Food and Chemical Toxicology 48 (2010) 441–447

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Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate / foodchemtox

Human cancer cell antiproliferative and antioxidant activities of Juglans regia L.

Márcia Carvalho a,b,*, Pedro J. Ferreira a, Vanda S. Mendes b, Renata Silva b, José A. Pereira c,Carmen Jerónimo d,e, Branca M. Silva a,f

a CEBIMED, Faculty of Health Sciences, University Fernando Pessoa, Porto, Portugalb REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugalc CIMO, Escola Superior Agrária, Instituto Politécnico de Bragança, Portugald Department of Genetics, Portuguese Oncology Institute, Porto, Portugale Department of Pathology and Molecular Immunology, ICBAS – Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugalf REQUIMTE, Department of Pharmacognosy, Faculty of Pharmacy, University of Porto, Porto, Portugal

a r t i c l e i n f o

Article history:Received 10 September 2009Accepted 28 October 2009

Keywords:Juglans regia L.LeafGreen huskWalnutAntiproliferative activityAntioxidant

0278-6915/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.fct.2009.10.043

* Corresponding author. Address: Faculdade de CiêFernando Pessoa, R. Carlos da Maia, 296, 4200-150225074630; fax: +351 225508269.

E-mail address: mcarv@ufp.edu.pt (M. Carvalho).

a b s t r a c t

Several studies suggest that regular consumption of nuts, mostly walnuts, may have beneficial effectsagainst oxidative stress mediated diseases such as cardiovascular disease and cancer. Walnuts containseveral phenolic compounds which are thought to contribute to their biological properties. The presentstudy reports the total phenolic contents and antioxidant properties of methanolic and petroleum etherextracts obtained from walnut (Juglans regia L.) seed, green husk and leaf. The total phenolic contentswere determined by the Folin–Ciocalteu method and the antioxidant activities assessed by the abilityto quench the stable free radical 2,20-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,20-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes.Methanolic seed extract presented the highest total phenolic content (116 mg GAE/g of extract) andDPPH scavenging activity (EC50 of 0.143 mg/mL), followed by leaf and green husk. In petroleum etherextracts, antioxidant action was much lower or absent. Under the oxidative action of AAPH, all methano-lic extracts significantly protected the erythrocyte membrane from hemolysis in a time- and concentra-tion-dependent manner, although leaf extract inhibitory efficiency was much stronger (IC50 of 0.060 mg/mL) than that observed for green husks and seeds (IC50 of 0.127 and 0.121 mg/mL, respectively). Walnutmethanolic extracts were also assayed for their antiproliferative effectiveness using human renal cancercell lines A-498 and 769-P and the colon cancer cell line Caco-2. All extracts showed concentration-dependent growth inhibition toward human kidney and colon cancer cells. Concerning A-498 renal can-cer cells, all extracts exhibited similar growth inhibition activity (IC50 values between 0.226 and0.291 mg/mL), while for both 769-P renal and Caco-2 colon cancer cells, walnut leaf extract showed ahigher antiproliferative efficiency (IC50 values of 0.352 and 0.229 mg/mL, respectively) than green huskor seed extracts. The results obtained herein strongly indicate that walnut tree constitute an excellentsource of effective natural antioxidants and chemopreventive agents.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Epidemiological studies have consistently shown that there is aclear significant positive association between regular consumptionof fruits, nuts and vegetables, and a reduced incidence of ischemicheart disease and some types of cancer, particularly stomach,oesophagus, lung, oral cavity and pharynx, endometrial, pancreasand colon cancers (Block et al., 1992; Heimendinger et al., 1996;Reddy et al., 2003; Mathew et al., 2004; Jenab et al., 2004). These

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antioxidant and chemopreventive properties have been attributedto their high content of natural antioxidants, especially ascorbic acid(vitamin C), tocopherols (vitamin E), b-carotene (provitamin A),anthocyanins and other polyphenols (Cao et al., 1996; Silva et al.,2004, 2008; Giada and Filho, 2006). Several studies showed thatphenolic compounds are the major bioactive phytochemicals withhuman health benefits (Cao et al., 1996; Sun et al., 2002; Silvaet al., 2004, 2008; Giada and Filho, 2006; Parry et al., 2006; Yanget al., 2009). In fact, many authors have reported a direct relationshipbetween total phenolic content and antioxidant activity in numer-ous seeds, fruits and vegetables (Sun et al., 2002; Silva et al., 2004,2008; Parry et al., 2006; Blomhoff et al., 2006; Yang et al., 2009).

The Juglans genus (family Juglandaceae) comprises several spe-cies and is widely distributed throughout the world. The walnut

442 M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447

tree (Juglans regia L.) is its well-known member, constituting animportant species of deciduous trees found primarily in temperateareas and cultivated commercially throughout southern Europe,northern Africa, eastern Asia, United States and western SouthAmerica. In Portugal, walnut trees can be found all over the coun-try, being the nuts very popular and largely consumed as part ofthe Mediterranean diet. Nevertheless, not only dry seeds (nuts)are used but also green walnuts, shells, bark, green husks (epi-carps) and leaves, which have been used in the cosmetic and phar-maceutical industries (Oliveira et al., 2008).

