLUDING REMARKS

The results of our studies described above clearly demonstrated that the combination of silica gel column chromatography and HSCCC was successfully used in the separation and purification of schizandrin, angeloylgomisin H, gomisin A, schisantherin C, deoxyschizandrin, γ-schisandrin and schisandrin C from the fruits of S. chinensis. It is proved that the

combined use of silica gel column chromatography and HSCCC is a good separation strategy that can also be used for the separation and purification of other lignans from natural products.

Acknowledgments

Financial supports from the Natural Science Foundation of China (20872083), scientific and tech-nological major special project (2010ZX09401-302-5-12) and the Key Science and Technology Program of Shandong Province (BS2009SW047) are gratefully acknowledged.

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YU SUN1,2

SHUANGSHUANG XU1

YANLING GENG1

XIAO WANG1

TIANYOU ZHANG2

1Shandong Analysis and Test Center, Shandong Academy of Sciences,

Jinan, China 2Shandong MingRen Freda

Pharmaceutical co., LTD, Jinan, Shandong, China

NAUČNI RAD

IZOLACIJA I PREČIŠĆAVANJE LIGNINA IZ Schisandra chinensis KOLONSKOM HROMATOGRAFIJOM NA SILIKAGELU KOMBINOVANOM SA HSCCC HROMATOGRAFIJOM

Kolonska hromatrografija na silikagelu kombinovana sa HSCCC hromatrografijom je uspe-

šno primenjena za razdvajanje šizandrina (I), angeloilgomisina H (II), gomisina A (III),

šisanderina C (IV), deoksišizandrina (V), γ-šisandrina (VI) i šisandrina C (VII) iz ploda

Schisandra chinensis (Turcz.) Baillona. Petroletarski ekstrakti ploda S. chinensis pret-

hodno razdvojeni na koloni sa silikagelom su podeljeni na dve frakcije: uzorak 1 i uzorak 2.

Uzorak 1 (260 mg) je razdvojen HSCCC hromatografijom koristeći petroletar-etil acetat-

–metanol-voda (10:8:10:8, v/v) kao dvofazni sistem rastvarača, pri čemu je dobijeno 18,2

mg šizandrina, 15,7 mg angeloilgomisina H, 16,5 mg gomisina A i 16,7 mg šisanderina C.

Uzorak 2 (230 mg) je razdvojen HSCCC hromatografijom koristeći petroletar-etil acetat-

–metanol-voda (10:0.5:10:1, v/v) kao dvofazni sistem rastvarača, pri čemu je dobijeno 19,7

mg deoksišizandrina, 23,4 mg γ-šisandrina i 18,2 mg šisandrina C. Čistoća izdvojenih

jedinjenja je veća od 94%, što je određeno HPLC metodom. Hemijske strukture ovih jedi-

njenja su dokazane ESI-MS i 1H-NMR metodama.

Ključne reči: Schisandra chinensis (Turcz.) Baillon., lignin, HSCCC hromato-grafija.

YU SUN ISOLATION AND PURIFICATION OF LIGNANS FROM … › CICEQ › 2103 › No3 › CICEQ_Vol19_ No3_p435-440... · 2017-02-03 · Baillon., lignans, high-speed counter-current chromatography.

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