Accepted Manuscript Title: Molecularly imprinted polymer for specific extraction of hypericin from Hypericum perforatum L herbal extract Author: Zhaozhou Li Cuili Qin Daomin Li Yuze Hou Songbiao Li Junjie Sun PII: DOI: Reference:

S0731-7085(14)00271-4 http://dx.doi.org/doi:10.1016/j.jpba.2014.05.031 PBA 9594

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

18-2-2014 20-5-2014 21-5-2014

Please cite this article as: Z. Li, C. Qin, D. Li, Y. Hou, S. Li, J. Sun, Molecularly imprinted polymer for specific extraction of hypericin from Hypericum perforatum L herbal extract, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.05.031 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Molecularly imprinted polymer for specific extraction of hypericin

2

from Hypericum perforatum L herbal extract

3

Zhaozhou Li a*, Cuili Qin a, Daomin Li a, Yuze Hou a, Songbiao Li a, Junjie Sun a

ip t

4 5

cr

6 7

a

8

Luoyang 471023, P. R. China.

us

College of Food and Bioengineering, Henan University of Science and Technology,

an

9 10

M

11

E-mail addresses of authors:

14

Zhaozhou Li: [email protected]

15

Daomin Li: [email protected]

16 17 18 19 20 21

te

d

13

Ac ce p

12

*Corresponding author. Telephone: +86 379 64282342; Fax: +86 379 64282342. E-mail: [email protected]

22

Full postal address: College of Food and Bioengineering, Henan University of Science

23

and Technology, NO. 263 Kaiyuan Avenue, Luolong District, Luoyang 471023, P. R.

24

China.

25

1

Page 1 of 52

25

Abstract The molecularly imprinted polymers (MIPs) were prepared by an oxidation-reduction

27

polymerization system using a non-covalent molecularly imprinting strategy with

28

hypericin as the template, acrylamide as the functional monomer and pentaerythritol

29

triacrylate as the cross-linker in the porogen of acetone. The UV spectrum revealed

30

that a cooperative hydrogen-bonding complex between hypericin and acrylamide

31

might be formed at the ratio of 1 : 6 in the prepolymerized system. Two classes of the

32

binding sites were produced in the resulting hypericin-imprinted polymer with the

33

dissociation constants of 16.61 µg L-1 and 69.35 µg L-1, and the affinity binding sites of

34

456.53 µg g-1 and 603.06 µg g-1, respectively. The synthesized MIPs were characterized

35

by scanning electron microscope, thermogravimetric and differential thermal analysis.

36

High-performance liquid chromatography was used to investigate the adsorption and

37

recognition properties of the MIPs. Selective binding of the template molecule was

38

demonstrated in comparison to the analog pseudohypericin. After the Hypericum

39

perforatum L plant being air dried and finely ground, an extract was prepared by

40

shaking the powder in a methanol-water solution (80 : 20, v/v), vacuum filtration

45

Ac ce p

te

d

M

an

us

cr

ip t

26

46

achieved with the recovery of 82.30%. The results showed that MISPE can be a useful

47

tool for specific isolation and effective clean-up of target compounds from natural products.

48

Keywords: Molecularly imprinted polymer; Solid-phase extraction; Hypericin; Hypericum

49

perforatum L

41 42 43 44

though a Büchner funnel, liquid-liquid extraction with ethyl ether and ethyl acetate, and evaporating on a rotary evaporator until dry. With the sorbents of the optimized MIPs, a molecularly imprinted solid-phase extraction (MISPE) procedure was developed for enrichment and separation of hypericin from the Hypericum extract in the presence of interfering substances. The selective extraction of hypericin from herbal medicine was

2

Page 2 of 52

50

1. Introduction Hypericin (4, 5, 7, 4', 5', 7'-Hexahydroxy-2, 2'-dimethylnaphthodianthrone) is a

52

naturally occurring substance found in Hypericum perforatum L (Saint John's wort) and

53

related species. There have been various biological activities, such as antitumor, antiviral

54

and antidepressant properties, found in hypericin and its structural analog (the structures

55

are shown in Fig. 1)[1, 2]. Emerging evidence clearly indicates that hypericin is a very

56

promising lead for developing a new type of drug to treat infections or cancers[3].

57

However, the chemosynthesis and biosynthesis of hypericin remain difficult due to the

58

low yields and complicated steps even though a great deal of effort has been paid.

59

Thus, it is still the focus of many research activities to develop the effective, desirable

60

and practicable enrichment materials or methods for hypericin separation and purification

61

from Hypericum perforatum L[4].

M

an

us

cr

ip t

51

Several methods were described for the isolation and purification of hypericin

63

and its structural analog from Hypericum perforatum L, which mainly consist of

64

preparative high-performance liquid chromatography (HPLC)[5], size exclusion column

66 67 68 69

te

Ac ce p

65

d

62

chromatography[6], high-speed counter-current chromatography[7, 8], and successive column chromatographies over silica[9, 10], polyamide[11] or Sephadex LH series[12, 13]. However, the main problem associated with these separation columns packed with ordinary stationary phases is the low selectivity of the retention mechanism. Actually, along with the desired analyte(s), many unwanted interfering substances of similar

70

hydophobicity/hydrophilicity are also retained and concentrated, due to the very limited

71

selectivity of the partition equilibria involved[14]. Therefore, it is necessary to develop

72

an efficient adsorbent material with high affinity for hypericin.

73

Molecular imprinting is known as a template polymerization method of producing

74

tailor-made and highly selective synthetic receptors for given molecules. Molecularly 3

Page 3 of 52

imprinted polymers (MIPs) are man-made polymers with a predetermined selectivity

76

towards a given analyte or a group of structurally related species [14-16]. In comparison

77

to their biological analogs, major advantages of the MIPs, other than possessing

78

antibody-like molecular selectivity, include physical robustness, resistance to elevated

79

temperatures and pressures, inertness to acids, bases, and organic solvents, as well as low

80

production cost and ease of preparation[17]. These attributes make MIPs ideal for

81

extensive application in solid-phase extraction[18, 19], biomimic sensors[20, 21],

82

immunoassay[22], drug delivery[23], catalysis or artificial enzymes[24]. Among these

83

applications, the one most widely used is solid-phase extraction (SPE), for which MIPs

84

are commercialized. MIPs used in SPE create a very promising variety of clean-up

85

method—molecular imprinting solid-phase extraction (MISPE). The imprinted polymer

86

exhibits high selectivity, reusability and adsorption capacity, which leads to extremely

87

high purity and recovery separation with low cost[25, 26]. In addition, owing to the

88

good stability of MIPs, the MISPE column can be used in extreme operating conditions

89

(for example, high temperatures and pressures, organic environments and extreme pH

90

values).

92 93 94 95

cr

us

an

M

d

te

Ac ce p

91

ip t

75

Although several methods have been reported for the preparation of MIPs, bulk

polymerization is the most popular and general method to prepare MIPs due to its attractive properties, such as rapidity and simplicity in preparation, with no requirement for sophisticated or expensive instrumentation, and purity in the produced MIPs[27, 28]. In the present work, the MIPs were prepared based on bulk polymerization, using

96

hypericin as the template, methacrylic acid (MAA), 4-vinylpyridine (4-VP) or

97

acrylamide (AM) as the functional monomer, and pentaerythritol triacrylate (PETRA) as

98

the cross-linker in the porogen of acetone. The synthesized MIPs were characterized via

99

scanning electron microscope (SEM), thermogravimetric (TG) and differential thermal

4

Page 4 of 52

analysis (DTA). High-performance liquid chromatography (HPLC) was used to evaluate

101

the adsorption and recognition properties of the MIPs. By using the MIPs as the sorbent,

102

the proposed MISPE procedure was firstly developed and proven to be applicable for

103

selective enrichment and determination of hypericin directly from the extract of

104

Hypericum perforatum L.