Walnut seeds (commonly named walnuts) are receiving increas-ing interest as nutraceutics mainly due to the fact that their regularconsumption has been reported to reduce the risk of coronary heartdisease (Blomhoff et al., 2006). The health benefits of these nuts areusually attributed to their chemical composition. Walnuts are goodsources of essential fatty acids (linoleic acid is its major fatty acid),tocopherols and tocotrienols, proteins, fibers, melatonin, sterols, fo-late, tannins and other polyphenols (Pereira et al., 2008). Amongseveral nut types, walnuts have the highest content of antioxidants,especially polyphenols and tocopherols, and most of phenolic com-pounds are located in the pellicles (Blomhoff et al., 2006). However,as far as we know, it is not clear which of these two classes of com-pounds is mainly responsible for walnut antioxidant activity.

Walnut leaves are considered a source of healthcare com-pounds, and have been widely used in traditional medicine fortreatment of skin inflammations, hyperhidrosis and ulcers andfor its antidiarrheic, anti-helmintic, antiseptic and astringent prop-erties (Bruneton, 1999). In Portugal, as in some other Europeancountries, especially in rural areas, dry walnut leaves are fre-quently used to prepare infusions (Amaral et al., 2004; Pereiraet al., 2007). Antiradicalar and antibacterial activities have alsobeen recently described for different J. regia cultivars (Pereiraet al., 2007; Almeida et al., 2008).

Walnut green husk is a by-product of the walnut production,being formed in large amounts. Probably due to its scarce utiliza-tion, this matrix is very little studied. Recently, aqueous extractof green husk was suggested as a low cost natural source of pheno-lic compounds with antiradicalar and antimicrobial activities (Oli-veira et al., 2008).

Interest in natural antioxidant sources prompted us to continueinvestigating the biological activity of J. regia phytochemicals.Thus, the aim of the present study was to determine the total phe-nolic content and to evaluate the antioxidant and human cancercell antiproliferative activities of J. regia (cultivar Franquette) seed,green husk and leaf. For these purposes, methanolic and petroleumether extracts were prepared and total phenolic contents weredetermined by Folin–Ciocalteu method. The antiradicalar proper-ties were studied for their ability to quench the stable free radical2,20-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,20-azo-bis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidativehemolysis of human erythrocytes. In addition, walnut methanolicextracts were tested for their potential antiproliferative propertieswith 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) bioassay on human renal (A-498 and 769-P) and colon(Caco-2) carcinoma cells. As far as we know, this is the first timethat antihemolytic activity and human renal cancer cell antiprolif-erative properties of walnut seed, green husk and leaf are evalu-ated. In addition, green husk human colon cancer cellantiproliferative activity has never been evaluated.

2. Materials and methods

2.1. Chemicals and reagents

Methanol and petroleum ether were purchased from Merck (Darmstadt, Ger-many). 2,20-Diphenyl-1-picrylhydrazyl (DPPH), 2,20-azobis(2-amidinopropane)dihydrochloride (AAPH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT) were purchased from Sigma (St. Louis, MO, USA). Folin–Ciocalteu’sphenol reagent was obtained from Fluka. All other chemicals were obtained fromSigma (St. Louis, MO, USA).

2.2. Samples

Walnut (J. regia L.) leaves from cultivar Franquette were harvested in May 2007in Bragança (Northeast of Portugal). Leaves were collected from the middle third ofbranches exposed to sunlight. Walnut fruits (cv. Franquette) were harvested in Sep-tember 2007 in the same region of Portugal. Fruits were handpicked from the soil,and green husks and nuts (seeds) were separated. Fruits were dried in a stove(Memmert Schwabach 854, 1994) at 30 �C, in the dark, for five days. All plant mate-rial was put in plastic bags, immediately frozen at �20 �C and then freeze-dried.Lyophilization was carried out using a Labconco 4.5 apparatus (Kansas City, MO).

2.3. Extraction procedure

1.5 g of freeze-dried plant material was thoroughly mixed with methanol orpetroleum ether (3 � 25 mL). The extracts were filtered and concentrated to dry-ness under reduced pressure (40 �C). The methanolic extraction efficiency in rela-tion to dry matter was variable as follows: 18.4%, 30.1% and 27.7% for seeds,green husks and leaves, respectively, while petroleum ether extraction mean yieldswere 36.1%, 0.6% and 1.1% for seeds, green husks and leaves, respectively.

