Isolation and biological activities of lyoniside from rhizomes and stems of Vaccinium myrtillus

Phytochemistry Letters 4 (2011) 138ndash143

Contents lists available at ScienceDirect

Phytochemistry Letters

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Isolation and biological activities of lyoniside from rhizomes and stems ofVaccinium myrtillus

Anna Szakiel a Laurence Voutquenne-Nazabadioko b Max Henry c

a Department of Plant Biochemistry Faculty of Biology University of Warsaw ul Miecznikowa 1 02-096 Warszawa Polandb Groupe Isolement et Structure Institut de Chimie Moleculaire de Reims (ICMR) CNRS UMR 6229 Bat 18 BP 1039 51687 Reims Cedex Francec Structure et Reactivite des Systemes Moleculaires Complexes (SRSMC) Nancy-Universite UMR7565 CNRS-UHP 5 rue Albert Lebrun BP 80403 54001 Nancy Cedex France

A R T I C L E I N F O

Article history

Received 8 September 2010

Received in revised form 18 January 2011

Accepted 7 February 2011

Available online 21 February 2011

Keywords

Vaccinium myrtillus

Ericaceae

Bilberry

Droplet counter-current chromatography

Lignan

Lyoniside

A B S T R A C T

A lignan glycoside identified as lyoniside (9-O-b-D-xylopyranosyl(+)lyoniresinol) was obtained from

ethanol extracts of the rhizomes and stems of bilberry (Vaccinium myrtillus L) on a preparative scale by

droplet counter-current chromatography The application of this method permitted the isolation of a

pure substance in only one chromatographical step The occurrence of lyoniside in bilberry is reported

for the first time Seasonal fluctuations in the content of this compound in plant organs were

demonstrated showing its highest levels in bilberry rhizomes and stems during the winter and their

subsequent decrease in the spring In vitro the purified lyoniside was evaluated for antioxidant

allelopathic and antifungal activities It showed significant radical scavenging properties in a 22-

diphenyl-1-picrylhydrazyl (DPPH) assay with IC50 of 23 mg ml1 Applied at concentration of

10 mg ml1 it suppressed by 75 the seedling radical growth of Lactuca sativa and Lepidium sativum

and exerted strong inhibitory effect (55) on germination of Larix decidua Moreover the synergistic

action of lyoniside and triterpene acids was demonstrated in inhibitory effect exerted on germination

and growth of Pinus sylvestris Among 5 tested fungi strains of Ascomycota the highest susceptibility was

shown by Fusarium oxysporum and Mucor hiemalis with mycelial growth inhibited by lyoniside

concentration of 50 mg ml1 by 78 and 80 respectively

2011 Phytochemical Society of Europe Published by Elsevier BV All rights reserved

1 Introduction

Bilberry (Vaccinium myrtillus L Ericaceae) is a perennial dwarfshrub native to Europe and Northern America widely known for itstasty fruit of high nutritive value and its use in folk medicine(Anonymous 2001 Camire 2002) The berries contain high levelsof phenolics ndash mainly anthocyanins (Prior et al 1998 Valentovaet al 2007) ndash which due to their antioxidative properties areconsidered to be the pharmacologically active and health-promoting constituents (Martin-Aragon et al 1999 Yao andVieira 2007) The detection of flavonoids and phenolic acids invarious bilberry organs including flowers (Riihinen et al 2008)and even litter accumulated in the soil around bilberry (Gallet andLebreton 1995) makes this shrub one of the most phenolic-richplants known However in comparison to berries and leaves thephenolic content of the rhizomes and stems remains still lesscharacterized The occurrence of new unusual p-coumarates in

Corresponding author Tel +48 225543316 fax +48 225543221

E-mail addresses szakalbioluwedupl (A Szakiel)

laurencevoutquenneuniv-reimsfr (L Voutquenne-Nazabadioko)

MaxHenrypharmauhp-nancyfr (M Henry)

1874-3900$ ndash see front matter 2011 Phytochemical Society of Europe Published by

doi101016jphytol201102002

bilberry stems has recently been reported (Hybelbauerova et al2009) suggesting that the unexplored organs of even well-knownplants could represent a source of interesting phytochemicals Thisreport describes the preparative isolation of a lignan glycosidelyoniside from the rhizomes and stems of bilberry the seasonalfluctuations of this compound in plant organs and the surroundingsoil and some of its biological activities pointing to participationin chemical protection and environmental interactions of thisplant

