An effective β-cyclodextrin polyurethane spherical adsorbent for the chromatographic enrichment of corilagin from Phyllanthus niruri L. extract

    An effective β-cyclodextrin polyurethane spherical adsorbent for the chromatographic enrichment of corilagin from Phyllanthus niruri L. extract Jun Zhao, Tian-Tian Liu, Guo Chen PII: DOI: Reference:

S1381-5148(16)30051-7 doi: 10.1016/j.reactfunctpolym.2016.03.019 REACT 3661

To appear in: Received date: Revised date: Accepted date:

24 November 2015 22 March 2016 23 March 2016

Please cite this article as: Jun Zhao, Tian-Tian Liu, Guo Chen, An effective β-cyclodextrin polyurethane spherical adsorbent for the chromatographic enrichment of corilagin from Phyllanthus niruri L. extract, (2016), doi: 10.1016/j.reactfunctpolym.2016.03.019

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ACCEPTED MANUSCRIPT An effective β-cyclodextrin polyurethane spherical adsorbent for the chromatographic enrichment of corilagin

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from Phyllanthus niruri L. extract

Department of Bioengineering and Biotechnology, College of Chemical Engineering,

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a

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Jun Zhao a,b,*, Tian-Tian Liu a, Guo Chen a

b

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Huaqiao University, 668 Jimei Avenue, Amoy, 361021, China Institute of Oil and Natural Products, Huaqiao University, 668 Jimei Avenue, Amoy,

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361021, China

* Corresponding author: Dr. Jun Zhao

(First name: Jun; Family name: Zhao) a

Department of Bioengineering and Biotechnology, College of Chemical Engineering,

Huaqiao University, 668 Jimei Avenue, Amoy, 361021, China; b

Institute of Oil and Natural Products, Huaqiao University, 668 Jimei Avenue, Amoy,

361021, China Tel.: +86-592-616-2300

Fax.: +86-592-616-2300

E-mail: [email protected]

Other authors: Tian-Tian Liu (First name: Tian-Tian; Family name: Liu) 1

ACCEPTED MANUSCRIPT a

Department of Bioengineering and Biotechnology, College of Chemical Engineering,

Huaqiao University, 668 Jimei Avenue, Amoy, 361021, China Tel.: +86-592-616-2300

Fax.: +86-592-616-2300

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E-mail: [email protected]

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Dr. Guo Chen (First name: Guo; Family name: Chen)

Department of Bioengineering and Biotechnology, College of Chemical Engineering,

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a

Huaqiao University, 668 Jimei Avenue, Amoy, 361021, China Fax.: +86-592-616-2300

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Tel.: +86-592-616-2300

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E-mail: [email protected]

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ACCEPTED MANUSCRIPT Abstract A novel β-cyclodextrin polyurethane (β-CD-PU) spherical adsorbent was prepared by the reversed-phase suspension crosslinking technique for the

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chromatographic enrichment of a polyphenolic compound corilagin. β-Cyclodextrin (β-CD) and PEG1000 were co-crosslinked with hexamethylene diisocyanate, and the

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polyurethane skeleton was formed and confirmed by FTIR, TGA and

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C CP/MAS

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NMR analysis. A chromatographic operation was carried out with the raw Phyllanthus niruri L. extract directly loaded onto the adsorbent, and one-step capture of corilagin had been realized. The ethanol aqueous solution of 20%(w/w) was favorable for

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corilagin elution with recovery and purity of 49.4% and 64.8%, respectively. The adsorption kinetic experiments showed that the pseudo-second order model provided

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a good fit with the data, but the values of equilibrium adsorption capacity deviated notably, revealing the complexity of the adsorption mechanism. It was considered that the formation of inclusion complex corilagin/β-CD played a significant role on

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corilagin adsorption and recovery, which was illustrated by the inclusion property investigation. The inclusion complex preferred 1:1 stoichiometry and the association

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constant was determined as 1.69×103 L⋅mol-1 at 298 K, indicating the β-CD cavity showed excellent affinity to corilagin. Moreover, there was evidence that the crystal

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pattern of β-CD-PU changed when it adsorbed corilagin by XRD analysis, which indirectly revealed the mechanism of adsorption. These results suggested that

β-CD-PU adsorbent was a promising candidate for the recovery and enrichment of corilagin or other polyphenolic natural products from plant materials. Keywords: Cyclodextrin polyurethane; Corilagin; Phyllanthus niruri L.; Adsorbent; Chromatography

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1. Introduction

β-Cyclodextrin (β-CD) is an oligosaccharide containing seven D-glucopyranose

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units all with α-(1→4) glucosidic bonds [1]. β-CD molecule possesses a cavity with the diameter of 0.60~0.65 nm that is suitable for the inclusion of various guest

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compounds as they insert into the cavity partially or entirely [2,3]. β-CD also has large amount of hydroxyl groups on the outer surface of the cyclic shape molecule

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that can be reacted with many crosslinking reagents to prepare cyclodextrin polymers (CDPs). CDPs preserve the property to encapsulate organic molecules due to the

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dramatic hydrophobic hollow cavity that belongs to the β-CD molecule [4]. Consequently CDPs may act as adsorbents suitable for specific applications in chromatographic separation, water treatment and pharmacy [5-7].

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Diisocyanates are a series of high-active crosslinker that are prone to react with the CDs’ hydroxyl groups to form CD-polyurethane (CD-PU) polymers. 1,6-Hexamethylene diisocyanate (HDI) and toluene-2,4-diisocyanate (TDI) are the

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most commonly used crosslinkers to prepare aliphatic- and aromatic-based

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polyurethane copolymers, respectively. The reactions are usually carried out in the solvent pyridine, DMF or DMSO, and it will be accelerated by conventional heating

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or microwave-assisted method [8]. Most of these polymers used are amorphous granules in shape ground from the large blocks. When filled in the column, these granules are close packed and compressed in high flow rate, resulting in high bed pressure drop. As a comparison, the spherical beads show good rigidity and may prevent excessive pressure drop in the column. Generally, these spherical beads can be obtained by the reversed-phase suspension crosslinking technique [9]. In this method, the hydrophilic monomers or pre-polymers are dispersed by comminuted into the hydrophobic solvents to form the embedded droplets, and afterward solidification occurs to the droplets by adding crosslinkers [10]. The stabilizer and porogenic agents are also added to improve the shape and structure of the beads, and the size distribution of the final beads can be controlled by the phase composition, the speed of agitator and the concentration of stabilizer used [11]. These spherical beads are specially designed for chromatographic usage. The CD-PU polymers retain the native CD moiety with dramatic host-guest recognition and the carbamate group in their skeleton [12]. They exhibit strong interactions with the compounds through π-π stacking, hydrogen bonding and even pore filling [13,14], therefore they are used for the separation of many organic 4

ACCEPTED MANUSCRIPT compounds. One of the most important applications is to adsorb phenols and their derivatives from aqueous solution [14,15]. It is considered that the phenol molecules may both entrapped into the CD cavities and inserted in the polyurethane skeleton, the

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latter is probably attributed to the hydrogen bonding between phenolic hydroxyl and N―H bond in the carbamate groups [16].

