Abstract
Naturally occurring oligostilbenes are receiving more attention because they exhibit several beneficial effects for health, including hepatoprotective, antitumor, anti-adipogenic, antioxidant, antiaging, anti-inflammatory, anti-microbial, antiviral, immunosuppressive and neuroprotective activities. Thus, they could be of some potentially therapeutic values for several diseases. In this study, we adopted the alkaline extraction–acid precipitation (AEAP) method for extraction of oligostilbenes from the seed kernel of Iris lactea. Then, the high-speed counter-current chromatography (HSCCC) was used for preparative isolation and purification of oligostilbenes from the AEAP extracts. Finally, three oligostilbenes, namely vitisin D (73 mg), ampelopsin B (25 mg) and cis-vitisin A (16 mg), were successfully fractionated by HSCCC with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (2:5:3:6, v/v/v/v) from 300 mg of the AEAP extracts in ∼190 min. The purities of the three isolated oligostilbenes were all over 95.0% as analyzed by high performance liquid chromatography. They all were isolated from I. lactea for the first time. The method of AEAP for the preparation of the oligostilbene-enriched crude sample was simple, and the HSCCC technique for the isolation and purification of oligostilbenes was efficient.
Introduction
Oligostilbenes, well-known polyphenols, have been found in some plant families, such as Leguminosae, Vitaceae, Dipterocarpaceae, Paeoniaceae, Cyperaceae, Gnetaceae and Iridaceae (1–7). Most phytochemical studies on oligostilbenes composition have been sufficiently made on the grapevines, and the structures are well-characterized by various spectroscopic methods (8–10). It has been previously reported that the oligostilbenes could cure hepatotoxin, enhance osteoblast proliferation and chemoprevent cancer (11–13). In addition, they also exhibit a wide range of pharmacological activities, including anti-adipogenic, antioxidant, antiaging, anti-inflammatory, antimicrobial, antiviral, neuroprotective and immunosuppressive (14–21). Oligostilbenes are naturally occurring with a very small amount in plants and not commercially available. As a consequence, they have been studied much less than the monomer.
In most of the published reports, the preparation process regarding the extraction and isolation of oligostilbenes is described as follows: (i) first, the plant material is extracted with ethanol, methanol or acetone and the combined extracts are concentrated to get the crude residue; (ii) then the crude sample is suspended to water and successively partitioned with different solvents (n-hexane, light petroleum, ethyl acetate or n-butyl alcohol) to afford the solvent-soluble fractions; (iii) the extracted fractions are further repeatedly subjected to chromatographies on Sephadex LH-20, silica or reverse-phase C18 to obtain pure oligostilbene compounds. These traditional methods are widely applied to the separation of various kinds of natural products. Recently, several literature studies reported the separation of stilbenes by high-speed counter-current chromatography (HSCCC) (22, 23). HSCCC is a form of liquid–liquid partition chromatography without the solid matrix. The advantages of HSCCC over the conventional separation technique are time-saving, simple process and high recovery.
Iris lactea Pall. var. chinensis (Fisch.) Koidz belongs to the family of Iridaceae. The dried seeds have been used as a folk medicine for the treatment of jaundice, diarrhea, leucorrhea, pharyngitis, inflammation and carbuncle swollen in traditional Chinese medicine. In the course of biochemical studies on I. lactea, four oligostilbenes, vitisin A, ɛ-viniferin, vitisin B and vitisin C, were separated by HSCCC from the crude ethanol extracts of the seed kernels (24).
In this study, we again focused on the oligostilbene composition presented in the seed kernel of I. lactea and wanted to develop a more practical process than the current method to isolate oligostilbene compounds. We attempted to use the alkaline extraction–acid precipitation (AEAP) method to extract oligostilbenes present in the seed kernels. And then, the HSCCC technique was applied for the separation and purification of oligostilbenes from the crude AEAP sample. The structures of the three isolated oligostilbenes, namely vitisin D, ampelopsin B and cis-vitisin A, are shown in Figure 1.
Figure 1.
Chemical structures of (1) vitisin D, (2) ampelopsin B and (3) cis-vitisin A.
