Accepted Manuscript Title: Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum by selective solid phase extraction and high performance liquid chromatography–diode array detector–quadrupole time-of-flight tandem mass spectrometry Author: Xin Hu Huading Zhao Shuyun Shi Hui Li Xiaoling Zhou Feipeng Jiao Xinyu Jiang Dongming Peng Xiaoqin Chen PII: DOI: Reference:

S0731-7085(15)00251-4 http://dx.doi.org/doi:10.1016/j.jpba.2015.04.018 PBA 10054

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

13-2-2015 8-4-2015 13-4-2015

Please cite this article as: X. Hu, H. Zhao, S. Shi, H. Li, X. Zhou, F. Jiao, X. Jiang, D. Peng, X. Chen, Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum by selective solid phase extraction and high performance liquid chromatographyndashdiode array detectorndashquadrupole time-of-flight tandem mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx.doi.org/10.1016/j.jpba.2015.04.018 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.

*Graphical Abstract

Graphical abstract Sensitive characterization of polyphenolic antioxidants in P. odoratum by selective Fe3O4@PDA extraction and HPLC‒DAD‒QTOF-MS/MS.

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HPLC-UV chromatograms of P. odoratum after Fe3O4@PDA extraction

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original after DPPH spiking test

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*Highlights (for review)

Highlights ► Antioxidants were screened by DPPH spiking combined with HPLC‒DAD. ► Fe3O4@PDA was prepared to selectively extract polyphenolic antioxidants.

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► Twenty-five antioxidants were efficiently characterized by HPLC‒QTOF-MS/MS.

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► Four antioxidants were first reported.

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*Revised Manuscript

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Sensitive characterization of polyphenolic antioxidants in Polygonatum odoratum

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by

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chromatography‒diode array detector‒quadrupole time-of-flight tandem mass

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spectrometry

selective

solid

phase

extraction

and

high

performance

liquid

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Xin Hua, Huading Zhaoa, Shuyun Shia,*, Hui Lia, Xiaoling Zhoub, Feipeng Jiaoa,

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Xinyu Jianga, Dongming Pengc,**, Xiaoqin Chena

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College of Chemistry and Chemical Engineering, Central South University,

Changsha 410083, PR China

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b

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c

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China

an

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Hunan Academy of Forest Sciences, Changsha 410004, PR China

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School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208,

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* Corresponding author. Tel.: +86 731 88879616; fax: +86 731 88879616

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**Corresponding Author. Tel.: +86 731 85555868; fax: +86 731 85555868

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E-mail address: [email protected] (S. Shi); [email protected] (D. Peng)

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ABSTRACT

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The complexity of natural products always leads to the co-elution of interfering

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compounds with bioactive compounds, which then has a detrimental effect on

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structural elucidation. Here, a new method, based on selective solid phase extraction

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combined with 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) spiking and high

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performance liquid chromatography‒diode array detector‒quadrupole time-of-flight

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tandem mass spectrometry (HPLC‒DAD‒QTOF-MS/MS), is described for sensitive

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screening, selective extraction and identification of polyphenolic antioxidants in

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Polygonatum odoratum. First, twenty-five polyphenolic antioxidants (1‒25) were

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screened by DPPH spiking with HPLC. Second, polydopamine coated Fe3O4

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microspheres (Fe3O4@PDA) were prepared to selectively extract target antioxidants

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with extraction efficiency from 55% to 100% when the amount of Fe3O4@PDA,

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extraction time, desorption solvent and time were 10 mg, 20 min, acetonitrile, and 5

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min. Third, twenty-five antioxidants (ten cinnamides and fifteen homoisoflavanones)

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were identified by HPLC‒DAD‒QTOF-MS/MS. Furthermore, the DPPH scavenging

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activities of purified compounds (IC50, 1.6‒32.8 μg/mL) validated the method. Among

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the identified antioxidants, four of them (12, 13, 18 and 19) were new compounds,

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four of them (2, 4, 8 and 14) were first obtained from family Liliaceae, five of them (1,

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3, 5, 7 and 9) were first reported in genus Polygonatum, while one compound (24)

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was first identified in this species. The results indicated that the proposed method was

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an efficient and sensitive approach to explore polyphenolic antioxidants from

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complex natural products.

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Keywords: Polygonatum odoratum; Antioxidant; Polyphenolic compound; Selective

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extraction; HPLC‒QTOF MS/MS 2

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1. Introduction Antioxidants have great interest due to their potential use in providing protection

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against many physiological and pathological processes caused by free-radical

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reactions [1‒2]. Natural products are good exogenous candidates for antioxidants [3],

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which are then widely investigated from the viewpoint of health-promoting

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functionalities. Polygonatum odoratum (family Liliaceae) is widely distributed in

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central and southwest areas of China, and its rhizomes have been widely used for

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removing dryness, promoting secretion of fluid and quenching thirst [4‒5]. Previous

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chemical investigations of P. odoratum have reported the existence of twenty-one

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steroidal saponins [6‒7], fourteen homoisoflavanones [8‒10], two cinnamides [11],

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and polysaccharides [12]. Surprisingly, researchers mainly focus on evaluating the

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antioxidant activities of crude extracts/fractionations of P. odoratum, and flavonoid

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rich extract exhibits the strongest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH)

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scavenging activity [9,12]. Up to now, only Wang and co-workers have reported the

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DPPH

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5,7,4'-trihydroxy-6-methyl

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methoxyl homoisoflavanone [9], however, the detailed composition and antioxidant

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efficacy of cinnamides (a class of good radical scavengers [13]) in P. odoratum

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remained unclear, not to mention the antioxidant profile of P. odoratum.

activities

of

homoisoflavanone

two and

homoisoflavanones,

i.e.

