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Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography
Journal of Chromatography B, 988 (2015) 127–134
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography Huanhuan Lv a,b , Honglun Wang a,∗ , Yanfeng He a,b , Chenxu Ding a , Xiaoyan Wang a , Yourui Suo a a b
Key Laboratory of Tibetan Medicine Research, Northwest Institution of Plateau Biology, Chinese Academy of Sciences, Xining, 810001, China University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 26 January 2015 Accepted 25 February 2015 Available online 3 March 2015 Keywords: HSCCC Iris lactea Oligostilbenes Preparation
a b s t r a c t A method of using high-speed counter-current chromatography (HSCCC) for preparative isolation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea Pall. var. chinensis (Fisch.) Koidz was established in this study. Four oligostilbenes were successfully separated and purified by HSCCC with two sets of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) in the head-to-tail elution mode for the first separation to mainly isolate vitisin A (58 mg), -viniferin (76 mg) and peak II (43 mg) from 300 mg of the crude ethanol extracts, and then light petroleum–ethyl acetate–methanol–water (5:5:3:6, v/v/v/v) in the tail-to-head elution mode for the second separation to isolate vitisin B (52 mg) and vitisin C (11 mg) from 100 mg of peak II. The purities of the isolated four oligostilbenes were all over 95.0% as determined by HPLC. Vitisin A, vitisin B and vitisin C, resveratrol tetramers, were isolated from Iris lactea for the first time. The preparation of crude sample was simple and the HSCCC method for the isolation and purification of four oligostilbenes was rapid, efficient and economical. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Iris lactea Pall. var. chinensis (Fisch.) Koidz, belongs to the family of Iridaceae, is an herbaceous perennid native to China, Korea, Japan and Russia. It has developed root system, high resistibility, adaptability and saline-tolerance. These futures determine that it is very suitable for the green transformation and the water and soil conservation in the regions with dry climate. The dried seeds of Iris lactea have the effects of clearing heat, eliminating dampness and stanching bleed. It has been used in the treatment of jaundice, diarrhea, vomiting blood, leucorrhea, pharyngitis, and carbuncle swollen in traditional Chinese medicine [1]. The photographs of the flowers, seeds and seed kernels of Iris lactea are shown in Fig. 1. The previous phytochemical studies of Iris species showed the presence of flavones, isoflavones, flavanones, xanthones, triterpenes, quinones, and stilbenes [2–4]. Interestingly, more attention has been focused on the naturally occurring oligostilbenes because these compounds exhibit a
∗ Corresponding author. Tel.: +86 971 6143857. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.jchromb.2015.02.035 1570-0232/© 2015 Elsevier B.V. All rights reserved.
wide range of pharmacological properties, such as neuroprotective, cytotoxic, anti-proliferative, anti-microbial, anti-inflammatory, antioxidant, antitumor and hepatoprotective activities [5–12]. The oligostilbenes are receiving much attention as potential therapeutic agents for several pathological diseases [13]. Before further pharmacological evaluation of the oligostilbenes from Iris lactea, development of an efficient method for the isolation of large quantity of pure oligostilbenes is critical and urgently needed. High-speed counter-current chromatography (HSCCC) is a form of support-free liquid-liquid partition chromatography [14]. Solute separation is based on partitioning between the mobile phase and stationary phase. Without any solid matrix, the stationary phase is retained in the column with the aid of a centrifugal force field, so it eliminates irreversible adsorption of the sample onto solid support and has high sample recovery. Therefore, HSCCC can provide a suitable alternative for the large scale separation of compounds without degrading the active compounds and causing the loss of sample. Due to the high efficiency, HSCCC has been widely used in the separation and purification of a diversity of natural products [15–18]. To the best of our knowledge, there is no published report about the separation and purification of oligostilbenes from Iris lactea with the use of HSCCC technique.
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Fig. 1. The photographs of (1) the flowers, (2) seeds, and (3) seed kernels of Iris lactea.
This study is the first attempt to use the HSCCC technique for the separation and purification of oligostilbenes from the seed kernel of Iris lactea. The objective of this study was to develop an efficient method to separate and purify four oligostilbenes from the ethanol extracts of seed kernel of Iris lactea by HSCCC. The structures of the four isolated oligostilbenes, namely vitisin A, -viniferin, vitisin B and vitisin C are shown in Fig. 2.
2. Experimental 2.1. Materials and reagents All organic solvents used for extraction and HSCCC separation were of analytical grade and purchased from Tianjin Baishi Chemical Co., Ltd. (Tianjin, China). Methanol and acetonitrile used for HPLC analyses were of chromatographic grade (Shandong Yuwang
Fig. 2. Chemical structures of (1) vitisin A, (2) -viniferin, (3) vitisin B and (4) vitisin C.
H. Lv et al. / J. Chromatogr. B 988 (2015) 127–134
Industrial Co., Ltd., Shandong, China). Ultrapure water (18.25 M) and pure water used in the present study were purified on a UPTI-10L system (Chengdu Ultra Pure Technology Co., Ltd., Chengdu, China). The seeds of Iris lactea were collected from Malian Lake of Alxa League of Inner Mongolia, China, in August 2013. 2.2. Apparatus and instruments The HSCCC separation was performed on a TBE-300 C high-speed counter-current chromatography instrument (Tauto Biotechnique Company, Shanghai, China). The apparatus was equipped with three preparative coils connected in series (the diameter of PTFE tube = 1.9 mm, total volume = 320 ml, including the 300 ml separation volume and a 20 ml sample loop). The revolution speed of the instrument was adjustable, ranging from 0 to 1000 rpm. The system was also equipped with a TBP5002 constant flow pump (Tauto Biotechnique Company, Shanghai, China), a model of UV2000D detector (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China), and a DC0506 low constant temperature bath (Tauto Biotechnique Company, Shanghai, China). EasyChrom-1000 chromatography workstation (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China) was employed to record the chromatograms. An Angilent 1260 HPLC system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) was equipped with a quaternary pump (G1311 C), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a diode array detector (G1315D), a Zorbax Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 m), and an Agilent HPLC workstation was used for data acquisition and processing. The nuclear magnetic resonance spectrometer was a Varian INOVA 600 NMR system (Varian, Palo Alto, CA, USA). 2.3. Preparation of the crude sample The dried seeds of Iris lactea were de-coated by a plant disintegrator and the seed kernels were extracted with 85% ethanol (3 × 25 L, each 3 hours) at 60 ◦ C in a HYY25L decocting machine (Huayankorea Machinery and Equipment Co., Ltd., Tianjin, China). The combined filtrate was concentrated to dryness under reduced pressure to afford the ethanol extracts. The extracts were collected as the crude sample for HSCCC separation. 2.4. Measurement of partition coefficient The partition coefficients of the target oligostilbenes in different two-phase solvent systems were determined by HPLC. About 4 ml of each phase of the pre-equilibrated two-phase solvent system was mixed with suitable amount of samples in a test tube. The test tube was shaken vigorously and left to stand at room temperature until the two phases have a clear separation layer. Then the two phases were separated, dried and re-dissolved in methanol, then analyzed by HPLC at 325 nm to obtain the partition coefficients of the target compounds respectively. The partition coefficient is expressed as the peak area of the target compound in the upper phase divided by that of the lower phase. 2.5. Preparation of the two-phase solvent system and sample solution