Instruments and reagents
The HSCCC separation was performed on a TBE-300C 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 1,000 rpm. The system was also equipped with a TBP5002 constant flow pump (Tauto Biotechnique Company), a model of UV2000D detector (Shanghai Sanotac Scientific Instrument Co., Ltd., Shanghai, China) and a DC0506 low constant temperature bath (Tauto Biotechnique Company). EasyChrom-1000 chromatography workstation (Shanghai Sanotac Scientific Instrument Co., Ltd.) was employed to record the chromatograms.
An Agilent 1260 HPLC system (Agilent Technologies Co., Ltd., Santa Clara, CA, USA) was equipped with a quaternary pump (G1311C), an auto-sampler (G1329B), a thermostated column compartment (G1316A), a diode array detector (G1315D) and a Zorbax Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 μm), 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).
All organic solvents used for extraction and HSCCC separation were of analytical grade and purchased from Tianjin Baishi Chemical Co., Ltd. (Tianjin, China). HCl and NaOH used for extraction were of analytical grade. Methanol used for HPLC analyses was of chromatographic grade (Shandong Yuwang Industrial Co., Ltd., Shandong, China). Ultrapure water (18.25 MΩ) used in this study was purified on a UPT-I-10L system (Chengdu Ultra Pure Technology Co., Ltd., Chengdu, China). The seeds of I. lactea were collected from Malian Lake of Alxa League of Inner Mongolia, China, in August 2013.
Experimental and methods
Preparation of the AEAP sample
The collected seeds of I. lactea were cleaned with water and dried in shade. The dried seeds were de-coated by a plant disintegrator, and the seed kernels were macerated with 2.0% NaOH aqueous solution at room temperature for 12 h. The extracts were filtrated and combined as the alkali-extracted solution. Then, the HCl solution was drop-wisely added to the alkali-extracted solution at room temperature with vigorous stirring to pH 3.0 and left to stand at room temperature until the precipitation process was completed. After centrifuged at 4,500 g for 10 min, the precipitations were separated from the supernatant and re-dissolved with ethanol to remove other impurities. The ethanol-soluble solution were collected and dried under reduced pressure to get the AEAP sample for the subsequent HSCCC separation.
Measurement of partition coefficients
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 the sample 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. The two phases were separated, dried and re-dissolved in methanol, then analyzed by HPLC at 280 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.
Preparation of the two-phase solvent system and sample solution
The two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (2:5:3:6, v/v/v/v) was prepared by adding all the solvents into a separation funnel at selected volume ratios. The solvent system was thoroughly equilibrated by shaking in a separation funnel at room temperature. After being equilibrated, the upper phase and lower phase were separated and degassed by sonication for ∼20 min prior to use.
The sample solution for the HSCCC separation was prepared as follows: 300 mg of the AEAP sample was dissolved in 10 mL of the lower phase of the solvent system n-hexane–ethyl acetate–methanol–water (2:5:3:6, v/v/v/v).
HSCCC separation procedure
The HSCCC separation was carried out with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water at the volume ratio of 2:5:3:6. 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 2.8 mL/min. When the hydrodynamic equilibrium was established in the column, ∼10 mL of the sample solution containing 300 mg of the AEAP sample 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 280 and 325 nm. Each peak fraction was manually gathered according to the chromatographic peak profile and evaporated under vacuum.
HPLC analyses and identification of HSCCC peak fractions
The HPLC analyses of the AEAP sample, 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 μm) with gradient elution throughout this study. The mobile phase was composed of methanol and water in a gradient elution mode as follows: 0–40 min, 40–70% methanol. The flow rate was maintained at 1.0 mL/min, and the column temperature was set at 30°C. The detection wavelength was 280 nm.
The identifications of the HSCCC peak fractions were carried out by 1H NMR (600 MHz), 13C NMR (150 MHz) and by comparing the NMR data to the values in the published literature.
Results
The extraction of oligostilbenes by AEAP
In this study, the seed kernels were macerated with 2.0% NaOH aqueous solution to get the alkali-extracted solution, and the HCl solution was added to the alkali-extracted solution to pH 3.0. Subsequently, the ethanol was used to partly remove the impurities. The ethanol-soluble solution was collected as the AEAP sample for HSCCC separation.
HPLC analysis of the AEAP extracts
When a gradient elution mode composed of methanol and water (0–40 min, 30–70% methanol), the separation with the best resolution and shortest analysis time was achieved. The flow rate was 1.0 mL/min, the column temperature was set at 30°C and 280 nm was selected as the detection wavelength. Under the selected condition, the three target compounds almost reached base-line separation as shown in Figure 2 and have a similar type of UV spectra with maximum absorbance at 280 nm.
