Polydopamine-Coated Magnetic Molecularly Imprinted Polymers with

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Polydopamine-coated magnetic molecularly imprinted polymers with fragment template for identification of Pulsatilla saponins metabolites in rat feces with UPLC-Q-TOF-MS Yuzhen Zhang, Jia-Wei Zhang, Chong-Zhi Wang, Lian-Di Zhou, qihui zhang, and Chun-Su Yuan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05747 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 20, 2017

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Journal of Agricultural and Food Chemistry

Polydopamine-coated magnetic molecularly imprinted polymers with fragment

template

for

identification

of

Pulsatilla

saponins

metabolites in rat feces with UPLC-Q-TOF-MS Yu-Zhen Zhang1, Jia-Wei Zhang1, Chong-Zhi Wang3, Lian-Di Zhou2,*, Qi-Hui Zhang1, 3,*, Chun-Su Yuan3

1

School of Chemistry and Chemical Engineering, Chongqing University, Chongqing 400044,

China; 2

Basic Medical College, Chongqing Medical University, Chongqing 400016, China;

3

Tang Center for Herbal Medicine Research and Department of Anesthesia & Critical Care,

University of Chicago, Chicago, IL 60637, U.S.A..

*Correspondence: 1* E-mail: [email protected] (Q.H. Zhang), Fax: (+86)-023-65102531; 2* E-mail: [email protected] (D.L. Zhou), Fax: (+86)-023-65714434.

1

ACS Paragon Plus Environment


ABSTRACT 1

In this work, a modified pretreatment method using magnetic molecularly

2

imprinted polymers (MMIPs) was successfully applied to study the metabolites of an

3

important botanical with ultra-performance liquid chromatography/quadrupole time-

4

of-flight mass spectrometry (UPLC-Q-TOF-MS). The MMIPs for glucoside-specific

5

adsorption was used to identify metabolites of Pulsatilla chinensis in rat feces.

6

Polymers were prepared by using Fe3O4 nanoparticles as the supporting matrix, D-

7

glucose as fragment template and dopamine as the functional monomer and cross-

8

linker. Results showed that MMIPs exhibited excellent extraction performance, large

9

adsorption capacity (5.65 mg/g), fast kinetics (60 min) and magnetic separation.

10

Furthermore, the MMIPs coupled with UPLC-Q-TOF-MS were successfully utilized

11

for the identification of 17 compounds including 15 metabolites from the Pulsatilla

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saponin metabolic pool. This study provides a reliable protocol for the separation and

13

identification of saponin metabolites in a complex biological sample, including those

14

from herbal medicines.

15

Keywords: Magnetic molecularly imprinted polymers; Metabolites study; P. chinensis;

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Fragment template.

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1. Introduction 17

Pulsatilla chinensis (Bunge) Regel is a medical and edible botanical, which has

18

been used for thousands of years in Asia. Ever-dwindling numbers of the wild plant

19

has forced the price of P. chinensis higher and higher, with the shortage replaced

20

through artificial cultivation.1 As an edible tonic tea, P. chinensis is widely consumed

21

alone or in combination with other botanicals for preventing viral infection.1,2 In

22

addition, P. chinensis is commonly used for treating intestinal amebiasis, malaria,

23

vaginal trichomoniasis, bacterial infections and malignant tumors.3,4 The triterpenoid

24

saponins have been demonstrated to be the main chemical constituents and major

25

active ingredients of P. chinensis.3 Anemoside B4 (AB4), the most important bioactive

26

triterpenoid saponin of P. chinensis, has been reported to possess anti-inflammatory

27

activity.5 It can significantly inhibit the proliferation of Hep G2 cells6 and has

28

prominent effects on the antitumor activities associated with cell proliferation,

29

apoptosis and oxidative stress.7,8

30

Recently, we reported the metabolic profile of AB4 associated with the microflora

31

in rat small and large intestines. Ten metabolites were detected and identified with

32

ultra-performance

33

spectrometry (UPLC-Q-TOF-MS).8 However, metabolic studies in complex

34

biological samples are still challenging analytically because of the low concentration

35

of analytes in complex biological matrices.9 Thus, developing powerful enrichment

36

and high selectivity techniques for the extraction of target components from complex

37

samples remains an important role.

liquid

chromatography/quadrupole

time-of-flight

mass

38

Molecularly imprinted polymers (MIPs), with three dimensional complementary

39

cavities engineered through templates, has attracted great attention in the fields of 3



40

complex matrices,10-13 drug delivery,9,14,15 biosensor,16-18 selective photocatalytic

41

degradation19,20 and chromatographic separation.21,22 Furthermore, magnetic surface

42

imprinted materials can be rapidly and conveniently separated from sample under

43

external magnetic fields without centrifugation or filtration procedures23 and can also

44

improve mass transfer and reduce permanent entrapment.24 Thus, the novel magnetic

45

MIPs (MMIPs) technique has widespread application in the pre-treatment of samples.

