Journal of Chromatography B, 969 (2014) 190–198
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Isolation of two new prenylated flavonoids from Sinopodophyllum emodi fruit by silica gel column and high-speed counter-current chromatography Yanjun Sun a,b , Yinshi Sun c , Hui Chen a,b , Zhiyou Hao a,b , Junmin Wang a,b , Yanbin Guan a,b , Yanli Zhang a,b , Weisheng Feng a,b,∗ , Xiaoke Zheng a,b,∗∗ a Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450046, China b School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450046, China c College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 28 March 2014 Received in revised form 14 June 2014 Accepted 9 August 2014 Available online 19 August 2014 Keywords: Sinopodophyllum emodi New prenylated flavonoid Silica gel column chromatography High-speed counter-current chromatography
a b s t r a c t Two new prenylated flavonoids, sinoflavonoids A–B, were isolated from the dried fruits of Sinopodophyllum emodi by silica gel column chromatography (SGCC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in water, respectively. The ethyl acetate fraction was pre-separated by SGCC with a petroleum ether–acetone gradient. The eluates containing target compounds were further separated by HSCCC with n-hexane–ethyl acetate–methanol–water (4:6:4:4, v/v). Finally, 17.3 mg of sinoflavonoid A and 25.9 mg of sinoflavonoid B were obtained from 100 mg of the pretreated concentrate. The purities of sinoflavonoid A and sinoflavonoid B were 98.47% and 99.38%, respectively, as determined by HPLC. Their structures were elucidated on the basis of spectroscopic evidences (HR-ESI-MS, 1 H-NMR, 13 C-NMR, HSQC, HMBC). The separation procedures proved to be efficient, especially for trace prenylated flavonoids. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sinopodophyllum emodi (Wall.) Ying (Berberidaceae) is distributed widely in the southwest of China [1]. The dried ripe fruit of S. emodi, called “Xiaoyelian” in Chinese, is used to regulate menstruation and promote the circulation of blood. As a traditional Tibetan medicine, it is clinically applied to the treatment of amenorrhea, dead fetus, and placental retaining. It has been recorded in Chinese Pharmacopoeia and Yue Wang Yao Zhen (Somaratsa), which was compiled in the mid-eighth century as the earliest literature on traditional Tibetan medicine [2]. Previous chemical investigations on S. emodi revealed the presence of lignans, flavonoids, steroids, phenolics [1–11]. The prenylated flavonoids, which were representative bioactive constituents from the genus Sinopodophyllum, possessed multiple biological activities including anti-oxidant, anti-cancer, anti-inflammatory, anti-bacterium,
∗ Corresponding author. Tel.: +86 371 65962746; fax: +86 371 65962746. ∗∗ Co-corresponding author. Tel.: +86 371 65962746; fax: +86 371 65962746. E-mail addresses: [email protected] (W. Feng), [email protected] (X. Zheng). http://dx.doi.org/10.1016/j.jchromb.2014.08.017 1570-0232/© 2014 Elsevier B.V. All rights reserved.
anti-osteoporosis, prevention of Alzheimer’s disease, anti-diabete, cardiovascular protection, and estrogen-like effect [12]. Conventional isolation strategies for prenylated flavonoids from S. emodi involved multiple chromatographic steps, which were time-consuming and resulted in a substantial loss of samples due to irreversible adsorption [2,10]. Therefore, it is indispensible to develop a rapid and efficient method for the purification of prenylated flavonoids from S. emodi. HSCCC is a peculiar liquid-liquid partition technology, which can eliminate some complications such as irreversible adsorption and deactivation of target compounds [13]. HSCCC has been widely used in the preparative isolation and purification of natural products with high recovery and loading capacity, low solvent consumption, acceptable efficiency, and the ease of scaling-up [14]. To the best of our knowledge, only flavonoid glycosides [15–17], isoflavones [18], polymethoxylated flavones [19,20] were successfully isolated from natural sources by SGCC and HSCCC. Although HSCCC has been developed for the purification normal flavonoid (only kaempferol) [21], there are no reports on the application of SGCC and HSCCC for the isolation of prenylated flavonoids. The present research established an optimal preparation method of two new prenylated flavonoids (sinoflavonoids A-B) (Fig. 1) from S. emodi with SGCC and HSCCC.