2 Results and discussion

21 Isolation and structure elucidation

Fractionation of a 70 EtOH extract of V myrtillus rhizomes bymeans of droplet counter-current chromatography DCCC with thesolvent system CHCl3MeOHH2O (433720 vvv) in the descend-ing mode separated the compounds into 8 distinct fractionscomprised of molecules of increasing polarity The separatefractions contained 2ndash3 compounds except one which containeda single pure substance that could be directly submitted tostructural analysis The fractionation of plant extracts usuallyrequires several steps of separation and purification which often

Elsevier BV All rights reserved

Table 11H (500 MHz) and 13C NMR (125 MHz) spectral data of lyoniside in CD3OD

Position dH (m J in Hz) dC

Lyoniresinol1 ndash 1391

2 645 s 1066

3 ndash 1486

4 ndash 1336

5 ndash 1486

6 645 s 1066

7 435 (d 63) 424

8 211 (tt 63ndash54) 462

9 347 (dd 99ndash41)

384 (dd 99ndash54)

714

10 ndash 1258

20 ndash 1471

30 ndash 1381

40 ndash 1484

50 371 s 1083

60 ndash 1304

70ax 261 (dd 152ndash116) 332

eq 278 (dd 154ndash46)

80 211 (dtt 116ndash64ndash44) 398

90 353 (dd 110ndash68)

366 (dd 110ndash43)

657

OCH3 336 s 605

2 OCH3 377 s 570

OCH3 389 s 568

Xylose100 432 (d 77) 1050

200 330 (dd 91ndash77) 744

300 342 (t 91) 773

400 357 (ddd 106ndash91ndash54) 706

500 325 (dd 115ndash54)

390 (dd 115ndash106)

664

[()TD$FIG]

0

005

01

015

02

025

20016012080400

Concentration (μgmiddotml-1

)

DP

PH

(μ

mo

lmiddotl-1

)

α-tocopherol

lyoniside

Fig 2 DPPH free radical scavenging activity of lyoniside a-Tocopherol was assayed

as the positive control Results are means of triplicates SD

A Szakiel et al Phytochemistry Letters 4 (2011) 138ndash143 139

results in low recoveries of final products due to their irreversibleadsorption or decomposition The application of a liquidndashliquidpartition technique without solid stationary phase allows non-destructive and high-yield isolation of even labile compoundsDCCC and its modified version HSCCC have been successfullyapplied in the preparative separation of various natural com-pounds including two novel anthocyanins from bilberry fruit (Duet al 2004) In the present study the application of this methodpermitted the isolation of a pure substance in only onechromatographical step The compound was obtained as needle-shaped white crystals [a]D

25 +342 (c 075 MeOH) the UV(MeOH) absorption maxima 210 and 278 nm ESIMS (positive ionmode) mz 5753 [M+Na]+ HRESIMS (positive ion mode) mz5752114 (calcd for C27H36O12Na 5752104) Analysis of 1D and2D NMR spectra (Table 1) identified the purified compound aslyoniside 9-O-b-D-xylopyranosyl(+)lyoniresinol (Fig 1) This is thefirst report of the isolation of this compound from V myrtillusLyoniside-containing fractions obtained from stems were found tobe more contaminated and required an additional purification step

[()TD$FIG]

H

OH

O O

OH

OCH3

H3CO

HO

H3CO

OCH3

HO

OH

OH

Fig 1 Chemical structure of lyoniside (9-O-b-D-xylopyranosyl(+)lyoniresinol)

on preparative TLC plates (silica gel tolueneacetone 8515 (vv))The simple isolation of lignans using preparative TLC withsatisfactory results has been reported previously (Elfahmi etal 2007) The presence of lyoniside was not detected in extractsobtained from bilberry leaves and fruit

Lyoniside is a phenyltetralin lignan which was originallyisolated and identified as a major component of the wood ofLyonia ovalifolia in 1960 (Kashima et al 2010) It was subsequentlydetected in woody parts (inner bark stem bark roots) of otherplants and occasionally also in sempervivent leaves According toour present findings the occurrence of lyoniside in V myrtillus isrestricted to the winter-persistent part of the plant includingaboveground stems and underground rhizomes

