Phenolic profiles and antioxidant and anticarcinogenic activities of prevention

Phenolic profiles and antioxidant and anticarcinogenic activities of Greek herbal infusions;balancing delight andchemoprevention?Andriana C.Kaliora a ,Dimitra A.A.Kogiannou a ,Panagiotis Kefalas b ,Issidora S.Papassideri c ,Nick Kalogeropoulos a ,⇑aDepartment of Science of Dietetics-Nutrition,Harokopio University,Athens,Greeceb Department of Food Quality and Chemistry of Natural Products,Mediterranean Agronomic Institute of Chania/Centre International de Hautes Etudes Agronomiques Méditerranéennes,Chania,Crete,Greece cDepartment of Cell Biology and Biophysics,Faculty of Biology,University of Athens,Athens,Greecea r t i c l e i n f o Article history:Received 22March 2013Received in revised form 21May 2013Accepted 11July 2013Available online 19July 2013Keywords:Herbal infusions LC-DAD-MS GC-MSPolyphenols Terpenic acids Colon cancer Prostate cancer Cell growth arrest Inflammation IL-8p65Subunita b s t r a c tTotal phenolic content,antioxidant activity and phenolic profiles of six herbal infusions –namely rose-mary,Cretan dittany,St.John’s Wort,sage,marjoram and thyme were assayed.Additionally,the infusion anticarcinogenic effect as to their ability to (a)scavenge free radicals,(b)inhibit cell growth,(c)decrease IL-8levels and (d)regulate p65subunit in epithelial colon cancer (HT29)and prostate (PC3)cancer cells was investigated.LC-DAD-MS and GC–MS analyses showed major qualitative and quantitative differ-ences in phenolic profiles of the infusions.All herbal infusions exhibited antiradical activity which corre-lated strongly with their total phenolic content.Infusions exhibited the potential to inhibit cell growth and to reduce IL-8levels in HT29colon and PC3prostate cancer cells.The regulation reported in p65sub-unit in HT29treated with St John’s Wort and in PC3treated with thyme might point to the NF-j B as the molecular target underlying the effect of these infusions.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionThe use of aromatic plants and herbs in medicines is since hu-mans inhabited earth.It was ‘‘since ever’’when humans were try-ing to find out which plants might be beneficial to human health,or even fight several pains and aches,or wounds.Herbs are the old-est drugs in the world.The NCI (National Cancer Institute)has examined more than 30,000plants with anticancer activity (Ipek et al.,2005).However,the use of plants as a means for treatment is still very limited.The main aromatic plants belong to the fami-lies Labiatae (or Lamiaceae ),Umbelliferae ,Lauracae ,Myrtacae and Compositae .The flora of Mediterranean countries presents highbiodiversity,being extremely rich in native aromatic plants,many of which are nowadays cultivated systematically.Carcinogenesis is generally recognised as a complex and multistep process in which inflammation plays a crucial role,and distinct molecular and cellu-lar alterations occur (Franco,Schoneveld,Georgakilas,&Panayiot-idis,2008;Kryston,Georgiev,Pissis,&Georgakilas,2011).The contribution of diet in all cancer-related death estimates is 30–35%in the environmental factors,being higher than tobacco,which is 25–30%.Colorectal cancer is strongly associated with diet,and is linked to 70%of cancer related-deaths,while prostate cancer has been associated with diet too (Georgakilas,2012).In the term of chemoprevention,many phenolic compounds,abundant in the Mediterranean diet,have been proven effective (Ramos,2008)act-ing on intracellular signaling network molecules involved (a)in the initiation,as ‘‘blocking agents’’,meaning they scavenge ROS or potentiate the antioxidant enzyme system or (b)in promotion and progression,as ‘‘suppressing agents’’,deregulating prolifera-tion,inducing cell-cycle arrest or apoptosis,as well as inhibiting inflammation,invasion or angiogenesis.Notably,phenolic com-pounds have been found to penetrate tissues such as prostate0308-8146/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.foodchem.2013.07.056Abbreviations:DPPH,2-diphenyl-1-picryhydrazyl;MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;IL-8,interleukin-8;NF-j B,nuclear factor-kappa B;DMEM,Dulbecco’s modified Eagle’s medium.