5,7,4'-trihydroxy-6-methyl-8-

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scavenging

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So far, the development technologies, based on high performance liquid

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chromatography (HPLC) analysis and free radical (e.g. DPPH) scavenging activity

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measurements, can afford HPLC peaks associated with their antioxidant activities (e.g.

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on-line HPLC‒DPPH assay [14‒16], DPPH spiking with HPLC analysis [17], and

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HPLC-based DPPH activity profiling [16]). Our previous comparative results have

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demonstrated that DPPH spiking with HPLC analysis offered the highest sensitivity 3

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and resolution [16]. However, in HPLC analysis, co-elution of interfering compounds

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with interesting bioactive compounds remains a common issue, which then affects the

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following mass detection and structural identification [18]. Pertinent sample clean-up

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is therefore of primordial importance before instrumental analysis. Matrix clean-up is

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more extensive with solid phase extraction (SPE), in which the separation materials

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are normal-phase silica gel, reversed-phase C18 or C8, molecularly imprinted polymers,

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etc. [19‒21]. Although they offer certain advantages (e.g. high reproducibility, fast

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sample treatment, compatibility with most analytical instrumentation), they also

77

encounter specific limitations, a feature that gives rise to the non-selective extraction

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of two or more types of bioactive compounds with different polarities and quantities.

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Polydopamine (PDA), formed by the oxidation of dopamine (DA) [22], is a relatively

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novel adsorbent for highly selective extraction of aromatic compounds via π-π

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stacking and hydrogen-bonding interactions [23‒24]. Notably, PDA coated Fe3O4

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microspheres (Fe3O4@PDA) possess significant advantages including excellent

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dispersibility in water, and easy magnetic separation [25]. Therefore, we used

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Fe3O4@PDA as an excellent magnetic SPE adsorbent for selective extraction of

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polyphenolic antioxidants (cinnamides and homoisoflavanones) from P. odoratum. To

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our knowledge, no relevant work has been reported.

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Therefore, the aim of the present work was to develop a novel sensitive method to

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screen and identify antioxidants in P. odoratum. DPPH spiking with HPLC analysis

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was used to screen polyphenolic antioxidants. Then polyphenolic antioxidants were

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selectively extracted by Fe3O4@PDA and identified by HPLC‒diode array

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detector‒quadrupole

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(HPLC‒DAD‒QTOF-MS/MS).

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antioxidants, including ten cinnamides (1‒10) and fifteen homoisoflavanones (11‒25),

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time-of-flight As

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tandem consequence,

mass twenty-five

spectrometry polyphenolic

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Page 6 of 36

were sensitively screened and identified. Interestingly, compounds 12, 13, 18 and 19

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were new compounds, compounds 2, 4, 8 and 14 were obtained from family Liliaceae

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for the first time, compounds 1, 3, 5, 7 and 9 were reported in the genus Polygonatum

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for the first time, while one compound (24) was first identified in this species. This is

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the first study of antioxidant profile of P. odoratum, and cinnamides and

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homoisoflavanones could be considered as the main antioxidants.

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2. Experimental

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2.1. Chemicals and reagents

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All solvents used for extraction and separation obtained from Chemical Reagent

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Factory of Hunan Normal University (Hunan, China) were of analytical grade.

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HPLC-grade

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(FeCl3·6H2O), polyethylene glycol 6000 (PEG 6000), anhydrous sodium acetate were

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purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

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Trihydroxymethylaminomethane (Tris) was acquired from Shanpu Chemical Reagent

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Co., Ltd. (Shanghai, China). Dopamine and butylated hydroxyanisole (BHA) were

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obtained from Xiya Reagent Co., Ltd. (Chengdu, China). Ultrapure water (18.2 MΩ)

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was purified and filtered using a Milli-Q water purification system (Millipore,

112

Bedford, MA, USA). DPPH (95%) was bought from Sigma-Aldrich (Shanghai

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Division), and DPPH radical solutions were freshly prepared in methanol every day

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and

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N-trans-p-coumaroyltyramine, N-cis-feruloyltyramine and N-trans-feruloyltyramine

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were purified from Fructus polygoni orientali by high speed counter current

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chromatography

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5,7,2',4'-Tetrahydroxyl

formic

acid,

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and

iron(III)

chloride

hexahydrate

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acetonitrile

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kept

protected

in

our

from

light.

laboratory

[26].

homoisoflavanone,

N-cis-p-coumaroyltyramine,

Six

main

5,7,4'-trihydroxyl

homoisoflavanones, homoisoflavanone,

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Page 7 of 36

5,7,2',4'-tetrahydroxy-6-methyl homoisoflavanone, 5,7,2',4'-tetrahydroxy-6-methoxy-

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8-methyl homoisoflavanone, 5,7,2'-trihydroxy-8,4'-dimethoxy homoisoflavanone, and

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5,7,4'-trihydroxy-6-methyl homoisoflavanone, were purified by us using Sephadex

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LH-20 (25–100 μm, Amersham Biosciences, Sweden) column chromatography

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eluting with pure methanol, and their structures were identified by comparing their

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DAD, MS and nuclear magnetic resonance (NMR) spectra with those reported in

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previous

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homoisoflavanone,

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5,7-dihydroxyl-6-methyl-8,4'-dimethoxyl homoisoflavanone were kindly supported

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by Prof. Bai, Shangdong Academy of Medical Sciences, Jinan, Shangdong [10].