22 Free radical scavenging activity

The exclusive occurrence of lyoniside in winter-persistentorgans suggests a possible role in protection against oxidativestress and in chemical defence against pathogens and herbivoresThe antioxidant potency of lyoniside was evaluated using theDPPH (22-diphenyl-1-picrylhydrazyl) method to measure freeradical scavenging ability (Fig 2) The scavenging effect on a01 mM DPPH solution exerted by lyoniside concentrations ofbetween 20 and 200 mg ml1 ranged from 48 to almost 90respectively The IC50 (antioxidant concentration required toquench 50 of the initial DPPH) was 23 mg ml1 At the highestconcentration tested the scavenging activity of lyoniside was 93of that of a-tocopherol thus this compound may be regarded as aneffective radical scavenger

23 Seasonal fluctuations in plant organs and the surrounding soil

Quantitative determinations of lyoniside present in bilberrytissues throughout the year showed some seasonal fluctuations(Fig 3) The highest levels were detected during winter withconcentrations of 112 mg g1 dry wt in rhizomes and 184 mg g1

in stems in December In spring the content of this compounddecreased by 24 (rhizomes) and 12 (stems) in March and evenmore by 33 (rhizomes) and 31 (stems) in May Levels increasedagain in September reaching 91 of the December values in bothrhizomes and stems The content of phenolic compounds inbilberry and other plants is known to change according to the

[()TD$FIG]

0

02

04

06

08

1

12

14

1211109876543

Month

mg

g-1

dry

wt

Rhizomes

Stems

Soil

Fig 3 Seasonal fluctuations of lyoniside in V myrtillus plant and the surrounding

soil Results are means of triplicates SD

A Szakiel et al Phytochemistry Letters 4 (2011) 138ndash143140

growth season physiological and developmental stage and also inresponse to environmental factors like light climate and soilfertility Regulation of the synthesis and accumulation of phenolicsin bilberry leaves by a complex interaction between intrinsic plantfactors and external abiotic and biotic stimuli has been proposedto explain the observed temporal fluctuations in these compounds(Witzell et al 2003) The parallel changes in the amounts oflyoniside in stems and rhizomes might also be explained byecological interactions between bilberry and other organisms(neighbouring plants natural enemies parasites pathogenicbacteria and fungi) as well as by variations due to the growthcycle and adaptation to winter conditions

The presence of high levels of lyoniside in bilberry rhizomes andstems during the winter and their subsequent decrease in thespring suggest that the accumulated compound may be exudatedinto the surrounding soil similarly to many other phenolicallochemicals Indeed lyoniside was detected in samples of soilobtained from the natural bilberry habitat with the highest level(011 mg g1 dry wt) found in May and December and the lowestlevel (005 mg g1) in July These amounts are lower than thosedescribed for other phenolics originating from falling decomposingleaves (Gallet and Lebreton 1995) but the possible contribution of

Table 2The influence of lyoniside on seed germination and growth of model plants Values ar

Plantcompound Germination Seedli

Number of germinating seeds Inhibition () Radicl

Lettuce

Control 291 2283

Lyoniside 192 34 571

Cress

Control 282 3813

Lyoniside 231 18 96

Pine

Control 263 5

Lyoniside 232 15 39

OLURa 192 27 41

Lyoniside + OLURa 51 80 18

Spruce

Control 232 628

Lyoniside 141 39 424

Larch

Control 222 712

Lyoniside 101 55 354

a The mixture of oleanolic and ursolic acids

lyoniside to known allelopathic activity of bilberry cannot be ruledout Like other phenolics lignans are regarded as effectiveallelochemicals due to their capacity to interfere with the activitiesof many proteins (enzymes transporters ion-channels receptors)involved in the transport of compounds and biosynthetic pathwaysin target plants (Wink 2003)