Figure 2.
HPLC chromatograms of the AEAP sample (1) and HSCCC peak fractions (2–4). (2) Vitisin D, (3) ampelopsin B and (4) cis-vitisin A. HPLC conditions: column: Eclipse XDB-C18 analytical column (4.6 mm × 250 mm, 5 μm); mobile phase: methanol–water (0–40 min, 40–70% methanol); flow rate: 1.0 mL/min; detection wavelength: 280 nm. This figure is available in black and white in print and in color at JCS online.
The separation of three oligostilbenes by HSCCC
The separation of the crude AEAP sample was achieved with good resolution, acceptable separation time, and the solvent system possessed 65.23% of the retention rate of the stationary phase with the flow rate of the mobile phase of 2.8 mL/min, the separation temperature of 30°C and the revolution speed of 900 rpm (Figure 3). The whole HSCCC separation procedure yielded 73 mg of Compound 1 (collected from 63 to 76 min), 25 mg of Compound 2 (collected from 171 to 189 min) and 16 mg of Compound 3 (collected from 126 to 141 min), at purities of 97.23, 99.05 and 96.34% from 300 mg of the crude AEAP sample in ∼190 min.
Figure 3.
HSCCC chromatogram of the AEAP sample. HSCCC conditions: solvent system: n-hexane–ethyl acetate–methanol–water (2:5:3:6, v/v/v/v); revolution speed: 900 rpm; flow rate: 2.8 mL/min; separation temperature: 30°C; sample size: 300 mg of the AEAP sample in 10 mL lower phase; detection wavelength: 280 and 325 nm; retention of stationary phase: 65.23%. This figure is available in black and white in print and in color at JCS online.
Structure identification
The structure identifications of the HSCCC peak fractions were performed by 1H NMR (600 MHz) and 13C NMR (150 MHz) and by comparison with the published data. 13C NMR and 1H NMR data of Compounds 1, 2 and 3 are shown in Tables I and II, respectively. Compound 1 was identified as vitisin D by comparison with the 1H NMR and 13C NMR data given in the literature (25). Comparison with the spectroscopic data given in the literature, Compound 2 was allowed to be identified as ampelopsin B (26). In addition, Compound 3 was identified as cis-vitisin A in agreement with the reported data (27).
Table I.
13C NMR Spectral Data of vitisin D, ampelopsin B and cis-vitisin A in CD3OD (150 MHz)
| Position | Vitisin D | Ampelopsin B | cis-Vitisin A |
|---|---|---|---|
| 1a | 131.7 | 131.1 | 133.2 |
| 2a | 130.5 | 130.1 | 128.2 |
| 3a | 116.1 | 116.1 | 116.1 |
| 4a | 158.3 | 158.8 | 158.4 |
| 5a | 116.1 | 116.1 | 116.1 |
| 6a | 130.5 | 130.1 | 128.2 |
| 7a | 89.2 | 89.0 | 94.7 |
| 8a | 48.7 | 48.6 | 57.4 |
| 9a | 141.7 | 142.8 | 147.4 |
| 10a | 120.96 | 119.6 | 107.2 |
| 11a | 157.6 | 158.7 | 159.6 |
| 12a | 101.6 | 101.5 | 101.9 |
| 13a | 156.1 | 157.0 | 159.6 |
| 14a | 106.5 | 105.3 | 107.2 |
| 1b | 136.2 | 135.5 | 128.4 |
| 2b | 129.3 | 128.9 | 132.8 |
| 3b | 115.3 | 115.6 | 132.5 |
| 4b | 155.6 | 155.9 | 154.9 |
| 5b | 115.3 | 115.6 | 114.7 |
| 6b | 129.3 | 128.9 | 127.7 |
| 7b | 40.5 | 34.0 | 125.5 |
| 8b | 43.8 | 36.3 | 131.1 |
| 9b | 141.4 | 138.6 | 136.1 |
| 10b | 121.00 | 123.6 | 120.5 |
| 11b | 156.7 | 157.1 | 162.4 |
| 12b | 96.2 | 95.7 | 96.4 |
| 13b | 160.1 | 160.5 | 158.8 |
| 14b | 110.5 | 109.2 | 109.5 |
| 1c | 134.6 | 130.3 | |
| 2c | 131.3 | 129.2 | |
| 3c | 132.7 | 116.1 | |
| 4c | 153.2 | 158.7 | |
| 5c | 114.2 | 116.1 | |
| 6c | 125.1 | 129.2 | |
| 7c | 36.2 | 89.0 | |
| 8c | 33.5 | 49.7 | |
| 9c | 138.3 | 142.5 | |
| 10c | 119.6 | 121.2 | |
| 11c | 158.1 | 158.8 | |
| 12c | 95.9 | 101.1 | |
| 13c | 160.1 | 156.8 | |
| 14c | 108.8 | 104.9 | |
| 1d | 131.4 | 134.0 | |
| 2d | 130.6 | 129.0 | |
| 3d | 116.3 | 115.5 | |
| 4d | 158.6 | 155.9 | |
| 5d | 116.3 | 115.5 | |
| 6d | 130.6 | 129.0 | |
| 7d | 89.0 | 41.2 | |
| 8d | 48.6 | 41.9 | |
| 9d | 143.1 | 141.7 | |
| 10d | 123.3 | 121.1 | |
| 11d | 158.7 | 158.3 | |
| 12d | 101.1 | 96.3 | |
| 13d | 156.6 | 160.3 | |
| 14d | 105.1 | 110.7 |
Table II.
