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UPLC-Q-Exactive Orbitrap/MS-Based Lipidomics Approach To Characterize Lipid Extracts from Bee Pollen and Their In Vitro Anti-Inflammatory Properties Qiangqiang Li, Xinwen Liang, Liang Zhao, Zhongyin Zhang, Xiaofeng Xue, Kai Wang, and Liming Wu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02285 • Publication Date (Web): 24 Jul 2017 Downloaded from http://pubs.acs.org on July 25, 2017

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Journal of Agricultural and Food Chemistry

UPLC-Q-Exactive Orbitrap/MS-Based Lipidomics Approach To Characterize Lipid Extracts from Bee Pollen and Their In Vitro Anti-Inflammatory Properties Qiangqiang Li a, b, c, Xinwen Liang a, b, c, Liang Zhao d, Zhongyin Zhang e, Xiaofeng Xue a, b, c, Kai Wang a, b, c * and Liming Wu a, b, c *

a

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing, 100093, China.

b

Risk Assessment Laboratory for Bee Products Quality and Safety of Ministry of Agriculture, Beijing,

100093, China. c

Bee Product Quality Supervision and Testing Center, Ministry of Agriculture, Beijing, 100093,

China. d

Beijing Key Laboratory of Functional Food from Plant Resources, College of Food Science &

Nutritional Engineering, China Agricultural University, Beijing 100083, China. e

Henan Institute of Science and Technology, Xinxiang, 453003, China.

Corresponding authors: *

Dr. Kai Wang. Institute of Apicultural Research, Chinese Academy of Agricultural Sciences, Beijing,

100093, China. Fax: +86 10 62594643. E-mail: [email protected] *

Dr. Liming Wu. Institute of Apicultural Research, Chinese Academy of Agricultural Sciences,

Beijing, 100093, China. Fax: +86 10 62594643. E-mail: [email protected]

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Abstract

2

Bee pollen (BP) is collected by honeybees from flower pollen mixed with nectar

3

and its secretions, with extensive nutritional and therapeutic properties. Lipids are

4

known to be critical contributors for the therapeutic effects of BP and vary depending

5

on different plant sources; however, lipid profiles of BP are not available. Here, an

6

UPLC-Q-Exactive Orbitrap/MS method was established for comprehensive

7

lipidomics analysis of BP derived from three major nectar plants (Brassica campestris

8

L., Camellia sinensis L. and Nelumbo nucifera Gaertn.). A total of nine lipid classes,

9

including phosphatidylcholine (41 species), phosphatidylethanolamine (43 species),

10

phosphatidylglycerol

(9

species),

phosphatidylserine

11

lysophosphatidylcholine (12 species), ceramide (8 species), diglyceride (27 species),

12

triglyceride (137 species) and fatty acids (47 species), were first identified and

13

quantified in the three BPs. In vitro anti-inflammatory activity was also discovered in

14

the lipid extracts of three BPs, which has potential relevance to the abundance of

15

phospholipids and unsaturated fatty acids in BP. Our comprehensive lipidomics

16

profiling and in vitro anti-inflammatory properties of BP provide evidence for its

17

future application.

18

Key words: Bee pollen; nectar plants; lipidomics; UPLC-Q-Exactive Orbitrap/MS;

19

anti-inflammatory.

2


(10

species),

Page 3 of 34


Abbreviations

20

Lipids

Abbr.

Lipids

Abbr.

Phosphatidylcholine

PC

Polyunsaturated fatty acids

PUFA

Phosphatidyl ethanolamine

PE

α-linolenic acid

ALA

Phosphatidylglycerol

PG

Eicosapentaenoic acid

EPA

Phosphatidylserine

PS

Docosahexaenoic acid

DHA

Lysophosphatidylcholine

LPC

Bee pollen from Camellia

BP-Cs

Ceramide

Cer

sinensis L.

Diglyceride

DG

Bee pollen from Nelumbo

Triglyceride

TG

nucifera Gaertn.

Saturated fatty acids

SFA

Bee pollen from Brassica

Unsaturated fatty acids

USFA

campestris L.

