Biological activity of carotenoids in red paprika, Valencia orange and Golden delicious apple

700 P. MOLNÁR ET AL.

Copyright © 2005 John Wiley & Sons, Ltd. Phytother. Res. 19, 700–707 (2005)

Copyright © 2005 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 19, 700–707 (2005)Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1735

Biological Activity of Carotenoids in RedPaprika, Valencia Orange and GoldenDelicious Apple

Péter Molnár1, Masami Kawase2*, Kazue Satoh3, Yoshitaka Sohara2, Toru Tanaka2,Satoru Tani2, Hiroshi Sakagami4, Hideki Nakashima5, Noboru Motohashi6, Nóra Gyémánt7

and Joseph Molnár7

1Department of Biochemistry and Medical Chemistry, University of Pécs Medical School, Pécs, H-7601 Hungary2Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama 350-0295, Japan3School of Medicine, Showa University, Shinagawa, Tokyo 142-8555, Japan4Division of Pharmacology, Department of Diagnostic and Therapeutic Sciences, Meikai University School of Dentistry, Sakado,Saitama 350-0283, Japan5Department of Microbiology, St Marianna University School of Medicine, 2-16-1 Sugao, Miyamae-ku, Kawasaki,Kanagawa 216-8511, Japan6Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588, Japan7Faculty of Medicine, Institute of Medical Microbiology, Albert Szent-Györgyi Medical Centrum, University of Szeged, Szeged,Dóm tér 10, H-6720, Hungary

Carotenoid fractions were extracted from red paprika, Valencia orange peel and the peel of Golden deliciousapple. Thus, hypophasic carotenoids of paprika (PM1), orange (PM3) and apple (PM4), and epiphasic extrac-tions of paprika (PM2) and apple (PM5) were obtained by extraction, saponification and partition betweenMeOH–H2O (9:1) (hypophasic) and hexane (epiphasic). A high content of capsanthin was quantified in hypo-phasic carotenoids (PM1) from red spice paprika, whereas the hypophasic fractions from orange (PM3) andapple (PM4) were mainly composed of violaxanthin, zeaxanthin and lutein. On the other hand, a high contentof βββββ,βββββ-carotene and βββββ-cryptoxanthin was found in epiphasic fractions (PM2 and PM5). The extracts werestudied for their anti-Helicobacter pylori (H. pylori), anti-human immunodeficiency virus (HIV), cytotoxic,multidrug resistance (MDR) reversal and radical scavenging activity. Among five PM extracts and βββββ,βββββ-carotene, PM4 showed potent anti-H. pylori activity (MIC50 ===== 36 µµµµµg/mL), comparable to metronidazole (MIC50

===== 45 µµµµµg/mL). The extracts were inactive against HIV. PM3 and PM4 showed slightly higher cytotoxic activityagainst three human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carci-noma HSG) and human promyelocytic leukemic HL-60 cells than against three normal human oral cells(gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF), suggesting a tumor-specificcytotoxic activity. PM1, PM3 and PM4 displayed much higher MDR-reversing activity than (±±±±±)-verapamil.ESR spectroscopy demonstrated that PM1-5 and βββββ,βββββ-carotene produced little or no detectable radical underalkaline conditions and did not scavenge the O2

−−−−− produced by the hypoxanthine and xanthine oxidase reaction.On the other hand, PM1 and PM2 scavenged efficiently 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, whereassinglet oxygen was also quenched efficiently by PM5 and PM2. The data suggest the potential importance ofcarotenoids as possible anti-H. pylori and MDR reversal agents. The active principles in the carotenoid extractmight differ, depending upon the types of fruits and vegetables. Copyright © 2005 John Wiley & Sons, Ltd.

Keywords: carotenoids; paprika; orange; apple; cytotoxic activity; anti-H. pylori; MDR; anti-HIV; radical intensity; HPLC.

Received 20 April 2004Accepted 16 June 2005

* Correspondence to: Dr Masami Kawase, Faculty of PharmaceuticalScience, Josai University, 1-1 keyakidai Sakado, Saitama 350-0295, Japan.E-mail: [email protected]/grant sponsor: Hungarian National Research Foundation;Contract/grant number: OTKA T 037441.