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Furfural was purchased from Sigma-Aldrich (St. Louis, MO, USA). The purities of

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them were determined to be over 98% by HPLC area normalization method.

[8‒10].

homoisoflavanone

and

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5,7,4'-trihydroxyl-6,8-dimethyl

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5,7,4'-Trihydroxyl-6-methyl-8-methoxyl

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An Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) has been

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selected to analysis of samples. Chromatographic separation was performed on a

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SunFireTM C18 (250 mm × 4.6 mm i.d., 5 µm, Waters, MA, USA) column in tandem

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with a Phenomenex C18 guard cartridge (4.0 mm × 3.0 mm, Phenomenex, Torrance,

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CA). The mobile phase consisted of A (0.4% acetic acid in water) and B (0.4% acetic

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acid in acetonitrile) was programmed as follows: 0–5 min, 5% B; 5–45 min, 5–65% B.

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The flow rate was 1.0 mL/min while the column temperature was set at 25°C.

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Chromatograms were acquired at 290 nm.

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Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa

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Clara, CA) equipped with an electrospray ionization (ESI) interface was coupled in

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parallel by splitting the mobile phase 1:4 using an adjustable high-pressure stream 6

Page 8 of 36

splitter (Valco Instrument Company, Houston, TX, USA). MS data were acquired

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across the range m/z 100–1200 in positive and negative ion modes. Mass spectrometer

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was calibrated by means of an automated calibrate delivery system. The operating

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conditions were as follows: nitrogen as dry gas at a flow rate of 5.0 L/min with

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temperature at 325 ºC; nitrogen as sheath gas at a flow rate of 12 L/min with

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temperature at 400 ºC; pressure of nebulizer, 55 psi; capillary voltage, 3500 V;

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skimmer, 65 V; OCT 1 RF Vpp, 750 V; fragmentor voltage, 130 V. Full-scan MS

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spectra and MS/MS spectra were acquired with collision energy at 30 eV to achieve

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the maximum number of characteristic fragments for structural elucidation.

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Multi-well plates (Greiner) and multi-well plates readers (Bio-Tek Instruments,

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USA) were used in DPPH scavenging experiments.

Transmission electron microscopy (TEM) (JEM2100F, JEOL, Japan) was used to

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observe the morphology of microspheres. The infrared spectra (4000400 cm-1) were

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performed on a Fourier transform infrared spectrometer (FT-IR) (Nicolet 6700,

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Thermo Nicolet Co., Waltham, MA, USA). The encapsulation efficiency of

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microspheres was carried out by thermo-gravimetric analysis (TGA SDTQ600, TA,

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USA). Magnetization was measured at room temperature in a vibration sample

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magnetometer (VSM7407, Lake Shore, USA).

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2.3. Preparation of P. odoratum extract

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The rhizomes of P. odoratum were collected from Shaoyang, Hunan province of

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China, in October 2013. The plant material was authenticated by Prof. Mijun Peng,

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Key laboratory of Hunan Forest Products and Chemical Industry Engineering, Jishou

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University, Zhangjiajie, China. A voucher specimen (No. PO201310) was deposited at

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the College of Chemistry and Chemical Engineering, Central South University, 7

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Changsha, Hunan, China. The freeze-dried and pulverized rhizomes of P. odoratum

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(90 g) were decocted by 75% ethanol (700 mL) at 85°C three times (each for 3 h).

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The combined filtrates were concentrated to dryness under vacuum by rotary

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evaporation at 60°C to afford crude extract (10.2 g), which was then suspended in

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water (70 mL) and submitted to a D101 macroporous resin column (10.0 cm × 100

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cm, contained 1.0 kg D101 macroporous resin) to get rid of polysaccharide by elution

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with 10% ethanol solution (1 L). After that, 80% ethanol fraction (1.7 g, named as P.

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odoratum extract) was stored at 4ºC for further experiments.

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2.4. DPPH radical scavenging assay

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The DPPH scavenging activity was performed as previously described [27]. In brief, samples with different concentrations (25 μL) mixed with DPPH solution (40 μL, 0.4

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mg/mL) and then made up with methanol to a final volume of 250 μL. Then the

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absorbance was measured at 517 nm after the mixtures were incubated at 37 °C for 30

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min. The DPPH solution served as a control. The antioxidant activity is expressed as

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percentage of DPPH radical elimination calculated according to the following formula:

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[(Ablank–Asample)/Ablank] × 100 %, where Ablank and Asample are the absorbance of the

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black DPPH solution and the DPPH solution after addition of sample, respectively.

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Sample concentration providing 50% inhibition (IC50) was calculated from the graph

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plotting inhibition percentage. BHA was used as positive standard. All tests were run

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in triplicate, and the average value was calculated.

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2.5. DPPH spiking with HPLC analysis

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P. odoratum extract (100 μL, 10.0 mg/mL) reacted with DPPH (400 μL, 36 mg/mL),

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then the mixtures were incubated at 37 °C for 30 min, and then passed through a 0.45 8

Page 10 of 36

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μm filter for HPLC analysis. P. odoratum extract (2.0 mg/mL) was used as a control.