2.4. Total phenolic content measurement

Phenolic compounds concentration in the obtained extracts was determined byusing the Folin–Ciocalteu’s phenol reagent, according to a previously described pro-cedure (Singleton and Rossi, 1965), with some modifications. Briefly, 1 mL of the ex-tract solution was mixed with 1 mL of Folin–Ciocalteu’s phenol reagent. After 3 min,1 mL of saturated sodium carbonate solution was added to the mixture and ad-justed to 10 mL with distilled water. The reaction was kept in the dark for90 min, after which the absorbance was read at 725 nm (Analytik Jena 200-2004spectrophotometer). Gallic acid was used for constructing the standard curve. Thecontents of total phenolics are expressed as mg of gallic acid equivalents (GAE)/gof extract.

2.5. Determination of DPPH radical scavenging activity

The capacity to scavenge the DPPH free radical was monitored according to amethod reported before (Hatano et al., 1988). Various concentrations of sample ex-tracts (0.3 mL) were mixed with 2.7 mL of methanolic solution containing DPPHradicals (6 � 10�5 M). The mixture was shaken vigorously and left to stand in thedark until stable absorption values were obtained. The reduction of the DPPH rad-ical was measured by monitoring continuously the decrease of absorption at517 nm. DPPH scavenging effect was calculated as percentage of DPPH discolour-ation using the equation: % scavenging effect = [(ADPPH � AS)/ADPPH] � 100, whereAS is the absorbance of the solution when the sample extract has been added at aparticular level and ADPPH is the absorbance of the DPPH solution. Three experi-ments were performed in triplicate. The antiradicalar activity was expressed interms of the amount of antioxidant necessary to decrease the initial DPPH absor-bance by 50% (EC50). The EC50 value for each extract was determined graphicallyby plotting the percentage of DPPH scavenging as a function of extractconcentration.

2.6. Measurement of inhibition of erythrocyte hemolysis mediated by peroxyl freeradicals

Blood (5–10 mL) was obtained from healthy non-smoking volunteers by veni-puncture, after written informed consent was obtained. Human erythrocytes fromcitrated blood were immediately isolated by centrifugation at 1500 rpm for10 min at 4 �C. After removal of plasma and buffy coat, the erythrocytes werewashed three times with phosphate-buffered saline (PBS; pH 7.4), and then resus-pended using the same buffer to the desired hematocrit level. In order to inducefree-radical chain oxidation in erythrocytes, aqueous peroxyl radicals were gener-ated by thermal decomposition of AAPH (dissolved in PBS; final concentration50 mM). To study the protective effects of walnut extracts against AAPH-inducedoxidative hemolysis, an erythrocyte suspension at 2% hematocrit was preincubatedwith the methanolic (50–200 lg/mL final concentrations) or petroleum ether ex-tracts (200–1000 lg/mL final concentrations) of walnut leaf, green husk and seed(dissolved in PBS) at 37 �C for 30 min, followed by incubation with and without50 mM AAPH. This reaction mixture was shaken gently while being incubated for4 h at 37 �C. In all experiments, a negative control (erythrocytes in PBS), as wellas extract controls (erythrocytes in PBS with each extract) were used.

The extent of hemolysis was determined spectrophotometrically as describedbefore by Ko et al. (1997). Briefly, aliquots of the reaction mixture were taken outat each hour of the 4 h of incubation, diluted with saline, and centrifuged at4000 rpm for 10 min to separate the erythrocytes. The percentage of hemolysiswas determined by measuring the absorbance of the supernatant (A) at 545 nm

M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447 443

and compared with that of complete hemolysis (B) by treating an aliquot with thesame volume of the reaction mixture with distilled water. The hemolysis percent-age was calculated using the formula: A/B � 100. The inhibitory concentration50% (IC50) at time 3 h was also calculated from dose–response curve obtained byplotting the percentage of hemolysis inhibition versus the extract concentration.Four independent experiments were used for these calculations.