INTRODUCTION

Epidemiological studies have indicated that a high con-sumption of fruits and vegetables reduces the risk oflifestyle-related diseases, including cancer (Sun et al.,2002). A possible mechanism mediating the protectiveeffect is related to bioactive components in fruits andvegetables that act mostly by interfering with reactionsof damaging oxidizing agents and free radicals (Woodallet al., 1997; Krinsky, 1998). Fruits and vegetables

contain at least several hundred different types ofantioxidants such as phenolic compounds, flavonoids,carotenoids, ascorbic acid and others. Among them,carotenoids have been recently studied widely and havebeen proven to play an important role in preventingoxidative damage which is caused by free radicals inage-related diseases such as cancer and ageing (Brittonet al., 1995; Motohashi et al., 2001; Motohashi et al.,2003; Tapiero et al., 2004). An intake of dietarysupplied nutrients rich in carotenoids also appears to bebeneficial in protecting the retinal tissues (Mozaffariehet al., 2003). Most current research is focused on aproposed role of carotenoids as lipid antioxidants whichcan protect against oxidation and other destructiveprocesses mediated by singlet oxygen and free radicals,though more specific effects on the immune system arenow under investigation (Britton et al., 1995).

BIOLOGICAL ACTIVITY OF CAROTENOIDS 701


Paprika (Deli et al., 2001; Deli and Molnár, 2002),orange (Melendez-Martinez et al., 2003a; Melendez-Martinez et al., 2003b) and apple (Wolfe et al., 2003)are commonly eaten worldwide and are good sourcesof carotenoids. This study investigated anti-Helicobacterpylori (H. pylori), anti-human immunodeficiency virus(HIV), cytotoxic, multidrug resistance (MDR) reversaland radical scavenging activity by the carotenoidextracts of red paprika, Valencia orange and Goldendelicious apple.

MATERIALS AND METHODS

The ripe fruits of red spice paprika, Capsicum annuum;Valencia orange and Golden delicious apple werepurchased on the local market (Pécs, Hungary).

The following chemicals and reagents were obtainedfrom the indicated companies: RPMI1640 mediumand Dulbecco’s modified Eagle medium (DMEM)(Gibco BRL, Grand Island, NY, USA); fetal bovineserum (FBS) (JRH Bioscience, Lenexa, KS, USA);3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), metronidazole, ascorbic acid, rosebengal and 2,2,6,6-tetramethyl-4-hydroxy-piperideine(4-OH TEMP) (Wako Pure Chemical Ind., Ltd.,Osaka, Japan); 3′-azido-2′,3′-dideoxythymidine (AZT),dideoxycytidine (ddC), hypoxanthine (HX), xanthineoxidase (XOD) and 1,1-diphenyl-2-picrylhydrazyl(DPPH) (Sigma Chemical Co., St Louis, MO, USA);dextran sulfate (DS, 8 kDa), (Kowa Chemical Co.,Tokyo, Japan); curdlan sulfate (CRDS, 300 kDa)(Ajinomoto, Tokyo, Japan); 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) (Dojin, Kumamoto, Japan);diethylethylenetriamine pentaacetic acid (DETAPAC)(Sigma Chemical Co., St Louis, MO, USA); β,β-carotene and gallic acid (Tokyo Kasei Co., Tokyo,Japan); clarithromycin (Taisho Pharmaceutical Co.,Tokyo, Japan). α-Tocopherol was a gift from Eisai Ltd(Tokyo, Japan). A strain of H. pylori (ATCC43504)was purchased from American Type Culture Collec-tion (Rockville, MD, USA).

UV/VIS spectra were acquired with a BeckmanDU-65 spectrophotometer.

The plant materials; preparation of extracts. The smallpieces of the plant material (ripe fruits of red spicepaprika, Capsicum annuum; peels of Valencia orangeand Golden delicious apple) were blended with MeOHand the suspension was left to stand overnight. After20 h, the mixture was filtered and the filter cake wasre-extracted twice with MeOH and then with Et2O. Theether extract was washed free from MeOH with waterin a separatory funnel. The three MeOH extracts werecombined, diluted with Et2O and washed free fromMeOH with water. The ether solutions were combinedand dried over anhydrous Na2SO4 (total extract). Thissolution was saponified with 30% KOH in MeOHin heterogeneous phase at room temperature for 18 h(overnight). After saponification, the ether solution waswashed free from alkali and evaporated to drynessin vacuo. The residue was partitioned between hexaneand MeOH–H2O (9:1). The hypophasic carotenoids(phase MeOH–H2O (9:1)) were dissolved in benzeneand crystallized or precipitated from benzene–hexane

(1:5). Hypophasic extractions of paprika (PM1), orange(PM3) and apple (PM4) were obtained. The epiphasiccarotenoids (dissolved in hexane) were transferredin benzene and were crystallized or precipitated frombenzene–MeOH (1:5) (Molnár et al., 1980; Deli et al.,2000). Epiphasic extractions of paprika (PM2) andapple (PM5) were obtained.

