Article pubs.acs.org/IECR
Simultaneous Purification of Pulchinenoside B4 and B5 from Pulsatilla chinensis Using Macroporous Resin and Preparative High Performance Liquid Chromatography Liming Wang,†,§ Qiongming Xu,†,§ Sheng Su,† Jiangyun Liu,*,† Yulin Feng,*,‡ Xiaoran Li,† Weifeng Zhu,‡ and Shilin Yang†,‡ †
College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, China College of Pharmacy, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
‡
S Supporting Information *
ABSTRACT: Pulchinenoside B4 and B5 (PB4, PB5) are two major triterpenoid saponins existing in the roots of Pulsatilla chinensis (Bunge) Regel. In this study, a systematic preparative process was developed for the simultaneous purification of PB4 and PB5 from the herb. The performance and separation characteristics of nine types of macroporous resins were critically evaluated. Static absorption/desorption experiments revealed that LX17 belonging to the polyacrylate class possessed superior separation properties. Further dynamic absorption/desorption experiments on LX17 column were conducted to obtain the optimal parameters. To obtain both compounds with high purity, a second stage procedure was developed using preparative reversed-phase high performance liquid chromatography with a dynamic axial compression column system. The separation process was high-efficiency and low-cost, which indicated potential for industrial applications. of target ingredients from complex extracts.12−18 On the other hand, polyacrylate resins, providing special hydrogen-bonding interactions with adsorbents, are suitable for the separation of phenolic acid and polyphenols,19−21 alkaloids,22 and dye ions, etc.23,24 As each type of resin has its own characteristics, it is critical to explore the suitable resins with specific features for the enrichment of target ingredients from various herbal extracts. To obtain pure ingredients frequently demanded in pharmaceutical applications, multiple-stage separation methods, such as various column chromatographies and crystallization techniques, are usually exploited.25−30 Preparative HPLC was applied as a powerful technique for the purification of natural products,31 owing to its high efficiency, selectivity, and degree of automation. HPLC columns with traditional packing became nonhomogenous with time during the process of scale-up separation due to progressive consolidations of column beds. This phenomenon is resulted from repeated thermal expansion/ contraction, pump pulsations, etc. In contrast, dynamic axial compression (DAC) column, which was axially compressed under constant pressure to maintain a stable bed, ran well with good efficiencies and repeatability. DAC columns have been widely adopted in preparative HPLC separation procedures.31,32 In this study, a systematic preparative method was developed for the simultaneous purification of PB4 and PB5 from the herb. The performance of nine types of MARs was critically evaluated by static and dynamic absorption/desorption experiments to select the optimal resin. A second stage separation process was conducted to obtain both target compounds with high purity by
1. INTRODUCTION The roots of Pulsatilla chinensis (Ranunculaceae) are used in traditional Chinese medicine for the treatments of intestinal amebiasis, malaria, vaginal trichomoniasis, and bacterial infections.1 Oleanane-type and lupinane-type triterpenoid saponins, the main ingredients of the herb,2−7 showed antiparasitic, antiseptic, anti-inflammatory, antitumor and immunological activities.5,8−10 Among these saponins, pulchinenoside B4 (Anemoside B4, PB4)2,4 and pulchinenoside B5 (PB5)7 are the two major bioactive compounds11 (Figure 1). Moreover, PB4 has been adopted as a reference substance for the quality control of Pulsatilla chinensis.11 As a result of our systematic investigations of the bioactive ingredients of Pulsatilla chinensis, PB4 and PB5 were revealed to possess antitumor effects.8,11 Therefore, innovative separation and purification technologies are needed to provide these ingredients with high purity for further pharmaceutical applications. The conventional methods for the separation of saponins including PB4 and PB5 from the crude extracts of Pulsatilla chinensis were performed by liquid−liquid extraction, followed by multistep silica gel column chromatography and semipreparative high performance liquid chromatography (HPLC).2−8 However, these methods are not suitable for large-scale production due to the need of bulk amounts of organic solvents and laborious work. In recent years, macroporous adsorption resins (MARs) have been successfully applied in the enrichment of target compounds from crude extracts in industrial practices due to their higher efficiency, easier regeneration, and environment-friendly features.12 Polystyrene and polyacrylate series with different functional groups are two commercially available MARs, which provide different characteristics, such as polar categories, mechanical strength, porous availability, surface areas, etc. Polystyrene resins are frequently used in processing different plant extracts, as they performed well for the enrichment © 2012 American Chemical Society
Received: Revised: Accepted: Published: 14859
August 11, 2012 October 15, 2012 October 18, 2012 October 18, 2012 dx.doi.org/10.1021/ie302165v | Ind. Eng. Chem. Res. 2012, 51, 14859−14866
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Figure 1. Chemical structures of PB4 and PB5.