⇑Corresponding author.Tel.:+302109549251;fax:+302109577050.E-mail addresses:akaliora@hua.gr (A.C.Kaliora),dimkog@hua.gr (D.A.A.Kogiannou),panos@maich.gr (P.Kefalas),ipapasid@biol.uoa.gr (I.S.Papassideri),nickal@hua.gr (N.Kalogeropoulos).(Henning et al.,2006)or colorectal tissues(Garcea et al.,2005). Rosemary and thyme(Ngo,Williams,&Head,2011;Sasaki et al., 2005)as well as individual constituents such as thymol and carva-crol(Sasaki et al.,2005)or carnosol(Arunasree,2010)have been proven to inhibit carcinogenesis.Except for the bioavailability con-cern when conducting in vitro experiments,an obvious additional weakness of the current state of research is that most of it has been conducted using extracts obtained from the dried aerial parts of plants by solvent extraction.Hence,aiming to a more realistic ap-proach from the consumers’point of view,this survey was de-signed to determine composition in six Greek aqueous herbal infusions by LC-DAD-MS and GC–MS.Further,we examined the infusion bioactivity in cell growth and in inflammation of HT29 and PC3cancer cell lines.2.Materials and methods2.1.Plant materialsWe studied six species of Greek herbs;Rosemary(Rosmarinus officinalis,Lamiaceae),Cretan Dittany(Origanum dictamnus,Lami-aceae),Thyme(Thymus vulgaris,Lamiaceae),Marjoram(Origanum majorana,Lamiaceae),Sage(Salvia officinalis,Lamiaceae)and St John’s Wort(Hypericum perforatum,Clusiaceae).Dried herbs were kindly donated from a specialized store in Nyvritos,island of Crete, except St John’s Wort,which was purchased from the women’s cooperative of Ano Poroia,in Central Macedonia,Northern Greece. All samples were provided wrapped in paper-cellophan bags and were stored in a cool place in dark.Only the leaves andflowers of the herbs were used for the preparation of herbal infusions. 2.2.Chemicals,standards and cell linesFolin–Ciocalteu reagent,DPPH(2,2-diphenyl-1-picryhydrazyl) stable radical,Trolox(6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid),phosphate buffer saline(PBS)tablets and3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) were obtained from Sigma–Aldrich;cinnamic acid,phloretic acid and oleanolic acid,were obtained from Aldrich;bis-(trimethyl-silyl)-trifluoroacetamide(BSTFA),p-(dimethylamino)-cinnamalde-hyde(DMACA),rutin,catechin,gallic acid,p-hydroxy-benzoic acid,p-hydroxy-phenylacetic acid,p-coumaric acid,o-coumaric acid,syringic acid,ferulic acid,chlorogenic acid,quercetin and ursolic acid were obtained from Sigma;protocatechuic acid,sina-pic acid,3,4-dihydroxyphenylacetic acid and caffeic acid were pur-chased from Fluka;vanillic acid from Serva;chrysin and genistein from Alfa-Aesar;epicatechin from Biochemika;rosmarinic acid, naringenin and kaempferol from Extrasynthése;thymol from Rie-del-de Haen.All the solvents used were of analytical grade and were obtained from Aldrich(Germany).Human HT29epithelial co-lon cancer and PC3prostate(hormone independent)cancer cell lines were obtained by American Tissue Culture Collection.Dul-becco’s Modified Eagle’s Medium(DMEM)was purchased from Biosera(UK).foetal bovine serum(FBS),penicillin/streptomycin and trypsin/EDTA were supplied by PAA Company(UK).Recombi-nant tumor necrosis factor-a(TNF-a)and quantikine sandwich ELISA kit for the measurement of interleukin-8(IL-8)levels were from R&D Systems(UK).PathScan sandwich ELISA kit for the measurement of p65particle was from Cell Signaling Systems (USA).2.3.Preparation of the herbal infusionsPreparation of herbal infusions and dehydration process were alike in all herbs.First,3g of individual herb were infused into 250ml of boiling water(equivalent to a teacup)on a preheated hot plate for a total of3min.Infusions were left to room temper-ature to cool for5min and