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2.6. Preparation and application of Fe3O4@PDA microspheres At first, Fe3O4 microspheres were synthesized by solvothermal method according to

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our previous work [20‒21]. Then Fe3O4 microspheres (200 mg) and DA (380 mg)

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were dispersed in 200 mL of Tris buffer (10 mM, pH 8.5), and then the mixtures were

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mechanically stirred for 24 hours at room temperature. The resultant Fe3O4@PDA

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microspheres were isolated magnetically, rinsed with ethanol and deionized water, and

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dried overnight under vacuum at 40 °C.

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Fe3O4@PDA microspheres (10 mg) were suspended in 2 mL of P. odoratum extract

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(2 mg/mL in water) for magnetic SPE. After shaking at room temperature for 20 min,

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Fe3O4@PDA was separated by a magnet. The adsorbed cinnamides and

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homoisoflavanones were then desorbed by 2 mL of acetonitrile for 5 min, and

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analyzed by HPLC‒DAD‒QTOF-MS/MS.

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3. Results and discussion

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3.1. Preparation and characterization of Fe3O4@PDA microspheres

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DA can be self-polymerized in alkaline to produce surface-adherent PDA on a wide

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variety of solid surfaces (e.g. Fe3O4 microspheres [25], plastic microtube [28]).

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Notably, the properties of hydrophilic PDA and magnetic Fe3O4 make Fe3O4@PDA

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excellent water dispersibility and easy magnetic separation.

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The core-shell Fe3O4@PDA can be obviously observed by TEM (Fig. 2), which

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showed regular spherical shape and good dispersibility. The particle diameter of

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Fe3O4@PDA was 200‒300 nm and the PDA coat is about 50 nm in thickness. In

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FT-IR spectra of Fe3O4 (Fig. 3a), the strong absorption peak at 582 cm–1 was 9

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characteristic of Fe–O vibration. For Fe3O4@PDA (Fig. 3b), the appearance of a

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broad stretching vibration band at 3410 cm–1 for catechol hydrogen groups and N–H

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groups, C=C resonance vibrations and C–N stretching vibration at about 1608, 1293,

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and 1511 cm–1 suggested that PDA was successfully coated on the surface of Fe3O4.

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The magnetic saturation of Fe3O4@PDA at about 70.5 emu/g (a remanence of 4.5

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emu/g and a coercivity of 44.5 Oe) (Fig. 4) showed that Fe3O4@PDA has high

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magnetic responsivity, and could be accumulated in water under conventional magnet

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and dispersed quickly within a slight shake once the magnetic field was removed.

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3.2. DPPH spiking with HPLC analysis

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Natural products are highly complex system with different polarities and quantities

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of components, proved to be a crucial and challenging task to thoroughly separate and

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identify them [27]. Then chromatographic conditions especially the compositions of

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mobile phase are very important to HPLC analysis. FeCl3 color reaction shows that P.

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odoratum extraction contains large quantity of polyphenolic components. The

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addition of acid to mobile phase could improve remarkably the separation efficiency

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and peak shape of polyphenolic components [27,30], therefore, in the course of

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optimizing separation conditions, mobile phase (methanol–water, acetonitrile–water

237

and different concentrations of acetic acid or formic acid in water), gradient program

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(gradient time, gradient shape and initial composition of the mobile phase), column

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temperature and detection wavelength (relatively higher absorption) were investigated.

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Finally, best resolution, shortest analysis time and lowest pressure variations were

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achieved when a gradient elution mode composed of solvent A (0.4% acetic acid in

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water) and B (0.4% acetic acid in acetonitrile) was programmed as follows: 0–5 min,

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5% B; 5–45 min, 5–65% B, and flow rate and column temperature were set at 1.0

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mL/min and 25 °C, respectively. HPLC chromatogram of P. odoratum acquired at 290

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nm is shown in Figure 1b. Compound I was positively identified as furfural by

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comparison of their chromatographic characteristics with standard, which probably

247

was a reaction product of polysaccharides from the high-temperature extraction

248

procedure [31]. Compounds 1‒10 had similar UV spectra with maximum absorbance

249

at 290–300 (shoulder) and 310–320 nm, presumably corresponding to cinnamides

250

[26]. Compounds 11‒25 had maximum UV absorptions at about 290 nm (band II)

251

with a weak shoulder at about 340 nm (band I), which is characteristic of

252

homoisoflavanones [32].

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DPPH is a kind of stable organic radical used for evaluating radical scavenging

254

abilities of antioxidants. P. odoratum extract showed potent capacity to scavenge

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DPPH with the IC50 value of 123.8 ± 14.4 μg/mL (Table 1), indicating the richness of

256

antioxidants. In the case of polyphenolic antioxidants, one or more hydrogen atoms

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will be transferred to DPPH after reaction with DPPH, and then their conjugated

258

structures are destroyed, and as a result, their HPLC peaks will disappeare. Fig. 1a

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showed the HPLC chromatogram of P. odoratum after DPPH spiking. By comparing

260

it with Fig. 1b, it is easy to see that peak areas of twenty-four compounds (1, 3‒25)

261

almost disappeared after spiking with DPPH solution, and peak area of one compound

262

(2) reduced obviously. Therefore, twenty-five compounds (1‒25) were the main

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antioxidants in P. odoratum.

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3.3. Selective extraction of antioxidants from P. odoratum

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Despite the superior chromatographic resolution of HPLC using C18 column,

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co-elution of interfering compounds with interesting compounds remained a common

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issue, which then affected the ESI droplet desolvation process and then the structural 11

Page 13 of 36

elucidation of interesting compounds, especially that of the minor ones. Therefore, a

270

clean-up procedure should be conducted before HPLC‒MS/MS analysis. The

271

dihydroxyindole, indoledione, and dopamine units in PDA coating had high affinity

272

for polyphenolic compounds through a combination of charge transfer, π-π stacking

273

and hydrogen-bonding interactions. Therefore, Fe3O4@PDA could be used as a kind

274

of SPE material to selectively extract polyphenolic compounds from natural products.