105

2. Materials and methods

106

2.1. Materials and reagents

us

cr

ip t

100

Hypericin and pseudohypericin were purchased from Alexis Corporation (Lausen,

108

Switzerland). Functional monomers (MAA, AM and 4-VP) were obtained from

109

Sigma-Aldrich. Cross-linker (PETRA) was purchased from Adamas-Beta, Ltd. Benzoyl

110

peroxide (BPO), N, N-dimethylaniline (DMA), methanol, acetonitrile, acetic acid, and

111

hydrochloric acid were obtained from Sinopharm Chemical Reagent Co., Ltd. Water

112

used was filtered from a Milli-Q Water purification system (Millipore Corporation,

113

Bedford, MA, USA).

M

d

te

Hypericum perforatum L was collected during the flowering stage in the region

119

Ac ce p

114

an

107

120

in the herbarium of this institute.

121

2.2. Apparatus and HPLC analysis

115 116 117 118

of Longnan city, Gansu province, P. R. China, and it had not been sprayed with herbicide, burnt, mowed, grazed by livestock, or mechanically cut, for several years prior to the experiment. The herb was authenticated by associate researcher Yongjiang Luo from the Lanzhou Institute of Animal Science and Veterinary Pharmaceutical Science, Chinese Academy of Agricultural Sciences. Voucher specimens were deposited

122

An Agilent 8453 double-beam spectrophotometer was used for recording UV

123

spectra and determining the absorbance. SEM imaging was carried out on a field 5

Page 5 of 52

124

emission SEM (JSM-6700F, JEOL Ltd., Japan), and thermal stability was evaluated

125

by a TG-DTA system (Pyris Diamond, Perkin-Elmer, USA). The determination of hypericin and pseudohypericin by HPLC was carried out

127

on a Varian liquid chromatographic system (Varian Company, USA) equipped with a

128

Prostar 210 pump, a LC workstation version 6.41 system software and a Prostar 325

129

UV-Vis detector. Chromatographic separation was performed on a C18 reversed-phase

130

column (5 µm, 4.6 mm×250 mm HC, Agilent Corporation, USA). The mobile phase

131

consisted of 15% ammonium acetate-acetic acid buffer (0.50 mol L-1, pH 3.70) and

132

85% methanol (by volume). Effluents were monitored at a wavelength of 590 nm. The

133

flow rate was 1 mL min-1, the injection volume was 20 µL, and the column temperature

134

was maintained at room temperature (22 °C) [29].

M

an

us

cr

ip t

126

The naphthodianthrones were determined under the optimized chromatographic

136

conditions. Retention times were 4.7 min for pseudohypericin and 6.8 min for hypericin.

137

Two linear calibration curves with r values of 0.9999 and 0.9997 were obtained for

138

hypericin and pseudohypericin, respectively. According to the signal-to-noise ratio

139

equal to 3.0, the limits of detection were 6.90 µg L-1 for hypericin and 8.31 µg L-1 for

141 142 143 144

te

Ac ce p

140

d

135

pseudohypericin. The limit of quantitation was determined as the lowest concentration of the analyte in extract that could be quantified with an inter-assay relative standard deviation (RSD) of less than 20% and an accuracy between 80% and 120% [30]. The respective values for hypericin and pseudohypericin were 14.70 µg L-1 and 22.40 µg L-1. 2.3. Preparation of polymers by oxidation-reduction initiating system

145

The polymerizations were performed using bulk polymerization techniques,

146

which is schematically depicted in Fig. 2. A glass tube was loaded with 0.05 mmol

147

template (hypericin), 10 mL porogen (acetone) and the 0.3 mmol functional monomer

148

(MAA, AM or 4-VP). The mixture was incubated at 4 °C for 12 h. Subsequently, the 6

Page 6 of 52

cross-linker (PETRA, 0.75 mmol) and initiator (BPO, 0.032 mmol; DMA, 0.032 mmol)

150

were added stepwise. The mixed solution was poured into a sonication bath for 5 min

151

and nitrogen purge for 5 min. After sealed the tube in a nitrogen atmosphere, the

152

reaction mixture was placed at room temperature (22 °C) for 12 h. The resulting bulk

153

polymers were ground, sieved and repeated floatation in acetone to collect 30 µm-50

154

µm particles. Then, the collected particles were put into the Soxhlet extractor, and the

155

template and unpolymerized monomers were removed using a methanol-acetic acid

156

mixture (90 : 10, v/v) for 12 h. Finally, the polymers were dried in a vacuum drying

157

oven at 60 °C until a constant weight and set aside for further study. Non-molecular

158

imprinted polymers (NIPs) were synthesized simultaneously following the same

159

procedure except for the addition of template.

160

2.4. Characterizing hypericin-imprinted polymers

161

2.4.1. Determination of static adsorption capacity

d

M

an

us

cr

ip t

149

To evaluate the adsorption capacity of MIPs (NIPs), 25 mg MIPs (NIPs) were

163

placed into a 10 mL plastic centrifuge tube and mixed with 5 mL of a hypericin acetone

165 166 167 168

Ac ce p

164

te

162

solution (0.01 mmol L-1). After 24 h adsorption at 25 °C, the substrate concentrations were quantified by a calibration curve developed with HPLC at the concentrations of 20 µg L-1-1000 µg L-1 of hypericin standard solutions, with 6 calibration levels. The adsorption capacity of MIPs (Q, µg g-1) was calculated with equation (1) [31].

Q = (Cs0 - Cs )  V

m

(1)

169

Here, Cs0 and Cs are concentrations of the substrates in the initial solution and in

170

the supernatant after treatment for a certain period of time (mmol L-1), respectively. V

171

is the volume of substrate solution (mL), and W is the weight of the dry MIPs used (g).

172

The imprinting efficiency of the imprinted polymer (I) was calculated by the 7

Page 7 of 52

173

following equation: (2)

Q NIP

Where QMIP and QNIP are the static adsorption amounts of MIP and NIP (µg g-1), respectively.

177

2.4.2. Dynamic adsorption test

cr

176

ip t

175

Q MIP

I

174

To investigate the adsorption kinetics of MIPs (NIPs), 50 mg of MIPs (NIPs)

179

were weighed into hypericin solution (0.01 mmol L-1, 5 mL) in a 10 mL centrifuge

180

tube. The tubes were sealed and then shaken in an air bath shaker (200 rpm) at 25 °C

181

for the different time intervals (for 0.5 h, 1 h, 2h, 4h, 8h and 12h, respectively). The

182

concentrations of free hypericin were determined by HPLC

183

2.4.3. Isotherm and Scatchard analysis

.

M

an

us

178

Seven batches of 50 mg polymers were weighed into plastic centrifuge tubes and

185

mixed with 5 mL of an acetone solution containing 0.005 mmol L-1 to 0.03 mmol L-1

186

hypericin. In the air bath shaker, the mixtures were shaken for 4 h at 25 °C, then

188 189 190 191

te

Ac ce p

187

d

184

centrifuged at 15000 rpm for 15 min, and the resulting supernates were determined by HPLC. The obtained data were fitted by equation (3), and the isotherm was plotted based on the concentrations of hypericin in supernatants versus the amount of substrate bound to MIPs.