werefiltered throughfilter paper.Next, infusions were dehydrated by lyophilization(Heto Lyolab3000, Heto-Holten,Allerod,Denmark)and the residues were weighed and stored atÀ40°C for further experiments.2.4.Phenolic profile determination by LC-DAD-MSChromatographic separation and identification of phenolic compounds in the extracts was performed on an LC-DAD-MS (ESI+)setup consisting of a Finnigan MAT Spectra System P4000 pump,coupled with a UV6000LP diode array detector and a Finn-igan AQA mass spectrometer.The separation was performed on a 125Â2mm,4l m,Superspher100–4(RP-18)column(Mache-rey–Nagel)kept at40°C,at aflow rate of0.30ml/min.For the gra-dient elution,the following program was used:(A)H2O(containing 2.5%AcOH);(B)MeOH;isocratic at100%A for2min,then0%A in 50min,followed by10min isocratic wash at0%A.The data were processed with the Xcalibur1.2software.The analysis was moni-tored at278and340nm and by ESI in the positive mode at a probe temperature of400°C,probe voltage of4.0kV and at20and70eV in the mass analyzer.For individual herbal infusions,three differ-ent preparations were run in duplicate.2.5.Simple polyphenol and terpenic acid extraction and determination by GC/MSSimple polyphenols and triterpenic acids were determined in freeze-dried infusions by GC–MS,after derivatization to trimethyl-silyl ethers with250l l BSTFA at70°C for20min(Soleas,Diaman-dis,Karumanchiri,&Goldberg,1997).A selective ion monitoring (SIM)GC/MS method was applied for the detection of27polyphe-nols,two terpenoid phenols and two terpenic acids,based on the ±0.05RT presence of target and qualifier ions of commercial stan-dards at the predetermined ratios(Kalogeropoulos,Konteles,Tro-ullidou,Mourtzinos,&Karathanos,2009).Quantification was carried out by employing3-(4-hydroxyphenyl)-1-propanol as internal standard and constructing reference curves for every pol-yphenol and terpenic acid by means of standard solutions.The tar-get and qualifier ions employed are given in Supplementary material A.For individual herbal infusions,3different preparations were run in duplicate.2.6.Determination of total polyphenols in herbal infusionsTotal polyphenol content was assayed in aqueous solutions of the freeze-dried infusions by the Folin–Ciocalteu reaction,using gallic acid as the reference standard.In brief,10l l of sample (4mg/ml for all samples,except for dittany of which working solu-tion was2mg/ml)was transferred into a1.5ml Eppendorf tube containing790l l deionized water and then50l l Folin–Ciocalteu reagent was added and mixed well.After one minute,150l l of sat-urated Na2CO3solution was added,mixed well and kept in dark for 120min,at room temperature.Finally,the absorption of the reac-tion product was measured at725nm using an Analytik Jena-SPE-CORD200spectrophotometer,and the results were expressed as mg GAE(gallic acid equivalents)per cup(Arnous,Makris,&Kef-alas,2002).Three individual preparations of each infusion were subjected to Folin–Ciocalteu reaction.2.7.Determination of totalflavanols in herbal infusionsTotalflavanols content was estimated by using the p-dimeth-ylaminocinnamaldehyde(DMACA)assay,as described by Arnous et al.(2002).Briefly,0.2ml of sample(4mg/ml for all samples,234 A.C.Kaliora et al./Food Chemistry142(2014)233–241except for dittany of which working solution was2mg/ml)was introduced in a 1.5ml Eppendorf tube,and added500l l HCl 0.24M and500l l DMACA solution0.2%w/v.The mixture was vor-texed and allowed to react for10min at room temperature.The absorbance at640nm was then read against a blank prepared sim-ilarly without DMACA.The concentration of totalflavanols was estimated from a calibration curve,constructed by rutin instead of catechin,and the results were expressed as mg rutin equivalents per cup.Three individual preparations of each infusion were sub-jected to DMACA assay.2.8.Antiradical activityThe antioxidant activity was evaluated in terms of radical scav-enging activity applying the DPPH free radical