275

In order to improve extraction efficiency, some essential factors were investigated,

276

i.e. the amount of Fe3O4@PDA, extraction time, desorption solvent and time, when

277

the concentration of P. odoratum extract was set at 2 mg/mL. The results indicated

278

that highest recoveries of all the interesting compounds (55%‒100%) were achieved

279

when the amount of Fe3O4@PDA, extraction time, desorption solvent and time were

280

10 mg, 20 min, acetonitrile, and 5 min, respectively. Fig. 1c showed the HPLC

281

chromatogram of P. odoratum after Fe3O4@PDA extraction under the optimal

282

conditions, which indicated that twenty-five objective antioxidants (1‒25) in P.

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odoratum extract (present in Fig. 1b) were extracted efficiently.

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3.4. Characterization and identification of antioxidants in P. odoratum High resolution MS could measure mass at an extremely high accuracy (< 2 ppm),

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which then could provide particular molecular formulas, and has been proven to be a

288

very powerful approach for structural characterization [33‒34]. Both positive and

289

negative ion modes were evaluated in MS detection. The results indicated that

290

consistent signals were generated in positive and negative ion modes, however, the

291

positive ion mode mainly produced [M+Na]+ ions, whilst the negative ion mode had a

292

higher intensity with [M‒H]‒ ions. Therefore, negative ion mode was selected. Fig. 5a

293

and b showed HPLC–MS/MS total ion current profiles of P. odoratum extract before

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Page 14 of 36

and after Fe3O4@PDA extraction. It was easy to see that cleaner ion signal, better

295

resolution, and lower background noise were achieved after Fe3O4@PDA extraction

296

because of the rid of interfering compounds. Finally, antioxidants 1–25 had been

297

identified or tentatively characterized, and the retention time, UV and MS data of

298

them are presented in Table 2, while the structures of these antioxidants are shown in

299

Supplementary Fig. S1.

300

At first, N-trans-feruloyltyramine (compound 10) was taken as an example to explain

301

fragmentation details of cinnamides. Fig. 6a revealed that 10 had a quasi-molecular

302

ion [M‒H]‒ at m/z 312.1241 (C18H18NO4, 1.6 ppm error). The fragment ion at m/z

303

190.0870 with a high intensity could be related to the deprotonated amide of

304

N-[2-(4-hydroxyphenyl)ethyl] acrylamide (C11H13NO2). In addition, the fragment ions

305

at m/z 178.0871 and 135.0448 corresponded to the deprotonated forms of

306

N-[2-(4-hydroxyphenyl)ethyl] acetamide (C10H13NO2) and dihidroxystyrene (C8H8O2),

307

respectively [35]. The proposed fragmentation pathways for 10 are given in Fig. 6b.

308

The isomeric compound N-cis-feruloyltyramine (compound 8) presented identical

309

fragment ions with those of 10. However, they exhibited different maximum UV

310

absorption (275 and 305 nm for 8, and red-shift to 292 and 318 nm for 10) (Table 2).

311

Moreover, 8 had shorter HPLC retention times than 10 (Fig. 1b). All the diagnostic

312

difference provided information for the structural identification of cis/trans isomers

313

[36]. Compounds 7 (tR, 26.11 min; λmax, 275 and 303 nm) and 9 (tR, 28.03 min; λmax,

314

291 and 308 nm) exhibited the same [M–H]– ions at m/z 282.1132 (C17H17NO3), 40

315

Da less than those of 8/10, which was consistent with the disappearance of a methoxyl

316

group. Furthermore, there existed three characteristic fragment ions at m/z 119 (16 Da

317

less than those of 8/10), 190 and 178 (the same with those of 8/10). Thus, structures

318

of compounds 7 and 9 were tentatively established as N-cis-p-coumaroyltyramine and

Ac

ce pt

ed

M

an

us

cr

ip t

294

13

Page 15 of 36

N-trans-p-coumaroyltyramine, which were further confirmed by comparing their

320

HPLC–DAD–MS/MS data with those of standards. Compounds 3 and 5 had similar

321

UV spectra with those of 7 and 9, respectively, and they exhibited [M–H]– ions at m/z

322

298 (C17H17NO4, 16 Da greater than those of 7/9). The abundant [M‒H‒H2O]‒

323

fragment ion was observed. Comparing the other information provided by MS/MS

324

spectra for 3/5 to 7/9, it was deduced that an octopamine moiety in 3/5 replaced the

325

tyramine moiety in 7/9. Then compounds 3 and 5 could be identified as

326

N-cis-p-coumaroyloctopamine and N-trans-p-coumaroyloctopamine [36]. Using

327

similar

328

N-cis-feruloyloctopamine and N-trans-feruloyloctopamine. Compounds 1 and 2

329

exhibited [M–H]– ions at m/z 162.0552 (C9H8NO2) and 192.0658 (C10H11NO3), which

330

both gave a [M‒H‒CONH]‒ fragment ion at m/z 119.0499 (C8H7O) and 149.0605

331

(C9H9O2). Taking into account the information provided by UV spectra, it was

332

deduced that compounds 1 and 2 were trans-p-coumaramide and trans-ferulamide. It

333

is noted that five cinnamides (1, 3, 5, 7 and 9) were reported in the genus

334

Polygonatum for the first time, and to date, this is the first report of the occurrence of

