Research ArticleHypericum perforatum Synthesis ofActive Principles during Flowering and FruitificationmdashNovelAspects of Biological Potential

Nebojša Kladar1 Jasminka MrZanoviT2 Goran AnaIkov3 Slavica ŠolajiT2 Neda GavariT1

Branislava SrZenoviT1 and Biljana BoDin1

1University of Novi Sad Faculty of Medicine Department of Pharmacy Hajduk Veljkova 3 21000 Novi Sad Serbia2University of Novi Sad Faculty of Medicine Oncology Institute of Vojvodina Put Dr Goldmana 4 21203 Sremska Kamenica Serbia3University of Novi Sad Faculty of Sciences Department of Biology and Ecology Trg Dositeja Obradovica 3 21000 Novi Sad Serbia

Correspondence should be addressed to Biljana Bozin biljanabozinmfunsacrs

Received 22 August 2017 Revised 18 October 2017 Accepted 29 October 2017 Published 22 November 2017

Academic Editor Maria Camilla Bergonzi

Copyright copy 2017 Nebojsa Kladar et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

St Johnrsquos wort is a widely usedmedicinal plantThe quality of herbal drug which is inmost of the cases collected fromnature variesTherefore the aim of the present study was detailed chemical characterization ofHypericum perforatum subsp perforatum samplescollected in close time intervals during flowering and fruitification with the purpose to state the phenological stage characterized bymaximum levels of active principlesThe antioxidant potential and potential to inhibit biologically important enzymes as well as thecytotoxicity and genotoxicity of the sample collected during the full flowering period were evaluated Data showed that the optimalperiod for the achieving of maximum level of active principles is the phenophase between floral budding and flowering stageSignificant antioxidant potential and the ability to inhibit biologically important enzymes (especially 120572-glucosidase) were recordedThe extract exhibited no genotoxicity in subcytotoxic concentrations while increased cytotoxicity recorded in cotreatment withbleomycin on malignant cell lines was especially significant

1 Introduction

The genus Hypericum includes more than 500 species clas-sified into 36 sections [1] The best known representativeof the genus is St Johnrsquos wort (Hypericum perforatum L1753 Hypericaceae) a species widely used in traditional andconventional medicine To date conducted in vitro and invivo as well as clinical studies suggest antioxidant antiviralantifungal antibacterial wound-healing antidepressant andmany more properties ofHyperici herba [2] Two basic formsof preparations based on H perforatum are being usedOil macerates are when applied externally intended fortreatment of different skin changes while internal applicationis recommended in the case of stomach and bile disordersinflammation of respiratory and urogenital systemmigrainediabetes and so forth However of particular importanceare water and water-alcoholic extracts which exhibit clini-cally proved antidepressant activity [3 4] Considering the

overall multitargeting therapeutic potential a constant raiseof demand for Hyperici herba is present on the worldmarket The most important secondary metabolites presentin the drug are phloroglucinols (hyperforin adhyperforin)naphtodianthrones (hypericin pseudohypericin) flavonoids(rutin quercetin quercitrin isoquercitrin hyperoside andamentoflavone) phenolic acids and small amounts of essen-tial oil The content of active principles in plants variesdepending on different ecological factors characteristic forplant habitat as well as plant development Usually Hperforatum is not being cultivated but rather collected fromnature which leads to differences of herbal drugs from theaspect of chemical quality The recommendation is thatthe collecting of plant material should be performed atopen habitats during the period of flowering However longpostfloral retention of corolla and differences in types ofvegetation induce the existence of different stages of flowering

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 2865610 11 pageshttpsdoiorg10115520172865610

2 Evidence-Based Complementary and Alternative Medicine

at the same time point which leaves a space for variationsof levels of active principles in herbal drug [5] As known tothe authors no studies which provide detailed descriptionof active principles variation in Hyperici herba related tothe phenological development of biological source wereconducted Usually only the differences in chemical profilesof the plants collected before during and after flowering arebeing reported [6]

Considering the overall safety of Hyperici herba theresults of in vitro tests suggesting new potential medi-cal indications of H perforatum are of high importancePrevious reports state the inhibitory activity of water-alcoholic extracts of different Hypericum species on acetyl-cholinesterase (AChE) suggesting the potential beneficialeffects in patients suffering from Alzheimerrsquos disease (AD)[5 7] Since AD is commonly associated with depressionpreparations based on Hyperici herba could have significanttherapeutic values Also the raise of incidence of diabetesmellitus (DM) on the world scale [8] points a great attentiontoward new drugs which can affect the metabolism ofsugars There are reports of Hypericum extracts inhibiting 120572-glucosidase and 120572-amylase which could lead to decrease inglucose absorption and consequently to lowering blood sugarlevels [5]

Cancer represents one of the most frequent causes ofmortality around the world with the prognosis of increase to70 until 2030 [9] This combined with so far unsatisfyingeffects of available chemotherapy and radiation therapy leadstoward constant search for new anticancer drugs especiallyof natural origin [10] The attention of researchers is directedto antiproliferative effects of herbal preparations used intraditional medicine as well as to the effects of combinedadministration of compounds of natural origin and anti-neoplastic drugs Possible benefits of such therapy couldbe reflected through lowering the dosage of conventionalmedicines and consequently lowering the toxicity of antineo-plastic drugs on healthy cells followed by higher cytotoxiceffects toward cancer cells It is well known that biologicallyactive compounds originating from plants can promote thegene expression which would result in multiplication ofconventionally used medicines [11]

The aim of the present study was detailed chemicalevaluation of secondary metabolites accumulation duringflowering and fruitification of St Johnrsquos wort The biologicalpotential of the water-alcoholic extract obtained from Hperforatum subsp perforatum collected at full flowering stagewas assessed through antioxidative potential and potential toinhibit120572-amylase120572-glucosidasemonoamine oxidasesA andB (MAO-A and MAO-B resp) and AChE Also cytotoxicpotential of the extract and combination of the extract andantineoplastic drug (bleomycin) on malignant cell lines wasevaluated since no reports describing this cotreatment werefound For the evaluation of potential DNA damage andoverall safety genotoxic potential of the evaluated extract onperipheral blood lymphocytes was estimated

2 Material and Methods

21 Plant Material and Obtaining of the Extracts Plant mate-rial included 22 specimens of Hypericum perforatum subspperforatum grown in wild near Padej Republic of Serbia(45∘501015840595310158401015840N 20∘91015840535110158401015840E) The vouchers are identifiedand deposited in BUNS (Herbarium of the Departmentof Biology and Ecology Faculty of Natural Sciences andMathematics University of Novi Sad) In close time intervalsstarting from 20 April until the 6 August 2014 aerial partsof the plants were collected Water-alcoholic extracts of 22plant samples (Table 1) are obtained by maceration with 70ethanol for 72 h The residues of solvent are evaporated anddry extracts (d e) are dissolved in absolute methanol prior tochemical characterization by liquid chromatography Sample12 collected at the stage of full flowering was extracted aspreviously described evaporated to dryness and d e wasdissolved in water (HP) for further evaluation of biologicalpotential cytotoxicity and genotoxicity and estimation oflevels of total phenolics and total flavonoids

22 Chemical Characterization of the Extracts

221 Total Phenolics and Flavonoids The amounts of totalphenolics and flavonoids in the HP are quantified as pre-viously described [5] and expressed as mg of gallic acidequivalents (GAE) per g of dry extract (d e) and mg ofquercetin equivalents (QE) per g of d e respectively

222 HPLC Analysis Two methods of liquid chromatog-raphy coupled with diode array detector (HPLC-DAD) areused for qualitative and quantitative profiling of plant extracts(Figure 1)The experiments are performedonAgilentHP 1100HPLC-diode array detection (DAD) system equipped withan autosampler (Agilent Waldbronn Germany) while thecomponents of interest were separated using reversed-phaseZorbax CB-C18 column (46 times 150mm i d 5 120583m particlesize) held at 25∘C Method I previously described by Bozinet al [3] was used for quantification of hypericin (Hpc)hyperforin (Hpf) apigenin (Ap) amentoflavone (Am) andnaringenin (NA) Method II was developed based on a reportby Ziakova and Brandsteterova [12] and used for determi-nation of quercetin (Qe) rutin (R) epicatechin (Ec) caffeic(CA) chlorogenic (CHA) ferulic (FA) gallic (GA) and p-hydroxybenzoic acid (PHB) Gradient elution was applied(325min 0 B 8min 12 B 15min 25 B 158min 30B 25min 90 B and 254min 100 B) with the flow rateof 1mLmin where solvent A was 01 solution of aceticacid in water and solvent B was 01 solution of acetic acidin acetonitrile Before the injection of extracts calibrationcurves of all chemical standards of quantified compoundswere obtained

23 Antioxidant Potential

231 Radical Scavenging Capacity (RSC) The ability of HPto neutralize 22-dipheny-l-picrylhydrazil (DPPH) hydroxyl

Evidence-Based Complementary and Alternative Medicine 3Ta

ble1Detailedchem

icalcharacteriz

ationof

Hperforatum

subspperfo

ratum

water-alco

holic

extractsby

HPL

C-D

AD

Dateo

fsam

plingph

enologicalstage

Com

poun

dHpf

Hpc

ApNA

EcR

Qe

Am

FAGA

CHA

CAPH

Bde

Sample

mggdh

20042014BFlowastlowast

1Meanvalue

04113

01277

000

4800378

nd

00871

00654

00083

nd

01735

00742

00613

00716

1766

SD000

0100026

000

0100016

nd

00033

00022

000

01n

d00011

00013

00025

00028

27042014BF

2Meanvalue

04565

01359

000

6000475

nd

01235

00721

00138

nd

01321

00733

006

00006

892357

SD00052

00057

000

0200023

nd

00059

00022

000

01n

d000

6500028

000

0900034

04052014BF

3Meanvalue

04325

01498

00057

00498

nd

01300

00975

00140

nd

00249

00622

00573

00753

2227

SD000

0300018

000

00000

06n

d000

4100021

000

01n

d000

0600015

00012

00031

11052014BF

4Meanvalue

04142

01389

000

6000496

nd

01363

00942

00117

nd

00251

00635

00552

00716

2317

SD00132

00018

000

0300018

nd

00031

000

40000

02n

d000

04000

0900013

00031

18052014BF

5Meanvalue

04569

01689

000

9500513

nd

01506

01155

00144

nd

00275

00766

00548

00818

2349

SD000

61000

47000

04000

06n

d000

4800024

000

05n

d000

0300019

00023

00020

25052014BF

6Meanvalue

04020

01918

00116

00523

nd

01050

01024

00149

nd

00146

00636

00533

00750

2323

SD00034

000

02000

0300026

nd

000

4600035

000

04n

d000

0000025

00025

00020

01062014FBlowastlowast

7Meanvalue

04305

02107

000

9000550

nd

00904

01237

00130

nd

00165

01283

00654

00807

2356

SD00161

00013

000

04000

04n

d00039

00014

000

05n

d000

0200012

00017

000

04

04062014FB

8Meanvalue

064

0103370

00057

00742

nd

00879

01359

00248

nd

00147

01255

00674

00774

2387

SD00205

00020

000

0200018

nd

00028

00031

00010

nd

000

0300034

00032

00023

08062014FB

9Meanvalue

08934

04987

00021

01259

nd

00836

01346

00456

nd

00266

01292

00746

00841

2047

SD004

4300128

000

0100055

nd

000

0500038

000

01n

d000

0100034

00034

000

05

11062014Flowastlowast

10Meanvalue

09237

05913

ndlowast

01206

nd

00800

01220

004

1700351

00275

01274

00707

00653

2180

SD00131

00012

nd

000

03n

d000

0200019

00020

000

05000

01000

4800029

00010

15062014F

11Meanvalue

09195

06632

nd

01253

nd

00799

0119

7004

0200350

00193

006

4200509

00580

1866

SD00355

00257

nd

000

41n

d00027

000

45000

09000

08000

0300030

00013

00022

18062014F

12Meanvalue

08666

06987

nd

01159

nd

00805

01224

00381

00369

00077

00636

004

8300515

1667

SD00210

00026

nd

00029

nd

00016

00029

000

07000

08000

0200025

00017

00013

22062014F

13Meanvalue

07541

07811

nd

01100

nd

00749

01104

00334

00369

nd

00615

00508

00533

1434

SD00114

00171

nd

000

45n

d000

04000

03000

02000

02n

d00024

000

04000

06

25062014F

14Meanvalue

07005

07857

nd

01086

nd

00737

00985

00326

004

01n

d00692

00450

00388

1421

SD00191

00148

nd

000

46n

d00023

00029

000

0400017

nd

000

06000

06000

08

03072

014AFlowastlowast

15Meanvalue

06379

08852

nd

01076

004

6900621

00887

00333

00369

nd

00736

00412

00390

1929

SD000

0200214

nd

00031

00020

000

0500032

00010

00010

nd

000

0400011

00014

06072

014AF

16Meanvalue

05489

06769

nd

00840

00599

006

0900799

00292

00415

nd

00633

00430

00337

1868

SD00168

00332

nd

000

0100026

00019

000

06000

01000

05n

d00013

00016

00012

13072

014AF

17Meanvalue

04740

05560

nd

00815

00587

00615

00712

00289

00351

nd

00580

00357

00339

1354

SD00192

00082

nd

00039

00011

000

08000

09000

0300015

nd

00025

00012

000

09

20072

014AF

18Meanvalue

05073

05450

nd

00834

00570

00587

00787

00287

00413

nd

00637

00331

00344

1410

SD00082

00072

nd

00028

00011

00013

00031

00011

000

00n

d00021

000

03000

08

27072

014AF

19Meanvalue

06582

05269

nd

00862

00490

00573

00778

00302

00369

nd

00568

00343

00344

1034

SD00019

00144

nd

00016

00024

00028

00014

00010

00016

nd

000

0400012

000

05

30072

014AF

20Meanvalue

06898

04987

nd

00854

00315

00615

00947

00308

00386

nd

00543

00321

00337

1076

SD00013

00182

nd

000

41000

0600021

000

0100012

00015

nd

00013

000

0600012

03082014AF

21Meanvalue

07568

05174

nd

00862

00315

00611

00990

00307

00369

nd

006

8600369

004

28122

SD00127

00165

nd

00012

00015

000

0500011

00013

000

01n

d00010

000

08000

00

06082014AF

22Meanvalue

08121

04786

nd

00853

00326

00561

01015

00291

00369

nd

00590

00345

00450

1233

SD00020

00054

nd

00023

000

0400012

00050

00012

000

02n

d00029

00013

000

03lowastn

dnot

detectedlowastlowastBF

beforefl

oweringFB

floralbu

ddingFflo

weringAFaft

erflo

wering

4 Evidence-Based Complementary and Alternative Medicine

(min)1 2 3 4 5 6 7 8 9

(mAU

)

0250500750

1000125015001750

(1) (2)

(3)(4)

(a)

(min)

(mAU

)

1 2 3 4 5 6 7 8 9minus100

0100200300400500600

(3)

(4)

(b)

(min)

(mAU

)

5 10 15 20 250

5001000150020002500

(5)(6) (7) (8) (9) (10)

(11)

(c)

Figure 1 HPLC-DAD chromatograms of sample 12 obtained by method I (a) detection at 270 nm and (b) detection at 590 nm and methodII (c) detection at 280 nm Detected compounds (1) NA (2) Am (3) Hpf (4) Hpc (5) R (6) GA (7) CHA (8) PHB (9) CA (10) FA and (11)Qe

