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Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxi...
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Yunnaneic acid B - a component of Pulmonaria officinalis extract prevents the peroxynitrite-induced oxidative stress in vitro Justyna Krzy#anowska-Kowalczyk, Joanna Kolodziejczyk-Czepas, Mariusz Kowalczyk, #ukasz Pecio, Pawel Nowak, and Anna Stochmal J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 28 Apr 2017 Downloaded from http://pubs.acs.org on April 30, 2017

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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.

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Journal of Agricultural and Food Chemistry

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Yunnaneic acid B - a component of Pulmonaria officinalis extract prevents the peroxynitrite-

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induced oxidative stress in vitro

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Justyna Krzyzanowska-Kowalczyk#, Joanna Kolodziejczyk-Czepas*§, Mariusz Kowalczyk#,

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Łukasz Pecio#, Pawel Nowak§, Anna Stochmal#

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#

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Cultivation, State Research Institute, Czartoryskich 8, 24-100 Puławy, Poland

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§

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Department of Biochemistry and Crop Quality, Institute of Soil Science and Plant

Department of General Biochemistry, Faculty of Biology and Environmental Protection,

University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland,

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*Corresponding author

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Dr. J. Kolodziejczyk-Czepas, Department of General Biochemistry, University of Lodz,

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Pomorska 141/143 90-236 Lodz, Poland. E-mail: [email protected], Phone/Fax: +48

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42 635 44 84

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Abstract

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Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of

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yunnaneic acid B. We studied antioxidant activity, cytotoxicity of this compound (1-50

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µg/ml) and its contents in various plant extracts. This is the first study confirming the

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presence of yunnaneic acid B in P. officinalis L., Pulmonaria obscura Dumort, and hence, in

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the Boraginaceae family.

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Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-

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scavenging efficacy in inorganic experimental systems provided the EC50 values of 7.14 and

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50.45 µg/ml, respectively. Then, we examined the antioxidant action of yunnaneic acid B in

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blood plasma, under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B

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effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it

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was able to prevent the peroxynitrite-induced decrease of the non-enzymatic antioxidant

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capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations

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≤50 µg/ml) towards peripheral blood mononuclear cells was excluded.

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Keywords: antioxidant; blood plasma; cytotoxicity; Pulmonaria; yunnaneic acid B

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Introduction

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The term “yunnaneic acids” is a collective name of several oligomeric derivatives of

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caffeic and rosmarinic acids, and originates from Salvia yunnanensis C. H. Wright, a plant,

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which was identified for the first time as a source of these compounds. So far, eight yunnaneic

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acids (signed with letters A-H) have been isolated from roots of this herb [1,2]. Yunnaneic

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acids C (C27H22O12) and D (C27H24O12) are trimers of caffeic acid. Yunnaneic acid A

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(C54H46O24) contains yunnaneic acids C and D, while yunnaneic acid B (C54H46O25) is a dimer

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of yunnaneic acid C, which comprises caffeic acid and the Diels-Alder adduct of rosmarinic

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acid (Fig. 1). Yunnaneic acid E (C27H24O14) and F (C29H26O14) are also derivatives of

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yunnaneic acid C. Yunnaneic acid G (C36H30O16) and H (C36H26O16) are arylnaphthalene-type

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lignan esters, formed as a result of oxidative coupling of two molecules of rosmarinic acid [3].

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Currently, S. yunnanensis and S. miltiorrhiza are considered as primary sources of

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yunnaneic acids, however, these polyphenolic oligomers may also be synthesized by other

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plant species. Contribution of yunnaneic acids to pharmacological properties of medicinal

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plants has not been established yet. Contrary to numerous reports confirming a wide range of

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biological activities of caffeic acid and its low-molecular derivatives (e.g. chlorogenic and

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rosmarinic acids) [4-7], physiological effects of yunnaneic acids still remain unknown.

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Thus, our study has evaluated biological properties of yunnaneic acid B, isolated from

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aerial parts of Pulmonaria officinalis L. (lungwort). We have confirmed the presence of this

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compound in two Pulmonaria species (P. officinalis and P. obscura) and studied its

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antioxidant activity using inorganic experimental conditions as well as in biological

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experimental system, (i.e. blood plasma, exposed to peroxynitrite (ONOO−)-induced oxidative

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stress in vitro). Additionally, cytotoxicity of yunnaneic acid B towards peripheral blood

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mononuclear cells (PBMCs) was examined.