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The high affinity to phenolic structure will make CD-PU a potential adsorbent for many phenol-contained natural products. There have been large amounts of researches

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focused on the inclusion properties of natural polyphenolic compounds with CDs until now [17]. They provide the feasibility of further study on their separation with CDPs.

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In the present work, a novel β-CD-PU spherical adsorbent will be produced by the reversed-phase suspension crosslinking technique and used to enrich corilagin from the raw extract of the herb Phyllanthus niruri L. with chromatographic operation

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(Scheme 1). Corilagin (β-1-O-galloyl-3,6-(R)-hexahydroxydiphenoyl-D-glucose) (Fig. 1) is a polyphenolic gallotannin contained in the plant Phyllanthus niruri L. [18], Dimocarpus longan L. [19], Geranium sibiricum L. [20], Lumnitzera racemosa L.

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[21], Terminalia catappa L. [22], etc. It acts as a bio-antioxidant to scavenge

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superoxide or peroxyl radicals in the organisms [23], and shows hepato-protective [22], anti-inflammatory [24,25], anti-tumor [26], and anti-hyperalgesic activity [18].

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Thanks to its versatile activities, it is a promising compound for clinical usage. Corilagin is usually obtained by solvent extraction from the herb plants. Sometimes enhanced techniques, like high pressure, microwave-assisted and enzymatic method is used to increase the extraction yield [20,27]. The further purification includes repetitive solvent extraction and chromatographic operation, and corilagin is enriched and purified stepwise during these tedious traditional procedures. The β-CD-PU spherical adsorbent will be a powerful candidate for corilagin separation instead. The properties of this adsorbent are characterized, and the feasibility of one-step enriching of corilagin will be evaluated as well. Moreover, the inclusion complex of corilagin with β-CD will also be studied, and the association constant of the inclusion complex will be determined to preliminarily discuss the adsorption mechanism of corilagin with the home-made β-CD-PU adsorbent. (Scheme 1. The spherical β-CD-PU adsorbent prepared by the reversed-phase suspension crosslinking for the chromatographic enrichment of corilagin from Phyllanthus niruri L.) (Fig. 1. The molecular structure of corilagin.)

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ACCEPTED MANUSCRIPT 2. Experimental 2.1. Materials The plant Phyllanthus niruri L. was picked from Kulangsu Island, Amoy, China,

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identified by Fujian Institute of Subtropical Botany. The whole plant was washed with deionized water, dried in the air and pulverized by a disintegrator and screened with

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standard sieves of 80 mesh. The fine powders were stored in sealed vessels and kept in the refrigerator at 4 oC .

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β-CD (DRAMAX®W7 Pharma) was provided by Maxdragon BioChem. Ltd. (Guangzhou, China). Corilagin (≥98%) was purchased from Shanghai DINGJIE

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Biotechnology Co., Ltd. (Shanghai, China). High-speed vacuum pump oil GS-1 was provided by Beijing SIFANG Special Oil Factory (Beijing, China). The

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nano-Mg(OH)2 (≥95%) with particle diameter not more than 200 nm was obtained from Hangzhou WANJING New Material Co., Ltd. (Hangzhou, China). Glycerol (≥99%), paraffin liquid and tween-80 were purchased from XILONG Chemical Co.,

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Ltd. (Shantou, China). Hexamethylene diisocyanate (HDI, ≥99.5%) and polyethylene

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glycol 1000 (PEG1000) were provided by Gracia Chemical Reagent Co., Ltd. (Chengdu, China). Dimethyl sulfoxide (DMSO, ≥99.5%) and orthophosphate (H3PO4,

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guaranteed reagent) were purchased from Sinopharm Chemical Reagent Co., Ltd. (shanghai, China). Acetonitrile and methanol obtained from Tedia Company Inc. (Fairfield, US) were of HPLC grade filtered with membrane of 0.22 µm. Other chemicals used are of reagent grade or higher quality. 2.2. Preparation of β-CD-PU adsorbent The β-CD-PU adsorbent was prepared by the reversed-phase suspension crosslinking technique [9]. Generally, 4 g β-CD, 2 g PEG1000, and 1 g glycerol were dissolved in 20 ml DMSO at 50 oC, and 8 ml HDI was added dropwise within 20 min with stirring. The mixture was kept stirring for a further 2 h at 50 oC. After that, 1 g nano-Mg(OH)2 powder, 0.5 g tween-80 and the extra 1 ml HDI were added, and the mixture was promptly dispersed in the mixed-oil containing 50 g paraffin liquid and 150 g pump oil GS-1 under agitation at 500 rpm. The reaction continued for 8 h at room temperature. Then the beads were filtered off and washed with 300 ml petroleum ether (b.p. 60-90 oC), and successively treated with 300 ml HCl (0.5 mol·L-1) to remove Mg(OH)2 that was filled inside the beads. Finally the beads were washed with water and screened with standard sieves. The component with a particle

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ACCEPTED MANUSCRIPT size range of 300-900 µm was retained for further use. 2.3. Characterization of β-CD-PU adsorbent

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The particle size distribution and mean particle diameter were determined by laser particle size analyzer MASTERSIZER 2000 (Malvern Instruments, UK).

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The surface morphology of adsorbent was observed with S-4800-ESEM scanning electron microscope (SEM, Hitachi Instruments, Japan). The emission current was 4.3

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mA, and the accelerating and decelerating voltage were 5 kV and 0 V respectively. The magnification was 1800. The sample was coated with gold for 2 min in a vacuum

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and the current was set as 10 mA.

Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a FTIR-8400S spectrometer (Shimadzu Instruments, Japan). The samples were mixed

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with KBr, ground and compressed into transparent disks prior to FTIR characterization. The samples were scanned from 4000 to 400 cm-1 using an average of 20 scans with a resolution of 1 cm-1.

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Thermogravimetric analysis (TGA) and differential thermal analysis (DTA) were

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carried out on an instruments DTG-60H (Shimadzu Instruments, Japan) with high-pure nitrogen purge. The flow rate of N2 was 50 ml⋅min-1. The sample (ca. 8 mg)

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was loaded in an open platinum pan and heated from room temperature to 800 oC at 10 oC⋅min-1. The

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C cross-polarization/magic angle spinning (CP/MAS) NMR spectrum was

obtained by using BRUKER AVANCE III 400 MHz solid-state NMR spectrometer from Bruker Corporation (Fällanden, Switzerland) with a 4 mm rotor at the rotate frequency of 12 kHz. Other parameters were as follows: 1000 scan times, sweep width = 50 kHz, delay time = 2 s, and contact time = 2 ms. The external reference compound was adamantane for 13C spectrum (38.1 ppm). Powder X-ray diffraction (XRD) was conducted with a RIGAKU Smartlab X-ray diffractometer (Rigaku, Tokyo, Japan) with Cu-Kα radiation (λ = 1.54 Å, 40 kV and 30 mA) at 25 oC. The samples were analyzed in the 2θ angle range from 5o to 70o with a fixed scan rate of 0.5o 2θ/s. Particularly, 40 mg β-CD-PU sample was put into 2 ml corilagin solution (5 mg⋅ml-1) with shaking in 2 h at 25 oC. The solid sample was filtered off and dried in a freeze drier. All powder samples were ground and then mounted on glass sample holders. The water content and shrinkage of the β-CD-PU adsorbent were measured with the method as reported [28]. The pore structural parameter, including specific surface 7

ACCEPTED MANUSCRIPT area and mean pore diameter, were measured with JW-BK nitrogen adsorption specific surface area analyzer (JWGB Instruments, China).

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2.4. Determination of association constant of the complex corilagin⊂β-CD The experiment was carried out in a 1 cm length cuvette with the mixture of corilagin and β-CD. The starting concentration of corilagin was 0.197 mmol⋅L-1, and

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the concentration of β-CD changed every time. The absorbance A was measured by a

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UV-1800 ultraviolet-visible spectrophotometer at 270 nm.

Suppose β-CD and corilagin form 1:1 complex, the inclusion association constant

K=

[CD-Cor ] [CD][ Cor ]

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K can be described as [29]

(1)

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where [CD], [Cor], and [CD-Cor] represent the equilibrium concentration of β-CD, corilagin and their complex, respectively. From Eq. (1), the starting concentration of

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β-CD ([CD]0) and corilagin ([Cor]0) is expressed as

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[CD]0 = [CD] + [CD-Cor] = [CD] + K [CD][Cor]

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[Cor]0 = [Cor] + [CD-Cor] = [Cor] + K [CD][Cor]

(2) (3)

Eq. (3) can be rearranged as

[Cor] =

[Cor]0 1 + K [CD]

(4)

In general, the starting corilagin concentration [Cor]0 is set much lower than that of

β-CD ([CD]0), so there is the relation of [CD]0>>[Cor]0. β-CD and corilagin are supposed to form 1:1 inclusion complex. When all corilagin molecules form inclusion complexes with β-CD, only a small part of β-CD molecules will be consumed, therefore the concentration of β-CD hardly changes, i.e. [CD]≈[CD]0 under the equilibrium state. So the concentration of corilagin [Cor] can be obtained from Eq. (4) as

[Cor] =

[Cor]0 1 + K [CD]0

(5)

According to Lambert-Beer's law, when the length of cuvette L is 1 cm, the absorbance A can be expressed as A = ε [CD-Cor]L = ε K [CD][Cor] = 8

ε K [CD]0 [Cor]0 1 + K [CD]0

(6)

ACCEPTED MANUSCRIPT where ε represents the molar extinction coefficient (L⋅mol-1⋅cm-1). Eq. (6) can be rewritten as the double-reciprocal form 1 1 1 1 = + A ε K [Cor]0 [CD]0 ε [Cor]0

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(7)

The slope and intercept values can be conveniently obtained from the plot of 1/A vs.

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1/[CD]0, and the association constant K is further calculated.

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2.5. Adsorption kinetics measurement

The adsorption kinetics of the β-CD-PU adsorbent toward corilagin was

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measured using a batch method. In general, 0.5 g hydrated β-CD-PU adsorbent was added into 50 ml corilagin aqueous solution with the initial concentration of [Cor]0 in

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a conical flask, and continuously stirred at 180 rpm in the oscillator at the temperature of 20 oC, 25 oC and 30 oC. 0.5 ml aliquots were removed at variable time intervals and immediately centrifuged at 8,000 rpm for 15 s. The supernatant was diluted and

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analyzed by a UV-1800 ultraviolet-visible spectrophotometer (Mapada Instruments, China) at 270 nm. The concentration of corilagin [Cor] was calculated from the

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calibration curve. The kinetic plots was represented with the relative concentration of corilagin [Cor]/[Cor]0 and the adsorption capacity Q vs. time. The adsorption ratio

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was calculated as

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Adsorption ratio = 1 − [ Cor ] / [ Cor ]0 × 100%

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The adsorption capacity Q was calculated as

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Q = [ Cor ]0 − [ Cor ] V0 / W

(9)

where V0 was the initial volume of corilagin solution and W was the mass of adsorbent. The data were fit by using pseudo-first order (PFO) and pseudo-second order (PSO) model according to Ref [30] as described by Eq. (10) and (11), respectively. Q = Qe (1 − e − k1t )

(10)

Qe2 k2t 1 + Qe k2t

(11)

Q=

where Qe represented the adsorption capacity at equilibrium. k1 and k2 were the rate constant for PFO model and PSO model, respectively. 2.6. Extraction and chromatographic operation

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ACCEPTED MANUSCRIPT A batch of 53.4 g Phyllanthus niruri L. powders was soaked in 250 ml 95%(w/w) ethanol with ultrasonic oscillating at 25 oC for 30 min. The mixture was filtrated and the filter residue was re-soaked twice with 50 ml 95% ethanol each time. The merged

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filtrate was evaporated in a Hei-VAP rotary evaporator (Heidolph Instruments, Germany). The evaporated residue obtained was re-dissolved in the minimum

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quantity of deionized water with ultrasonic oscillating, filtrated through 0.22 µm membrane prior to chromatographic operation.