assay.Individual aqueous samples(2mg/ml,25l l)were mixed with975l l of 0.1mM DPPHÅin a1.5ml Eppendorf tube.Absorbance of the reac-tion mixture was measured in t=0and t=30min(kept in dark)at 515nm using an Analytik Jena-SPECORD200spectrophotometer.A calibration curve of Trolox(analogue of vitamin E)was prepared, and the results were expressed as mg Trolox equivalents/cup (Arnous et al.,2002).Three individual preparations of each infusion were subjected to DPPH radical scavenging assay.1.9.Estimation of reducing powerThe reducing power of dried infusions was determined by the ferric reducing antioxidant potential(FRAP)assay as described by Arnous et al.(2002).Briefly,50l l of each sample(2mg/ml aque-ous solutions of dry infusion diluted2–4times)was mixed with 50l l FeCl3solution(3mM in5mM HCl)in Eppendorf tubes,vor-texed,incubated at37°C for30min,and then900l l TPTZ solution (1mM in0.05M HCl)was added.The absorbance of the product of the reaction between Fe2+and TPTZ was measured at620nm against a blank for each sample,containing distilled water instead of FeCl3.Ascorbic acid was used as the calibration standard and the results were expressed as mg ascorbic acid equivalents(mg AAE)/ cup.Three individual preparations of each infusion were subjected to FRAP assay.2.10.HT29and PC3culture conditionsMonolayers of cells were grown in T75flasks and maintained in DMEM supplemented with10%(v/v)foetal bovine serum(FBS),1% antibiotic solution and incubated at37°C in a humidified atmosphere of5%CO2in air.The cells were dispersed with tryp-sin–EDTA(0.05%trypsin and0.02%EDTA)and plated in eitherflat bottom96-well plates for cell proliferation assay or inflat bottom 12-well plates for TNF-a stimulation.2.11.Cell growth arrestCells were counted in exponential growth phase applying the MTT assay.Briefly,in aflat bottom96well plate7Â103HT29 cells/well or5Â103PC3cells/well were placed to afinal volume of0.2ml and were incubated for24h until adherence.Next,the medium was changed and dried residues of individual lyophilized infusions were diluted in DMEM,filter sterilized and added in cul-ture in different concentrationsfinal in the well(1l g/l L,0.6l g/l L and0.2l g powder/l L of medium).Cells were measured using MTT after24,48and72h in culture.For this purpose,20l l MTT(5mg/ ml dissolved in PBS)was added to cell culture and subsequently cells were incubated for4h at37°C.At the end of incubation, MTT was discarded and replaced by DMSO.Optical density was measured on a PowerWave XS2,Microplate Spectrophotometer, BioTek,at550nm.As a background value,a well containing only culture medium plus MTT plus DMSO was used.Each experiment was carried out in triplicate.The blanks gave values close to0.1 (±0.02).The number of cells used in the assay was within the linear portion of the plot and yielded an absorbance of0.35–0.80Abs.The MTT assay for cell growth arrest was conducted in at leastfive indi-vidual cultures treated with each one of the infusions in the above mentioned concentrations.2.12.TNF-a stimulationCells were placed to polystyrene12-well plates(105HT29or PC3cells/well)with culture medium.Cells were incubated for 24h to allow adhering and subsequently,after medium refresh-ment,filter sterilized solutions of individual lyophilized infusions were added in culture in DMEM.Next,the proliferating cells were treated with individual herbal infusions(concentrationfinal in the well0.2l g powder/l L of medium)for12h.After treatment,super-natants were discarded and cultured cells were stimulated with TNF-a(50ng/ml).The supernatants were collected for measure-ment of IL-8and monolayers were collected for measurement of p65subunit.Cultures for TNF-a stimulation and inhibition by infu-sions were run in three replicates.2.13.Quantification of IL-8proteinLevels of IL-8protein were assessed in three independent cell cultures(see Section2.12)by quantitative,sandwich ELISA accord-ing to the manufacturer’s instructions.Sensitivity of IL-8ELISA was