Adsorption properties and preparative separation of phenylethanoid glycosides from Cistanche deserticola by use of macroporous resins

Journal of Chromatography B, 937 (2013) 84–90

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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb

Adsorption properties and preparative separation of phenylethanoid glycosides from Cistanche deserticola by use of macroporous resins Boyan Liu a,b , Jie Ouyang b , Xiaofan Yuan a,∗ , Liwei Wang a , Bing Zhao a a b

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190 Beijing, PR China Department of Food Science and Engineering, College of Biological Science and Biotechnology, Beijing Forestry University, 100083 Beijing, PR China

a r t i c l e

i n f o

Article history: Received 7 May 2013 Accepted 12 August 2013 Available online 18 August 2013 Keywords: Macroporous resin Phenylethanoid glycosides Echinacoside Acteoside Cistanche deserticola Y. C. Ma Separation

a b s t r a c t A simple and efficient chromatographic method for large-scale preparative separation of phenylethanoid glycosides (mainly contain echinacoside and acteoside) from Cistanche deserticola was developed. The adsorption properties of eight macroporous resins were evaluated. Three selected resins were further screened depending on the adsorption kinetics curves, in which HPD300 resin showed the best separation efficiency. The adsorption isotherm data on HPD300 resin were fitted to the Freundlich equation in certain concentration range. The dynamic adsorption and desorption experiments were carried out on columns packed with HPD300 resin to optimize the separation process. The breakthrough curves showed that acteoside had a higher affinity to the resin than echinacoside. The contents of echinacoside and acteoside in the product increased from 1.79% and 1.43% in the crude extracts to 16.66% and 15.17%, with recovery yields of 80.41% and 90.17%, respectively. The purity of total phenylethanoid glycosides in the product was 76.58%. © 2013 Elsevier B.V. All rights reserved.

1. Introduction The stem of Cistanche deserticola Y. C. Ma is a common traditional Chinese medicine mainly used for the treatment of kidney deficiency, body weakness and constipation. It is officially recorded in Chinese Pharmacopoeia [1]. Phenylethanoid glycosides (PhGs), including echinacoside, acteoside (Fig. 1), isoacteoside and 2 -acetylacteoside, etc., are regarded as the major bioactive constituents of Cistanche species [2]. PhGs are a group of water soluble natural products and mainly isolated from medicinal plants. The structural characteristic of PhGs is that cinnamic acid and hydroxyphenylethyl moieties are attached to a ␤-glucopyranose through ester and glycosidic linkages respectively [3]. Pharmacological studies show that PhGs from Cistanche have medicinal functions such as antifatigue [4], neuroprotective effects [5,6], hepatoprotective effects [7,8] and enhancing antibody production [9]. Many conventional and modern methods for the separation and purification of PhGs from Cistanche have been developed including silica gel column chromatography [10], polyamide chromatography [11], high-speed counter-current chromatography [12–14] and preparative high-performance liquid chromatography [15]. However, these methods are not suitable for large-scale preparation. The employing of macroporous resins is an efficient method in

∗ Corresponding author. Tel.: +86 13611350422. E-mail address: [email protected] (X. Yuan). 1570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jchromb.2013.08.018

bio-separation. The macroporous resins have a high surface area, a large average pore diameter and high mechanical strength. It is economical, environmental friendly and easy regeneration [16,17]. PhGs possess benzene rings and hydrogen groups and can be adsorbed by resins with different polarities. Macroporous resins have been applied to the separation of PhGs from Cistanche extracts, but the adsorption properties have not been detailed or the purity of total PhGs are not detected [18,19]. Because the solvent, temperature and the physical properties of resin all play important roles in the adsorption and desorption process and the extracts solution is a multicomponent system, the adsorption behavior cannot be easily predicted [20]. The objective of this paper is to develop an efficient method for the preparative separation of total PhGs from C. deserticola with macroporous resin. The simultaneous adsorption and desorption behaviors of echinacoside and acteoside on the optimal macroporous resin were studied.