Table 1. Chemical and Physical Properties of Nine Macroporous Resins in Test resin
structure
particle size (mm)
specific surface area (m2/g)
pore size (nm)
moisture content (%)
polarity
D101 AB-8 HPD100 HPD450 HPD600 LX60 LX68G XDA-8 LX17
polystyrene polystyrene polystyrene polystyrene polystyrene polystyrene polystyrene polystyrene polyacrylate
0.3151.25 0.3151.25 0.3151.25 0.3151.25 0.31.2 0.3151.25 0.3150.8 0.3151.25 0.3151.25
600 600 500 500−550 550−600 850 1300 1200 ≥460
5.0 13−14 8.5−9.0 9−11 8.0 7.0 3.0 2.5 30.0
69.8 50.0 38.5 52.9 32.2 63.7 63.7 61.5 64.2
nonpolar weak nonpolar weak polar middle middle polar middle
2.3. Preparation of the Crude Extracts of Pulsatilla chinensis. Roots of Pulsatilla chinensis were obtained from Suzhou city, Jiangsu province in November 2009, and were authenticated by Professor Xiaoran Li at Soochow University. A voucher specimen (No. PC20091101) was deposited there. The roots of Pulsatilla chinensis (1.0 kg) were ground into powders, and then extracted with 70% aqueous ethanol (7.5 L) under reflux for 2 h, repeated two times. The extracted liquids were combined and concentrated to 1.0 L solution (with the ratio of raw material to extract volume to be 1.0 g/mL), which contained 51.56 mg/mL of PB4 and 17.90 mg/mL of PB5. The sample solution was stored at 4 °C before use, and deionized water was added to prepare solutions of different concentrations as needed. 2.4. HPLC Analysis of PB4 and PB5. Quantitative analysis was carried out by HPLC on a Shimadzu Prominence LC-20A liquid chromatographic system (Shimadzu instruments company, Japan) composed of binary pumps, a variable wavelength detector, a Shimadzu ELSD-II low temperature-evaporative light-scattering detector (ELSD) and LC solution software. A Cosmosil C18 column (250 mm × 4.6 mm i.d., 5 μm) and a Cosmosil C18 guard column (10.0 mm × 4.6 mm i.d., 5 μm) were used at a column temperature of 35 °C. The mobile phase was methanol−water (65:35, v/v), and the flow rate was set at 1.0 mL/min. The retention times of PB4 and PB5 were determined at 7.52 and 8.95 min, respectively. The chromatographic peaks were identified by comparing their respective retention times with those of reference standards, which were eluted in parallel under the same conditions. The working calibration curves showed good linearity over the range of 8.376−27.22 μg/mL for PB4 and 5.454−28.00 μg/mL for PB5. The regression curves for
preparative reversed-phase HPLC using a DAC column system. The separation method developed in this study provides a typical case report for preparative purification of triterpenoid saponins from natural resources.