24 Allelopathic potential

The allelopathic activity of lyoniside was estimated byexamining its influence on seed germination and subsequentseedling growth in two commonly used dicotyledon model plants(lettuce and cress) conifer trees (pine spruce) which occur inforests with bilberry as the dominant understorey and also larchwhich is not a neighbour of bilberry in its natural habitat Theconcentration of lyoniside applied was comparable to thatdetected in the soil obtained from the bilberry habitat The resultsare presented in Table 2 Lyoniside inhibited the germination ofdicotyledonous plants with a moderate effect on lettuce (34) anda weaker effect on cress (18) but it strongly suppressed theseedling radicle growth of both plants by 75 Among the testedconifers the strongest inhibitory effect was exerted on larchwhich showed considerable inhibition of both germination (55)and radicle and hypocotyl growth (50 and 46 respectively) Theinfluence on pine and spruce was much smaller which demon-strates that lyoniside alone cannot be responsible for knownallelopathic potential of bilberry against these trees The relation-ships between plants in their natural habitats are highly complexdue to their long-term coevolution and cannot rely exclusively onthe action of a single compound or even one class of compounds(Jha et al 2006 Mallik and Pellisier 2000) Therefore weexamined the simultaneous influence of lyoniside and twoisomeric triterpenoids oleanolic and ursolic acids which alsooccur in V myrtillus rhizomes and are detected in soil samples onseed germination and seedling growth of pine Separate applica-tion of these triterpene acids and lyoniside exerted only a slightinhibitory effect on the germination and growth of seedlingsHowever a much stronger effect was exerted by their mixturewhich inhibited germination by 80 and suppressed growth byapproximately 70 (radicles by 64 hypocotyl by 75) thuspointing to synergism between lyoniside and triterpene acidsPhenylpropanoids and triterpenoids occur constitutively in manyplant species acting as antimicrobial phytoprotectants that form acommon line of defence in the first chemical barrier to infection

e means of triplicates SD

ng growth

e length (mm) Inhibition () Hypocotyl length (mm) Inhibition ()

201 1067215

224 75 814287 24

205 245155

074 75 104370 58

28 2 036

063 22 15 01 25

057 18 1 002 50

046 64 05 002 75

16 284 062

235 32 185 02 35

126 308 089

147 50 165 032 46

A Szakiel et al Phytochemistry Letters 4 (2011) 138ndash143 141

Moreover they can also be considered as potent allelochemicalssupporting the competition with neighbouring plants Our resultsprovide further proof of the synergistic action of compoundswithin these two large classes of natural products Free triterpeneacids were previously found to act directly on membranes of thetarget plants (Szakiel and Kabacinska 2009) and this alteredmembrane permeability might allow more polar compounds likelyoniside to enter the cytoplasm However the strong effect of thiscompound on larch which is not a natural environmentalneighbour of bilberry shows that lyoniside by itself can be aneffective allelochemical against a plant that has not evolvedmechanisms of tolerance or detoxification

25 Antifungal activity

The purified lyoniside was tested for its fungicidal activityagainst 5 strains of Ascomycota (Table 3) Mycelial growth of alltested fungi was markedly influenced by lyoniside although thedynamics of inhibition varied (Table 2) Lignans are known to bepotent antimycotics influencing fungal cell synthesis (Hwang etal 2007) although various strains differ appreciably in theirsensitivity to particular compounds The growth of Aspergillus niger

and Trichoderma viridae was almost completely inhibited at alyoniside concentration of 50 ml ml1 during first 3 days ofincubation but subsequently the mycelia spread rapidly andafter 7 days both strains had developed full colonies covering theentire dish surface (data not shown) In contrast the inhibition ofgrowth of Alternaria brassicicola Fusarium oxysporum and Mucor

hiemalis by lyoniside was less than 100 after 3 days of incubationbut it remained significant until the end of experiment (64 78 and80 respectively) and continued even when the incubation wasprolonged for more than 7 days Thus lyoniside can be regarded asan antifungal agent that is particularly active against phytopatho-gens such as F oxysporum and M hiemalis with higher efficacy thanthat of synthetic fungicide captan commonly used in agriculture

Lignans are compounds possessing a diverse spectrum ofbiological properties The findings of this study suggest thatlyoniside is an example of a multifunctional plant secondary

Table 3The influence of lyoniside on mycelial growth of tested fungi Commercial fundicide cap

diameter of colony without inoculation plug Values are means of triplicates SD

Fungi Treatment

concentration (mg ml1)