less than3.5pg/ml.2.14.Total NF-j B p65To measure p65subunit,we applied a sandwich ELISA Kit according to the manufacturer’s instructions in cell lysates in three independent cell cultures(see Section2.11).2.15.Statistical analysisAll statistical analyses were performed with SPSS Statistics19.0. For all measures,descriptive statistics were calculated.Data are ex-pressed as means±SD.One-way ANOVA using Tukey test was ap-plied after checking for normal distribution.Differences between the means were considered statistically significant at P<0.05.3.Results and discussionIn this study,an effort to determine the phenolic profile and the chemopreventive properties of herbal infusions in HT29and PC3 cell cultures was carried out.In an attempt to interpret results to real dietary habits in the Mediterranean,the infusion preparation method was done according to tradition in Greece(Liolios,Gortzi, Lalas,Tsaknis,&Chinou,2009).The quantities of dried residues yielded after lyophilisation of herbal infusions prepared with a standard method in all herbs are given in Table1.Rosemary yielded the lower quantity,followed by sage,while St John’s Wort yielded the highest quantity.The total phenolic content of the her-bal infusions presented herein showed quantitative variation(Ta-ble1).The phenol concentration was lower in infusions prepared from rosemary,followed by this prepared from sage,thyme,Cretan dittany and marjoram,while the highest was reported for St John’s Wort.Research on the phenolic content of medicinal plants is im-mense and most of it focused to plants of Lamiaceae family due to the large body of biological effects they demonstrate(Fecka&Tur-ek,2008).To compare our results with the total phenolic contents reported currently in literature is rather vain,as in most studiesA.C.Kaliora et al./Food Chemistry142(2014)233–241235organic solvents have been applied to extract polyphenols from dried parts of the plants(Yoo,Lee,Lee,Moon,&Lee,2008).In the study of Atoui,Mansouri,Boskou,and Kefalas(2005),the pro-tocol applied for preparation of herbal infusions was identical to ours;however the reported phenolic content for Cretan dittany infusion was slightly higher(109+3.2vs83.0±1.3mg GAE/cup), probably because Folin–Ciocalteu assay was applied in ethyl ace-tate extracts of the infusions(Atoui et al.2005)while in the pres-ent study no extraction of the freeze–dried infusion was undertaken.Regarding the antioxidant activity of the herbal infu-sions studied,St John’s Wort exhibited the highest antioxidant activity and again rosemary the lower while the activities exhib-ited by marjoram and Cretan dittany were alike(Table1).The cor-relation between the total phenolic content or total phenolic acids orflavonoids and the DPPHÅscavenging activity was found signif-icantly positive(p<0.01).Also,significantly positive correlations (p<0.01)were observed between antiradical activity and p-OH-benzoic acid,protocatechuic acid,gallic acid,chlorogenic acid,epi-Table1Dry matter yield by freeze–drying,total phenolics,totalflavanols,antiradical capacity and reducing power of the herbal infusions studied.Common name Dried infusion(mg/cup a)Total phenolics(mg GAE b/cup)Totalflavanols(mg rutin/cup)Radical scavenging(mg TE d/cup)Reducing power(mg AAE c/cup)Rosemary72.7±6.38.5±0.1 3.2±0.107.7±0.80.52±0.07 Cretan ditanny360.0±11.283.0±1.311.9±0.04141.7±5.617.8±0.72 Thyme441.9±20.458.7±2.418.6±0.1270.5±4.3 6.79±0.15 Marjoram496.2±18.598.4±0.435.5±0.24149.6±2.418.9±1.0 St.John’s Wort624.7±25.7152.8±1.763.2±0.45430.8±8.466.5±1.5 Sage182.7±16.334.5±0416.1±0.2252.8±0.6 4.72±0.41a1cup=200mL.b GAE stands for gallic acid equivalents.c TE stands for Trolox equivalents.d AAE stands for ascorbic acid equivalents.Table2Simple polyphenols and terpenic acids(l g/cup a)in the infusions of the aromatic plants studied.Compounds Rosemary Cretan dittany Thyme Marjoram St John’s Wort SageMean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SDPhenolic