Q

C

=

Q max

Kd

Q

Kd

(3)

192

Kd is the equilibrium dissociation constant, C is the free equilibrium concentration of

193

hypericin (mmol L-1), and Qmax is the apparent maximum number of binding sites (µg g-1).

194

2.4.4. Determination of hypericin selection absorption

8

Page 8 of 52

The selectivity test was performed for the template molecule itself and the analog

196

pseudohypericin. A 10 mL plastic centrifuge tube was loaded with 50mg MIPs and an

197

acetone solution containing hypericin and pseudohypericin at the concentrations of

198

0.02 mmol L-1 of each substance. After 12 h oscillation in the air bath shaker at 25 °C,

199

the mixtures were centrifuged at 15000 rpm for 15 min. Subsequently, the concentrations

200

of the free molecules were detected by HPLC. The adsorptive parameters of static

201

distribution coefficient (KD), separation factor (α) and relative separation factor (β)

202

were calculated by equation (4), (5) and (6), respectively. CS

us

CP

(4)

an

KD =

203

cr

ip t

195

Where Cp is the concentration of substrate binding on the MIP (mmol g-1), and Cs

205

is the concentration of substrate in the solution (mmol L-1). KD reflects static adsorption

206

amount. The adsorption capacity increases with increasing values of KD.

d

  K D1 K

(5) D2

te

207

M

204

Where KD1 and KD2 are the static distribution coefficients of target molecule and

209

competitive one. The parameter α embodies the adsorption selectivity between target

213

Ac ce p

208

214

stronger selectivity resulted from the molecular imprinting.

215

2.5. Preparation of the MISPE column

210 211 212

molecule and the competitive one.

  1 

(6) 2

Where α1 and α2 are the separation factors of MIP and NIP, respectively. The β

value suggests selective difference between MIP and NIP. The larger β value is meant

216

A 0.8 g of either MIP or NIP was suspended in a methanol-isopropanol solution

217

(2 : 1, v/v). The resulted slurry was packed in an empty home-made SPE column (150 9

Page 9 of 52

mm × 10 mm) with two wool-glass frits at the top and bottom to form a regular

219

sorbent bed. Before use, the cartridge was washed three times with 6 mL of acetone.

220

The capability of the MISPE column was determined with the calibration curve

221

developed by HPLC at the known concentrations of the hypericin and pseudohypericin

222

standard solutions.

223

2.6. Evaluation of the MISPE selectivity

cr

ip t

218

In order to investigate the selectivity of the MISPE protocol, a mixture containing

225

hypericin and its analog pseudohypericin was prepared in acetone. One and a half

226

milliliter of the mixture (50 µg mL-1 for each) was loaded onto the SPE cartridge. The

227

cartridge was then consecutively rinsed with 5 mL acetone to eliminate the molecules

228

retained by non-specific interactions with the sorbent, and eluted by 10 mL

229

methanol-acetic acid (90 : 10, v/v). The collected loading solution, rinsing solution and

230

eluate were analyzed by HPLC.

231

2.7. Extraction of hypericin from Hypericum perforatum L extract by MISPE

233 234 235 236 237

an

M

d

te

The Hypericum perforatum L plant (10 g) was air dried at the room temperature

Ac ce p

232

us

224

indoors and away from sunlight. After being finely ground, an extract was prepared by extraction of the biomass (0.5 dry weight mL-1) with a methanol-water solution (80 : 20, v/v). A homogeneous suspension was obtained by shaking on a flat-bed orbital shaker for 6 h. Then, we filtered the solution by vacuum filtration through a Büchner funnel containing a filter paper. The filter cake was washed with methanol (15 mL, three

238

times). The combined filtrate was defatted at the room temperature with n-hexane (30

239

mL, three times) and it was sequentially fractionated by liquid-liquid extraction with

240

ethyl ether and ethyl acetate (50 mL of each solvent, three times). All the solvents

241

were previously saturated with H2O. The resulting upper phases were concentrated on

10

Page 10 of 52

a rotary evaporator until completely dry, then dissolved in 1.5 mL acetone and

243

percolated through the MISPE cartridge with the optimized procedure. The resulting

244

fractions were dried and dissolved in the mobile phase and then submitted for HPLC

245

analysis.

246

3. Results and discussion

247

3.1. Spectrophotometric analysis

cr

ip t

242

The principle of molecular imprinting technology is the fixation of the host-guest

249

complex formed by the molecular interaction between template and the selected

250

monomer through different types of intramolecular forces. In order to elucidate the

251

recognition mechanism on a molecular level, spectrophotometric analysis was employed

252

in the hypericin imprinting process.

M

an

us

248

A series solutions were prepared in acetone, in which the molar ratio of hypericin

254

and the functional monomer varied at 1 : 0, 1 : 2, 1 : 6, 1 : 10, 1 : 14 and 1 : 18,

255

respectively. After equilibrium for 12 h, the changes in absorbance and difference

256

absorption spectra of the mixture were measured with an Agilent 8453 double-beam

261

Ac ce p

te

d

253

262

interaction has been formed between hypericin and AM. However, the other monomers

263

did not show any interactions with the template molecule.

257 258 259 260

264 265

spectrophotometer using pure acetone as the reference. Three different functional monomers MAA, AM and 4-VP were investigated for

the formation of complementary intermolecular interactions with the template. The maximum absorption wavelength of hypericin was significantly shifted when AM was used as the functional monomer (as shown in Fig. 3), which demonstrated a strong

The complex reaction of template (A) with the functional monomer (B) can be described by the following reaction[32]:

11

Page 11 of 52

268 269

As the concentration of B (b0) is much greater than that of A (a0), so the concentration of the complex (c) can be calculated according to: c

a 0 b 0n K （1  b 0n K）

(8)

ip t

267

(7)

A + nB = C

266

Where K refers to association constant, n=1, 2, 3, …

271

The absorbance of the mixture can be determined at the maximum wavelength

274 275

A = A A +A B +A C =[(a 0 -c) A +(b0 -nc) B +c C ]l

When b0=0, the absorbance is:

A 0 = a 0 A l

M

d

A  A  A 0  (a 0  c) A l  c Cl  a 0 A l  c l

(11)

Where    C   A . Substituting equation (11) into (8) yields:

A b 0n  KA  K a 0l

(12)

284

Ac ce p

279

(10)

The absorbance difference measured is:

te

278

(9)

Where εA, εB and εC are the molecular absorptivities of A, B and C, respectively.

276 277

us

273

and can be expressed as follows:

an

272

cr

270

285

mixture, and the value of K calculated from the slop of the equation was -265.58 mol-2

286

l2. Fig. 2 shows the imprinting process with the optimized molar ratio of the template

287

hypericin to the monomer AM.