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The chromatographic operation was carried out in a short glass column (φ 10 mm × 15 cm) connected to an ÄKTA Explorer 10 chromatographic system (GE Healthcare & Amersham Biosciences, Sweden). Proper column vertical alignment was assured in

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all experiments. A batch of 8 ml β-CD-PU adsorbent was packed in the column and sedimented under the column pressure of 0.3 MPa. The final sedimented bed height

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was 10.1 cm at the set flow rate 1 ml⋅min-1. After equilibration with deionized water, the raw extract solution 60 ml was loaded and adsorbed. Afterward the packed-bed was washed with deionized water until the absorbance at 270 nm in the effluent

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returned to the base line. The corilagin adsorbed was successively eluted by 20%(w/w)

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and 95%(w/w) ethanol aqueous solution. The effluents for each stage were collected respectively for further HPLC analysis. Finally, the adsorbent was cleaned with 0.1

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mol⋅L-1 NaOH and afterwards regenerated with deionized water. 2.7. HPLC analysis

The contents of corilagin in the raw extract or eluent were determined by HPLC analysis. The samples were concentrated, dried below 45 oC, re-dissolved and then diluted to 10 ml with 50%(w/w) methanol aqueous solution. Any aliquot of each sample was filtered through 0.22 µm polyvinylidene fluoride (PVDF) membrane prior to HPLC analysis. The Agilent 1200 HPLC system (Agilent Instruments, US) consisted of a quaternary pump, a variable wavelength UV-visible detector and a chromatography workstation. An Agilent C18 HPLC column (250 mm × 4.6 mm I.D., 5 µm) placed in a 25 oC thermostat was used. The flow rate was set as 1.0 ml·min-1 and the injection volume was 20 µl for each sample. The mobile phase was a mixture of acetonitrile and 0.1%(w/v) H3PO4 with the initial ratio of 15:85(v/v) within the first 10 min. A gradient elution program was adopted by increasing acetonitrile concentration from 15%(v/v) to 20% within the next 5 min, from 20% to 25% within the third 5 min, and maintaining the ratio 25:75 after that. The detection wavelength was set as 280 nm. 10

ACCEPTED MANUSCRIPT The external standard method was used to determine the amount of corilagin, and the regression equation were obtained as Y=2.75×104C

(12)

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with a coefficient of determination (R2) of 0.99. Where C represented corilagin concentration (mg⋅ml-1) and Y was the peak area (mAU·s). The retention time of

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corilagin was 8.71 min.

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3. Results and discussion

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3.1. Preparation of β-CD-PU spherical adsorbent

The spherical β-CD-PU adsorbent is specially designed for chromatography usage. For this purpose, the reversed-phase suspension crosslinking technique is

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introduced. HDI is a commonly used crosslinking reagent that forms carbamate structure in the polymer chain between CD molecules. PEG1000 is added in order to improve the mechanical strength of the beads, while nano-Mg(OH)2 acts as the

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porogenic agent. The pre-crosslinking reaction proceeds rapidly and the mixture turns

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to a viscous milk-white solution.

The dispersing medium used is very similar to our previous work [9]. It is found

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that the mass ratio paraffin liquid to pump oil GS-1 of 1:3 is suitable for the suspension crosslinking and further solidification to form fine spherical beads. The size distribution of the beads is strongly influenced by the agitating speed. The bead size diminishes as agitation becomes more violent, but at high agitation speed the beads becomes unexpected irregular shapes. As a result, the proper agitating speed is chosen as 500-600 rpm.

3.2. The main properties of β-CD-PU adsorbent The appearance and the surface SEM photograph of the β-CD-PU adsorbent are showed in Fig. 2. It can be seen that the adsorbent has fine spherical shape without conglutinated beads. The surface of adsorbent prepared is very rough with many protuberant lumps, folds and a few large pores, which may be caused by the shrinkage of the polymer skeleton. (Fig. 2. The morphology of β-CD-PU adsorbent (a) and the SEM photograph (b).) The particle size of the adsorbent follows good logarithmic normal distribution 11

ACCEPTED MANUSCRIPT after sieving (Fig. 3). The adsorbent has a particle size range of 300-900 µm and the mean diameter 570 µm.

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(Fig. 3. Size distribution of β-CD-PU adsorbent.) The water content and shrinkage of β-CD-PU adsorbent are 73.1% and 64.3%

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respectively, indicating the adsorbent has good water absorbing capacity. In wet condition, so large amount of water molecules may distribute around the polymer

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skeleton and occupy the CD inner cavity of the adsorbent. All these water molecules will escape during the drying process, bringing about the remarkable shrinkage of the

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polymer skeleton. As a result, the pores will become small in dry condition. Through the nitrogen adsorption-desorption experiment, it is found that β-CD-PU adsorbent

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belongs to the mesoporous material with its mean pore diameter of 3.41 nm. Most of the pores are amorphous, with a wide range of pore distribution from 3 nm to 10 nm and even larger, as described in Fig. 4(a). The mean pore diameter of the spherical adsorbent is much smaller than that of CD-PUs reported (14~29 nm) [31]. However, it

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is noted that a kind of β-CD polymer crosslinked by HDI (1:8, mol/mol) reported by

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Ma and Li has the pore diameter less than 2.5 nm [32]. The pore size can be influenced and regulated by the additives, such as PEG. The PEG chains are probable

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to intertwine with the polyurethane skeleton to form compact network and the degree of crosslinking rises as a result. The porogenic agent nano-Mg(OH)2 can generate large pores but it does not change the structure of the inner meso-pores. (Fig. 4. The nitrogen gas adsorption and desorption experiment results. (a) Pore distribution of β-CD-PU adsorbent; (b) The N2 adsorption and desorption isotherm of β -CD-PU.)