2. MATERIAL AND METHODS 2.1. Materials and Reagents. PB4 and PB5 standards were purified and identified by MS, NMR, and UV adsorption spectra in the authors’ lab.11 The purities of these two compounds were both higher than 99.5% as determined by HPLC. Methanol (HPLC grade), ethanol (analytical grade) and all other chemicals and reagents (analytical grade) were purchased from Shanghai Chemical Reagents Company (Shanghai, China). Deionized water was purified by a DW100 purification system from Shanghai HiTech Instruments Company (Shanghai, China). 2.2. Adsorbents. MARs including D101, AB-8, LX17, LX60, LX68G, XDA-8, and LX2000 were purchased from Xi’an Sunresin Technology Company (Shanxi province, China), and MARs including HPD100, HPD450, and HPD600 were purchased from Cangzhou Bon Absorbent Technology Company (Hebei province, China). Before the adsorption experiments, the resins were weighed and pretreated by soaking in ethanol overnight. Subsequently they were washed once by 4% HCl, distilled water and 4% NaOH solution, respectively, and finally they were washed by distilled water thoroughly to remove the monomers and porogenic agents trapped inside the pores during the synthesis process. The moisture contents of the tested resins were determined by drying the beads (1.0 g in wet weight) at 80 °C for over 24 h to constant weight. The chemical and physical properties of these MARs were summarized in Table 1. 14860
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PB4 and PB5 were Y = 1.4880X + 9.7104 (R2 = 0.9995, n = 6), and Y = 1.4869X − 9.6754 (R2 = 0.9994, n = 6), respectively, where Y is the logarithm of peak area of the analyte and X is the logarithm of injected quantity (μg) of PB4 or PB5. 2.5. Procedure for the Static Absorption and Desorption Tests. 2.5.1. Static Adsorption Resins Screening. All macroporous resins were screened through static adsorption tests, which were performed as follows: The hydrated resins (1.0 g in dry mass) were put into an Erlenmeyer flask and 10 mL of sample solutions (one-fold dilution with stock solution at concentrations of 25.93 mg/mL PB4 and 9.240 mg/mL PB5) were added. The flasks were then shaken (180 rpm) for 10 h at 25 °C to reach adsorption equilibrium. The solutions after absorption were analyzed by HPLC. After reaching absorption equilibrium, the resins were first washed by deionized water and then desorbed with 10 mL of 95% ethanol (v/v) in water. The flasks were shaken (180 rpm) for 5 h at 25 °C. The desorbed solutions were then analyzed by HPLC. Each experiment was done in triplicate. The preliminary choices of the resins were evaluated by their capacities of absorption/desorption and desorption ratios. The following equations were used to quantify the absorption and desorption capacities.25 Absorption capacity: Qe =
Equation of intraparticle diffusion kinetics model: Q t = k idt 1/2 + C
(5)
where, Qe and Qt are the adsorption capacity at equilibrium and at any time t (mg/g, dry resin), respectively. The parameters k1 (min−1), k2 (g/(mg min)) and kid (mg/(g min1/2)) are the rate constants of the pseudo-first-order, pseudo-second-order and intraparticle diffusion models for the adsorption process, respectively. C (mg/g), the constant, represents boundary layer thickness. Plotting log(Qe − Qt) against t for the pseudo-first-order equation, t/Qt against t for the pseudo-second-order equation and Qt versus t1/2 for the intraparticle diffusion equation, can get straight lines and corresponding parameters, respectively. 2.5.3. Adsorption Isotherms on LX17 Resin. Stock solutions were diluted to get seven different sample solutions in sequences, with concentrations ranging 51.56 mg/mL for PB4 and from 0.89 mg/mL to 17.90 mg/mL for PB5. Each sample solution (10 mL) was contacted with 1.0 g pretreated resins in shakers and was shaken (180 rpm) for 6 h at 25 °C. The initial and equilibrium concentrations were determined by HPLC. The Langmuir and Freudlich equations were used to reveal the linearity fitting and to describe interactions between solutes and resins.25 Freundlich equation:
(C0 − Ce)Vi W
(1)
Q e = KFCe1/ n
(6)
where Qe represents the absorption capacity at absorption equilibrium (mg/g resin); C0 and Ce are the initial and equilibrium concentrations of solutes in the solutions, respectively (mg/mL); Vi is the volume of the initial feed solution (mL) and W is the weight of the dried adsorbent (g).
where KF is the Freundlich constant that indicates the adsorption capacity, and 1/n is an empirical constant related to the magnitude of the adsorption driving force.