Y. Sun et al. / J. Chromatogr. B 969 (2014) 190–198
191
Fig. 1. The chemical structures of two new flavonoids from Sinopodophyllum emodi.
2. Experimental
2.3. Preparation of the crude extract
2.1. Apparatus
The fruits of S. emodi (4 kg) were dried constantly at 60 ◦ C, then grilled to powder, and passed through a 40-mesh sieve. The powder was ultrasonically extracted by 10-fold amounts of 95% ethanol at 55 ◦ C for 60 min. The working frequency and power were fixed at 40 kHz and 200 W, respectively. The extraction procedure was then repeated twice. After combination and removal of the ethanol under reduced pressure, the extract was suspended in 5 L distilled water, and subjected to a series of solvent extractions (1:1, v/v) with petroleum ether (b.p. 60–90 ◦ C), dichloromethane, ethyl acetate, and finally n-butanol. After concentration and freeze-drying, the dried fractions were stored at −10 ◦ C.
Ultrasonic-assisted extraction (UAE) was performed on a digitally controlled ultrasonic apparatus (KQ 5200V, Kunshan Ultrasonic Instruments Manufacture Co. Ltd., China). The HSCCC experiments were carried out by a Model GS-10A high-speed counter-current chromatography (Beijing Institute of New Technology Application, Beijing, China). The instrument was equipped with a polytetrafluoroethylene multilayer coil column (i.d. of the tubing = 1.6 mm, total volume = 230 mL) and a manual sample injection valve with a 10 mL loop. The revolution radius between the holder axis and central axis of the centrifuge (R) was 5 cm, and the ˇ value of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal (ˇ = r/R, where r was the distance from the coil to the holder shaft, and R was the revolution radius or the distance between the holder axis and central axis of the centrifuge). The revolution speed of the apparatus could be regulated by a speed controller ranging from 0 to 1000 rpm. The HSCCC system was also equipped with BF-2002 CT11 signal collection cell (Chromatography Center of Beifenruili Group Company, Beijing, China), a Model NS-1007A constant-flow pump and a Model 8823B-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. The data were collected with HW-2000 chromatography workstation (Qianpu Software Co. Ltd., Shanghai, China). The analytical HPLC data were measured on a Waters Alliance 2489 separations module equipped with a Waters 2695 UV/visible Detector and Empower pro data handling system (Waters Co., Milford, USA). The structures of the target compounds were determined by high resolution electrospray ionization mass (HR-ESI-MS) spectrometer (Shimadzu LC-MS 2010, Japan) and nuclear magnetic resonance (NMR) spectrometer (Bruker AM 500, Switzerland). 2.2. Materials and reagents Methanol used for HPLC was of chromatographic grade (Siyou Biology Medical Tech Co. Ltd., Tianjin, China), and water was purified by means of a water purifier (18.2 M) (Wanjie Water Treatment Equipment Co. Ltd., Hangzhou, China). All organic solvents used for preparation of crude fractions, SGCC and HSCCC separation were of analytical grade (Fuyu Chemical Reagent Co. Ltd., Tianjin, China). The chromatographic silica gel was produced from Qingdao Ocean Chemical Factory (Qingdao, China). The fruits were collected in Deqin, Yunnan province, People’s Republic of China, in September 2012, and were identified as the fruits of S. emodi (Wall.) Ying by Professor Chengming Dong (School of Pharmacy, Henan University of Traditional Chinese Medicine).