2. Materials and methods 2.1. Chemicals and reagents Echinacoside was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acteoside was obtained from Tauto Biotechnology Corporation (Shanghai, China). Acetonitrile was of HPLC grade (Fisher, USA). The ethanol of analytical grade was purchased from Beijing

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Fig. 1. Chemical structures of echinacoside (A) and acteoside (B).

Chemistry Corporation (Beijing, China). Deionized water was purified by a Milli-Q Water Purification system (Millipore, USA). The stem of C. deserticola Y. C. Ma was collected from Alashan League, Inner Mongolia of China.

2.4.2. Determination of concentration of total PhGs The standard curve of PhGs content was measured using echinacoside as the standard at 333 nm using an ultraviolet (UV) spectrophotometer. The standard curve was Y = 0.0292X − 0.0031 (R2 = 0.9997), where Y is the absorbance of the solution and X is the concentration of PhGs (␮g/mL).

2.2. Adsorbents Macroporous resins including ADS-7, ADS-17, AB-8, D101, HPD100, HPD300, HPD600 and HPD722 were purchased from Cang Zhou Bon Adsorber Technology Co., Ltd. (Hebei, China). Their physical properties are summarized in Table 1. The resins were soaked in 95% ethanol for 24 h and then washed thoroughly with deionized water before using.

2.3. Preparation of C. deserticola extracts The dried stem of C. deserticola (1 kg) was ground to powder and ultrasonically extracted with 20 L of 50% (v/v) ethanol at 50 ◦ C for 60 min. The working frequency and power were 40 kHz and 700 W, respectively. The extracts were filtered and then concentrated under reduced pressure with a rotary evaporator to dryness. The contents of echinacoside and acteoside in the dry extracts were 1.79% and 1.43%, respectively. The crude extracts were stored at −20 ◦ C for further separation.

2.4. Analytical methods 2.4.1. HPLC analysis of echinacoside and acteoside The concentrations of echinacoside and acteoside were analyzed by HPLC on Shimadzu LC-20AT system with a SPD-M20A diode array detector. Analysis was performed on a Waters XTerra C18 column (4.6 mm × 250 mm, 5 ␮m). The mobile phase was consisted of 0.2% formic acid aqueous solution (A) and acetonitrile (B). The gradient elution program was as follows: 0–23 min, hold 14% of B; 23–24 min, 14–17% of B; 25–35 min, 17% of B; 35–36 min, 17–20% of B; 36–60 min, 20% of B. The detection wavelength was 330 nm, the flow rate was 0.8 mL/min, the injection volume was 10 ␮L and the oven temperature was set at 30 ◦ C.

2.5. Static adsorption and desorption tests 2.5.1. Adsorption and desorption properties of the resins Static adsorption and desorption tests were performed in order to select the most suitable macroporous resin. In adsorption experiment, 35 mL of crude extracts solutions (initial concentrations of echinacoside and acteoside were 0.54 and 0.43 mg/mL, respectively) with a certain amount of wet resins (equal to 0.35 g of dry resin) were added into flasks. The flasks were shaken (100 rpm) in a constant temperature oscillator at 25 ◦ C for 12 h. After fully adsorption, the resins were washed with deionized water for three times and then desorbed with 35 mL of 50% (v/v) ethanol. The flasks were then shaken (100 rpm) for 24 h at 25 ◦ C. The solutions before and after desorption were analyzed by HPLC. The adsorption capacity, desorption capacity and desorption ratio of the resins were calculated according to the following equations: qe =

(C0 − Ce )Vi W

(1)

qd =

Cd Vd W

(2)

D=

Cd Vd × 100% (C0 − Ce )Vi

(3)

where qe is the adsorption capacity (mg/g), C0 and Ce are the initial and equilibrium concentrations of echinacoside or acteoside (mg/mL), Vi is the volume of the initial sample solution (mL), W is the weight of the tested dry resin (g), qd is the desorption capacity (mg/g), Cd is the concentration of echinacoside or acteoside in desorption solution (mg/mL), Vd is the volume of the desorption solution (mL) and D is the desorption ratio (%).

