International Network Environmental Management Conflicts http://www.igetecon.org/revista/index.php/inicio/index International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Qualitative Phytochemical Screening and Antimicrobial Activity Evaluation of the Bulb Extracts of Gladiolus psittacinus Hook (Iridaceae)

Franois Munyemana PhD, Professor Auxiliar, Departamento de Qumica, UniversidadeEduardo Mondlane, Maputo, Moambique E-mail: munyfranc@yahoo.co.uk ou francoism@uem.mz Ana Paula Mondego Mestre, Assistente, Curso de Farmcia, Instituto Superior de Cincias e Tecnologia de Moambique, Maputo, Moambique Paulo Cumbane Licenciado, Assistente Estagirio, Curso de Farmcia, Instituto Superior de Cincias e Tecnologia de Moambique, Maputo, Moambique Abstract InMozambique,thebulbofGladioluspsittacinusHookisusedintreatingdiarrhea,dysentery, gonorrhea, renal and rheumatic pains, etc. In this study, was evaluated the antimicrobial activity ofdifferentextractsandfractionsofdriedandfreshbulbagainst6bacterialstrains: StaphylococcusaureusATTC25923,EscherichiacoliATCC25922,Klebsiellapneumoniae ATCC15380,PseudomonasaeruginosaATCC27953,ShigellaflexneriATCC12022and2 fungi:CandidaalbicansandSaccharomycescerevisiae,andtheinteractionCiprofloxacin Extract. Most of the bulb extracts and fractions showed strong inhibitory activity against Candida albicans, Saccharomyces cerevisiae and Pseudomonas aeruginosa. The aqueous extract revealed antagonism with ciprofloxacin while the juice (from the fresh bulb) showed additive effect. Keywords: Gladiolus psittacinus; Iridaceae; Antimicrobial; Bulb. Introduction Sincetheantiquity,themanusesnaturalresourcessuchasvegetables,forvarious purposes, mainly food and medicine. In this constant man-environment interaction, the need has becomeanimportantfactorinthedevelopmentoffolkmedicine.Plantshavebeenusedasthe basisofmanytraditionalmedicinesystemsthroughouttheworldforthousandsofyears.They continue to provide mankind withnew remedies. Everyregion has itsown history of traditional medicine,forexampletraditionalChinesemedicine,ArabictraditionalmedicineandAfrican traditional medicine. 15 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Suchpracticesaretraditionalbecausetheyaredeeplyrootedinaspecificsocio-cultural context,whichvariesfromonecommunitytoanother.Eachcommunityhasitsownparticular approach to health and disease even at the level of ethno-pathogenic perceptions of diseases and therapeutic behavior (Rukangira; Ochoa; Ocampo; Muoz, 2001).In Africa, herbal medicine gained popularity as alternative and complementary therapies, largelyasaresultofculturaltraditionsandexcessivecostofmodernmedicines.Traditional remediesmadefromplantsplayanimportantroleinthehealthofmillionsofpeople.Low-incomepeoplesuchassubsistencefarmers,peopleofsmallisolatedvillagesandnative communities use folk medicine for the treatment of common infections (Rojas et al, 2006).The interest in medicinal plants has grown considerably in recent years because they have been a valuable source of products for maintaining human health, becoming potential candidates formanyapplicationsinthepharmaceuticalindustry.Medicinalplantsarethebestsourcesto obtain a variety of drugs (Santos; Ferreira; Damiao, 2010). Mozambique,withitsrichandvariedflora,isoneofthecountrieswherepeoplerely much on the use of traditional knowledge and medicinal plants to treat various diseases.GladioluspsittacinusHook(synonymsinsouthernAfricacollections:Gladiolusnatalensisand Gladiolusdalenii)isanherbaceousandbulbousplantbelongingtothefamilyIridaceaewhich comprisesabout1800speciesgroupedin88genus.Ofthesegenus,GladiolusandIrisarethe mostrepresentativewithabout260and250speciesrespectively(Machado,2007&Manning, 2004).ThegreatesteconomicimportanceoftheIridaceaefamilyresidesinornamentalsand medicinal use.Phytochemical study of several species of the family Iridaceae has led to the isolation of severalsecondarymetaboliteswithhighbiologicalactivity,especially,antimicrobial, antiinflammatory, antidiarrhoeal, antidysenteric, antioxidant, antinociceptive, antifungal etc.In the Iridaceae family occur mainly the following secondary metabolites: Flavonoids, alkaloids, saponins, terpenoids, anthraquinones, naphthoquinones and coumarins. TheplantsofthegenusGladiolusareusedindifferentpharmacopoeiasfortreating variousdiseases.InsomeregionsinsouthernAfricaplantsofthegenusGladiolusareusedto treatavarietyofdiseases,includingacutediarrhea,intestinalparasiticinfections,asthma, diabetes, constipation, impotency, headaches, relieve rheumatic pains and hemorrhoids. 