Subscriber access provided by UNIV OF NEWCASTLE
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 34
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] 1
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
1
Page 2 of 34
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
ACS Paragon Plus Environment
(10
species),
Page 3 of 34
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
13
. Nevertheless, lipid
Journal of Agricultural and Food Chemistry
Page 4 of 34
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
ACS Paragon Plus Environment
Page 5 of 34
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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