Desorption ratio:
Langmuir equation:
D% =
CdVd × 100 (C0 − Ce)Vi
Qe =
(2)
Equation of pseudo-first-order kinetics model: k1t + log Q e 2.303
(3)
Equation of pseudo-second-order kinetics model: t t 1 = + 2 Qt Qe Q e k2
1 + KLCe
(7)
where KL is the adsorption equilibrium constant, Q0 is the theoretical maximum adsorption capacity (mg/g resin). Qe is the same as mentioned above. 2.6. Dynamic Adsorption/Desorption Experiments. Dynamic adsorption/desorption experiments were carried out in a glass column (30 cm × 1.5 cm i.d.) packed with LX17 resin (10.0 g dry weight). The bed volume (BV) of the wet-packed resin was 15 mL. The adsorption process was performed by loading feed solution into the pretreated glass column. After adsorption equilibrium, desorption was completed by gradient elution with solutions of water and ethanol at different ratios. Each part of the eluents was analyzed by HPLC. Each experiment was done in triplicate. Several variables, such as the concentration of feed solution, the volume of feed solution for adsorption process, the percentage of ethanol in solutions (0, 15, 30, 50, 60, 70, 80, 90, 100, v/v), the flow rate (5, 10, 20, BV/h) and the volume of the eluents for desorption process at 25 °C were systematically investigated. For the sake of the convenience of investigation, the other parameters were kept constant except the one under investigation each time. 2.7. Scale-up Enrichment of PB4 and PB5 from Crude Extracts of Pulsatilla chinensis. Scale-up separation was conducted by about 135 fold as that of lab levels. A glass column with a BV of 2.0 L (1000 mm × 100 mm i.d.) was slurry packed with LX17 resin (1350 g, dry weight). The 5.5 L aqueous sample solution (containing 21.45 mg/mL PB4 and 7.52 mg/mL PB5) was applied to the column. After sample
where D is the desorption ratio (%); Cd is the concentration of the solutes in the effluent (mg/mL); Vd is the volume of the effluent; C0, Ce, and Vi are the same as in eq 1. 2.5.2. Adsorption Kinetics on LX17 Resin. The adsorption kinetics curves of PB4 and PB5 on the preliminarily selected LX17 resins were studied according to the following process: 2.8 g of pretreated resins (equal to 1.0 g dry resin) and 10 mL of sample solutions (contained 25.93 mg/mL PB4 and 9.240 mg/mL PB5) were added to each flask with a lid, and then the mixtures were shaken for 5.5 h at 25 °C (180 rpm). Then the concentrations of PB4 and PB5 in the adsorption process were determined by HPLC at certain time intervals after pretest (30, 60, 90, 150, 210, 270, and 330 min). To better illustrate the adsorption mechanism, the following kinetics models were adopted to describe the adsorption process: pseudo-first-order, pseudo-second-order, and intraparticle diffusion kinetics models.25
log(Q e − Q t) = −
Q 0KLCe
(4) 14861
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Table 2. Adsorption Capacity, Desorption Capacity and Desorption Ratio of Different Resins for PB4 and PB5 adsorption (mg/g) resin D101 AB-8 HPD100 HPD450 HPD600 LX60 LX68G XDA-8 LX17
PB4 75.2 16.7 33.2 50.4 76.6 38.0 121.4 185.3 221.9
± ± ± ± ± ± ± ± ±
desorption (mg/g) PB5
5.1 1.6 2.5 3.4 4.3 2.2 8.7 10.4 15.3
37.6 11.3 25.8 23.9 42.5 20.9 55.7 80.6 87.5
± ± ± ± ± ± ± ± ±
PB4 1.9 0.5 1.2 0.9 2.1 1.5 2.8 3.7 3.5
47.8 5.6 23.8 21.9 41.3 27.1 76.7 172.5 211.6
± ± ± ± ± ± ± ± ±
desorption ratio (%) PB5
2.5 0.5 1.4 1.3 1.8 1.7 4.5 12.6 14.8
20.9 2.5 10.8 10.8 16.0 11.7 34.0 66.6 85.5
± ± ± ± ± ± ± ± ±
1.6 0.3 1.0 1.2 0.8 0.8 1.7 3.3 4.1
PB4
PB5
63.5 33.7 71.5 43.9 53.5 71.5 63.3 93.2 95.4
55.6 21.9 41.8 45.2 37.7 55.9 60.7 82.6 97.6
Table 3. Dynamic Adsorption Capacities, Purities and Recoveries of PB4 and PB5 on LX17 and XDA-8 Resins adsorption capacity (mg/g)
purity (%)
recovery (%)
resin
PB4
PB5
PB4
PB5
PB4
PB5
LX17 XDA-8
70.01 ± 3.04 36.30 ± 1.89
24.95 ± 1.29 12.94 ± 0.56
40.61 ± 0.96 36.02 ± 1.24
13.38 ± 0.26 10.69 ± 0.33
90.44 70.56
92.68 72.67