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lower phase were separated and degassed by sonication for about 20 min prior to use. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) was used for the first HSCCC separation and light petroleum-ethyl acetate-methanolwater (5:5:3:6, v/v/v/v) for the second separation run. The sample solution for the first HSCCC separation run was prepared by dissolving 300 mg of the crude ethanol extracts in 10 ml lower phase of the solvent system n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v). The eluent of Peak II in the first separation run was manually collected and evaporated to dryness at 60 ◦ C under vacuum. Then the sample solution for the second separation run was prepared by dissolving 100 mg of Peak II in 10 ml of the upper phase of the solvent system light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v). 2.6. HSCCC separation procedure The first HSCCC separation run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-methanolwater at the volume ratio of 3:6:4.2:5.5 in the head-to-tail elution mode (the upper phase as the stationary phase). The coil column was entirely filled with the upper phase and then the apparatus was rotated at 900 rpm. After 30 min, the lower phase was pumped into the column at a flow rate of 1.5 ml/min. When the hydrodynamic equilibrium was established in the column, about 10 ml sample solution containing 300 mg of the crude ethanol extracts was injected through the injection valve. The separation temperature was controlled at 30 ◦ C. The eluents from the outlet of the column were continuously monitored with a UV detector at 325 nm. Each peak fraction was manually collected according to the elution profile and evaporated under vacuum. The eluent of Peak II in the first HSCCC separation is a mixed compound. Therefore, a second HSCCC separation run was employed for the separation of Peak II. Another solvent system composed of light petroleum–ethyl acetate–methanol–water at the volume ratio of 5:5:3:6 in the tail-to-head elution mode (the lower phase as the stationary phase) were employed in the second HSCCC separation. 2.7. HPLC analysis and identification of HCCC peak fractions The HPLC analysis of the ethanol extracts, partition coefficients and each peak fraction obtained from HSCCC were conducted on a reversed-phase Zorbax Eclipse XDB C18 column (4.6 mm × 250 mm, 5 m) with gradient elution throughout this study. The mobile phase was composed of methanol, acetonitrile and water in a gradient elution mode as follows: 0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile. The flow rate was maintained at 1.0 ml/min and the column temperature was set at 30 ◦ C. The detection wavelength was 325 nm. The identifications of the HSCCC peak fractions were carried out by 1 H NMR (600 MHz) and 13 C NMR (600 MHz). The assignments were confirmed by HSQC and HMBC. Deuterated DMSO was used as the solvent and the reference compound tetramethylsilane (TMS) was used as internal standard for the determination of chemical shifts. 3. Results and discussion 3.1. Optimization of HPLC method
The selected two sets of two-phase solvent systems were respectively prepared by adding all the solvents into a separation funnel at selected volume ratios. Each solvent system was thoroughly equilibrated by shaking repeatedly in a separation funnel at room temperature. After being equilibrated, the upper phase and
In the course of optimizing the HPLC conditions, the system conditions including the composition of mobile phase (methanol–water, methanol–acetonitrile–water, and acetonitrile–water), gradient program, column temperature and
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Fig. 3. HPLC chromatograms of (A) the ethanol extracts, (B) the eluent of Peak II and (C–F) the HSCCC peak fractions, i.e. (C): vitisin A, (D): -viniferin, (E): vitisin B, (F): vitisin C. HPLC conditions: Column: Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 m); mobile phase: methanol–acetonitrile–water (0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile); flow rate: 1.0 ml/min; detection wavelength: 325 nm; column temperature: 30 ◦ C.
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Table 1 The partition coefficients (K) of vitisin A, -viniferin, vitisin B and vitisin C in different solvent systems. Solvent systems(v/v)
n-hexane–ethyl acetate–methanol–water
light petroleum–ethyl acetate–methanol–water
K-values
4:6:3:7 4:6:5:5 3:6.4:5 2.5:6:4.5:5 3:6.5:4:5.5 3:6.5:4.2:5.5 3:6.5:4.5:5.7 3:5:3:5 5:5:3:5 5:5:3:7 5:5:3:6
Vitisin A
-viniferin
Vitisin B
Vitisin C
1.27 0.05 0.14 0.82 0.43 0.38 0.26 0.64 0.03 0.64 0.11
6.47 0.18 0.76 3.13 2.16 1.89 1.11 3.13 1.09 3.13 1.34
9.95 0.10 0.51 3.55 2.03 1.47 0.91 3.59 0.62 2.13 0.82
13.12 0.11 0.63 4.42 2.51 1.64 1.08 4.70 0.84 2.99 1.26
3.2. Selection of the two-phase solvent and other conditions of HSCCC
Fig. 4. HSCCC chromatogram of the ethanol extracts. Peak 1: vitisin A; Peak II: mixture of vitisin B and vitisin C; Peak 2: -viniferin. HSCCC conditions: solvent system: n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v); stationary phase: upper phase; mobile phase: lower phase; revolution speed: 900 rpm; separation temperature: 30 ◦ C; flow rate: 1.5 ml/min; sample size: 300 mg ethanol extracts in 10 ml mobile phase; detection wavelength: 325 nm; retention of stationary phase: 68.75%.
detection wavelength were all investigated in this study. The final results showed that the separation with good resolution and short analysis time were achieved when a gradient elution mode composed of methanol, acetonitrile, and water was programmed as follows: 0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile. The flow rate was 1.0 ml/min, the column temperature was set at 30 ◦ C, and 325 nm was selected as the detection wavelength. Spectra were recorded from 190 to 400 nm. Under the optimum gradient elution, the target compounds in the crude ethanol extracts of seed kernels reached almost base-line separation as shown in Fig. 3.
HSCCC is a very useful and efficient technique for the separation and purification of natural products. The selection of the two-phase solvent system is the most important step in HSCCC separation. A successful HSCCC separation is mainly based on choosing a solvent system that can provide suitable partition coefficients (K-values) for the target compounds. In the present study, two sets of twophase solvent system (n-hexane-ethyl acetate-methanol-water and light petroleum-ethyl acetate-methanol-water) at different volume ratios were tested. The K-values of the target compounds were measured and summarized in Table 1. When the volume ratio of n-hexane-ethyl acetate-methanol-water was at 4:6:5:5, it was too hydrophobic because it offered smaller K values for all the four compounds (K1 = 0.05, K2 = 0.18, K3 = 0.10, K4 = 0.11). When the volume ratio was set at 4:6:3:7, the K values of compound 2, 3, and 4 (K2 = 6.47, K3 = 9.95, K4 = 13.12) were too large. Therefore, it is necessary to find a suitable polarity range. Consequently, five other two-phase solvent systems composed of n-hexane-ethyl acetatemethanol-water at different volume ratios were tested. The solvent system of n-hexane-ethyl acetate-methanol-water (3:6.5:4.2:5.5, v/v/v/v) was selected and used to isolate the crude ethanol extracts because it provided suitable range of K-values for the target compounds (K1 = 0.38, K2 = 1.89, K3 = 1.47, K4 = 1.64). The flow rate of the mobile phase, the separation temperature and the revolution speed were also important factors that can affect the efficiency of the whole HSCCC separation. Therefore, the flow rate, the temperature and the revolution speed were also investigated in this study in order to obtain the optimum separation. As reported in literature [19], the small flow rate can produce good
Fig. 5. HSCCC chromatogram of the eluent of Peak II. Peak 3: vitisin B; Peak 4: vitisin C; HSCCC conditions: solvent system: light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v); (A) stationary phase: upper phase; mobile phase: lower phase, elution mode: head-to-tail mode; revolution speed: 900 rpm; flow rate: 2.0 ml/min; retention of stationary phase: 69.00%; (B) stationary phase: lower phase; mobile phase: upper phase; elution mode: tail-to-head mode; revolution speed: 800 rpm; flow rate: 3.0 ml/min; retention of stationary phase: 76.67%; other conditions: separation temperature: 30 ◦ C; detection wavelength: 325 nm; sample size: 100 mg Peak II in 10 ml mobile phase.