Diameter of colo

Days of incubati

15

Alternaria brassicicola Control 35198

Lyoniside 20 2 082

Lyoniside 50 0

Captan 50 0

Aspergillus niger Control 1516

Lyoniside 20 4 052

Lyoniside 50 0

Captan 50 0

Fusarium oxysporum Control 14186

Lyoniside 20 10104

Lyoniside 50 6125

Captan 50 8102

Mucor hiemalis Control 40236

Lyoniside 20 12146

Lyoniside 50 2 010

Captan 50 8120

Trichoderma viridae Control 21205

Lyoniside 20 4 090

Lyoniside 50 1 020

Captan 50 0

metabolite that can be involved solely or synergically in variousmechanisms of plant chemical protection and in environmentalinteractions Apart from their functions in the host plant theimportance of lignans for humans is due to their potentialapplication in the fields of pharmacy and nutrition Previousstudies have suggested possible therapeutic uses for lyoniside dueto its antiinflammatory anticancer and antioxidant activities (Jinet al 2006 Sadhu et al 2007 Song et al 2007) perhaps it can beuseful also in the treatment of some neural disorders (Arai et al2009)

3 Experimental

31 Plant material

Whole plants of V myrtillus L were collected from a naturalforest habitat in central Poland The identity of a voucher specimenwas confirmed by the taxonomist Dr Maja Graniszewska anddeposited in the herbarium of the University of Warsaw (accessionno WA 0000017594)

32 Extraction and purification

Air-dried and powdered rhizomes (60 g) were extracted in aSoxhlet apparatus initially for 10 h with diethyl ether to removethe lipophilic compounds and then for 24 h with 70 aqueousEtOH to obtain the polar compounds After evaporation at 60 8Cunder reduced pressure the latter extract yielded a gummyresidue (663 g) 1 g of which was submitted to fractionation byDCCC (droplet counter-current chromatography) using the solventsystem CHCl3MeOHH2O (433720 vvv) in the descendingmode DCCC was performed with a Tokyo Rikakikai Eyelamodel 300 DCCC chromatograph equipped with 95 tubes(400 mm 2 mm) connected in series The sample was dissolvedin a mixture consisting of 10 ml of lower phase and 10 ml of upperphase of the applied solvent system and injected using a Merckpump DuramatR through the 16 mm injection loop when themobile phase front had emerged and the hydrostatic equilibrium

tan was used as the reference compond The growth of mycelium is measured as a

ny (mm) Inhibition ()

on

3 5 15 3 5

50 50

16128 21145 94 68 58

12142 18198 100 76 64

4 082 6102 100 92 88

40110 50

10 095 33168 73 75 44

0 19123 100 100 62

2 088 4108 100 95 92

32378 50

18175 28206 29 44 44

7103 11163 57 78 78

16208 21322 46 50 58

50 50

20202 25378 70 60 50

4 068 10210 95 92 80

22318 31282 80 56 38

50 50

6108 29305 81 88 42

2 075 20186 95 96 60

2 082 11148 100 96 78

A Szakiel et al Phytochemistry Letters 4 (2011) 138ndash143142

was established in all the tubes The flow rate of the mobile phasewas set to 1 ml min1 Fractions were collected in glass test tubesusing a Gilson MicrocolR TDC 80 automatic fraction collector andanalyzed by TLC (thin layer chromatography) using CHCl3MeOHH2O (61327 vvv) TLC analyses were performed on plastic-backed silica gel 60 (020 mm thickness) plates (Merck) chroma-tograms were visualized by spraying the plates with 10 H2SO4 inMeOH followed by heating at 110 8C A total of 370 fractions of7 ml each were collected during 40 h and those containingcompounds with similar Rf values were combined into 8 mainfractions After evaporation of CHCl3 fractions 15ndash31 (mainfraction 3) yielded from MeOHH2O a 7 mg of a pure crystalizedcompound which was subjected to structure elucidation

33 Spectral analysis

Optical rotations were measured in MeOH using a PerkinElmer341 polarimeter 1H and 13C NMR spectra were recorded with aBruker Avance DRX 500 (1H at 500 MHz and 13C at 125 MHz) 2Dexperiments were performed using standard Bruker micropro-grams the spectra were acquired in CD3OD at 293 K HRESIMS andESIMS were recorded using a Finningan LCQ deca quadripole iontrap mass spectrometer (Finnigan MAT San Jose USA) Thesamples were introduced by direct infusion in a MeOH solution at arate of 5 ml min1