(OH) and nitroso (NO) radicals is estimated spectropho-tometrically as previously described [5] Briefly differentconcentrations of the extract are added to theDPPH∙ solutionand the disappearance of purple color was followed at 515 nmIn the OH-test free radicals are formed in Fentonrsquos reactionand degradation of 2-deoxy-D-ribose followed by formationof malonyl-dialdehyde (MDA) was evaluated spectropho-tometrically at 532 nm Nitroso radicals are generated afteraddition of sodium nitroprusside in the reaction mixturewhile the antioxidant potential was estimated upon additionof Griessrsquos reagent which forms purple complex with NO∙

232 Inhibition of Lipid Peroxidation (LP) The potential ofHP to inhibit the process of lipid peroxidation is estimatedas in previously published research by Kladar et al [5]Liposomes are used as a test-model of biological membraneswhile OH∙ are formed in Fentonrsquos reaction

The obtained results for the HP were compared withascorbic acid and synthetic antioxidants such as butylatedhydroxytoluene (BHT) propyl gallate (PG) and quercetindihydrate (QDH)

233 Ferric Reduction Antioxidant Potential The ability ofHP to reduce Fe3+ to Fe2+ is estimated based on a method byLesjak et al [13] inwhich Fe2+ with 246-tripyridyl-S-triazine(TPTZ) forms a colored complex with the absorbance maxi-mumatwavelength 593 nmThe same procedurewas used fordetermination of ferric reduction potential of ascorbic acidsince the antioxidant potential of the extract was quantifiedas the milligrams of the ascorbic acid equivalent per gram ofd e (mg AAEg d e) All measurements are performed intriplicate

24 Inhibition of Biologically Important Enzymes

241 Inhibition of Monoamine Oxidase A and MonoamineOxidase B The inhibitory potential of the HP against humanrecombinantMAO-A andMAO-B is estimated similarly as inthe study by Samoylenko et al [14] Deamination of kynure-nine which is used as a substrate and consequent formationof 4-hydroxyquinoline are followed spectrofluorimetrically(Ex = 320 nm Em = 380 nm) In the MAO-A inhibitionassay the final concentrations of the enzyme and substrate inthe reaction mixture were 5 120583gmL and 80120583M respectivelywhile in the case ofMAO-B inhibition the concentrations ofenzyme and substrate were 10 120583gmL and 50120583M respectivelyThe water solution of the examined extract was added ina single concentration to the reaction mixtures to estimatethe percentage of the enzyme inhibition compared to thecontrol which contained only the enzyme and substrateStandards of moclobemide and selegiline are used as positivecontrols for MAO-A andMAO-B inhibition respectively Allmeasurements are performed in triplicate

242 Inhibition of Acetylcholinesterase Anticholinesterasepotential of the HP is estimated bymodified Ellmanrsquos method[3] Final activity of the enzyme in the reaction mixture was815UL Two concentrations of the examined extract areadded in the reaction mixture The percentage of inhibitionwas calculated compared to the reaction mixture whichcontained no extract Galantamine is used as a positivecontrol All measurements are performed in triplicate

243 Inhibition of 120572-Amylase Inhibition of 120572-amylase activ-ity by the HP is evaluated as previously described [5] Reac-tion mixtures contained Starch azure (substrate) porcine120572-amylase (final reaction mixture activity 06UmL) and

Evidence-Based Complementary and Alternative Medicine 5

sodium phosphate buffer (pH = 72) with NaCl Two concen-trations of the extract are added in test tubes The percentageof inhibition is calculated compared to the absorbance ofreactionmixtures containing no extract Acarbose is used as apositive control All the measurements are done in triplicate

244 Inhibition of 120572-Glucosidase The potential of the exam-ined extract to inhibit 120572-glucosidase is estimated by themod-ified method of Sigma-Aldrich [15] The assay mixture con-tained potassium phosphate buffer (pH = 68) glutathione(reduced solution) and 120572-glucosidase from Saccharomycescerevisiae obtained from Sigma-Aldrich and p-nitrophenyl-120572-D-glucoside (PNP-Gluc) which was used as a substrateTwo different concentrations of the HP are added in thereaction mixture After the incubation of 20min at 37∘Cthe reaction was stopped by addition of Na

2CO3and the

absorbance of solutionwas spectrophotometricallymeasuredat 400 nm Reaction mixture without the examined extractwas considered as a 100 enzyme activity Final activity ofthe enzyme in the reaction mixture was 76UL Acarbosewas used as a positive control All the measurements areperformed in triplicate

25 Cytotoxicity and Genotoxicity

251 Growth and Culture of Cells The estimation of cellgrowth activity is performed on one untransformed humancell line MRC-5 (fetal lung fibroblast ECACC 84101801) andthree human malignant transformed cell lines HeLa (cervixepithelioid carcinoma ECACC number 93021013) HT-29(colon adenocarcinoma ECACC number 91072201) and Hs-294T (melanoma ATCCHTB-140)The cell lines were grownand maintained in DMEM (Sigma-Aldrich USA) mediumsupplemented with FCS (10) penicillin (100 unitsmL)and streptomycin (100120583gmL) being referred to as completemedium Furthermore the cells were cultured in 25 cm2flasks at 37∘C in the atmosphere of 5CO

2andhigh humidity

and subcultured twice a week A single cell suspension wasobtained using 01 trypsin with 004 EDTA

Heparinized whole blood collected by venous puncturefrom 40-year-old healthy female donor not having beenexposed to any chemical or physical agent during the last6 months is used for the analysis of genotoxic potentialBriefly 05mL of the whole blood was added to 5mL ofthe Lymphochrome lymphocytes culture medium (LonzaBioWhittaker) supplemented with phytohemagglutininLymphocyte cell culture of peripheral blood was incubatedat 37∘C for 72 h in 5 CO

2atmosphere with 95 humidity

252 Cytotoxicity Assay Cytotoxicity of HP is evaluated bythe colorimetric sulforhodamine B (SRB) assay as previouslydescribed [16] Cell lines are plated into 96-well microtiterplates at different seeding density of 5 times 103 cells per wellfor MRC-5 and Hs-294T 4 times 103 cells for HeLa and 6 times 103cells for HT-29 in a volume of 180 120583L and preincubated incomplete medium supplemented with 5 FCS at 37∘C for24 h For the evaluation of the cell growth activity the extractwas diluted in NaCl to obtain final concentrations in range of

01ndash600120583gmL while the control group contained onlyNaClAll cell lines were previously treated with bleomycin (BLM)in order to determine EC

50values During the examination

of combined treatment (HP and BLM) effects on cell cul-tures the final concentration of BLM was 100 120583gmL Serialdilutions of HP and BLM (20120583Lwell) were added to achieverequired final concentrationsMicroplates are then incubatedat 37∘C for an additional 48 h The development of colorwas spectrophotometrically measured at 540 nm against620 nm as background The results of cell growth activitywere expressed as mean plusmn SD of experiments performedin octuplicate The effect on cell growth was expressed as apercent of the control and calculated as

(AtAc) times 100 () (1)

where At is the absorbance of the test sample and Ac is theabsorbance of the control Using EC

50values obtained in

a nontumor cell line and in the respective tumor cell linenontumortumor EC

50ratios (NTT) were calculated for the

extract drug and combination of the extract and drug

253 Genotoxicity Genotoxicity of HP is evaluated bycytochalasin block micronuclei assay (CBMN) which wasperformed according to published procedures [17] withminor modifications The peripheral blood lymphocytesculture was set up in triplicate vessels per concentration foreach experimental treatment condition According to the rec-ommendations for testing the genotoxic effects of compounds[18] the extract was added after 20 h of preincubationThe final concentrations of the extract in peripheral bloodculture were 50 100 and 200120583gmL After 44 h of culturesetup the medium was replaced with fresh one containingcytochalasin-B (CytB) at a final concentration of 6120583gmL andincubation continued for the next 24 h Thereafter cells wereexposed to a cold hypotonic solution (056 KCl) and fixedthree times with methanol glacial acetic acid (3 1 vv) Air-dried slides were stained with 2 Giemsa in distilled waterfor 9min The CBMN assay was performed analyzing morethan 1000 cells for each sample Standard criteria were usedfor the identification of micronuclei (MN) [19] Monitoredvalues included number of mononucleated binucleated andmultinucleated cells incidence of micronuclei and nucleardivision index (NDI) Micronucleus incidence was presentedas a number of micronuclei per 1000 examined binuclearcells

26 Statistical Analysis The data were presented as meanvalues plusmn SD Concentrations of the extracts needed for theneutralization of 50 of free radicals and LP inhibition of50 of the enzyme activity or inhibition of 50 of cell growth(RSC50 IC50 and EC

50 resp) were determined by regression

analysis from concentration-effect curve using MicrosoftExcel v2010 One-way Anova and Fisher LSD test appliedin genotoxicity assay as well as principle component analysis(PCA) were performed by Statistica 130 StatSoft

Evidence-Based Complementary and Alternative Medicine 3Ta

ble1Detailedchem

icalcharacteriz

ationof

Hperforatum

subspperfo

ratum

water-alco

holic

extractsby

HPL

C-D

AD

Dateo

fsam

plingph

enologicalstage

Com

poun

dHpf

Hpc

ApNA

EcR

Qe

Am

FAGA

CHA

CAPH

Bde

Sample

mggdh

20042014BFlowastlowast

1Meanvalue

04113

01277

000

4800378

nd

00871

00654

00083

nd

01735

00742

00613

00716

1766

SD000

0100026

000

0100016

nd

00033

00022

000

01n

d00011

00013

00025

00028

27042014BF

2Meanvalue

04565

01359

000

6000475

nd

01235

00721

00138

nd

01321

00733

006

00006

892357

SD00052

00057

000

0200023

nd

00059

00022

000

01n

d000

6500028

000

0900034

04052014BF

3Meanvalue

04325

01498

00057

00498

nd

01300

00975

00140

nd

00249

00622

00573

00753

2227

SD000

0300018

000

00000

06n

d000

4100021

000

01n

d000

0600015

00012

00031

11052014BF

4Meanvalue

04142

01389

000

6000496

nd

01363

00942

00117

nd

00251

00635

00552

00716

2317

SD00132

00018

000

0300018

nd

00031

000

40000

02n

d000

04000

0900013

00031

18052014BF

5Meanvalue

04569

01689

000

9500513

nd

01506

01155

00144

nd

00275

00766

00548

00818

2349

SD000

61000

47000

04000

06n

d000

4800024

000

05n

d000

0300019

00023

00020

25052014BF

6Meanvalue

04020

01918

00116

00523

nd

01050

01024

00149

nd

00146

00636

00533

00750

2323

SD00034

000

02000

0300026

nd

000

4600035

000

04n

d000

0000025

00025

00020

01062014FBlowastlowast

7Meanvalue

04305

02107

000

9000550

nd

00904

01237

00130

nd

00165

01283

00654

00807

2356

SD00161

00013

000

04000

04n

d00039

00014

000

05n

d000

0200012

00017

000

04

04062014FB

8Meanvalue

064

0103370

00057

00742

nd

00879

01359

00248

nd

00147

01255

00674

00774

2387

SD00205

00020

000

0200018

nd

00028

00031

00010

nd

000

0300034

00032

00023

08062014FB

9Meanvalue

08934

04987

00021

01259

nd

00836

01346

00456

nd

00266

01292

00746

00841

2047

SD004

4300128

000

0100055

nd

000

0500038

000

01n

d000

0100034

00034

000

05

11062014Flowastlowast

10Meanvalue

09237

05913

ndlowast

01206

nd

00800

01220

004

1700351

00275

01274

00707

00653

2180

SD00131

00012

nd

000

03n

d000

0200019

00020

000

05000

01000

4800029

00010

15062014F

11Meanvalue

09195

06632

nd

01253

nd

00799

0119

7004

0200350

00193

006

4200509

00580

1866

SD00355

00257

nd

000

41n

d00027

000

45000

09000

08000

0300030

00013

00022

18062014F

12Meanvalue

08666

06987

nd

01159

nd

00805

01224

00381

00369

00077

00636

004

8300515

1667

SD00210

00026

nd

00029

nd

00016

00029

000

07000

08000

0200025

00017

00013

22062014F

13Meanvalue

07541

07811

nd

01100

nd

00749

01104

00334

00369

nd

00615

00508

00533

1434

SD00114

00171

nd

000

45n

d000

04000

03000

02000

02n

d00024

000

04000

06

25062014F

14Meanvalue

07005

07857

nd

01086

nd

00737

00985

00326

004

01n

d00692

00450

00388

1421

SD00191

00148

nd

000

46n

d00023

00029

000

0400017

nd

000

06000

06000

08

03072

014AFlowastlowast

15Meanvalue

06379

08852

nd

01076

004

6900621

00887

00333

00369

nd

00736

00412

00390

1929

SD000

0200214

nd

00031

00020

000

0500032

00010

00010

nd

000

0400011

00014

06072

014AF

16Meanvalue

05489

06769

nd

00840

00599

006

0900799

00292

00415

nd

00633

00430

00337

1868

SD00168

00332

nd

000

0100026

00019

000

06000

01000

05n

d00013

00016

00012

13072

014AF

17Meanvalue

04740

05560

nd

00815

00587

00615

00712

00289

00351

nd

00580

00357

00339

1354

SD00192

00082

nd

00039

00011

000

08000

09000

0300015

nd

00025

00012

000

09

20072

014AF

18Meanvalue

05073

05450

nd

00834

00570

00587

00787

00287

00413

nd

00637

00331

00344

1410

SD00082

00072

nd

00028

00011

00013

00031

00011

000

00n

d00021

000

03000

08

27072

014AF

19Meanvalue

06582

05269

nd

00862

00490

00573

00778

00302

00369

nd

00568

00343

00344

1034

SD00019

00144

nd

00016

00024

00028

00014

00010

00016

nd

000

0400012

000

05

30072

014AF

20Meanvalue

06898

04987

nd

00854

00315

00615

00947

00308

00386

nd

00543

00321

00337

1076

SD00013

00182

nd

000

41000

0600021

000

0100012

00015

nd

00013

000

0600012

03082014AF

21Meanvalue

07568

05174

nd

00862

00315

00611

00990

00307

00369

nd

006

8600369

004

28122

SD00127

00165

nd

00012

00015

000

0500011

00013

000

01n

d00010

000

08000

00

06082014AF

22Meanvalue

08121

04786

nd

00853

00326

00561

01015

00291

00369

nd

00590

00345

00450

1233

SD00020

00054

nd

00023

000

0400012

00050

00012

000

02n

d00029

00013

000

03lowastn

dnot

detectedlowastlowastBF

beforefl

oweringFB

floralbu

ddingFflo

weringAFaft

erflo

wering

4 Evidence-Based Complementary and Alternative Medicine

(min)1 2 3 4 5 6 7 8 9

(mAU

)

0250500750

1000125015001750

(1) (2)

(3)(4)

(a)

(min)

(mAU

)

1 2 3 4 5 6 7 8 9minus100

0100200300400500600

(3)

(4)

(b)

(min)

(mAU

)

5 10 15 20 250

5001000150020002500

(5)(6) (7) (8) (9) (10)

(11)

(c)

Figure 1 HPLC-DAD chromatograms of sample 12 obtained by method I (a) detection at 270 nm and (b) detection at 590 nm and methodII (c) detection at 280 nm Detected compounds (1) NA (2) Am (3) Hpf (4) Hpc (5) R (6) GA (7) CHA (8) PHB (9) CA (10) FA and (11)Qe

(OH) and nitroso (NO) radicals is estimated spectropho-tometrically as previously described [5] Briefly differentconcentrations of the extract are added to theDPPH∙ solutionand the disappearance of purple color was followed at 515 nmIn the OH-test free radicals are formed in Fentonrsquos reactionand degradation of 2-deoxy-D-ribose followed by formationof malonyl-dialdehyde (MDA) was evaluated spectropho-tometrically at 532 nm Nitroso radicals are generated afteraddition of sodium nitroprusside in the reaction mixturewhile the antioxidant potential was estimated upon additionof Griessrsquos reagent which forms purple complex with NO∙