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separation, but needs long separation time and more mobile phase. The temperature affects the retention of stationary phase, the K values and the mutual solvency of the two phases. The revolution speed mainly has the effects on the retention of stationary phase. Considering these aspects, the flow rate of the mobile phase was selected as 1.5 ml/min, the separation temperature was set at 30 ◦ C, and the revolution speed was of 900 rpm in the first HSCCC separation run. Under the optimum condition, the crude ethanol extracts was separated by HSCCC with the retention of stationary phase of 68.75%. The HSCCC chromatogram is given in Fig. 4. As shown in Fig. 4, compound 1 and compound 2 were successfully isolated and with high purity as determined by HPLC. Restricted by the limited peak capacity of the first HSCCC separation run, the eluent of Peak II is a mixture of compound 3 and 4 as shown in Fig. 3. Compound 3 and compound 4, of similar structure, eluted together as one peak in the first HSCCC separation run. The first HSCCC separation procedure yielded 58 mg compound 1, 76 mg compound 2, and 43 mg peak II from 300 mg crude ethanol extracts. Therefore, a second HSCCC separation run was needed to isolate Peak II. The solvent system composed of light petroleum-ethyl acetatemethanol-water at different volume ratios were tested for mainly separating Peak II. When the volume ratio was changed to 5:5:3:6, the appropriate K values for compound 3 (K3 = 0.82) and compound 4 (K4 = 1.26) were obtained. As seen in Fig. 5A, the two compounds were not completely separated in the head-to-tail elution mode. While in Fig. 5B, the results indicated that it gave much better separation for compound 3 and compound 4 in a tail-to-head mode. Finally, the solvent system composed of light petroleumethyl acetate-methanol-water at a volume ratio of 5:5:3:6 in the tail-to-head elution mode with the flow rate of mobile phase of 3.0 ml/min, the revolution speed of 800 rpm, and separation temperature of 30 ◦ C were used to separate peak II after the first HSCCC separation step. The second HSCCC separation procedure yielded 52 mg compound 3 and 11 mg compound 4 from 100 mg peak II. In addition, the purities of the four isolated compounds were 98.23%, 99.05%, 97.00% and 95.34%, respectively as determined by HPLC (Fig. 3). Their UV spectra are shown in Fig. 6. 3.3. The structural identification The structures of the four compounds isolated by HSCCC were identified by different spectroscopic analysis, including the use of UV spectra, 1D (1 H NMR and 13 C NMR) and 2D NMR technique (notably 1 H–1 H COSY, HMBC and HSQC spectra), and by comparing the NMR data to the values in published literature. The 1 H NMR spectrum of compound 1 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.12 (2H, d, J = 8.4 Hz) and ␦ 6.75 (2H, d, J = 8.4 Hz), ␦ 7.02 (2H, d, J = 8.4 Hz) and ␦ 6.68 (2H, d, J = 8.4 Hz), ␦ 6.87 (2H, d, J = 8.4 Hz) and ␦ 6.57 (2H, d, J = 8.4 Hz); one
3,5-dihydroxybenzene moiety at ␦ 6.01 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, d, J = 2.2 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.30 (1H, d, J = 6.2 Hz) and ␦ 4.34 (1H, d, J = 5.2 Hz), ␦ 5.10 (1H, d, J = 4.2 Hz) and ␦ 5.25 (1H, d, J = 4.2 Hz), ␦ 5.79 (1H, d, J = 11.7 Hz) and ␦ 4.01 (1H, d, J = 11.7 Hz); two trans olefinic protons at ␦ 6.25 (1H, d, J = 16.2 Hz) and ␦ 6.27 (1H, d, J = 16.2 Hz); nine aromatic protons at ␦ 6.17 (1H, d, J = 2.2 Hz) and ␦ 6.39 (1H, d, J = 2.2 Hz), ␦ 5.98 (1H, d, J = 2.2 Hz) and ␦ 5.89 (1H, d, J = 2.2 Hz), ␦ 5.95 (1H, d, J = 2.2 Hz) and ␦ 5.99 (1H, d, J = 2.2 Hz), ␦ 5.85 (1H, d, J = 2.0 Hz), ␦ 6.68 (1H, d, J = 8.4 Hz) and ␦ 6.76 (1H, dd, J = 2.2, 1.8 Hz). The structure of compound 1 was in agreement with that of the reported vitisin A, which was previously isolated from Vitis coignetiae [20]. Compared the 1 H NMR and 13 C NMR data with the literature values, compound 2 was identified as -viniferin [21]. The 1 H NMR spectrum exhibited signals for two 4-hydroxybenzene moieties at ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.73 (2H, d, J = 8.4 Hz), ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.65 (2H, d, J = 8.4 Hz); one 3,5-dihydroxybenzene moieties at ␦ 6.03 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, t, J = 2.2 Hz); two aliphatic protons of a dihydrobenzofuran ring at ␦ 5.32 (1H, d, J = 5.2 Hz) and ␦ 4.40 (1H, d, J = 5.2 Hz); two trans olefinic protons at ␦ 6.81 (1H, d, J = 16.2 Hz) and ␦ 6.56 (1H, d, J = 16.2 Hz); two aromatic protons at ␦ 6.58 (1H, d, J = 2.2 Hz) and ␦ 6.22 (1H, d, J = 2.2 Hz). The 1 H NMR spectrum of compound 3 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.18 (2H, d, J = 8.3 Hz) and ␦ 6.78 (2H, d, J = 8.1 Hz), ␦ 6.45 (2H, d, J = 8.5 Hz) and ␦ 6.47 (2H, d, J = 8.5 Hz), ␦ 7.10 (2H, d, J = 8.3 Hz) and ␦ 6.72 (2H, d, J = 8.3 Hz); two 3,5-dihydroxybenzene moieties at ␦ 6.02 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, t, J = 2.2 Hz), ␦ 5.97 (2H, d, J = 2.2 Hz) and ␦ 6.00 (1H, t, J = 2.2 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.31 (1H, d, J = 4.9 Hz) and ␦ 4.49 (1H, d, J = 4.9 Hz), ␦ 5.43 (1H, d, J = 5.2 Hz) and ␦ 4.11 (1H, d, J = 5.2 Hz), ␦ 5.27 (1H, d, J = 5.5 Hz) and ␦ 4.37 (1H, d, J = 5.5 Hz); two trans olefinic protons at ␦ 6.52 (1H, d, J = 16.1 Hz) and ␦ 6.76 (1H, d, J = 16.1 Hz); seven aromatic protons at ␦ 6.64 (1H, d, J = 2.2 Hz), ␦ 6.81 (1H, d, J = 2.2 Hz), ␦ 6.93 (1H, dd, J = 2.2 Hz), ␦ 6.22 (1H, d, J = 2.2 Hz), ␦ 6.54 (1H, d, J = 2.2 Hz), ␦ 6.17 (1H, d, J = 2.2 Hz) and ␦ 5.97 (1H, d, J = 2.2 Hz). These data are in accordance with those reported in published literature [22], compound 3 was identified as vitisin B, which was once isolated from Vitis vinifera ‘Kyohou’. The 1 H NMR spectrum of compound 4 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.11 (2H, d, J = 8.2 Hz) and ␦ 6.97 (2H, d, J = 8.3 Hz), ␦ 6.98 (2H, d, J = 8.4 Hz) and ␦ 6.74 (2H, d, J = 8.4 Hz), ␦ 7.11 (2H, d, J = 8.2 Hz) and ␦ 6.74 (2H, d, J = 8.4 Hz); two 3,5-dihydroxybenzene moieties at ␦ 5.81 (2H, d, J = 2.2 Hz) and ␦ 6.00 (1H, t, J = 2.2 Hz), ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.65 (1H, t, J = 8.4 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.26 (1H, d, J = 4.2 Hz) and ␦ 4.45 (1H, d, J = 4.2 Hz), ␦ 5.30 (1H, d, J = 8.9 Hz) and ␦ 3.67 (1H, d, J = 8.9 Hz), ␦ 5.12 (1H, d, J = 5.0 Hz) and ␦ 4.17 (1H, d, J = 5.0 Hz); two trans olefinic protons at ␦ 6.68 (1H,
Fig. 6. UV spectra of the HSCCC peak fractions. (A): vitisin A, (B): -viniferin, (C): vitisin B, (D): vitisin C.
H. Lv et al. / J. Chromatogr. B 988 (2015) 127–134 Table 2 13 C NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C in DMSO-d6. Position 1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c 12c 13c 14c 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d
Vitisin A C-NMR
13
131.7 127.2 115.2 157.3 115.2 127.2 92.3 55.1 145.7 105.5 158.6 101.0 158.6 105.5 127.5 131.3 131.5 154.4 114.6 126.7 122.0 129.3 134.7 118.1 160. 7 95.8 158.4 102.9 129.1 128.9 115.2 157.6 115.2 128.9 40.0 40.1 140.5 121.1 156.8 95.1 155.7 109.0 133.6 127.2 115.1 154.8 115.1 127.2 39.9 48.0 139.9 118.5 157.6 99.8 158.6 103,3
-viniferin 13
C-NMR
131.7 127.0 115.2 157.3 115.2 127.0 92.3 55.1 146.0 105.5 158.6 101.1 158.6 105.5 128.0 127.8 115.5 157.3 115.5 127.7 128.9 122.0 134.8 118.4 160.7 95.9 158.5 103.1
Table 3 1 H NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C in DMSO-d6.