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Table 1 Physical properties of macroporous resins. Surface area (m2 /g)

Name

Polarity

ADS-7 ADS-17 AB-8 D101 HPD100 HPD300 HPD600 HPD722

Polar Moderate-polar Weak-polar Non-polar Non-polar Non-polar Polar Weak-polar

≥100 90–150 480–520 480–520 650–700 800–870 500–550 485–530

250–300 250–300 130–140 100–110 90–100 50–55 100–120 130–140

2.5.2. Static adsorption kinetics on HPD100, HPD300 and D101 Static adsorption kinetics data were obtained by adding preweighed amounts of selected resins (equal to 0.35 g of dry resin) into 50 mL crude extracts solutions (initial concentrations of echinacoside and acteoside were 0.58 and 0.46 mg/mL, respectively) and shaking (100 rpm) at 25 ◦ C for 8 h. The concentrations of echinacoside and acteoside in the solutions were determined every 30 min. 2.5.3. Adsorption isotherms Pre-weighed amounts of HPD300 resin (equal to 0.35 g of dry resin) were added into 35 mL of crude extracts solutions at different initial concentrations. The flasks were shaken (100 rpm) for 12 h at 25 ◦ C, 35 ◦ C and 45 ◦ C, respectively. The initial and equilibrium solutions were determined by HPLC. Langmuir [21] and Freundlich [22] models were employed to describe the adsorption behaviors. Langmuir equation :

Freundlich equation :

qe =

qm KL Ce 1 + KL Ce 1/n

qe = KF Ce

Average pore diameter (Å)

Particle diameter (mm) 0.3–1.25 0.3–1.2 0.3–1.25 0.3–1.25 0.3–1.25 0.3–1.2 0.3–1.25 0.3–1.25

50% (v/v) ethanol desorption solution was collected and dried. The purity of echinacoside, acteoside and total PhGs were calculated. 3. Results and discussion 3.1. Adsorption and desorption capacities, desorption ratios of the resins Eight kinds of macroporous resins, ranging from non-polarity to polarity, were tested to select the appropriate resins for adsorbing and separating echinacoside and acteoside from C. deserticola crude extracts. The adsorption and desorption capacities, desorption ratios of the resins are shown in Fig. 2. The interactions of the adsorbate, adsorbent and solvent, involving a physical action through van der Waals force or hydrogen bonding, affect the adsorption process. Based on the theory

(4)

(5)

where qe is the adsorption capacity (mg/g), Ce is the equilibrium concentration (mg/mL), qm is the theoretically calculated maximum adsorption capacity (mg/g resin), KL is the Langmuir constant, KF and 1/n are the Freundlich constants. 2.6. Dynamic adsorption and desorption Dynamic adsorption and desorption experiments were carried out on a column (1.6 cm × 30 cm) wet-packed with HPD300 resin and the bed volume (BV) was 20 mL. The crude-extract solution (the concentrations of echinacoside and acteoside were 2.0 and 1.6 mg/mL, respectively) was loaded onto the resin column at a flow rate of 1 BV/h. The concentrations of echinacoside and acteoside were monitored until the breakthrough point of echinacoside was indicated. To select the suitable loading flow rate and volume, the solutions was loaded onto the columns at different flow rates of 1, 2, and 4 BV/h and the breakthrough curves were examined. The dynamic desorption tests were performed as follows: the columns were first washed by 4 BV of deionized water after adsorption equilibrium and then eluted by ethanol-water solutions (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, v/v) at 2 BV/h, respectively. The solution after desorption was analyzed by HPLC. All of the dynamic adsorption and desorption experiments were performed at room temperature. 2.7. Laboratory preparative-scale separation 1.6 L extracts solution (echinacoside 2 mg/mL and acteoside 1.6 mg/mL) was subjected to a column (4.0 cm × 40.0 cm) with a bed volume of 400 mL HPD300 resin by 1 BV/h. The column was firstly washed by 4 BV of deionized water and then 3 BV of 10% (v/v) ethanol to remove the high polar components. 4 BV of 50% (v/v) ethanol was used for desorption at the flow rate of 2 BV/h. The

Fig. 2. Adsorption and desorption capacities and desorption ratio of echinacoside (A) and acteoside (B) on macroporous resins.