1H NMR Spectral Data of vitisin D, ampelopsin B and cis-vitisin A in CD3OD (600 MHz)
| Position | Vitisin D | Ampelopsin B | cis-Vitisin A |
|---|---|---|---|
| 1a | |||
| 2a | 7.14 (d, J = 8.4Hz) | 7.04 (d, J = 8.5 Hz) | 7.12 (d, J = 8.8 Hz) |
| 3a | 6.74 (d, J = 8.4 Hz) | 6.71 (d, J = 8.5 Hz) | 6.99 (d, J = 8.8 Hz) |
| 4a | |||
| 5a | 6.74 (d, J = 8.4 Hz) | 6.71 (d, J = 8.5 Hz) | 6.99 (d, J = 8.8 Hz) |
| 6a | 7.14 (d, J = 8.4 Hz) | 7.04 (d, J = 8.5 Hz) | 7.12 (d, J = 8.8 Hz) |
| 7a | 5.75 (d, J = 11.6 Hz) | 5.68 (d, J = 11.5 Hz) | 5.21 (d, J = 5.1 Hz) |
| 8a | 4.01 (d, J = 11.9 Hz) | 4.07 (d, J = 11.5 Hz) | 3.94 (d, J = 5.1 Hz) |
| 9a | |||
| 10a | 6.00 (d, J = 2.2 Hz) | ||
| 11a | |||
| 12a | 5.86 (br s) | 6.32 (d, J = 2.0 Hz) | 6.18 (d, J = 2.2 Hz) |
| 13a | |||
| 14a | 5.87 (br s) | 6.11 (d, J = 2.0 Hz) | 6.00 (t, J = 2.2 Hz) |
| 1b | |||
| 2b | 6.97 (d, J = 8.4 Hz) | 6.92 (d, J = 8.5 Hz) | 5.93 (d, J = 2.2 Hz) |
| 3b | 6.62 (d, J = 8.4 Hz) | 6.63 (d, J = 8.5 Hz) | |
| 4b | |||
| 5b | 6.62 (d, J = 8.4 Hz) | 6.63 (d, J = 8.5 Hz) | 6.44 (d, J = 8.1 Hz) |
| 6b | 6.97 (d, J = 8.4 Hz) | 6.92 (d, J = 8.5 Hz) | 6.68 (dd, J = 8.1, 2.2 Hz) |
| 7b | 5.59 (d, J = 3.7 Hz) | 5.16 (t, J = 4.0 Hz) | 5.78 (br s) |
| 8b | 5.27 (d, J = 3.7 Hz) | 3.19 (dd, J = 18.0, 4.0 Hz) | 5.78 (br s) |
| 3.55 (dd, J = 18.0, 4.0 Hz) | |||
| 9b | |||
| 10b | |||
| 11b | |||
| 12b | 6.02 (br s) | 6.04 (d, J = 2.0 Hz) | 6.28 (d, J = 2.2 Hz) |
| 13b | |||
| 14b | 5.77 (br s) | 6.29 (d, J = 2.0 Hz) | 6.17 (d, J = 2.2 Hz) |
| 1c | |||
| 2c | 5.90 (br s) | 7.05 (d, J = 8.8 Hz) | |
| 3c | 6.77 (d, J = 8.8 Hz) | ||
| 4c | |||
| 5c | 6.52 (d, J = 7.4 Hz) | 6.77 (d, J = 8.8 Hz) | |
| 6c | 6.53 (br d, J = 7.4 Hz) | 7.05 (d, J = 8.8 Hz) | |
| 7c | 4.66 (br t, J = 4.5 Hz) | 5.84 (d, J = 11.7 Hz) | |
| 8c | 2.93 (br d, J = 16.6 Hz) | 4.22 (d, J = 11.7 Hz) | |
| 3.14 (br d, J = 16.6 Hz) | |||
| 9c | |||
| 10c | |||
| 11c | |||
| 12c | 6.06 (br s) | 6.06 (d, J = 2.2 Hz) | |
| 13c | |||
| 14c | 6.06 (br s) | 6.18 (d, J = 2.2 Hz) | |
| 1d | |||
| 2d | 7.09 (d, J = 8.4 Hz) | 7.03 (d, J = 8.1 Hz) | |
| 3d | 6.86 (d, J = 8.4 Hz) | 6.60 (d, J = 8.1 Hz) | |
| 4d | |||
| 5d | 6.86 (d, J = 8.4 Hz) | 6.60 (d, J = 8.1 Hz) | |
| 6d | 7.09 (d, J = 8.4 Hz) | 7.03 (d, J = 8.1 Hz) | |
| 7d | 5.58 (d, J = 11.0 Hz) | 5.52 (d, J = 3.7 Hz) | |
| 8d | 3.39 (d, J = 11.1 Hz) | 5.40 (d, J = 3.7 Hz) | |
| 9d | |||
| 10d | |||
| 11d | |||
| 12d | 6.19 (br s) | 6.02 (d, J = 2.2 Hz) | |
| 13d | |||
| 14d | 5.99 (br s) | 6.05 (d, J = 2.2 Hz) |
Discussion
Selection of extraction methods
