MS-Based Lipidomics Approach To

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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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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

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

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

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

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

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

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

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

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

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phosphatidylglycerol

(9

species),

phosphatidylserine

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

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

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

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

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phospholipids and unsaturated fatty acids in BP. Our comprehensive lipidomics

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

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future application.

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

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anti-inflammatory.

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species),

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Abbreviations

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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

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

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

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product for human health due to its nutritional and therapeutic properties

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

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

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

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

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

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available.

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2-4

. The

6-8

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

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

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

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

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

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

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

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. Nevertheless, lipid

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

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

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different nectar plants.

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

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

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

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

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

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

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

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cyclotron

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

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

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identify potential biomarkers for preventing human diseases 15.

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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

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includes sterols analysis by TLC

.

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

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

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

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sensitive analytical method enabling identification and quantification of lipids at the

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

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

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

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fragment ion scanning

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

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

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

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

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

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time and capable of detecting low-abundance lipid compounds

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

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

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separation of polar isomeric lipid molecular species

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. It has been successfully used for lipidomics profiling in

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21

. They also

. In addition, Liu et al.

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successfully applied this technique into the identification of bovine milk

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different infant formulas

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

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lipid composition analysis.

24, 25

and

based on the differences in lipid molecular species

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

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

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

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sample preparation, chromatographic conditions and mass parameters. The potential

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

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LPS-stimulated RAW 264.7 cells were also investigated.

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2 Materials and Methods

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2.1 Reagents

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

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

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

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

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

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

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

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

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

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chemicals were of analytical grade and purchased from Sangon Biotechnology

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(Shanghai, China).

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2.2 Sample preparation

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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triplicate.

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2.3 Instruments and methods

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The lipidomics profiling was accomplished using a UPLC system tandem

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

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

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

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

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

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negative and positive ionization mode, respectively.

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

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

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

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

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

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

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

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

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

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

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previous reports

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

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

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MS1 and MS2, with an MS1 mass error of