2.4. Pre-separation of the crude extract by SGCC The ethyl acetate fraction (50 g) was dissolved in acetone (80 mL), and added to silica gel (60 g, 200–300 mesh) by constant stirring. Acetone was evaporated in a rotary evaporator under vacuum at 45 ◦ C. The fraction impregnated silica gel was cooled down to room temperature and kept in a desiccator until required. The silica gel chromatography column was packed as follows: the exit of the chromatography column was plugged with glass wool to retain solids. Silica gel (1.94 kg, 200–300 mesh) was suspended in petroleum ether (7.5 L), and then transferred to the column (140 cm lenth × 8 cm i.d.). The column was rinsed with 7.5 L of petroleum ether-acetone (10:1, v/v). Prior to sample application, the level was lowered 20 cm above the stationary phase. The fraction impregnated silica gel was added to the top of the column, and elution was performed with a petroleum ether–acetone gradient (10:1, 10:3, 10:5, 15 L each) at a constant flow rate of 15 mL min−1 . Fractions of 100 mL each were analyzed by TLC. TLC analyses were performed on GF254 silica gel plates at room temperature, using petroleum ether–acetone (1:1, v/v) as developing reagent. Spots were visualized under an ultraviolet lamp at 254 nm. The eluates containing target compounds were pooled and concentrated under reduced pressure. The concentrate was stored at −10 ◦ C for the subsequent HSCCC separation.
2.5. Further separation by HSCCC 2.5.1. Determination of the partition coefficient The partition coefficient (K) of target compounds in different two-phase solvent systems was determined by HPLC as follows: 25 mg of the pretreated concentrate and 1 mL of the equilibrated two-phase solvent were added into a 5 mL centrifuge tube. The centrifuge tube was then stoppered and vortically mixed for 1 min to thoroughly equilibrate the sample between the two phases. Then an aliquot of each phase (10 L) was analyzed by HPLC. The K value was expressed as the ratio of the peak area of a given compound in the upper phase divided that in the lower phase.
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Fig. 2. HSCCC chromatogram of the pretreated concentrate from SGCC. Two-phase solvent system: n-hexane–ethyl acetate–methanol–water (4:6:4:4, v/v); mobile phase: the lower phase; stationary phase: the upper phase; flow rate: 1.7 mL min−1 ; revolution speed: 800 rpm; detection wavelength: 254 nm; sample size: 100 mg pretreated concentrate was dissolved in the solvent mixture of n-hexane–ethyl acetate–methanol–water (5 mL for each phase); the retention percentage of the stationary phase: 67%.
2.5.2. Preparation of two-phase solvent system and sample solution A solvent system consisting of n-hexane–ethyl acetate– methanol–water (4:6:4:4, v/v) was prepared by adding the solvents to a separation funnel according to the volume ratios and thoroughly equilibrated by shaking repeatedly. Then the two phases were separated and degassed by sonication for 30 min before use. The sample solution was prepared as follows: 100 mg pretreated concentrate was dissolved in the solvent mixture of nhexane–ethyl acetate–methanol–water (5 mL for each phase). 2.5.3. HSCCC separation procedure The separations were initiated by filling the coiled column with the upper phase (stationary phase). The lower phase (mobile phase) was pumped into the column in a head-to-tail mode at a flow rate of 1.7 mL min−1 , when the apparatus was rotated at 800 rpm. Approximately 10 mL of the sample solution containing 100 mg of the pretreated concentrate was injected into the head of the column through the injection valve after hydrodynamic equilibrium was established in the column, as indicated by the mobile phase eluting from the tail outlet. The eluates from the column outlet were continuously monitored by a UV detector at 254 nm. The fractions during 120–150 min (peak 1) and 170–200 min (peak 2) were collected respectively according to the elution profile (Fig. 2). All fractions of the same pure compound were combined and evaporated under reduced pressure. The purified compounds were stored at −20 ◦ C before HPLC and NMR analyses. 2.5.4. HPLC analysis and identification of HSCCC peak fractions The pretreated concentrate from SGCC and each HSCCC peak fraction were analyzed by HPLC (Fig. 3A–C). Analyses were accomplished on a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm) at 35 ◦ C. Methanol (A) −0.1% trifluoroacetic acid (v/v) (B) was used as the mobile phase in gradient elution mode as follows: 30–85% A at 0–30 min, 85–100% A at 30–40 min. The flow rate of the mobile phase was 1.0 mL min−1 . The eluates were monitored at 254 nm by a UV/visible detector. Based on the peak area normalized to all observed HPLC peak areas, the purities of the isolated flavonoids were determined.