335

three phenylethylamides (2, 4 and 8) in family Liliaceae.

compounds

4

and

6

were

plausibly

identified

as

ce pt

ed

M

an

principle,

us

cr

ip t

319

The fragmentation behaviors of homoisoflavonoids correlated with the saturation

337

status of C2‒3 bond and the type of substituted groups on B-ring [37‒38]. When C2‒3

338

bond was saturated and B-ring was substituted with a hydroxyl group, the

339

predominant fragmentation was the cleavage of C3‒9 bond by loss of the benzyl part of

340

the B-ring together with the acquisition of a hydrogen to give [M‒H‒CH2‒B-ring+H]‒,

341

which was then followed by the neutral loss of a carbonyl group to attain

342

[M‒H‒CH2‒B-ring+H‒CO]‒. Up to now, homoisoflavonoids isolated from P.

343

odoratum all contained a saturated C2‒3 bond, and most of them had a hydroxyl group

Ac

336

14

Page 16 of 36

[8‒10].

344

on

7

revealed

345

homoisoflavanone (compound 22) had a quasi-molecular ion [M‒H]‒ at m/z 313.1074

346

(C18H18O5, –0.6 ppm error), and it showed prominent fragment ions at m/z 207.0654

347

(C11H11O4,

348

[M‒H‒CH2‒B-ring+H‒CO]‒). Based on the characteristic fragmentation ions and

349

comparison with standards, compounds 11, 14, 15, 16, 17, 20, 21 and 25 were

350

unequivocally

351

5,7,4'-trihydroxyl

352

homoisoflavanone,

353

5,7,2'-trihydroxy-8,4'-dimethoxy

354

homoisoflavanone,

355

5,7-dihydroxyl-6-methyl-8,4'-dimethoxyl homoisoflavanone. The mass spectra of

356

compound 12 displayed parent ion [M–H]– at m/z 331.0815 (C17H16O7, 14 Da less

357

than that of 17) with the fragment ions at m/z 209.0547, 195.0290, 181.0497 and

358

167.0340 (the same with those of 17), indicating that compound 12 and 17 contained

359

the same A-ring, and the difference was that compound 12 had two hydroxyl groups

360

in B-ring, whilst compound 17 only had a hydroxyl group and a methoxyl group in

361

B-ring.

362

5,7,2',4'-tetrahydroxy-8-methoxyl homoisoflavanone. Compounds 19, 23 and 24

363

displayed the same fragment ions with those of 11/14, 15/20, 16/21, then using similar

364

principles, compounds 19, 23 and 24 were preliminarily characterized as

365

5,7,2'-trihydroxy-4'-methoxyl

366

methoxyl

367

homoisoflavanone. Compounds 13 and 15 were a pair of isomers with the same parent

368

ions and fragment ions, therefore, the difference between them was the substituted

[M‒H‒CH2‒B-ring+H]‒)

identified

as

that

and

5,7,4'-trihydroxyl-6,8-dimethyl

m/z

179.0705

5,7,2',4'-tetrahydroxyl

homoisoflavanone,

5,7,2',4'-tetrahydroxy-6-methyl

us

homoisoflavanone,

(C10H11O3,

ip t

Fig.

cr

B-ring

5,7,2',4'-tetrahydroxy-6-methoxy-8-methyl

5,7,4'-trihydroxy-6-methyl

an

homoisoflavanone,

homoisoflavanone,

ce pt

ed

M

5,7,4'-trihydroxyl-6-methyl-8-methoxyl homoisoflavanone and

compound

12

could

be

identified

as

Ac

Therefore,

homoisoflavanone

homoisoflavanone, and

5,7,2'-trihydroxy-8-methyl-4'-

5,7,2'-trihydroxy-6-methyl-8,4'-dimethoxyl

15

Page 17 of 36

369

position of methyl group. Compound 13 then could be tentatively established as

370

5,7,2',4'-tetrahydroxy-8-methyl-homoisoflavanone.

371

observed between compounds 18 and 20, and compound 18 was plausibly identified

372

as 5,7,4'-trihydroxy-8-methyl homoisoflavanone. By comparison with previously

373

reported literature, we found that compounds 12, 13, 18 and 19 did not correspond to

374

any of the known homoisoflavanones and may be new compounds. Compound 14

375

was found in family Liliaceae for the first time, and compound 24, previously

376

reported in P. cyrtonema [39], had been identified in this species for the first time.

was

cr

ip t

phenomenon

us

377

3.5. Evaluation of DPPH scavenging activity

an

378

Similar

Antioxidant activities of screened compounds should be evaluated to validate the

380

DPPH spiking method. Only three compounds (10, 20 and 21) had been reported in

381

previous studies to possess DPPH scavenging activities [9, 40]. Due to the lack of

382

commercial standards and the difficulty to isolate some minor or unstable compounds

383

with high purities, thirteen purified compounds (7‒10, 11, 14‒17, 20‒22 and 25) were

384

selected to evaluate their DPPH scavenging activities (Table 1). Cinnamides (7‒10)

385

reflected significant DPPH radical scavenging activities when compared to the

386

standard BHA. Cinnamides (7‒10) exhibited stronger DPPH radical scavenging

387

activities than homoisoflavanones (11, 14‒17, 20‒22 and 25).