In most literature studies, the preferred method for extraction of oligostilbenes from plant materials was organic solvent extraction, which consists of different selective solvents, such as ethanol, methanol or acetone. After yielding the crude extracts, successive partition and repeat column chromatographies on silica, Sephadex LH-20 or reverse-phase C18 are always needed in order to get the pure oligostilbene compounds. However, the tedious and repetitious processes of organic solvent extraction and chromatographic isolation greatly increase the costs of oligostilbenes production. AEAP as a classic method has been used in the extraction of lignins, hemicelluloses and phenolic compounds (28–30). The AEAP method presents many properties: environmentally friendly, easy operation, cost saving and product safety. Oligostilbenes, having phenolic hydroxyl groups in the structure, exhibit certain weak acidity. When combined with alkali, the water solubility of the oligostilbenes can be improved and making them insoluble in acidic solution. Then with acid treatment, the insolubles can be precipitated. According to the distinct features of polyphenols, the AEAP method for the extraction of oligostilbenes from I. lactea was developed in this study. As described in method part of Preparation of the AEAP sample a specific pH value would be of much importance for acid precipitation. After a series of primary experiments have been conducted, pH 3.0 was finally selected because of the amount of precipitates was mostly generated. Subsequently, we used the ethanol to re-dissolve the precipitations and the ethanol-soluble solution was collected in order to enrich the oligostilbenes to get the AEAP sample. It was no need to further partition the AEAP sample by different organic solvents as in the reported methods (1, 2). Therefore, the AEAP sample could be directly used as a crude sample for the subsequent HSCCC separation without further treatment. Consequently, the AEAP method for extraction of oligostilbenes from I. lactea was a much simpler process.
Optimization of HPLC conditions
Analyses of the crude sample, selection of the two-phase solvent system and determination of the purities of the HSCCC fractions were all performed by HPLC. Thus, it was important to firstly establish a proper HPLC analyses method. In the course of optimizing the HPLC conditions, the composition of mobile phase, gradient program, column temperature and detection wavelength were all investigated in this study. Under the selected conditions, the three target compounds almost reached base-line separation. From Figure 2, it can be seen that the three target compounds have a similar type of UV spectra with maximum absorbance at 280 nm. The formerly isolated four oligostilbenes (vitisin A, ɛ-veniferin, vitisn B and vitisin C) have the maximum absorbance at 325 nm, thus dual-wavelength detection was adopted in the later HSCCC separation.