HPLC analysis of carotenoid extracts (PM1-5). HPLCwas performed by using Gynkotek Model 300B pump,a Gynkotek gradient former, a Hewlett-Packard1050 detector, with HP ChemStation software, and aWaters 991 photodiode array detector. Carotenoidswere analysed on a 250 mm × 4.6 mm i.d., 6 µmparticle, endcapped Chromsyl C18 column. Gradientelution was performed with a mixture of mobile phasesas follows: A, 12% H2O in MeOH; B, MeOH: andC, 30% CH2Cl2 in MeOH. The gradient programwas 0–2 min, 100% A; 2–10 min, to 80% A 20% B;10–18 min, to 50% A 50% B; 18–25 min, to 100% B;25–27 min, 100% B, 27–34 min, to 100% C; 34–41 min,10% C (in linear steps). The flow rate was 1.2 cm3/min(Deli et al., 2000).

Measurement of anti-H. pylori activity. The micro-dilution broth method was used to determine theminimum inhibitory concentration (MIC). Brain heartinfusion (BHI) broth containing 10% fetal bovineserum (FBS) (Biofluid, Inc. Rockville, MD, USA) and0.1% glucose was used as the medium and was cul-tured in a jar conditioned with AnaeroPack (Campylo)(Mitsubishi Gas Chemical Co., Inc.). Briefly, H. pyloriwas inoculated in the medium and cultured at 37 °Cfor 2 days. The collected bacterial suspension was dilutedto about 107 colony forming unit (CFU)/mL with themedium. The fractions were dissolved in DMSO, andthen diluted with the medium. To the solution of thefractions, a suspension of bacteria was added to make106 CFU/150 µL/well. The mixture was incubated at37 °C for 5 days. The MIC values of the fractions weredetermined by observation (Kawase et al., 2003b).

Assay for anti-HIV activity. Human T cell leukemiavirus 1 (HTLV1)-bearing CD4 positive human T celllines (MT-4 cells) were infected with HIV-1IIIB at amultiplicity of infection (m.o.i.) of 0.01. HIV- or mock-infected MT-4 cells (1.5 × 105/mL, 200 µL) were placedinto 96-well microtiter plates in RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calfserum (FCS) and incubated in the presence of vary-ing concentrations of the compounds tested. Afterincubation for 5 days at 37 °C in a CO2 incubator, thecell viability was quantified by a colorimetric assay(at 540 nm and 690 nm), monitoring the ability ofviable cells to reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to a blue formazanproduct (Nakashima et al., 1992). All data representthe mean values of triplicate measurements. The valuesare translated into percentage cytotoxicity and percent-age antiviral protection, from which 50% cytotoxicconcentration (CC50) and 50% effective concentration(EC50) are calculated. The selectivity index (SI) wasdefined as follows: SI = CC50/EC50.

Cell culture. Human squamous cell carcinoma (HSC-2)cells and human salivary gland tumor (HSG) cells



were maintained as monolayer cultures at 37 °C inDulbecco’s modified Eagle medium (DMEM) (GibcoBRL, Grand Island, NY, USA) supplemented with10% heat-inactivated FBS in a humidified 5% CO2

atmosphere, and subcultured by trypsinization. Humangingival fibroblasts (HGF) were isolated from theperiodontal tissue of healthy gingival biopsy of a 10-year-old female, as described previously (Sakagamiet al., 2000), according to the guideline of MeikaiUniversity Ethic Committee, after obtaining informedconsent from the patients. Cells between the fifth andseventh passages were used.