16 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. In Mozambique the plant Gladiolus psittacinus Hook is used to treat diarrhoea, dysentery, gonorrhea, regulate the blood flow of women and as a sedative. For the treatment of diarrhea and dysentery it is an infusion that can be administered orally or enema. As sedative, the plant is used to calm patients with mental disorders in case of extreme agitation: two drops of fresh juice of the bulb are applied in the nostrils of the patient. In the present study was carried out the qualitative phytochemical screening of the bulbs extractsofGladioluspsittacinusHookgrowninMozambiqueandevaluatedtheantimicrobial activityofdifferentextractsandfractionsofdriedandfreshbulbagainstgram-positive,gram-negative bacteria and fungi. Materials and Methods Plant Material and Extraction Fresh samples of Gladiolus psittacinus Hook bulbs were collected in Chiboene, Moamba district, Maputo province in July 2006 and in Bilene district, Gaza province in March 2011. The samplesauthenticationwasmadeintheherbariumoftheDepartmentofBiologyEduardo MondlaneUniversityandintheherbariumoftheBotanyDepartmentInstituteofagricultural Research of Mozambique (voucher specimen Nr. 48529). Samples preparation Fresh Juice:Aftercollection,thefreshbulbswerewashed,cutintosmallpiecesandmechanicallycrushed with the help of a centrifuge juice in order to extract the fresh juice.Dried powdered bulb: Thefreshbulbswerewashed,cutintosmallpiecesandweredriedintheovenfor7daysata temperature of about 50 C and then ground with the help of an electric grinder. 17 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Preparation of extracts Crudeextractswerepreparedbymaceration,percolationorSoxhletextractionusing followingsolvents:Methanol90%,Methanol,Dichloromethanemethanol(1:1),Distilled water.Fractions were obtained by solvent-solvent partition with increasing polarities (hexane or petroleum ether, Chloroform, ethyl acetate, n-butanol). Extraction of dried sample by percolation 300gofdriedpowderedbulbwaspercolatedfor4daysina500mlpercolatorusingas solvent a mixture of methanol and water (9:1). The resulting extract (3 l) was filtered with suction on a borosilicate glass funnel fitted with a filter membrane and stored at 4 C in a fridge for 48 hours for precipitation of the fatty material.Theextractwithprecipitatedmaterialwasleftatroomtemperaturefor1handthen decantedandfiltratedwithWhatmanfilterpaper(No.1)andconcentratedunderreduced pressure in a rotary evaporator BUCHI at 40 C until the formation of a pasty mass (methanol extract). 100 g of the methanol extract was suspended in a volume of 200 ml of mixture methanol / water (1:1) in 250 ml Beaker and quantitatively transferred to a separatory funnel of 500 ml and thensubmittedtoliquid/liquidpartitionusingsolventswithincreasingpolarities(n-hexane, chloroform, ethyl acetate and n-butanol).The hexane fraction (HEX) was obtained by liquid / liquid partition using 3x100 ml of n-hexane.Andthenseparatedintoa500mlflaskandconcentratedinarotaryevaporatorunder reducedpressureat40oC.Thechloroformfraction(CHL)wasalsoobtainedbyliquid/liquid partition with3x100ml of chloroform. And then separated into a 500 ml flask and concentrated todrynessinarotaryevaporatorat40o C.Thehydromethanolicsolutionwastransferredtoa round bottom flask of 500 ml and concentrated until elimination of methanol.The aqueous extractwasfurtherentirelysubjectedtoliquid / liquid partition with 3x100 mlofethylacetate,followedby3x100mlofn-Butanol,yieldingthreefractions:ethylacetate