288

3.2. Optimum of polymerized components

280 281 282 283

K may be calculated by plotting A b 0n versus A . It is found that the plot

showed good linear relationship with r of 0.9956 at n = 6 and the linear regression equation was calculated as A b06  268.58A  18.71 . The result indicated a 1 : 6 cooperative hydrogen-bonding complex might be predominating in the pre-polymerization

12

Page 12 of 52

The imprinting effect has close relationships with the conditions of polymerization,

290

which includes template, monomer, initiator, porogen and washing solvents etc. It is

291

crucial to choose a suitable functional monomer that can form stable host-guest

292

complexes with the template prior to polymerization [33]. Subsequently, three imprinted

293

polymers were prepared using the above three monomers for the rebinding study, and

294

the results demonstrated that the adsorption capacities were perfectly consistent with

295

the spectrophotometric analysis.

cr

ip t

289

Porogenic solvent has a critical effect on the pore properties and recognition abilities,

297

since it not only brings all the components into one phase but also creates macropore

298

structures in the imprinted polymer. For the non-covalent imprinting system, solvent

299

plays an essential role in the steps of pre-organization and polymerization. It influences

300

the types and strength of the interactions occurring. In addition, the solvent generates

301

pores and affects the material characteristics of the MIPs (morphology, shape, size and

302

distribution of the cavities). In the preliminary experiments, solvents used for preparing

303

hypericin-MIPs were firstly tested. Organic solvents with different polarity (acetone,

304

ethanol and methanol) were employed for the preparation of MIPs. It is believed that

309

Ac ce p

te

d

M

an

us

296

310

shows the surface morphologies of MIPs and NIPs via SEM. Particles of the MIP (Fig.

311

4a and b) exhibited a more porous, larger pore size, and rough structure than that of

312

the NIP (Fig. 4c and d). The MIP with uniform and more open structure is obviously

313

favorable for the embedding of the template and mass transfer.

305 306 307 308

the low polar solvent was favorable for the stability of pre-polymer and the interaction between template and functional monomer. In view of this, acetone was finally chosen as the porogen solvent, and it was also used in characterizing the properties of MIPs to avoid the influence of swelling process. Moreover, the selected porogen acetone provided sufficient rigidity and desirable surface properties for highly specific recognition. Fig. 4

13

Page 13 of 52

Owing to its ease of use, radical polymerization is the most frequent exploited

315

method to produce the synthetic polymers. The radicals can be generated at 60 °C (2,

316

2-azobis-isobutyronitrile), 45 °C (azobis valeronitrile), 22 °C (peroxide initiator), or 0

317

°C upon ultraviolet photolysis (2, 2-azobis-isobutyronitrile, 365nm). Comparative

318

researches on recognition specificity have shown that the low-temperature approach

319

gives the most specific material, which can reduce the heat destabilization of pre-polymer

320

in maximal degree[34]. However, the template hypericin is sensitive to light and heat,

321

so the polymer was initialed by peroxide initiator at relatively low temperature.

us

cr

ip t

314

Extraction of the template leaves a three-dimensional material in which the cavity

323

shapes and functional group locations are complementary to the guest molecule [35]. In

324

the washing step, the washing efficiency is largely dependent on the extraction solvent.

325

Thus the solvents with high polarity and acidity (methanol-acetic acid, 90 : 10, v/v;

326

methanol-acetic acid, 95 : 5, v/v; methanol) were tested in the removal process. It was

327

found that the extraction time of MIPs was shortest by using a methanol-acetic acid

328

mixture (90 : 10, v/v). So we selected this mixed liquid as the washing solvent.

329

However the template trapped by high cross-linked polymer could be leaked out in

331 332 333 334

M

d

te

Ac ce p

330

an

322

recognized process, especially in bulk MIPs. According to the reported literatures, MIPs prepared by surface imprinting, or other template removal methods may be the key to this problem[36].

3.3. Binding characteristics of the polymer 3.3.1. Adsorption parameters of hypericin-imprinted polymers

335

Adsorption parameters of hypericin-MIPs are shown in Table 1. The polymer of

336

highest imprinting efficiency was synthesized with the functional monomer AM,

337

which displayed the most specific recognition of the template. The adsorption capabilities

14

Page 14 of 52

of P1-M and P1-N were 645.70 µg g-1 and 181.60 µg g-1, respectively. It could be seen

339

that the hypericin-imprinted polymer exhibited strong affinity to the template itself.

340

Fig. 5 shows the relationship between Q and adsorptive time. The adsorption amounts

341

of P1 were increased quickly in the period of 0 h-2 h, then the increments were

342

reduced on the stage of 2 h-4 h, and the saturated binding was observed after 4 h.

343

3.3.2. Affinity analysis

cr

ip t

338

Fig. 6a shows the binding isotherms for hypericin on the MIP and NIP. The

345

binding amounts were increased gradually with increasing hypericin concentration in

346

the solution. The amounts of substrate bound to the MIP were more than that to the

347

NIP, which could be attributed to the imprinting effect. In the Scarchard analysis, the

348

experimental binding isotherm is replotted according to the formula 3. The Scatchard

349

plot falls on straight line in the homogeneous system that containing only one type of

350

binding site. In contrast, the curved Scatchard plot has been cited as the evidence for

351

the existence of heterogeneous binding site. The heterogeneity can still be accommodated

353 354 355

an

M

d

te

Ac ce p

352

us

344

using the Scatchard analysis by modeling the curved isotherm as two separate straight lines, which is a graphical method for applying the bi-Langmuir isotherm. The steeper and flatter lines measure the high- and low-affinity sites, respectively[37]. As shown in Fig. 6b, there were two classes of binding sites in the MIP, one of which includes

356

high-affinity sites (Kd1 = 4913.34 µg L-1, Qmax1 = 9872.09 µg g-1), and the other includes

357

low-affinity sites (Kd2 = 201.78 µg L-1, Qmax2 = 6547.76 µg g-1). But there were only the

358

low-affinity sites in the NIP (Kd = 21459.30 µg L-1, Qmax = 8515.12 µg g-1). The value

359

of dissociation constant Kd of NIP was much higher than MIP that exposes the low

15

Page 15 of 52

binding strength of NIP. The difference in hypericin binding affinity to the MIP and

361

NIP clearly indicated the role of the imprinting process in the formation of specific

362

binding sites. The high-affinity sites are specific for template due to the imprinting

363

process. However, the low-affinity sites are non-specific but inherent to the polymer

364

system or the preparation process[38].

365

3.3.3. Selective adsorption for hypericin

us

cr

ip t

360

In the selective adsorption test, the target molecule hypericin and the competitive

367

one pseudohypericin possess similar structure and co-exist in Hypericum extract as

368

bioactive components. The parameters of selective evaluation are shown in Table 2.

369

Based on the values of KD, there was no significant difference of rebinding hypericin

370

and pseudohypericin with NIP (α = 0.79). However, MIP had obvious adsorption

371

selectivity for hypericin (α = 5.89). The selectivity of MIP was 7.41 times higher than

372

that of NIP, which suggested that the imprinting process significantly improved

373

adsorption selectivity to the template.

374

3.3.4. Thermal stability

376 377 378 379

M

d

te

Ac ce p

375

an

366

TG and DTA of the hypericin-imprinted polymer (P1-M) in the dynamic nitrogen

atmosphere have been performed to determine its mode of decomposition. The TG plot shows that the decomposed temperature was 277.68 °C, and the mass-loss ratio was 99.88% when the temperature went up to 455.97 °C. With the initial temperature (228.48 °C) of thermal absorption, the DTA curve demonstrates that the melting point

380

temperature and enthalpy of melting (△H) were 387.02 °C and 6717.36 J g-1, respectively.

381

In Fig. 7, the endothermic process was accompanied with the MIP decomposed, and

382

so the weight-loss may have resulted from the heat absorption.