3. Results and discussions 3.1. Pre-separation by SGCC As seen in the HPLC chromatogram (Fig. 4), the high concentration of flavonoids and lignans, and a trace of sinoflavonoids A (peak 1, 70.1 min) and B (peak 2, 78.9 min), were present in the ethyl acetate fraction. To improve the preparative separation efficiency, SGCC was employed for pre-separation, eluting with a petroleum ether–acetone gradient. The chromatographic parameters on separation efficiency, including flow rate, mobile phase composition, and loading amount were investigated to produce optimum separation. Three kinds of binary solvent systems were tested, includdichloromethane–methanol, dichloromethane–acetone, ing and petroleum ether–acetone. With the mixed solvents of dichloromethane–methanol and dichloromethane–acetone, the eluates contained a lot of impurities such as citrusinol (peak 3, 15.9 min) and 8-prenylkaempferol (peak 4, 22.0 min) (Fig. 3D and E), which possessed the similar polarities as target compounds. Selection for ideal mobile phase depended on the recovery of target compounds, the amount of eluent required, eluting time, and satisfactory chemical separation [22]. Based on the aforementioned criterions, the binary solvents of petroleum ether–acetone were selected here. The different ratios of petroleum ether–acetone (v/v) were also investigated systematically. The target compounds could not be eluted by petroleum ether. With petroleum ether–acetone (10:1), it could be eluted but a large amount of eluent was needed. With petroleum ether–acetone (10:5), the target compounds moved down too quickly and could not be separated from other components. Therefore, the target compounds were separated in a gradient elution mode (10:1, 10:3, 10:5). The separation efficiency on the silica gel column decreased and the recovery increased with the increase of loading amount as it exceeded column loading capacity [22]. As the close polarities of prenylated flavonoids from S. emodi, their separations are inherently difficult. In addition, the prenylated flavonoids can make strong interactions on silica gel due to the existence of polar functional groups (OH). With the decrease of required silica gel, the adsorption of prenylated flavonoids on silica gel decreased
Y. Sun et al. / J. Chromatogr. B 969 (2014) 190–198
193
Fig. 3. (A) HPLC chromatogram of the pretreated concentrate from SGCC, which were isolated with the mixed solvents of petroleum ether–acetone; (B) HPLC chromatogram and UV spectrum of HSCCC peak fraction 1 in Fig. 2; (C) HPLC chromatogram and UV spectrum of HSCCC peak fraction 2 in Fig. 2; (D) HPLC chromatogram of the eluates containing the target compounds from SGCC, which were isolated with the mixed solvents of dichloromethane–methanol; (E) HPLC chromatogram of the eluates containing the target compounds from SGCC, which were isolated with the mixed solvents of dichloromethane–acetone. Experimental conditions: column, a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm); mobile phase, methanol (A) and 0.1% trifluoroacetic acid (B) at the gradient (30–85% A at 0–30 min, 85–100% A at 30–40 min); flow rate, 1.0 mL min−1 ; detection wavelength, 254 nm; column temperature, 35 ◦ C.
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Fig. 4. HPLC chromatogram of the ethyl acetate fraction from Sinopodophyllum emodi. Experimental conditions: column, a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm); mobile phase, methanol (A) and 0.1% trifluoroacetic acid (B) at the gradient (20–22% A at 0–5 min, 22–25% A at 5–12 min, 25–45% A at 12–55 min, 45–65% A at 55–71 min, 65–100% A at 71–110 min); flow rate, 1.0 mL min−1 ; detection wavelength, 254 nm; column temperature, 35 ◦ C.