acidsi.Hydroxybenzoic acidsVannilic acid14.8 1.211.2 1.655.7 3.749.4 1.559.0 2.410.6 1.8 Protocatechuic acid 2.40.438.9 3.631.7 2.458.5 1.9274.016.522.0 2.9 Syringic acid 5.50.424.6 2.983.0 6.588.4 4.535.6 3.413.0 1.8 Gallic acid––15.0 2.5––25.1 2.8100.67.1––p-Hydroxy-phenylacetic acid––––––16.5 1.3––––3,4-Dihydroxy-phenylacetic acid––73.417.325.60.485.7 3.4––7.00.9ii.Hydroxycinnamic acidsCinnamic acid 2.00.2 5.80.58.9 1.410.5 2.611.80.6 3.00.3 Phloretic acid–––––––––– 2.70.4 o-Coumaric acid––10.10.833.9 3.933.8 2.8––10.2 1.9 p-Coumaric acid13.60.718.6 2.9––79.1 5.477.07.116.5 2.3 Ferulic acid9.6 1.1––37.2 3.780.3 2.547.8 3.617.1 1.7 Caffeic acid61.3 2.667.8 6.8310.412.5630.128.5183.921.1262.870.5 Sinapic acid 5.80.9––26.0 1.655.8 4.3––12.90.3 Chlorogenic acid––37.0 4.327.4 3.9––10.5 3.316.4 2.3 Rosmarinic acid669.278.57244322.57007.31039674.4253.8––8082.799.8FlavonoidsChrysin––28.90.8––68.6 2.263.18.513.0 1.9 Epicatechin 4.80.336.2 1.921.2 1.539.5 5.329275.41240.99.40.3 Naringenin––22.0 1.033.6 3.258.2 4.4––18.7 5.1 Catechin8.1 1.232.4 6.438.2 1.861.57.53448.2394.515.9 1.0 Genistein––31.4 1.829.8 1.6––––21.3 4.2 Kaempferol––––––44.7 6.0165.417.911.5 1.4 Quercetin––15.7 2.9––43.7 5.33134.4298.110.90.8Terpenoid phenolsThymol––––80655.63184.91011.674.1––1859.083.2 Carvacrol362.338.936416.92317.0182138.05651.72976.5125.62245.6202.73745.3290.4Terpenic acidsOleanolic acid17.7 1.3––––32.7 2.8––66.2 5.7 Ursolic acid12.20.7––––48.20.7––40.6 6.0SumsTotal simple polyphenols1159.4126.444129.92697.5270563.58987.715191.9545.739132.32227.714181.9475.2 Phenolic acids784.256.87546.4365.77647.1143.010887.6315.3800.265.18476.9186.9 Flavonoids12.9 1.5166.67.3122.8 5.7316.225.636086.51240.6100.712.5 Terpenoid phenols362.338.936416.92317.0262793.63997.93988.151.52245.6202.75604.3355.6 Terpenic acids29.9 1.5––––80.9 3.5––106.811.6a1cup=200mL.236 A.C.Kaliora et al./Food Chemistry142(2014)233–241Table3LC-DAD-MS characteristics of phenols identified in the herbal infusions studied.I:rosemary;II:Cretan dittany;III:thyme;IV:marjoram;V:St.John’s Wort;VI:sage.No.RT(min)k max(nm)Molecularion(M+H)+(m/z)Fragment ions(m/z)Proposed structure I II III IV V VI1 6.88244,320,290sh*–377(354+Na+),163Unknown derivative of chlorogenicacids s s s d s29.40310–361(M+Na+),147Quinate of p-coumaric acid s s s s d s 310.12264453Unknown s s s s s d 411.66246,320,328sh579317,163Teucrol succinoyl glucoside s s s s d s513.60240,278603373,291Unknown s s s s d s 616.76236,272,336595617(M+Na+),449,287Luteolin,4’-O-rutinoside s s s s s d 716.80272,338595617(M+Na+),337,325,295,273Nisenin-2(6,8-di-c-glucopyranoapigenin)1s d s s s s 816.91270,340595433,271Apigenin diglucoside s s s d s s 919.13250,286,320457457Unknown s s s d s s 1020.36240,292451473(M+Na+),305,1793-O-Rhamnoside of3,4,20,60tetrahydroxy,4-0methoxydihydrochalcones s s s d s1120.79240,348463287Luteolin30-O-glucuronide s s s s s d 1221.26256,354465487(M+Na+),303Coeluting compounds:quercetin3-O-galactoside(hyperoside),quercetin3-O-glucoside(isoquercitrin)s s s s d s1322.14–22.83240,244,288sh,328,330361383(M+Na+),743(MÂ2+Na+),163Rosmarinic acid d d d d s d1422.18248,288sh,330361743(MÂ2+Na+),383(M+Na+),163Isomer of rosmarinic acid s s s s s d1522.25266,328–307,409Unknown d s s s s s 1622.55254,354435457(M+Na+),303Pentoside of3or40-O-quercetin s s s s d s 1722.74–22.83242,290sh,330355163Chlorogenic acid s d d s s s 1823.76254,350449472(M+Na+),303Quercitrin s s s s d s 1924.02244,290sh,318539561(M+Na+),433,341,297Lithospermic acid2s d s s s s 2024.44254,270,334463485(M+Na+),301,286Homoplantagenin1s s s s s d 2124.45240,332–247,307Unknown d s s s s s 2224.59252,264,348609631(M+Na+),463,30130-O-Rutinoside of7-methylatedflavones s d s s s 2325.59268,34446330130-O-Glucoside of luteolin,7-O-methylatedd s s s s s2425.71246,290sh,334539561(M+Na+),433,341,297Isomer of lithospermic acid s d s s s s2526.76244,308,318539561(M+Na+),373,341Isomer of lithospermic acid s d s s s s 2628.29240,346577599(M+Na+),515,317,3016-Hydroxy,7-O-methyl,luteolin,30-O-glucosyl