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Materials and methods

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Plant material

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Plant material was obtained from the following national suppliers of herbs: Pulmonariae

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herba from Flos (Mokrsko, Poland), Dary Natury (Koryciny, Poland), Herbapol (Kraków,

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Poland), aerial parts of Pulmonaria obscura Dumort. were collected from natural habitats in

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Puławy, Poland; roots of Salvia miltiorrhiza were from Nanga (Złotów, Poland), Boraginis

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herba and Salviae folium were from Dary Natury. Aerial parts of Pulmonaria officinalis L.

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used for yunnaneic acid B isolation as well as quantity analysis were purchased from Kania

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(Częstochowa, Poland) herb supplier. The voucher samples are deposited at the Department

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of Biochemistry and Crop Quality of Institute of Soli Science and Plant Cultivation, Puławy,

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Poland.

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Chemicals

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The synthesis of peroxynitrite was carried out according to the protocol described by Pryor et

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al. [8] DPPH (1,1-diphenyl-2-picrylhydrazyl), Trolox® as well as caffeic, rosmarinic and

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thiobarbituric acids were purchased from Aldrich (Sigma-Aldrich, St. Louis, USA). PBMCs

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were isolated with the use of Histopaque® 1077 medium (Sigma-Aldrich, St. Louis, USA).

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The cytotoxicity assay was conducted using equipment and materials produced by

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NanoEnTek Inc. (Seoul, Korea). All other organic and inorganic reagents (of an analytical

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grade) were provided by local or international suppliers.

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Isolation of yunnaneic acid B

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Pulmonaria officinalis L. aerial parts were finely ground and defatted with chloroform in

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Soxhlet apparatus. The methanol extract (80% v/v methanol in water) was purified in a

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stepwise manner by different chromatographic methods. The extract was applied to 4 ACS Paragon Plus Environment

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preconditioned RP-C18 column (80×100 mm, Cosmosil 140C18-PREP, 140 µm), followed by

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removal of polar constituents, while flavonoids and other phenolic compounds were eluted

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with 50% methanol. This fraction was further purified by low-pressure chromatography on

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Sephadex LH-20 column (48×400 mm), as well as a reversed phase column (32×300 mm,

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Cosmosil 40C18-PREP, 40 µm), followed by semi-preparative HPLC (10×250 mm, Atlantis

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T3 Prep OBD, 5 µm). That last purification step was performed on a Glison chromatographic

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system (Gilson Inc., Middleton, WI, USA) equipped with an evaporative light scattering

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detector (ESLD, Gilson PrepELS II). The drift tube of ELSD detector was maintained at 65°C

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and the pressure of the nebulizer gas (nitrogen) was 47 psi. Fraction was separated

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isocratically, using 20% aqueous acetonitrile solutions which containing 0.1% formic acid.

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The mobile phase flow was 4 ml/min, the column temperature was maintained at 35 °C. The

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effluent from HPLC system was diverted through a passive splitter to ELSD with a split ratio

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1:100.

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The chromatographic separation yielded a cream-colored substance, subsequently identified

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as yunnaneic acid B by means of HR-QTOF-MS/MS and NMR techniques (1D and 2D) and

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compared with literature data [1,9]. Its purity was determined to be higher than 80% by means

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of UHPLC analysis with charged aerosol detection.

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NMR spectra were acquired in CD3OD (with 0.1% of trifluoroacetic acid) at 25°C on an

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Avance III HD 500 MHz instrument (1H: 500.20 MHz;

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Rheinstetten, Germany) equipped with an inverse BBI probe. Standard pulse sequences and

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parameters were used to obtain 1D 1H, 1D selective TOCSY, 1D selective NOESY, 1D

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UDEFT

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gH2BC, gHMBC, band-selective HSQC and CT-HMBC spectra. Chemical shift referencing

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was carried out using the internal solvent resonances at δH 3.31 and δC 49.0 (calibrated to

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TMS at 0.00 ppm). Chemical shifts are in ppm and the J values in Hz. Data processing was

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C 125.78 MHz; Bruker BioSpin,

C [10] and DEPT-135, g-DQF-COSY, gNOESY (mixing time 400 ms), gHSQC,

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performed with the Topspin software (version 3.5pl2, Bruker BioSpin, Rheinstetten,

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Germany).

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Extraction and purification of plant extracts

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Five grams of each plant material was extracted in Soxhlet apparatus using chloroform or, in

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case of root samples from Salvia miltiorrhiza, hexane to remove non-polar constituents.

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Defatted plant material (100 mg) was extracted using an automated accelerated solvent

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extractor, ASE 200 (Dionex, Sunnyvale, CA). Conditions of the extraction process were as

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follows: extraction solvent 80% (v/v) methanol in water, solvent pressure 1500 psi,

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temperature of extraction cells 100 °C, 3 cycles of the extraction each 2 min. To test the

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impact of temperature on the extraction efficiency, preliminary extractions using different

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temperature conditions (40, 70 and 100 °C) were performed.