Selection of solvent system and other HSCCC conditions
In recent years, HSCCC has been widely used for the separation of various natural products. Selection of a suitable two-phase solvent system was the most important part for a successful HSCCC separation (31). The partition coefficients (K-values) of the target compounds were obtained and analyzed by the liquid–liquid extraction experiments and HPLC. K-values of the target compounds should be in the range of 0.5–2.0 and the separation factor between two compounds ought to be >1.5 (α = K2/K1, K2 > K1). The settling time of the solvent system should be no more than 30 s. According to the polarity of the target compounds, n-hexane–ethyl acetate–methanol–water at the volume ratios of 3:5:3:5, 3:5:3:7 and 2:5:3:6, , respectively, were tested. As shown in Table III, the solvent system composed of n-hexane–ethyl acetate–methanol–water at the volume ratio of 3:5:3:5 provided K1= 0.12, K2= 1.01 and K3= 0.46 for the three target compounds. Compounds 1 and 3 would be eluted out with the solvent front as for the smaller K-values. The two-phase solvent system n-hexane–ethyl acetate–methanol–water (3:5:3:7, v/v/v/v) provided a small K-value for Compound 1 (K1= 0.30). Thus, these two solvent systems were not suitable for the separation of the three target compounds. The solvent system n-hexane–ethyl acetate–methanol–water (2:5:3:6, v/v/v/v) provided suitable K-values (K1= 0.62, K2= 2.39 and K3= 1.27) for the target compounds, and the separation factors were α1= 2.05 and α2 = 1.88. Other factors such as the flow rate of the mobile phase, the separation temperature and the revolution speed were also investigated in this study. Finally, the flow rate of the mobile phase was 2.8 mL/min, the separation temperature was 30°C and the revolution speed was adjusted at 900 rpm.
Table III.
K-values of the Three Target Compounds Measured in Different Solvent Systems
| Solvent systems (v/v) |
K-values |
||
|---|---|---|---|
| Compound 1 | Compound 2 | Compound 3 | |
| n-Hexane–ethyl acetate–methanol–water (3:5:3:5) | 0.12 | 1.01 | 0.46 |
| n-Hexane–ethyl acetate–methanol–water (3:5:3:7) | 0.30 | 2.23 | 0.73 |
| n-Hexane–ethyl acetate–methanol–water (2:5:3:6) | 0.62 | 2.39 | 1.27 |
Conclusion
In this study, AEAP was used as a sample preparation method in the extraction of oligostilbenes from the I. lactea seed kernels. The AEAP method has the following advantages: environmentally friendly, easy operation, cost saving and product safety. Three oligostilbenes, vitisin D, ampelopsin B and cis-vitisin A, were yielded in only one HSCCC run with high purity. The HSCCC technique has the properties of time-saving, simple process and high recovery. Therefore, this study provided a convenient and efficient method for extraction and fractionation of oligostilbenes from I. lactea. The results of this study also indicated that I. lactea presented a potentially rich and important source of oligostilbenes.
Acknowledgments
This work was supported by the National Science Foundation of China (31470426, 31300292), the International Cooperation Project of Qinghai Province (2013-H-802), Qinghai Provincial Natural Science Foundation (2014-ZJ-765, 2015-ZJ-728), Taishan Scholar Program of Shangdong Province (tshw201502046) and Youth Innovation Promotion Association, CAS.