maleate1s s s s d s2728.41244,288,316539561(M+Na+),269(297-CO),181Isomer of lithospermic acid s d s s s s 2828.72268,334593615(M+Na+),491,28540-O-Rutinoside of7-O-methylatedapigenins s d s s s2929.07242,286,318–741(M+Na+),553,521Derivative of lithospermic acid and3-(3,4-Dihydroxyphenyl)-2-hydroxypropanoic acid,3s d s s s s 3030.17238,288303177Hesperetin s s s d s s3130.28240,340,322sh 715451,429,401,379C,O-diglucoside,tentatively:apigenin,6-C-glucosyl benzoate,40-O-glucoside4s s s s d s3230.67256,334–Unknown s d s s s s 3330.72240,340711427,399,185Luteolin6,8-di-C-glucoside succinate s s s s d s 3431.01268,334539467,327Unknown s s s s s d3532.58240,280,414345711(MÂ2+Na+),689(MÂ2+H+),399(M+CH3OH+Na+),377(M+CH3OH+H+),367(M+Na+),362(M+H2O)Pentahydroxy aurone,3-Omethylatedd s s s s s3632.73338315337(M+Na+),247Cirsimaritrin s s s s d s3733.12242,274,332,375771(MÂ2+Na+),457(M+CH3COOH+Na+),429(M+Na++CH3OH)(M+H2O)Hexahydroxyflavonetetra-O-methylatedd s s s s s3834.27274-323(M+Na+),181Glucose p-hydroxy benzoate s d d s s s 3935.17240,280,336345711(MÂ2+Na+),367(M+Na+)Methylatedflavone at OH-405s d s s s s 4036.90266,344285269(radical positive;methyl loss)7-O-Methylated apigenin d s s s s s(continued on next page)A.C.Kaliora et al./Food Chemistry142(2014)233–241237catechin,catechin,kaempherol and quercetin individually.Antiox-idant power of phytochemicals is significant,as reactive oxygen species constitute a major source of DNA damage leading to muta-tion and cancer and consequently antioxidant phytochemicals might restrain the carcinogenic process(Surh,2003).The total phenolic content has been found to positively correlate with anti-oxidant activity of herbs(Djeridane et al.,2006;Katalinic,Milos, Kulisic,&Jukic,2006).Herein,the association suggests that the antioxidant activity of herbal infusions can be attributed largely to their total phenolic content.The phenolic profile of the infusions is presented in Tables2and 3.GS-MS analysis of the infusions showed differences in phenolic acid profile;in detail,six hydroxybenzoic acids,nine hydroxycin-namic acids,sevenflavonoids,two terpenoid phenols and two ter-penic acids were determined(Table2).All the hydroxybenzoic acids were present in marjoram,while all hydroxycinnamic acids were present in sage.Similarly,allflavonoids,terpenoid phenols and terpenic acids were present in sage.According to GC–MS data, the main components of all infusions were carvacrol and rosmari-nic acid,which predominated among simple phenolic species in all infusions except St John’s Wort that lacks rosmarinic acid while it contains primarilyflavonoids–epicatechin,catechin and quercetin (Table2).The combination of diode array detection(DAD)and po-sitive electrospray ionisation mass spectrometry(ESI+),coupled to the HPLC using reverse phase silica provided an accurate method for the structure elucidation of individual phenolics.Under the conditions used,all the compounds analysed had an intense signal corresponding to the pseudo-molecular ion[M+H]+.To a lesser extent,water adducts[M+18]+and sodium adducts[M+23]+ were also demonstrated(Kiehne&Engelhardt,1996).The MS and UV characteristics of the identified phenolics in each extract are given in Table3,while the tentative structures of phenolic spe-cies are shown in Supplementary material B.Infusions did not show identical phenolic profile.Overall,approximately44phenolic species were detected,phenolic acids and their derivatives being present in all herbal infusions.Whilst the presence offlavonoids varied in our samples,sage infusion clearly contained mostlyflav-ones.Identification of the individual phenolic compounds was achieved by comparison of the UV–vis absorption spectra and MS data with the literature(Fang,Yu,&Prior,2002;Sakakibara,Hon-da,Nakagawa,Ashida,&Kanazawa,2003).In the study of Atoui et al.