2.7. Measurement of inhibition of A-498, 769-P and Caco-2 cell proliferation by walnutextracts

Human renal epithelial cancer cells A-498 (ATCC HTB-44) and 769-P (ATCC CRL-1933) were cultured in RPMI 1640 medium (GlutaMax Media, Gibco BRL Life Tech-nologies) with 25 mM HEPES containing 10% heat-inactivated fetal bovine serum,100 U/mL penicillin and 100 lg/mL streptomycin. Human colon cancer Caco-2(ATCC HTB-37) cells were grown as a monolayer in Dulbecco’s Modified Eagle Med-ium (DMEM) with Glutamax-1 (Gibco BRL Life Technologies) and supplementedwith 10% fetal bovine serum, 1% non-essential amino acids, 100 U/mL penicillin,100 lg/mL streptomycin, and 2.5 lg/mL amphotericin B. For the assay, cells weregrown in T-75 culture flasks in a humidified incubator at 37 �C under 5% CO2/95%air mixture. After trypsin detachment, A-498, 769-P and Caco-2 cells were counted,subcultured at 5 � 103 cells per well of a 96-well microplates and incubated to al-low cell attachment. After 24 h incubation, the cells were treated with differentconcentrations of walnut leaf, green husk and seed methanolic extracts. Each ex-tract was initially solubilized in an amount of dimethylsulfoxide (DMSO) to obtain50 mg extract/mL stock solutions and further diluted in medium prior to addition toplates. Cells were exposed to 200 lL medium with final concentrations of 31.25,62.5, 125, 250 and 500 lg extract/mL. By these serial dilutions, the DMSO final con-centration was never higher than 1% and the same concentration was used for sol-vent-control wells. The plates were incubated for 48 h under the same conditions.At the end of exposure time, cell proliferation was measured using the mitochon-drial MTT assay as previously described (Carvalho et al., 2002). MTT is a yellowwater-soluble dye that is reduced in viable cells to an insoluble purple colouredproduct, MTT-formazan (3-[4,5-dimethylthiazol-2-yl]-3,5-diphenylformazan) bythe mitochondrial enzyme succinate dehydrogenase. After exposure to the extracts,the medium was removed and the cells were washed once with fresh culture med-ium. Subsequently the cultures were incubated at 37 �C in a humidified atmospherecontaining 5% CO2 for 4 h with 200 lL of 0.5 mg/mL MTT (final concentration). MTTsolution was then removed and intracellular MTT-formazan was solubilized in100 lL dimethyl sulfoxide and the absorbance measured at 550 nm in a microplatereader. Cell viability was measured as the percentage of absorbance compared tocontrol. The 50% inhibitory concentration (IC50) values, defined as the amount of ex-tract that inhibits 50% of cell growth, were calculated from concentration–responsecurves following a 48 h exposure time. Three independent experiments performedin triplicate were used for these calculations.

2.8. Statistical analysis

Statistic analysis was performed using the Statistical Package for Social Sciences(SPSS, version 16.0) for Windows. Comparisons between two groups were per-formed by unpaired t-test. Multiple comparisons between more than two groupswere performed by one-way ANOVA supplemented with Tukey’s HSD post hoc test.Significance was accepted at P lower than 0.05. The regression analysis between to-tal phenolic contents and EC50 values for DPPH scavenging activity was conductedusing the same statistical package.

3. Results and discussion

3.1. Total phenolic content

Nuts chemical composition is well-known. It contains severaltherapeutically active constituents, especially polyphenols. Totalphenolic contents of methanolic and petroleum ether extracts ofwalnut seed, green husk and leaf are shown in Table 1. As expected,methanolic extracts obtained from all walnut parts presented ahigher phenolic content than petroleum ether extracts. This factcorrelates with the remarkable differences in the polarity of theextraction solvents used and solubility of phenolic compounds inthem. In fact, methanol (polar solvent) is considered as one ofthe best solvents for phenolics extraction (Bruneton, 1999), whilepetroleum ether (mixture of alkanes, e.g., pentane, hexane, andheptane) is a non-polar solvent frequently used for the extractionof less polar constituents, such as tocopherols and tocotrienols,carotenoids and chlorophylls. Therefore, the obtained walnutmethanolic extracts are likely enriched in phenolic acids, flavo-noids and tannins, while petroleum ether extracts are very poor

in phenolic compounds and mainly enriched in non-polar constit-uents such as tocopherols and tocotrienols, sterols and fatty acids.

Among methanolic extracts, seed presented the highest totalphenolic content (mean value of 116.22 ± 3.76 mg of GAE/g of ex-tract), followed by leaf (94.39 ± 5.63 mg of GAE/g of extract) andgreen husk (50.18 ± 2.69 mg of GAE/g of extract). Taking into ac-count the extraction yields, the total phenolic concentration inwalnut seed is 2143 mg/100 g, which is in agreement with previ-ous studies showing walnuts (seeds) as a good dietary source ofphenolics (Blomhoff et al., 2006; Chen and Blumberg, 2008; Yanget al., 2009).

Most phenolic compounds commonly identified in walnut seedsare phenolic acids, namely gallic, ellagic, syringic, 5-O-caffeoylqui-nic, caffeic, p-coumaric, ferulic and sinapic acids, and tannins, suchas glansrins A, B and C, casuarinin, stenophyllarin, between others(Fukuda et al., 2003; Colaric et al., 2005). On the other hand, stud-ies about walnut green husks are very scarce and its chemical com-position has never been determined. In relation to walnut leaves,its phenolic composition has already been studied by someresearchers. Amaral et al. (2004) determined the phenolic profileof walnut leaves of several cultivars, including cv. Franquette. Thisvariety was characterized by the presence of, at least, nine phenoliccompounds: three hydroxycinnamic acid derivatives, the 3-O-caf-feoylquinic, 3-O-p-coumaroylquinic and 4-O-p-coumaroylquinicacids, and six flavonol heterosides, the quercetin 3-O-galactoside(its major compound), a quercetin 3-O-pentoside derivative, quer-cetin 3-O-arabinoside, quercetin 3-O-xyloside, quercetin 3-O-ram-noside and a kaempferol 3-O-pentoside (Amaral et al., 2004). Morerecently Pereira et al. (2007) identified another two hydroxycin-namic acid derivatives, the 5-O-caffeoylquinic and p-coumaricacids.