232 Inhibition of Lipid Peroxidation (LP) The potential ofHP to inhibit the process of lipid peroxidation is estimatedas in previously published research by Kladar et al [5]Liposomes are used as a test-model of biological membraneswhile OH∙ are formed in Fentonrsquos reaction

The obtained results for the HP were compared withascorbic acid and synthetic antioxidants such as butylatedhydroxytoluene (BHT) propyl gallate (PG) and quercetindihydrate (QDH)

233 Ferric Reduction Antioxidant Potential The ability ofHP to reduce Fe3+ to Fe2+ is estimated based on a method byLesjak et al [13] inwhich Fe2+ with 246-tripyridyl-S-triazine(TPTZ) forms a colored complex with the absorbance maxi-mumatwavelength 593 nmThe same procedurewas used fordetermination of ferric reduction potential of ascorbic acidsince the antioxidant potential of the extract was quantifiedas the milligrams of the ascorbic acid equivalent per gram ofd e (mg AAEg d e) All measurements are performed intriplicate

24 Inhibition of Biologically Important Enzymes

241 Inhibition of Monoamine Oxidase A and MonoamineOxidase B The inhibitory potential of the HP against humanrecombinantMAO-A andMAO-B is estimated similarly as inthe study by Samoylenko et al [14] Deamination of kynure-nine which is used as a substrate and consequent formationof 4-hydroxyquinoline are followed spectrofluorimetrically(Ex = 320 nm Em = 380 nm) In the MAO-A inhibitionassay the final concentrations of the enzyme and substrate inthe reaction mixture were 5 120583gmL and 80120583M respectivelywhile in the case ofMAO-B inhibition the concentrations ofenzyme and substrate were 10 120583gmL and 50120583M respectivelyThe water solution of the examined extract was added ina single concentration to the reaction mixtures to estimatethe percentage of the enzyme inhibition compared to thecontrol which contained only the enzyme and substrateStandards of moclobemide and selegiline are used as positivecontrols for MAO-A andMAO-B inhibition respectively Allmeasurements are performed in triplicate

242 Inhibition of Acetylcholinesterase Anticholinesterasepotential of the HP is estimated bymodified Ellmanrsquos method[3] Final activity of the enzyme in the reaction mixture was815UL Two concentrations of the examined extract areadded in the reaction mixture The percentage of inhibitionwas calculated compared to the reaction mixture whichcontained no extract Galantamine is used as a positivecontrol All measurements are performed in triplicate

243 Inhibition of 120572-Amylase Inhibition of 120572-amylase activ-ity by the HP is evaluated as previously described [5] Reac-tion mixtures contained Starch azure (substrate) porcine120572-amylase (final reaction mixture activity 06UmL) and

Evidence-Based Complementary and Alternative Medicine 5

sodium phosphate buffer (pH = 72) with NaCl Two concen-trations of the extract are added in test tubes The percentageof inhibition is calculated compared to the absorbance ofreactionmixtures containing no extract Acarbose is used as apositive control All the measurements are done in triplicate

244 Inhibition of 120572-Glucosidase The potential of the exam-ined extract to inhibit 120572-glucosidase is estimated by themod-ified method of Sigma-Aldrich [15] The assay mixture con-tained potassium phosphate buffer (pH = 68) glutathione(reduced solution) and 120572-glucosidase from Saccharomycescerevisiae obtained from Sigma-Aldrich and p-nitrophenyl-120572-D-glucoside (PNP-Gluc) which was used as a substrateTwo different concentrations of the HP are added in thereaction mixture After the incubation of 20min at 37∘Cthe reaction was stopped by addition of Na

2CO3and the

absorbance of solutionwas spectrophotometricallymeasuredat 400 nm Reaction mixture without the examined extractwas considered as a 100 enzyme activity Final activity ofthe enzyme in the reaction mixture was 76UL Acarbosewas used as a positive control All the measurements areperformed in triplicate

25 Cytotoxicity and Genotoxicity

251 Growth and Culture of Cells The estimation of cellgrowth activity is performed on one untransformed humancell line MRC-5 (fetal lung fibroblast ECACC 84101801) andthree human malignant transformed cell lines HeLa (cervixepithelioid carcinoma ECACC number 93021013) HT-29(colon adenocarcinoma ECACC number 91072201) and Hs-294T (melanoma ATCCHTB-140)The cell lines were grownand maintained in DMEM (Sigma-Aldrich USA) mediumsupplemented with FCS (10) penicillin (100 unitsmL)and streptomycin (100120583gmL) being referred to as completemedium Furthermore the cells were cultured in 25 cm2flasks at 37∘C in the atmosphere of 5CO

2andhigh humidity

and subcultured twice a week A single cell suspension wasobtained using 01 trypsin with 004 EDTA

Heparinized whole blood collected by venous puncturefrom 40-year-old healthy female donor not having beenexposed to any chemical or physical agent during the last6 months is used for the analysis of genotoxic potentialBriefly 05mL of the whole blood was added to 5mL ofthe Lymphochrome lymphocytes culture medium (LonzaBioWhittaker) supplemented with phytohemagglutininLymphocyte cell culture of peripheral blood was incubatedat 37∘C for 72 h in 5 CO

2atmosphere with 95 humidity

252 Cytotoxicity Assay Cytotoxicity of HP is evaluated bythe colorimetric sulforhodamine B (SRB) assay as previouslydescribed [16] Cell lines are plated into 96-well microtiterplates at different seeding density of 5 times 103 cells per wellfor MRC-5 and Hs-294T 4 times 103 cells for HeLa and 6 times 103cells for HT-29 in a volume of 180 120583L and preincubated incomplete medium supplemented with 5 FCS at 37∘C for24 h For the evaluation of the cell growth activity the extractwas diluted in NaCl to obtain final concentrations in range of

01ndash600120583gmL while the control group contained onlyNaClAll cell lines were previously treated with bleomycin (BLM)in order to determine EC

50values During the examination

of combined treatment (HP and BLM) effects on cell cul-tures the final concentration of BLM was 100 120583gmL Serialdilutions of HP and BLM (20120583Lwell) were added to achieverequired final concentrationsMicroplates are then incubatedat 37∘C for an additional 48 h The development of colorwas spectrophotometrically measured at 540 nm against620 nm as background The results of cell growth activitywere expressed as mean plusmn SD of experiments performedin octuplicate The effect on cell growth was expressed as apercent of the control and calculated as

(AtAc) times 100 () (1)

where At is the absorbance of the test sample and Ac is theabsorbance of the control Using EC

50values obtained in

a nontumor cell line and in the respective tumor cell linenontumortumor EC

50ratios (NTT) were calculated for the

extract drug and combination of the extract and drug

253 Genotoxicity Genotoxicity of HP is evaluated bycytochalasin block micronuclei assay (CBMN) which wasperformed according to published procedures [17] withminor modifications The peripheral blood lymphocytesculture was set up in triplicate vessels per concentration foreach experimental treatment condition According to the rec-ommendations for testing the genotoxic effects of compounds[18] the extract was added after 20 h of preincubationThe final concentrations of the extract in peripheral bloodculture were 50 100 and 200120583gmL After 44 h of culturesetup the medium was replaced with fresh one containingcytochalasin-B (CytB) at a final concentration of 6120583gmL andincubation continued for the next 24 h Thereafter cells wereexposed to a cold hypotonic solution (056 KCl) and fixedthree times with methanol glacial acetic acid (3 1 vv) Air-dried slides were stained with 2 Giemsa in distilled waterfor 9min The CBMN assay was performed analyzing morethan 1000 cells for each sample Standard criteria were usedfor the identification of micronuclei (MN) [19] Monitoredvalues included number of mononucleated binucleated andmultinucleated cells incidence of micronuclei and nucleardivision index (NDI) Micronucleus incidence was presentedas a number of micronuclei per 1000 examined binuclearcells

26 Statistical Analysis The data were presented as meanvalues plusmn SD Concentrations of the extracts needed for theneutralization of 50 of free radicals and LP inhibition of50 of the enzyme activity or inhibition of 50 of cell growth(RSC50 IC50 and EC

50 resp) were determined by regression

analysis from concentration-effect curve using MicrosoftExcel v2010 One-way Anova and Fisher LSD test appliedin genotoxicity assay as well as principle component analysis(PCA) were performed by Statistica 130 StatSoft

4 Evidence-Based Complementary and Alternative Medicine

(min)1 2 3 4 5 6 7 8 9

(mAU

)

0250500750

1000125015001750

(1) (2)

(3)(4)

(a)

(min)

(mAU

)

1 2 3 4 5 6 7 8 9minus100

0100200300400500600

(3)

(4)

(b)

(min)

(mAU

)

5 10 15 20 250

5001000150020002500

(5)(6) (7) (8) (9) (10)

(11)

(c)

Figure 1 HPLC-DAD chromatograms of sample 12 obtained by method I (a) detection at 270 nm and (b) detection at 590 nm and methodII (c) detection at 280 nm Detected compounds (1) NA (2) Am (3) Hpf (4) Hpc (5) R (6) GA (7) CHA (8) PHB (9) CA (10) FA and (11)Qe

(OH) and nitroso (NO) radicals is estimated spectropho-tometrically as previously described [5] Briefly differentconcentrations of the extract are added to theDPPH∙ solutionand the disappearance of purple color was followed at 515 nmIn the OH-test free radicals are formed in Fentonrsquos reactionand degradation of 2-deoxy-D-ribose followed by formationof malonyl-dialdehyde (MDA) was evaluated spectropho-tometrically at 532 nm Nitroso radicals are generated afteraddition of sodium nitroprusside in the reaction mixturewhile the antioxidant potential was estimated upon additionof Griessrsquos reagent which forms purple complex with NO∙

232 Inhibition of Lipid Peroxidation (LP) The potential ofHP to inhibit the process of lipid peroxidation is estimatedas in previously published research by Kladar et al [5]Liposomes are used as a test-model of biological membraneswhile OH∙ are formed in Fentonrsquos reaction

The obtained results for the HP were compared withascorbic acid and synthetic antioxidants such as butylatedhydroxytoluene (BHT) propyl gallate (PG) and quercetindihydrate (QDH)

233 Ferric Reduction Antioxidant Potential The ability ofHP to reduce Fe3+ to Fe2+ is estimated based on a method byLesjak et al [13] inwhich Fe2+ with 246-tripyridyl-S-triazine(TPTZ) forms a colored complex with the absorbance maxi-mumatwavelength 593 nmThe same procedurewas used fordetermination of ferric reduction potential of ascorbic acidsince the antioxidant potential of the extract was quantifiedas the milligrams of the ascorbic acid equivalent per gram ofd e (mg AAEg d e) All measurements are performed intriplicate

24 Inhibition of Biologically Important Enzymes

241 Inhibition of Monoamine Oxidase A and MonoamineOxidase B The inhibitory potential of the HP against humanrecombinantMAO-A andMAO-B is estimated similarly as inthe study by Samoylenko et al [14] Deamination of kynure-nine which is used as a substrate and consequent formationof 4-hydroxyquinoline are followed spectrofluorimetrically(Ex = 320 nm Em = 380 nm) In the MAO-A inhibitionassay the final concentrations of the enzyme and substrate inthe reaction mixture were 5 120583gmL and 80120583M respectivelywhile in the case ofMAO-B inhibition the concentrations ofenzyme and substrate were 10 120583gmL and 50120583M respectivelyThe water solution of the examined extract was added ina single concentration to the reaction mixtures to estimatethe percentage of the enzyme inhibition compared to thecontrol which contained only the enzyme and substrateStandards of moclobemide and selegiline are used as positivecontrols for MAO-A andMAO-B inhibition respectively Allmeasurements are performed in triplicate

242 Inhibition of Acetylcholinesterase Anticholinesterasepotential of the HP is estimated bymodified Ellmanrsquos method[3] Final activity of the enzyme in the reaction mixture was815UL Two concentrations of the examined extract areadded in the reaction mixture The percentage of inhibitionwas calculated compared to the reaction mixture whichcontained no extract Galantamine is used as a positivecontrol All measurements are performed in triplicate

243 Inhibition of 120572-Amylase Inhibition of 120572-amylase activ-ity by the HP is evaluated as previously described [5] Reac-tion mixtures contained Starch azure (substrate) porcine120572-amylase (final reaction mixture activity 06UmL) and

Evidence-Based Complementary and Alternative Medicine 5

sodium phosphate buffer (pH = 72) with NaCl Two concen-trations of the extract are added in test tubes The percentageof inhibition is calculated compared to the absorbance ofreactionmixtures containing no extract Acarbose is used as apositive control All the measurements are done in triplicate

244 Inhibition of 120572-Glucosidase The potential of the exam-ined extract to inhibit 120572-glucosidase is estimated by themod-ified method of Sigma-Aldrich [15] The assay mixture con-tained potassium phosphate buffer (pH = 68) glutathione(reduced solution) and 120572-glucosidase from Saccharomycescerevisiae obtained from Sigma-Aldrich and p-nitrophenyl-120572-D-glucoside (PNP-Gluc) which was used as a substrateTwo different concentrations of the HP are added in thereaction mixture After the incubation of 20min at 37∘Cthe reaction was stopped by addition of Na

2CO3and the

absorbance of solutionwas spectrophotometricallymeasuredat 400 nm Reaction mixture without the examined extractwas considered as a 100 enzyme activity Final activity ofthe enzyme in the reaction mixture was 76UL Acarbosewas used as a positive control All the measurements areperformed in triplicate

25 Cytotoxicity and Genotoxicity

251 Growth and Culture of Cells The estimation of cellgrowth activity is performed on one untransformed humancell line MRC-5 (fetal lung fibroblast ECACC 84101801) andthree human malignant transformed cell lines HeLa (cervixepithelioid carcinoma ECACC number 93021013) HT-29(colon adenocarcinoma ECACC number 91072201) and Hs-294T (melanoma ATCCHTB-140)The cell lines were grownand maintained in DMEM (Sigma-Aldrich USA) mediumsupplemented with FCS (10) penicillin (100 unitsmL)and streptomycin (100120583gmL) being referred to as completemedium Furthermore the cells were cultured in 25 cm2flasks at 37∘C in the atmosphere of 5CO

2andhigh humidity

and subcultured twice a week A single cell suspension wasobtained using 01 trypsin with 004 EDTA

Heparinized whole blood collected by venous puncturefrom 40-year-old healthy female donor not having beenexposed to any chemical or physical agent during the last6 months is used for the analysis of genotoxic potentialBriefly 05mL of the whole blood was added to 5mL ofthe Lymphochrome lymphocytes culture medium (LonzaBioWhittaker) supplemented with phytohemagglutininLymphocyte cell culture of peripheral blood was incubatedat 37∘C for 72 h in 5 CO

2atmosphere with 95 humidity

252 Cytotoxicity Assay Cytotoxicity of HP is evaluated bythe colorimetric sulforhodamine B (SRB) assay as previouslydescribed [16] Cell lines are plated into 96-well microtiterplates at different seeding density of 5 times 103 cells per wellfor MRC-5 and Hs-294T 4 times 103 cells for HeLa and 6 times 103cells for HT-29 in a volume of 180 120583L and preincubated incomplete medium supplemented with 5 FCS at 37∘C for24 h For the evaluation of the cell growth activity the extractwas diluted in NaCl to obtain final concentrations in range of

01ndash600120583gmL while the control group contained onlyNaClAll cell lines were previously treated with bleomycin (BLM)in order to determine EC