while satisfied desorption ratios were achieved on both of them either. The performances of MARs are related to the adsorbates and chemical features and physical properties of the resins. Both PB4 and PB5 possess triterpene aglycones for hydrophilic interactions and two oligosaccharide chains for strong-polar interactions with polystyrene adsorbents. By comparisons of different polystyrene resins, polar resin HPD600 showed higher adsorption capacities than HPD450 and HPD100, indicating a preferred polar interaction with the adsorbates. Nonpolar resin D101 demonstrated similar adsorption capacities to HPD600, while weak-polar resin AB-8 gave poor adsorption capacities, suggesting that a smaller pore size for adsorption is favorable. As the specific surface areas doubled, the adsorption capacities of LX68G and XDA-8 increased over 2-fold than those of HPD450 and HPD600 with similar polarity. Among all the tested polystyrene resins, the adsorption and desorption capacities of XDA-8 resin showed the best performance owing to its strong polarity, high surface area with small pore size. Interestingly, LX17 resin (similar as Amberlite XAD-7, Rohm and Haas Company) belonging to the polyacrylate class also exhibited excellent adsorption efficiency of 221.9 mg/g for PB4 and 87.5 mg/g for PB5, which was even higher than that from XDA resin. This could be attributed to their physical and chemical properties of different types of resins. Compared with polystyrene resins, polyacrylate resins have different adsorption behaviors and mechanisms.20,23 The main force between styrene resins and adsorbates is the hydrophobic interaction, while the main forces between the polyacrylate resins and adsorbates are electrostatic attraction and hydrogen-bonding interactions. Abundant hydroxyl groups of two oligosaccharide units of PB4 and PB5 can directly act as hydrogen-bonding donator and form hydrogen bonds with the oxygen atom of the ester group of LX17. Accordingly, LX17 and XDA-8 resins were selected for further dynamic separation investigation. 3.1.2. Dynamic Separation Performance of Two Selected Resins. Dynamic separation properties of the two selected resins toward PB4 and PB5 are shown in Table 3. The adsorption capacity of LX17 is about twice as much as that of XDA-8. In terms of the purity and recovery of PB4 and PB5 after enrichment with resins, LX17 were also superior to those of
loading and adsorption equilibrium, desorption was performed successively with 6 BV of water, 3.7 BV of 60% ethanol at a flow rate of 6.5 L/h. The 60% ethanol effluent was collected and subjected to analytical HPLC to determine the contents and the recoveries of PB4 and PB5. 2.8. Preparative Separation of PB4 and PB5 by Reversed-Phase HPLC. Before preparative HPLC, the refined extract prepared by LX17 column was further processed using a LX2000 resin column (100 cm ×8 cm i.d.). The refined extract (50 g) was dissolved in water, loaded on the LX2000 column, eluted with water followed by 30%, 70%, and 100% methanol. The 70% methanol fraction was collected and dried to give the concentrated extract (24.5 g), which was then submitted to preparative HPLC. The preparative HPLC was carried out on a DAC column system (150 cm × 10 cm i.d., HB-DAC-100, Jiangsu hanbon science and technology company, China), filled with Dubke ODS gel (10 μm, 1.8 kg). The column was extensively washed with methanol before use. The preparative HPLC separation was performed as follows: The DAC column was pre-equilibrated with 65% methanol. The concentrated extract (12.0 g) was dissolved in 65% methanol (100 mL), filtered with a 0.45 μm membrane, and then loaded to the column by a pump at a flow rate of 100 mL/min. The column was then eluted with 65% methanol (40 min) to get PB4 and PB5, followed by washing with 100% methanol (10 min) for regeneration; all the eluting experiments were conducted at a flow rate of 300 mL/min, monitored at a UV wavelength of 203 nm at ambient temperature. The peaks corresponding to PB4 and PB5 were collected and subjected to analytical HPLC to determine the contents and the product recoveries.