389

ed

ce pt

Ac

388

M

379

4. Conclusion

390

In this study, a new method, based on selective solid phase extraction combined

391

with DPPH spiking and HPLC‒DAD‒QTOF-MS/MS, has been successfully

392

developed for sensitive screening and identification of polyphenolic antioxidants in P.

393

odoratum. DPPH spiking with HPLC method exhibited good reliability and 16

Page 18 of 36

sensitivity to screen antioxidants from complex mixtures. By removal of interferences

395

by Fe3O4@PDA, twenty-five polyphenolic antioxidants were sensitively identified by

396

matching their characteristic UV spectra, accurate mass signals and key diagnostic

397

fragment ions with standards and previously reported compounds, and among them,

398

twenty-two compounds were reported for the first time to possess antioxidant

399

activities. It is important to highlight that, to our best knowledge, this is the first study

400

available to systematically investigate antioxidant profile of P. odoratum. Our results

401

would help to explain why P. odoratum can be used to treat human disease associated

402

with oxidative stress. Moreover, the application of this method provided a new

403

promising alternative for rapid and sensitive screening, analysis and identification of

404

polyphenolic antioxidants from other natural products and pharmaceutical

405

formulations by slightly varying the operation conditions.

M

an

us

cr

ip t

394

407

Acknowledgments

ed

406

This work was supported by the National Natural Science Foundation of China

409

(21275163), the Natural Science Foundation of Hunan Province, China (13JJ3099)

410

and the Shenghua Yuying project of Central South University, China.

Ac

ce pt

408

17

Page 19 of 36

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482

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520

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525

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liquid chromatography-diode array detector-mass spectrometry for purification,

527

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528

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524

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530

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532

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529

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534

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536

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537

[37] J. Qi, D.R. Xu, Y.F. Zhou, M.J. Qin, B.Y. Yu, New features on the fragmentation

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546

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545

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ed

544

ionization

ce pt

543

detection/electrospray

Ac

542

chromatography/diode-array

23

Page 25 of 36

Figure captions

550

Fig. 1. HPLC‒UV chromatograms of P. odoratum after (a) and before (b) DPPH

551

spiking, and after Fe3O4@PDA extraction (c).

552

Fig. 2. TEM image of Fe3O4@PDA.

553

Fig. 3. FT-IR spectra of Fe3O4 (a) and Fe3O4@PDA (b).

554

Fig. 4. Magnetization curve of Fe3O4@PDA.

555

Fig. 5. HPLC–MS/MS total ion current profiles of P. odoratum before (a) and after (b)

556

Fe3O4@PDA extraction.

557

Fig. 6. MS/MS spectrum (a) and the main fragmentation pathway (b) of

558

N-trans-feruloyltyramine.

559

Fig. 7. MS/MS spectra of 5,7,4'-trihydroxyl-6,8-dimethyl homoisoflavanone and its

560

fragmentation pathway.

Ac

ce pt

ed

M

an

us

cr

ip t

549

24

Page 26 of 36

Table 1 Antioxidant activities of P. odoratum extract and thirteen purified compounds in DPPH assay. DPPH (IC50, μg/mL)a

P. odoratum extract

123.8 ± 14.4

7

2.9 ± 0.5

8

2.0 ± 0.4

9

2.7 ± 0.6

10

1.6 ± 0.3

11

4.9 ± 0.6

14

15.2 ± 1.9

15

9.1 ± 1.1

16

3.9 ± 0.5

17

6.9 ± 0.8

20

29.6 ± 3.4

ce pt

ed

M

an

us

cr

ip t

Samples

12.1 ± 2.0

21

7.4 ± 1.0

22

32.8 ± 4.2

25

Ac

BHAb

8.2 ± 1.5

a

Each value is mean ± SD (n = 3).

b

Used as control.

25

Page 27 of 36

ip t cr us

Table 2 List of fully/partially identified polyphenolic antioxidants from P. odoratum. tR

λmax

[M–H]– (m/z)

Molecular

Fragment ions

(min)

(nm)

(Δ ppm)

formula

(m/z)

an

No.

(neutral form)

Identification

19.19

293, 308

162.0552 (–1.9)

C9H8NO2

119.0499

trans-p-coumaramideb

2

20.84

294, 318

192.0658 (–1.6)

C10H11NO3

149.0605, 135.0447

trans-ferulamidec

3

21.36

275, 303

298.1084 (1.7)

C17H17NO4

280.0988, 188.0715, 119.0499

N-cis-p-coumaroyloctopamineb

4

22.14

275, 305

328.1188 (0.9)

C18H19NO5

310.1081, 188.0716, 135.0448

N-cis-feruloyloctopaminec

5

22.85

292, 307

298.1081 (0.7)

C17H17NO4

280.0987, 118.0713, 119.0498

N-trans-p-coumaroyloctopamineb

6

23.42

293, 318

328.1191 (1.8)

C18H19NO5

310.1084, 118.0715, 135.0447

N-trans-feruloyloctopaminea

7

26.11

275, 303

282.1132 (0.7)

C17H17NO3

190.0871, 178.0872, 119.0499

N-cis-p-coumaroyltyramineb

8

27.15

275, 305

312.1241 (1.6)

C18H19NO4

190.0870, 178.0873, 135.0449

N-cis-feruloyltyraminec

9

28.03

291, 308

282.1132 (0.7)

C17H17NO3

190.0871, 178.0873, 119.0499

N-trans-p-coumaroyltyramineb

10

28.44

292, 318

312.1241 (1.6)

C18H19NO4

190.0870, 178.0871, 135.0448

N-trans-feruloyltyraminea

11

31.85

289

301.0714 (0.7)