46

Dopamine (DA) is a small-molecule mimicking adhesive proteins, which contains

47

catechol and amine functional groups. At a weak alkaline pH, it can produce an

48

adherent polydopamine (PDA) by self-polymerization.25 Recently, it has been

49

reported that the self-polymerization of the functional monomer dopamine is used in

50

the preparation of imprinted nanoparticles.26,27 Thus, we innovatively developed a

51

facile, simple and green approach to imprint templates on the surface coating of

52

magnetic nanoparticles with a thin layer of PDA polymers.

53

However, some template molecules, such as purified natural products, are so

54

expensive that they limit the application of the MMIPs technique. Imprinted polymers

55

prepared with fragment templates (partial structures of templates) could solve the

56

problem of these expensive and rare templates.13 Moreover, MMIPs using fragment

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templates can avoid template leakage28 and adsorb a number of active ingredients.29

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Thus, it is of importance to extract the saponin metabolites of Pulsatilla and generate

59

comprehensive metabolite information.

60

Herein, we describe the synthesis of MMIPs based PDA polymer using candidate

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fragment templates, which derive from the four components of AB4, D-glucose, L-

62

rhamnose, L-arabinose and oleanolic acid. In addition, we describe the application of

63

MMIPs in the isolation of metabolites of triterpenoid saponins in rat feces after oral

64

administration of P. chinensis. In this present study, we incorporate aspects of green 4


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chemistry in the synthesis of MMIPs and determine the suitability and sensitivity of

66

our technique as a new option for identifying metabolites from a metabolic pool in

67

complex biological systems.

2. Materials and methods 2.1. Chemicals and reagents 68

Anemoside B4 standard (>98%) was obtained from Sichuan Vic's Biological

69

Technology Co., Ltd (Sichuan, China). D-glucose, L-rhamnose, L-arabinose, oleanolic

70

acid and dopamine hydrochloride (DA·HCl) were acquired from Adamas Reagent Co.,

71

Ltd (Basel, Switzerland). Iron (III) chloride hexahydrate (FeCl3·6H2O), sodium

72

acetate

73

(Na3C6H5O7·2H2O), hydrochloric acid (HCl), methanol (MeOH), anhydrous ethanol

74

(EtOH) and acetic acid (AcOH) were purchased from Chengdu ke Long chemical

75

reagent factory (Chengdu, China). Acetonitrile (ACN) and formic acid of HPLC grade

76

were from Merck (Darmstadt, Germany). The ultrapure water used in experiments

77

was prepared by a Milli-Q system (Millipore, Milford, MA, USA). Other reagents

78

were of analytical grade.

anhydrous

(NaAc),

ethylene

glycol

(CH2OH)2,

sodium

citrate

2.2. Instruments 79

Scanning electron microscope (SEM) and Transmission electron microscopy (TEM)

80

images were adopted by merlin compact (ZEISS, Germany) and JEM 2100F (JEOL,

81

Japan). FT-IR spectra were obtained by an IR Affinity-1 Fourier Transform Near IR

82

spectrometer (Shimadzu, Japan). The thermo gravimetric analysis was performed by

83

TGA/DSL1/1600LF (Mettler Toledo, Switzerland). UPLC analysis was carried out on

84

a Nexera UPLC LC-30A system (Shimadzu, Kyoto, Japan) equipped with a binary

85

pump, an online degasser, an autoinjector and a thermostatically controlled column 5



86

compartment. The Agilent Zorbax Eclipse plus C18 column (2.1×100 mm, 1.8 μm)

87

was used for chromatographic separation. 2.3. Synthesis of MMIPs nanoparticles

88

The synthetic routine of MMIPs is shown in Figure 1. First, magnetic Fe3O4

89

nanoparticles (Fe3O4 NPs) were synthesized by solvothermal method. Typically, 1.35

90

g of FeCl3·6H2O were dissolved in 40 mL of ethylene glycol and magnetically stirred

91

at room temperature until forming a clear solution. Subsequently, 3.1 g of sodium

92

acetate anhydrous and 0.4 g of sodium citrate were added. After that, the mixtures

93

were vigorously stirred at 150 °C for 30 min, and then the homogeneous black

94

solution was transferred to a Teflon-lined stainless steel autoclave. The autoclave was

95

maintained at 200 °C for 8 h. Finally, the obtained black materials were rinsed with

96

anhydrous ethanol and ultrapure water, and then dried under vacuum at 50 °C for 8 h.