383

3.4. Optimization of the MISPE procedure 16

Page 16 of 52

384

3.4.1. Particle size, breakthrough volume and mass capacity The optimum particle size of MIPs loaded in SPE cartridge is 30 µm-50 µm,

386

whether bigger or smaller will lead to a decrease in extraction efficiency. So the particles

387

must be floated repeatedly in acetone and sieved by grading screen after ground. The

388

different amounts of MIPs, 0.4 g, 0.8 g and 1.2 g, were packed in an empty home-made

389

SPE column (150 mm × 10 mm), respectively. The extraction test under the optimized

390

conditions above indicated an increased recovery and good permeability by packing the

391

polymer of 0.8 g. Additionally, wet packing method gave more homogeneous density

392

than dry packing did.

an

us

cr

ip t

385

In the consideration of the loading step, several solvents including methanol,

394

acetone, ethanol, acetonitrile and tetrahydrofuran were tested for the suitable loading

395

solvent. Keeping a constant analyte load (75 µg) and adjusting correspondingly the

396

concentrations of the analyte, the load volume was increased from 0.5 mL to 2.5 mL

397

acetone without any breakthough. However, some leaching was observed when other

398

loading solvents used. The possible reason was that MIPs exhibit enhanced selectivity

399

for the template in the application environments similar to those of the polymerisation

401 402 403

d

te

Ac ce p

400

M

393

conditions. That is, the binding capacity was enhanced in the polymerisation solvent (porogen). Therefore, the loading solvent was determined as acetone, and that the optimal loading volume was selected as 1.5 mL considering both extraction time and the accuracy of result.

404

The cartridge mass capacity was expressed as the absolute amount of analyte

405

loaded into the column in conditions in which more than 1% (w/w) of the sample is

406

not retained by the stationary phase [39]. Serial amounts of hypericin, ranging from

407

0.01 µmol to 1 µmol in 1.5 mL of acetone, were tested for the determination of the

408

mass capacity of the MISPE column. It was found that 1% (w/w) of total was leached 17

Page 17 of 52

out of the cartridge when 1.5 mL of acetone containing 302.67 µg hypericin was

410

loaded. So, the mass capacity of the hypericin-imprinted sorbent was calculated as

411

378.34 µg g-1. The imprinted polymer can be regarded as a strong adsorptive material

412

that is suitable for retaining large amounts of the analyte.

413

3.4.2. MISPE cartridge washing

ip t

409

To optimize the process of selective MISPE, the parameters of washing step were

415

investigated. Hypericin in acetone (50 µg mL-1, 1.5 mL) was loaded into the cartridge

416

as described above and then rinsed with different volumes of acetone, methanol,

417

ethanol and acetonitrile. The washing performance could be observed in Fig. 8. It was

418

found that acetone had little effect on the column adsorption of the target analyte.

419

With increasing volume of acetone, the breakthrough of the analyte was no significant

420

alteration. In view of the hypericin recovery (Fig. 8) and washing capacity of the

421

solvent for impurities in the real sample, 5 mL of acetone was selected as the rinsing

422

solvent.

423

3.4.3. Elution of hypericin from MISPE cartridge

425 426 427 428

us

an

M

d

te

Ac ce p

424

cr

414

The optimization of the elution step was carried out using water, tetrahydrofuran

and methanol-acetic acid (90 : 10 and 95 : 5, v/v) as the eluent, respectively. It is shown the release percentage for the elution in Fig. 8. The same hypericin solution was loaded, and different volumes of the eluent between 5 mL and 10 mL were tested. Good recoveries of hypericin can be attributable to progressively increased eluting

429

strength. The best recovery was obtained when using 10 mL methanol-acetic acid (90 :

430

10, v/v) as the elution solution.

431

3.5. Selectivity of the MISPE column

432

The prepared hypericin-imprinted polymer with the highest imprinting efficiency 18

Page 18 of 52

was employed as the sorbent of the SPE column for the pretreatment of hypericin and

434

structurally similar pseudohypericin. Fig. 9 shows the HPLC-UV chromatograms of

435

the standard mixtures pretreated by the MISPE column. Good baseline separation was

436

obtained without the SPE procedure, and hypericin was eluted second (Fig. 9a). No

437

apparent peak is observed in Fig. 9b in the measurement of the solutions after MISPE,

438

which indicates that hypericin and its analog were almost completely adsorbed onto

439

the MIPs. Fig. 9c shows that almost all the pseudohypericin and only minor amounts of

440

hypericin were washed from the MISPE column with acetone. When using the more

441

polar eluting solution, namely methanol-acetic acid (90 : 10, v/v), hypericin was

442

almost completely eluted (as shown in Fig. 9d). The elution ratio of hypericin following

443

MISPE was 91.58% (as shown in Table 3), while pseudohypericin was only partially

444

recovered (5.56%), due to the limited retention during the washing step. This result

445

can be easily elucidated when considering the recognition sites and spatial structure of

446

the imprinted polymer are specific complementary to the template hypericin. However,

447

the longer side-chain length of pseudohypericin could cause steric hindrance to access

448

the recognition sites of the imprinted polymer. The MISPE column exhibited highly

450 451 452 453

cr

us

an

M

d

te

Ac ce p

449

ip t

433

selective binding affinity for hypericin, which can be attributed to the well-defined three-dimentional cavities with spatially oriented functional groups in the highly cross-linked polymer network and the non-covalent interactions between the template and the functional monomer.

3.6. MISPE of hypericin from Hypericum extract

454

The selected MISPE conditions (as described in Section 3.4) were applied for the

455

extraction of hypericin from Hypericum perforatum L herbal extract. We observed no

456

interfering peaks around the retention times of hypericin and pseudohypericin in Fig.

457

10b, which indicated that the components in the herbal extract were almost adsorbed 19

Page 19 of 52

completely onto the MISPE column. It can be seen from Fig. 10c, although most of the

459

components were discarded, hypericin was strongly retained on the column. The losses

460

of hypericin and pseudohypericin were 7.0% and 12.0% of the loading amounts,

461

respectively. When using more polar eluting solutions, namely methanol containing

462

10% acetic acid, hypericin was eluted almost completely (as shown in Fig. 10d). It was

463

obvious that the naphthodianthrones were separated individually in the extract with

464

the MISPE procedure. The recovery of analyte after the MISPE procedure was

465

estimated as the ratio of the amount of the analyte eluted using acidic solvent and the

466

amount loaded on the MISPE column. As reported in Table 4, a high yield of

467

hypericin with 82.30% satisfactory recovery was obtained from the extract by the

468

proposed MISPE. However, the pseudohypericin recovery was only 4.40% owing to

469

the non-specific adsorption by the MISPE column. This efficient procedure allowed

470

interfering peaks arising from the complex biological matrix to be suppressed and

471

made it possible to obtain a cleaner extract.

te

d

M

an

us

cr

ip t

458

A literature survey revealed that a number of studies concerning the application

473

of column chromatography for separation and purification of hypericin from Hypericum

478

Ac ce p

472

479

cleaning of the separation media proved to be a tedious and labour-intensive work.

480

Instead, the MISPE cartridge displayed a better performance. The advantage of the

481

MISPE protocol was the high selectivity, resulting in less interferences in the separation

482

process [40, 41]. The proposed method is a rapid, efficient and non-expensive method

474 475 476 477

extract [9-13]. Successive column chromatography with Sephadex LH 20 and Sephadex LH 60 has been proved the most convenient method. The overall recovery of hypericin and pseudohypericin was calculated as 43%. However, its potential use seems limited because of the low plant material loaded (approximately 2 g), complicated procedure, time-consuming (at least 1 week needed) and high cost of the resins and reagents. The

20

Page 20 of 52

483

(for the amount of consumed solvents and instrumentation used), which could be applied

484

to a greater scale for the selective enrichment of hypericin from Hypericum extract.