50values During the examination

of combined treatment (HP and BLM) effects on cell cul-tures the final concentration of BLM was 100 120583gmL Serialdilutions of HP and BLM (20120583Lwell) were added to achieverequired final concentrationsMicroplates are then incubatedat 37∘C for an additional 48 h The development of colorwas spectrophotometrically measured at 540 nm against620 nm as background The results of cell growth activitywere expressed as mean plusmn SD of experiments performedin octuplicate The effect on cell growth was expressed as apercent of the control and calculated as

(AtAc) times 100 () (1)

where At is the absorbance of the test sample and Ac is theabsorbance of the control Using EC

50values obtained in

a nontumor cell line and in the respective tumor cell linenontumortumor EC

50ratios (NTT) were calculated for the

extract drug and combination of the extract and drug

253 Genotoxicity Genotoxicity of HP is evaluated bycytochalasin block micronuclei assay (CBMN) which wasperformed according to published procedures [17] withminor modifications The peripheral blood lymphocytesculture was set up in triplicate vessels per concentration foreach experimental treatment condition According to the rec-ommendations for testing the genotoxic effects of compounds[18] the extract was added after 20 h of preincubationThe final concentrations of the extract in peripheral bloodculture were 50 100 and 200120583gmL After 44 h of culturesetup the medium was replaced with fresh one containingcytochalasin-B (CytB) at a final concentration of 6120583gmL andincubation continued for the next 24 h Thereafter cells wereexposed to a cold hypotonic solution (056 KCl) and fixedthree times with methanol glacial acetic acid (3 1 vv) Air-dried slides were stained with 2 Giemsa in distilled waterfor 9min The CBMN assay was performed analyzing morethan 1000 cells for each sample Standard criteria were usedfor the identification of micronuclei (MN) [19] Monitoredvalues included number of mononucleated binucleated andmultinucleated cells incidence of micronuclei and nucleardivision index (NDI) Micronucleus incidence was presentedas a number of micronuclei per 1000 examined binuclearcells

26 Statistical Analysis The data were presented as meanvalues plusmn SD Concentrations of the extracts needed for theneutralization of 50 of free radicals and LP inhibition of50 of the enzyme activity or inhibition of 50 of cell growth(RSC50 IC50 and EC

50 resp) were determined by regression

analysis from concentration-effect curve using MicrosoftExcel v2010 One-way Anova and Fisher LSD test appliedin genotoxicity assay as well as principle component analysis(PCA) were performed by Statistica 130 StatSoft

Evidence-Based Complementary and Alternative Medicine 5

sodium phosphate buffer (pH = 72) with NaCl Two concen-trations of the extract are added in test tubes The percentageof inhibition is calculated compared to the absorbance ofreactionmixtures containing no extract Acarbose is used as apositive control All the measurements are done in triplicate

244 Inhibition of 120572-Glucosidase The potential of the exam-ined extract to inhibit 120572-glucosidase is estimated by themod-ified method of Sigma-Aldrich [15] The assay mixture con-tained potassium phosphate buffer (pH = 68) glutathione(reduced solution) and 120572-glucosidase from Saccharomycescerevisiae obtained from Sigma-Aldrich and p-nitrophenyl-120572-D-glucoside (PNP-Gluc) which was used as a substrateTwo different concentrations of the HP are added in thereaction mixture After the incubation of 20min at 37∘Cthe reaction was stopped by addition of Na

2CO3and the

absorbance of solutionwas spectrophotometricallymeasuredat 400 nm Reaction mixture without the examined extractwas considered as a 100 enzyme activity Final activity ofthe enzyme in the reaction mixture was 76UL Acarbosewas used as a positive control All the measurements areperformed in triplicate

25 Cytotoxicity and Genotoxicity

251 Growth and Culture of Cells The estimation of cellgrowth activity is performed on one untransformed humancell line MRC-5 (fetal lung fibroblast ECACC 84101801) andthree human malignant transformed cell lines HeLa (cervixepithelioid carcinoma ECACC number 93021013) HT-29(colon adenocarcinoma ECACC number 91072201) and Hs-294T (melanoma ATCCHTB-140)The cell lines were grownand maintained in DMEM (Sigma-Aldrich USA) mediumsupplemented with FCS (10) penicillin (100 unitsmL)and streptomycin (100120583gmL) being referred to as completemedium Furthermore the cells were cultured in 25 cm2flasks at 37∘C in the atmosphere of 5CO

2andhigh humidity

and subcultured twice a week A single cell suspension wasobtained using 01 trypsin with 004 EDTA

Heparinized whole blood collected by venous puncturefrom 40-year-old healthy female donor not having beenexposed to any chemical or physical agent during the last6 months is used for the analysis of genotoxic potentialBriefly 05mL of the whole blood was added to 5mL ofthe Lymphochrome lymphocytes culture medium (LonzaBioWhittaker) supplemented with phytohemagglutininLymphocyte cell culture of peripheral blood was incubatedat 37∘C for 72 h in 5 CO

2atmosphere with 95 humidity

252 Cytotoxicity Assay Cytotoxicity of HP is evaluated bythe colorimetric sulforhodamine B (SRB) assay as previouslydescribed [16] Cell lines are plated into 96-well microtiterplates at different seeding density of 5 times 103 cells per wellfor MRC-5 and Hs-294T 4 times 103 cells for HeLa and 6 times 103cells for HT-29 in a volume of 180 120583L and preincubated incomplete medium supplemented with 5 FCS at 37∘C for24 h For the evaluation of the cell growth activity the extractwas diluted in NaCl to obtain final concentrations in range of

01ndash600120583gmL while the control group contained onlyNaClAll cell lines were previously treated with bleomycin (BLM)in order to determine EC

50values During the examination

of combined treatment (HP and BLM) effects on cell cul-tures the final concentration of BLM was 100 120583gmL Serialdilutions of HP and BLM (20120583Lwell) were added to achieverequired final concentrationsMicroplates are then incubatedat 37∘C for an additional 48 h The development of colorwas spectrophotometrically measured at 540 nm against620 nm as background The results of cell growth activitywere expressed as mean plusmn SD of experiments performedin octuplicate The effect on cell growth was expressed as apercent of the control and calculated as

(AtAc) times 100 () (1)

where At is the absorbance of the test sample and Ac is theabsorbance of the control Using EC

50values obtained in

a nontumor cell line and in the respective tumor cell linenontumortumor EC

50ratios (NTT) were calculated for the

extract drug and combination of the extract and drug

253 Genotoxicity Genotoxicity of HP is evaluated bycytochalasin block micronuclei assay (CBMN) which wasperformed according to published procedures [17] withminor modifications The peripheral blood lymphocytesculture was set up in triplicate vessels per concentration foreach experimental treatment condition According to the rec-ommendations for testing the genotoxic effects of compounds[18] the extract was added after 20 h of preincubationThe final concentrations of the extract in peripheral bloodculture were 50 100 and 200120583gmL After 44 h of culturesetup the medium was replaced with fresh one containingcytochalasin-B (CytB) at a final concentration of 6120583gmL andincubation continued for the next 24 h Thereafter cells wereexposed to a cold hypotonic solution (056 KCl) and fixedthree times with methanol glacial acetic acid (3 1 vv) Air-dried slides were stained with 2 Giemsa in distilled waterfor 9min The CBMN assay was performed analyzing morethan 1000 cells for each sample Standard criteria were usedfor the identification of micronuclei (MN) [19] Monitoredvalues included number of mononucleated binucleated andmultinucleated cells incidence of micronuclei and nucleardivision index (NDI) Micronucleus incidence was presentedas a number of micronuclei per 1000 examined binuclearcells

26 Statistical Analysis The data were presented as meanvalues plusmn SD Concentrations of the extracts needed for theneutralization of 50 of free radicals and LP inhibition of50 of the enzyme activity or inhibition of 50 of cell growth(RSC50 IC50 and EC

50 resp) were determined by regression

analysis from concentration-effect curve using MicrosoftExcel v2010 One-way Anova and Fisher LSD test appliedin genotoxicity assay as well as principle component analysis(PCA) were performed by Statistica 130 StatSoft

6 Evidence-Based Complementary and Alternative Medicine

3 Results and Discussion

31 Chemical Characterization of the H perforatum Subspperforatum Extracts Preliminary chemical characterizationof the HP showed that the amounts of total phenolics andflavonoids were 19331 plusmn 785mg GAEg d e and 3585 plusmn084mg QEg d e respectively which is in order withprevious studies of this species [6 7 20] The amounts ofcompounds extractable from plant samples collected duringselected phenological stages varied from 1034 to 2357(Table 1) and generally were decreasing during the period ofplant development Detailed chemical characterization andquantification of selected secondary metabolites by liquidchromatography (HPLC-DAD) reveal several patterns ofaccumulation (Table 1) Hypericinmarked as one of themainactive principles reached its maximum values in samples12ndash15 which were collected during the period of floweringThese values varied from 070 to 088mgg of dry herb (d h)and were similar as in the previously conducted studies [3 621 22] The termination of flowering period (samples 13ndash17)was followed by the decrease of hypericin levels which cor-responds to previously reported results suggesting its highestaccumulation in plantmaterial with fully opened flowers [23ndash26] However we have observed different trend of hyperforinaccumulation The highest content (089ndash092mgg d h)was detected in samples 9ndash11 obtained from plant materialcollected at floral budding stage andwas similar to previouslyreported results [3 6 20] This is also in accordance with thestudy conducted by Gioti et al [20] and may be explainedby the general instability and photosensitivity of hyperforinmolecule [27] Opposite are the results of the study conductedby Cirak et al [25] and Cirak et al [28] who report themaximum of hyperforin content in the period of blossomand a study conducted by Filippini et al [6] who statethe maximum level of this secondary metabolite during theperiod of fruit development although without specifying theH perforatum subspecies examined However it is notablethat in our research the content of hyperforin decreasesafter opening of flowers but three weeks after again starts toincrease which might partially be correlated with fruiting orwith the development of new flowersThe levels of rutin reachtheir maximum value of 015mgg d h at the period beforeflowering (sample 5) after which they decrease as alreadyreported by Bagdonaite et al [29] who state the maximumlevels of rutin at the floral budding stage The rest of thequantified flavonoids for example naringenin quercetinand amentoflavone reached the maximum levels during thefloral budding or in the stage of opening of flowers Thelevel of apigenin decreased during the development of plantswhile epicatechin is being detected only in samples collectedin periods after flowering The amounts of chlorogenic andcaffeic acid reached their maximum values during the floralbudding stage while the content of gallic acid generallydecreased during plant development possibly as a result oftannins formation

Performed PCA indicates that the first componentexplains 54 of variance and together with the secondcomponent covers more than 80 of variance The maincompounds affecting the grouping of samples based on the

load values of the first component are hypericin apigeninrutin ferulic and p-hydroxybenzoic acid However in thespace defined by the second axis the grouping is mainlya result of different quantities of hyperforin quercetinand chlorogenic acid in the examined samples Particularlysignificant is the correlation between the detected levels ofhyperforin hypericin amentoflavone and naringenin whichaccumulate at the flowering period and are some of the mainactive principles ofHyperici herba (Figure 2(a))The positionof the analyzed samples in the space defined by the first andthe second principal component clearly indicates four differ-ent phenological stages of H perforatum subsp perforatum(Figure 2(b)) It is obvious that the samples collected at theend of floral budding stage and the beginning of floweringstage contain the highest quantities of hyperforin followed byconsiderable amount of hypericin As the plant developmentcontinues the levels of hypericin further increase but theaccumulation of hyperforin decreases confirming what waspreviously stated that the optimal period for harvesting ofplant material is the transition between floral budding andflowering stage

32 Antioxidant Potential of the Examined H perforatumWater-Alcoholic Extract Reactive oxygen species (ROS) areinvolved in pathogenesis of various diseases and syndromesGenerally it is proved that plants possess significant antiox-idant potential mainly addressed to the presence of differ-ent phenolic and flavonoid compounds [5] Furthermoremodern trends suggest the use of natural products in foodand cosmetic industry as a replacement for synthetic antioxi-dants However considering the complexity of plant extractscomposition and antioxidant mechanisms of action the useof several assays for examination of ROS neutralizationpotential is highly recommended [3] Therefore in thepresent study five assays were performed for the estimationof HP antioxidant potential (Table 2) In all of the appliedtest-systems the examined extract managed to reach 50of ROS neutralization and the obtained results for IC

50

values corresponded to those previously reported [3 7 22]Regarding DPPH∙ scavenging assay HP exhibited weakerbut comparable antioxidant potential to QDH and PG It isimportant to point out that DPPH∙ is a synthetic compoundnot occurring in the human body and represents a goodchoice only for preliminary screening of antioxidant potential[3] Significantly lower potential of the HP compared tosynthetic antioxidants was noticed in the neutralization ofOH∙ and NO∙ and inhibition of LP but in the FRAP-testnotable antioxidant activity was reported (21226mg AAEgd e) However it must be emphasized that comparison ofantioxidant potential in the present study was performedbetween pure compounds and plant extract which is amixture of different secondary metabolites whereby some ofthem do not possess potential to scavenge ROS Consideringthe relevance of in vitro recorded antioxidant potential inconcentrations up to 10120583M the examined HP represents apromising free radical scavenging agent [30]

Evidence-Based Complementary and Alternative Medicine 7

Hpf

Hpc

Ap

Ec

R

Qe

Am

FAGA

CHACA

PHB

minus10 minus05 00 05 10PCA 1 5400

minus10

minus05

00

05

10PC

A2

27

45

NA

(a)

0 1 2 3 4PCA 1

minus6

minus5

minus4

minus3

minus2

minus1

minus5 minus4 minus3 minus2 minus1

0

1

2

3

PCA

2

1

10

2221

20

191817

16

15

14

1312

11

9

8

7

5 6

43

2

Floral budding

Before flowering

Flowering

After flowering

Hyperforin

Hypericin

(b)

Figure 2 Principal component analysis Projection of the examined variables compounds (a) and case samples (b) in the space defined bythe first and the second principal component

33 Inhibition of Biologically Important Enzymes Clini-cally proved antidepressant activity of H perforatum water-alcoholic extracts is so far ascribed to several mechanismswhich by most of the authors include inhibition of synap-tosomal reuptake of noradrenaline dopamine and serotoninand inhibition of MAO-A and MAO-B [5] Generally scien-tific studies gave the advantage to the hyperforin rather thanhypericin as the main active principle responsible for theantidepressant potential of St Johnrsquoswort-based preparationsIn the present study significantly higher potential of theexamined extract to inhibit MAO-A was observed (Table 2)However when comparing this activity with moclobemidea synthetic MAO-A inhibitor it may be concluded thatHP is several times less potent Since most of the patientssuffering from AD also show symptoms of depression itwould be significant that H perforatum which was alreadyproved as efficient in treatment of mild to moderate forms ofdepression also shows therapeutic effects inAD Inhibitors ofacetylcholinesterase are one of the several recognized groupsof medicines used in the treatment of AD Galantamine analkaloid isolated fromGalanthus woronowii Amaryllidaceaepresents one of themost potent inhibitors of AChE Howeverscreening of plants and other products of natural originwhich possess anticholinesterase activity is constantly inthe focus In our study the examined extract applied inconcentrations of 475 and 800 120583gmL inhibited 1722 and4231 of AChE activity respectively (Table 2) These resultsare comparable to the results reported by Bozin et al [3]and Altun et al [7] but significantly less promising whencompared to the anticholinesterase potential of galantamine(IC50= 856120583gmL)

