New antibacterial and cytotoxic activities of falcarindiol isolated in Crithmum maritimum L.leaf extract
Laetitia Meot-Duros a,*,Stéphane Cérantola b ,Hélène Talarmin c ,Christophe Le Meur d ,Gaëtan Le Floch d ,Christian Magnéa,*
a
Laboratoire d’Ecophysiologie et de Biotechnologie des Halophytes et des Algues Marines,EA 3877(LEBHAM),Institut Universitaire Européen de la Mer,Universitéde Bretagne Occidentale,Technopôle Brest Iroi,Place Nicolas Copernic,29280Plouzané,France b
Service de Résonance Magnétique Nucléaire,Universitéde Bretagne Occidentale,29238Brest Cedex 3,France c
Unitéde Physiologie Comparée et Intégrative,EA 3879,Brest Cedex 3,France d
Laboratoire de Biodiversitéet d’Ecologie Microbienne,EA 3882,ESMISAB,Universitéde Bretagne Occidentale,Technopôle Brest-Iroi,29280Plouzané,France
a r t i c l e i n f o Article history:
Received 15July 2009
消防器材使用培训
什么新什么异Accepted 9November 2009
Keywords:Antibacterial
Crithmum maritimum L.Cytotoxicity Falcarindiol
a b s t r a c t
Bioassay-guided fractionation of a chloroformic extract obtained from Crithmum maritimum leaves led to the chemical isolation of falcarindiol,a polyacetylene widely distributed within the Apiaceae family.Structure of this compound was confirmed by NMR and activity was tracked using a screening microplate bioassay.Falcarindiol strongly inhibited the growth of Micrococcus luteus and Bacillus cereus ,with a min-imum inhibitory concentration (MIC)value of 50l g ml À1.Moreover,this compound showed cytotoxicity against IEC-6cells with an IC 50value of 20l M after 48h of exposition.The results suggest that Crith-mum maritimum could be potentially ud in food manufactures and cosmetology as prervative agents and biopesticides,or in medicine as new antibiotics,confirming the interest in studying halophytic spe-cies as sources of bioactive compounds.
Ó2009Elvier Ltd.All rights rerved.
1.Introduction
三打两建Becau of the increasing pathogen resistance to antibiotics due to the massive u of commercial prervatives,there is a growing demand for new antimicrobial drugs.With that respect,natural crude extracts and biologically active compounds isolated from plant species can be valuable resources for such new drugs (Al-Fatimi et al.,2007).
Crithmum maritimum L.,commonly known as a fennel or rock samphire,is a perennial member of the Apiaceae family.This halophytic plant grows in sand hills and is often found on rocky cliffs.It has been attributed many interests in folk medicine,including diuretic,antiscorbutic,digestive and purgative proper-ties (Guil-Guerrero et al.,1996),and can be consumed as a condi-ment (Atia et al.,2006).Various authors have studied C.maritimum esntial oil,revealing antioxidant and antibiotic activities (Flamini et al.,1999;Ozcan,2000;Ruberto et al.,2000;Marongiu et al.,
2007).In a recent study,we showed that a fennel apolar fraction had strong antimicrobial activity against Micrococcus luteus ,Salmonella arizonae ,Erwinia carotovora ,Pudomonas fluorescens ,P.aeruginosa ,P.marginalis ,Bacillus cereus and Candida albicans with minimum inhibitory concentra
tion values ranging from 1to 100l g ml À1(Meot-Duros et al.,2008).In the prent work,we report the purification and identification of falcarindiol,an antibac-terial and cytotoxic compound in C.maritimum leaf apolar extract.This polyacetylene exhibits various biological activities,such as anti-inflammatory,antiplatelet-aggregatory and antimutagenic properties (Miyazawa et al.,1996;Christenn and Brandt,2006).Falcarindiol has also shown antimicrobial and cytotoxic activities (Stavri and Gibbons,2005;Zidorn et al.,2005).Moreover,polyacet-ylenes prent beneficial effects at nontoxic concentrations (Purup et al.,2009).Thus,the results confirm the interest of this halo-phytic species as a source of bioactive compounds.2.Experimental
2.1.Plant material
Leaves of Crithmum maritimum L.were collected along the shoreline at ‘‘Pointe du Toulinguet”(Brittany,France)during Au-gust 2006.After sampling,the leaves were cleaned with deionized
0278-6915/$-e front matter Ó2009Elvier Ltd.All rights rerved.doi:10.1016/j.fct.2009.11.031
出入库流程Abbreviations:ATCC,american type culture collection;BCC,brittany culture collection;MIC,minimum inhibitory concentration;VLC,vacuum liquid chromatography.