C16H14O6

179.0342, 151.0395

5,7,2',4'-tetrahydroxyl homoisoflavanonea

12

33.48

290

C17H16O7

209.0547, 195.0290, 181.0497,

5,7,2',4'-tetrahydroxy-8-methoxyl homoisoflavanonee

34.25

293

14

35.45

289

15

35.69

292

16

36.17

296

17

37.49

291

d

ep te

Ac c

13

M

1

331.0815 (–0.9)

167.0340

315.0863 (–1.9)

C17H16O6

193.0498, 165.0546

5,7,2',4'-tetrahydroxy-8-methyl homoisoflavanonee

285.0761 (–0.7)

C16H14O5

179.0342, 151.0394

5,7,4'-trihydroxyl homoisoflavanonec

315.0864 (–1.6)

C17H16O6

193.0497, 165.0546

5,7,2',4'-tetrahydroxy-6-methyl-homoisoflavanonea

345.0969 (–1.5)

C18H18O7

223.0605, 209.0444, 195.0657,

5,7,2',4'-tetrahydroxy-6-methoxy-8-methyl

181.0499

homoisoflavanonea

209.0548, 195.0292, 181.0498,

5,7,2'-trihydroxy-8,4'-dimethoxy homoisoflavanonea

345.0970 (–1.2)

C18H18O7

26

Page 28 of 36

ip t cr us

167.0341 37.92

294

299.0916 (–1.0)

C17H16O5

193.0499, 165.0547

5,7,4'-trihydroxy-8-methyl homoisoflavanonee

19

38.29

289

315.0864 (–1.6)

C17H16O6

179.0342, 151.0393

5,7,2'-trihydroxy-4'-methoxyl homoisoflavanonee

20

39.44

293

299.0915 (1.3)

C17H16O5

193.0497, 165.0547

5,7,4'-trihydroxy-6-methyl homoisoflavanonea

21

40.09

297

329.1091 (–1.8)

C18H18O6

223.0604, 209.0446, 195.0657,

5,7,4'-trihydroxy-6-methyl-8-methoxyl homoisoflavanonea

an

18

181.0500 296

313.1074 (–0.6)

C18H18O5

23

41.70

291

329.1021 (–1.3)

C18H18O6

24

42.50

296

359.1128 (–0.8)

C19H20O7

a

44.09

296

343.1186 (1.2)

C19H20O6

ep te

25

207.0654,179.0705

5,7,4'-trihydroxyl-6,8-dimethyl homoisoflavanonea

193.0499, 165.0548

5,7,2'-trihydroxy-8-methyl-4'-methoxyl homoisoflavanonea

223.0604, 209.0447, 195.0655,

5,7,2'-trihydroxy-6-methyl-8,4'-dimethoxyl

181.0500

homoisoflavanoned

222.0529, 208.0375, 194.0581,

5,7-dihydroxyl-6-methyl-8,4'-dimethoxyl homoisoflavanonea

M

41.34

d

22

180.0424

compounds have been previously reported in P. odoratum.

b

compounds were isolated from genus Polygonatum for the first time.

c

compounds were isolated from family Liliaceae for the first time.

d

novel compounds.

Ac c

compounds were found in genus Polygonatum but not in P. odoratum.

e

27

Page 29 of 36

Figure(s)

Fig. 1

300

6

250

5 4

I

50

1

c b a

0

10

3

7

8

20 18-19 2122 12 14-16 23 13 17 25 24

20 30 Time (min)

40

Ac

ce pt

ed

M

an

0

9 10

2

cr

100

11

us

150

ip t

mAU

200

Page 30 of 36

Ac

ce pt

ed

M

an

us

cr

ip t

Fig. 2

Page 31 of 36

Fig. 3

a

1292.9 1510.9 1608.4

3410.1

ip t

100

b

cr

50

0

3000 2000 -1 Wavenumber (cm )

1000

ce pt

ed

M

an

4000

us

583.8

Ac

Transmittance (%)

150

Page 32 of 36

Fig. 4

80

ip t

0

cr

M(emu/g)

40

-80 0 H(Oe)

5000

10000

an

-5000

Ac

ce pt

ed

M

-10000

us

-40

Page 33 of 36

Fig. 5

×106

6

a

5

ip t

3 2

cr

Intensity

4

us

1 0

6

20 30 Time (min)

40

12

b

M

×106

10

an

0

5

11

ed

21 13 14-16

6

7

3

1

4

ce pt

Intensity

4

2

23

1

Ac

0

0

10

5

8

20 22

910

20 30 Time (min)

23 25 17-19

24

40

Page 34 of 36

Fig. 6

190.0870 178.0871

100 a

135.0448

40

200

m/z

250

us

150

cr

-

[M-H] 312.1241

20

0 100

ip t

60

300

an

Relative intensity/%

80

350

O

-C7H6O2

M

b O OH

NH

m/z 190

m/z 312

H

-C8H6O2

-C9H9NO2

NH

OH

H

m/z 178 H3CO

HO

H

HO

HO

H

m/z 135

Ac

ce pt

OH

H

OH

O

ed

OCH3

NH

Page 35 of 36

Fig. 7

207.0654

100

CH3 HO

207 O

OH

B 3

H3C

9 OH

40

O

ip t

60

179

-

[M-H] 313.1074

200

m/z

250

300

an

150

350

ce pt

ed

M

0 100

us

20

cr

179.0705

Ac

Relative intensity/%

80

Page 36 of 36