3.2. DPPH free radical scavenging activity

Fig. 1 shows the scavenging effect of walnut seed, green huskand leaf methanolic and petroleum ether extracts on the DPPH rad-ical. All methanolic extracts presented a strong concentration-dependent antiradicalar activity. Among them, seed extract, whichcontained the highest content of phenols, exhibited the highestDPPH free radical scavenging capacity, followed by leaf and greenhusk extracts. The calculated EC50 values were respectively0.143 ± 0.020, 0.199 ± 0.023 and 0.412 ± 0.025 mg of methanolicextract/mL (Table 1). Our results are in agreement with previousreports showing the antiradicalar properties of extracts from wal-nut seeds (Fukuda et al., 2003), green husks (Oliveira et al., 2008)and leaves (Pereira et al., 2007). In addition, the highest EC50 valuethat corresponds to the lowest scavenging activity on DPPH radi-cals was observed for petroleum ether extracts. EC50 values ofpetroleum ether extracts obtained from walnut leaf and green huskwere respectively 2.921 ± 0.740 and 1.479 ± 0.117 mg/mL (Table1). At the range of concentrations tested in this experiment itwas not possible to determine the EC50 value of the walnut seedpetroleum ether extracts. These results indicate that walnut poly-phenols have stronger antioxidant capacity than non-polar antiox-idants present in the lipid fraction, such as tocopherols andtocotrienols, being the main responsible for the antiradicalar pro-tection provided by walnut derivatives.

Phenolic compounds constitute a group of secondary metabo-lites that are quite widespread in nature with several therapeuticalproperties. Their antioxidant activity is mainly due to their redoxproperties, which allow them to act as reducing agents, hydrogendonors, free radicals scavengers, singlet oxygen quenchers, andmetal chelators (Costa et al., 2009). Many authors have reporteda direct relationship between total phenolic content and antioxi-dant activity in numerous seeds, fruits and vegetables (Sun et al.,2002; Silva et al., 2004, 2008; Parry et al., 2006; Blomhoff et al.,

Table 1Extraction yield (in percentage), total phenolic content (mg GAE/g extract), EC50 values (mg extract/mL) determined for the DPPH free radical scavenging capacity and IC50 values(mg extract/mL) calculated for the antihemolytic activity of methanolic and petroleum ether extracts of walnut leaves, green husks and seeds after 3 h of incubation with AAPH.Means marked with different letters, within each column, are significantly different (P < 0.05) (ND, not detected; � without activity).

Samples Extraction yield (%) Phenolic content (mg GAE/g) DPPH scavenging activity EC50 (mg/mL) Antihemolytic activity IC50 (mg/mL)

Methanolic extractSeed 18.44 ± 0.26a 116.22 ± 3.76a 0.143 ± 0.020a 0.121 ± 0.027aGreen husk 30.07 ± 0.75b 50.18 ± 2.69b 0.412 ± 0.025b 0.127 ± 0.014aLeaf 27.70 ± 3.51a,b 94.39 ± 5.63c 0.199 ± 0.023c 0.060 ± 0.005b

Petroleum ether extractSeed 36.14 ± 1.11a ND � 0.225 ± 0.057Green husk 0.59 ± 0.04b 25.06 ± 2.26a 1.479 ± 0.117a �Leaf 1.07 ± 0.34b 27.92 ± 1.40b 2.921 ± 0.740b �

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Fig. 1. Scavenging effect of walnut seed, green husk and leaf methanolic (A) andpetroleum ether (B) extracts on the DPPH radical. Each value represents themean ± SD of three independent assays.

444 M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447

2006; Yang et al., 2009). In agreement with those reports, in thisstudy the EC50 values of methanolic extracts obtained from theDPPH assay were correlated significantly and inversely with the to-tal phenolic contents (R2 = 0.943, P < 0.05), suggesting that pheno-lics in these extracts comprise an important portion of theantioxidant activity. As expected, for walnut petroleum ether ex-tracts no significantly correlation was established. Of note, theDPPH radical scavenging capacity of the lipid fraction of the walnutkernel and walnut oil has already been evaluated (Savage et al.,1999; Espín et al., 2000), and it was suggested that antioxidantcapacity was mainly due to the presence of tocopherols.