34 Quantitative determination

Lyoniside was determined spectrophotometrically by absor-bance at l = 278 nm using a Shimadzu UV-2401PC spectropho-tometer A calibration curve was prepared using MeOH solutions ofpure crystallized lyoniside at concentrations ranging from 10 to250 mg ml1 Extracts of plant organs and soil samples collected inMarch May July September and December 2008 were fractionat-ed by DCCC as described in Section 32 Fractions containinglyoniside obtained from rhizomes were subjected directly tospectrophotometric quantitative determination while fractionsobtained from the stems and soil were first purified by preparativeTLC in the solvent system tolueneacetone 8515 (vv) PreparativeTLC separation was carried out using 20 cm 20 cm glass platescovered with a 025 mm thickness layer of silica gel 60 H (Merck)purified compound was localized by spraying with water andeluted from the gel with MeOH

35 Free radical scavenging activity

The DPPH (22-diphenyl-1-picrylhydrazyl) method was used tomeasure free radical-scavenging activity 2 ml of 01 mM DPPH inMeOH was added to 2 ml of MeOH containing different amounts oflyoniside to produce final concentrations of 0 (control) 20 40 60120 and 200 mg ml1 The absorbance at 517 nm was measuredafter 10 20 and 30 min a-Tocopherol in the same concentrationswas used as the reference compound Triplicates of each samplewere run and the mean values calculated The scavenging of DPPHradical [] was calculated according to the formula [(A0 A1)A0 100] where A0 is the absorbance of the control reaction and A1

is the absorbance of reactions containing lyoniside or a-tocopherol

36 Allelopathic bioassays

Sheets of Whatman No1 filter paper were placed in Petri dishes(100 mm diameter) and impregnated with 10 ml of 10 mg ml1

lyoniside in EtOH The solvent was evaporated and 30 seeds oftested plants (lettuce Lactuca sativa cress Lepidium sativum pinePinus sylvestris spruce Picea abies or larch Larix decidua) were

distributed evenly on the prepared sheets moisturized with 10 mlof pure sterile water Control dishes without lyoniside wereprepared in parallel In the bioassays examining possible syner-gism between lyoniside and a mixture of oleanolic and ursolicacids the tested compounds were applied to 30 seeds of P

sylvestris either separately or mixed at the final concentration of10 mg ml1 All dishes were then closed and placed in the dark in athermostat (22 8C) Germinating seeds of lettuce and cress werecounted after 3 days and those of pine spruce and larch after 7days the radicle and hypocotyl lengths were measured after 7 and15 days

37 Antifungal bioassays

Fungi strains obtained from the Institute of FermentationTechnology and Microbiology Technical University of Łodz weregrown on Sabouraud agar (A niger A brassicicola and M hiemalis)or potato dextrose agar (F oxysporum and T viridae) Petri dishes(55 mm) filled with 6 ml of sterile medium containing lyoniside atconcentrations of 20 or 50 mg ml1 were inoculated with 5 mmagar plugs containing mycelia and incubated at 25 8C in darknessfor 5 days Three replicates were prepared for each lyonisideconcentration Commercial fungicide captan (Kaptan 50WPAgrecol) at concentration of 50 mg ml1 was used as the referencecompound The radial growth of mycelium (colony diameter) wasmeasured after 15 3 and 5 days for each culture and comparedwith controls without lyoniside and captan

References

Anonymous 2001 Monograph Vaccinium myrtillus (bilberry) Alternative Medici-nal Reviews 6 500ndash504

Arai MA Masada A Ohtsuka T Kageyama R Ishibashi M 2009 The first Hes1dimer inhibitors from natural products Bioorganic and Medicinal ChemistryLetters 19 5778ndash5781

Camire ME 2002 Phytochemicals in the Vaccinium family bilberries blue-berries and cranberries In Meskin MS Bidlack WR Davies AJ OmayeST (Eds) Phytochemicals in Nutrition and Health CRC Press Boca Raton p289

Du Q Jerz G Winterhalter P 2004 Isolation of two anthocyanin sambubiosidesfrom bilberry (Vaccinium myrtillus) by high-speed counter-current chromatog-raphy Journal of Chromatography A 1045 59ndash63