Monounsaturated fatty acids

MUFA

Lipids extracts of bee pollen

BP-Nn

BP-Bc

BPL

21 22 23

1 Introduction

24

Bee pollen (BP) is defined as a mixture of plant pollen agglutinated with nectar

25

and honeybee enzymes 1. It has been promoted as a functional food or therapeutic

26

product for human health due to its nutritional and therapeutic properties

27

functional properties, including antioxidant, anti-inflammatory, anti-carcinogenic,

28

hepatoprotective, anti-bacterial and anti-fungicidal activities, are largely ascribed to

29

the specific composition of BP, such as amino acids, lipids, vitamins, minerals, and

30

flavonoids 5. The chemical composition of BP varies from different nectar plants

31

Nevertheless, comparisons of the effects of nectar variations on BP profiles are not

32

available.

33

2-4

. The

6-8

.

Lipids are an extremely important component of BP, with a proportion of 1～13 %

34

(dry weight ratio) 1, 9, 10. Barbosa et al. found that the lipophilic fractions of BP from

35

Cistus ladanifer, Castanea sativa and Rubus sp. can resist several Gram-positive

36

bacteria 11. Wu et al. concluded that a steroid fraction of chloroform extracts from BP

37

of Brassica campestris induced apoptosis in prostate cancer PC-3 cells 12. Ishikawa et

38

al. demonstrated that the lipid-soluble components of BP exerted an anti-allergic

39

effect by inhibiting IgE-mediated mast cell activation in vivo 3


13

. Nevertheless, lipid


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40

profiles of BP are unavailable, which limits the further applications of BP. Thus, there

41

is an urgent need to identify lipid components and characterize differences in BP from

42

different nectar plants.

43

Lipidomics aims to characterize and quantify the range of intact lipid molecules

44

in biological samples, allowing correlations of the lipid composition to diet and

45

disease 14. Based on its chemically functional backbones and biochemical principles,

46

lipids are classified into fatty acids, glycerides, phospholipids, and sphingolipids,

47

among others. Recent years witnessed the development of new mass spectrometry

48

techniques, such as electrospray ionization tandem mass spectrometry (ESI-MS/MS),

49

matrix-assisted laser desorption/ionization combined with Fourier transform ion

50

cyclotron

51

(MALDI-TOF-MS), which promoted impressive advances. Lipidomics can provide

52

insights into the specific functions of lipid species on human health and can be used to

53

identify potential biomarkers for preventing human diseases 15.

54

resonance

MS

(MALDI-FTICR-MS)

and

time-of-flight

MS

The previous lipid analysis research in BP is relatively stagnant, but generally 16

and fatty acids analysis by GC or GC-MS

10, 17-19

55

includes sterols analysis by TLC

.

56

Specific lipidomics profiling at the species level remains elusive. Moreover, due to

57

the complicated lipid structure and numerous lipid isomers, it is extremely exhausting

58

to recognize various lipids by normal mass spectrometry. Therefore, a reliable and

59

sensitive analytical method enabling identification and quantification of lipids at the

60

species level is required for evaluating the lipid composition of bee pollen as

61

influenced by its nectar plants. Q-Exactive Orbitrap mass spectrometry, with

62

extremely high resolution, sensitivity, and mass accuracy, is a powerful technique for

63

fragment ion scanning

64

complex matrices such as animal tissues, plasma, cells, even agro-products. Yamada

65

et al. developed a practical workflow for high-throughput and exhaustive lipid

66

profiling and identified over 400 lipid compounds in mouse plasma by combining

67

reverse-phase liquid chromatography coupled to Q-Exactive Orbitrap mass

68

spectrometry with below 2 % relative standard deviation (RSD) of relative retention

69

time and capable of detecting low-abundance lipid compounds

70

developed a supercritical fluid chromatography (SFC) method with a single

71

octadecylsilyl column coupled to Q-Exactive Orbitrap mass spectrometry for better

72

separation of polar isomeric lipid molecular species

20

. It has been successfully used for lipidomics profiling in

22

21

. They also

. In addition, Liu et al.

4


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23

73

successfully applied this technique into the identification of bovine milk

74

different infant formulas

75

distribution. These developed methods make the lipidomic analysis feasible on the

76

lipid composition analysis.

24, 25

and

based on the differences in lipid molecular species

77

Here, based on the high-throughput properties and reliability of lipidomics

78

profiling by UPLC-Q-Exactive Orbitrap mass spectrometry, we first applied this

79

method to the lipidomics profiling of BP from three nectar plants with optimization of

80

sample preparation, chromatographic conditions and mass parameters. The potential

81

anti-inflammatory properties of the lipid extracts of bee pollen (BPL) in

82

LPS-stimulated RAW 264.7 cells were also investigated.