The specific surface area can be calculated as 2.053 m2⋅g-1 based on the assumption of monolayer adsorption under the condition of middle and low pressure. The value is less than that of CD-PUs crosslinked with 4,4'-diphenylmethane diisocyanate, 1,4-phenylene diisocyanate or 1,5-naphthalene diisocyanate, but higher than the polymers crosslinked with 4,4'-dicyclohexylmethane diisocyanate [31]. Moreover, the specific surface area is strongly connected with the molar ratio diisocyanate:CD. Increasing this ratio can enlarge the surface area of adsorbent, and the optimal ratio exists; but on the contrary, excessive crosslinking may reduce the surface area. In this work, the molar ratio of HDI to CD is 16:1, the specific surface area is much higher than that of adsorbent with the ratio 2:1, but lower than that of 8:1 12

ACCEPTED MANUSCRIPT [16], indicating high degree of crosslinking for β-CD-PU synthesized in our work. It is interesting that capillary condensation phenomenon will happen when the relative partial pressure of N2 increases above 0.85 (Fig. 4(b)). It illustrates multilayer

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adsorption will dominate under high pressure, and in this condition, N2 molecules will closely packed inside the pores. Another interesting phenomenon is that adsorption

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hysteresis occurs at the relative partial pressure above 0.6, that is, the desorption isotherm is upper compared with the adsorption isotherm (as shown in Fig. 4(b)).

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Unfortunately, the mechanism is not clear, though it is attributed to the complicated structure of pores.

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3.3. FTIR spectrum

The FTIR spectra of β-CD and β-CD-PU are depicted in Fig. 5. For both samples,

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the wide peak nearby 3343 cm-1 is well-known for symmetric and antisymmetric OH stretching modes. It is so strong that it covers the stretching bands of NH group. The asymmetric and symmetric methylene stretching bands are located at 2932

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and 2858 cm-1 respectively, and the stretching bands of the methylene units become

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sharper when β-CD is co-crosslinked with PEG. The peaks at 1627 and 1570 cm-1 are both attributed to the absorbance of the urethane moiety, the former corresponds to the

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stretching vibration of carbonyl group and the latter corresponds to the scissoring bending vibration of NH group. The peak at 1259 cm-1 is related to the vibration absorbance of the C=OC bonds between β-CD and the carbonyl group of the urethane moiety. The appearance of these three peaks as new signals that are not observed in the β-CD demonstrates that HDI has reacted with the hydroxyl groups of

β-CD to form urethane moiety in the adsorbent β-CD-PU. Moreover, the peak at 1033 cm-1 is in agreement with the stretching vibration of COC bond of β-CD molecules and PEG. (Fig. 5. FTIR spectra of β -CD and β -CD-PU.)

3.4. Thermogravimetric and differential thermal analysis Fig. 6 describes the TGA and DTA curves of the adsorbent β-CD-PU. The TGA curve obtained is quite similar to that of β-CD/HDI polymer reported [8]. There is weight loss of ca. 6% at temperatures lower than 200 oC mostly due to evaporation of water adsorbed around the polymer skeleton. As the temperature increases, the remarkable weight loss of β-CD-PU is observed within the range of 280-320 oC due 13

ACCEPTED MANUSCRIPT to the disruption of β-CD according to Hedges et al. [33] The polyurethane moiety will decompose when the temperature further increases above 300 oC, and the DTA curve appears a notable exothermic peak at around 325 oC correspondingly [34]. A

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small peak at 400 oC in the DTA curve may be related to the weight loss of PEG, but not remarkable in TGA curve. Overall, the thermal stability of the adsorbent is good

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enough for chromatography usage below 200 oC.

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(Fig. 6. TGA and DTA curves of β -CD-PU.)

3.5. 13C CP/MAS NMR spectrum

therefore, the

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The spherical β-CD-PU adsorbent is insoluble in water or any organic solvents, C CP/MAS NMR was used instead of conventional NMR

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measurement in the solution. The spectrum is shown in Fig. 7, and the assignments have been made by directly labeling the position of the carbon in the specific molecule of CD (C), HDI (H) and PEG (P) in the spectrum. The remarkable signal at

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159.56 ppm is assigned to the urethane carbon atoms of HDI (H1). The strong peaks at

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42.58 and 30.49 ppm are assigned to the six methylene groups in the molecule HDI [8]. The former shifts to lower field induced by the carbamate group, and the latter is

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an overlapped peak containing H3 and H4 of HDI. These strong signals confirm the occurrence of crosslinking reaction, and indicate the high-content of urethane moiety in β-CD-PU adsorbent.

(Fig. 7. 13C CP/MAS NMR spectrum of β -CD-PU. C = CD; H = HDI; P = PEG. Each subscript corresponds to the position of the carbon in the specific molecule.)

According to Endo et al. [35], the

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C NMR chemical shifts of six carbon atoms

in β-CD molecule are 102.58 (C1), 72.67 (C2), 73.89 (C3), 81.94 (C4), 72.89 (C5), and 61.17 ppm (C6). The chemical shift of 101.36 ppm is assigned to the C1 of β-CD, which approximately agrees with the value reported. However, the chemical shifts of C2, C3 and C5 in β-CD molecule have been overlapped with each other, and thus only one peak appears at 70.79 ppm. This strong peak also covers the methylene group signals in PEG chain (P2 and P3) at around 70 ppm [36,37]. The peak at 63.63 ppm corresponds to both C6 in β-CD molecule and the PEG’s methylene group that is connected with the end hydroxyl group (P1). Moreover, the chemical shift of 81.54 ppm is related to the C4 of β-CD. The three peaks are partially overlapped, since the resolution of CP/MAS is much lower than the conventional NMR measurement in the 14

ACCEPTED MANUSCRIPT solution. 3.6. XRD analysis

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The XRD pattern of the β-CD-PU adsorbent shows distinctive peaks at about 2θ = 10.2o, 20.9o, 23.3o and 38.1o (Fig. 8). The first two 2θ values are quite similar with

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that reported by Mohamed et al. [30], and the peak at 2θ = 23.3o is assigned to the PEG moiety in the polymer [38]. The representative sharp peaks of β-CD from 5.0o to

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28.0o (main 2θ values are 9.0 o, 10.6 o, 12.4 o, 15.4 o, 17.1 o, 18.9 o, 19.5 o, 22.7 o etc.) hardly appear in the spectrum of β-CD-PU, and such broad diffraction bands indicate

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the non-crystalline pattern of this solid polymer.

(Fig. 8. XRD spectra of β-CD-PU, corilagin, the mixture of them and the β -CD-PU after

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adsorbing corilagin.)