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Crude extracts were purified by solid phase extraction (SPE). After evaporation to dryness

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under reduced pressure at 40 °C, extracts were dissolved in Milli-Q water (Millipore Corp.,

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Billerica, MA) and loaded on Oasis HLB 3cc (60mg) cartridges (Waters) preconditioned with

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methanol (2ml) and water (2 ml). Secondary metabolites fractions were eluted using 80%

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methanol in 0.1% HCOOH (2 ml). Prior to estimation of phenolic acids contents, samples

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were diluted ten times with 80% (v/v) methanol in water. All samples were prepared in

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triplicate.

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Estimation of yunnaneic acid B and rosmarinic acid concentrations in plant extracts

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LC-ESI-QTOF-MS estimation of phenolic acids contents was carried out using the Thermo

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Ultimate 3000 RS (Thermo Fischer Scientific, Waltham, MS, USA) chromatographic system,

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hyphenated to a Bruker Impact II HD (Bruker, Billerica, MA, USA) quadrupole-time of flight

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(QTOF) mass spectrometer. Separations were performed on a Waters CSH C18 column (2.1 x 6 ACS Paragon Plus Environment

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100 mm, 1.7µm, Milford, MA, USA), with mobile phase A consisting of 0.1% (v/v) formic

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acid in water, and the mobile phase B consisting of 0.1% (v/v) formic acid in acetonitrile. A

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linear gradient from 10% to 35% of phase B in phase A over 15 minutes was used to separate

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phenolic acids. The flow rate was 0.55 ml/min and the column was held at 50 °C.

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Linear (centroid) spectra were acquired in a negative ion mode over a mass range from m/z 50

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to m/z 2000 with 5 Hz frequency. Operating parameters of the ESI ion source were as follows:

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capillary voltage 3 kV; dry gas flow 6 l/min; dry gas temperature 200 °C; nebulizer pressure

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0.7 bar; collision RF 700.0 V; transfer time 100.0 µs; prepulse storage 7.0 µs. Ultra-pure

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nitrogen was used as drying and nebulizer gas, argon was used as a collision gas. Collision

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energy was set automatically from 15 to 75 eV depending on the m/z of fragmented ion.

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Acquired data were calibrated internally with sodium formate introduced to the ion source at

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the beginning of each separation via a 20 µl loop.

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Processing of spectra was performed using Bruker DataAnalysis 4.3 software. Base peak ion

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chromatograms were extracted from the full scan data with 0.01 Da width for ions m/z

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353.0878, m/z 359.0772 and m/z 1093.2255, corresponding to deprotonated molecules of

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chlorogenic, rosmarinic and yunnaneic B acid. These traces were then smoothed using

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Savitzky-Golay algorithm (window width 5 points, one iteration) and peaks at RT 2.5, 6.8 and

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9.0 min were integrated. Identity of analytes was confirmed by comparing their retention time

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and MS/MS spectra with these of authentic analytical standards.

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Calibration curves were prepared from dilutions of stock solutions using 1 ml volumetric

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flasks. Each calibration level was prepared in duplicate and analyzed three times. The relation

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of concentration to peak area was linear in the range from 10 to 150 pmol/µl (10 to 80

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pmol/µl for yunnaneic acid B), details of calibration curves are shown in Table 1.

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Concentrations of phenolic acids in plant material were expressed in µg/mg of dry weight

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Analysis of antioxidant activity in inorganic experimental systems

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a) peroxynitrite scavenging assay

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The ability of examined substances to scavenge peroxynitrite was determined by

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measurements of the inhibition of Evans blue bleaching (after 30 min of incubation, 100 µl of

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reagent mixture per microplate well, λ=611 nm). The reaction mixture contained yunnaneic

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acid B or reference compounds (1-100 µg/ml), 50 µM Evans blue, 0.1 mM

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diethylenetriaminepentaacetic acid (DTPA), 90 mM NaCl, 5 mM KCl and 1 mM ONOO−,

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suspended in 50 mM phosphate-bufered saline (pH 7.4). The ONOO−-induced decrease of

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Evans blue color was calculated using the following equation: % of sample bleaching = 100 ×

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(A0−A1)/A0. Absorbance of control samples (untreated with ONOO−) was assumed as A0

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value, while A1 was the absorbance after 30 min of incubation of reaction mixtures,

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containing 1 mM ONOO− and the examined acid or reference antioxidants. Parameters of

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samples treated with ONOO− in the absence of the antioxidants were then assumed as 100%

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of Evans blue dye oxidation (bleaching).