3. RESULTS AND DISCUSSION 3.1. The Preliminary Choice of the Resins. 3.1.1. Batch Static Adsorption/Desorption Experiments. In the static adsorption/desorption tests, adsorption/desorption properties of nine MARs for target compounds were studied at 25 °C, as shown in Table 2. MARs in this survey demonstrated a wide range of adsorption capacities, ranging from 16.7 mg/g to 221.9 mg/g for PB4, and from 11.3 mg/g to 87.5 mg/g for PB5. The adsorption capacities of PB4 and PB5 on two resins, XDA-8 and LX17, were especially higher than those of other resins 14862
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3.2.2. Adsorption Isotherms on LX17 Resin. Equilibrium adsorption isotherms on LX17 were investigated with different solution concentrations at 25 °C. As shown in Figure 2B. The adsorption capacity increased with the initial concentration, and reached the saturation plateau when the initial concentration reached 21.45 mg/mL (7.52 mg/mL) for PB4 (PB5). Equilibrium adsorption isotherms at higher temperatures were not undertaken, as it was well-known that the increase in temperature is a disadvantage for a physical adsorption process between acrylic ester resins and adsorbates.23 Two common theoretical models for modeling adsorption equilibrium data, the Langmuir and Freundlich isotherms were applied (Supporting Information, Table S2). The calculated correlation coefficient of the Langmuir equation (with R2 higher than 0.999 for both adsorbates) was higher than that of the Freundlich equation, which implied that the Langmuir isotherm could reasonably explain the adsorption process. This result suggested the monolayer coverage of PB4 and PB5 onto the resin. The theoretical maximum adsorption capacity Q0 of PB4 (PB5), determined from the Langmuir equation, was 192.31 mg/g (68.49 mg/g). In the Freundlich equation, the adsorption easily takes place when the 1/n value is between 0.1 and 0.5 (0.2222 and 0.2019 for PB4 and PB5, respectively), which indicated that the LX17 resin is applicable for the separation of PB4 and PB5.19 3.3. Dynamic Adsorption and Desorption on LX17 Resin. 3.3.1. Effects of Feeding Concentration and Feeding Volume on Adsorption. In the process of dynamic adsorption, the effects of feeding concentration and feeding volume on adsorption capacity were investigated. The breakthrough point generally refers to the circumstance under which the adsorbate concentration in the eluent reaches 5% of that in the sample solution applied. The results of leakage curve at different concentrations were calculated (Supporting Information, Table S3). The adsorption capacities slightly increased but fell-back with the increment of initial feed concentrations, which may be due to competition adsorption of impurities and limitation of diffusivity of triterpenoid saponins into the microspores of LX17. The highest adsorption capacity was achieved when the initial concentration of PB4 (PB5) was 20.62 mg/mL (7.16 mg/mL), and the feed volume was 41 mL (= 2.73 BV). 3.3.2. Effects of the Proportions of Ethanol Solutions for Desorption Process. After adsorption equilibrium at the optimal condition as mentioned in section 3.3.1, the adsorbate-laden column was first washed with deionized water, and then desorbed by gradient elutions with 15%−100% ethanol (5 BV for each fraction) at a flow rate of 5 BV/h. Figure 3 showed that PB4 and PB5 could be desorbed with an initial 15% ethanol and could be efficiently desorbed by 60% aqueous ethanol. After sample loading and adsorption equilibrium, the procedure was thus set by elution with water at first to remove impurities, followed by 60% aqueous ethanol. 3.3.3. Effects of the Eluent Volume and Flow Rate on Desorption. The flow rate and the volume of eluent on desorption were investigated under the optimized separation conditions as described above. After the adsorption equilibrium reached for more than 150 min, the adsorbate-laden column was first washed with deionized water to remove impurities, and then desorbed by 60% ethanol (5 BV) at a flow rate of 5, 10, and 20 BV/h, respectively. The concentrations of PB4 and PB5 in the eluents were plotted versus eluent volumes at different flow rates, and the elution curves were shown in Figure 4. The time to reach maximum concentration at 5 BV/h was obviously longer than that at other flow rates, indicating that decreasing