References
- 1.Jin Q., Han X.H., Hong S.S., Lee C., Choe S., Lee D. et al. ; Antioxidative oligostilbenes from Caragana sinica; Bioorganic & Medicinal Chemistry Letters, (2012); 22(2): 973–976. [DOI] [PubMed] [Google Scholar]
- 2.Ha do T., Kim H., Thuong P.T., Ngoc T.M., Lee I., Hung N.D. et al. ; Antioxidant and lipoxygenase inhibitory activity of oligostilbenes from the leaf and stem of Vitis amurensis; Journal of Ethnopharmacology, (2009); 125(2): 304–309. [DOI] [PubMed] [Google Scholar]
- 3.Wibowo A., Ahmat N., Hamzah A.S., Latif F.A., Norrizah J.S., Khong H.Y. et al. ; Identification and biological activity of secondary metabolites from Dryobalanops beccarii; Phytochemistry Letters, (2014); 9: 117–122. [Google Scholar]
- 4.Riaz N., Malik A., Rehman A.U., Ahmed Z., Muhammad P., Nawaz S.A. et al. ; Lipoxygenase inhibiting and antioxidant oligostilbene and monoterpene galactoside from Paeonia emodi; Phytochemistry, (2004); 65(8): 1129–1135. [DOI] [PubMed] [Google Scholar]
- 5.Ito T., Endo H., Shinohara H., Oyama M., Akao Y., Iinuma M.; Occurrence of stilbene oligomers in Cyperus rhizomes; Fitoterapia, (2012); 83(8): 1420–1429. [DOI] [PubMed] [Google Scholar]
- 6.Sri-in P., Sichaem J., Siripong P., Tip-pyang S.; Macrostachyols A–D, new oligostilbenoids from the roots of Gnetum macrostachyum; Fitoterapia, (2011); 82(3): 460–465. [DOI] [PubMed] [Google Scholar]
- 7.Farag S.F., Takaya Y., Niwa M.; Stilbene glucosides from the bulbs of Iris tingitana; Phytochemistry Letters, (2009); 2(4): 148–151. [Google Scholar]
- 8.Chaher N., Arraki K., Dillinseger E., Temsamani H., Bernillon S., Pedrot E. et al. ; Bioactive stilbenes from Vitis vinifera grapevine shoots extracts; Journal of the Science of Food and Agriculture, (2014); 94(5): 951–954. [DOI] [PubMed] [Google Scholar]
- 9.Ha do T., Chen Q.C., Hung T.M., Youn U.J., Ngoc T.M., Thuong P.T. et al. ; Stilbenes and oligostilbenes from leaf and stem of Vitis amurensis and their cytotoxic activity; Archives of Pharmacal Research, (2009); 32(2): 177–183. [DOI] [PubMed] [Google Scholar]
- 10.Jeong H.Y., Kim J.Y., Lee H.K., Ha do T., Song K.S., Bae K. et al. ; Leaf and stem of Vitis amurensis and its active components protect against amyloid beta protein (25–35)-induced neurotoxicity; Archives of Pharmacal Research, (2010); 33(10): 1655–1664. [DOI] [PubMed] [Google Scholar]
- 11.Yabe N., Matsui H.; Ampelopsis brevipedunculata (Vitaceae) extract inhibits a progression of carbon tetrachloride-induced hepatic injury in the mice; Phytomedicine, (2000); 7(6): 493–498. [DOI] [PubMed] [Google Scholar]
- 12.Kwak J.H., Lee S.R., Park H.J., Byun H.E., Sohn E.H., Kim B.O. et al. ; Kobophenol A enhances proliferation of human osteoblast-like cells with activation of the p38 pathway; International Immunopharmacology, (2013); 17(3): 704–713. [DOI] [PubMed] [Google Scholar]
- 13.Kim J.A., Kim M.R., Kim O., Phuong N.T., Yun J., Oh W.K. et al. ; Amurensin G inhibits angiogenesis and tumor growth of tamoxifen-resistant breast cancer via Pin1 inhibition; Food and Chemical Toxicology, (2012); 50(10): 3625–3634. [DOI] [PubMed] [Google Scholar]
- 14.Kim S.H., Park H.S., Lee M.S., Cho Y.J., Kim Y.S., Hwang J.T. et al. ; Vitisin A inhibits adipocyte differentiation through cell cycle arrest in 3T3-L1 cells; Biochemicl and Biophysical Research Communication, (2008); 372(1): 108–113. [DOI] [PubMed] [Google Scholar]
- 15.Lee J.P., Min B.S., An R.B., Na M.K., Lee S.M., Lee H.K. et al. ; Stilbenes from the roots of Pleuropterus ciliinervis and their antioxidant activities; Phytochemistry, (2003); 64(3): 759–763. [DOI] [PubMed] [Google Scholar]