Cytotoxic activity. Cells were incubated for 24 h withthe indicated concentrations of test samples in culturemedium, and the viable cell number was determined byMTT method (Sakagami et al., 2000). In brief, the cellswere washed with PBS, and incubated for 4 h with freshculture medium containing 0.2 mg/mL MTT (SigmaChemical Ind., St Louis, MO). After removing themedium, cells were lysed with 100 µL DMSO and theabsorbance at 540 nm of the cell lysate was measuredwith Labsystems MultiskanR (Biochromatic) with Star/DOT Matrix printer JL-10. The A540 values of controlHSC-2, HSG and HGF cells were 1.519, 0.884 and 0.283,respectively. The 50% cytotoxic concentration (CC50)was determined from the dose-response curve.

Radical intensity. The radical intensity of the samplewas determined at 25 °C in the indicated buffer, usingelectron spin resonance (ESR) spectrometer (JEOLJES REIX, X-band, 100 KHz modulation frequency).Instrument settings: center field, 336.0 ± 5.0 mT; micro-wave power, 8 mW; modulation amplitude, 0.1 mT;gain, 630; time constant, 0.03 s; scanning time, 2 min.The radical intensity was defined as the ratio of peakheights of these radicals to that of MnO (Satoh et al.,1997).

Radical scavenging activity against superoxide anion(O2

−−−−−) and hydroxyl radical (•OH). O2− was generated by

hypoxanthine (HX) and xanthine oxidase (XOD) reac-tion (total volume: 200 µL) [2 mm HX in 0.1 m phos-phate buffer (PB) (pH 7.4) 50 µL, 0.5 mM DETAPAC20 µL, DMPO (5%) 30 µL, sample (in H2O) 50 µL,XOD (0.5 U/mL in PB) 50 µL] (in a flat cell). The gainand time constant were changed to 320 and 0.1 s, re-spectively. The radical intensity was determined by ESRspectroscopy 1 min after mixing. The O2

− scavengingactivity was expressed as superoxide dismutase (SOD)unit/mg sample, by calibration with standard curve ofSOD.

The •OH was produced by Fenton reaction (200 µL)(1 mM FeSO4 containing 0.2 mM DETAPAC) 50 µL,PB (0.1 mM, pH 7.4)] 50 µL, 92 mM DMPO 20 µL,sample (in H2O) 50 µL, 1 mM H2O2, 30 µL]. The gainwas changed to 160.

DPPH scavenging assay. For the determination ofDPPH radical, 30 µL of 300 µM DPPH in ethylalcohol were added to 60 µL of sample in 40% DMSOsolution (in a capillary tube) and analysed 1 min aftermixing. The microwave power, gain and time constantwere changed to 5, 400 and 0.1 s, respectively. The thirdpeak height of five DPPH signals was used as the ratioto that of MnO.

1O2 scavenging assay. The sample was added tothe reaction mixture of 20 mM 2,2,6,6-tetramethyl-4-piperidinol (4-OH TEMP) and 20 µM rose bengalin 20% DMSO solution and the mixture in flat cell wasirradiated for 5 min at 550 nm (light emitting diodegreen) and analysed by using ESR spectrometer. Themicrowave power, modulation width, the gain andtime constant were changed to 5, 0.2, 200 and 0.1 s.Singlet oxygen was measured by oxidation of 4-OHTEMP to 2,2,6,6-tetramethyl-4-hydroxy-1-piperidinyloxy(4-OH TEMPO) radical.

Cell and fluorescence uptake. The MDR1/A expressingcells were selected by culturing the infected cells with60 ng/mL colchicine to maintain the expression of theMDR phenotype (Kessel, 1989). The L5178 MDR celland the L5178 Y parent cell were grown in McCoy’s5A medium supplemented with 10% heat-inactivatedhorse serum, l-glutamine and antibiotics. The cellswere adjusted to a concentration of 2 × 106/mL andresuspended in serum-free McCoy’s 5A medium, and0.5 mL aliquot of the cell suspension was distributedinto each Eppendorf centrifuge tube. Then, 10 µL of2 mg/mL test compounds was added and incubated for10 min at room temperature. Then, 10 µL rhodamine123 (R123) as indicator of drug accumulation was addedto the extracts (5.2 µM final concentration) and the cellswere incubated for a further 20 min at 37 °C, washedtwice and resuspended in 0.5 mL PBS (pH 7.4) for ana-lysis. The fluorescence intensity of cell population wasmeasured by flow cytometry using Beckton DickinsonFACScan instrument. (±)-Verapamil was used as thepositive control in R123 accumulation experiments(Weaver et al., 1993). The R123 accumulation wascalculated from fluorescence of one height values. Then,the percentage of mean fluorescence intensity wascalculated in treated MDR1 and parental cells, com-pared with untreated cells. The fluorescence activityratio was calculated by the following equation (Kessel,1989; Weaver et al., 1993):

MDR1 reversal activity = (MDR1 treated/MDR1 control)/(parental treated/parental control).