Vitisin B C-NMR
Vitisin C 13 C-NMR
position
131.7 127.4 115.4 157.3 115.4 127.4 92.4 54.7 145.7 105.5 159.1 101.3 159.1 105.5 131.4 125.0 130.5 158. 5 109.9 125.2 122.9 128.9 134.7 118.5 160.7 96.1 158.2 103.4 131.4 126.2 114.9 156.7 114.9 126.2 89.9 50.4 141.0 118.2 160.6 95.5 159.1 105.6 131.6 127.1 115.3 157.4 115.3 127.1 93.0 55.0 146.0 105.6 158.7 101.7 158.7 105.6
131.8 127.4 115.1 157.2 115.1 127.4 92.5 54.0 145.6 105.1 158.8 101.2 158.8 105.1 130.5 124.5 129.6 157.5 109.5 125.6 123.2 128.9 134.7 119.7 160.8 96.1 159.2 103.7 130.5 127.0 115.2 159.2 115.2 127.0 92.3 52.6 139.3 118.3 160.4 95.7 158.5 106.4 131.3 127.4 115.2 157.4 115.2 127.4 92.9 54.8 146.2 105.4 158.6 101.0 158.6 105.4
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c 12c 13c 14c 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d
13
133
Vitisin A 1 H-NMR
-viniferin
7.12(d,8.4) 6.75(d,8.4)
Vitisin B 1 H -NMR
Vitisin C 1 H -NMR
7.11(d,8.4) 6.73(d,8.4)
7.18(d,8.3) 6.78(d,8.1)
7.11(d,8.2) 6.97(d,8.3)
6.75(d,8.4) 7.12(d,8.4) 5.30(d,5.2) 4.34(d,5.2)
6.73(d,8.4) 7.11(d,8.4) 5.32(d,5.2) 4.40(d,5.2)
6.78(d,8.1) 7.18(d,8.3) 5.31(d,4.9) 4.49(d,4.9)
6.97(d,8.3) 7.11(d,8.2) 5.26(d,4.2) 4.45(d,4.2)
6.01(d,2.2)
6.04(d,2.2)
6.02(d,2.2)
5.81(d,2.2)
6.04(t,2.2)
6.04(t,2.2)
6.04(t,2.2)
6.00(t,2.2)
6.01(d,2.2)
6.04(d,2.2)
6.02(d,2.2)
5.81(d,2.2)
5.85(d, 2.0)
7.11(d,8.4) 6.65(d,8.4)
6.64(d,2.2)
6.68(d,2.2)
6.68(d,8.4) 6.76(dd, 8.4,1.8) 6.25(d, 16.2) 6.27(d, 16.2)
6.65(d,8.4) 7.11(d,8.4) 6.81(d,16.2) 6.56(d,16.2)
6.81(d,8.4) 6.93(dd,8.4,2.0) 6.52(d,16.1) 6.76(d,16.1)
6.73(d,8.3) 6.90(dd,8.4,2.0) 6.58(d,16.1) 6.83(d,16.1)
6.17(d,2.2)
6.22(d, 2.2)
6.22(d,2.2)
6.23(d,2.2)
6.39(d,2.2)
6.58(d,2.2)
6.54(d,2.2)
6.54(d,2.2)
7.02(d,8.4) 6.68(d,8.4)
6.45(d,8.5) 6.47(d,8.5)
6.98(d,8.4) 6.74(d,8.4)
6.68(d,8.4) 7.02(d,8.4) 5.10(d,4.2) 5.25(d,4.2)
6.47(d,8.5) 6.45(d,8.5) 5.43(d,5.2) 4.11(d,5.2)
6.74(d,8.4) 6.98(d,8.4) 5.30(d,8.9) 3.67(d,8.9)
5.98(d,2.2)
6.17(d,2.2)
6.14(d,2.2)
5.89(d,2.2)
5.97(d,2.2)
6.06(d,2.2)
6.87(d,8.4) 6.57(d,8.4)
7.10(d,8.5) 6.72(d,8.3)
7.11(d,8.2) 6.74(d,8.4)
6.57(d,8.4) 6.87(d,8.4) 5.79(d,11.7) 4.01(d,11.7)
6.72(d,8.3) 7.10(d,8.5) 5.27(d,5.5) 4.37(d,5.5)
6.74(d,8.4) 7.11(d,8.2) 5.12(d,5.0) 4.17(d,5.0)
5.97(d,2.2)
6.02(d,2.2)
5.95(d,2.2)
6.00(d,2.2)
6.03(d.2.2)
5.99(d, 2.2)
5.97(d,2.2)
6.02(d,2.2)
1
H-NMR
4. Conclusion d, J = 16.1 Hz) and ␦ 6.83 (1H, d, J = 16.1 Hz); seven aromatic protons at ␦ 6.68 (1H, d, J = 2.2 Hz), ␦ 6.73 (1H, d, J = 2.2 Hz), ␦ 6.90 (1H, dd, J = 8.4, 2.0 Hz), ␦ 6.23 (1H, d, J = 2.2 Hz), ␦ 6.54 (1H, d, J = 2.2 Hz), ␦ 6.14 (1H, d, J = 2.2 Hz) and ␦ 6.06 (1H, d, J = 2.2 Hz). Compared with the given data in literature [23], compound 4 was identified as vitisin C, which was also isolated from Vitis vinifera ‘Kyohou’. The 1 H NMR features of compound 4 were similar to those of vitisin B. Vitisin C was a stereoisomer of vitisin B at the position of C8c. The detailed 13 C NMR and 1 H NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C are listed in Tables 2 and 3, respectively.
The present study demonstrated that the two-step HSCCC separation is an effective method which is suitable for the preparation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea. The whole process yielded four oligostilbenes, namely vitisin A, -viniferin, vitisin B and vitisin C at high purity. Vitisin A, vitisin B and vitisin C were all resveratrol tetramers and isolated from Iris lactea for the first time, among them, vitisin B and vitisin C are epimerides. In general, both the extraction and separation procedure are eco-friendly and simple approaches. Therefore, these methods can be applied to large-scale extraction and largequantity preparation of oligostilbenes from Iris lactea.
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Acknowledgements This work was supported by the National Science Foundation of China (31470426), the National Spark Plan Project (2012GA870001), and the International Cooperation Project of Qinghai Province (2013-H-802). References [1] J.S. Wen, J.Q. Min, S. Pan, F.Z. Chao, Two new C-glycosylflavones from the leaves of Iris lactea var. chinensis, Chin. Chem. Lett. 19 (2008) 821. [2] M.I.C. Atta-ur-Rahman, M. Nur-e-Alam, P.Ö. Ndognii, T. Badarchiin, G. Purevsuren, Two new quinones from Iris bungei, Chem. Pharm. Bull. 48 (2000) 738. [3] K. Seki, K. Haga, R. Kaneko, Phenols and a dioxotetrahydrodibenzofuran from seeds of Iris pallasii, Phytochemistry 38 (1995) 965. [4] B.B. Lin, G.K. Wang, Q. Wang, C.Y. Ge, M.J. Qin, A new belamcandaquinone from the seeds of Iris bungei Maxim, Fitoterapia 82 (2011) 1137. [5] J.Y. Kim, H.Y. Jeong, H.K. Lee, S.H. Kim, B.Y. Hwang, K.H. Bae, Y.H. Seong, Neuroprotection of the leaf and stem of Vitis amurensis and their active compounds against ischemic brain damage in rats and excitotoxicity in cultured neurons, Phytomedicine 19 (2012) 150. [6] D.T. Ha, Q.C. Chen, T.M. Hung, U.J. Youn, T.M. Ngoc, P.T. Thuong, H.J. Kim, Y.H. Seong, B.S. Min, K.H. Bae, Stilbenes and oligostilbenes from leaf and sem of Vitis amurensis and their cytotoxic activity, Arch. Pharm. Res. 32 (2009) 177. [7] D. Colin, A. Lancon, D. Delmas, G. Lizard, J. Abrossinow, E. Kahn, B. Jannin, N. Latreffe, Antiproliferative activities of resveratrol and related compounds in human hepatocyte derived HepG2 cells are associated with biochemical cell disturbance revealed by fluorescence analyses, Biomedicine 90 (2008) 1674. [8] S.C. Peng, C.Y. Cheng, F. Sheu, C.H. Su, The antimicrobial activity of heyneanol A extracted from the root of Taiwanese wild grape, J. Appl. Mcrobiol. 105 (2008) 485. [9] M.G. Dilshara, K.T. Lee, H.J. Kim, H.J. Lee, Y.H. Choi, C.M. Lee, L.K. Kim, G.Y. Kim, Anti-inflammatory mechanism of ␣-viniferin regulates lipopolysaccharideinduced release of proinflammatory mediators in BV2 microglial cells, Cell. Immunol. 290 (2014) 21.