*Corresponding authors.Tel.:+33(0)298498669;fax:+33(0)298498772(L.Meot-Duros).
E-mail @univ-brest.fr (L.Meot-Duros),christian.magne@univ-brest.fr (C.Magné).
Food and Chemical Toxicology 48(2010)
553–557
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water,rapidly soaked,stored atÀ25°C and then freeze dried.The dry material was ground to afine powder.
2.2.Extraction and purification of bioactive compound
Five grams of powder were homogenized with150ml chloro-form under magnetic stirring at4°C for24h.Afterfiltration over glass wool,the chloroformic extract was concentrated by rotary evaporation at30°C.The residue(400mg)was dissolved in hexane for purification.
Hexane extract was subjected to vacuum liquid chromatogra-phy(VLC)on silica gel60(0.063–0.200m
m,Merck).Washing was performed with100ml hexane,and elution proceeded through increasing solvent polarity(25%increments of ethyl ace-tate).Finally,resin was washed with pure methanol to yield six fractions.Each fraction was tested for antibacterial activity and the active fraction was kept for further purification work.
Purification of the active compound was made by column chro-matography(Sephadex LH-20,Sigma–Aldrich),adapting the proto-col of Seger et al.(2006).Twenty sub-fractions of2ml were obtained from the active fraction upon elution with26ml of dichloromethane,followed by14ml of acetone.After evaporation to dryness,residues were dissolved in methanol.Purity was mon-itored by TLC on pre-coated plates(Alugram SIL G/UV254,Mache-rey–Nagel,Germany).Hexane–ethyl acetate(7:3)was ud as the mobile pha and spots were visualid under UV at365nm. Fractions with the same UV profile were gathered before testing for antimicrobial activity.The bioactive fraction was analyd by NMR to identify the antibacterial compound.
2.3.Identification of bioactive compound
Identification of Crithmum maritimum antibacterial compound was made by Nuclear Magnetic Resonance.1D1H-and13C-NMR, HMBC,HMQC and J-MOD experiments were carried out on a Brü-
ker ADVANCE500spectroscope equipped with a5l m diameter cryoprobe.Samples were dissolved in650l l of MeOD.Homonu-clear and heteronuclear NMR spectra were recorded at25°C.
2.4.Antimicrobial tests
Antimicrobial activity was tested against a panel of micro-organisms,including food-borne micro-organisms and clinical iso-lates(Table1):gram positive bacteria(cocci and bacilli),gram neg-ative bacteria and one yeast.All the micro-organisms were obtained from American Type Culture Collection(ATCC)or were from Brittany Culture Collection(BCC).Strains were grown in li-quid nutritive broth at37°C overnight before being ud for anti-bacterial test.
Methanolic solution of falcarindiol isolated from a fennel extract was dropped in sterile96-well plate(NUNC microplate, Fisher Bioblock)at thefinal concentrations of10,20and 50l g mlÀ1.Six repetitions were made for each concentration.After evaporation of the solvent,100l l of microorganism suspensions (102cells mlÀ1)were added in the wells.Antibiotic solution(mix-ture of streptomycin and penicillin G at5and10mg mlÀ1,respec-tively)was dropped in the prence of micro-organisms for positive control.The microplate was aptically aled and incu-bated at30°C for24h.After agitation,microorganism growth was estimated by reading the absorbance at405nm with 司马衍
a micro-plate spectrophotometer(Multiskan MCC/340,Titertek).The MIC was defined as the lowest concentration of plant extracts able to inhibit microorganism growth.
2.5.Cytotoxic activity
IEC-6cells,normal rat small intestine cell line(ATCC CRL1592), were grown in DMEM(BE12-604F,Biowhittaker)supplemented with5%foetal bovine rum(FBS),insulin(10mg lÀ1),penicillin (100mg lÀ1)and streptomycin(100mg lÀ1)at37°C,in5%humid-ified air(Upreti et al.,2007).The viability of the cultures was as-sd by trypan blue exclusion(0.4%solution,Sigma),prior to start every experiment.The batches showing more than95%cell viability were ud in the study.