RESULTS AND DISCUSSION

Characterization of carotenoid fractions

Paprika, orange and apple contain carotenoids whichare esterified with fatty acids, as well as unesterifiedcarotenoids. These extracts were saponified and thepartitions of the total extracts between 90% H2O-MeOH and hexane gave two fractions, hypophasicand epiphasic fractions, respectively. Therefore, fivecarotenoids fractions (PM1-5) such as hypophasic (PM1)and epiphasic (PM2) carotenoids were obtained fromred spice paprika, hypophasic carotenoids (PM3) fromValencia orange peel, hypophasic (PM4) and epiphasiccarotenoids (PM5) from Golden delicious apple peel.The HPLC analyses of hypophasic (PM1, 3, 4) andepiphasic (PM2, 5) fractions have been performed andtheir HPLC chromatographs are shown in Figs 1–4.The peak assignment of the carotenoids in PM extractswas based on comparison of HPLC retention times and



Figure 1. HPLC analysis of hypophasic carotenoids (PM1) fromred paprika. 1, capsorubin; 2, capsanthin-5,6-epoxide; 3, 5,6-diepikarpoxanthin; 4, capsanthin-3,6-epoxide; 5, capsanthin;6, capsanthone; 7, antheraxanthin; 8, mutatoxanthin; 9, (9Z)-and/or (9’Z)-capsanthin; 10, zeaxanthin; 11, β-cryptoxanthin;12: β,β-carotene.

Figure 2. HPLC analysis of epiphasic carotenoids (PM2) fromred spice paprika. 1, (Z )-isomers of capsanthin; 2, cryptocapsin;3, β-cryptoxanthin; u: unidentified; 4, β,β-carotene; 5, (Z )-isomers of β,β-carotene.

Figure 3. HPLC analysis of hypophasic carotenoids (PM3)from Valencia orange peel. 1, Apo-carotenoids and 5,8-epoxides (furanoid oxides); 2, (Z )-isomers of neoxanthin;3, (all-E )-violaxanthin; 4, luteoxanthin; 5, (9Z)-violaxanthin; 6,(di-Z )-isomers of violaxanthin; 7, antheraxanthin; 8, lutein;9, (Z )-isomers of lutein; 10, β-cryptoxanthin (in traces).

Figure 4. HPLC analysis of hypophasic carotenoids (PM4) fromthe peel of Golden delicious apple. 1, unidentified; 2, (all-E )-neoxanthin; 3, (9’Z)-neoxanthin; 4, (all-E)-violaxanthin; 5,luteoxanthin; 6, (9Z)-violaxanthin; 7, antheraxanthin; 8, lutein;9, (Z )-isomers of lutein.

well known that a number of carbonyl carotenoids,many of epoxy character, are found in orange peel(Molnár and Szabolcs, 1980). Major carotenoids in ahypophasic fraction (PM3) of orange peel were (9Z)-violaxanthin and (all-E)-violaxanthin (Fig. 3). These twocarotenoids were also found as the main carotenoidsin a hypophasic fraction (PM4) of apple peel (Fig. 4).The epiphasic fraction (PM5) contained very smallamounts of carotenoids, but with a large amount ofsteroids and other compounds: β-cryptoxanthin 5,6-epoxide, β-cryptoxanthin and β,β-carotene were onlydetected in PM5.