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Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography Huanhuan Lv a,b , Honglun Wang a,∗ , Yanfeng He a,b , Chenxu Ding a , Xiaoyan Wang a , Yourui Suo a a b
Key Laboratory of Tibetan Medicine Research, Northwest Institution of Plateau Biology, Chinese Academy of Sciences, Xining, 810001, China University of Chinese Academy of Sciences, Beijing 100049, China
a r t i c l e
i n f o
Article history: Received 12 September 2014 Received in revised form 26 January 2015 Accepted 25 February 2015 Available online 3 March 2015 Keywords: HSCCC Iris lactea Oligostilbenes Preparation
a b s t r a c t A method of using high-speed counter-current chromatography (HSCCC) for preparative isolation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea Pall. var. chinensis (Fisch.) Koidz was established in this study. Four oligostilbenes were successfully separated and purified by HSCCC with two sets of two-phase solvent system, n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) in the head-to-tail elution mode for the first separation to mainly isolate vitisin A (58 mg), -viniferin (76 mg) and peak II (43 mg) from 300 mg of the crude ethanol extracts, and then light petroleum–ethyl acetate–methanol–water (5:5:3:6, v/v/v/v) in the tail-to-head elution mode for the second separation to isolate vitisin B (52 mg) and vitisin C (11 mg) from 100 mg of peak II. The purities of the isolated four oligostilbenes were all over 95.0% as determined by HPLC. Vitisin A, vitisin B and vitisin C, resveratrol tetramers, were isolated from Iris lactea for the first time. The preparation of crude sample was simple and the HSCCC method for the isolation and purification of four oligostilbenes was rapid, efficient and economical. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Iris lactea Pall. var. chinensis (Fisch.) Koidz, belongs to the family of Iridaceae, is an herbaceous perennid native to China, Korea, Japan and Russia. It has developed root system, high resistibility, adaptability and saline-tolerance. These futures determine that it is very suitable for the green transformation and the water and soil conservation in the regions with dry climate. The dried seeds of Iris lactea have the effects of clearing heat, eliminating dampness and stanching bleed. It has been used in the treatment of jaundice, diarrhea, vomiting blood, leucorrhea, pharyngitis, and carbuncle swollen in traditional Chinese medicine [1]. The photographs of the flowers, seeds and seed kernels of Iris lactea are shown in Fig. 1. The previous phytochemical studies of Iris species showed the presence of flavones, isoflavones, flavanones, xanthones, triterpenes, quinones, and stilbenes [2–4]. Interestingly, more attention has been focused on the naturally occurring oligostilbenes because these compounds exhibit a
∗ Corresponding author. Tel.: +86 971 6143857. E-mail address: [email protected] (H. Wang). http://dx.doi.org/10.1016/j.jchromb.2015.02.035 1570-0232/© 2015 Elsevier B.V. All rights reserved.
wide range of pharmacological properties, such as neuroprotective, cytotoxic, anti-proliferative, anti-microbial, anti-inflammatory, antioxidant, antitumor and hepatoprotective activities [5–12]. The oligostilbenes are receiving much attention as potential therapeutic agents for several pathological diseases [13]. Before further pharmacological evaluation of the oligostilbenes from Iris lactea, development of an efficient method for the isolation of large quantity of pure oligostilbenes is critical and urgently needed. High-speed counter-current chromatography (HSCCC) is a form of support-free liquid-liquid partition chromatography [14]. Solute separation is based on partitioning between the mobile phase and stationary phase. Without any solid matrix, the stationary phase is retained in the column with the aid of a centrifugal force field, so it eliminates irreversible adsorption of the sample onto solid support and has high sample recovery. Therefore, HSCCC can provide a suitable alternative for the large scale separation of compounds without degrading the active compounds and causing the loss of sample. Due to the high efficiency, HSCCC has been widely used in the separation and purification of a diversity of natural products [15–18]. To the best of our knowledge, there is no published report about the separation and purification of oligostilbenes from Iris lactea with the use of HSCCC technique.
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Fig. 1. The photographs of (1) the flowers, (2) seeds, and (3) seed kernels of Iris lactea.
This study is the first attempt to use the HSCCC technique for the separation and purification of oligostilbenes from the seed kernel of Iris lactea. The objective of this study was to develop an efficient method to separate and purify four oligostilbenes from the ethanol extracts of seed kernel of Iris lactea by HSCCC. The structures of the four isolated oligostilbenes, namely vitisin A, -viniferin, vitisin B and vitisin C are shown in Fig. 2.
2. Experimental 2.1. Materials and reagents All organic solvents used for extraction and HSCCC separation were of analytical grade and purchased from Tianjin Baishi Chemical Co., Ltd. (Tianjin, China). Methanol and acetonitrile used for HPLC analyses were of chromatographic grade (Shandong Yuwang
Fig. 2. Chemical structures of (1) vitisin A, (2) -viniferin, (3) vitisin B and (4) vitisin C.
H. Lv et al. / J. Chromatogr. B 988 (2015) 127–134
Industrial Co., Ltd., Shandong, China). Ultrapure water (18.25 M) and pure water used in the present study were purified on a UPTI-10L system (Chengdu Ultra Pure Technology Co., Ltd., Chengdu, China). The seeds of Iris lactea were collected from Malian Lake of Alxa League of Inner Mongolia, China, in August 2013. 2.2. Apparatus and instruments The HSCCC separation was performed on a TBE-300 C high-speed counter-current chromatography instrument (Tauto Biotechnique Company, Shanghai, China). The apparatus was equipped with three preparative coils connected in series (the diameter of PTFE tube = 1.9 mm, total volume = 320 ml, including the 300 ml separation volume and a 20 ml sample loop). The revolution speed of the instrument was adjustable, ranging from 0 to 1000 rpm. The system was also equipped with a TBP5002 constant flow pump (Tauto Biotechnique Company, Shanghai, China), a model of UV2000D detector (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China), and a DC0506 low constant temperature bath (Tauto Biotechnique Company, Shanghai, China). EasyChrom-1000 chromatography workstation (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China) was employed to record the chromatograms. An Angilent 1260 HPLC system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) was equipped with a quaternary pump (G1311 C), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a diode array detector (G1315D), a Zorbax Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 m), and an Agilent HPLC workstation was used for data acquisition and processing. The nuclear magnetic resonance spectrometer was a Varian INOVA 600 NMR system (Varian, Palo Alto, CA, USA). 2.3. Preparation of the crude sample The dried seeds of Iris lactea were de-coated by a plant disintegrator and the seed kernels were extracted with 85% ethanol (3 × 25 L, each 3 hours) at 60 ◦ C in a HYY25L decocting machine (Huayankorea Machinery and Equipment Co., Ltd., Tianjin, China). The combined filtrate was concentrated to dryness under reduced pressure to afford the ethanol extracts. The extracts were collected as the crude sample for HSCCC separation. 2.4. Measurement of partition coefficient The partition coefficients of the target oligostilbenes in different two-phase solvent systems were determined by HPLC. About 4 ml of each phase of the pre-equilibrated two-phase solvent system was mixed with suitable amount of samples in a test tube. The test tube was shaken vigorously and left to stand at room temperature until the two phases have a clear separation layer. Then the two phases were separated, dried and re-dissolved in methanol, then analyzed by HPLC at 325 nm to obtain the partition coefficients of the target compounds respectively. The partition coefficient is expressed as the peak area of the target compound in the upper phase divided by that of the lower phase. 2.5. Preparation of the two-phase solvent system and sample solution