485

3.7. Validation of the MISPE method The method of standard addition was used to evaluate the recovery of the MISPE

487

process. By mixing 0.5 mL standard solutions (500 µg L-1) with 1 mL of the acetone

488

extract of Hypericum perforatum L, the recoveries were calculated as 92.37% for

489

hypericin and 95.44% for pseudohypericin (n = 3). The instrument precision was

490

evaluated by analysis of 5 replicate samples containing the known concentrations of the

491

naphthodianthrones. It was found to be satisfactory (RSD = 0.51%). After repetitive

492

use of the MIP cartridge for 5 times, the RSD values for hypericin and pseudohypericin

493

were respectively calculated to be 2.41% and 3.72%, indicating a good reproducibility

494

of the MISPE column.

495

4. Conclusions

te

d

M

an

us

cr

ip t

486

This study shows the possibility of using MIPs for the efficient and selective

497

extraction of hypericin from Hypericum extract. The MIPs were prepared with hypericin

498 499 500 501

Ac ce p

496

as template, AM as functional monomer and PETRA as cross-linker. Optimal imprinting parameters for enhanced recognition properties of MIPs toward hypericin were attained. The spectrophotometric analysis demonstrated that the most stable complex could be formed when using AM as the functional monomer at the molar ratio of 1:6

502

between the template and the functional monomer. The morphology and thermal

503

stability of the resulting MIPs were characterized using SEM, TG and DTA. After

504

evaluation of the imprinting effect and selectivity, the obtained MIPs showed strong

505

adsorption capacity (645.70 µg g-1 for the MIP), high imprinting efficiency (3.56) and

506

relative separation factor (7.41). It was suggested that the MIPs exhibited a high 21

Page 21 of 52

selectivity toward hypericin in comparison to the analog. Furthermore, the Scatchard

508

analysis demonstrated that both the high- and low-affinity sites were produced in the

509

MIP. However, there were only the low-affinity sites in the NIP. With the sorbent of the

510

optimized hypericin imprinted polymer, a MISPE protocol was firstly developed for

511

selective clean-up of hypericin from the Hypericum extract after a series sample

512

preparation steps. The high recovery (82.30%) from the sample proved the procedure

513

was suitable for selective enrichment and purification hypericin from complex

514

samples. However, the isolated amount varied with the hypericin contents in different

515

plant materials. It will be expected to reach a few milligrams after repeated the MISPE

516

column. Overall, the proposed MISPE method is efficient, rapid and inexpensive (for the

517

amount of consumed solvents and instrumentation used). These characteristics make

518

this imprinted polymer a very promising candidate for scaled-up SPE experiments and

519

therefore potentially suitable for many industrial applications.

cr

us

an

M

d te Ac ce p

520

ip t

507

22

Page 22 of 52

521

Acknowledgements

This study was supported by Doctoral Research Initiation Grant from Henan University

523

of Science and Technology (No. 09001609), National Student Research Training Program

524

(No. 201310464007), Youth Science Foundation of Henan University of Science and

525

Technology (No. 2013QN021) and Key Science and Technology Program of Educational

526

Commission of Henan Province, China (No. 14B550015).

us

cr

ip t

522

527

an

528

Ac ce p

te

d

M

529

23

Page 23 of 52

References

531

[1] G. Stojanovic, A. Dordevic, A. Smelcerovic, Do other hypericum species have

532

medical potential as St. John's Wort (Hypericum perforatum)?, Curr. Med. Chem., 20

533

(2013) 2273-2295.

534

[2] J. Asgarpanah, Phytochemistry, pharmacology and medicinal properties of

535

Hypericum perforatum L, Afr. J. Pharm. Pharmacol., 6 (2012) 1387-1394.

536

[3] A. Karioti, A.R. Bilia, Hypericins as potential leads for new therapeutics, Int. J.

537

Mol. Sci., 11 (2010) 562-594.

538

[4] X. Cao, Q. Wang, Y. Li, G. Bai, H. Ren, C. Xu, Y. Ito, Isolation and purification of

539

series bioactive components from Hypericum perforatum L. by counter-current

540

chromatography, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 879 (2011)

541

480-488.

542

[5] S. Puri, G. Handa, A.K. Kalsotra, V.K. Gupta, A.S. Shawl, O.P. Suri, G.N. Qazi,

543

547

Ac ce p

548

[7] F.B. Williams, L.C. Sander, S.A. Wise, J. Girard, Development and evaluation of

549

methods for determination of naphthodianthrones and flavonoids in St. John's wort, J.

550

Chromatogr. A, 1115 (2006) 93-102.

551

[8] A. Smelcerovic, H. Laatsch, Z. Lepojevic, S. Djordjevic, The separation of hypericine

544 545 546

te

d

M

an

us

cr

ip t

530

Preparative high-performance liquid chromatographic separation of naphthodianthrones from St. John's Wort, J. Chromatogr. Sci., 44 (2006) 177-180. [6] A. Karioti, F.F. Vincieri, A.R. Bilia, Rapid and efficient purification of naphthodianthrones from St. John's wort extract by using liquid-liquid extraction and SEC, J. Sep. Sci., 32 (2009) 1374-1382.

24

Page 24 of 52

and pseudohypericine from Hypericum perforatum L, Pharmazie, 57 (2002) 178-180.

553

[9] A. Kubin, G. Alth, R. Jindra, G. Jessner, R. Ebermann, Wavelength-dependent

554

photoresponse of biological and aqueous model systems using the photodynamic plant

555

pigment hypericin, J. Photochem. Photobiol. B: Biol., 36 (1996) 103-108.

556

[10] D. Kacerovská, K. Pizinger, F. Majer, F. Šmíd, Photodynamic therapy of

557

nonmelanoma skin cancer with topical Hypericum perforatum extract-A pilot study,

558

Photochem. Photobiol., 84 (2008) 779-785.

559

[11] S. Sattler, U. Schaefer, W. Schneider, J. Hoelzl, C.M. Lehr, Binding, uptake, and

560

transport of hypericin by Caco-2 cell monolayers, J. Pharm. Sci., 86 (1997) 1120-

561

1126.

562

[12] V. Butterweck, V. Christoffel, A. Nahrstedt, F. Petereit, B. Spengler, H. Winterhoff,

563

Step by step removal of hyperforin and hypericin: activity profile of different Hypericum

564

preparations in behavioral models, Life Sci., 73 (2003) 627-639.

565

[13] Z. Li, X. Wang, G. Shi, Y. Bo, X. Lu, X. Li, R. Shang, L. Tao, J. Liang,

567 568 569

cr

us

an

M

d

te

Ac ce p

566

ip t

552

Enzyme-assisted extraction of naphthodianthrones from Hypericum perforatum L. by 12

C6+-ion beam-improved cellulases, Sep. Purif. Technol., 86 (2012) 234-241.

[14] L. Peng, Y. Wang, H. Zeng, Y. Yuan, Molecularly imprinted polymer for solid-phase extraction of rutin in complicated traditional Chinese medicines, Analyst,

570

136 (2011) 756-763.

571

[15] C.H. Lu, W.H. Zhou, B. Han, H.H. Yang, X. Chen, X.R. Wang, Surface-imprinted

572

core-shell nanoparticles for sorbent assays, Anal. Chem., 79 (2007) 5457-5461.