Elfahmi Ruslan K Batterman S Bos R Kayser O Woerdenbag HJ Quax WJ2007 Lignan profile of Piper cubeba an Indonesian medicinal plant BiochemicalSystematics and Ecology 35 397ndash402

Gallet C Lebreton P 1995 Evolution of phenolic patterns in plants and associatedlitters and humus of a mountain forest ecosystem Soil Biology and Biochemis-try 27 157ndash165

Hwang EI Lee YM Lee SM Yeo WH Moon JS Kang TH Park KD Kim SU2007 Inhibition of chitin synthase 2 and antifungal activity of lignans from thestem bark of Lindera erythrocarpa Planta Medica 73 679ndash682

Hybelbauerova S Sejbal J Dracinsky M Rudowska I Koutek B 2009 Unusualp-coumarates from the stems of Vaccinium myrtillus Helvetica Chimica Acta 922795ndash2801

Jha S Jha PK Gewali MB 2006 Allelopathic potential of some herbaceousforage species at Biratnagar Nepal Pakistan Journal of Plant Sciences 12103ndash113

Jin UH Lee DY Kim DS Lee IS Kim CH 2006 Induction of mitochondria-mediated apoptosis by methanol fraction of Ulmus davidiana Planch (Ulmaceae)in U87 glioblastoma cells Environmental Toxicology and Pharmacology 22136ndash141

Kashima K Sano K Yun YS Ina H Kunugi A Inoue H 2010 Ovafolinins AndashEfive new lignans from Lyonia ovalifolia Chemical and Pharmaceutical Bulletin58 191ndash194

Mallik AU Pellisier F 2000 Effect of Vaccinium myrtillus on spruce regenerationtesting the notion of coevolutionary significance in allelopathy Journal ofChemical Ecology 26 2197ndash2209

Martin-Aragon S Basabe B Benedi J Villar A 1999 In vitro and in vivoantioxidant properties of Vaccinium myrtillus Pharmaceutical Biology 37109ndash113

Prior RL Cao G Martin A Sofic E McEwen J OrsquoBrien C Lischner NEhlenfeldt M Kalt W Krewer G Mainland CM 1998 Antioxidant capac-ity as influenced by total phenolic and anthocyanin content maturity andvariety of Vaccinium species Journal of Agricultural and Food Chemistry 462686ndash2693

Riihinen K Jaakola L Karenlampi S Hohtola A 2008 Organ-specific distributionof phenolic compounds in bilberry (Vaccinium myrtillus) and lsquonorthbluersquo

A Szakiel et al Phytochemistry Letters 4 (2011) 138ndash143 143

blueberry (Vaccinium corymbosum V angustifolium) Food Chemistry 110156ndash160

Sadhu SK Khatun A Phattanawasin P Ohtsuki T Ishibashi M 2007 Lignanglycosides and flavonoids from Saraca asoca with antioxidant activity Journal ofNatural Medicines 61 480ndash482

Song IK Kim KS Suh SJ Kim MS Kwon DY Kim SL Kim CH 2007 Anti-inflammatory effect of Ulmus davidiana Planch (Ulmaceae) on collagen-inducedinflammation in rats Environmental Toxicology and Pharmacology 23 102ndash110

Szakiel A Kabacinska B 2009 Triterpenoids in allelopathic potential of plants ofVaccinium genus Acta Biochimica Polonica 56 (Suppl 2) 76ndash77

Valentova K Ulrichova J Cvak L Simanek V 2007 Cytoprotective effect of abilberry extract against oxidative damage of rat hepatocytes Food Chemistry101 912ndash917

Wink M 2003 Evolution of secondary metabolites from an ecological and molec-ular phylogenetic perspective Phytochemistry 64 3ndash19

Witzell J Gref R Nasholm T 2003 Plant-part specific and temporal variation inphenolic compounds of boreal bilberry (Vaccinium myrtillus) plants Biochemi-cal Systematics and Ecology 31 115ndash127

Yao Y Vieira A 2007 Protective activities of Vaccinium antioxidants with poten-tial relevance to mitochondrial dysfunction and neurotoxicity NeuroToxicol-ogy 28 93ndash100