UV-visible absorption spectra with those of authenticsamples (Molnár and Szabolcs, 1980). The carotenoidfractions (PM1-5) contained overlapping but alsosignificantly different varieties of carotenoids. Thus, itshould be expected that hypophasic fractions (PM1,3 and 4) contained oxygenated carotenoids, whileepiphasic fractions (PM2 and 5) have less oxygenated,more lipophilic carotenoids. The main carotenoids werecapsanthin and zeaxanthin in hypophasic carotenoids(PM1) extracted from red spice paprika (Fig. 1). Onthe other hand, the epiphasic fraction (PM2) containedmainly β,β-carotene and β-cryptoxanthin (Fig. 2). It is



Anti-H. pylori activity

Antibacterial agents from a diet rich in fruit and veg-etable may be superior as H. pylori control agents, whencompared with many non-natural products (Kawaseand Motohashi, 2004). A high intake of carotenoidshas been shown to prevent the development of H.pylori-associated disease (Zhang et al., 2000; Correaet al., 2000; Nair et al., 2000). As free radicals play animportant role in the pathogenesis of gastroduodenalmucosal inflammation, peptic ulcer disease and prob-ably even gastric cancer, various micronutrients areconsidered to protect the gastric mucosa by scavengingthe free radicals. Among PM1-5 and β,β-carotene, PM4displayed potent anti-H. pylori activity (MIC50 = 36 µg/mL), comparable to metronidazole (MIC50 = 45 µg/mL)(Table 1). Other PM extracts and β,β-carotene did notexhibit potent anti-H. pylori activity (MIC50 > 200 µg/mL) (Table 1).

Anti-HIV activity

It is shown that the level of carotenoids are decreasedin HIV-infected people and administration of β,β-carotene improves immune function (Singhal and Austin,2002; Coodley, 1995). The inhibition of HIV-inducedcytopathic effects by PM1-5 and β,β-carotene wasstudied (Table 1). However, there was no significantinhibition by PM1-5 and β,β-carotene for the cytopathiceffects of HIV infection in MT4 cells using effectiveconcentrations of >200 µg/mL (selectivity index (SI) < 1),compared with four positive controls – dextran sulfate(DS) (SI > 273), curdlan sulfate (CRDS) (SI > 5397),AZT (SI > 4006) and dideoxycytidine (ddC) (SI > 1172).

Cytotoxic activity

Carotenoids showed potent in vitro anti-tumorpromoting activity (Garewal, 1993). A higher intakeof carotenoids-rich foods is associated with a reducedrisk of the development of cancer. Among carotenoids,β,ε-carotene, lutein, zeaxanthin, lycopene and β,β-carotene is being investigated as the possible cancerpreventive agents, because these carotenoids are com-monly found in vegetables and fruits (Nishino et al.,1999). β-Cryptoxanthin was found to show antitumor-initiating activity, as well as antitumor-promoting act-ivity. Capsanthin and related carotenoids isolated fromthe fruits of red paprika exhibited potent antitumor-promoting activity (Maoka et al., 2001). Cytotoxic act-ivities of PM1-5 and β,β-carotene against four humantumor cells (HSC-2, HSC-3, HSG and HL-60) andthree normal cells (HGF, HPC and HPLF) were invest-igated (Table 2). The cytotoxicity of all PM extractsagainst tumor cells was much weaker than that of gallicacid. PM3 and PM4 were relatively cytotoxic to HSC-2 and HL-60, when compared with normal cells andshowed tumor-specific cytotoxicity in which their safetyindex [(sum of CC50 of three normal cells/sum of CC50

of four tumor cells) × 4/3] values were 1.7 and >2.1,respectively.

Table 1. Anti-H. pylori and anti-HIV activity of carotenoids(PM1-5)

Anti-H.pylori Anti-HIV activity

MIC50 CC50 EC50 SIa

Extract (µg/mL) (µg/mL) (µg/mL) (CC50/EC50)

PM1 >200 = 20.472 >40 <1PM2 >200 = 21.549 >40 <1PM3 >200 = 17.455 >40 <1PM4 36 = 55.254 >200 <1PM5 >200 >1000 >1000 ><1β,β-Carotene >200 = 93.385 >200 <1Clarithromycin 4.1 × 10−2 – – –Metronidazole 45 – – –DS – = 725.770 = 2.662 = 273CRDS – >1000 = 0.185 >5397AZT (µM) – = 68.443 = 0.0171 = 4006ddC (µM) – >5000 = 4.268 >1172

a SI = CC50/EC50

Table 2. Cytotoxic activity of carotenoids (PM1-5) against tumor and normal cells