Considering the widespread use of Hyperici herbascreening of new biological activities of this plant is morethan welcome Diabetes is one of the syndromes with rapidly

rising incidence Predictions state that the prevalence ofdiabetes will double in 2000ndash2030 period in all age groups[8] Increased blood sugar level is the initial symptom whichlater progresses toward different complications Thereforeinhibition of enzymes included in sugar metabolism is oneof the established therapy approaches of diabetes Severalapproved medicines utilize this pharmacological mechanismof action but the question iswhether someof the phytoprepa-rations also possess this biological activity The present studyis one of the first reports dealing with the potential of Hperforatum extract to inhibit 120572-amylase and 120572-glucosidaseFor both of the enzymes at tested concentrations IC

50

values were reached (Table 2) However when compared toacarbose whichwas used as positive controlmore significantis inhibition of 120572-glucosidase This leaves a space for furtherevaluation of H perforatum extracts as potent antihyper-glycemic agents

34 Cytotoxicity The antitumor effects of HP and a combi-nation of HP with BLM were evaluated in vitro by SRB assayusing human nontumor MRC-5 and three tumor cell linesHeLa Hs-294T and HT-29 Considering the inhibition oftumor cells growth moderate activity of HP was recorded(EC50= 34094ndash46894 120583gmL) (Table 2) which corresponds

to the previously published results [31] The ratio of selec-tive cytotoxicity toward nontumor and tumor-cells was low(NTT= 102ndash140) Furthermore themost susceptible tumorcell line was HeLa followed by HT-29 while the lowestcytotoxic potential was exhibited on Hs-294T cell line

The cytotoxicity of the HP could be correlated to thereported antiproliferative activity of hyperforin hypericinquercetin and chlorogenic acid Hyperforin represents apromising anticancer agent which suppresses proliferationof a number of cancer cell lines including mammary

8 Evidence-Based Complementary and Alternative Medicine

Table 2 Biological potential of the examined extract HP antioxidant potential inhibition of biologically important enzymes and cytotoxicpotential

(a) Antioxidant potential

Radical scavenging capacity (RSC) LP FRAPDPPH∙ OH∙ NO∙

RSC50(120583gmL)

HP 368 plusmn 012 6699 plusmn 315 13979 plusmn 598 47172 plusmn 1125 21226 plusmn 359BHT 003 plusmn 001 732 plusmn 056

Ascorbic acid 205 plusmn 011 QDH 097 plusmn 002 PG 069 plusmn 004 909 plusmn 059 927 plusmn 039

(b) Inhibition of biologically important enzymes

MAO-A MAO-B AChE 120572-Amylase 120572-Glucosidase of inhibition

Conc (120583gmL) 409 7353 475 800 400 800 832 1664HP 2046 plusmn 159 8669 plusmn 347 1722 plusmn 136 4231 plusmn 418 5110 plusmn 329 9078 plusmn 297 4133 plusmn 219 7101 plusmn 367

IC50(120583gmL)

Moclobemide 064 plusmn 004 Selegiline 023 plusmn 002

Galantamine 856 plusmn 077 Acarbose 493 plusmn 033 4287 plusmn 343

(c) Cytotoxic potential

MRC-5 HeLa Hs-294 T HT-29 HeLa Hs-294 T HT-29EC50(120583gmL) NTT

HP 48062 plusmn 2015 34094 plusmn 1274 46894 plusmn 1447 34246 plusmn 1167 140 102 140HP + BLM 276 plusmn 021 002 plusmn 000 005 plusmn 001 421 plusmn 014 10804 5360 065BLM 803 plusmn 457 772 plusmn 514 4518 plusmn 234 16531 plusmn 589 104 177 048Note NTT nontumortumor cells EC50 ratio

carcinoma squamous cell carcinoma melanoma gliomafibrosarcoma chronic myeloid leukemia and lymphoma[32] The hyperforin cytotoxicity is often explained by themechanism of apoptosis [33 34] Schempp et al [32] statethat hyperforin induces early apoptotic events for examplerapid loss of mitochondrial transmembrane potential andsubsequent cytochrome c release which consequently leadstoward reducing of size of experimental colon carcinomametastases and inhibits proliferation in peripheral bloodmononuclear cellsMolecularmechanism activated by hyper-forin in chronic lymphoid leukemia and acute myeloidleukemia cells known for their resistance to chemotherapyis activation of the proapoptotic Bcl-2 family protein Badand Noxa [35] Antiproliferative effect of hyperforin in vivo isexplained by inhibition of angiogenesis [5] Studies supportthis by stating the comparability with antineoplastic drugpaclitaxel followedwith the absence of any signs of acute tox-icity [32]The second abundant biologically active compoundin the examined extract exhibiting antiproliferative actionon numerous human cancer cell lines was hypericin Theproposed mechanism of hypericin antiproliferative effects isalso apoptosis [5] Beyond hyperforin and hypericin it wasalso reported that flavonoids such as quercetin inhibit can-cer cells growth [36] However cytotoxicity of H perforatumextracts in our research presents the result of joined action of

major constituents hyperforin and hypericin as well as othersecondary metabolites present in the investigated extract

On the other hand significant antitumor activity wasrecorded during the treatment of selected cell cultures byseries of concentrations of combination of HP and BLM(100 120583gmL) (Table 2 Figure 3) Selective cytotoxicity ofthis combined treatment was considerably high on cervixcancer cells (HeLa) as well as on melanoma cancer cells(Hs-294T) while it was low on colon cancer cells (HT-29)By comparing the EC

50values of different tumor cell lines

it can be noticed that HeLa are the most susceptible to thetreatment by combination of the HP and BLM followed byHs-294T while HT-29 are the most resistant It is known thatproducts of natural origin in combination with conventionalmedicines can affect the efficacy of the applied therapyby interfering in signaling cascades of transport proteinsand enzymes and up- or downregulating the transport andmetabolism [11] Bleomycin is radiomimetic which inducesDNA damage as ionizing radiation (IR) As a result of itsaction formed DNA strain breaks lead toward the pro-duction of DNA adducts and excess ROS which induceoxidative stress mitochondrial leakage and apoptosis [37]It is important to highlight that BLM belongs to the groupof drugs associated with radiation sensitivity [38] Previousreports suggest that hypericin increases the sensitivity ofcancer cells to ionizing radiation which puts it in a group of

Evidence-Based Complementary and Alternative Medicine 9

0

20

40

60

80

100

120

0 600 300 100 1 01Concentration (gmL)

Hs-294 T

HH + BLM

ce

ll gr

owth

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HT-29

HH + BLM

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HeLa

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

MRC-5

Concentration (gmL)

ce

ll gr

owth

Figure 3 Cytotoxicity of HP and combination of HP and BLM (100 120583gmL) during 48 h treatment The results are expressed as mean plusmn SDperformed in octuplicate

potential new agents in combination with radiation therapyof malignant gliomas [39] Furthermore significant level ofselectivity toward cervix carcinoma and melanoma cell linesagainst healthy cells was achieved described by NTT [40]Altogether it is obvious that the cellular events leading to theincrement of cytostatic effect as a result of cotreatment by HPand BLM are complex and should be investigated further

35 Genotoxicity Genotoxicity assays enable the assessmentof mutagenic and cancerogenic potential of different extractsof medicinal plants and their protective effects as well asmechanisms of their biological activities These assays areused to identify the extracts with the ability to react withnucleic acids in subcytotoxic concentrations If the substancedamages the genome it is manifested by the incrementof incidence of micronuclei Genotoxicity of the HP wastested by micronucleus assay on lymphocytes of peripheralblood during the 24 h treatment The results of micronucleiincidence are shown in Figure 4 During the treatmentHP induced decreasing of micronuclei incidence which was

lower compared to the control value Statistically significantdecrease of micronuclei (MN 355 versus 980 119901 = 0008)was observed at concentration of 200 120583gmL Ramos et al[41] reported thatH perforatum exhibits antigenotoxic effectson HT-29 cells which corresponds to the result of our studyNamely it was stated that the extracts of H perforatum haveprotective effect against oxidative DNA damage followed byincreasing of the repair of alkylating DNA damage by baseexcision repair pathway These protective properties wereascribed to the high amount of phenolic compounds Inanother in vitro study of St Johnrsquos wort extracts rich inflavonoids exhibited strong antioxidant activity and radicalscavenging characteristics [42] Considering that the currentstudy confirms notable antioxidant potential of HP decreas-ing of the micronuclei incidence is the expected result

4 Conclusions

The results of the conducted study concerning the chemicalprofiling ofH perforatum subsp perforatumduring floweringand fruitification suggest that the maximum accumulation of

10 Evidence-Based Complementary and Alternative Medicine

98

355

899 926

000

200

400

600

800

1000

1200

0 200 100 50Concentration (gmL)

Num

ber o

f MN

1000

lowast

Figure 4 Incidence of micronuclei by HP during 24 h treatmentMN micronuclei BN binucleated cells The results are shown asmean plusmn SD performed in triplicate Concentrations were expressedin 120583gmL lowastStatistically significantly different from the control(Fisher LSD test 119901 lt 005)

hypericin and hyperforin is present in the transition periodbetween floral budding and full flowering stage Hypericiherba collected at aforementioned time interval will alsocontain significant amounts of naringenin rutin quercetinamentoflavone chlorogenic caffeic and p-hydroxybenzoicacid The evaluation of biological potential of the preparedwater-alcoholic extract from the plant material collectedat the stage of full flowering suggests notable antioxidantpotential comparable to synthetic antioxidants Also thepotential of the extract to inhibit biologically importantenzymes cannot be neglected especially in the case of 120572-glucosidase which opens a place for further evaluation of StJohnrsquos wort as antihyperglycemic agent The combination ofHP at concentrations higher than 1 120583gmL and bleomycin(100 120583gmL) leads to significant increment of antiproliferativeeffects on cancer cell lines while the examined extract doesnot show signs of genotoxicity at subcytotoxic concentra-tions (lt200120583gmL)However during evaluation of biologicalpotential of different plant extracts the fact that the evaluatedsubject (in this case the Strsquo Johnrsquos wort extract) is a mixture ofcompounds with different characteristics exhibiting variousbiological effects must be always taken into the account

Conflicts of Interest

All of the authors declare no conflicts of interest

Acknowledgments

The Ministry of Science and Technological DevelopmentRepublic of Serbia (Grant no OI 172058) supported thisresearch work

References

[1] NK B Robson ldquoStudies in the genusHypericumLGuttiferaerdquoBulletin of the British Museum National History vol 8 pp 55ndash226 1981

[2] N Kladar B Srđenovic N Grujic et al ldquoSt JohnrsquoS Wort(Hypericum Spp) - Relation between the biological sourceand medical propertiesrdquoHypericum Botanical Sources MedicalProperties and Health Effects pp 53ndash80 2015

[3] B Bozin N Kladar N Grujic et al ldquoImpact of origin andbiological source on chemical composition anticholinesteraseand antioxidant properties of some St Johnrsquos wort species(Hypericum spp hypericaceae) from the central BalkansrdquoMolecules vol 18 no 10 pp 11733ndash11750 2013

[4] A Brattstrom ldquoLong-term effects of St Johnrsquos wort (Hypericumperforatum) treatment A 1-year safety study in mild to moder-ate depressionrdquo Phytomedicine vol 16 no 4 pp 277ndash283 2009

[5] N Kladar B Srđenovic N Grujic et al ldquoEcologically andontogenetically induced variations in phenolic compounds andbiological activities of Hypericum maculatum subsp macula-tum Hypericaceaerdquo Revista Brasileira de Botanica vol 38 no4 pp 703ndash715 2015

[6] R Filippini A Piovan A Borsarini and R Caniato ldquoStudyof dynamic accumulation of secondary metabolites in threesubspecies of Hypericum perforatumrdquo Fitoterapia vol 81 no2 pp 115ndash119 2010

[7] M L Altun B S Yilmaz I E Orhan and G S CitogluldquoAssessment of cholinesterase and tyrosinase inhibitory andantioxidant effects of Hypericum perforatum L (St Johnrsquoswort)rdquo Industrial Crops and Products vol 43 no 1 pp 87ndash922013

[8] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[9] J Ferlay I Soerjomataram R Dikshit et al ldquoCancer incidenceand mortality worldwide sources methods and major patternsin GLOBOCAN 2012rdquo International Journal of Cancer vol 136no 5 pp E359ndashE386 2015

[10] G M Cragg and D J Newman ldquoPlants as a source of anti-cancer agentsrdquo Journal of Ethnopharmacology vol 100 no 1-2pp 72ndash79 2005

[11] Y Li D Kong A Ahmad B Bao and F H Sarkar ldquoAugmentingthe efficacy of chemo- and radio-therapy by nutraceuticalsEvidence from pre-clinical and clinical trialsrdquo Nutraceuticalsand Cancer vol 9789400726307 pp 355ndash376 2012

[12] A Ziakova and E Brandsteterova ldquoValidation of HPLC deter-mination of phenolic acids present in some Lamiaceae familyplantsrdquo Journal of Liquid Chromatography amp Related Technolo-gies vol 26 no 3 pp 443ndash453 2003

[13] M M Lesjak I N Beara D Z Orcic et al ldquoJuniperussibirica Burgsdorf as a novel source of antioxidant and anti-inflammatory agentsrdquo Food Chemistry vol 124 no 3 pp 850ndash856 2011

[14] V Samoylenko M M Rahman B L Tekwani et al ldquoBan-isteriopsis caapi a unique combination of MAO inhibitoryand antioxidative constituents for the activities relevant toneurodegenerative disorders and Parkinsonrsquos diseaserdquo Journalof Ethnopharmacology vol 127 no 2 pp 357ndash367 2010

[15] A Munrsquoim A Andriani K F Mahmudah and M MashitaldquoScreening of a-glucosidase inhibitory activity of some Indone-sian medicinal plantsrdquo in Proceedings of the Screening of 120572-glucosidase inhibitory activity of some Indonesian medicinalplants vol 3 pp 144ndash150 2013

[16] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Lemon Balm Kombuchardquo Food and BioprocessTechnology vol 5 no 5 pp 1756ndash1765 2012

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 7

Hpf

Hpc

Ap

Ec

R

Qe

Am

FAGA

CHACA

PHB

minus10 minus05 00 05 10PCA 1 5400

minus10

minus05

00

05

10PC

A2

27

45

NA

(a)

0 1 2 3 4PCA 1

minus6

minus5

minus4

minus3

minus2

minus1

minus5 minus4 minus3 minus2 minus1

0

1

2

3

PCA

2

1

10

2221

20

191817

16

15

14

1312

11

9

8

7

5 6

43

2

Floral budding

Before flowering

Flowering

After flowering

Hyperforin

Hypericin

(b)

Figure 2 Principal component analysis Projection of the examined variables compounds (a) and case samples (b) in the space defined bythe first and the second principal component

33 Inhibition of Biologically Important Enzymes Clini-cally proved antidepressant activity of H perforatum water-alcoholic extracts is so far ascribed to several mechanismswhich by most of the authors include inhibition of synap-tosomal reuptake of noradrenaline dopamine and serotoninand inhibition of MAO-A and MAO-B [5] Generally scien-tific studies gave the advantage to the hyperforin rather thanhypericin as the main active principle responsible for theantidepressant potential of St Johnrsquoswort-based preparationsIn the present study significantly higher potential of theexamined extract to inhibit MAO-A was observed (Table 2)However when comparing this activity with moclobemidea synthetic MAO-A inhibitor it may be concluded thatHP is several times less potent Since most of the patientssuffering from AD also show symptoms of depression itwould be significant that H perforatum which was alreadyproved as efficient in treatment of mild to moderate forms ofdepression also shows therapeutic effects inAD Inhibitors ofacetylcholinesterase are one of the several recognized groupsof medicines used in the treatment of AD Galantamine analkaloid isolated fromGalanthus woronowii Amaryllidaceaepresents one of themost potent inhibitors of AChE Howeverscreening of plants and other products of natural originwhich possess anticholinesterase activity is constantly inthe focus In our study the examined extract applied inconcentrations of 475 and 800 120583gmL inhibited 1722 and4231 of AChE activity respectively (Table 2) These resultsare comparable to the results reported by Bozin et al [3]and Altun et al [7] but significantly less promising whencompared to the anticholinesterase potential of galantamine(IC50= 856120583gmL)