3.3. Inhibition of AAPH-induced oxidative hemolysisby walnut extracts

Erythrocytes are considered as major targets for free radical at-tack owing to the presence of both high membrane concentrationof polyunsaturated fatty acids and the oxygen transport associatedwith redox active hemoglobin molecules, which are potent pro-

moters of reactive oxygen species (ROS) (Ajila and Rao, 2008).Thus, in vitro oxidative hemolysis of human erythrocytes was usedhere as a model to study the free radical-induced damage of bio-logical membranes and the protective effect of walnut methanolicand petroleum ether extracts. For this study, AAPH was used as thefree-radical initiator to induce oxidative damage in erythrocytes.Thermal decomposition at physiological temperature of AAPH gen-erates peroxyl radicals (ROO�) in the aqueous phase (Niki, 1990),which can attack the erythrocyte membrane to induce lipid perox-idation. Since peroxidation of membrane lipids is a free-radicalchain reaction, the erythrocyte membrane is quickly damaged,leading to hemolysis.

Fig. 2 shows the antihemolytic effects of walnut seed, greenhusk and leaf methanolic extracts (50–200 lg/mL) on humanerythrocytes exposed to the water-soluble radical initiator AAPH.Erythrocytes incubated at 37 �C as a 2% suspension in PBS (controlsamples) were stable, with little hemolysis observed within 4 h.When AAPH was added to the erythrocyte suspension, hemolysisinduction was time-dependent. The hemolysis is lagged, indicatingthat endogenous antioxidants in the erythrocytes, namely glutathi-one, tocopherol, ascorbate and enzymes, such as catalase andsuperoxide dismutase, can efficiently quench radicals to protectthem against free radical-induced hemolysis, as described previ-ously (Zou et al., 2001). All methanolic extracts significantly pro-tected the erythrocyte membrane from hemolysis induced byAAPH in a concentration- and time-dependent manner (Fig. 2),being leaf extract the most efficient one (IC50 value of0.060 ± 0.005 mg/mL; P < 0.05) (Table 1). Of note that when thecells were incubated with extracts of walnut seed, green husk orleaf alone (without AAPH) at the highest concentration tested(200 lg/mL), hemolysis was maintained at a background level sim-ilar to that in the control samples (data not shown). Comparison ofresults based on 50% hemolysis inhibitory effectiveness did not re-veal any statistically significant difference between walnut seedand green husk extracts after 3 h of incubation (0.121 ±0.027 mg/mL and 0.127 ± 0.014 mg/mL, respectively).

As shown in Fig. 3, petroleum ether extract obtained from wal-nut seed presented a weaker protective effect against AAPH-in-duced hemolysis (IC50 value of 0.225 ± 0.057 mg/mL, Table 1) incomparison with walnut methanolic extracts. For the tested con-centrations (200–1000 lg/mL) both green husk and leaf petroleumether extracts provided no protection against AAPH-induced oxi-dative hemolysis. We also performed additional experiments usinghigher concentrations (1000–4000 lg/mL) of green husk and leafpetroleum ether extracts, but those higher concentrations inducedsignificant hemolysis when compared to control samples (data notshown). These results are in accordance with those of DPPH assay,indicating that walnut polyphenolic compounds have strongerantioxidant properties than the non-polar antioxidants (mainlytocopherols) present in the lipid extract.

It is known that polyphenols enhance red blood cell resistanceto oxidative stress both in vitro and in vivo (Youdim et al., 2000;

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Fig. 2. Effects of walnut seed (A), green husk (B) and leaf (C) methanolic extracts onAAPH-induced hemolysis in erythrocytes. An erythrocyte suspension at 2% hemat-ocrit was preincubated with extracts at the indicated concentrations for 30 min at37 �C. The cell suspension was then incubated with 50 mM AAPH for 4 h at 37 �C. Inall experiments, control erythrocytes (incubated with PBS only) and AAPH-treatederythrocytes (incubated with 50 mM AAPH) were used. Values are expressed as themean ± SEM of four independent experiments. *P < 0.05, as compared with AAPH atrespective time, #P < 0.05, as compared with control at respective time.

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Fig. 3. Effect of walnut seed petroleum ether extract on AAPH-induced hemolysis inerythrocytes. An erythrocyte suspension at 2% hematocrit was preincubated withthe extracts at the indicated concentrations for 30 min at 37 �C. The cell suspensionwas then incubated with 50 mM AAPH for 4 h at 37 �C. In all experiments, controlerythrocytes (incubated with PBS only) and AAPH-treated erythrocytes (incubatedwith 50 mM AAPH) were used. Values are expressed as mean ± SEM of fourindependent experiments. *P < 0.05, as compared with AAPH at respective time,#P < 0.05, as compared with control at respective time.

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ol)

Fig. 4. Inhibition of proliferation of A-498, 769-P and Caco-2 human cancer celllines by methanolic extracts of walnut seed, green husk and leaf. Cells were exposedto extracts at 500 lg/mL for 48 h. Values are expressed as mean ± SD of threeindependent experiments, performed in triplicate. Bars marked with differentletters, within each cancer cell line, are significantly different (P < 0.05).

M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447 445

Costa et al., 2009). Antioxidant properties of polyphenols areattributed mainly to their free radical scavenging and metal-che-

lating properties (Bors et al., 1990; van Acker et al., 1998). In ourerythrocyte cell model, these phytochemicals present in the incu-bation medium may quench peroxyl radicals in the aqueous phasebefore these radicals attack the lipid molecules of the erythrocytemembrane, thereby break the free-radical chain reaction and ulti-mately oxidative hemolysis.

Walnuts are rich in substances such as ellagic acid, a knownpolyphenol, tocopherols (essentially c-tocopherol), fiber, melato-nin, phytosterols, folate, tannins, and other polyphenols (Andersonet al., 2001; Reiter et al., 2005; Li et al., 2006). The high content ofpolyunsaturated fatty acids (linoleic and linolenic acid) and vita-min E in walnuts has been proposed to exert anti-atherogenic ef-fect and to decrease the risk of heart disease (Zambón et al.,2000; Pereira et al., 2008). However, there has been very littleinvestigation into the contribution of walnut non-nutrient phyto-chemicals to antioxidative protection and reduction in cardiovas-cular risk (Blomhoff et al., 2006). Our results strengthen thatpotential of walnut phenolics to act as preventive agents in cardio-vascular diseases should be furthermore investigated.

3.4. Human cancer cell antiproliferative activity of walnut extracts

The most effective extracts in antioxidant assays were furtherstudied for antiproliferative activities. Therefore, walnut seed,green husk and leaf methanolic extracts were evaluated for theirability to inhibit the growth of human renal (A-498 and 769-P)and colon (Caco-2) cancer cell lines. There was a considerable dif-ference in the sensitivity of these cell lines to walnut extracts(500 lg/mL) as shown in Fig. 4. The A-498 cells were in averageinhibited to a higher degree than 769-P and Caco-2 cells. The inhib-itory effect for the highest concentration of the extracts was in therange of 78–91% inhibition compared to controls (average 83%) forthe A-498 cells, of 33–68% inhibition (average 52%) for the 769-Pcells, and 23–78% inhibition (average 47%) for the Caco-2 cells.Fig. 5 presents the concentration effectiveness of walnut extractson viability of A-498, 769-P and Caco-2 cells. Five different concen-trations of each extract (31.25, 62.5, 125, 250, and 500 lg of ex-tract/mL) were applied. All extracts inhibited human renal andcolon cancer cell growth in a concentration-dependent manner,although there was no significant difference between the threelowest concentrations (31.25–125 lg/mL). Treatment of A-498cells with walnut leaf extracts produced the greatest concentra-tion-dependent response, from 16% to 91% cell growth inhibitionat concentrations ranging from 31.25 to 500 lg/mL, followed bythose of green husk extracts (from 17% to 79% inhibition), and seedextracts (from 22% to 78% inhibition) (Fig. 5A). In 769-P cells, treat-ment with these extracts resulted in levels of cell growth inhibition

A498 Cell line

Seed Green husk Leaf0

20

40

60

80

100

12031.25 μg/mL62.50 μg/mL125 μg/mL250 μg/mL500 μg/mL

aa

aa

b

a a a

b

aba

aa,b

c

Cell

prol

ifera

tion

(% o

f con

trol)

769P Cell line

Seed Green husk Leaf0

20

40

60

80

100

12031.25 μg/mL62.50 μg/mL125 μg/mL250 μg/mL500 μg/mL

aa a,b

a,b

b

aa,b

a,bb

c

a a,ba,bb,cc

Cel

l pro

lifer

atio

n (%

of c

ontr

ol)

Caco-2 Cell line

Seed Green husk Leaf0

20

40

60

80

100

12031.25 μg/mL62.50 μg/mL125 μg/mL250 μg/mL500 μg/mL

a a aa,b

b

a aa

b

c

a a,ba,b b

b,c

Cel

l pro

lifer

atio

n (%

of c

ontr

ol)

A

B

C

Fig. 5. Concentration effectiveness of walnut methanolic extracts on proliferationof A-498 (A), 769-P (B) and Caco-2 (C) cells. Five concentrations of extracts (31.25,62.5, 125, 250, and 500 lg of extract/mL) were applied for 48 h. Values arepresented as mean ± SD of three independent experiments, performed in triplicate.Bars marked with different letters, within each sample, are significantly different(P < 0.05).

Table 2IC50 values (mg of methanolic extract/mL) for antiproliferative activity of seed, greenhusk and leaf extracts towards A-498, 769-P and Caco-2 cells. Values are presented asmean ± SEM of three independent experiments, performed in triplicate.

Samples Antiproliferative activity IC50 (mg/mL)

A-498 769-P Caco-2

Seed 0.291 ± 0.018 >0.500 >0.500Green husk 0.285 ± 0.051 0.496 ± 0.071 >0.500Leaf 0.226 ± 0.053 0.352 ± 0.055 0.229 ± 0.014

446 M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447

ranging from 1% to 68% with leaf extracts, followed by 8–54% withgreen husk extracts and 4–33% with seed extracts (Fig. 5B). InCaco-2 cells, the level of cell growth inhibition observed with leafextracts ranged from 13% to 78%, followed by 15–41% with greenhusk extracts, and 0–23% with seed extracts at concentrationsranging from 31.25 to 500 lg/mL for 48 h (Fig. 5C). The IC50 valuesobtained for antiproliferative activities of seed, green husk and leafextracts toward the growth of A-498, 769-P, and Caco-2 cellsin vitro are presented in Table 2. All extracts exhibited similargrowth inhibition activity toward A-498 cells (IC50 values between226 and 291 lg/mL), while walnut leaf extracts showed lower IC50

values signifying a higher antiproliferative efficiency toward both769-P and Caco-2 cells (IC50 values of 352 and 229 lg/mL, respec-tively) than green husk or seed extracts (Table 2). The IC50 valuestoward 769-P cells of seed extracts and toward Caco-2 cells ofgreen husk and seed extracts could not be determined at the con-centrations of extracts tested in this experiment (found to be great-er than 500 lg/mL).

J. regia species has been claimed to possess anticancer activities(Kaur et al., 2003; Hardman and Ion, 2008). Yang et al. (2009) dem-onstrated recently a strong antiproliferative capacity of walnut

seed extract against human HepG2 liver and Caco-2 colon cancercells. The anticancer effects of these nuts are usually attributedto their chemical composition. Walnuts (seeds) contain compo-nents that may slow or prevent cancer growth including omega 3fatty acids (Berquin et al., 2008), vitamin E (mainly the c-tocoph-erol form) (Fleshner, 2002; Spaccarotella et al., 2008), phytosterols(Bradford and Awad, 2007), ellagic acid (Han et al., 2006; Lossoet al., 2004), gallic acid and flavonoids namely quercetin (Yáñezet al., 2004; Kawaii et al., 1999), carotenoids (Bhuvaneswari andNagini, 2005), and melatonin (Srinivasan et al., 2008). Recent stud-ies conducted on cell cultures and animal models seem to indicatethat polyphenols are the main phytochemicals with antioxidantand antiproliferative properties of higher plants (Silva et al.,2004, 2008; Giada and Filho, 2006; Fiorentino et al., 2008; Zhanget al., 2008; Mertens-Talcott et al., 2006). These molecules mightact as cancer-blocking agents, preventing initiation of the carcino-genic process and as cancer-suppressing agents, inhibiting cancerpromotion and progression (Russo, 2007). In detail, polyphenolscan block cancer either through: (i) suppression of nuclear fac-tor-kB (NF-kB) activation; NF-kB is a nuclear transcription factorthat regulates the expression of various genes involved in inflam-mation and carcinogenesis and its activation induces transcrip-tional upregulation of the genes involved in cell-cycleprogression; (ii) suppression of activator protein-1 (AP-1) tran-scription factor activation; AP-1 activity is increased by several tu-mor promoting agents and its inhibition is a recognized moleculartarget in chemoprevention; (iii) suppression of mitogen activatedprotein kinases (MAPK); (iv) suppression of protein kinases (PK),namely PKC; (v) suppression of growth-factor receptor (GFR)-med-iated pathways; (vi) cell cycle arrest and induction of apoptosis;(vii) antioxidant (protecting pre-malignant cells from oxidativebreakage of cellular DNA) and anti-inflammatory effects; and (viii)suppression of angiogenesis (Bonfili et al., 2008; Fresco et al.,2006). In this study, the inhibition of cancer cell proliferation bywalnut extracts was not correlated with the total phenolic con-tents in the extracts tested, suggesting that a specific phytochem-ical or a class of phytochemicals in extracts may be responsible fortheir antiproliferative activities. Alternatively, taking into consid-eration the complexity of the mechanisms proposed for their che-mopreventive properties, it is likely that anticarcinogenic effectsattributed to polyphenols may be based on synergistic, additive,or antagonistic interactions of many compounds present in thesefoods (Mertens-Talcott et al., 2005; Liu, 2003). Corroborating this,currently available data suggest that some extracts may showgreater effects than an individual constituent, implying that com-binations of constituents present in plant extracts may be highlyimportant in the final biological activity.

In conclusion, the results of this study represent the first evi-dence that walnut seed, green husk and leaf methanolic extractspossess effective antihemolytic and human renal cancer cell anti-proliferative activities. These properties seem more likely relatedto its phenolic constituents. It is therefore suggested that J. regiamay be used as an inexpensive and easily accessible source ofeffective natural antioxidants and chemopreventive agents, and fu-ture clinical investigations on this medicinal plant should beencouraged.

M. Carvalho et al. / Food and Chemical Toxicology 48 (2010) 441–447 447

Conflict of Interest

The authors declare that there are no conflicts of interest.

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