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lower phase were separated and degassed by sonication for about 20 min prior to use. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v) was used for the first HSCCC separation and light petroleum-ethyl acetate-methanolwater (5:5:3:6, v/v/v/v) for the second separation run. The sample solution for the first HSCCC separation run was prepared by dissolving 300 mg of the crude ethanol extracts in 10 ml lower phase of the solvent system n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v). The eluent of Peak II in the first separation run was manually collected and evaporated to dryness at 60 ◦ C under vacuum. Then the sample solution for the second separation run was prepared by dissolving 100 mg of Peak II in 10 ml of the upper phase of the solvent system light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v). 2.6. HSCCC separation procedure The first HSCCC separation run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-methanolwater at the volume ratio of 3:6:4.2:5.5 in the head-to-tail elution mode (the upper phase as the stationary phase). The coil column was entirely filled with the upper phase and then the apparatus was rotated at 900 rpm. After 30 min, the lower phase was pumped into the column at a flow rate of 1.5 ml/min. When the hydrodynamic equilibrium was established in the column, about 10 ml sample solution containing 300 mg of the crude ethanol extracts was injected through the injection valve. The separation temperature was controlled at 30 ◦ C. The eluents from the outlet of the column were continuously monitored with a UV detector at 325 nm. Each peak fraction was manually collected according to the elution profile and evaporated under vacuum. The eluent of Peak II in the first HSCCC separation is a mixed compound. Therefore, a second HSCCC separation run was employed for the separation of Peak II. Another solvent system composed of light petroleum–ethyl acetate–methanol–water at the volume ratio of 5:5:3:6 in the tail-to-head elution mode (the lower phase as the stationary phase) were employed in the second HSCCC separation. 2.7. HPLC analysis and identification of HCCC peak fractions The HPLC analysis of the ethanol extracts, partition coefficients and each peak fraction obtained from HSCCC were conducted on a reversed-phase Zorbax Eclipse XDB C18 column (4.6 mm × 250 mm, 5 m) with gradient elution throughout this study. The mobile phase was composed of methanol, acetonitrile and water in a gradient elution mode as follows: 0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile. The flow rate was maintained at 1.0 ml/min and the column temperature was set at 30 ◦ C. The detection wavelength was 325 nm. The identifications of the HSCCC peak fractions were carried out by 1 H NMR (600 MHz) and 13 C NMR (600 MHz). The assignments were confirmed by HSQC and HMBC. Deuterated DMSO was used as the solvent and the reference compound tetramethylsilane (TMS) was used as internal standard for the determination of chemical shifts. 3. Results and discussion 3.1. Optimization of HPLC method
The selected two sets of two-phase solvent systems were respectively prepared by adding all the solvents into a separation funnel at selected volume ratios. Each solvent system was thoroughly equilibrated by shaking repeatedly in a separation funnel at room temperature. After being equilibrated, the upper phase and
In the course of optimizing the HPLC conditions, the system conditions including the composition of mobile phase (methanol–water, methanol–acetonitrile–water, and acetonitrile–water), gradient program, column temperature and
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Fig. 3. HPLC chromatograms of (A) the ethanol extracts, (B) the eluent of Peak II and (C–F) the HSCCC peak fractions, i.e. (C): vitisin A, (D): -viniferin, (E): vitisin B, (F): vitisin C. HPLC conditions: Column: Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 m); mobile phase: methanol–acetonitrile–water (0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile); flow rate: 1.0 ml/min; detection wavelength: 325 nm; column temperature: 30 ◦ C.
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Table 1 The partition coefficients (K) of vitisin A, -viniferin, vitisin B and vitisin C in different solvent systems. Solvent systems(v/v)
n-hexane–ethyl acetate–methanol–water
light petroleum–ethyl acetate–methanol–water
K-values
4:6:3:7 4:6:5:5 3:6.4:5 2.5:6:4.5:5 3:6.5:4:5.5 3:6.5:4.2:5.5 3:6.5:4.5:5.7 3:5:3:5 5:5:3:5 5:5:3:7 5:5:3:6
Vitisin A
-viniferin
Vitisin B
Vitisin C
1.27 0.05 0.14 0.82 0.43 0.38 0.26 0.64 0.03 0.64 0.11
6.47 0.18 0.76 3.13 2.16 1.89 1.11 3.13 1.09 3.13 1.34
9.95 0.10 0.51 3.55 2.03 1.47 0.91 3.59 0.62 2.13 0.82
13.12 0.11 0.63 4.42 2.51 1.64 1.08 4.70 0.84 2.99 1.26
3.2. Selection of the two-phase solvent and other conditions of HSCCC
Fig. 4. HSCCC chromatogram of the ethanol extracts. Peak 1: vitisin A; Peak II: mixture of vitisin B and vitisin C; Peak 2: -viniferin. HSCCC conditions: solvent system: n-hexane-ethyl acetate-methanol-water (3:6:4.2:5.5, v/v/v/v); stationary phase: upper phase; mobile phase: lower phase; revolution speed: 900 rpm; separation temperature: 30 ◦ C; flow rate: 1.5 ml/min; sample size: 300 mg ethanol extracts in 10 ml mobile phase; detection wavelength: 325 nm; retention of stationary phase: 68.75%.
detection wavelength were all investigated in this study. The final results showed that the separation with good resolution and short analysis time were achieved when a gradient elution mode composed of methanol, acetonitrile, and water was programmed as follows: 0–20–35 min, 30%–50%–65% methanol; 0–20–35 min, 5%–5%–5% acetonitrile. The flow rate was 1.0 ml/min, the column temperature was set at 30 ◦ C, and 325 nm was selected as the detection wavelength. Spectra were recorded from 190 to 400 nm. Under the optimum gradient elution, the target compounds in the crude ethanol extracts of seed kernels reached almost base-line separation as shown in Fig. 3.
HSCCC is a very useful and efficient technique for the separation and purification of natural products. The selection of the two-phase solvent system is the most important step in HSCCC separation. A successful HSCCC separation is mainly based on choosing a solvent system that can provide suitable partition coefficients (K-values) for the target compounds. In the present study, two sets of twophase solvent system (n-hexane-ethyl acetate-methanol-water and light petroleum-ethyl acetate-methanol-water) at different volume ratios were tested. The K-values of the target compounds were measured and summarized in Table 1. When the volume ratio of n-hexane-ethyl acetate-methanol-water was at 4:6:5:5, it was too hydrophobic because it offered smaller K values for all the four compounds (K1 = 0.05, K2 = 0.18, K3 = 0.10, K4 = 0.11). When the volume ratio was set at 4:6:3:7, the K values of compound 2, 3, and 4 (K2 = 6.47, K3 = 9.95, K4 = 13.12) were too large. Therefore, it is necessary to find a suitable polarity range. Consequently, five other two-phase solvent systems composed of n-hexane-ethyl acetatemethanol-water at different volume ratios were tested. The solvent system of n-hexane-ethyl acetate-methanol-water (3:6.5:4.2:5.5, v/v/v/v) was selected and used to isolate the crude ethanol extracts because it provided suitable range of K-values for the target compounds (K1 = 0.38, K2 = 1.89, K3 = 1.47, K4 = 1.64). The flow rate of the mobile phase, the separation temperature and the revolution speed were also important factors that can affect the efficiency of the whole HSCCC separation. Therefore, the flow rate, the temperature and the revolution speed were also investigated in this study in order to obtain the optimum separation. As reported in literature [19], the small flow rate can produce good
Fig. 5. HSCCC chromatogram of the eluent of Peak II. Peak 3: vitisin B; Peak 4: vitisin C; HSCCC conditions: solvent system: light petroleum-ethyl acetate-methanol-water (5:5:3:6, v/v/v/v); (A) stationary phase: upper phase; mobile phase: lower phase, elution mode: head-to-tail mode; revolution speed: 900 rpm; flow rate: 2.0 ml/min; retention of stationary phase: 69.00%; (B) stationary phase: lower phase; mobile phase: upper phase; elution mode: tail-to-head mode; revolution speed: 800 rpm; flow rate: 3.0 ml/min; retention of stationary phase: 76.67%; other conditions: separation temperature: 30 ◦ C; detection wavelength: 325 nm; sample size: 100 mg Peak II in 10 ml mobile phase.