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Fig. 3. Adsorption kinetics curves for echinacoside (A) and acteoside (B) on HPD100, HPD300 and D101 resins at 25 ◦ C.

of similarity and intermiscibility, the non-polar resin had small adsorption capacity for the strong polarity impurities like inorganic salt and sugar and a high adsorption capacity for relatively low polarity substance like PhGs. It can be found that the resins got better adsorption capacity along with the decrease of polarity. The non-polar resins HPD100, HPD300 and D101 were suitable for adsorbing echinacoside and acteoside. All of the resins had a good and similar desorption ratio except ADS-7 and ADS-17. Therefore, HPD100, HPD300 and D101 were selected to further investigate the adsorption kinetics.

3.2. Static adsorption kinetics on HPD100, HPD300 and D101 Adsorption kinetics curves of echinacoside and acteoside on HPD100, HPD300 and D101 resins at 25 ◦ C are shown in Fig. 3. The adsorption equilibration time of echinacoside and acteoside on HPD300 resin were 1.5 and 2 h, respectively. HPD300 took the shortest time and got the highest adsorption capacity to reach equilibrium among the three resins. This may due to the surface area of HPD300 is larger than other resins. Previous studies also showed that the resin with large surface area has good adsorption behavior [20,23]. So, HPD300 resin was selected for the separation of PhGs.

87

Fig. 4. Adsorption isotherms of echinacoside (A) and acteoside (B) on HPD300 resin at 25, 35 and 45 ◦ C.

3.3. Adsorption isotherms The adsorption isotherms are shown in Fig. 4. Echinacoside and acteoside adsorption capacities on HPD300 resin increased with decreasing temperature. With the increase of equilibrium concentration, the adsorption capacity for acteoside increased and reached saturation status. The adsorption capacity for echinacoside decreased after reaching the peak. It is possible that the adsorption potency of acetoside with HPD300 resin is larger than that of echinacoside. For the competitive adsorption, a small amount of echinacoside was absorbed at high concentration. A similar phenomenon was observed for competitive adsorption of organic cations on clays [24] and the adsorption of gold and copper onto ion-exchange resins [25]. The Langmuir and Freundlich adsorption models were applied to fit the experimental data below the equilibrium concentration of 0.8 mg/mL. As shown in Table 2, for Langmuir equation, most of the R2 values were lower than 0.9, while for Freundlich equation, nearly all the R2 values exceed 0.95. The adsorption data suggesting that the experimental results fitted better to the Freundlich equation in the experimental concentration range. In most cases, the n value in Freundlich equation between 1 and 10 shows beneficial adsorption [26]. The values of n summarized in Table 2 indicated that the adsorption on HPD300 resin could take place easily.

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Table 2 Langmuir and Freundlich parameters of echinacoside and acteoside on HPD300 resin below the equilibrium concentration of 0.8 mg/mL. Temperature (◦ C)

Echinacoside 25 35 45 Acteoside 25 35 45

Langmuir equation

Freundlich equation

qm

KL

R2

KF

n

R2

38.7297 34.1064 28.8517

335.6451 485.3132 486.5074

0.8583 0.7388 0.6047

45.2136 41.2158 37.1336

7.9690 7.8009 6.4809

0.9389 0.9668 0.9981

41.1353 38.4468 34.5543

69.3236 78.4814 86.9072

0.9058 0.8626 0.8761

49.7195 46.7325 41.3231

5.0724 5.0832 5.2913

0.9573 0.9697 0.9643

3.4. Dynamic adsorption and desorption tests 3.4.1. Dynamic breakthrough curves on HPD300 resin The breakthrough curves of echinacoside and acteoside at different loading flow rates are shown in Fig. 5. The leakage point in this study was defined as the concentration of leakage solution was 5% of the initial concentration [27,28]. Echinacoside and acteoside had different leakage points and echinacoside reached leakage point earlier. HPD300 resin is a non-polar adsorbent and easily adsorb non-polar compounds rather than polar compounds. With the increasing of the loading amount, echinacoside desorbed from resin and the value of C/C0 exceeded 1. It is possible that PhGs with lower