fraction(EA),n-butanolfraction(Bu)andaqueousfraction(Aq.Fr.)respectively.Thetwo organic fractions were also concentrated in a rotary evaporator under reduced pressure at 40 C. 18 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. All fractions were stored in dry desiccator until constant weight is obtained, and after 7 days the yield of extraction was determined. Extraction of the dried sample by Soxhlet 20 g of the dried powdered bulb was introduced into a cellulose cartridge Whatman(30 mm x 100 mm) and subjected to Soxhlet extraction with 400 ml of the mixture dichloromethane - methanol(1:1).After24hourstheextractwasfilteredbysuctiononaglassfunnelwitha membrane filter, then through a filter paper, and concentrated in rotary evaporator at low pressure untildryness.Thedriedextractwasplacedinadesiccatoruntilobtainingconstantweight,and after 7 days the yield of extraction was determined Extraction of the fresh sample by Soxhlet 30gof the fresh bulb, pre-treatedwith distilled waterandcut into smallfragments,was milledinKenwoodblenderandtransferredquantitativelytothecellulosecartridge.Thereafter, the material was extracted in a Soxhlet using as solvent 400 ml of distilled water. After 24 hours, theextractwasrecoveredfromtheflask,cooledtoroomtemperatureandfilteredwithcotton woolinglassfunnel,followedbyfilterpaperandtakentocarryoutbiologicaltesting,aftera preliminary purification by filtration with sterile cellulose acetate filter (GVS Filter Technology, USA) 0.45 mm in porosity and 25-mm diameter. Extraction of juice in fresh bulb 50 g of the fresh bulb treated with fresh distilled water was mixed with 50 g of water and milled in Kenwood blender. The resulting sample was transferred to a 250 ml Beaker and filtered withcottonwoolinaglassfunnel.Thefiltratewascentrifugedandtheresultingextractwas purifiedwithcelluloseacetatefilter(GVSFilterTechnology,USA)porosityof0.45mmand diameter of 25mm, andtransferred to a sterileglass container andtaken to carry out biological testing. 19 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Solvent-Solvent Partition of the fresh juice Inthatextraction,20goffreshjuicewastreatedwithmethanol(600ml)for24hours. Afterfiltration,thefiltratewasevaporatedtodrynessinrotaryevaporator,leadingtoayellow residue after total evaporation of the solvent. This residue was dissolved in distilled water (20ml) andthenproceededtothesuccessiveextractions(3timesrespectively),firstwiththesolvent petroleumether(PE),thenwithchloroform(CHL),followedbyethylacetate(EA)andfinally with n-butanol (Bu) with equal volumes of 100ml each. Petroleum ether (PE), chloroform (CHL), ethylacetate(EA)andn-butanol(Bu)fractionswereconcentratedontherotaryevaporatorto givepetroleumetherresidue(white),chloroformresidue(paleyellow),ethylacetateresidue (yellowandbutanolic residue (intenseyellow color),which have been subjected to preliminaryphytochemical tests. Phytochemical Analysis The extracts and fractions were subjected to qualitative phytochemical tests for alkaloids, tannins (hydrolysable and condensed), coumarins, iridoids, anthraquinones, flavonoids, saponins, steroidsandtriterpenoids,aminoacids,proteinsandcarbohydratesadoptingtheprocedures described by Gomes & Silva (2007), Duarte; Fonte & Santos (2010) and Khan et al (2010). Antimicrobial assay

Bacterialculturesreferencestandard(ATCC)andtheyeastCandidaalbicans(isolated from vaginal discharge) were obtained from the Microbiology Laboratory of the Central Hospital of Maputo. The yeast Saccharomyces cerevisiae was obtained from commercial packaging yeast. The tests were performed using a standard method recommended for this purpose (NCCLS). Six bacterialstrainswereusedinthetests,Staphylococcusaureus(ATTC25923),Escherichiacoli (ATCC25922),Klebsiellapneumoniae(ATCC15380),Pseudomonasaeruginosa(ATCC 27953),Shigellaflexneri(ATCC12022)andSalmonellatyphimurium(ATCC14028).The culture media used were Nutrient broth, Sabouraud Dextrose Broth and Mueller Hinton Agar. 20 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Bacterial