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separation, but needs long separation time and more mobile phase. The temperature affects the retention of stationary phase, the K values and the mutual solvency of the two phases. The revolution speed mainly has the effects on the retention of stationary phase. Considering these aspects, the flow rate of the mobile phase was selected as 1.5 ml/min, the separation temperature was set at 30 ◦ C, and the revolution speed was of 900 rpm in the first HSCCC separation run. Under the optimum condition, the crude ethanol extracts was separated by HSCCC with the retention of stationary phase of 68.75%. The HSCCC chromatogram is given in Fig. 4. As shown in Fig. 4, compound 1 and compound 2 were successfully isolated and with high purity as determined by HPLC. Restricted by the limited peak capacity of the first HSCCC separation run, the eluent of Peak II is a mixture of compound 3 and 4 as shown in Fig. 3. Compound 3 and compound 4, of similar structure, eluted together as one peak in the first HSCCC separation run. The first HSCCC separation procedure yielded 58 mg compound 1, 76 mg compound 2, and 43 mg peak II from 300 mg crude ethanol extracts. Therefore, a second HSCCC separation run was needed to isolate Peak II. The solvent system composed of light petroleum-ethyl acetatemethanol-water at different volume ratios were tested for mainly separating Peak II. When the volume ratio was changed to 5:5:3:6, the appropriate K values for compound 3 (K3 = 0.82) and compound 4 (K4 = 1.26) were obtained. As seen in Fig. 5A, the two compounds were not completely separated in the head-to-tail elution mode. While in Fig. 5B, the results indicated that it gave much better separation for compound 3 and compound 4 in a tail-to-head mode. Finally, the solvent system composed of light petroleumethyl acetate-methanol-water at a volume ratio of 5:5:3:6 in the tail-to-head elution mode with the flow rate of mobile phase of 3.0 ml/min, the revolution speed of 800 rpm, and separation temperature of 30 ◦ C were used to separate peak II after the first HSCCC separation step. The second HSCCC separation procedure yielded 52 mg compound 3 and 11 mg compound 4 from 100 mg peak II. In addition, the purities of the four isolated compounds were 98.23%, 99.05%, 97.00% and 95.34%, respectively as determined by HPLC (Fig. 3). Their UV spectra are shown in Fig. 6. 3.3. The structural identification The structures of the four compounds isolated by HSCCC were identified by different spectroscopic analysis, including the use of UV spectra, 1D (1 H NMR and 13 C NMR) and 2D NMR technique (notably 1 H–1 H COSY, HMBC and HSQC spectra), and by comparing the NMR data to the values in published literature. The 1 H NMR spectrum of compound 1 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.12 (2H, d, J = 8.4 Hz) and ␦ 6.75 (2H, d, J = 8.4 Hz), ␦ 7.02 (2H, d, J = 8.4 Hz) and ␦ 6.68 (2H, d, J = 8.4 Hz), ␦ 6.87 (2H, d, J = 8.4 Hz) and ␦ 6.57 (2H, d, J = 8.4 Hz); one
3,5-dihydroxybenzene moiety at ␦ 6.01 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, d, J = 2.2 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.30 (1H, d, J = 6.2 Hz) and ␦ 4.34 (1H, d, J = 5.2 Hz), ␦ 5.10 (1H, d, J = 4.2 Hz) and ␦ 5.25 (1H, d, J = 4.2 Hz), ␦ 5.79 (1H, d, J = 11.7 Hz) and ␦ 4.01 (1H, d, J = 11.7 Hz); two trans olefinic protons at ␦ 6.25 (1H, d, J = 16.2 Hz) and ␦ 6.27 (1H, d, J = 16.2 Hz); nine aromatic protons at ␦ 6.17 (1H, d, J = 2.2 Hz) and ␦ 6.39 (1H, d, J = 2.2 Hz), ␦ 5.98 (1H, d, J = 2.2 Hz) and ␦ 5.89 (1H, d, J = 2.2 Hz), ␦ 5.95 (1H, d, J = 2.2 Hz) and ␦ 5.99 (1H, d, J = 2.2 Hz), ␦ 5.85 (1H, d, J = 2.0 Hz), ␦ 6.68 (1H, d, J = 8.4 Hz) and ␦ 6.76 (1H, dd, J = 2.2, 1.8 Hz). The structure of compound 1 was in agreement with that of the reported vitisin A, which was previously isolated from Vitis coignetiae [20]. Compared the 1 H NMR and 13 C NMR data with the literature values, compound 2 was identified as -viniferin [21]. The 1 H NMR spectrum exhibited signals for two 4-hydroxybenzene moieties at ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.73 (2H, d, J = 8.4 Hz), ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.65 (2H, d, J = 8.4 Hz); one 3,5-dihydroxybenzene moieties at ␦ 6.03 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, t, J = 2.2 Hz); two aliphatic protons of a dihydrobenzofuran ring at ␦ 5.32 (1H, d, J = 5.2 Hz) and ␦ 4.40 (1H, d, J = 5.2 Hz); two trans olefinic protons at ␦ 6.81 (1H, d, J = 16.2 Hz) and ␦ 6.56 (1H, d, J = 16.2 Hz); two aromatic protons at ␦ 6.58 (1H, d, J = 2.2 Hz) and ␦ 6.22 (1H, d, J = 2.2 Hz). The 1 H NMR spectrum of compound 3 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.18 (2H, d, J = 8.3 Hz) and ␦ 6.78 (2H, d, J = 8.1 Hz), ␦ 6.45 (2H, d, J = 8.5 Hz) and ␦ 6.47 (2H, d, J = 8.5 Hz), ␦ 7.10 (2H, d, J = 8.3 Hz) and ␦ 6.72 (2H, d, J = 8.3 Hz); two 3,5-dihydroxybenzene moieties at ␦ 6.02 (2H, d, J = 2.2 Hz) and ␦ 6.04 (1H, t, J = 2.2 Hz), ␦ 5.97 (2H, d, J = 2.2 Hz) and ␦ 6.00 (1H, t, J = 2.2 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.31 (1H, d, J = 4.9 Hz) and ␦ 4.49 (1H, d, J = 4.9 Hz), ␦ 5.43 (1H, d, J = 5.2 Hz) and ␦ 4.11 (1H, d, J = 5.2 Hz), ␦ 5.27 (1H, d, J = 5.5 Hz) and ␦ 4.37 (1H, d, J = 5.5 Hz); two trans olefinic protons at ␦ 6.52 (1H, d, J = 16.1 Hz) and ␦ 6.76 (1H, d, J = 16.1 Hz); seven aromatic protons at ␦ 6.64 (1H, d, J = 2.2 Hz), ␦ 6.81 (1H, d, J = 2.2 Hz), ␦ 6.93 (1H, dd, J = 2.2 Hz), ␦ 6.22 (1H, d, J = 2.2 Hz), ␦ 6.54 (1H, d, J = 2.2 Hz), ␦ 6.17 (1H, d, J = 2.2 Hz) and ␦ 5.97 (1H, d, J = 2.2 Hz). These data are in accordance with those reported in published literature [22], compound 3 was identified as vitisin B, which was once isolated from Vitis vinifera ‘Kyohou’. The 1 H NMR spectrum of compound 4 exhibited signals for three 4-hydroxybenzene moieties at ␦ 7.11 (2H, d, J = 8.2 Hz) and ␦ 6.97 (2H, d, J = 8.3 Hz), ␦ 6.98 (2H, d, J = 8.4 Hz) and ␦ 6.74 (2H, d, J = 8.4 Hz), ␦ 7.11 (2H, d, J = 8.2 Hz) and ␦ 6.74 (2H, d, J = 8.4 Hz); two 3,5-dihydroxybenzene moieties at ␦ 5.81 (2H, d, J = 2.2 Hz) and ␦ 6.00 (1H, t, J = 2.2 Hz), ␦ 7.11 (2H, d, J = 8.4 Hz) and ␦ 6.65 (1H, t, J = 8.4 Hz); six aliphatic protons of a dihydrobenzofuran ring at ␦ 5.26 (1H, d, J = 4.2 Hz) and ␦ 4.45 (1H, d, J = 4.2 Hz), ␦ 5.30 (1H, d, J = 8.9 Hz) and ␦ 3.67 (1H, d, J = 8.9 Hz), ␦ 5.12 (1H, d, J = 5.0 Hz) and ␦ 4.17 (1H, d, J = 5.0 Hz); two trans olefinic protons at ␦ 6.68 (1H,
Fig. 6. UV spectra of the HSCCC peak fractions. (A): vitisin A, (B): -viniferin, (C): vitisin B, (D): vitisin C.