Considering the widespread use of Hyperici herbascreening of new biological activities of this plant is morethan welcome Diabetes is one of the syndromes with rapidly

rising incidence Predictions state that the prevalence ofdiabetes will double in 2000ndash2030 period in all age groups[8] Increased blood sugar level is the initial symptom whichlater progresses toward different complications Thereforeinhibition of enzymes included in sugar metabolism is oneof the established therapy approaches of diabetes Severalapproved medicines utilize this pharmacological mechanismof action but the question iswhether someof the phytoprepa-rations also possess this biological activity The present studyis one of the first reports dealing with the potential of Hperforatum extract to inhibit 120572-amylase and 120572-glucosidaseFor both of the enzymes at tested concentrations IC

50

values were reached (Table 2) However when compared toacarbose whichwas used as positive controlmore significantis inhibition of 120572-glucosidase This leaves a space for furtherevaluation of H perforatum extracts as potent antihyper-glycemic agents

34 Cytotoxicity The antitumor effects of HP and a combi-nation of HP with BLM were evaluated in vitro by SRB assayusing human nontumor MRC-5 and three tumor cell linesHeLa Hs-294T and HT-29 Considering the inhibition oftumor cells growth moderate activity of HP was recorded(EC50= 34094ndash46894 120583gmL) (Table 2) which corresponds

to the previously published results [31] The ratio of selec-tive cytotoxicity toward nontumor and tumor-cells was low(NTT= 102ndash140) Furthermore themost susceptible tumorcell line was HeLa followed by HT-29 while the lowestcytotoxic potential was exhibited on Hs-294T cell line

The cytotoxicity of the HP could be correlated to thereported antiproliferative activity of hyperforin hypericinquercetin and chlorogenic acid Hyperforin represents apromising anticancer agent which suppresses proliferationof a number of cancer cell lines including mammary

8 Evidence-Based Complementary and Alternative Medicine

Table 2 Biological potential of the examined extract HP antioxidant potential inhibition of biologically important enzymes and cytotoxicpotential

(a) Antioxidant potential

Radical scavenging capacity (RSC) LP FRAPDPPH∙ OH∙ NO∙

RSC50(120583gmL)

HP 368 plusmn 012 6699 plusmn 315 13979 plusmn 598 47172 plusmn 1125 21226 plusmn 359BHT 003 plusmn 001 732 plusmn 056

Ascorbic acid 205 plusmn 011 QDH 097 plusmn 002 PG 069 plusmn 004 909 plusmn 059 927 plusmn 039

(b) Inhibition of biologically important enzymes

MAO-A MAO-B AChE 120572-Amylase 120572-Glucosidase of inhibition

Conc (120583gmL) 409 7353 475 800 400 800 832 1664HP 2046 plusmn 159 8669 plusmn 347 1722 plusmn 136 4231 plusmn 418 5110 plusmn 329 9078 plusmn 297 4133 plusmn 219 7101 plusmn 367

IC50(120583gmL)

Moclobemide 064 plusmn 004 Selegiline 023 plusmn 002

Galantamine 856 plusmn 077 Acarbose 493 plusmn 033 4287 plusmn 343

(c) Cytotoxic potential

MRC-5 HeLa Hs-294 T HT-29 HeLa Hs-294 T HT-29EC50(120583gmL) NTT

HP 48062 plusmn 2015 34094 plusmn 1274 46894 plusmn 1447 34246 plusmn 1167 140 102 140HP + BLM 276 plusmn 021 002 plusmn 000 005 plusmn 001 421 plusmn 014 10804 5360 065BLM 803 plusmn 457 772 plusmn 514 4518 plusmn 234 16531 plusmn 589 104 177 048Note NTT nontumortumor cells EC50 ratio

carcinoma squamous cell carcinoma melanoma gliomafibrosarcoma chronic myeloid leukemia and lymphoma[32] The hyperforin cytotoxicity is often explained by themechanism of apoptosis [33 34] Schempp et al [32] statethat hyperforin induces early apoptotic events for examplerapid loss of mitochondrial transmembrane potential andsubsequent cytochrome c release which consequently leadstoward reducing of size of experimental colon carcinomametastases and inhibits proliferation in peripheral bloodmononuclear cellsMolecularmechanism activated by hyper-forin in chronic lymphoid leukemia and acute myeloidleukemia cells known for their resistance to chemotherapyis activation of the proapoptotic Bcl-2 family protein Badand Noxa [35] Antiproliferative effect of hyperforin in vivo isexplained by inhibition of angiogenesis [5] Studies supportthis by stating the comparability with antineoplastic drugpaclitaxel followedwith the absence of any signs of acute tox-icity [32]The second abundant biologically active compoundin the examined extract exhibiting antiproliferative actionon numerous human cancer cell lines was hypericin Theproposed mechanism of hypericin antiproliferative effects isalso apoptosis [5] Beyond hyperforin and hypericin it wasalso reported that flavonoids such as quercetin inhibit can-cer cells growth [36] However cytotoxicity of H perforatumextracts in our research presents the result of joined action of

major constituents hyperforin and hypericin as well as othersecondary metabolites present in the investigated extract

On the other hand significant antitumor activity wasrecorded during the treatment of selected cell cultures byseries of concentrations of combination of HP and BLM(100 120583gmL) (Table 2 Figure 3) Selective cytotoxicity ofthis combined treatment was considerably high on cervixcancer cells (HeLa) as well as on melanoma cancer cells(Hs-294T) while it was low on colon cancer cells (HT-29)By comparing the EC

50values of different tumor cell lines

it can be noticed that HeLa are the most susceptible to thetreatment by combination of the HP and BLM followed byHs-294T while HT-29 are the most resistant It is known thatproducts of natural origin in combination with conventionalmedicines can affect the efficacy of the applied therapyby interfering in signaling cascades of transport proteinsand enzymes and up- or downregulating the transport andmetabolism [11] Bleomycin is radiomimetic which inducesDNA damage as ionizing radiation (IR) As a result of itsaction formed DNA strain breaks lead toward the pro-duction of DNA adducts and excess ROS which induceoxidative stress mitochondrial leakage and apoptosis [37]It is important to highlight that BLM belongs to the groupof drugs associated with radiation sensitivity [38] Previousreports suggest that hypericin increases the sensitivity ofcancer cells to ionizing radiation which puts it in a group of

Evidence-Based Complementary and Alternative Medicine 9

0

20

40

60

80

100

120

0 600 300 100 1 01Concentration (gmL)

Hs-294 T

HH + BLM

ce

ll gr

owth

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HT-29

HH + BLM

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HeLa

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

MRC-5

Concentration (gmL)

ce

ll gr

owth

Figure 3 Cytotoxicity of HP and combination of HP and BLM (100 120583gmL) during 48 h treatment The results are expressed as mean plusmn SDperformed in octuplicate

potential new agents in combination with radiation therapyof malignant gliomas [39] Furthermore significant level ofselectivity toward cervix carcinoma and melanoma cell linesagainst healthy cells was achieved described by NTT [40]Altogether it is obvious that the cellular events leading to theincrement of cytostatic effect as a result of cotreatment by HPand BLM are complex and should be investigated further

35 Genotoxicity Genotoxicity assays enable the assessmentof mutagenic and cancerogenic potential of different extractsof medicinal plants and their protective effects as well asmechanisms of their biological activities These assays areused to identify the extracts with the ability to react withnucleic acids in subcytotoxic concentrations If the substancedamages the genome it is manifested by the incrementof incidence of micronuclei Genotoxicity of the HP wastested by micronucleus assay on lymphocytes of peripheralblood during the 24 h treatment The results of micronucleiincidence are shown in Figure 4 During the treatmentHP induced decreasing of micronuclei incidence which was

lower compared to the control value Statistically significantdecrease of micronuclei (MN 355 versus 980 119901 = 0008)was observed at concentration of 200 120583gmL Ramos et al[41] reported thatH perforatum exhibits antigenotoxic effectson HT-29 cells which corresponds to the result of our studyNamely it was stated that the extracts of H perforatum haveprotective effect against oxidative DNA damage followed byincreasing of the repair of alkylating DNA damage by baseexcision repair pathway These protective properties wereascribed to the high amount of phenolic compounds Inanother in vitro study of St Johnrsquos wort extracts rich inflavonoids exhibited strong antioxidant activity and radicalscavenging characteristics [42] Considering that the currentstudy confirms notable antioxidant potential of HP decreas-ing of the micronuclei incidence is the expected result

4 Conclusions

The results of the conducted study concerning the chemicalprofiling ofH perforatum subsp perforatumduring floweringand fruitification suggest that the maximum accumulation of

10 Evidence-Based Complementary and Alternative Medicine

98

355

899 926

000

200

400

600

800

1000

1200

0 200 100 50Concentration (gmL)

Num

ber o

f MN

1000

lowast

Figure 4 Incidence of micronuclei by HP during 24 h treatmentMN micronuclei BN binucleated cells The results are shown asmean plusmn SD performed in triplicate Concentrations were expressedin 120583gmL lowastStatistically significantly different from the control(Fisher LSD test 119901 lt 005)

hypericin and hyperforin is present in the transition periodbetween floral budding and full flowering stage Hypericiherba collected at aforementioned time interval will alsocontain significant amounts of naringenin rutin quercetinamentoflavone chlorogenic caffeic and p-hydroxybenzoicacid The evaluation of biological potential of the preparedwater-alcoholic extract from the plant material collectedat the stage of full flowering suggests notable antioxidantpotential comparable to synthetic antioxidants Also thepotential of the extract to inhibit biologically importantenzymes cannot be neglected especially in the case of 120572-glucosidase which opens a place for further evaluation of StJohnrsquos wort as antihyperglycemic agent The combination ofHP at concentrations higher than 1 120583gmL and bleomycin(100 120583gmL) leads to significant increment of antiproliferativeeffects on cancer cell lines while the examined extract doesnot show signs of genotoxicity at subcytotoxic concentra-tions (lt200120583gmL)However during evaluation of biologicalpotential of different plant extracts the fact that the evaluatedsubject (in this case the Strsquo Johnrsquos wort extract) is a mixture ofcompounds with different characteristics exhibiting variousbiological effects must be always taken into the account

Conflicts of Interest

All of the authors declare no conflicts of interest

Acknowledgments

The Ministry of Science and Technological DevelopmentRepublic of Serbia (Grant no OI 172058) supported thisresearch work

References

[1] NK B Robson ldquoStudies in the genusHypericumLGuttiferaerdquoBulletin of the British Museum National History vol 8 pp 55ndash226 1981

[2] N Kladar B Srđenovic N Grujic et al ldquoSt JohnrsquoS Wort(Hypericum Spp) - Relation between the biological sourceand medical propertiesrdquoHypericum Botanical Sources MedicalProperties and Health Effects pp 53ndash80 2015

[3] B Bozin N Kladar N Grujic et al ldquoImpact of origin andbiological source on chemical composition anticholinesteraseand antioxidant properties of some St Johnrsquos wort species(Hypericum spp hypericaceae) from the central BalkansrdquoMolecules vol 18 no 10 pp 11733ndash11750 2013

[4] A Brattstrom ldquoLong-term effects of St Johnrsquos wort (Hypericumperforatum) treatment A 1-year safety study in mild to moder-ate depressionrdquo Phytomedicine vol 16 no 4 pp 277ndash283 2009

[5] N Kladar B Srđenovic N Grujic et al ldquoEcologically andontogenetically induced variations in phenolic compounds andbiological activities of Hypericum maculatum subsp macula-tum Hypericaceaerdquo Revista Brasileira de Botanica vol 38 no4 pp 703ndash715 2015

[6] R Filippini A Piovan A Borsarini and R Caniato ldquoStudyof dynamic accumulation of secondary metabolites in threesubspecies of Hypericum perforatumrdquo Fitoterapia vol 81 no2 pp 115ndash119 2010

[7] M L Altun B S Yilmaz I E Orhan and G S CitogluldquoAssessment of cholinesterase and tyrosinase inhibitory andantioxidant effects of Hypericum perforatum L (St Johnrsquoswort)rdquo Industrial Crops and Products vol 43 no 1 pp 87ndash922013

[8] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[9] J Ferlay I Soerjomataram R Dikshit et al ldquoCancer incidenceand mortality worldwide sources methods and major patternsin GLOBOCAN 2012rdquo International Journal of Cancer vol 136no 5 pp E359ndashE386 2015

[10] G M Cragg and D J Newman ldquoPlants as a source of anti-cancer agentsrdquo Journal of Ethnopharmacology vol 100 no 1-2pp 72ndash79 2005

[11] Y Li D Kong A Ahmad B Bao and F H Sarkar ldquoAugmentingthe efficacy of chemo- and radio-therapy by nutraceuticalsEvidence from pre-clinical and clinical trialsrdquo Nutraceuticalsand Cancer vol 9789400726307 pp 355ndash376 2012

[12] A Ziakova and E Brandsteterova ldquoValidation of HPLC deter-mination of phenolic acids present in some Lamiaceae familyplantsrdquo Journal of Liquid Chromatography amp Related Technolo-gies vol 26 no 3 pp 443ndash453 2003

[13] M M Lesjak I N Beara D Z Orcic et al ldquoJuniperussibirica Burgsdorf as a novel source of antioxidant and anti-inflammatory agentsrdquo Food Chemistry vol 124 no 3 pp 850ndash856 2011

[14] V Samoylenko M M Rahman B L Tekwani et al ldquoBan-isteriopsis caapi a unique combination of MAO inhibitoryand antioxidative constituents for the activities relevant toneurodegenerative disorders and Parkinsonrsquos diseaserdquo Journalof Ethnopharmacology vol 127 no 2 pp 357ndash367 2010

[15] A Munrsquoim A Andriani K F Mahmudah and M MashitaldquoScreening of a-glucosidase inhibitory activity of some Indone-sian medicinal plantsrdquo in Proceedings of the Screening of 120572-glucosidase inhibitory activity of some Indonesian medicinalplants vol 3 pp 144ndash150 2013

[16] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Lemon Balm Kombuchardquo Food and BioprocessTechnology vol 5 no 5 pp 1756ndash1765 2012

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Evidence-Based Complementary and Alternative Medicine

Table 2 Biological potential of the examined extract HP antioxidant potential inhibition of biologically important enzymes and cytotoxicpotential

(a) Antioxidant potential

Radical scavenging capacity (RSC) LP FRAPDPPH∙ OH∙ NO∙

RSC50(120583gmL)

HP 368 plusmn 012 6699 plusmn 315 13979 plusmn 598 47172 plusmn 1125 21226 plusmn 359BHT 003 plusmn 001 732 plusmn 056

Ascorbic acid 205 plusmn 011 QDH 097 plusmn 002 PG 069 plusmn 004 909 plusmn 059 927 plusmn 039

(b) Inhibition of biologically important enzymes

MAO-A MAO-B AChE 120572-Amylase 120572-Glucosidase of inhibition

Conc (120583gmL) 409 7353 475 800 400 800 832 1664HP 2046 plusmn 159 8669 plusmn 347 1722 plusmn 136 4231 plusmn 418 5110 plusmn 329 9078 plusmn 297 4133 plusmn 219 7101 plusmn 367

IC50(120583gmL)