The interaction between β-CD-PU adsorbent and the adsorbate corilagin is also

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investigated by XRD analysis (Fig. 8), and the XRD spectra may provide some

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beneficial information to the adsorption process. When 40 mg adsorbent is physically mixed with 10 mg corilagin in solid, the superposition of corilagin specific peaks

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from 12o to 30o is observed, illustrating no remarkable interaction between them in the solid state. However, the crystal pattern of corilagin changes after being adsorbed onto

β-CD-PU, for the reason that nearly all corilagin peaks disappear in the spectrum. It is noted that the concentration 5 mg⋅ml-1 is very high as for corilagin, and further raising its concentration is quite difficult. So it reveals that the crystal pattern change of corilagin is regardless of its concentration. Moreover, the diffraction band in the range of 20o to 24o in spectrum (c) notably changes and the small peak at 2θ = 38.1o disappears, compared with the spectrum (a). The phenomena indicate that the crystal type of adsorbent-adsorbate complex shows some difference from the original adsorbent, and the polymer may become more amorphous after adsorption. The crystal pattern change of adsorbent relates to the amount of the absorbed corilagin. In our experiments, the amount of corilagin adsorbed was considerable, so the XRD spectrum (c) showed notable difference from spectrum (a). However, when β-CD-PU adsorbs corilagin from diluted solution (0.1 mg⋅ml-1, data not shown), its spectrum will be nearly the same as spectrum (a). It is proposed that this change may result from the existence of interactions between adsorbent and adsorbate, probably due to the formation of binary inclusion complex, and the amount of complex will directly determine proportion of the crystal pattern change. 15

ACCEPTED MANUSCRIPT 3.7. Association constant of the binary inclusion complex β-CD/corilagin Thanks to the dramatic property derived from CD’s hydrophobic hollow cavity, CD oligomers or polymers can act as excellent adsorbents for some natural products

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by forming inclusion complexes with them [39-41]. It is reported that β-CD shows good affinity to polyphenolic compounds, e.g., Xu et al. determined the association

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constant of epigallocatechin gallate⊂β-CD complex by NMR spectroscopy as (2.514±0.008)×103 L⋅mol-1 at 308 K [39]. Cai et al. pointed out that the galloyl ester

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played an important role in the recognition and inclusion process of polyphenol with

β-CD cavity, especially galloylation occurred at C-3 for phenolic flavan-3-ols [42].

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Corilagin is a typical polyphenolic glucoside with three galloyl ester groups attaching to the glucose moiety (Fig. 1). When it is encapsulated with β-CD, the value of the association constant is calculated as 1.69×103 L⋅mol-1 at 298 K with R2 > 0.99 based

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on Eq. (8). The high coefficient of determination (R2) value demonstrates the linear relation of 1/A vs. 1/[CD]0, indeed, it is confirmed that β-CD forms 1:1 complex with corilagin. The association constant value obtained in this work is just a bit lower but

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retains in the same order of magnitude compared with the value of epigallocatechin

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gallate reported, indicating the β-CD cavity shows excellent affinity to galloyl ester groups. Associating with the XRD spectrum change of β-CD-PU after adsorbing

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corilagin (Fig. 8), we propose that the inclusion complex formation plays an important role during the adsorption process. 3.8. Adsorption kinetics

The adsorption kinetics of corilagin adsorbed onto β-CD-PU is described in Fig. 9. In Fig. 9(a), the plots of [Cor]/[Cor]0 vs. time show that the adsorption rate is very fast during the initial process (within 10 min) but becomes slower later. It reaches equilibrium at about 120 min at 25 oC, regardless of different concentration of corilagin, and the equilibrium adsorption ratios are very close with each other at about 48%. Unlike the adsorption ratios, the equilibrium adsorption capacity Qe gradually increases with the increase of corilagin concentration. The values of Qe are listed in Table 1. Adsorption process is notably influenced by temperature, as shown in the plots of [Cor]/[Cor]0 vs. time in Fig. 9(b). As temperature rises to 30 oC, the adsorption process accelerates in the initial stage, and it reaches equilibrium at about 90 min, less than the value at 25 oC. As a comparison, it takes much longer time to reach equilibrium at 20 oC (more than 150 min), indicating a considerable decrease of the 16

ACCEPTED MANUSCRIPT adsorption rate at lower temperature. It is interesting that three kinetic curves intercross near the time point of 40 min, and then the positions of the curves are inverted dramatically when adsorption proceeds. The final equilibrium adsorption

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ratios are 56%, 48% and 45% at the temperature of 20 oC, 25 oC and 30 oC, respectively. It demonstrates that higher temperature will accelerate the diffusion

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between the solute and adsorbent, resulting in higher adsorption rate. However higher temperature is unfavorable for Qe, a thermodynamic quantity, indicating the

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adsorption of corilagin on β-CD-PU is an exothermic process.

(Fig. 9. Adsorption kinetics of corilagin adsorbed on β -CD-PU adsorbent. (a) Different initial

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corilagin concentration at 25 oC; (b) Different temperature as corilagin concentration of 0.1

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mg⋅ml-1. The plots of Q vs. time are fitted with pseudo-second order (PSO) model.) (Table 1. Adsorption kinetic parameters calculated by using pseudo-first order (PFO) or pseudo-second order (PSO) model.)

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The parameters including Qe, k1 and k2 are calculated by using PFO and PSO

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model for comparison (Table 1). In general, the PSO model provides a better fit with the experimental results, as the R2 values are higher than the values calculated by PFO

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model. The results are consistent with that of HDI-2-D and HDI-2-R reported by Wilson’s group [30]. It is thought that crosslinked CD polymers usually have at least two types of adsorption sites. The adsorbate locates either inside the CD cavity or in the polymer skeleton interstitial region [43], and the PSO model is more suitable to describe the adsorbent with two types of biding sites. However, the values of equilibrium adsorption capacity obtained in our experiments (Qe,exp) are not consistent with that calculated using both models. The Qe,exp values are between the values calculated from the two models, but more close to that of PFO model (shown in Table 1). This phenomenon reveals the complexity of adsorption mechanism, and it may correlate with the type of adsorption sites and the pore structure. Probably, the two types of adsorption sites show different adsorption rate toward corilagin, and one of them play a major role for the adsorption. As mentioned in Section 3.2, the adsorbent β-CD-PU is mesoporous material with the mean pore diameter of 3.41 nm and a wide pore distribution. Corilagin is a bulky molecule with three aromatic rings and one glucose moiety (Fig. 1). So the adsorption rate will be affected due to small size of the meso-pores that cannot be regulated by porogenic agent. In the initial stage of adsorption process, corilagin 17

ACCEPTED MANUSCRIPT molecules promptly travel through the large pores and then bind to the interstitial region of polymer skeleton. When the bulky molecules transport into inner pores, the diffusion rate reduces, and adsorption decelerates as a result. As a comparison, a type

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of CD-epichlorohydrin polymer CroCD-TuC had larger mean pore diameter of 70 nm, and thus it adsorbed rutin (the molecule size is approximately equal to corilagin)

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much faster than β-CD-PU [41]. Therefore it is speculated that the diffusing limitation plays a significant role during adsorption process in our opinion, and the pore size of

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the adsorbent will greatly affect the adsorption performance.

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3.9. Enrichment of corilagin from Phyllanthus niruri L. extract The chromatogram for the separation of corilagin from Phyllanthus niruri L. is shown in Fig. 10, and the HPLC chromatograms of each elution fractions and HPLC

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analysis results are described as Fig. 11 and Table 2, respectively. There are large amount of components in the raw extract from Phyllanthus niruri L., and the percentage of corilagin is 9.9% among them from HPLC analysis. The dark yellow

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raw extract with total corilagin concentration 5.87 mg·ml-1 is directly loaded onto the

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column. Although the UV-absorbance of overload effluent is very high, corilagin is almost undetectable in the effluent according to Fig. 11(b). It indicates that corilagin is

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adsorbed effectively by β-CD-PU adsorbent, probably due to the formation of inclusion complex with β-CD cavity (as mentioned above) or hydrogen bonding with amide group of urethane skeleton [16]. Another reason may attribute to the micro amorphous pore of adsorbent (as mentioned above) that will cause a long eluting path of such small molecules. A part of water soluble impurities can be washed out from adsorbent and thus the mixture adsorbed is separated stepwise. Moreover, corilagin may re-equilibrated between solid and aqueous phases since the inclusion of corilagin in β-CD cavity is reversible, as a result, a significant amount of corilagin (ca. 27.6%) is also detected in the effluents in washing step (Fig. 11(c)). (Fig. 10. Elution chromatogram for the enrichment of corilagin.) (Table 2. HPLC analysis results of corilagin from each collected eluent.) (Fig. 11. HPLC chromatograms of raw extract (a) and the eluent (b)~(e) from the column.)

The corilagin adsorbed is prone to be eluted by organic solution, and ethanol is preferred as the eluent due to its cheapness and low toxicity. It is found in Fig. 11(d) that 20%(w/w) ethanol aqueous solution is favorable for the eluting operation. The recovery of corilagin in this step is 49.4%. The purity and purification factor of 18

ACCEPTED MANUSCRIPT corilagin can reach to 64.8% and 6.55 respectively, calculated by peak-area normalization method. It means that one-step capture of corilagin has been realized. However, the amount of impurity will gradually increase as the elution being carried

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out. It illustrates that the elution process may be adsorption kinetics controlled. Ethanol of higher concentration (such as 95%(w/w)) is not suitable for corilagin

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elution since many lower-polarity components are eluted as well (Fig. 11(e)), though a further amount of corilagin (ca. 17.5%) is determined in the eluent.

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Due to the strong solubility of many polyphenolic compounds, NaOH solution is used for column cleaning. These polyphenols dissociate and further are oxidized in

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NaOH solution accompanied by color deepened from dark yellow to brown, thus the absorbance at 270 nm is abnormally high in Fig. 10. The eluent is not collected for the reason of corilagin decomposition in alkali solution. After that, deionized water (at

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least 20 column volume) is loaded through the whole system to clean the channels and regenerate the adsorbent for next chromatographic operation. In the second operation, the recovery of corilagin was 48.1%, and the purity and

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purification factor were 48.8% and 5.31, respectively. The column efficiency shows a

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bit lower than the first operation. It is found that the adsorbent turns yellow, probably due to the pigmentation effect. The pigments from raw extract are insoluble in organic

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solvents or NaOH solution. It is quite difficult to wash a trace of pigment off the adsorbent, therefore the column efficiency will be in decline as repeated use. Nevertheless, the adsorbent can be off-line cleaned by ultrasonic oscillating in organic solvents, and then washed several times, saved in 20% ethanol solution.

4. Conclusions

A kind of spherical β-CD-PU adsorbent with the particle size range of 300-900 µm has been prepared using reversed-phase suspension crosslinking technique. It is a meso-pore material with mean pore diameter of 3.41 nm and a wide range of pore distribution from 3 nm to 10 nm. The FTIR, TGA and

13

C CP/MAS NMR spectra

have confirmed the formation of carbamate group, and the retention of β-CD in this adsorbent. The XRD spectrum has revealed the existence of strong interactions between corilagin and β-CD-PU. It is speculated as the formation of inclusion complex. The association constant of the binary inclusion complex corilagin⊂β-CD has been determined as 1.69×103 L⋅mol-1 at 298 K, indicating the β-CD cavity shows excellent affinity to corilagin. The adsorption kinetic data can be well fitted with the pseudo-second order model, but the values of equilibrium adsorption capacity 19

ACCEPTED MANUSCRIPT deviated notably. It is probable that the two types of adsorption sites have different adsorption rate toward corilagin, and one of the sites play a major role for the adsorption. The diffusion of corilagin molecules into inner meso-pores is a

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rate-limiting step during the adsorption process. In this work, one-step capture of corilagin from Phyllanthus niruri L. raw extract

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on home-made spherical β-CD-PU adsorbent has been evaluated and shown to be an efficient operation for primary enrichment. Nearly one half of corilagin can be

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recovered by one chromatographic operation with the purity of 64.8%. These results suggest that β-CD-PU adsorbent is a promising candidate for the recovery and

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enrichment of specific natural products directly from plant materials.

Acknowledgment

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This work was supported by Fujian Provincial Department of Science and Technology (Science Foundation for Young Scientists of Fujian Province, China, 2013J05028), National Natural Science Foundation of China (21576108) and the

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Scheme Captions Scheme 1. The spherical β-CD-PU adsorbent prepared by the reversed-phase

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suspension crosslinking for the chromatographic enrichment of corilagin from

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Phyllanthus niruri L.

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Scheme 1.

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Figure Captions

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Fig. 1. The molecular structure of corilagin.

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Fig. 2. The morphology of β-CD-PU adsorbent (a) and the SEM photograph (b).

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Fig. 3. Size distribution of β-CD-PU adsorbent.

Fig. 4. The nitrogen gas adsorption and desorption experiment results. (a) Pore distribution of β-CD-PU adsorbent; (b) The N2 adsorption and desorption isotherm of

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β-CD-PU.

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Fig. 5. FTIR spectra of β-CD and β-CD-PU. Fig. 6. TGA and DTA curves of β-CD-PU.

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Fig. 7. 13C CP/MAS NMR spectrum of β-CD-PU. C = CD; H = HDI; P = PEG. Each

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subscript corresponds to the position of the carbon in the specific molecule.

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Fig. 8. XRD spectra of β-CD-PU, corilagin, the mixture of them and the β-CD-PU after adsorbing corilagin.

Fig. 9. Adsorption kinetics of corilagin adsorbed on β-CD-PU adsorbent. (a) Different initial corilagin concentration at 25 oC; (b) Different temperature as corilagin concentration of 0.1 mg⋅ml-1. The plots of Q vs. time are fitted with pseudo-second order (PSO) model.

Fig. 10. Elution chromatogram for the enrichment of corilagin. Fig. 11. HPLC chromatograms of raw extract (a) and the eluent (b)~(e) from the column.

26

ACCEPTED MANUSCRIPT Fig. 1.

HO O O

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OH

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OH O

OH

HO

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OH

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HO

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Fig. 2.

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ACCEPTED MANUSCRIPT Fig. 3. 20

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After sieving Before sieving

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1000

Particle size (µm)

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0 100

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Volume (%)

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ACCEPTED MANUSCRIPT Fig. 4.

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0.010 0.008

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(b) Adsorption Desorption

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Quantity Adsorbed (cm g )

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Pore diameter (nm) 10

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∆V/∆(logd) (cm .g .nm )

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Relative N2 partial pressure p/p0

30

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Fig. 5.

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O 1 N

4

N

1

H2

O

2

HO

C2,3,5

O

3

H

P:

3 O

O n-2

P2,3

OH

H1

C4

C6

P1

120

80

δ (ppm)

40

AC CE P

TE

D

MA

160

NU

C1

200

PT

5

RI

*

SC

C:

33

0

ACCEPTED MANUSCRIPT Fig. 8.

PT

SC

RI

Intensity (counts)

(a) β-CD-PU (b) Corilagin / β-CD-PU mixture (c) β-CD-PU adsorbing corilagin (d) Corilagin

(a)

(d)

20

30

40

2θ (degree)

50

60

AC CE P

TE

D

MA

10

NU

(b) (c)

34

70

ACCEPTED MANUSCRIPT Fig. 9.

0.9

10

RI

0.8

8

0.7

6 4

0.6

2 0 20

40

60

80

9 8

0.9

TE

6 5 4

AC CE P

-3

1.0

D

7

-1

Q (10 mmol.g )

120

MA

Time (min)

100

3

0.5

Solid line: Q Dash line: [Cor]/[Cor]0 o 20 C o 25 C o 30 C

0.8 0.7 0.6

2

0.5

1 0

0

20

40

60

0.4 80

100

120

Time (min)

35

140

[Cor]/[Cor]0

0

(b)

1.0

SC

-3

-1

Q (10 mmol.g )

12

Solid line: Q Dash line: [Cor]/[Cor]0

PT

-1

0.05 mg.ml -1 0.10 mg.ml -1 0.15 mg.ml

[Cor]/[Cor]0

14

NU

(a)

ACCEPTED MANUSCRIPT

Overloading

1000

Eluting with 95% ethanol Washing with water

0 0

400

800

1200

NU

Eluting with 20% ethanol

500

SC

4000 1500

RI

Cleaning with NaOH

1600

TE

D

MA

Elution volume (ml)

36

PT

5000

AC CE P

Absorbance at 270 nm (mAU)

Fig. 10.

2000

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

Fig. 11.

37

ACCEPTED MANUSCRIPT

Table Captions Table 1. Adsorption kinetic parameters calculated by using pseudo-first order (PFO)

PT

or pseudo-second order (PSO) model.

AC CE P

TE

D

MA

NU

SC

RI

Table 2. HPLC analysis results of corilagin from each collected eluent.

38

ACCEPTED MANUSCRIPT

Table 1. Adsorption kinetic parameters calculated by using pseudo-first order (PFO) or pseudo-second order (PSO) model. PSO model

Qe,exp* (×10-3 mmol⋅g-1)

Qe (×10-3 mmol⋅g-1)

k1 (min-1)

R2

Qe (×10-3 mmol⋅g-1)

k2 (g⋅mmol-1⋅min-1)

R2

25 25 25 20 25 30

0.05 0.10 0.15 0.10 0.10 0.10

3.87 7.50 11.29 8.78 7.50 6.75

3.63 7.29 10.79 8.36 7.29 6.52

0.1344 0.0717 0.0742 0.0421 0.0717 0.0875

0.9638 0.9833 0.9815 0.9884 0.9833 0.9859

3.98 8.36 12.35 9.82 8.36 7.37

45.32 10.59 7.49 4.93 10.59 15.22

0.9894 0.9961 0.9945 0.9967 0.9961 0.9979

NU

SC

RI

PT

[Cor]0 (mg⋅ml-1)

TE

D

MA

The adsorption capacity at equilibrium obtained in the experiments.

AC CE P

*

PFO model

T (oC)

39

ACCEPTED MANUSCRIPT

Table 2. HPLC analysis results of corilagin from each collected eluent. Raw extract

Overload effluent

Washed by water

20% ethanol elution

95% ethanol elution

Loading volume (ml) Peak Area (mAU) Total corilagin amount (mg) Recovery (%) Purity of corilagin (%) Purification factor

/ 1934.9 352.2 / / /

60 837.2 15.2 4.33 1.4 /

840 1777.6 97.1 27.56 14.3 1.44

500 9558.6 174.0 49.40 64.8 6.55

250 3381.1 61.5 17.47 12.9 1.30

RI

SC

NU MA D TE AC CE P 40

PT

Operation Stage