Cell viability was determined by the MTT method(Upreti et al., 2007).IEC-6cells(1Â104/well in100l l medium)were eded in 96-well plates and allowed to adhere for24h at37°C in5%CO2–95%atmosphere.Medium was aspirated and replaced with med-ium containing falcarindiol at afinal concentration of0.2to 20l M,and plates were incubated for24and48h at37°C.Some cultures were also incubated with only the vehicle solvent(0.5% DMSO).Then,0.5mg mlÀ1of MTT was added to each well and plates were let for2h at37°C.The reaction mixture was centri-fuged,carefully taken out and cells were dissolved with100l l of DMSO.Finally,absorbance was measured at550nm.Experiments w
ere carried out at least three times and the data prented are the mean±SD from independent experiments.Comparisons were made with appropriate controls employing Student’s‘‘t”test. Asterisks were ud to indicate a significant difference,at the 0.05level.
3.Results and discussion
A previous screening showed that Crithmum maritimum chloro-formic extract had strong antibacterial activity against veral mi-cro-organisms including Micrococcus luteus(Meot-Duros et al., 2008).Bioassay-guided fractionation by VLC and purification by LH-20column chromatography led to the isolation of one active compound.Signals obtained in1H(Fig.1)and13C-NMR spectra led to the identification of two acetylenic and two hydroxyl func-tions(Table2).In particular,the four quaternary carbons at d c=68.7,70.1,79.7and81.0ppm indicated the prence of two tri-ple bonds.Following further2D NMR experiments(COSY,HMBC, HMQC),this compound was unambiguously attributed to falcarin-diol(Fig.2),a polyacetylene widely distributed in the Apiaceae family(Christenn and Brandt,2006)and previously identified in Crithmum maritimum leaves(Cunsolo and Ruberto,1993).
A previous screening of C.maritimum antimicrobial activity indicated that its apolar fraction was active against Micrococcus lu-teus,Bacillus cereus,Salmonella arizonae,Pudomonasfluorescens,
Pudomonas marginalis,Escherichia coli,Erwinia carotovora and Candida albicans(Meot-Duros et al.,2008).After isolation and
Table1
Bacterial strains and yeast ud in this study.
Organism, gram morphology Species Culture collection,
identification number
Cocci
Gram positive Micrococcus luteus ATCC10240
Bacilli
Gram positive Bacillus cereus BCC3.05.002,
Gram negative Salmonella enterica
subsp.arizonae
BBC3.08.012,ATCC13314
Pudomonasfluorescens BCC3.05.001,ATCC15453
Pudomonas marginalis BCC3.08.030
Escherichia coli BCC3.08.001,ATCC4157
Erwinia carotovara
subsp.carotovora
春节晚会歌曲
BCC3.08.031
Yeast Candida albicans BCC3.08.036
554L.Meot-Duros et al./Food and Chemical Toxicology48(2010)553–557
identification of falcarindiol,this compound was tested against the micro-organisms,to check whether this polyacetylene could be responsible for C.maritimum antimicrobial activity.Results ob-tained with microplate bioassay are prented in Table 3.It ap-peared that falcarindiol did not prent
the same activity against all the micro-organisms tested.Indeed,only Micrococcus luteus and Bacillus cereus exhibited growth inhibition under 50l g ml À1falcarindiol,the six other micro-organisms being unaffected by this polyacetylene.
The antimicrobial activity of falcarindiol has already been dem-onstrated in various studies.Stavri and Gibbons (2005)showed
that this compound had antimycobacterial properties against Mycobacterium fortuitum ,M.smegmatis ,M.phlei ,M.aurum and M.abscessus .Moreover,falcarindiol was active against Mycobacterium tuberculosis (Kobaisy et al.,1997)and it inhibited the growth of bacteria such as Pudomonas aeruginosa ,Bacillus subtilis ,Staphylo-coccus aureus (Kobaisy et al.,1997;Lechner et al.,2004;Chou et al.,2006).In our study,falcarindiol purified from Crithmum maritimum did not affect Erwinia carotovora growth,confirming results from Garrod et al.(1979).Falcarindiol did not affect Escherichia coli growth,confirming the results of Lee et al.(2000)but not tho of Kobaisy et al.(1997).The authors also studied the effects of this polyacetylene on the yeast Candida albicans ,showing a strong activity with a MIC of 25l g ml À1.This result differed with tho obtained in our study,where none of the falcarindiol concentra-tions tested inhibited the yeast growth.For the first time,we dem-onstrated that falcarindiol (so apolar C.maritimum extract)exhibited a strong antibacterial activity,wi
th MIC of 50l g ml À1,against M.luteus,considered as an emerging nosocomial pathogen in immunocompromid patient and B.cereus ,responsible for food-borne illness.