and their contents in the eluent increased. As it was loaded at >25 mg g−1 , it led to overlapping of neighboring peaks. Normally, separation efficiency will improve by increasing the ratio of adsorbent to sample due to higher interaction surface area available [23]. The satisfactory separation efficiency of target compounds was observed with from 0 to 25 mg g−1 . These results showed that the optimal loading amount on the silica gel column was determined as 25 mg g−1 . Mobile phase flow rate is an important influencing factor for the separation efficiency [24]. At flow rates
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Isolation of two new prenylated flavonoids from Sinopodophyllum emodi fruit by silica gel column and high-speed counter-current chromatography Yanjun Sun a,b , Yinshi Sun c , Hui Chen a,b , Zhiyou Hao a,b , Junmin Wang a,b , Yanbin Guan a,b , Yanli Zhang a,b , Weisheng Feng a,b,∗ , Xiaoke Zheng a,b,∗∗ a Collaborative Innovation Center for Respiratory Disease Diagnosis and Treatment & Chinese Medicine Development of Henan Province, Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450046, China b School of Pharmacy, Henan University of Traditional Chinese Medicine, Zhengzhou, Henan 450046, China c College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, China
a r t i c l e
i n f o
Article history: Received 28 March 2014 Received in revised form 14 June 2014 Accepted 9 August 2014 Available online 19 August 2014 Keywords: Sinopodophyllum emodi New prenylated flavonoid Silica gel column chromatography High-speed counter-current chromatography
a b s t r a c t Two new prenylated flavonoids, sinoflavonoids A–B, were isolated from the dried fruits of Sinopodophyllum emodi by silica gel column chromatography (SGCC) and high-speed counter-current chromatography (HSCCC). The 95% ethanol extract was partitioned with petroleum ether, dichloromethane, ethyl acetate, and n-butanol in water, respectively. The ethyl acetate fraction was pre-separated by SGCC with a petroleum ether–acetone gradient. The eluates containing target compounds were further separated by HSCCC with n-hexane–ethyl acetate–methanol–water (4:6:4:4, v/v). Finally, 17.3 mg of sinoflavonoid A and 25.9 mg of sinoflavonoid B were obtained from 100 mg of the pretreated concentrate. The purities of sinoflavonoid A and sinoflavonoid B were 98.47% and 99.38%, respectively, as determined by HPLC. Their structures were elucidated on the basis of spectroscopic evidences (HR-ESI-MS, 1 H-NMR, 13 C-NMR, HSQC, HMBC). The separation procedures proved to be efficient, especially for trace prenylated flavonoids. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Sinopodophyllum emodi (Wall.) Ying (Berberidaceae) is distributed widely in the southwest of China [1]. The dried ripe fruit of S. emodi, called “Xiaoyelian” in Chinese, is used to regulate menstruation and promote the circulation of blood. As a traditional Tibetan medicine, it is clinically applied to the treatment of amenorrhea, dead fetus, and placental retaining. It has been recorded in Chinese Pharmacopoeia and Yue Wang Yao Zhen (Somaratsa), which was compiled in the mid-eighth century as the earliest literature on traditional Tibetan medicine [2]. Previous chemical investigations on S. emodi revealed the presence of lignans, flavonoids, steroids, phenolics [1–11]. The prenylated flavonoids, which were representative bioactive constituents from the genus Sinopodophyllum, possessed multiple biological activities including anti-oxidant, anti-cancer, anti-inflammatory, anti-bacterium,
∗ Corresponding author. Tel.: +86 371 65962746; fax: +86 371 65962746. ∗∗ Co-corresponding author. Tel.: +86 371 65962746; fax: +86 371 65962746. E-mail addresses: [email protected] (W. Feng), [email protected] (X. Zheng). http://dx.doi.org/10.1016/j.jchromb.2014.08.017 1570-0232/© 2014 Elsevier B.V. All rights reserved.
anti-osteoporosis, prevention of Alzheimer’s disease, anti-diabete, cardiovascular protection, and estrogen-like effect [12]. Conventional isolation strategies for prenylated flavonoids from S. emodi involved multiple chromatographic steps, which were time-consuming and resulted in a substantial loss of samples due to irreversible adsorption [2,10]. Therefore, it is indispensible to develop a rapid and efficient method for the purification of prenylated flavonoids from S. emodi. HSCCC is a peculiar liquid-liquid partition technology, which can eliminate some complications such as irreversible adsorption and deactivation of target compounds [13]. HSCCC has been widely used in the preparative isolation and purification of natural products with high recovery and loading capacity, low solvent consumption, acceptable efficiency, and the ease of scaling-up [14]. To the best of our knowledge, only flavonoid glycosides [15–17], isoflavones [18], polymethoxylated flavones [19,20] were successfully isolated from natural sources by SGCC and HSCCC. Although HSCCC has been developed for the purification normal flavonoid (only kaempferol) [21], there are no reports on the application of SGCC and HSCCC for the isolation of prenylated flavonoids. The present research established an optimal preparation method of two new prenylated flavonoids (sinoflavonoids A-B) (Fig. 1) from S. emodi with SGCC and HSCCC.