XDA-8. Accordingly, LX17 was selected as the optimal resin for the following adsorption/desorption separation processes. Usually, triterpene saponins can be adsorbed with satisfaction by polystyrene resins.12 Polyacrylate resins were known to perform well in adsorbing phenolic acids and alkaloids.19−22 Similar observations were also reported that polyacrylate resins performed better than polystyrene resins for the enrichment of dyes.23,24 In this case, LX17 showed high selectivity to adsorb PB4 and PB5. This may be attributed to the strong hydrogenbonding interactions between adsorbates and polyacrylate resins. 3.2. Static Experiments on LX17 Resin. 3.2.1. Adsorption Kinetics on LX17 Resin. The adsorption kinetics curve on LX17 was shown in Figure 2A. The adsorption capacity rapidly
Figure 2. Adsorption kinetic curves (A) and adsorption isotherm (B) for PB4 and PB5 on LX17 resin at 25 °C. Concentration (B) was expressed as ratio of raw material (g) to crude extract volume (mL).
increased in the first 60 min, then slowed down and reached equilibrium after 150 min (for PB4) and 100 min (for PB5), indicating that LX17 belongs to the slow adsorption resin type.26 Hence, the duration of adsorption was set to be longer than 150 min in the following study. Adsorption kinetics parameters for PB4 and PB5 on LX17 were calculated (Supporting Information, Table S1). A pseudo-second-order kinetics model was suitable to describe the entire adsorption process on the resin, with calculated correlation coefficient (R2) higher than 0.998 for both adsorbates. The principle of pseudo-secondorder kinetics implied that concentrations of both adsorbates and adsorbents were involved in rate determining steps during the adsorption process.19 The results indicated that the adsorption process was controlled by two or more rate-limiting steps such as external diffusion, boundary layer diffusion, and intraparticle diffusion.19 14863
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55 mL (refers to the circumstance under which the adsorbate concentration in the eluate was less than 1% of that in the sample solution applied) in this case. Therefore, 55 mL (3.67 BV) and 10 BV/h were selected as the proper volume of elution and flow rate for desorption. 3.4. Scale-up Enrichment of PB4 and PB5. Figure 5A,B shows the HPLC analysis of the fractions corresponding to PB4
Figure 3. Dynamic desorption curve of PB4 and PB5 on LX17 at different eluting ethanol concentration.
Figure 5. HPLC chromatograms of represented samples: (A) crude extract; (B) refined extract by LX17 column; (C) PB4 and (D) PB5 purified by preparative HPLC.
and PB5 after conducting separation on a scale-up column. Through only one cycle treatment by LX17, the contents of PB4 and PB5 increased from 17.2% and 6.0% in the crude extract to 39.9% and 12.5% in the refined extract, with recoveries of 96.9% and 86.8% for PB4 and PB5, respectively. This was in agreement with former experimental data (Table 3). 3.5. Preparative Purification of PB4 and PB5 by Reversed-Phase HPLC. To increase the loading amount, a purification step by LX2000 column was executed. In this step, most nonpolar and strong polar impurities were removed by water and pure methanol, separately. PB4 and PB5 were coeluted by 70% methanol. The extract was twice enriched again. Methanol was selected as the elution solvent in the preparative HPLC process, as it gave satisfactory separation in prior HPLC analysis and economic acceptance. To further optimize the mobile phase system, two different elution procedures
Figure 4. Dynamic desorption curve of PB4 and PB5 on LX17 eluting with 60% ethanol at different loading flow rates.