(2005)rosmarinic acid was not found in sage,though ex-pected,something attributed by the authors to the fact that ethyl acetate,known as a medium polarity solvent,was used for the recovery of phenolics.This is indicative of the effect of solvent extraction in LC-DAD-MS analysis.Herein,rosmarinic acid was predominant in marjoram comprising approximately89%of sim-ple phenolics,followed by rosemary and sage comprising approx-imately57%of simple phenolics.Infusions of thyme and dittany were with lower concentrations of rosmarinic acid,whereas terpe-noid phenols comprised97%and85%of their simple phenols, respectively(Table2).The effect of the herbal infusions in HT29and in PC3cell growth is presented in Fig.1.Different concentrations and differ-ent time intervals(24,48and72h)were tested.In the case of HT29(Fig.1A)the suppression in growth was remarkable for Cre-tan dittany reaching approximately95%.Similarly,rosemary infu-sion exhibited a remarkable suppression mainly in24h.Thyme exhibited a dose suppressive activity in higher concentrations. Overall,marjoram,sage and St John’s Wort were less potent.In the case of thyme the effect of0.2l g/l L at24h inhibited growth approximately70%and approximately35%after48h with the same treatment,indicative of the fact that cell growth is revers-ible.The same applies to sage and St John’s Wort at48h and mar-joram at72h in the same cell line.When treating PC3prostate cancer cells with equal concentrations of herbal infusions and for identical time intervals(Fig.1B),the sequence in antiprolifer-ative activity was modified as:thyme<St John’s Wort<rose-mary<sage<marjoram<Cretan dittany.Notably,marjoram was found remarkably effective even as early as24h,but Cretan dit-tany most effectively suppressed growth approximately80%after 24h and90%after48h.As in the case of HT29,in PC3treatment with rosemary and thyme cell growth appears reversible.With a holistic approach,in both HT29and PC3cell lines the most bioac-tive in cell growth arrest was the infusion obtained from Cretan dittany.Marjoram was found very efficient against PC3growth, but not against HT29cell growth,while contrary thyme was more effective against HT29rather than against PC3growth.The high-est concentration of rosmarinic acid was found in marjoram, while concentrations in dittany and thyme were alike and slightly lower to this in marjoram.Carvacrol was very high in both thyme and dittany.Both carvacrol and rosmarinic acid have been found with antiproliferative activity against human prostate cancer cell lines(Patel,Shah,&Bavadekar,2012;Yesil-Celiktas,Sevimli, Bedir,&Vardar-Sukan,2010)and against colon cancer cell lines(Encalada et al.2011;Savini,Arnone,Catani,&Avigliano, 2009).Apart from cell growth,inflammation is another critical event that supports cancer progression and IL-8is a proinflammatory cytokine found elevated in many types of cancer,with a critical role in angiogenesis,tumor progression and neutrophil chemotaxis (Vandercappellen,Van Damme,&Struyf,2008).Cancer develop-ment is linked to chronic inflammation,for example in obese peo-ple(Khandekar,Cohen,&Spiegelman,2011).Levels of IL-8are elevated in both prostate(Begley et al.2008)and colon cancer (Seaton et al.2008).In the present survey,the effect of the lowest concentration of herbal infusions(0.2l g/l L)in IL-8of stimulated HT29and PC3cells are presented in Fig.2.Treatment of HT29withTable3(continued)No.RT(min)k max(nm)Molecularion(M+H)+(m/z)Fragment ions(m/z)Proposed structure I II III IV V VI4137.50240,274,350,414465487(M+Na+),433Monomethyl anthranol s s d s s s4237.93242,316–415(383+CH3OH),383(M-H2O+CH3OH+Na+),329Rosmanol s s s s d s4342.00278,334301M+CH3OH+H+6-Methoxy-apigenin s s s s d s 4446.50330,544505Hypericin s s s s s d d points the detection of the compound in infusion;s points the absence of the compound in infusion.*Sh=shoulder.1Comparison with in-house data.2,3,4Tentative structures provided in Supplementary material B.55,6-Dihydroxy,7,8,40-trimethoxyflavone or5,6-dihydroxy,7,30,40trimethoxyflavone.238 A.C.Kaliora et al./Food Chemistry142(2014)233–241。