Y. Sun et al. / J. Chromatogr. B 969 (2014) 190–198
191
Fig. 1. The chemical structures of two new flavonoids from Sinopodophyllum emodi.
2. Experimental
2.3. Preparation of the crude extract
2.1. Apparatus
The fruits of S. emodi (4 kg) were dried constantly at 60 ◦ C, then grilled to powder, and passed through a 40-mesh sieve. The powder was ultrasonically extracted by 10-fold amounts of 95% ethanol at 55 ◦ C for 60 min. The working frequency and power were fixed at 40 kHz and 200 W, respectively. The extraction procedure was then repeated twice. After combination and removal of the ethanol under reduced pressure, the extract was suspended in 5 L distilled water, and subjected to a series of solvent extractions (1:1, v/v) with petroleum ether (b.p. 60–90 ◦ C), dichloromethane, ethyl acetate, and finally n-butanol. After concentration and freeze-drying, the dried fractions were stored at −10 ◦ C.
Ultrasonic-assisted extraction (UAE) was performed on a digitally controlled ultrasonic apparatus (KQ 5200V, Kunshan Ultrasonic Instruments Manufacture Co. Ltd., China). The HSCCC experiments were carried out by a Model GS-10A high-speed counter-current chromatography (Beijing Institute of New Technology Application, Beijing, China). The instrument was equipped with a polytetrafluoroethylene multilayer coil column (i.d. of the tubing = 1.6 mm, total volume = 230 mL) and a manual sample injection valve with a 10 mL loop. The revolution radius between the holder axis and central axis of the centrifuge (R) was 5 cm, and the ˇ value of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal (ˇ = r/R, where r was the distance from the coil to the holder shaft, and R was the revolution radius or the distance between the holder axis and central axis of the centrifuge). The revolution speed of the apparatus could be regulated by a speed controller ranging from 0 to 1000 rpm. The HSCCC system was also equipped with BF-2002 CT11 signal collection cell (Chromatography Center of Beifenruili Group Company, Beijing, China), a Model NS-1007A constant-flow pump and a Model 8823B-UV Monitor (Beijing Institute of New Technology Application, Beijing, China) at 254 nm. The data were collected with HW-2000 chromatography workstation (Qianpu Software Co. Ltd., Shanghai, China). The analytical HPLC data were measured on a Waters Alliance 2489 separations module equipped with a Waters 2695 UV/visible Detector and Empower pro data handling system (Waters Co., Milford, USA). The structures of the target compounds were determined by high resolution electrospray ionization mass (HR-ESI-MS) spectrometer (Shimadzu LC-MS 2010, Japan) and nuclear magnetic resonance (NMR) spectrometer (Bruker AM 500, Switzerland). 2.2. Materials and reagents Methanol used for HPLC was of chromatographic grade (Siyou Biology Medical Tech Co. Ltd., Tianjin, China), and water was purified by means of a water purifier (18.2 M) (Wanjie Water Treatment Equipment Co. Ltd., Hangzhou, China). All organic solvents used for preparation of crude fractions, SGCC and HSCCC separation were of analytical grade (Fuyu Chemical Reagent Co. Ltd., Tianjin, China). The chromatographic silica gel was produced from Qingdao Ocean Chemical Factory (Qingdao, China). The fruits were collected in Deqin, Yunnan province, People’s Republic of China, in September 2012, and were identified as the fruits of S. emodi (Wall.) Ying by Professor Chengming Dong (School of Pharmacy, Henan University of Traditional Chinese Medicine).
2.4. Pre-separation of the crude extract by SGCC The ethyl acetate fraction (50 g) was dissolved in acetone (80 mL), and added to silica gel (60 g, 200–300 mesh) by constant stirring. Acetone was evaporated in a rotary evaporator under vacuum at 45 ◦ C. The fraction impregnated silica gel was cooled down to room temperature and kept in a desiccator until required. The silica gel chromatography column was packed as follows: the exit of the chromatography column was plugged with glass wool to retain solids. Silica gel (1.94 kg, 200–300 mesh) was suspended in petroleum ether (7.5 L), and then transferred to the column (140 cm lenth × 8 cm i.d.). The column was rinsed with 7.5 L of petroleum ether-acetone (10:1, v/v). Prior to sample application, the level was lowered 20 cm above the stationary phase. The fraction impregnated silica gel was added to the top of the column, and elution was performed with a petroleum ether–acetone gradient (10:1, 10:3, 10:5, 15 L each) at a constant flow rate of 15 mL min−1 . Fractions of 100 mL each were analyzed by TLC. TLC analyses were performed on GF254 silica gel plates at room temperature, using petroleum ether–acetone (1:1, v/v) as developing reagent. Spots were visualized under an ultraviolet lamp at 254 nm. The eluates containing target compounds were pooled and concentrated under reduced pressure. The concentrate was stored at −10 ◦ C for the subsequent HSCCC separation.
2.5. Further separation by HSCCC 2.5.1. Determination of the partition coefficient The partition coefficient (K) of target compounds in different two-phase solvent systems was determined by HPLC as follows: 25 mg of the pretreated concentrate and 1 mL of the equilibrated two-phase solvent were added into a 5 mL centrifuge tube. The centrifuge tube was then stoppered and vortically mixed for 1 min to thoroughly equilibrate the sample between the two phases. Then an aliquot of each phase (10 L) was analyzed by HPLC. The K value was expressed as the ratio of the peak area of a given compound in the upper phase divided that in the lower phase.
192
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Fig. 2. HSCCC chromatogram of the pretreated concentrate from SGCC. Two-phase solvent system: n-hexane–ethyl acetate–methanol–water (4:6:4:4, v/v); mobile phase: the lower phase; stationary phase: the upper phase; flow rate: 1.7 mL min−1 ; revolution speed: 800 rpm; detection wavelength: 254 nm; sample size: 100 mg pretreated concentrate was dissolved in the solvent mixture of n-hexane–ethyl acetate–methanol–water (5 mL for each phase); the retention percentage of the stationary phase: 67%.
2.5.2. Preparation of two-phase solvent system and sample solution A solvent system consisting of n-hexane–ethyl acetate– methanol–water (4:6:4:4, v/v) was prepared by adding the solvents to a separation funnel according to the volume ratios and thoroughly equilibrated by shaking repeatedly. Then the two phases were separated and degassed by sonication for 30 min before use. The sample solution was prepared as follows: 100 mg pretreated concentrate was dissolved in the solvent mixture of nhexane–ethyl acetate–methanol–water (5 mL for each phase). 2.5.3. HSCCC separation procedure The separations were initiated by filling the coiled column with the upper phase (stationary phase). The lower phase (mobile phase) was pumped into the column in a head-to-tail mode at a flow rate of 1.7 mL min−1 , when the apparatus was rotated at 800 rpm. Approximately 10 mL of the sample solution containing 100 mg of the pretreated concentrate was injected into the head of the column through the injection valve after hydrodynamic equilibrium was established in the column, as indicated by the mobile phase eluting from the tail outlet. The eluates from the column outlet were continuously monitored by a UV detector at 254 nm. The fractions during 120–150 min (peak 1) and 170–200 min (peak 2) were collected respectively according to the elution profile (Fig. 2). All fractions of the same pure compound were combined and evaporated under reduced pressure. The purified compounds were stored at −20 ◦ C before HPLC and NMR analyses. 2.5.4. HPLC analysis and identification of HSCCC peak fractions The pretreated concentrate from SGCC and each HSCCC peak fraction were analyzed by HPLC (Fig. 3A–C). Analyses were accomplished on a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm) at 35 ◦ C. Methanol (A) −0.1% trifluoroacetic acid (v/v) (B) was used as the mobile phase in gradient elution mode as follows: 30–85% A at 0–30 min, 85–100% A at 30–40 min. The flow rate of the mobile phase was 1.0 mL min−1 . The eluates were monitored at 254 nm by a UV/visible detector. Based on the peak area normalized to all observed HPLC peak areas, the purities of the isolated flavonoids were determined.