Moclobemide 064 plusmn 004 Selegiline 023 plusmn 002

Galantamine 856 plusmn 077 Acarbose 493 plusmn 033 4287 plusmn 343

(c) Cytotoxic potential

MRC-5 HeLa Hs-294 T HT-29 HeLa Hs-294 T HT-29EC50(120583gmL) NTT

HP 48062 plusmn 2015 34094 plusmn 1274 46894 plusmn 1447 34246 plusmn 1167 140 102 140HP + BLM 276 plusmn 021 002 plusmn 000 005 plusmn 001 421 plusmn 014 10804 5360 065BLM 803 plusmn 457 772 plusmn 514 4518 plusmn 234 16531 plusmn 589 104 177 048Note NTT nontumortumor cells EC50 ratio

carcinoma squamous cell carcinoma melanoma gliomafibrosarcoma chronic myeloid leukemia and lymphoma[32] The hyperforin cytotoxicity is often explained by themechanism of apoptosis [33 34] Schempp et al [32] statethat hyperforin induces early apoptotic events for examplerapid loss of mitochondrial transmembrane potential andsubsequent cytochrome c release which consequently leadstoward reducing of size of experimental colon carcinomametastases and inhibits proliferation in peripheral bloodmononuclear cellsMolecularmechanism activated by hyper-forin in chronic lymphoid leukemia and acute myeloidleukemia cells known for their resistance to chemotherapyis activation of the proapoptotic Bcl-2 family protein Badand Noxa [35] Antiproliferative effect of hyperforin in vivo isexplained by inhibition of angiogenesis [5] Studies supportthis by stating the comparability with antineoplastic drugpaclitaxel followedwith the absence of any signs of acute tox-icity [32]The second abundant biologically active compoundin the examined extract exhibiting antiproliferative actionon numerous human cancer cell lines was hypericin Theproposed mechanism of hypericin antiproliferative effects isalso apoptosis [5] Beyond hyperforin and hypericin it wasalso reported that flavonoids such as quercetin inhibit can-cer cells growth [36] However cytotoxicity of H perforatumextracts in our research presents the result of joined action of

major constituents hyperforin and hypericin as well as othersecondary metabolites present in the investigated extract

On the other hand significant antitumor activity wasrecorded during the treatment of selected cell cultures byseries of concentrations of combination of HP and BLM(100 120583gmL) (Table 2 Figure 3) Selective cytotoxicity ofthis combined treatment was considerably high on cervixcancer cells (HeLa) as well as on melanoma cancer cells(Hs-294T) while it was low on colon cancer cells (HT-29)By comparing the EC

50values of different tumor cell lines

it can be noticed that HeLa are the most susceptible to thetreatment by combination of the HP and BLM followed byHs-294T while HT-29 are the most resistant It is known thatproducts of natural origin in combination with conventionalmedicines can affect the efficacy of the applied therapyby interfering in signaling cascades of transport proteinsand enzymes and up- or downregulating the transport andmetabolism [11] Bleomycin is radiomimetic which inducesDNA damage as ionizing radiation (IR) As a result of itsaction formed DNA strain breaks lead toward the pro-duction of DNA adducts and excess ROS which induceoxidative stress mitochondrial leakage and apoptosis [37]It is important to highlight that BLM belongs to the groupof drugs associated with radiation sensitivity [38] Previousreports suggest that hypericin increases the sensitivity ofcancer cells to ionizing radiation which puts it in a group of

Evidence-Based Complementary and Alternative Medicine 9

0

20

40

60

80

100

120

0 600 300 100 1 01Concentration (gmL)

Hs-294 T

HH + BLM

ce

ll gr

owth

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HT-29

HH + BLM

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HeLa

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

MRC-5

Concentration (gmL)

ce

ll gr

owth

Figure 3 Cytotoxicity of HP and combination of HP and BLM (100 120583gmL) during 48 h treatment The results are expressed as mean plusmn SDperformed in octuplicate

potential new agents in combination with radiation therapyof malignant gliomas [39] Furthermore significant level ofselectivity toward cervix carcinoma and melanoma cell linesagainst healthy cells was achieved described by NTT [40]Altogether it is obvious that the cellular events leading to theincrement of cytostatic effect as a result of cotreatment by HPand BLM are complex and should be investigated further

35 Genotoxicity Genotoxicity assays enable the assessmentof mutagenic and cancerogenic potential of different extractsof medicinal plants and their protective effects as well asmechanisms of their biological activities These assays areused to identify the extracts with the ability to react withnucleic acids in subcytotoxic concentrations If the substancedamages the genome it is manifested by the incrementof incidence of micronuclei Genotoxicity of the HP wastested by micronucleus assay on lymphocytes of peripheralblood during the 24 h treatment The results of micronucleiincidence are shown in Figure 4 During the treatmentHP induced decreasing of micronuclei incidence which was

lower compared to the control value Statistically significantdecrease of micronuclei (MN 355 versus 980 119901 = 0008)was observed at concentration of 200 120583gmL Ramos et al[41] reported thatH perforatum exhibits antigenotoxic effectson HT-29 cells which corresponds to the result of our studyNamely it was stated that the extracts of H perforatum haveprotective effect against oxidative DNA damage followed byincreasing of the repair of alkylating DNA damage by baseexcision repair pathway These protective properties wereascribed to the high amount of phenolic compounds Inanother in vitro study of St Johnrsquos wort extracts rich inflavonoids exhibited strong antioxidant activity and radicalscavenging characteristics [42] Considering that the currentstudy confirms notable antioxidant potential of HP decreas-ing of the micronuclei incidence is the expected result

4 Conclusions

The results of the conducted study concerning the chemicalprofiling ofH perforatum subsp perforatumduring floweringand fruitification suggest that the maximum accumulation of

10 Evidence-Based Complementary and Alternative Medicine

98

355

899 926

000

200

400

600

800

1000

1200

0 200 100 50Concentration (gmL)

Num

ber o

f MN

1000

lowast

Figure 4 Incidence of micronuclei by HP during 24 h treatmentMN micronuclei BN binucleated cells The results are shown asmean plusmn SD performed in triplicate Concentrations were expressedin 120583gmL lowastStatistically significantly different from the control(Fisher LSD test 119901 lt 005)

hypericin and hyperforin is present in the transition periodbetween floral budding and full flowering stage Hypericiherba collected at aforementioned time interval will alsocontain significant amounts of naringenin rutin quercetinamentoflavone chlorogenic caffeic and p-hydroxybenzoicacid The evaluation of biological potential of the preparedwater-alcoholic extract from the plant material collectedat the stage of full flowering suggests notable antioxidantpotential comparable to synthetic antioxidants Also thepotential of the extract to inhibit biologically importantenzymes cannot be neglected especially in the case of 120572-glucosidase which opens a place for further evaluation of StJohnrsquos wort as antihyperglycemic agent The combination ofHP at concentrations higher than 1 120583gmL and bleomycin(100 120583gmL) leads to significant increment of antiproliferativeeffects on cancer cell lines while the examined extract doesnot show signs of genotoxicity at subcytotoxic concentra-tions (lt200120583gmL)However during evaluation of biologicalpotential of different plant extracts the fact that the evaluatedsubject (in this case the Strsquo Johnrsquos wort extract) is a mixture ofcompounds with different characteristics exhibiting variousbiological effects must be always taken into the account

Conflicts of Interest

All of the authors declare no conflicts of interest

Acknowledgments

The Ministry of Science and Technological DevelopmentRepublic of Serbia (Grant no OI 172058) supported thisresearch work

References

[1] NK B Robson ldquoStudies in the genusHypericumLGuttiferaerdquoBulletin of the British Museum National History vol 8 pp 55ndash226 1981

[2] N Kladar B Srđenovic N Grujic et al ldquoSt JohnrsquoS Wort(Hypericum Spp) - Relation between the biological sourceand medical propertiesrdquoHypericum Botanical Sources MedicalProperties and Health Effects pp 53ndash80 2015

[3] B Bozin N Kladar N Grujic et al ldquoImpact of origin andbiological source on chemical composition anticholinesteraseand antioxidant properties of some St Johnrsquos wort species(Hypericum spp hypericaceae) from the central BalkansrdquoMolecules vol 18 no 10 pp 11733ndash11750 2013

[4] A Brattstrom ldquoLong-term effects of St Johnrsquos wort (Hypericumperforatum) treatment A 1-year safety study in mild to moder-ate depressionrdquo Phytomedicine vol 16 no 4 pp 277ndash283 2009

[5] N Kladar B Srđenovic N Grujic et al ldquoEcologically andontogenetically induced variations in phenolic compounds andbiological activities of Hypericum maculatum subsp macula-tum Hypericaceaerdquo Revista Brasileira de Botanica vol 38 no4 pp 703ndash715 2015

[6] R Filippini A Piovan A Borsarini and R Caniato ldquoStudyof dynamic accumulation of secondary metabolites in threesubspecies of Hypericum perforatumrdquo Fitoterapia vol 81 no2 pp 115ndash119 2010

[7] M L Altun B S Yilmaz I E Orhan and G S CitogluldquoAssessment of cholinesterase and tyrosinase inhibitory andantioxidant effects of Hypericum perforatum L (St Johnrsquoswort)rdquo Industrial Crops and Products vol 43 no 1 pp 87ndash922013

[8] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[9] J Ferlay I Soerjomataram R Dikshit et al ldquoCancer incidenceand mortality worldwide sources methods and major patternsin GLOBOCAN 2012rdquo International Journal of Cancer vol 136no 5 pp E359ndashE386 2015

[10] G M Cragg and D J Newman ldquoPlants as a source of anti-cancer agentsrdquo Journal of Ethnopharmacology vol 100 no 1-2pp 72ndash79 2005

[11] Y Li D Kong A Ahmad B Bao and F H Sarkar ldquoAugmentingthe efficacy of chemo- and radio-therapy by nutraceuticalsEvidence from pre-clinical and clinical trialsrdquo Nutraceuticalsand Cancer vol 9789400726307 pp 355ndash376 2012

[12] A Ziakova and E Brandsteterova ldquoValidation of HPLC deter-mination of phenolic acids present in some Lamiaceae familyplantsrdquo Journal of Liquid Chromatography amp Related Technolo-gies vol 26 no 3 pp 443ndash453 2003

[13] M M Lesjak I N Beara D Z Orcic et al ldquoJuniperussibirica Burgsdorf as a novel source of antioxidant and anti-inflammatory agentsrdquo Food Chemistry vol 124 no 3 pp 850ndash856 2011

[14] V Samoylenko M M Rahman B L Tekwani et al ldquoBan-isteriopsis caapi a unique combination of MAO inhibitoryand antioxidative constituents for the activities relevant toneurodegenerative disorders and Parkinsonrsquos diseaserdquo Journalof Ethnopharmacology vol 127 no 2 pp 357ndash367 2010

[15] A Munrsquoim A Andriani K F Mahmudah and M MashitaldquoScreening of a-glucosidase inhibitory activity of some Indone-sian medicinal plantsrdquo in Proceedings of the Screening of 120572-glucosidase inhibitory activity of some Indonesian medicinalplants vol 3 pp 144ndash150 2013

[16] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Lemon Balm Kombuchardquo Food and BioprocessTechnology vol 5 no 5 pp 1756ndash1765 2012

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 9

0

20

40

60

80

100

120

0 600 300 100 1 01Concentration (gmL)

Hs-294 T

HH + BLM

ce

ll gr

owth

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HT-29

HH + BLM

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

ce

ll gr

owth

HeLa

Concentration (gmL)

0

20

40

60

80

100

120

0 600 300 100 1 01

MRC-5

Concentration (gmL)

ce

ll gr

owth

Figure 3 Cytotoxicity of HP and combination of HP and BLM (100 120583gmL) during 48 h treatment The results are expressed as mean plusmn SDperformed in octuplicate

potential new agents in combination with radiation therapyof malignant gliomas [39] Furthermore significant level ofselectivity toward cervix carcinoma and melanoma cell linesagainst healthy cells was achieved described by NTT [40]Altogether it is obvious that the cellular events leading to theincrement of cytostatic effect as a result of cotreatment by HPand BLM are complex and should be investigated further

35 Genotoxicity Genotoxicity assays enable the assessmentof mutagenic and cancerogenic potential of different extractsof medicinal plants and their protective effects as well asmechanisms of their biological activities These assays areused to identify the extracts with the ability to react withnucleic acids in subcytotoxic concentrations If the substancedamages the genome it is manifested by the incrementof incidence of micronuclei Genotoxicity of the HP wastested by micronucleus assay on lymphocytes of peripheralblood during the 24 h treatment The results of micronucleiincidence are shown in Figure 4 During the treatmentHP induced decreasing of micronuclei incidence which was

lower compared to the control value Statistically significantdecrease of micronuclei (MN 355 versus 980 119901 = 0008)was observed at concentration of 200 120583gmL Ramos et al[41] reported thatH perforatum exhibits antigenotoxic effectson HT-29 cells which corresponds to the result of our studyNamely it was stated that the extracts of H perforatum haveprotective effect against oxidative DNA damage followed byincreasing of the repair of alkylating DNA damage by baseexcision repair pathway These protective properties wereascribed to the high amount of phenolic compounds Inanother in vitro study of St Johnrsquos wort extracts rich inflavonoids exhibited strong antioxidant activity and radicalscavenging characteristics [42] Considering that the currentstudy confirms notable antioxidant potential of HP decreas-ing of the micronuclei incidence is the expected result

4 Conclusions

The results of the conducted study concerning the chemicalprofiling ofH perforatum subsp perforatumduring floweringand fruitification suggest that the maximum accumulation of

10 Evidence-Based Complementary and Alternative Medicine

98

355

899 926

000

200

400

600

800

1000

1200

0 200 100 50Concentration (gmL)

Num

ber o

f MN

1000

lowast

Figure 4 Incidence of micronuclei by HP during 24 h treatmentMN micronuclei BN binucleated cells The results are shown asmean plusmn SD performed in triplicate Concentrations were expressedin 120583gmL lowastStatistically significantly different from the control(Fisher LSD test 119901 lt 005)

hypericin and hyperforin is present in the transition periodbetween floral budding and full flowering stage Hypericiherba collected at aforementioned time interval will alsocontain significant amounts of naringenin rutin quercetinamentoflavone chlorogenic caffeic and p-hydroxybenzoicacid The evaluation of biological potential of the preparedwater-alcoholic extract from the plant material collectedat the stage of full flowering suggests notable antioxidantpotential comparable to synthetic antioxidants Also thepotential of the extract to inhibit biologically importantenzymes cannot be neglected especially in the case of 120572-glucosidase which opens a place for further evaluation of StJohnrsquos wort as antihyperglycemic agent The combination ofHP at concentrations higher than 1 120583gmL and bleomycin(100 120583gmL) leads to significant increment of antiproliferativeeffects on cancer cell lines while the examined extract doesnot show signs of genotoxicity at subcytotoxic concentra-tions (lt200120583gmL)However during evaluation of biologicalpotential of different plant extracts the fact that the evaluatedsubject (in this case the Strsquo Johnrsquos wort extract) is a mixture ofcompounds with different characteristics exhibiting variousbiological effects must be always taken into the account

Conflicts of Interest

All of the authors declare no conflicts of interest

Acknowledgments

The Ministry of Science and Technological DevelopmentRepublic of Serbia (Grant no OI 172058) supported thisresearch work

References

[1] NK B Robson ldquoStudies in the genusHypericumLGuttiferaerdquoBulletin of the British Museum National History vol 8 pp 55ndash226 1981

[2] N Kladar B Srđenovic N Grujic et al ldquoSt JohnrsquoS Wort(Hypericum Spp) - Relation between the biological sourceand medical propertiesrdquoHypericum Botanical Sources MedicalProperties and Health Effects pp 53ndash80 2015

[3] B Bozin N Kladar N Grujic et al ldquoImpact of origin andbiological source on chemical composition anticholinesteraseand antioxidant properties of some St Johnrsquos wort species(Hypericum spp hypericaceae) from the central BalkansrdquoMolecules vol 18 no 10 pp 11733ndash11750 2013