573

[16] F. Chen, R. Wang, Y. Shi, Molecularly imprinted polymer for the specific solid-phase

25

Page 25 of 52

extraction of kirenol from Siegesbeckia pubescens herbal extract, Talanta, 89 (2012)

575

505-512.

576

[17] A. Opik, A. Menaker, J. Reut, V. Syritski, Molecularly imprinted polymers: a new

577

approach to the preparation of functional materials, Proc. Est. Acad. Sci., 58 (2009)

578

3-11.

579

[18] H. Zeng, Y. Wang, X. Liu, J. Kong, C. Nie, Preparation of molecular imprinted

580

polymers using bi-functional monomer and bi-crosslinker for solid-phase extraction of

581

rutin, Talanta, 93 (2012) 172-181.

582

[19] W. Cheng, Z. Liu, Y. Wang, Preparation and application of surface molecularly

583

imprinted silica gel for selective extraction of melamine from milk samples, Talanta,

584

116 (2013) 396-402.

585

[20] P.S. Sharma, F. D’Souza, W. Kutner, Molecular imprinting for selective chemical

586

sensing of hazardous compounds and drugs of abuse, Trends Analyt. Chem., 34 (2012)

587

59-77.

589 590 591

cr

us

an

M

d

te

Ac ce p

588

ip t

574

[21] B. Wu, Z. Wang, D. Zhao, X. Lu, A novel molecularly imprinted impedimetric sensor for melamine determination, Talanta, 101 (2012) 374-381. [22] T. Fodey, P. Leonard, J. O’Mahony, R. O’Kennedy, M. Danaher, Developments in the production of biological and synthetic binders for immunoassay and sensor-based

592

detection of small molecules, Trends Analyt. Chem., 30 (2011) 254-269.

593

[23] F. Puoci, G. Cirillo, M. Curcio, O.I. Parisi, F. Iemma, N. Picci, Molecularly

594

imprinted polymers in drug delivery: state of art and future perspectives, Expert Opin.

595

Drug Deliv., 8 (2011) 1379-1393.

26

Page 26 of 52

[24] G. Wulff, J. Liu, Design of biomimetic catalysts by molecular imprinting in

597

synthetic polymers: the role of transition state stabilization, Acc. Chem. Res., 45 (2012)

598

239-247.

599

[25] L.X. Yi, R. Fang, G.H. Chen, Molecularly imprinted solid-phase extraction in the

600

analysis of agrochemicals, J. Chromatogr. Sci., 51 (2013) 608-618.

601

[26] R. Garcia, Maria João Cabrita, A.M.C. Freitas, Application of molecularly imprinted

602

polymers for the analysis of pesticide residues in food—A highly selective and innovative

603

approach, Am. J. Anal. Chem., 2 (2011) 16-25.

604

[27] L. Chen, B. Li, Application of magnetic molecularly imprinted polymers in

605

analytical chemistry, Anal. Methods, 4 (2012) 2613-2621.

606

[28] L. Chen, S. Xu, J. Li, Recent advances in molecular imprinting technology: current

607

status, challenges and highlighted applications, Chem. Soc. Rev., 40 (2011) 2922-

608

2942.

609

[29] U. Ruckert, K. Eggenreich, W. Likussar, R. Wintersteiger, A. Michelitsch, A

611 612 613

cr

us

an

M

d

te

Ac ce p

610

ip t

596

high-performance liquid chromatography with electrochemical detection for the determination of total hypericin in extracts of St. John's wort, Phytochem. Anal., 17 (2006) 162-167.

[30] S. Bauer, E. Störmer, H.J. Graubaum, I. Roots, Determination of hyperforin, hypericin,

614

and pseudohypericin in human plasma using high-performance liquid chromatography

615

analysis with fluorescence and ultraviolet detection, J Chromatogr. B Biomed. Sci.

616

Appl., 765 (2001) 29-35.

617

[31] H. Yavuz, V. Karakoç, D. Türkmen, R. Say, A. Denizli, Synthesis of cholesterol

27

Page 27 of 52

imprinted polymeric particles, Int. J. Biol. Macromol., 41 (2007) 8-15.

619

[32] J. Zhou, X. He, Study of the nature of recognition in molecularly imprinted

620

polymer selective for 2-aminopyridine, Anal. Chim. Acta, 381 (1999) 85-91.

621

[33] K. Karim, F. Breton, R. Rouillon, E.V. Piletska, A. Guerreiro, I. Chianella, S.A.

622

Piletsky, How to find effective functional monomers for effective molecularly imprinted

623

polymers?, Adv. Drug Del. Rev., 57 (2005) 1795-1808.

624

[34] H. Yan, K. Row, Characteristic and synthetic approach of molecularly imprinted

625

polymer, Int. J. Mol. Sci., 7 (2006) 155-178.

626

[35] J. Marty, M. Mauzac, Molecular imprinting: State of the art and perspectives, in:

627

H. Ito, A. Abe, A.C. Albertsson (Eds.) Microlithography · Molecular Imprinting,

628

Springer-Verlag, Berlin, Heidelberg, 2005, pp. 1-35.

629

[36] J. Luo, L. Zhang, D. Chen, P. Wang, J. Zhao, Y. Peng, S. Du, Z. Zhang, Molecularly

630

imprinted layer-coated monodisperse spherical silica microparticles toward affinity-

631

enrichment of isoflavonoid glycosides from Radix puerariae, Analyst, 137 (2012)

633 634 635

cr

us

an

M

d

te

Ac ce p

632

ip t

618

2891-2902.

[37] R.J. Umpleby II, S.C. Baxter, A.M. Rampey, G.T. Rushton, Y. Chen, K.D. Shimizu, Characterization of the heterogeneous binding site affinity distributions in molecularly imprinted polymers, J. Chromatogr. B Analyt. Technol. Biomed. Life.

636

Sci., 804 (2004) 141-149.

637

[38] J. Chen, L.Y. Bai, K.F. Liu, R.Q. Liu, Y.P. Zhang, Atrazine molecular imprinted

638

polymers: comparative analysis by far-infrared and ultraviolet induced polymerization,

639

Int. J. Mol. Sci., 15 (2014) 574-587.

28

Page 28 of 52

[39] C. Baggiani, P. Baravalle, G. Giraudi, C. Tozzi, Molecularly imprinted solid-phase

641

extraction method for the high-performance liquid chromatographic analysis of fungicide

642

pyrimethanil in wine, J. Chromatogr., 1141 (2007) 158-164.

643

[40] Z.F. Guo, T.T. Guo, M.F. Guo, Preparation of molecularly imprinted adsorptive

644

resin for trapping of ligustrazine from the traditional Chinese herb Ligusticum chuanxiong

645

Hort, Anal. Chim. Acta, 612 (2008) 136-143.

646

[41] C. Lopez, B. Claude, P. Morin, J.P. Max, R. Pena, J.P. Ribet, Synthesis and study

647

of a molecularly imprinted polymer for the specific extraction of indole alkaloids

648

from Catharanthus roseus extracts, Anal. Chim. Acta, 683 (2011) 198-205.

an

us

cr

ip t

640

Ac ce p

te

d

M

649

29

Page 29 of 52

Figure captions：

651

Fig. 1. The chemical structures of hypericin (a) and its structural analog pseudohypericin

652

(b).

653

Fig. 2. Schematic representation of hypericin imprinted polymers.