3. Results and discussions 3.1. Pre-separation by SGCC As seen in the HPLC chromatogram (Fig. 4), the high concentration of flavonoids and lignans, and a trace of sinoflavonoids A (peak 1, 70.1 min) and B (peak 2, 78.9 min), were present in the ethyl acetate fraction. To improve the preparative separation efficiency, SGCC was employed for pre-separation, eluting with a petroleum ether–acetone gradient. The chromatographic parameters on separation efficiency, including flow rate, mobile phase composition, and loading amount were investigated to produce optimum separation. Three kinds of binary solvent systems were tested, includdichloromethane–methanol, dichloromethane–acetone, ing and petroleum ether–acetone. With the mixed solvents of dichloromethane–methanol and dichloromethane–acetone, the eluates contained a lot of impurities such as citrusinol (peak 3, 15.9 min) and 8-prenylkaempferol (peak 4, 22.0 min) (Fig. 3D and E), which possessed the similar polarities as target compounds. Selection for ideal mobile phase depended on the recovery of target compounds, the amount of eluent required, eluting time, and satisfactory chemical separation [22]. Based on the aforementioned criterions, the binary solvents of petroleum ether–acetone were selected here. The different ratios of petroleum ether–acetone (v/v) were also investigated systematically. The target compounds could not be eluted by petroleum ether. With petroleum ether–acetone (10:1), it could be eluted but a large amount of eluent was needed. With petroleum ether–acetone (10:5), the target compounds moved down too quickly and could not be separated from other components. Therefore, the target compounds were separated in a gradient elution mode (10:1, 10:3, 10:5). The separation efficiency on the silica gel column decreased and the recovery increased with the increase of loading amount as it exceeded column loading capacity [22]. As the close polarities of prenylated flavonoids from S. emodi, their separations are inherently difficult. In addition, the prenylated flavonoids can make strong interactions on silica gel due to the existence of polar functional groups (OH). With the decrease of required silica gel, the adsorption of prenylated flavonoids on silica gel decreased
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Fig. 3. (A) HPLC chromatogram of the pretreated concentrate from SGCC, which were isolated with the mixed solvents of petroleum ether–acetone; (B) HPLC chromatogram and UV spectrum of HSCCC peak fraction 1 in Fig. 2; (C) HPLC chromatogram and UV spectrum of HSCCC peak fraction 2 in Fig. 2; (D) HPLC chromatogram of the eluates containing the target compounds from SGCC, which were isolated with the mixed solvents of dichloromethane–methanol; (E) HPLC chromatogram of the eluates containing the target compounds from SGCC, which were isolated with the mixed solvents of dichloromethane–acetone. Experimental conditions: column, a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm); mobile phase, methanol (A) and 0.1% trifluoroacetic acid (B) at the gradient (30–85% A at 0–30 min, 85–100% A at 30–40 min); flow rate, 1.0 mL min−1 ; detection wavelength, 254 nm; column temperature, 35 ◦ C.
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Fig. 4. HPLC chromatogram of the ethyl acetate fraction from Sinopodophyllum emodi. Experimental conditions: column, a YMC-Pack ODS A column (5 m, 250 mm × 4.6 mm); mobile phase, methanol (A) and 0.1% trifluoroacetic acid (B) at the gradient (20–22% A at 0–5 min, 22–25% A at 5–12 min, 25–45% A at 12–55 min, 45–65% A at 55–71 min, 65–100% A at 71–110 min); flow rate, 1.0 mL min−1 ; detection wavelength, 254 nm; column temperature, 35 ◦ C.
and their contents in the eluent increased. As it was loaded at >25 mg g−1 , it led to overlapping of neighboring peaks. Normally, separation efficiency will improve by increasing the ratio of adsorbent to sample due to higher interaction surface area available [23]. The satisfactory separation efficiency of target compounds was observed with from 0 to 25 mg g−1 . These results showed that the optimal loading amount on the silica gel column was determined as 25 mg g−1 . Mobile phase flow rate is an important influencing factor for the separation efficiency [24]. At flow rates