flow rate resulted in the increase of the external mass transfer resistance. On the other hand, eluent consumption was largest at a flow rate of 20 BV/h when target compounds were completely desorbed. In general, overincreasing flow rate negatively affected the dynamic desorption process because eluent molecules have no sufficient time to undergo interactions with adsorbates at the surface of resins. The total recovery of PB4 and PB5 at 10 BV/h was calculated to be 92.25%, which was more favorable than that at 5 BV/h (75.09%) or 20 BV/h (78.94%), and the corresponding desorption volume was approximately 14864
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PB5 on LX17 resin at 25 °C (Table S2). Breakthrough volume and mass of PB4 and PB5 adsorbed on LX17 resin at different feed concentrations (Table S3). This material is available free of charge via the Internet at http://pubs.acs.org.
were tested during preparative separation: (1) eluting with 60% methanol at the first 20 min to remove impurities, then with 65% methanol for 30 min, and regenerated with 100% methanol for 10 min; (2) eluting with 65% methanol for the first 40 min, then regenerated with 100% methanol for the next 10 min. Both conditions gave satisfactory separation, and procedure (2) was selected for convenience. After the gradient optimization, PB4 and PB5 are baseline separated with satisfactory resolution as depicted in Figure 6. Chromatograms
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AUTHOR INFORMATION
Corresponding Author
*(J.L.) E-mail:
[email protected]. Tel.: +86-512-65884301. Fax: +86-512-65882089. (Y.F.) E-mail:
[email protected]. Tel.: +86-791-7119631. Author Contributions §
Liming Wang and Qiongming Xu contributed equally to this work. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Professor Hongliang Jiang for his help with the manuscript. This work was financially supported by the Creating New Drugs National Science and Technology Major Projects of China (No. 2011ZX11102), a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Major Science & Technology Project of Jiangxi Province (No. 2010AZD00301), Science & Technology Development Project of Jiangxi Province, (No.2010DQB01700) and Natural Science Foundation of Jiangxi Province (No.20114BAB215045).
Figure 6. Preparative HPLC chromatograms of purification of PB4 and PB5 from concentrated extract using a DAC column system. The monitor wavelength was set at 203 nm.
regarding HPLC analysis of the representative fractions corresponding to PB4 and PB5 are shown in Figure 5C,D, respectively. After the chromatographic purification process, the average contents of PB4 and PB5 in final products were 97.8% and 98.1%, respectively. Their recoveries were 90.3% and 85.4%, respectively. Through the entire purification procedure, 40.6 g of PB4 and 12.6 g of PB5 with the purity of 97.1% and 98.2% were finally obtained from 1.0 kg of herb powder. The total recovery of the process is 76.4% and 69.1% for PB4 and PB5, respectively.
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4. CONCLUSIONS A method for the simultaneous purification of PB4 and PB5 from Pulsatilla chinensis was established. Macroporous resins were used in the initial enrichment step to remove impurities. Among the different types of macroporous resins tested, LX17 resin belonging to the polyacrylate class demonstrated superior separation properties. The absorption equilibrium of LX17 was fitted best to the pseudo-second-order kinetics model and Langmuir isotherm model. Further dynamic absorption and desorption experiments on LX17 resin columns were conducted to obtain the optimal parameters. The contents of PB4 and PB5 in the refined extract were increased from 17.2% and 6.0% to 39.9% and 12.5%, respectively, after a single run in a large-scale test. A second stage process was conducted by preparative reversed-phase HPLC with a DAC column. Pure PB4 and PB5 were successfully obtained. To the best of our knowledge, this is the first case report of the superior selectivity of polyacrylate resins toward enrichment of triterpenoid saponins. The separation process was high-efficiency and low-cost which indicated potential for industrial applications.
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ASSOCIATED CONTENT
S Supporting Information *
Adsorption kinetics parameters for PB4 and PB5 on LX17 resin (Table S1). Langmuir and Freundlich parameters of PB4 and 14865
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