sensitivity test by disc diffusion method Theassaywasperformedusingthediskdiffusionmethodwhereeachsterilediskof Watman filter paper of 6.0 mm in diameter was impregnated with 10 l aliquots of the extract at concentrations of 1 mg / disk to the total extract and the respective fractions. TheaqueousextractandjuicewereplacedinPetridishescontainingsterilepaperdiscs and allowed to soak for two hours prior to testing. The suspensions containing the bacteria were preparedseparatelysuspendingacolonyofeachmicroorganismtakenfromstocksampleswith the aid of a platinum loop in test tubes containing nutrient broth and incubated for 24 hours at 37o C. Afterwereadjustedaccordingtotheturbidityof0.5inMcFarlandscale,byvisual comparisonwithastandardtubecontainingastandardizedsuspension. TheadjustedsuspensionswereseparatelydispersedinaPetridishcontainingMuellerHinton Agar. The Petri dish was divided into four equal portions and placed aseptically one disc of each impregnatedwithplantextractinthecenterofeachdivision. Plateswerealsoplacedinthedisksimpregnatedwithciprofloxacin,DMSOandpuredistilled waterrespectively.Theplateswereincubatedfor24hat37Candthenthereadingofthe diameter of zones of inhibition. All tests were done in triplicate. Fungal sensitivity test by disc diffusion method ThesuspensionsoftheyeastsCandidaalbicansandSaccharomycescerevisiaewere preparedseparatelybysuspendingacolonyofeachmicroorganismintesttubescontaining Sabouraud Dextrose Agar (SDB) incubated at 37 C for 48 hours, then standardized according to the turbidity 0 5 in McFarland scale for visual comparison with standard test tube. The dispersion ofthesuspensionintheplatecontainingtheMuellerHintonAgar,wascleanedwithsterile cottonswabacrosssizeofPetridish(90mm)uptoobtainauniforminoculum.Theyeast Saccharomycescerevisiaewasisolatedbysuspending1gofyeastin20mlofsugarsolution. Was allowed to stand for five minutes to allow the activation of yeast and subsequently taken 200 l ofit to a test tube containing 15ml of SDBand incubated for 48h. Assays were performed 21 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. usingthesameprocedurefollowedforantibacterialtests,withtheexceptionofthepositive control, having been used in this nystatin. Interaction Extract Ciprofloxacin Theaqueousextract(Aq.Extr.)andjuicewereevaluatedfortheabilitytointeractwith ciprofloxacin. Disks were initially impregnated to saturation with the extracts and dried in sterile medium for 30 minutes. Thereafter foi taken 10 uL of ciprofloxacin 2 mg / ml and was also added on thedisks containing extracts and aseptically transferred to Petri dishes previously inoculated with bacteria and incubated for 24 h at 37 C. All tests were done in triplicates and the respective percentages of interaction determined using the following formula: Interaction% = (Di-Dii) / Dii x100 where: Di: mean zone of inhibition of ciprofloxacin combined with extract. Dii: mean zone of inhibition of ciprofloxacin alone. Results and discussion Phytochemical Screening The phytochemical tests carried out on the extracts, fractions andfresh juice of the bulb, allowedtheidentificationofthefollowingmetabolites:alkaloids,anthraquinones,flavonoids, coumarins,saponins,hydrolysabletannins,steroidsandtriterpenoids,aminoacids,proteinsand reducing sugars. Thephytochemicalscreeningresultsshowedthatthereisnotmuchdifferencebetween the chemical composition of juice and powdered dry bulb, but in practice it has been proved to be easier to work with the dry powdered bulb than the juice in particular during the solvent / solvent partition.The extraction solvent / solvent of the juiceextracted from the fresh bulb and powdered dry bulb allowed to obtain less complex fractions aiming semi-purification of substances through their polarity and solubility.22 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Theresultsofthephytochemicalscreeningofthecrudeextractsaresummarizedinthe table1,andtheresultsofthephytochemicalscreeningofthefractionsfromsolvent-Solvent partition were summarized in table 2.