83

2 Materials and Methods

84

2.1 Reagents

85

The standards for PC, PE, PG, PS, and LPC were obtained from Avanti Inc.

86

(Shelby, AL, USA), and Cer, DG, TG, and FA were obtained from Sigma-Aldrich Inc.

87

(Saint Louis, MO, USA). β-carotene and α/β/γ/δ-tocopherol were purchased from

88

J&K Inc. (Beijing, China). Chloroform, acetonitrile (ACN), isopropanol (IPA),

89

methanol, ethanol and formic acid were purchased from Fisher Scientific Inc.

90

(Pittsburgh, PA, USA). All the solvents for lipid extraction or used as mobile phases

91

were of chromatographic grade. The ultrapure water was from a Millipore Milli-Q

92

water purification system (Millipore, Bedford, MA, USA). LPS (Escherichia coli

93

0111:B4) was purchased from Sigma-Aldrich Inc. (Saint Louis, MO, USA). Other

94

chemicals were of analytical grade and purchased from Sangon Biotechnology

95

(Shanghai, China).

96

2.2 Sample preparation

97

Three samples of fresh BP from Brassica campestris L. (BP-Bc), Nelumbo

98

nucifera Gaertn. (BP-Nn), and Camellia sinensis L. (BP-Cs) were acquired from the

99

beekeeping bases of the Chinese Academy of Agricultural Sciences. The collected

100

samples were ground and freeze-dried into a powder and then stored at -20 °C. 26, 27

101

The lipids extraction procedure was based on the Folch and Bligh methods

102

with some minor modifications. First, 5.0 g of BP powder was mixed with 30 mL

103

CHCl3:MeOH (2:1, V/V) in a glass tube and then fully vortexed for 10 min at room

104

temperature. The mixture was allowed to separate into the extraction solution and the

105

BP residue, and the residue was extracted twice more according to the procedure 5



106

described above. The extraction solution was combined and then mixed with 10 mL

107

ultrapure water in order to more easily layer the mixture. After centrifugation at 1,160

108

rpm for 15 min, the lower organic phase was transferred to a clean glass tube and

109

dried under a stream of nitrogen. The sample was then re-dissolved in 2 mL

110

CHCl3:MeOH (2:1, V/V) for UPLC-Q-Exactive Orbitrap/MS analysis. For the in vitro

111

studies, the dried bee pollen lipid extract (BPL) was dissolved in ethanol to prepare a

112

stock solution at a concentration of 100 mg/mL. The samples were prepared in

113

triplicate.

114

2.3 Instruments and methods

115

The lipidomics profiling was accomplished using a UPLC system tandem

116

Q-Exactive Orbitrap mass spectrometer (Thermo Fisher, CA, USA) equipped with a

117

heated electrospray ionization (HESI) probe. The lipid extracts of bee pollen from

118

Camellia sinensis L. (BPL-Cs), Nelumbo nucifera Gaertn. (BPL-Nn), and Brassica

119

campestris L. (BPL-Bc) were separated on a XSelect CSH C18 100 × 2.1 mm 2.5 µm

120

column (Waters) and CORTECS C18 100 × 2.1 mm 2.7 µm column (Waters) in

121

negative and positive ionization mode, respectively.

122

The binary solvent system consisted of mobile phase A (ACN and H2O at a

123

proportion of 60:40 V/V, containing 10 mM ammonium acetate), and mobile phase B

124

(IPA and ACN at a proportion of 90:10 V/V, containing 10 mM ammonium acetate).

125

The samples were eluted with a linear gradient from 37 % B to 98 % B over 20 min,

126

followed by 98 % B for 8 min and 7 min re-equilibration with 37 % B at a flow rate of

127

250 µL/min. The column chamber and sample tray were held at 45 °C and 10 °C,

128

respectively. The data were acquired in both negative and positive ionization mode

129

with dependent MS/MS acquisition at ranges of m/z 200-2000 and m/z 240-2000,

130

respectively. The full scan and fragment spectra were collected at a resolution of

131

70,000 and 17,500, respectively. The source parameters were set according to our

132

previous reports

133

software Lipidsearch 4.0 (Thermo Fisher, CA). Using the information for retention

134

time and characteristic product ions, nine classes of lipids were identified based on

135

MS1 and MS2, with an MS1 mass error of

MS-Based Lipidomics Approach To

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