50% cytotoxic concentration (CC50, µg/mL)SIa =

Human tumor cell Human normal cellCC50(normal)/

Compound HSC-2 HSC-3 HSG HL-60 HGF HPC HPLF CC50(tumor)

PM1 155 78 >200 66 >200 >200 >200 ><1.60PM2 133 173 162 143 >200 >200 >200 >1.31PM3 111 134 118 30 157 176 158 1.67PM4 98 102 136 52 >200 196 >200 >2.05PM5 105 168 >200 >200 >200 200 >200 ><1.19β,β-carotene 193 >200 >200 >200 >200 >200 >200 ><1.0Gallic acid 19 29 38 24 81 75 49 2.48A540 1.137 0.917 0.345 1.8 × 106/mL 0.431 0.372 0.482

Near confluent cells were incubated for 24 h without or with various concentrations of eachcompound, and the relative viable cell number (absorbance at 540 nm of the MTT-stained celllysate) was determined by the MTT method. The viable cell number of HL-60 cells was deter-mined by trypan blue exclusion. The CC50 was determined from the dose-response curve. Eachvalue represents the mean from duplicate determinations.a Determined by the equation: SI = [(CC50)HGF + (CC50)HPLF + (CC50)HPC/(CC50)HSC-2 + (CC50)HSC-3 + (CC50)HSG + (CC50)HL-60] × 4/3.



MDR reversal on tumor cells

The P-glycoprotein (P-gp) inhibitory effects ofseveral dietary constituents have been extensivelystudied (Kawase and Motohashi, 2003a; Defermeand Augustijns, 2003). Molnár and coworkers haverecently examined MDR modulating activity of severalcarotenoids and shown that capsanthin and capsorubinenhanced the drug accumulation most potently. Lutein,antheraxanthin and violaxanthin enhanced moderatelythe drug accumulation, while β,β-carotene showedlittle effect on the reversal of MDR in the tumor cells(Molnár et al., 2004). The ability of PM1-5 to inhibitthe P-gp-mediated R-123 efflux was measured in theMDR mouse T cell lymphoma L5178 transfected byhuman MDR1 gene, which was cultured in colchicine-containing medium (Table 3). PM1, 3 and 4 showedpromising levels of MDR-reversing activity and higherMDR reversing activity than (±)-verapamil. PM1, 3 and4 might restore the drug accumulation in cancer cellsby inhibiting the P-gp-mediated efflux pump (Szabóet al., 2000; Kawase and Motohashi, 2003a). PM2moderately enhanced the drug accumulation, while PM5and β,β-carotene did not show potent MDR modula-tory activity. The content of capsorubin, capsanthin,lutein and violaxanthin may have conferred to the frac-tions (PM1, 3 and 4) a high MDR modulating activity.These results are in agreement with the previous study(Molnár et al., 2004).

Radical scavenging activity

Carotenoids are involved in the scavenging of twoof reactive oxygen species (ROS), singlet molecularoxygen and peroxy radicals generated in the process oflipid peroxidation (Sies et al., 1993). ESR spectroscopydemonstrated that PM1-5 and β,β-carotene producedlittle or no detectable radical under alkaline conditions(pH 10.5 and 12.5) (data not shown). On the other

Table 3. Effect of carotenoids (PM1-5) on the multi-drug resistance of L-5178 cells

Concentration FluorescenceCompound (µg/mL) FSCa SSCa FL-1a activity ratio

Par (control)b – 458.12 109.14 869.36MDR +++++ R123 (mean)c – 544.02 125.71 1.64 –MDR +++++ R123 – 444.36 189.84 8.56 –(Dl)-verapamil 10 472.35 224.11 22.90 2.67DMSO 20 455.33 188.73 9.09 1.06PM1 4 490.03 214.93 962.12 112.39

40 469.13 264.01 1738.35 203.07PM2 4 483.48 205.77 65.29 7.62

40 487.50 198.06 414.44 48.41PM3 4 492.10 209.85 1439.72 168.19

40 407.08 200.70 809.44 94.56PM4 4 483.20 200.85 396.50 46.32

40 493.89 197.99 1612.27 188.34PM5 4 467.61 199.24 10.44 1.21

40 474.48 197.75 10.51 1.22β,β-Carotene 4 450.17 201.70 10.59 1.23

40 467.43 199.50 24.18 2.82

a FSC, forward scatter count; SSC, side scatter count; FL-1, fluorescence intensity.b Par, a parental cell without MDR gene.c MDR, a parental cell transfected with MDR gene.