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b) determination of DPPH• scavenging ability

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Methanol solution of DPPH• (500 µM) was diluted several times (with methanol) in order to

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obtain a working solution with the absorbance of 1.2 (λ=517 nm). Then, stock solutions of the

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examined substances were added to the working DPPH• solution (to their final concentration

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range of 1-50 mg/ml). After 30 min of incubation (dark place, at room temperature) the

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absorbance was recorded. Results were expressed as % of radical scavenging activity (% of

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RSA), calculated from the following equation: RSA (%) = 100 × (A0-A1)/A0. The A0

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parameter was the initial absorbance, while A1 was the absorbance after 30 min of incubation

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of the reaction mixture.

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In vitro evaluation of antioxidant action of yunnaneic acid B in biological experimental

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systems

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a) preparation of blood plasma and fibrinogen samples

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All blood units used in this study were commercially available and purchased from the

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Regional Centre of Blood Donation and Blood Treatment in Lodz, Poland. Stock solutions of

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the examined substances were prepared using 20% DMSO (dimethyl sulfoxide). The final

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concentration of DMSO in samples of plasma and PMBCs suspensions was ≤0.02%. Blood

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plasma samples were preincubated for 15 min at 37°C with yunnaneic acid B (1-50 µg/ml) or

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reference oxidants, i.e. rosmarinic acid and Trolox® (5 µg/ml). Then, the samples were

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exposed to ONOO−, added to the final concentration of 150 µM (the FRAP assay) or 100 µM

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(other antioxidant assays). Samples containing plasma treated with ONOO− in the absence of

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yunnaneic acid B or reference compounds were also prepared. Control plasma was treated

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with neither the investigated acid/reference antioxidants nor ONOO−, but these samples

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contained 0.02% DMSO (a vehicle for the used compounds).

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Fibrinogen was isolated from blood plasma using the cold ethanol precipitation

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technique, described by Doolittle et al. [11]. Then, the protein preparation was diluted in

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0.05M TBS (Tris-buffered saline, pH 7.4) to the final concentration of 2 mg/ml. Analogously

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to plasma samples, fibrinogen was preincubated with the examined substances, exposed to

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ONOO− and assayed in order to detect 3-nitrotyrosine during a competitive ELISA. All values

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were expressed as equivalents of nitrated standard protein, i.e. 3-nitrotyrosine containing

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fibrinogen [µM 3-NT-Fg].

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b) determination of antioxidant capacity using the ferric reducing ability of blood plasma

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(FRAP) assay

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The assay was based on evaluation of the non-enzymatic antioxidant capacity (NEAC) of

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blood plasma, conducted by measurements of its ability to reduce of ferric ions (Fe3+) to

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ferrous ions (Fe2+). Experiments were performed according to protocol, described previously

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by Bartosz & Janaszewska [12]. The standard curve was prepared from FeSO4.

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c) measurements of protein and lipid oxidation biomarkers in blood plasma

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Protective action of yunnaneic acid B against ONOO−-induced oxidation of blood plasma

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proteins was evaluated using protein thiol level [13] and 3-nitrotyrosine [14] as biomarkers of

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oxidative stress. Peroxidation of plasma lipids was determined using thiobarbituric acid-

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reactive substances (TBARS) as a biomarker, according to the method described previously

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by Wachowicz and Kustron [15].

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d) cytotoxicity assay

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PBMCs were isolated from fresh human blood according to the protocol provided by the

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manufacturer and suspended in 0.02 M phosphate-buffered saline (PBS) for obtaining the cell

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count of 1 × 106/ml. Cells were incubated with the examined acid (1-50 µg/ml) at 37ºC, for 1-

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4 hrs. Cytotoxicity evaluation was carried out in a microchip type automatic cell counter

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Adam-MC DigitalBio (NanoEnTek Inc., Seoul, Korea), using propidium iodide as a

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fluorescent dye.

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Statistical analysis

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Uncertain data were eliminated by the use of the Q-Dixon test. Antioxidant action of

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yunnaneic acid B and reference compounds was established in comparison to samples treated 10 ACS Paragon Plus Environment

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with peroxynitrite in the absence of the examined compounds. Evaluation of statistical

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significance was performed with the use of t-Student’s and Wilcoxon tests. All samples were

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prepared at least in duplicate, i.e. at least two independent pre-incubations of the examined

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phenolic acid (or reference antioxidants) with blood plasma or PBMCs, isolated from each

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donor were performed. All values were expressed as mean±SD; p