654

Fig. 3. Difference absorption spectra of hypericin in the presence of AM in methanol.

655

Concentration of hypericin: 0.1 mmol L-1; concentration of AM for lines 1-5: 0.2 mmol L-1,

656

0.4 mmol L-1, 0.6 mmol L-1, 1.2 mmol L-1, 1.8 mmol L-1; corresponding pure AM solutions

657

as blanks.

658

Fig. 4. Scanning electron micrographs of the hypericin-MIP and NIP. (a) hypericin-MIP,

659

(b) an enlarged view of image a, (c) NIP and (d) an enlarged view of image c.

660

Fig. 5. The kinetic adsorption curves of hypericin MIPs.

661

Fig. 6. Isotherms (a) and Scatchard plots (b) of hypericin bound on MIP and NIP.

662

Fig. 7. The spectra of TG and DTA for hypericin MIP.

663

Fig. 8. Release percentage of hypericin of increasing volumes of different washing

664

solvents through the MIP cartridge.

665

Fig. 9. Chromatograms and UV-Vis spectra of the standard mixtures. (a) Initial solution

667 668 669 670

cr

us

an

M

d

te

Ac ce p

666

ip t

650

before MISPE, (b) solution after loading, (c) solution after washing and (d) eluate after rinsing with methanol–acetic acid (90 : 10, v/v). Column, C18 reversed-phase column (5 µm, 4.6 mm×250 mm HC, Agilent Corporation, USA); mobile phase: ammonium acetate-acetic acid buffer (0.50 mol L-1, pH 3.70) and methanol, 15 : 85 (v/v), detection wavelength, 590 nm; flow rate, 1 mL min-1; injection volume, 20 µL;

671

column temperature, room temperature (22 °C).

672

Fig. 10. Chromatograms and UV-Vis spectra of Hypericum perforatum L extract. (a) Initial

673

solution before MISPE, (b) solution after loading, (c) solution after washing and (d)

674

eluates after rinsing with methanol–acetic acid (90 : 10, v/v). Column, C18 reversed-phase

30

Page 30 of 52

column (5 µm, 4.6 mm×250 mm HC, Agilent Corporation, USA); mobile phase:

676

ammonium acetate- acetic acid buffer (0.50 mol L-1, pH 3.70) and methanol, 15 : 85

677

(v/v), detection wavelength, 590 nm; flow rate, 1 mL min-1; injection volume, 20 µL;

678

column temperature, room temperature (22 °C).

ip t

675

679

cr

680 681

Ac ce p

te

d

M

an

us

682

31

Page 31 of 52

682

Table 1

683

The adsorption capability and imprinting efficiency of hypericin bulk MIPs. a Monomer

Q (µg g-1)

I

P1-M

AM

645.70

3.56

P1-N

AM

181.60

P2-M

4-VP

575.07

P2-N

4-VP

554.90

P3-M

MAA

443.92

P3-N

MAA

423.74

ip t

MIPs

cr

1.04

us

1.05

684

a

685

imprinting efficiency, I = QMIP/QNIP; Cs0 and Cs represent concentrations of the substrates in the

686

initial solution and in the supernatant after treatment for a certain period of time (mmol L-1),

687

respectively. QMIP and QNIP are the static adsorption amounts of MIP and NIP (µg g-1), respectively.

M

an

Q, adsorption capacity, Q = (Cs0-Cs)×(loading solution volume [mL]/adsorbent mass [g]); I,

691 692 693 694 695 696 697

te

690

Ac ce p

689

d

688

698 699 700 701 702

32

Page 32 of 52

703

Table 2

704

The selectivity capability of bulk MIPs prepared under the optimal conditions for

705

hypericin and its structural analog. a KD

P1-M

Hypericin

1.38

Pseudohypericin

0.23

Hypericin

0.16

Pseudohypericin

0.20

β

5.89

7.41

0.79

us

P1-N

α

ip t

Recognized molecule

cr

MIPs

706

a

707

(mmol g-1), and Cs is the concentration of substrate in the solution (mmol L-1); α, separation factor,

708

α = KD1/KD2, where KD1 and KD2 are the static distribution coefficients of target molecule and

709

competitive one; β, relative separation factor, β = α1/α2, where α1 and α2 are the separation factors

710

of MIP and NIP, respectively.

M

an

KD, distribution coefficient, KD = Cp/Cs, Cp is the concentration of substrate binding on the MIP

714 715 716 717 718 719 720

te

713

Ac ce p

712

d

711

721 722 723 724 725 33

Page 33 of 52

726

Table 3

727

Elution ratios of hypericin and pseudohypericin after MISPE. a C1 (µg

C2 (µg

C3 (µg

Q1 (µg

Q2 (µg

Q3 (µg

Elution

mL-1)

mL-1)

mL-1)

mL-1)

g-1)

g-1)

g-1)

ratio (%)

hypericin

50.00

2.50

1.20

6.53

89.06

7.50

81.56 91.58±2.54

pseudohypericin

50.00

5.00

1.28

0.38

84.38

79.69

4.69

ip t

C0 (µg

5.56±1.83

cr

Compound

728

a

729

[mL]/adsorbent mass [g]); Q2, rinsing capacity, Q2=C2×rinsed solution volume [mL]/adsorbent

730

mass [g]; Q3, elution capacity, Q3=C3×elution solution volume [mL]/adsorbent mass [g], where C0

731

refers to the concentration of the initial solution before MISPE; C1, C2 and C3 represent the

732

loading, rinsing and elution solution concentrations, respectively.

an

us

Data are shown as means±RSD. Q1, adsorption capacity, Q1=(C0-C1)×(loading solution volume

M

733 734

738 739 740

te

737

Ac ce p

736

d

735

34

Page 34 of 52

740

Table 4

741

Recoveries of hypericin and pseudohypericin from Hypericum perforatum L. extract

742

by MISPE. a Lost during

Lost during

Eluted

Recovery

cartridge (µg)

load (µg)

wash (µg)

(µg)

(%)

hypericin

14.41

1.01

1.59

11.86

82.30±3.14

pseudohypericin

45.32

5.44

38.07

cr

ip t

Loaded on the Compound

1.99

4.40±0.12

743

a

744

and pseudohypericin loaded on the cartridge were determined in the extract before MISPE.

us

Data are shown as means±RSD. With the method illustrated in Section 2.2, the amounts of hypericin

an

745 746

M

747 748

752 753 754 755 756 757 758

te

751

Ac ce p

750

d

749

759 760 761

35

Page 35 of 52

Highlights

761 762

►Preparation of hypericin imprinted polymers by oxidation-reduction initiating system.

764

►A molecularly imprinted solid-phase extraction (MISPE) procedure was developed.

765

►The selective extraction of hypericin was achieved with recovery of 82.30%.

766

►MISPE is a useful tool for specific isolation of hypericin from Hypericum extract.

us

cr

ip t

763

767

an

768 769

Ac ce p

te

d

M

770

36

Page 36 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 1a

Page 37 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 1b

Page 38 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 2

Page 39 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 3

Page 40 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 4a

Page 41 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 4b

Page 42 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 4c

Page 43 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 4d

Page 44 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 5

Page 45 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 6a

Page 46 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 6b

Page 47 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 7

Page 48 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 8

Page 49 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 9

Page 50 of 52

Ac

ce

pt

ed

M

an

us

cr

i

Fig 10

Page 51 of 52

Ac

ce

pt

ed

M

an

us

cr

i

*Graphical Abstract

Page 52 of 52