hand, the ESR intensity of DMPO-OOH, which wasa spin adduct of O2

− generated from the HX-XODsystem, was not influenced in the presence of PM1-5and β,β-carotene at 400 µg/mL concentration each (datanot shown). Therefore, PM1-5 and β,β-carotene hadlittle or no O2

− scavenging activity.1,1-Diphenyl-2-picrylhydrazyl (DPPH) is a stable free

radical and its free radical character is neutralized inthe presence of molecules having the ability to donatea hydrogen atom. It has often been used to estimatethe antiradical activity of antioxidants. The DPPHradical scavenging assay of PM1-5 and β,β-carotenewas tested, using two well-known natural antioxidants,ascorbic acid (EC50 = 3.9 µg/mL) and α-tocopherol(EC50 = 10.2 µg/mL) as positive controls (Table 4).DPPH produced five peaks of ESR signals as has beenreported previously. PM1-5 and β,β-carotene reducedthe radical intensity of all these peaks at 800 µg/mL.PM1 and PM2 were the most effective DPPH radical

Table 4. Radical scavenging activity of carotenoids (PM1-5)

DPPH radical Singlet oxygenscavenging activity scavenging activity

Concentration ConcentrationExtract (µg/mL) % (µg/mL) %

Control 0 100 0 100PM1 800 14.1 400 33.3PM2 800 10.3 400 16.8PM3 800 39.0 400 42.2PM4 800 72.1 400 71.1PM5 800 83.4 400 6.3β,β-Carotene 800 85.2 400 45.4Ascorbic acid 7.0 – 35.2 7.6

5.3 16.6 17.6 82.93.5 59.7 – –

α-Tocopherol 3.6 86.2 43 >10010 51.0 – –18 9.3 – –



scavengers, PM3 being the less effective. In DPPHassay, the electron-donating capacity was strongest inthe following order: PM2 > PM1 > PM3 > PM4 > PM5> β,β-carotene.

Singlet oxygen was also quenched by carotenoids (Sieset al., 1993). An 1O2 scavenging effect of PM1-5 wasobserved in the order of PM5 > PM2 > PM1 > PM3 >β,β-carotene > PM4. However, the activities of PM5are more likely due to the other compounds.

CONCLUSION

Carotenoids are important dietary nutrients havingantioxidant potential and are consequently being con-sidered as important preventive strategic molecules.Antioxidants may protect against oxidative damagesboth by directly neutralizing reactive oxidants and bymodulating gene expression contributing to oxidativestress (Svilaas et al., 2004). However, the interventiontrials with single antioxidants in pharmacological doseshave not supported a protective effect. One reason forthe ineffectiveness may be that the protective effects offruits and vegetables result from a concerted action ofthe numerous different antioxidants present in foods.Thus, antioxidants with different chemical charac-teristics may work in an integrated and complementary

network. If many antioxidants work in a network,‘total antioxidants’ may be a better concept than indi-vidual dietary antioxidants. It is pointed out that totalintake of antioxidants was well correlated with plasmalutein, zeaxanthin and lycopene (Svilaas et al., 2004).As these carotenoids are only minor contributors tothe total intake of antioxidants, dietary antioxidantsother than these carotenoids may save, recharge orsalvage them when they have been used in a redoxreaction (Svilaas et al., 2004). Thus, a mixture ofcarotenoids with different chemical structures anddifferent antioxidants might be a promising agent forthe medicinal use rather than individual carotenoids.

The present study demonstrates that carotenoidswere not toxic to both oral tumor and normal cells.PM3 and PM4 were relatively cytotoxic to tumor cellsand showed the tumor-specific cytotoxicities. It is inter-esting to identify the anti-H. pylori-related compoundsin PM4. The data suggest a potential importance ofcarotenoids as anti-H. pylori agents in gastric ulcer andpossible resistance modifiers in cancer chemotherapy.

Acknowledgement

This study was supported by the grant from OTKA T 037441(Hungarian National Research Foundation). The authors aregrateful to Professor J. Deli for his collaboration in the isolation ofpaprika phytoxanthins and in the HPLC analysis.

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Biological activity of carotenoids in red paprika, Valencia orange and Golden delicious apple

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