Fig. 6. Desorption ratio of echinacoside and acteoside using different concentrations of ethanol aqueous.

polarity, such as acteoside, adsorbed strongly onto the resin and replaced echinacoside. This phenomenon was also found on other adsorbents [29–32]. The increase of the loading flow had a negative effect on adsorption capacities [33,34]. As shown in Fig. 5, the leakage point reached earlier and the competitive adsorption rate decreased when the flow rate increased, and the shape of breakthrough curves was flat. This may be due to the decrease of the contact time to reach saturation [31]. The adsorption flow rate was optimized as 1 BV/h.for echinacoside and acteoside which exhibited better adsorption performance.

Fig. 5. Dynamic breakthrough curves of echinacoside (A) and acteoside (B) packed with HPD300 resin at different flow rates.

Fig. 7. Dynamic desorption curves of echinacoside and acteoside packed with HPD300 resin.

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reached 16.66% with a recovery yield of 80.41% and acteoside content reached 15.17% with a recovery yield of 90.17%. The purity of total PhGs in the product was 76.58%. 4. Conclusions In this study, a resin column chromatography method for separation of PhGs form C. deserticola was established. HPD300 resin was selected among the eight resins by static adsorption test, desorption test and static adsorption kinetics. The equilibrium adsorption of acteoside on HPD 300 resin fitted to Freundlich isotherm model well. The dynamic adsorption and desorption properties of HPD300 resin for echinacoside and acteoside were demonstrated in detail. Competitive adsorption behaviors were observed and acteoside showed a higher affinity to the resin than echinacoside. 50% (v/v) ethanol was selected as the optimal elution solvent. Under the optimal condition, the contents of echinacoside and acteoside in the product were 9.31-fold and 10.61-fold increased with recovery yields of 80.41% and 90.17%, respectively. The content of PhGs was 76.58%. The results indicated that macroporous resin is efficient for the large-scale production of PhGs from C. deserticola. Acknowledgements This work was financially supported by the Science and Technology Department of Inner Mongolia Autonomous Region and Science and Technology Bureau of Alashan League. References

Fig. 8. Chromatograms of crude extract (A) and product eluted by 50% (V/V) ethanol (B).

The breakthrough volumes of echinacoside and acteoside were 4 and 5 BV at the initial concentration of 2.0 and 1.6 mg/mL, respectively. A sample loading of 4 BV was selected for dynamic adsorption experiments for a high recovery of echinacoside. 3.4.2. Effect of ethanol aqueous solution on desorption tests Ethanol is suitable desorbent because it is easily removed from the solution and no toxicity to the samples [27]. As shown in Fig. 6, little echinacoside and acteoside were desorbed with 10% (v/v) ethanol and the best eluent was 50% (v/v) ethanol. Therefore 10% (v/v) ethanol was used to remove the impurities and 50% (v/v) ethanol was selected as desorption solution. 3.4.3. Dynamic desorption curve on HPD300 resin After washed by 10% (v/v) ethanol, the fully saturated columns were eluted with 50% (v/v) ethanol at the flow rate of 2 BV/h. As demonstrated in Fig. 7, echinacoside and acteoside were totally desorbed in 3 BV and 3.5 BV. 3.5. Laboratory preparative-scale separation The crude extract from the stem of C. deserticola was purified on a HPD300 resin column. Fig. 8 shows the chromatograms of samples before and after separation with HPD300 resin. Compared with the PhGs fingerprint obtained in literatures [35,36], it can be concluded that most kinds of PhGs are reserved. Echinacoside content

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