97

After that, 75 mg of synthesized magnetic nanoparticles were dispersed into 30 mL

98

of Tris–HCl buffer solution (pH=8.5, 10 mM) under ultrasonic vibration. Then 75 mg

99

of D-glucose was added and the mixture solution was shaken at room temperature for

100

30 min, followed by the addition of 75 mg of DA·HCl. The resulting mixtures were

101

reacted by self-polymerization in the dark and mechanically stirred at room

102

temperature for 12 h. The harvested products were collected using a magnet and

103

washed with acetonitrile-acetic acid (9:1, v/v) several times to remove D-glucose and

104

then dried under vacuum for further use. As a control, the corresponding MNIPs were

105

prepared in the identical manner without of D-glucose. 2.4. Adsorption experiments

106 107

The adsorption capacity of MMIPs/MNIPs was evaluated using AB4, as the test compound.

6


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108

The static adsorption experiments were carried out at 30 °C. 15 mg of

109

MMIPs/MNIPs were added to 3 mL of different concentrations (10 µg/mL-100

110

µg/mL) of AB4, which dissolved in Tris–HCl buffer solution (pH=8.5, 10 mM). Then,

111

the solution was shaken on an oscillator at 90 rmp for 4 h. The adsorption capacity of

112

MMIPs/MNIPs (Q, mg/g) was calculated based on following equation:

113

Q = (C0-Ce) · V/m

114

Where C0 (μg/mL) is the concentration of the initial solution, Ce (μg/mL) is the

115

equilibrium concentration of AB4 solution, V (mL) is the volume of AB4 solution, and

116

m means the mass of MIPs/NIPs (mg).

117

To further estimate the adsorption capacity of MMIPs/MNIPs, kinetic adsorption

118

experiments were also done at 30 °C. 15 mg of MMIPs/MNIPs were mixed with 3 mL

119

of 80 µg/mL Tris–HCl (pH=8.5, 10 mM) AB4 solution. Afterwards, the mixtures were

120

shaken on an oscillator at 90 rmp for 5, 10, 20, 30, 40, 60 and 90 min. The

121

equilibrium adsorption capacity of MMIPs/MNIPs (Q, mg/g) at different times was

122

calculated with the following equation:

123 124

Q = (C0-Ct) · V/m Here, Ct (μg/mL) is equilibrium concentration of AB4 solution at different times. 2.5. UPLC-MS analysis

125

UPLC analysis was operated on a Nexera UPLC-LC-30A system (Shimadzu,

126

Kyoto, Japan). Chromatographic separation was carried out on an Agilent Zorbax

127

Eclipse plus C18 column (2.1×100 mm, 1.8 μm) with flow rate of 0.3 mL/min at 30 °C.

128

Gradient mobile phase consisted of A ( 0.1% formic acid in water ) and B ( 0.1%

129

formic acid in acetonitrile ) was as follows: 10–25% B at 0–2 min, 25–70% B at 3–20

130

min, 70–95% B at 21–25 min, 95–95% B at 26–27 min, 95-10% B at 28–30 min. The

131

sample volume injected was set at 2 μL, while spectra were acquired at 203 nm. 7



132

Electrospray ionization (ESI) source was used for the ionization on AB Sciex

133

Triple TOF™ 5600+ system. The following MS/MS conditions were optimized as

134

follows: ion spray voltage, 4.5 kV; the turbo spray temperature, 550 °C; collision

135

energy, -55 eV. The scan range of m/z 100-1500 was chosen in negative modes.

136

Nitrogen was used as the nebulizer gas, and the nebulizer gas (gas 1) and the heater

137

gas (gas 2) were set to 60 and 60 ips, respectively. A real time multiple mass defect

138

filter and dynamic background subtraction performed on AB Sciex software

139

(Analyst® TF 1.6 software), which was used to screen the profile and provide a whole

140

production scan to avoid the omission of minor metabolites. In addition, an automated

141

calibration delivery system was used to regulate MS and MS/MS behavior in the

142

experiment. Data were evaluated by PeakView™ 1.2 and Metabolite Pilot™ 1.5

143

software (AB Sciex, Foster City, CA, USA). The theoretical mass tolerance was set at

144

the mass accuracy of

Polydopamine-Coated Magnetic Molecularly Imprinted Polymers with

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