Falcarindiol appeared to be cytotoxic against IEC-6cells,but only at the highest concentration (20l M)after 24h of exposure (Fig.3).Cell viability decread by 13%at this do.It has to
be
Fig.1.1H-NMR spectrum in MeOD of the bioactive compound isolated with LH-20column chromatography from Crithmum maritimum apolar leaf extract (solvent signals at 3.2and 4.8ppm;(Ã)ethylenic carbons;(w )alkenic carbons;(d )hydroxyl functions).
Table 21
H-and 13C-NMR assignments for the active compound,bad on COSY,HMQC and HMBC experiments,with MeOD as solvent.C.No.Substitution level of carbons d 13C (ppm)d 1H (ppm)1CH 2116.6 4.862CH 138 5.373CH 63.8 5.174C 79.7–5C 68.7–6C 70.1–7C 81–8CH 58.8 5.199CH 134 5.5310CH 129.8 5.4711CH 228.5 2.1212CH 233 1.3913CH 230.2 1.3914CH 230.4 1.3915CH 230.2 1.3916CH 223.7 1.3917
CH 3
14.4
0.89
Fig.2.Structure of falcarindiol.
Table 3
Antimicrobial activity of falcarindiol isolated from Crithmum maritimum leaves.((À)no inhibition;(+)significant inhibition of microorganism growth (>75%)).Bacterial strains
Falcarindiol from Crithmum maritimum (l g ml À1)102050Micrococcus luteus ÀÀ+Bacillus cereus
ÀÀ+Salmonella enterica subsp.arizonae ÀÀÀPudomonas fluorescens ÀÀÀPudomonas marginalis ÀÀÀEscherichia coli
ÀÀÀErwinia carotovora subsp.carotovora ÀÀÀCandida albicals
À
À
À
L.Meot-Duros et al./Food and Chemical Toxicology 48(2010)553–557555
noted that 0.5%DMSO did not affect the growth of IEC-6,compared with untreated control cultures.After a 48h exposure to the high-est concentration of falcarindiol (20l M),cell viability of IEC-6cells prented a 50%reduction,compared to control (Fig.4).DMSO did not affect the viability of the cells,both at 24and 48h of exposure.Cytotoxic properties of falcarindiol have already been reported in previous studies.This polyacetylene prented a wide range of IC 50,depending to the tested cell lines.Thus,falcarindiol prented a cytotoxic activity after 48h in vitro exposure against L1210(mou lymphocytic leukemia),CEM-C7H2(lymphocytic leuke-mia)and RPMI-8226(human myeloma)cell lines,with IC 50from 2.87to 31.8l M (Zidorn et al.,2005;Chen et al.,2007).
Falcarindiol prented a relatively low cytotoxicity against the tested cell lines.Our results were similar with tho obtained by Purup et al.(2009)on ll line (normal human intestinal cells),where falcarindiol was cytotoxic at 34l M.Tho authors had shown that this compound had a lower cytotoxic activity com-pared to that of two other polyacetylenes,falcarinol and panaxy-dol.They had explained this lower reactivity with the chemical properties of falcarindiol,which was able to generate two active centers for nucleophilic attack,thus reducing its lipophilic properties.
疯狂数钱
In addition to its antibacterial property,this polyacetylene has been shown to exhibit various biological activities.Indeed,falca-rindiol appeared to have antifungal properties,inhibiting spore germination of different fungi in concentrations ranging from 20to 200l g ml À1(Christenn and Brandt,2006).Moreover it inhib-
ited nitric oxide production (Matsuda et al.,1998;Kim et al.,2003)and was highly related to bitterness in carrot (Kreutzmann et al.,2008).
Considering the potential health benefits of falcarindiol,the re-sults of the investigations suggest that C.maritimum could be ud as food prervatives and antibiotics,as this plant can be eas-ily cultivated and quickly produces an important biomass,compat-ible with its exploitation.This study confirms the capacity of halophytic species to produce bioactive compounds and the possi-bility for using them as a source of medicinal agents.
Conflict of interest
The authors declare that there are no conflicts of interest.Acknowledgement
This rearch was supported by the Brest Metropole Oceane (BMO)through a Ph.D fellowship (L.M.).References
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