- 16.Kasiotis K.M., Pratsinis H., Kletsas D., Haroutounian S.A.; Resveratrol and related stilbenes: their anti-aging and anti-angiogenic properties; Food and Chemical Toxicology, (2013); 61: 112–120. [DOI] [PubMed] [Google Scholar]
- 17.Li Y.T., Yao C.S., Bai J.Y., Lin M., Cheng G.F.; Anti-inflammatory effect of amurensin H on asthma-like reaction induced by allergen in sensitized mice; Acta Pharmacological Sinica, (2006); 27(6): 735–740. [DOI] [PubMed] [Google Scholar]
- 18.Yim N., Ha do T., Trung T.N., Kim J.P., Lee S., Na M. et al. ; The antimicrobial activity of compounds from the leaf and stem of Vitis amurensis against two oral pathogens; Bioorganic & Medicinal Chemistry Letters, (2010); 20(3): 1165–1168. [DOI] [PubMed] [Google Scholar]
- 19.Nguyen T.N.A., Dao T.T., Tung B.T., Choi H., Kim E., Park J. et al. ; Influenza A (H1N1) neuraminidase inhibitors from Vitis amurensis; Food Chemistry, (2011); 124(2): 437–443. [Google Scholar]
- 20.Kim J.Y., Jeong H.Y., Lee H.K., Kim S., Hwang B.Y., Bae K. et al. ; 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, (2012); 19(2): 150–159. [DOI] [PubMed] [Google Scholar]
- 21.Feng L.L., Wu X.F., Liu H.L., Guo W.J., Luo Q., Tao F.F. et al. ; Vaticaffinol, a resveratrol tetramer, exerts more preferable immunosuppressive activity than its precursor in vitro and in vivo through multiple aspects against activated T lymphocytes; Toxicology and Applied Pharmacology, (2013); 267(2): 167–173. [DOI] [PubMed] [Google Scholar]
- 22.Ko J., Choi J., Bae S.K., Kim J., Yoon K.D.; Separation of five oligostilbenes from Vitis amurensis by flow-rate gradient high-performance counter-current chromatography; Journal of Separation Science, (2013); 36(24): 3860–3865. [DOI] [PubMed] [Google Scholar]
- 23.Jeon J.S., Kim C.Y.; Preparative separation and purification of flavonoids and stilbenoids from Parthenocissus tricuspidata stems by dual-mode centrifugal partition chromatography; Separation and Purification Technology, (2013); 105: 1–7. [Google Scholar]
- 24.Lv H.H., Wang H.L., He Y.F., Ding C.X., Wang X.Y., Suo Y.R.; Separation and purification of four oligostilbenes from Iris lactea Pall. var. chinensis (Fisch.) Koidz by high-speed counter-current chromatography; Journal of Chromatography B, (2015); 988: 127–134. [DOI] [PubMed] [Google Scholar]
- 25.Shinoda K., Takaya Y., Ohta T., Niwa M., Hisamichi K., Takeshita M. et al. ; Vitisins D and E, novel oligostilbenes from Vitis coignetiae stem barks; Heterocycles, (1997); 46: 169–172. [Google Scholar]
- 26.Oshima Y., Ueno Y., Hikino H., Yang L.L., Yen K.Y.; Ampelopsins A, B and C, newoligostilbenes of Ampelopsis brevipedunculata var. hancei; Tetrahedron, (1990); 46(15): 5121–5126. [Google Scholar]
- 27.Lto J., Gobaru K., Shimamura T., Niwa M.; Absolute configurations of some oligostilbenes from Vitis coignetiae and Vitis vinifera ‘Kyohou’; Tetrahedron, (1998); 54(24): 6651–6660. [Google Scholar]
- 28.Li X.N., Huang J.L., Wang Z.D., Jiang X.D., Yu W.C., Zheng Y.M. et al. ; Alkaline extraction and acid precipitation of phenolic compounds from longan (Dimocarpus longan L.) seeds; Separation and Purification Technology, (2014); 124: 201–206. [Google Scholar]
- 29.Sun S.L., Wen J.L., Ma M.G., Sun R.C.; Successive alkali extraction and structural characterization of hemicelluloses from sweet sorghum stem; Carbohydrate Polymers, (2013); 92(2): 2224–2231. [DOI] [PubMed] [Google Scholar]
- 30.Wang G., Chen H.; Fractionation of alkali-extracted lignin from steam-exploded stalk by gradient acid precipitation; Separation and Purification Technology, (2013); 105: 98–105. [Google Scholar]
- 31.Ito Y.; Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography; Journal of Chromatography A, (2005); 1065(2): 145–168. [DOI] [PubMed] [Google Scholar]