H. Lv et al. / J. Chromatogr. B 988 (2015) 127–134 Table 2 13 C NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C in DMSO-d6. Position 1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c 12c 13c 14c 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d
Vitisin A C-NMR
13
131.7 127.2 115.2 157.3 115.2 127.2 92.3 55.1 145.7 105.5 158.6 101.0 158.6 105.5 127.5 131.3 131.5 154.4 114.6 126.7 122.0 129.3 134.7 118.1 160. 7 95.8 158.4 102.9 129.1 128.9 115.2 157.6 115.2 128.9 40.0 40.1 140.5 121.1 156.8 95.1 155.7 109.0 133.6 127.2 115.1 154.8 115.1 127.2 39.9 48.0 139.9 118.5 157.6 99.8 158.6 103,3
-viniferin 13
C-NMR
131.7 127.0 115.2 157.3 115.2 127.0 92.3 55.1 146.0 105.5 158.6 101.1 158.6 105.5 128.0 127.8 115.5 157.3 115.5 127.7 128.9 122.0 134.8 118.4 160.7 95.9 158.5 103.1
Table 3 1 H NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C in DMSO-d6.
Vitisin B C-NMR
Vitisin C 13 C-NMR
position
131.7 127.4 115.4 157.3 115.4 127.4 92.4 54.7 145.7 105.5 159.1 101.3 159.1 105.5 131.4 125.0 130.5 158. 5 109.9 125.2 122.9 128.9 134.7 118.5 160.7 96.1 158.2 103.4 131.4 126.2 114.9 156.7 114.9 126.2 89.9 50.4 141.0 118.2 160.6 95.5 159.1 105.6 131.6 127.1 115.3 157.4 115.3 127.1 93.0 55.0 146.0 105.6 158.7 101.7 158.7 105.6
131.8 127.4 115.1 157.2 115.1 127.4 92.5 54.0 145.6 105.1 158.8 101.2 158.8 105.1 130.5 124.5 129.6 157.5 109.5 125.6 123.2 128.9 134.7 119.7 160.8 96.1 159.2 103.7 130.5 127.0 115.2 159.2 115.2 127.0 92.3 52.6 139.3 118.3 160.4 95.7 158.5 106.4 131.3 127.4 115.2 157.4 115.2 127.4 92.9 54.8 146.2 105.4 158.6 101.0 158.6 105.4
1a 2a 3a 4a 5a 6a 7a 8a 9a 10a 11a 12a 13a 14a 1b 2b 3b 4b 5b 6b 7b 8b 9b 10b 11b 12b 13b 14b 1c 2c 3c 4c 5c 6c 7c 8c 9c 10c 11c 12c 13c 14c 1d 2d 3d 4d 5d 6d 7d 8d 9d 10d 11d 12d 13d 14d
13
133
Vitisin A 1 H-NMR
-viniferin
7.12(d,8.4) 6.75(d,8.4)
Vitisin B 1 H -NMR
Vitisin C 1 H -NMR
7.11(d,8.4) 6.73(d,8.4)
7.18(d,8.3) 6.78(d,8.1)
7.11(d,8.2) 6.97(d,8.3)
6.75(d,8.4) 7.12(d,8.4) 5.30(d,5.2) 4.34(d,5.2)
6.73(d,8.4) 7.11(d,8.4) 5.32(d,5.2) 4.40(d,5.2)
6.78(d,8.1) 7.18(d,8.3) 5.31(d,4.9) 4.49(d,4.9)
6.97(d,8.3) 7.11(d,8.2) 5.26(d,4.2) 4.45(d,4.2)
6.01(d,2.2)
6.04(d,2.2)
6.02(d,2.2)
5.81(d,2.2)
6.04(t,2.2)
6.04(t,2.2)
6.04(t,2.2)
6.00(t,2.2)
6.01(d,2.2)
6.04(d,2.2)
6.02(d,2.2)
5.81(d,2.2)
5.85(d, 2.0)
7.11(d,8.4) 6.65(d,8.4)
6.64(d,2.2)
6.68(d,2.2)
6.68(d,8.4) 6.76(dd, 8.4,1.8) 6.25(d, 16.2) 6.27(d, 16.2)
6.65(d,8.4) 7.11(d,8.4) 6.81(d,16.2) 6.56(d,16.2)
6.81(d,8.4) 6.93(dd,8.4,2.0) 6.52(d,16.1) 6.76(d,16.1)
6.73(d,8.3) 6.90(dd,8.4,2.0) 6.58(d,16.1) 6.83(d,16.1)
6.17(d,2.2)
6.22(d, 2.2)
6.22(d,2.2)
6.23(d,2.2)
6.39(d,2.2)
6.58(d,2.2)
6.54(d,2.2)
6.54(d,2.2)
7.02(d,8.4) 6.68(d,8.4)
6.45(d,8.5) 6.47(d,8.5)
6.98(d,8.4) 6.74(d,8.4)
6.68(d,8.4) 7.02(d,8.4) 5.10(d,4.2) 5.25(d,4.2)
6.47(d,8.5) 6.45(d,8.5) 5.43(d,5.2) 4.11(d,5.2)
6.74(d,8.4) 6.98(d,8.4) 5.30(d,8.9) 3.67(d,8.9)
5.98(d,2.2)
6.17(d,2.2)
6.14(d,2.2)
5.89(d,2.2)
5.97(d,2.2)
6.06(d,2.2)
6.87(d,8.4) 6.57(d,8.4)
7.10(d,8.5) 6.72(d,8.3)
7.11(d,8.2) 6.74(d,8.4)
6.57(d,8.4) 6.87(d,8.4) 5.79(d,11.7) 4.01(d,11.7)
6.72(d,8.3) 7.10(d,8.5) 5.27(d,5.5) 4.37(d,5.5)
6.74(d,8.4) 7.11(d,8.2) 5.12(d,5.0) 4.17(d,5.0)
5.97(d,2.2)
6.02(d,2.2)
5.95(d,2.2)
6.00(d,2.2)
6.03(d.2.2)
5.99(d, 2.2)
5.97(d,2.2)
6.02(d,2.2)
1
H-NMR
4. Conclusion d, J = 16.1 Hz) and ␦ 6.83 (1H, d, J = 16.1 Hz); seven aromatic protons at ␦ 6.68 (1H, d, J = 2.2 Hz), ␦ 6.73 (1H, d, J = 2.2 Hz), ␦ 6.90 (1H, dd, J = 8.4, 2.0 Hz), ␦ 6.23 (1H, d, J = 2.2 Hz), ␦ 6.54 (1H, d, J = 2.2 Hz), ␦ 6.14 (1H, d, J = 2.2 Hz) and ␦ 6.06 (1H, d, J = 2.2 Hz). Compared with the given data in literature [23], compound 4 was identified as vitisin C, which was also isolated from Vitis vinifera ‘Kyohou’. The 1 H NMR features of compound 4 were similar to those of vitisin B. Vitisin C was a stereoisomer of vitisin B at the position of C8c. The detailed 13 C NMR and 1 H NMR spectral data of vitisin A, -viniferin, vitisin B and vitisin C are listed in Tables 2 and 3, respectively.
The present study demonstrated that the two-step HSCCC separation is an effective method which is suitable for the preparation and purification of oligostilbenes from the ethanol extracts of seed kernel of Iris lactea. The whole process yielded four oligostilbenes, namely vitisin A, -viniferin, vitisin B and vitisin C at high purity. Vitisin A, vitisin B and vitisin C were all resveratrol tetramers and isolated from Iris lactea for the first time, among them, vitisin B and vitisin C are epimerides. In general, both the extraction and separation procedure are eco-friendly and simple approaches. Therefore, these methods can be applied to large-scale extraction and largequantity preparation of oligostilbenes from Iris lactea.
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