[4] A Brattstrom ldquoLong-term effects of St Johnrsquos wort (Hypericumperforatum) treatment A 1-year safety study in mild to moder-ate depressionrdquo Phytomedicine vol 16 no 4 pp 277ndash283 2009

[5] N Kladar B Srđenovic N Grujic et al ldquoEcologically andontogenetically induced variations in phenolic compounds andbiological activities of Hypericum maculatum subsp macula-tum Hypericaceaerdquo Revista Brasileira de Botanica vol 38 no4 pp 703ndash715 2015

[6] R Filippini A Piovan A Borsarini and R Caniato ldquoStudyof dynamic accumulation of secondary metabolites in threesubspecies of Hypericum perforatumrdquo Fitoterapia vol 81 no2 pp 115ndash119 2010

[7] M L Altun B S Yilmaz I E Orhan and G S CitogluldquoAssessment of cholinesterase and tyrosinase inhibitory andantioxidant effects of Hypericum perforatum L (St Johnrsquoswort)rdquo Industrial Crops and Products vol 43 no 1 pp 87ndash922013

[8] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[9] J Ferlay I Soerjomataram R Dikshit et al ldquoCancer incidenceand mortality worldwide sources methods and major patternsin GLOBOCAN 2012rdquo International Journal of Cancer vol 136no 5 pp E359ndashE386 2015

[10] G M Cragg and D J Newman ldquoPlants as a source of anti-cancer agentsrdquo Journal of Ethnopharmacology vol 100 no 1-2pp 72ndash79 2005

[11] Y Li D Kong A Ahmad B Bao and F H Sarkar ldquoAugmentingthe efficacy of chemo- and radio-therapy by nutraceuticalsEvidence from pre-clinical and clinical trialsrdquo Nutraceuticalsand Cancer vol 9789400726307 pp 355ndash376 2012

[12] A Ziakova and E Brandsteterova ldquoValidation of HPLC deter-mination of phenolic acids present in some Lamiaceae familyplantsrdquo Journal of Liquid Chromatography amp Related Technolo-gies vol 26 no 3 pp 443ndash453 2003

[13] M M Lesjak I N Beara D Z Orcic et al ldquoJuniperussibirica Burgsdorf as a novel source of antioxidant and anti-inflammatory agentsrdquo Food Chemistry vol 124 no 3 pp 850ndash856 2011

[14] V Samoylenko M M Rahman B L Tekwani et al ldquoBan-isteriopsis caapi a unique combination of MAO inhibitoryand antioxidative constituents for the activities relevant toneurodegenerative disorders and Parkinsonrsquos diseaserdquo Journalof Ethnopharmacology vol 127 no 2 pp 357ndash367 2010

[15] A Munrsquoim A Andriani K F Mahmudah and M MashitaldquoScreening of a-glucosidase inhibitory activity of some Indone-sian medicinal plantsrdquo in Proceedings of the Screening of 120572-glucosidase inhibitory activity of some Indonesian medicinalplants vol 3 pp 144ndash150 2013

[16] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Lemon Balm Kombuchardquo Food and BioprocessTechnology vol 5 no 5 pp 1756ndash1765 2012

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Evidence-Based Complementary and Alternative Medicine

98

355

899 926

000

200

400

600

800

1000

1200

0 200 100 50Concentration (gmL)

Num

ber o

f MN

1000

lowast

Figure 4 Incidence of micronuclei by HP during 24 h treatmentMN micronuclei BN binucleated cells The results are shown asmean plusmn SD performed in triplicate Concentrations were expressedin 120583gmL lowastStatistically significantly different from the control(Fisher LSD test 119901 lt 005)

hypericin and hyperforin is present in the transition periodbetween floral budding and full flowering stage Hypericiherba collected at aforementioned time interval will alsocontain significant amounts of naringenin rutin quercetinamentoflavone chlorogenic caffeic and p-hydroxybenzoicacid The evaluation of biological potential of the preparedwater-alcoholic extract from the plant material collectedat the stage of full flowering suggests notable antioxidantpotential comparable to synthetic antioxidants Also thepotential of the extract to inhibit biologically importantenzymes cannot be neglected especially in the case of 120572-glucosidase which opens a place for further evaluation of StJohnrsquos wort as antihyperglycemic agent The combination ofHP at concentrations higher than 1 120583gmL and bleomycin(100 120583gmL) leads to significant increment of antiproliferativeeffects on cancer cell lines while the examined extract doesnot show signs of genotoxicity at subcytotoxic concentra-tions (lt200120583gmL)However during evaluation of biologicalpotential of different plant extracts the fact that the evaluatedsubject (in this case the Strsquo Johnrsquos wort extract) is a mixture ofcompounds with different characteristics exhibiting variousbiological effects must be always taken into the account

Conflicts of Interest

All of the authors declare no conflicts of interest

Acknowledgments

The Ministry of Science and Technological DevelopmentRepublic of Serbia (Grant no OI 172058) supported thisresearch work

References

[1] NK B Robson ldquoStudies in the genusHypericumLGuttiferaerdquoBulletin of the British Museum National History vol 8 pp 55ndash226 1981

[2] N Kladar B Srđenovic N Grujic et al ldquoSt JohnrsquoS Wort(Hypericum Spp) - Relation between the biological sourceand medical propertiesrdquoHypericum Botanical Sources MedicalProperties and Health Effects pp 53ndash80 2015

[3] B Bozin N Kladar N Grujic et al ldquoImpact of origin andbiological source on chemical composition anticholinesteraseand antioxidant properties of some St Johnrsquos wort species(Hypericum spp hypericaceae) from the central BalkansrdquoMolecules vol 18 no 10 pp 11733ndash11750 2013

[4] A Brattstrom ldquoLong-term effects of St Johnrsquos wort (Hypericumperforatum) treatment A 1-year safety study in mild to moder-ate depressionrdquo Phytomedicine vol 16 no 4 pp 277ndash283 2009

[5] N Kladar B Srđenovic N Grujic et al ldquoEcologically andontogenetically induced variations in phenolic compounds andbiological activities of Hypericum maculatum subsp macula-tum Hypericaceaerdquo Revista Brasileira de Botanica vol 38 no4 pp 703ndash715 2015

[6] R Filippini A Piovan A Borsarini and R Caniato ldquoStudyof dynamic accumulation of secondary metabolites in threesubspecies of Hypericum perforatumrdquo Fitoterapia vol 81 no2 pp 115ndash119 2010

[7] M L Altun B S Yilmaz I E Orhan and G S CitogluldquoAssessment of cholinesterase and tyrosinase inhibitory andantioxidant effects of Hypericum perforatum L (St Johnrsquoswort)rdquo Industrial Crops and Products vol 43 no 1 pp 87ndash922013

[8] S Wild G Roglic A Green R Sicree and H King ldquoGlobalprevalence of diabetes estimates for the year 2000 and projec-tions for 2030rdquoDiabetes Care vol 27 no 5 pp 1047ndash1053 2004

[9] J Ferlay I Soerjomataram R Dikshit et al ldquoCancer incidenceand mortality worldwide sources methods and major patternsin GLOBOCAN 2012rdquo International Journal of Cancer vol 136no 5 pp E359ndashE386 2015

[10] G M Cragg and D J Newman ldquoPlants as a source of anti-cancer agentsrdquo Journal of Ethnopharmacology vol 100 no 1-2pp 72ndash79 2005

[11] Y Li D Kong A Ahmad B Bao and F H Sarkar ldquoAugmentingthe efficacy of chemo- and radio-therapy by nutraceuticalsEvidence from pre-clinical and clinical trialsrdquo Nutraceuticalsand Cancer vol 9789400726307 pp 355ndash376 2012

[12] A Ziakova and E Brandsteterova ldquoValidation of HPLC deter-mination of phenolic acids present in some Lamiaceae familyplantsrdquo Journal of Liquid Chromatography amp Related Technolo-gies vol 26 no 3 pp 443ndash453 2003

[13] M M Lesjak I N Beara D Z Orcic et al ldquoJuniperussibirica Burgsdorf as a novel source of antioxidant and anti-inflammatory agentsrdquo Food Chemistry vol 124 no 3 pp 850ndash856 2011

[14] V Samoylenko M M Rahman B L Tekwani et al ldquoBan-isteriopsis caapi a unique combination of MAO inhibitoryand antioxidative constituents for the activities relevant toneurodegenerative disorders and Parkinsonrsquos diseaserdquo Journalof Ethnopharmacology vol 127 no 2 pp 357ndash367 2010

[15] A Munrsquoim A Andriani K F Mahmudah and M MashitaldquoScreening of a-glucosidase inhibitory activity of some Indone-sian medicinal plantsrdquo in Proceedings of the Screening of 120572-glucosidase inhibitory activity of some Indonesian medicinalplants vol 3 pp 144ndash150 2013

[16] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Lemon Balm Kombuchardquo Food and BioprocessTechnology vol 5 no 5 pp 1756ndash1765 2012

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Evidence-Based Complementary and Alternative Medicine 11

[17] J Mrđanovic S Solajic V Bogdanovic K Stankov G Bog-danovic and A Djordjevic ldquoEffects of fullerenol nano particlesC60 (OH) 24 on micronuclei and chromosomal aberrationsrsquofrequency in peripheral blood lymphocytesrdquo Digest Journal ofNanomaterials and Biostructures vol 7 pp 673-386 2012

[18] M Fenech ldquoCytokinesis-block micronucleus cytome assayrdquoNature Protocols vol 2 no 5 pp 1084ndash1104 2007

[19] L Muller and H J Martus ldquoGenetic toxicology testing guide-lines and regulationsrdquo in Cancer risk assessment chemicalcarcinogenesis hazard evaluation and risk quantification JohnWiley amp Sons New Jersey NJ USA 2010

[20] E M Gioti Y C Fiamegos D C Skalkos and C D StalikasldquoAntioxidant activity and bioactive components of the aerialparts of Hypericum perforatum L from Epirus Greecerdquo FoodChemistry vol 117 no 3 pp 398ndash404 2009

[21] C Cirak J Radusiene B (Saglam) Karabuk and V JanulisldquoVariation of bioactive substances and morphological traits inHypericum perforatum populations from Northern TurkeyrdquoBiochemical Systematics and Ecology vol 35 no 7 pp 403ndash4092007

[22] B A Silva J O Malva and A C P Dias ldquoSt Johnrsquos Wort(Hypericum perforatum) extracts and isolated phenolic com-pounds are effective antioxidants in several in vitro models ofoxidative stressrdquo Food Chemistry vol 110 no 3 pp 611ndash6192008

[23] D Tekelova M Repcak E Zemkova and J Toth ldquoQuantitativechanges of dianthrones hyperforin and flavonoids contentin the flower ontogenesis of Hypericum perforatumrdquo PlantaMedica vol 66 no 8 pp 778ndash780 2000

[24] O Kacar and N Azkan ldquoEffects of different climate factorsontogenetic and diurnal variability on the hypericin contentof Hypericum perforatum Lrdquo Journal of Agricultural Faculty ofUludag University vol 42 no 2 p 23 2005

[25] C Cirak J Radusiene B Karabuk V Janulis and L IvanauskasldquoVariation of bioactive compounds in Hypericum perforatumgrowing in Turkey during its phenological cyclerdquo Journal ofIntegrative Plant Biology vol 49 no 5 pp 615ndash620 2007

[26] C Cirak B Saglam A K Ayan and K Kevseroglu ldquoMorpho-genetic and diurnal variation of hypericin in some Hypericumspecies from Turkey during the course of ontogenesisrdquo Bio-chemical Systematics and Ecology vol 34 no 1 pp 1ndash13 2006

[27] C Y W Ang L Hu T M Heinze et al ldquoInstability ofSt Johnrsquos wort (Hypericum perforatum L) and degradationof hyperforin in aqueous solutions and functional beveragerdquoJournal of Agricultural and Food Chemistry vol 52 no 20 pp6156ndash6164 2004

[28] C Cirak J Radusiene V Janulis and L Ivanauskas ldquoPseu-dohypericin and hyperforin in Hypericum perforatum fromNorthern Turkey Variation among populations plant parts andphenological stagesrdquo Journal of Integrative Plant Biology vol 50no 5 pp 575ndash580 2008

[29] E Bagdonaite P Martonfi M Repcak and J Labokas ldquoVari-ation in concentrations of major bioactive compounds inHypericumperforatumL fromLithuaniardquo Industrial Crops andProducts vol 35 no 1 pp 302ndash308 2012

[30] P S Corredor-Sanchez Natural Antioxidants Their electro-chemistry and determination in plant material and biologicalfluids Purdue University 2007

[31] D Cenic-Milosevic Z Tambur S Ivancajic et al ldquoAntiprolif-erative effects of Tanaceti partheni Hypericum perforatum andpropolis on HeLa cellsrdquo Archives of Biological Sciences vol 66no 2 pp 705ndash712 2014

[32] CM Schempp V Kirkin B Simon-Haarhaus et al ldquoInhibitionof tumour cell growth by hyperforin a novel anticancer drugfrom St Johnrsquos wort that acts by induction of apoptosisrdquoOncogene vol 21 no 8 pp 1242ndash1250 2002

[33] M Dona I DellrsquoAica E Pezzato et al ldquoHyperforin inhibitscancer invasion and metastasisrdquo Cancer Research vol 64 no17 pp 6225ndash6232 2004

[34] M Weller M Trepel C Grimmel et al ldquoHypericin-inducedapoptosis of human malignant glioma cells is light- dependentindependent of bcl-2 expression and does not requirewild-typep53rdquo Neurological Research vol 19 no 5 pp 459ndash470 1997

[35] C Billard F Merhi and B Bauvois ldquoMechanistic insights intothe antileukemic activity of hyperforinrdquo Current Cancer DrugTargets vol 13 no 1 pp 1ndash10 2013

[36] K Hostanska J Reichling S Bommer M Weber and RSaller ldquoHyperforin a constituent of St Johnrsquos wort (Hypericumperforatum L) extract induces apoptosis by triggering acti-vation of caspases and with hypericin synergistically exertscytotoxicity towards human malignant cell linesrdquo EuropeanJournal of Pharmaceutics and Biopharmaceutics vol 56 no 1pp 121ndash132 2003

[37] L F Povirk ldquoProcessing of Damaged DNA Ends for Double-Strand Break Repair in Mammalian Cellsrdquo ISRN MolecularBiology vol 2012 pp 1ndash16 2012

[38] E Alley R Green and L Schuchter ldquoCutaneous toxicities ofcancer therapyrdquo Current Opinion in Oncology vol 14 no 2 pp212ndash216 2002

[39] W Zhang L Anker R E Law et al ldquoEnhancement ofradiosensitivity in human malignant glioma cells by hypericinin vitrordquo Clinical Cancer Research vol 2 no 5 pp 843ndash8461996

[40] D D Cetojevic-Simin A S Velicanski D D Cvetkovic et alldquoBioactivity of Meeker andWillamette raspberry (Rubus idaeusL) pomace extractsrdquo Food Chemistry vol 166 pp 407ndash4132015

[41] A A Ramos F Marques M Fernandes-Ferreira and CPereira-Wilson ldquoWater extracts of tree Hypericum sps protectDNA from oxidative and alkylating damage and enhance DNArepair in colon cellsrdquo Food and Chemical Toxicology vol 51 no1 pp 80ndash86 2013

[42] Y Zou Y Lu and D Wei ldquoAntioxidant activity of a flavonoid-rich extract of Hypericum perforatum L in vitrordquo Journal ofAgricultural and Food Chemistry vol 52 no 16 pp 5032ndash50392004

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom