antioxidant, anti-inflammatory, and antiproliferative activities of organic fractions from the...
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Antioxidant, anti-inflammatory, andantiproliferative activities of organic fractions fromthe Mediterranean brown seaweed Cystoseirasedoides
Lamia Mhadhebi, Audrey Laroche-Clary, Jacque Robert, andAbderrahman Bouraoui
Abstract: The present study was conducted to evaluate the antioxidant, anti-inflammatory, and antiproliferative activities oforganic fractions from Cystoseira sedoides (Desfontaines) C. Agardh. Various fractions of C. sedoides (chloroform (F-CHCl3),ethyl acetate (F-AcOEt), and methanol (F-MeOH)) were screened for total phenol content, as well as antioxidant activ-ity, using the stable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), and assays for determining the reducing power ofthese fractions. The anti-inflammatory properties of these fractions were assessed using the carrageenan-induced rat pawoedema model. The antiproliferative activity of C. sedoides fractions was evaluated on normal Madin–Darby caninekiney (MDCK), and fibroblast cells and on cancer cell lines (A549, MCF7, and HCT15), using the ability of the cellsto metabolically reduce 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) formazan dyes. The F-CHCl3and F-AcOEt fractions showed significant total phenolic content at 55.09 and 61.30 mg gallic-acid equivalent/g driedsample, respectively. Using the DPPH method, the F-CHCl3 and the F-AcOEt fractions exhibited the strongest radicalscavenging activity, with IC50 120 µg/mL for F-CHCl3 and 121 µg/mL for F-AcOEt, which approaches the activity ofthe powerful antioxidant standard, Trolox (IC50 = 90 µg/mL). The reducing power of the samples was in the followingorder: F-AcOEt > F-CHCl3 > F-MeOH fraction. The F-CHCl3 and F-AcOEt fractions of C. sedoides tested at differentdoses (25 and 50 mg/kg, intraperitoneally (i.p)), exhibited a dose-dependent reduction of rat paw oedema. The percent-age of inhibition of oedema, 3 h after carrageenan injection, ranged from 67.71% to 73.49% and from 67.74% to74.58%, for F-CHCl3 and F-AcOEt, respectively. Their effects are comparable with that of lysine acetylsalicylate(300 mg/kg body mass; i.p.), which is used as a reference drug with the ability to inhibit oedema by 66.14%. Our re-sults revealed that the F-CHCl3 and F-AcOEt fractions from C. sedoides showed important antiproliferative propertiestowards all of the cancer cell lines studied here, as judged by their IC50 values, which ranged from 52.6 to 66.5 µg/mL forA549; 22.4 to 70.2 µg/mL for MCF7, and 250.6 to 255.3 µg/mL for HCT15. Moreover, no visible destruction or alter-ation of normal cells was observed, even at 500 µg/mL F-CHCl3 or F-AcOEt. These results suggest that C. sedoidesfractions might be used as a potential source of natural antioxidant, anti-inflammatory, and antitumor agents. The purifi-cation and determination of the chemical structures of the compounds in these active fractions are under investigation.The results could provide a compound(s) with a promising role in future medicines and nutrition, when used either asa drug or a dietary supplement.
Key words: Cystoseira sedoides, antioxidant, anti-inflammatory activity, antiproliferative activity.
Résumé : La présente étude a eu pour objectif d’évaluer les activités antioxydantes, anti-inflammatoires et antiprolifératives deCystoseira sedoides. Nous avons examiné diverses fractions de C. sedoides, chloroforme (F-CHCl3), acétate d’éthyle (F-AcOEt)et méthanol (F-MeOH), afin de déterminer la teneur phénolique totale, l’activité antioxydante au moyen du radicalstable 1,1-diphényl-2-picrylhydrazyl (DPPH) et le pouvoir réducteur. Nous avons examiné les effets de ces fractionscontre l’inflammation en utilisant le modèle d’œdème de la patte de rat provoqué par le carragénine. Nous avons déter-miné l’activité antiproliférative des fractions de C. sedoides sur des lignées de cellules normales (MDCK et fibroblastes)et des lignées de cellules cancéreuses (A-549, MCF7 et HCT15) par la capacité des cellules à réduire métaboliquement lescolorants 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-formazan. Les fractions F-CHCl3 et F-AcOEtont montré une forte teneur phénolique totale de 55,09 et 61,30 mg d’equivalent acide Gallique/g d’échantillon sec, res-
Received 11 May 2011. Accepted 21 September 2011. Published at www.nrcresearchpress.com/cjpp on 24 November 2011.
L. Mhadhebi. Unité de Recherche des Substances Actives Marines (URSAM), Laboratoire de Pharmacologie, Faculté de Pharmacie deMonastir, Avenue Avicenne, 5000 Monastir, Tunisie; Laboratoire de Pharmacologie des Médicament Anticancéreux, Université VictorSegalen Bordeaux 2, Institut Bergonié, 229 Cours de l’Argonne, 33076 Bordeaux CEDEX, France.A. Laroche-Clary and J. Robert. Laboratoire de Pharmacologie des Médicament Anticancéreux, Université Victor Segalen Bordeaux 2,Institut Bergonié, 229 Cours de l’Argonne, 33076 Bordeaux CEDEX, France.A. Bouraoui. Unité de Recherche des Substances Actives Marines (URSAM), Laboratoire de Pharmacologie, Faculté de Pharmacie deMonastir, Avenue Avicenne, 5000 Monastir, Tunisie.
Corresponding author: Lamia Mhadhebi (e-mail: [email protected]).
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Can. J. Physiol. Pharmacol. 89: 911–921 (2011) doi:10.1139/Y11-093 Published by NRC Research Press
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pectivement. Dans la méthode DPPH, ces fractions ont démontré la plus importante activité de piégeage des radicauxavec une CI50 = 120 µg/mL et CI50 = 121 µg/mL, respectivement, ce qui s’est approché de l’activité du puissant antio-xydant de référence, Trolox (CI50 = 90 µg/mL). Le pouvoir réducteur a été le suivant : F-AcOEt > F-CHCl3 > F-MeOH.Les fractions F-CHCl3 et F-AcOEt, testées à différentes doses (25 et 50 mg/kg par voie intrapéritonéale (i.p.)), ont ré-duit l’œdème de la patte de rat en fonction de la dose administrée. Le pourcentage d’inhibition de l’œdème, 3 h aprèsl’injection de carragénine, s’est situé entre 67,71 % et 73,49 % et entre 67,74 % et 74,58 %, respectivement. Leurs ef-fets ont été comparables à ceux du médicament de référence, acétylsalicylate de lysine (300 mg/kg, i.p.), qui a causé61,14 % de l’inhibition de l’œdème. Nos résultats ont révélé que les fractions F-CHCl3 et F-AcOEt de C. sedoides ontdémontré un fort potentiel antiprolifératif sur toutes les lignées de cellules cancéreuses examinées, comme indiqué parleur CI50 et leurs valeurs, situées entre 52,6 et 66,5 µg/mL, 22,4 et 70,2 µg/mL, et 250,6 et 255,3 µg/mL, respective-ment. De plus, nous n’avons observé aucune destruction ou modification visible sur les lignées de cellules normales,même à 500 µg/mL. Ces résultats donnent à penser que les fractions de C. sedoides pourraient être utilisées commeune source potentielle d’agent naturel antioxydant, anti-inflammatoire et antitumoral. La purification et la déterminationdes structures chimiques des composés de ces fractions actives sont à l’étude. Elles pourraient avoir un rôle prometteuren médecine et en nutrition comme médicament ou additif alimentaire.
Mots‐clés : Cystoseira sedoides, antioxydant, activité anti-inflammatoire, activité antiproliférative.
[Traduit par la Rédaction]
Introduction
Although enormous effort has been invested in the detec-tion and isolation of natural products of terrestrial plant ori-gin, very little effort has gone into the exploration of the seaas source of potential drugs. Recently, marine animals havebeen reported to contain new biologically active substancesnot found in terrestrial animals (Gunaskera et al. 1990).Moreover, seaweeds and inverterbrates may contain specialdefense factors that differ from the immune systems of highlydeveloped vertebrates (da Silva et al. 2006). In the last 3 dec-ades, secondary metabolites produced by marine organismshave been investigated for their biological and pharmacologi-cal potential (Tziveleka et al. 2003; Bhadury et al. 2006), andhave received much attention from pharmaceutical companiesfor use in drug development.Seaweeds are an excellent source of bioactive compounds
that demonstrate a broad range of biological activities, e.g.,anti-inflammatory, antibiotics, antiviral, cytotoxic, and anti-mitotic activities (Naqvi et al.1980; Bhosale et al. 2002).Among these compounds, polyphenols (Naqvi and Glom-
bitza 1986), polysaccharides (Kwon and Nam 2007), mero-terpenoids (Valls et al. 1993), and terpenoids (Culioli et al.2004) are considered as promising bioactive molecules in thesearch for potentially therapeutic drugs.Substances that currently receive the most attention from
pharmaceutical companies or from academic researchers fordrug development or for drug design include fucoidans, agroup of sulfated polysaccharides purified from brown algae,possessing a variety of pharmacological effects, including anti-cancer and anti-inflammatory properties (Myers et al. 2010;Culioli and Abu-Ghannam 2011). Other substances biosynthe-sized by algae with an economic impact on food science and inhuman health include carotenoids, natural pigments used asantioxidant compounds reducing the incidence of many dis-eases, especially those mediated by light (Cantrell et al. 2003).Many compounds such as terpenoids, alkaloids, and steroidshave been isolated from different species of the Mediterraneanbrown algae of the genus Cystoseira, but few studies on thepharmacological properties of these compounds have been
published (Combaut et al. 1980; Banaigs et al. 1983; Amicoet al. 1990). However, despite the diversity in quality andquantity of the Tunisian Mediterranean-coast flora, with itslarge numbers of marine organisms and seaweeds, most ofthem have not yet been investigated for pharmacologicaland biological activities (Ismail et al. 2008; Dellai et al.2010). Therefore, the objective of this research was to in-vestigate the antioxidant, anti-inflammatory, and antiprolifer-ative activities of the organic fractions from brown seaweedCystoseira sedoides (Desfontaines) C. Agardh, namely, thechloroform (F-CHCl3), ethyl acetate (F-AcOEt), and metha-nol (F-MeOH) extracted fractions.
Materials and methods
Sample collectionCystoseira sedoides was collected from the Mediterranean
in various areas of the coastal region of Monastir (Tunisia),in June 2007, from a depth of 1–3 m. Identification of speci-mens was carried out in the National Institute of Marine Sci-ences and Technologies (INSTM), Salambô, Tunisia. Avoucher specimen has been deposited in the Department ofPharmacology, Monastir University. After collection, the sea-weeds were rinsed with fresh water to remove associated de-bris and epiphytes. The cleaned material was then air dried inthe shade at 30 °C. The fully dried samples were then finelypowdered and stored at –20 °C until use.
Chemicals and reagentsCarrageenan (BDH Chemicals, Ltd., Poole, UK), lysine
acetylsalicylate (ASL), dimethylsulfoxide (DMSO), Dulbecco’smodified Eagle’s minimum essential medium (DMEM),fetal bovine serum (FBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), cisplatin, penicillin,and streptomycin, were purchased from Sigma Chemicals(Berlin, Germany). DPPH (1,1-diphenyl-2-picrylhydrazyl),Folin–Ciocalteu reagent, gallic acid (GA), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylicacid) wereobtained from Sigma–Aldrich Chimie (Saint-Quentin-Fallavier,France).
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Preparation of the fractionsFor extraction of the bioactive compounds from shade-
dried seaweeds, 600 g of finely powdered algal material waspacked in small bags (5 cm × 10 cm) of Whatman filter pa-per No. 1, and all bags were sealed and soaked in a methanolbath for 48 h. The organic fractions were concentrated to“solvent free” by evaporation in a rotary vacuum evaporator(Buchi, B-480) at 40 °C.To localize the active fraction, the methanol crude extract
was submitted to subsequent extraction with different organicsolvents in order of increasing polarity: chloroform, ethylacetate, and methanol, to give the 3 fractions (i) chloroform(F-CHCl3), (ii) ethyl acetate (F-AcOEt), and (iii) methanol(F-MeOH). Solvents were removed from recovered fractionsusing a rotating evaporator, at 40 °C. The fractions werestored at –20 °C until use. The different fractions were dilutedto the desired final concentration immediately prior to use.
Antioxidant activity
Total phenolic contentThe total phenolic content (TPC) of the organic fractions
of C. sedoides were estimated following the method of Tagaet al. (1984). Briefly, a 100 µL aliquot of sample was mixedwith 2.0 mL of 2% Na2CO3 and allowed to stand for 2 min at
room temperature. After incubation, 100 µL of 50% Folin–Ciocalteu’s phenol reagents were added, and the reactionmixture was mixed thoroughly and allowed to stand for30 min at room temperature in the dark. Absorbance of allsample solutions was measured at 720 nm using a spectro-photometer (Jenway 6505 UV–Vis). A calibration curve ofgallic acid ranging from 0.05 to 1 mg/mL was prepared, andthe TPC was standardized against gallic acid and expressedin milligrams of gallic acid equivalent per gram of sampleon a dry mass basis. All determinations were performed intriplicate.
DPPH radical scavenging activityAntioxidant activity (AOA) of the organic fractions was
measured in terms of radical scavenging ability by using thestable radical 1,1-diphenyl-2-picrylhydrazyl (DPPH), as de-scribed by (Kim et al. 2002). Each sample stock solution(1 mg/mL) was diluted to a final concentration of 500, 250,100, 50, or 10 µg/mL in ethanol. A total of 0.5 mL of30 mmol/L DPPH–ethanol solution was added to 0.5 mL ofsample solution at different concentrations, and allowed to re-act at room temperature. After 30 min, the absorbance (A)was measured at 520 nm. Radical scavenging activity was ex-pressed as the inhibition percentage and was calculated usingthe following formula:
½1� %Radical scavenging ¼ 1� ½ðAsample � Asample blankÞ=Acontrol� � 100:
where the Acontrol is the absorbance of the control (DPPH so-lution without sample), the Asample is the absorbance of thetest sample (DPPH solution plus test sample), and the absor-bance sample blank is the absorbance of the sample only(sample without DPPH solution). The synthetic antioxidantTrolox was used as positive control. The concentration offraction required to reduce DPPH radicals by 50% (IC50)was calculated by linear regression of the plots, where theabscissa represented the concentration of tested marine algalfractions, and the ordinate represented the average percent ofscavenging capacity. DPPH was expressed in terms of Troloxequivalent antioxidant capacity (TEAC), which was calcu-lated based on its concentration of fraction required to reduceDPPH radicals by 50% (IC50), as follows: TEAC (mg Trolox/100 g) = IC50 (Trolox) / IC50 sample × 100. The mean va-lues were obtained from experiments conducted in triplicate.
Ferric-reducing antioxidant powerThe ferric reducing power of the 3 organic fractions of
C. sedoides was determined following the method of Oyaizu(1986). Briefly, 1.0 mL of each sample dissolved in distilledwater was mixed with 2.5 mL of phosphate buffer (0.2 mol/L,pH 6.6) and 2.5 mL potassium ferricyanide (1%). The reac-tion mixture was incubated for 20 min at 50.0 °C. After in-cubation, 2.5 mL of trichloacetic acid (10%) was added andthe mixture was centrifuged for 10 min. Finally, 2.5 mL ofthe upper layer was mixed with 2.5 mL of distilled waterand 0.5 mL of FeCl3 (0.1%). The solution was incubated atambient temperature for 30 min for colour development.Absorbance of all the sample solutions was measured at700 nm and compared with a gallic acid calibration curve.
The data were presented as milligrams of gallic-acid equiv-alent per gram of seaweed material (mg GAE/g dried sam-ple). A greater value of GAE related to greater reducingpower of the sample.
Anti-inflammatory activity
AnimalsFor anti-inflammatory evaluation of the organic fractions
of F-CHCl3, F-AcOEt, and F-MeOH, male adult Wistar ratsweighing 150–170 g were obtained from the Pasteur Institute(Tunis, Tunisia). The rats were housed in polypropylenecages, and were kept for 2 weeks prior to the study, for accli-matization to the conditions in the animal room: a 12 h(light) – 12 h (dark) cycle at 22 ± 2 °C, on a standard ratpellet diet and water, ad libitum.Before the day of assay, Wistar rats were fasted overnight
with free access to water. Housing conditions and in vivo ex-periments were conducted according to the guidelines estab-lished by the European Union on Animal Care (CFE Council(86/609).
Carrageenan induced rat paw oedemaThe anti-inflammatory activity of our organic fractions (F-
CHCl3, F-AcOEt, and F-MeOH) on carrageenan-induced pawoedema was determined according to the methods of Winteret al. (1962). Oedema was induced by injecting 0.05 mL of1% carrageenan subcutaneously into the subplantar region ofthe left hind paw.
Wistar rats were divided into 3 groups of 6 animals. Thetest group received the organic fractions from C. sedoides
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(doses of 25 or 50 mg/kg). F-CHCl3, F-AcOEt, and F-MeOH, were dissolved in 1% DMSO – 99% saline water,and administered intraperitoneally (i.p.). The reference groupreceived ASL (300 mg/kg, i.p). The control group receivedthe vehicle (2.5 mL/kg; 1% DMSO – 99% saline water, i.p.).All samples were administered 30 min before the injection ofcarrageenan. Measurement of paw size was done by means ofa volume-displacement technique, using a plethysmometer(model 7150, Ugo Basile, Italy), immediately before carra-geenan injection and 1, 2, 3, 4, and 5 h after carrageenan in-jection. Percent inhibition from our anti-inflammatory testswere obtained for each group using the following ratio:
½2� ½ðV t � VoÞcontrol � ðV t � VoÞtreated� � 100=ðV t � VoÞcontrolWhere Vt is the average volume for each group at different
hours after treatment, and Vo is the average volume obtainedfor each group before any treatment (Lanhers et al. 1991).
Antiproliferative activity
Cell lines and culture conditionsThe human tumor cell lines A549 (lung cell carcinoma),
HCT15 (colon cell carcinoma), and MCF7 (breast adenocar-cinoma), as well as normal cells (Mardin–Darby canine kid-ney (MDCK) and rat fibroblast) were purchased from theAmerican Type Culture Collection (ATCC; Manassas, Vir-ginia, USA).Freshly trypsinized cells were seeded and grown in Dul-
becco’s modified Eagle’s minimum essential medium(DMEM) supplemented with 10% (v/v) fetal bovine serum(FBS), and 1% penicillin–streptomycin, all obtained from Bi-ochrom AG (Berlin, Germany). They were grown in flasks(Nunc, Denmark) at 37 °C in a humidified atmosphere con-taining 5% CO2.Cells were replicated every 2–4 days, and the medium was
changed between replications. An aliquot of each fraction wasdissolved and sterilized by filtering through 0.22 µm microbio-logical filters (Whatman, UK) and kept at 4.0 °C until analysis.
Viability assayThe potential effects of the fractions on cell viability were
investigated according to previously reported conditions(Mosmann 1983; Hu and Robert 1995) using the MTT assay(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-mide; Sigma–Aldrich) as an indicator of metabolically activecells (Varache-Lembège et al. 2008). However, the develop-ment of this rapid colorimetric assay, which relies on theability of mitochondrial dehydrogenase enzymes to convertMTT to a purple formazan precipitate, has simplified largescale screening of cells and drugs. The formazan crystals aredissolved, and the optical density measured using a microplatereader. The use of MTT has thus become the method of choicebecause of its simplicity and adaptability to automation.Concentrations ranging from 25–500 µg/mL DMEM of the
3 organic fractions of C. sedoides were prepared from thestock solutions by serial dilution in DMEM to give a volumeof 200 µL in each well of a microplate reader (96-well plate).The final concentration of DMSO in the culture medium wasmaintained at 1% (v/v) to avoid toxic effects from the solvent.A known number (103) of A549, HCT15, or MCF7 cells
was transferred into 96-well plates (Nunc, Denmark) in200 µL of culture medium, and incubated for 24 h before theaddition of test compounds. After 24 h, cells were exposed,at 37 °C, to known concentrations of the different organicfractions to be tested.After drug exposure, cells were washed with phosphate-
buffered saline water (PBS) and then reincubated in freshculture medium for a further 48 h, then the culture mediumwas removed and 200 µL of MTT reagent (diluted in culturemedium, 0.5 mg/mL) was added. Following incubation for4 h, the MTT medium was removed and DMSO (200 µL)was added to dissolve the formazan crystals.Absorbance values were measured with a microplate
reader (Bio Tek EL 340, USA) using a test wavelength of570 nm and a reference wavelength of 630 nm. Results wereevaluated by comparing the absorbance of the treated cellswith the absorbance of wells containing cell treated by thesolvent control.Conventionally, cell viability was estimated to be 100% in
the solvent control. All experiments were performed at leasttwice in triplicate.The concentration of substance required for the IC50 was
estimated as that resulting in 50% decrease in absorbance,compared with controls incubated simultaneously but withouttest substances.
Data and statistical analysisData were expressed as the mean ± SEM, and statistical sig-
nificance was evaluated using Student’s t test for paired dataand differences were considered significant when p < 0.05.
Results
Evaluation of antioxidant activity
Total phenolic contentThe total phenolic content was measured by the Folin–Cio-
calteu method, and varied widely in the organic fractions ofC. sedoides, ranging from 35.08 ± 0.07 to 61.30 ± 0.05 mgGAE/g dried sample (Table 1). The highest levels of totalphenolic contents were found in F-CHCl3 and F-AcOEt frac-tions of C. sedoides, with 61.30 ± 0.05 and 55.0 ± 0.11 mgGAE/g dried sample, respectively. Whereas, the TPC in theF-MeOH fraction was only 35.0 ± 0.07 mg GAE/g driedsample. The TPC of the samples decreased in the followingorder: F-AcOEt > F-CHCl3 > F-MeOH fraction.
DPPH radical scavenging activityTo examine the antioxidant effect of organic fractions of
C. sedoides, the free radical scavenging activity was meas-ured using the 1,1-diphenyl-2-picrylhydrazyl free radical(DPPH); a stable, free radical, which was added to the totalextract from C. sedoides.These organic fractions were able toreduce the violet DPPH to yellow-coloured diphenyl prilhy-drazine, and the IC50 values were calculated and are pre-sented in Table 1.The F-CHCl3 and F-AcOEt fractions of C. sedoides exhib-
ited significant antioxidant activity with IC50 values of 120and 121 µg/mL, respectively, whereas, F-MeOH exhibitedless activity (IC50 value of 285 µg/mL). The F-CHCl3 and F-AcOEt fractions of C. sedoides have the highest free radical
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scavenging activity, which was found to be comparable withthat of Trolox (IC50 = 90 ± 0.02 µg/mL).
Ferric-reducing antioxidant powerTable 1 shows that greater differences were found in the
total antioxidant capacity among the 3 organic fractions,when measured by the ferric-reducing antioxidant power(FRAP) method. FRAP values for the 3 organic fractionswere found within the range 1.26 ± 0.01 and 4.02 ±0.04 mg GAE/g dried sample. The F-AcOEt fraction of C.sedoides has the highest ability to reduce Fe3+, followed bythe F-CHCl3 fraction. The FRAP values were 4.02 ± 0.04and 3.83 ± 0.08 mg GAE/g dried sample, respectively. TheFRAP value of the F-MeOH fraction of C. sedoides was con-siderably lower (1.26 ± 0.01 mg GAE/g dried sample), aswas the TPC (Table 1).
Anti-inflammatory activityThe anti-inflammatory properties of the organic fractions
of C. sedoides was investigated using the carrageenan in-duced rat paw oedema model. Carrageenan has been widelyused as an inflammagen capable of inducing experimental in-flammation for the screening of compounds possessing anti-inflammatory activity. This phlogistic agent, when injectedlocally into the rat hind paw of the control group, induced asevere inflammatory reaction, discernible within 30 min andpersisting until the end of the measurement duration. Themaximum peak was observed between 3 and 5 h after admin-istration (injection).As shown in Table 2, the organic fractions of C. sedoides
(F-CHCl3, F-AcOEt, and F-MeOH) showed significant anti-inflammatory activity when administered intraperitoneally inthe carrageenan-induced rat paw oedema test.The F-CHCl3 and F-AcOEt fractions from C. sedoides
tested at 25 and 50 mg/kg, i.p., exhibited, in a dose-dependentmanner, a significant inhibitory effect on the rat-paw oe-dema. The percentage of inhibition of oedema 3 h aftercarrageenan injection ranged from 67.71% to 73.49% forthe F-CHCl3 fraction, and from 67.74% to 74.58% for theF-AcOEt fraction. The F-MeOH fraction at 25 and 50 mg/kg,i.p., was less potent than either the F-CHCl3 or the F-AcOEtfractions; for the F-MeOH fraction, the percentages of in-hibition of oedema at 3 h after injection were 50.81% (25mg/kg) and 59.68% (50 mg/kg) . The reference drug ASL,
at 300 mg/kg body mass, i.p, produced 67% inhibition ofrat paw oedema (Table 2).
Antiproliferative activityThe results presented in Table 3, show that the F-CHCl3
fraction possesses a lower IC50 compared with that of theF-MeOH fraction of C. sedoides when tested on the 2 normaland 3 cancer cell lines (p < 0.01). However, the IC50 of theF-CHCl3 fraction of C. sedoides showed significantly higherantiproliferative activity than either the F-AcOEt fraction orcisplatin on the 2 normal and the 3 cancer cell lines (p <0.01).The lower the IC50, the higher the potency of a compound
with respect to the inhibition of cell, growth, cell toxicity,and cell death. Comparison of the evaluated IC50 from thenormal and cancer cell lines treated with the F-CHCl3 andF-MeOH fractions of C. sedoides with that for F-AcOEt andcisplatin (Table 3) showed that the IC50 of the samples on the5celllinesincreasedasfollows:cisplatin<F-AcOEt<F-CHCl3 <F-MeOH. The IC50 values for the 5 cell lines treated withcisplatin decreased as follows: MDCK > fibroblasts >MCF7 > HCT15 > A549. However, the lowest and highestIC50 values belonged to cisplatin and the F-MeOH fraction.Also, the IC50 values for the 5 cell lines treated with theF-MeOH fraction decreased as follows: MDCK > fibro-blast > HCT15 > MCF7 > A549.On the other hand, the IC50 value for the 5 cell lines treated
with the F-AcOEt fraction for increased as follows: fibro-blast < MDCK < MCF7 < HCT15 < A549. As a result, thehighest and lowest cytotoxicity of the F-AcOEt fraction wasdemonstrated towards the A549 cell line (IC50 = 22.4 µg/mL)and fibroblasts (IC50 = 150.3 µg/mL), respectively.The IC50 values for the 5 cell lines treated with the F-MeOH
fraction of C. sedoides increased as follows: MDCK < fi-broblast < MCF7 < HCT15 < A549. However, the highestand lowest cytotoxicity of the F-MeOH of C. sedoides wasdemonstrated towards the A 549 (IC50 = 250.6 µg/mL) andMDCK (IC50 = 420.2 µg/mL) cell lines, respectively.Thus, it can be seen that treatment with organic fractions
of C. sedoides induced concentration-dependent growth in-hibition in cancer cell-lines. Further, after 2 days of treat-ment, no microscopically visible alteration of normal cellswas observed, even at a dose 500 µg/mL. In addition, the vi-ability assay showed no destruction of cell layers.
Table 1. Total phenolic content and the antioxidant activity of the F-CHCl3, F-AcOEt, and F-MeOH fractions from Cystoseira sedoides.
Antioxidant activity
FractionTPC(mg GAE/g dried sample) DPPH* (IC50; µg/mL)
TEAC(mg Trolox/100 g dried sample)
FRAP(mg GAE/g dried sample)
F-CHCl3 55.09±0.11 120±0.14 75.0±0.15 3.83±0.18F-AcOEt 61.30±0.15 121±0.11 74.38±0.19 4.02±0.14F-MeOH 35.08±0.17 285±0.15 31.57±0.12 1.26±0.11ControlTrolox — 90±0.02 — —Note: F-CHCL, chloroform-extracted fraction; F-AcOET, ethyl-acetate-extracted fraction; F-MeOH, methanol-extracted fraction; TPC, total phenolic con-
tent; GAE, gallic-acid equivalent; DPPH, 1,1-diphenyl-2-picrylhydrazyl; TEAC, Trolox equivalent antioxidant capacity; FRAP, ferric-reducing antioxidantpower. Results are expressed as the mean ± SD (n = 3).*DPPH is expressed in terms of Trolox equivalents.
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Figures 1, 2, and 3 represent, respectively, the concentra-tion effectiveness of the F-CHCl3, F-AcOEt, and F-MeOHfractions of C. sedoides on the viability of the cancer celllines A549, HCT15, and MCF7, using an MTT assay. A mi-tochondrial enzyme in living cells, succinate-dehydrogenase,cleaves the tetrazolium ring, converting the MTT to an in-soluble purple formazan. Therefore, the amount of formazanproduced is directly proportional to the number of viablecells. Five different concentrations of each fraction (25, 50,100, 250, and 500 µg/mL) were applied. F-CHCl3 and F-AcOEt fractions of C. sedoides inhibited cell growth in A549,HCT15, and MCF7 cell lines in a concentration-dependentmanner.Treatment with the F-CHCl3 fraction of C. sedoides at con-
centrations ranging from 25 to 500 µg/mL (Fig. 1) producedsignificant cell growth inhibition in A549 (from 26% to 89%)and HCT15 (32% to 91%) cell lines. Whereas, the inhibitionof cell growth for the MCF7 line ranged from 36% to 69%.Treatment with the F-AcOEt fraction of C. sedoides in-
hibited cell growth from 39% to 90% for HCT15 cells, fol-lowed by 25% to 71% inhibition in MCF7 cells, and 27%to 64% inhibition in the A549 cells (Fig. 2). The level ofcell growth inhibition observed with the F-MeOH fractionof C. sedoides, at concentrations ranging from 25 to500 µg/mL, for 24 h (Fig. 3), ranged from 24% to 51% in-hibition in the HCT15 cell line, followed by 29% to 49%inhibition in the MCF7 cell line, and 11% to 50% inhibi-tion in the A549 cell line.
DiscussionThe present study was conducted to evaluate the antioxi-
dant, anti-inflammatory, and antiproliferative activities of or-ganic fractions from C. sedoides. In this study, the F-CHCl3and F-AcOEt fractions from C. sedoides showed remarkableantioxidant activity. In addition, these fractions from C. se-doides showed significant DPPH radical and ferric reducingproperties. Different findings for the amount of the total phe-nolic content in brown seaweeds have been reported, e.g.,from 1.23 to 3.28 mg GAE/g for extracts from Stypocaulonscoparium (López et al. 2011), and from 2.78 to 26 mgGAE/g of the organic fractions from 3 species of brown sea-weed from India (Chandini et al. 2008). Thus, the TPC in thefractions could explain their high antioxidant activities; thepositive correlation between total phenolic contents of analga and its antioxidant activity is well documented (Karawitaet al. 2005). Many researchers have investigated the antioxi-dant properties of seaweeds. Several compounds were identi-fied as antioxidants, including some protective enzymes(Nakano et al. 1995), ascorbic acid (Morgan et al. 1980), lip-ophilic antioxidants (Takamatsu et al. 2003), phlorotannins(Jiménez-Escrig et al. 2001), and catechins (Yoshie et al.2000). Increasing scientific evidence shows that polyphenolsare good antioxidants, are effective in preventing cardiovas-cular and inflammatory diseases, and can also be used aschemoprevention agents for cancer. These molecules mightact as cancer-blocking agents, preventing initiation of the car-cinogenic process and may also act as cancer-suppressing
Table 2. Anti-inflammatory effect from intraperitoneal administration of organic fractions (F-CHCl3, F-AcOEt, and F-MeOH) from Cysto-seira sedoides, and the reference drug lysine acetylsalicylate (ASL) in the carrageenan-induced rat paw oedema test.
Oedema (mL×10–2; mean ± SEM) Oedema inhibition (%)
Samples Dose (mg/kg) 1 h 3 h 5 h 1 h 3 h 5 hControl — 23.67±1.51 63.50±1.97 66.83±1.17 — — —ASL 300 17.38±1.06*** 21.50±1.85*** 19.88±0.83** 26.57 66.14 70.25FractionF-CHCl3 25 18.50±1.38*** 20.50±1.52*** 21.33±1.21** 21.84 67.71 68.80
50 10.67±1.75*** 16.83±1.17** 15.67±0.82*** 54.92 73.49 76.55F-AcOEt 25 18.82±1.53** 20.67±0.58** 24.33±1.53** 20.49 67.74 63.59
50 10.75±0.75** 16.14±1.85*** 15.71±1.70*** 54.58 74.58 76.49F-MeOH 25 18.33±1.63** 31.23±2.66** 31.57±0.72** 22.56 50.81 52.76
50 16.17±0.95*** 25.60±0.82*** 26.50±1.05*** 31.68 59.68 60.34
Note: Control, 2.5 mL/kg of 1% DMSO – 99% saline water; F-CHC3, chloroform-extracted fraction; F-AcOET, ethyl-acetate-extracted fraction; F.MeOH,methanol-extracted fraction. The values represent the mean difference in paw volume ± SEM; n = 6. **, p < 0.01; ***, p < 0.001 compared with thecontrol.
Table 3. In vitro growth inhibition by organic fractions from Cystoseira sedoides ( F-CHCl3; F-AcOEt; and F-MeOH)against 3 human tumor cell lines (A549, lung cell carcinoma; HCT15, colon cell carcinoma; MCF7, breast adenocarci-noma) and normal cells (MDCK, Mardin–Darby canine kidney; rat fibroblast).
Cancer cell lines (IC50±SD, µg/mL) Normal cell lines (IC50±SD, µg/mL)
Samples A549** HCT15** MCF7** MDCK** Fibroblasts**F-CHCl3 52.6±0.003 66.5±0.001 63.2±0.001 182.1±0.025 172.4±0.012F-AcOEt 22.4±0.001 52.5±0.003 70.2±0.001 180.5±0.032 150.3±0.019F-MeOH 250.6±0.005 255.3±0.002 253.8±0.004 420.2±0.027 350.6±0.024Cisplatin 0.1±0.077 1.8±0.013 2.1±0.005 4.4±0.011 3.5±0.021
Note: IC50, 50% inhibition of cell growth; F-CHCl3, chloroform-extracted fraction; F-AcOET, ethyl-acetate-extracted fraction; F.MeOH,methanol-extracted fraction; **, p < 0.01 IC50 for fractions compared with cisplatin.
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Fig. 1. Effects of the chloroform fraction (F-CHCl3) from Cystoseira sedoides on the viability of 3 human tumor cell lines (A549, lung cellcarcinoma; HCT15, colon cell carcinoma, and MCF7, breast adenocarcinoma). Data expressed as percent cell viability compared with thecontrol. Values are the mean ± SEM (n = 3). Statistical significance is based on comparison with the control cells (cancer cells withoutF-CHCl3); **, p < 0.01; ***, p < 0.001.
Fig. 2. Effects of the ethyl acetate fraction (F-AcOEt) from Cystoseira sedoides on the viability of 3 human tumor cell lines (A549, lung cellcarcinoma; HCT15, colon cell carcinoma; and MCF7, breast adenocarcinoma). Data expressed as percent cell viability compared with thecontrol. Values are the mean ± SEM (n = 3). Statistical significance is based on comparison with the control cells (cancer cells withoutF-AcOET); **, p < 0.01; ***p < 0.001.
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agents, inhibiting cancer promotion and progression (Russo2007).In addition, free radicals play a crucial role in the patho-
physiology of human diseases such as inflammation, cancer,and neurodegenerative diseases, and considerable attentionhas been focused on ways to protect against these diseases.Many antioxidants are being identified as anticarcinogens(Ferguson 1994). Using human cervical adenocarcinomacells, Yuan and Walsh (2006) have demonstrated the antioxi-dant and antiproliferative properties of extracts from a varietyof marine algae. Bravo (1998) also noted that there should bea close correlation between TPC and antioxidant and anti-cancer properties.The findings from this study revealed that organic fractions
from C. sedoides, namely, F-CHCl3 and F-AcOEt (at dosesof 25 and 50 mg/kg) produced a marked inhibition oncarrageenan-induced rat paw oedema when compared with ASL(300 mg/kg), which was used as a standard anti-inflammatorydrug for comparison. However the F-MeOH fraction fromC. sedoides was less potent than this reference drug. It isknown that the oedema induced by carrageenan involvesdifferent phases, with the participation of different chemicalmediators, such as histamine, serotonine, kinine prostanoids,and leucotriens (Ward 1994). The anti-inflammatory effectexhibited by the organic fractions of C. sedoides suggeststhat the properties of these fractions could possibly interferewith some of the mediators of inflammation, by eitherinhibiting their production or antagonizing their actions(Olajide et al. 1999). The anti-inflammatory potency of theF-AcOEt and F-CHCl3 fractions of C. sedoides may be due,in part, to a critical TPC contained in these fractions: 61.30and 55.10 mg GAE/g dried sample, respectively.Chemical and biological studies have indicated that the
main substances biosynthesized by brown algae with anti-inflammatory potential include sulfated polysaccharides,phlororotanins, and carotenoids, and it is possible that someof these bioactive compounds present in the organic fractionsare responsible, at least in part, for the anti-inflammatoryproperties of these extracts (Cardozo et al. 2007). Indeed,several pharmacological studies have reported that the sul-fated polysaccharides, fucoidans, present in brown algaepossess anti-inflammatory properties. These compounds arepotent selectin blockers, and have been used experimentallyto prevent inflammatory damage after ischemic events (Rit-ter et al. 1998). Fucoidans have also been shown to inhibitphospholipase A2, an important enzyme in the inflamma-tion cascade (Lomonte et al. 2003), and also appear to in-hibit the functions of macrophages, a predominant sourceof pro-inflammatory factors (Wijesekara et al. 2010). Kimet al. (2009) reported that ethanolic extract of the brown al-gae Ishige okamurae, was effective in inhibiting the produc-tion of 22 inflammatory mediators, such as TNF-a, Il-1b,Il-6, and PG-E2, in Raw 264.7 macrophage cells and by in-activation of NF-cB transcription factor in macrophagesstimulated by lipopolysaccharide.In an open label combined phase I and phase II pilot-scale
study in osteoarthritis of the knee, Myers et al. (2010)showed that when a formulation containing a blend of ex-tracts from 3 different species of brown algae was taken or-ally by patients over a 12 week period, the symptoms ofosteoarthritis decreased, in a dose-dependent manner.Brown seaweed extracts have also been demonstrated to
contain phlorotanins; polyphenolic compounds with anti-inflammatory activity (Myers et al. 2010). Investigationsconducted by Sugiura et al. (2009) demonstrated thatMeOH/CHCl3 extract from Eisenia arborea inhibited in-
Fig. 3. Effects of the methanolic fraction (F-MeOH) from Cystoseira sedoides on the viability of 3 human tumor cell lines (A549, lung cellcarcinoma; HCT15, colon cell carcinoma; and MCF7, breast adenocarcinoma). Data expressed as percent cell viability compared with thecontrol. Values are the mean ± SEM (n = 3). Statistical significance is based on the difference when compared with the control cells (cancercells without F-MeOH); **, p < 0.01; ***, p < 0.001.
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flammatory mediator (histamine and eicosanoids: LTB4,PGD2) release from rat basic leukemia (RBL) cells, andthat phlorotanins and MeOH/CHCl3 extract inhibited the ac-tivity of enzymes (phospholipase A2, cyclooxygenase A2,lipoxygense) involved in eicosanoid synthesis in the arachid-onate cascade.In addition to the polar components (fucoidans and phlor-
otanins), brown algae also produce nonpolar components,such as carotenoids, with anti-inflammatory potential. Re-cently, it has been claimed that fucoxanthin, one of the mostabundant carotenoids isolated from brown algae, exerts anti-inflammatory effects via inhibition of nitric oxide production,in lipopolysaccharide-induced Raw 264.7 macrophage cells(Heo et al. 2010). Considering this and previous studies, wecan deduce that the anti-inflammatory properties of the or-ganic fractions of C. sedoides, as seen acting against experi-mental inflammation induced by carrageenan, are probablyowing to the synergistic effects of polar and nonpolar compo-nents produced by this brown algae.According to our results, the IC50 values for the F-CHCl3,
F-AcOEt, and F-MeOH fractions of C. sedoides, as well asthe anticancer drug cisplatin, were higher for normal celllines than for cancer cell lines. Both the F-CHCl3 andF-AcOEt fractions of C. sedoides exhibited significant anti-proliferative effects against all of the cancer cell lines studied.Chemical and biological investigations have indicated that
the main substances biosynthesized by brown algae that haveantiproliferative and antitumor potential include sulfatedpolysaccharides, phlororotanins, and terpenes. The possibilityexists that some of these bioactive compounds present in theorganic fractions are responsible, at least in part, for the anti-proliferative properties of these extract. Indeed, several stud-ies have reported that fucoidans, the sulfated polysaccharidespresent in brown algae, display antiproliferative properties to-wards cancer cell lines in vitro, as well as inhibitory proper-ties towards tumors grown in mice (Wijesekara et al. 2010),and that they have antimetastatic properties by virtue ofblocking the interactions between cancer cell lines and thebasement membrane (Rocha et al. 2005). Alekseyenko et al.(2007) reported that fucoidans isolated from the brown algaeFucus evanescens and administered at a dose of 10 and25 mg/kg to C57B1/6 mice with transplanted Lewis lung ad-enocarcinoma, potentiated the antimetastatic and antitumoractivities of cyclophosphamide, respectively. It has also beenshown that sulfated polysaccharides inhibit the growth of hu-man gastric adenocarcinoma cells by inducing autophagy, aswell as apoptosis (Myers et al. 2010). The mechanismsthrough which fucoidans exert their antiproliferative effectsare not completely understood because of their remarkablestructural diversity, which entails multiple interactions (El-louali et al. 1993).In addition to its anti-inflammatory properties, phlorotanins,
the polyphenolic compounds of brown seaweeds, also possessantiproliferative properties. Indeed, Kong et al. (2009) reportedthat isolated phloroglucinol derivatives from Ecklonia cavademonstrated a high level of concentration-dependent apoptosisand antiproliferative activity in MCF7 human cancer cells.Using MTT assay, extracts from the brown algae Lamina-
ria japonica have been shown to induce inhibition of humanhepatocellular carcinoma cells (BEL7402) and murine leuke-mic cells (P388). The IC50 of these extracts were >120 µg/mL.
The antiproliferative activity of this extract is associatedwith the total phlorotanin algal content (Yang et al. 2010).In addition to the polar components (fucoidans and phloro-tanins), brown algae also produce nonpolar components,such as terpenes, with antiproliferative potential: the hydro-quinone diterpene mediterraneol from Cystoseira mediterra-nea is an inhibitor of mitotic cell divisions (Francisco et al.1985) and meroterpenes (usneoidone E and Z from Cysto-seira usneoides) have antitumor properties (Urones et al.1992; Fisch et al. 2003). Diterpenes isolated from the meth-anol extract of Padina pavonia collected from the Red Sea,have been reported to show antitumor activities against lungcarcinoma (H460) and liver carcinoma (HepG2) human celllines (Awad et al. 2008). Some authors (Blunt et al. 2005)reported that Cystoseira myrica collected in the Gulf ofSuez, yielded 4 hydroazulene diterpenes: (i) dictyone ace-tate, (ii) dictyol F monoacetate, (iii) isodictytriol monoace-tate, and (iv) cystoseirol monoacetate. Considering thefindings of the reports mentioned above, it is possible thatditerpene compounds from C. sedoides could be responsiblefor the antiproliferative activity measured in our study. Yetothers (Thun et al. 1991; DuBois et al. 1996) have reportedthat there is a close relationship between inflammation andcancer, in which tumor promoters recruit inflammatory cellsto the application site, and cancer development may also actby aggravating inflammation in the tissue and vice versa,and that inflammatory cells are capable of inducing geno-toxic effects (Rosin et al. 1994).
Conclusion
This study revealed that the F-CHCl3 and AcOEt fractionsof C. sedoides possess interesting anti-inflammatory proper-ties associated with significant antiproliferative activity, andthat these particular properties of both the F-CHCl3 andAcOEt fractions of C. sedoides are associated with TPC.These findings confirm other results that have been reportedrecently, relating to different species of brown algae and theirbioactive compounds.In light of these results, it is considered that further chemical
investigations are needed to identify the polar compounds,such as sulphated polysaccharides, in different fractions ofC. sedoides,as well as the nonpolar compounds such as ter-penes and carotenoides associated with anti-inflammatory andantiproliferative properties.
AcknowledgementsThe authors would like to thank Dr. Rafik Ben Said (Na-
tional Institute of Science and Marine Technology (INSTM)of Tunis, Tunisia) for identifying the specimens.
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