Yunnaneic Acid B, a Component of Pulmonaria ... - ACS Publications

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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

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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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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

Yunnaneic Acid B, a Component of Pulmonaria ... - ACS Publications

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