Table 1: Phytochemical tests of Crude extractsPhytoconstituents Dichloromethane-Methanol(1:1) Extract (24h soxhlet dried bulb) Methanol 90% Extract ( fresh bulb) Aqueous extract (24h Soxhlet fresh bulb) Juice (fresh bulb) Alkaloids++++ Anthraquinones++-- Coumarins-+-- Flavonoids++-- Saponins++-+ Tannins++-- Amino acids++++ Proteins---+ Reducing Sugars-+++ +: present; -: absent Table 2: Phytochemical tests of fractions from solvent solvent PartitionPhytoconstituents Juice(FreshBulb)Powdereddrybulb PE/HEXCHLEABuPE/HEXCHLEABu Alkaloids-+---++- Anthraquinones--+--++- Coumarins-++--++- Flavonoids-+++-+++ Iridoids-------- Saponins---+--++ Steroids/Terpenoids++++++++ +:present,-:absent,PE:petroleumetherfraction,HEX:hexanefraction,CHL:chloroformic fraction, EA: ethyl acetate fraction, Bu: butanolic fraction. Results of antimicrobial assay The results of the antimicrobial screening realized in this study with extracts and fractions less complex of Gladiolus psittacinus Hook against Escherichiacoli, Pseudomonas aeruginosa, Staphylococcusaureus,Shigellaflexneri,Salmonellatyphimurium,Klebsiellapneumoniae, CandidaalbicansandSaccharomycescerevisiaeshowedinterestingantimicrobialactivitiesof this plant as shown in table 3a and 3b. 23 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Table 3a. Results of antimicrobial activity tests mean diameter of inhibition zone (mm). Micro-organisms NTCIPMETHEXCHLEABU Candida albicans 180,6200,6280,6250,6240,6230,6 E.coli ATCC25922 320,6----- P. aeruginosa ATCC 27953 320,0180,6221,0230,3250,0200,1 K. pneumoniae ATCC 15380 301,0----- S.aureus ATCC 25923 320,6----70,0 Saccharomyces cerevisiae 100,6--200,3130,6 S. typhimurium ATCC 14028 320,0BE-BEBEBE Shigella flexneri ATCC 12022 330,6--BEBE- (_ _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) No zone of inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT ) 3.2 cg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction; CHL:chloroformfraction;EA:ethylacetatefraction;BU:butanolfraction;Aq.frac.:aqueousfraction; Aq.Extr.:aqueousextract;BE-bacteriostaticactivity.TheyeastSaccharomycescerevisiaehadnot control. Table 3b. Results of antimicrobial activity tests (cont.)Mean diameter of inhibition zone (mm) Micro-organismsNTCIPDMSO CH2Cl2- CH3OH Aq.Frac.JuiceAq.Extr. Candida albicans180,6-210,3-160,3- E.coli ATCC25922320,6---190,4160,1 P. aeruginosa ATCC 27953 320,0-151,0-121,0140,3 K. pneumoniae ATCC 15380 301,0----- S.aureus ATCC 25923 320,6----- Saccharomyces cerevisiae -120,3-150,0- S. typhimurium ATCC 14028 320,0----- Shigella flexneri ATCC 12022 330,6----- (_ _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) : No zone of inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT ) 3.2 cg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction; CHL:chloroformfraction;EA:ethylacetatefraction;BU:butanolfraction;Aq.Frac.:aqueousfraction; Aq.Extr.:aqueousextract;BE:bacteriostaticactivity.TheyeastSaccharomycescerevisiaehadnot control. 24 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. PreparationsofthebulbofGladioluspsittacinusHookareusedintraditionalmedicine forthetreatmentofvariousdiarrhealdiseases.Thepresenceofsecondarymetabolitesactive against microbial pathogens in the extracts of this plant has been reported. Amehetal(2011)demonstratedantimicrobialactivityagainstPseudomonasaeruginosa andAspergillusniger ofthe aqueous extract ofGladiolus bulb.Thephytochemical screening of thesameextractresultedintheidentificationofsomemetabolitesnamely:alkaloids,tannins, cardiotonic glycosides, flavonoids and carbohydrates. Nguedia et al (2004) showed antifungal activity of the hydroethanolic extract of the bulb of Gladiolusgregasius Bakeragainst Candida albicans and Candida krusei. However, the same extractwasnotactiveagainstEscherichiacoli,Pseudomonasaeruginosa,Proteusvulgaris, StaphylococcusaureusandStreptococcusfaecalis.Thephytochemicalscreeningrevealedthe presence of saponins, glycosides, polyphenols, phenols, triterpenes and steroids. ThecrudemethanolicextractexhibitedanantibacterialeffectagainstPseudomonas aeruginosa with an inhibition zone of 18 0.6 mm and a bacteriostatic effect against Salmonella typhimurium.AnantifungaleffectwasshownagainsttheyeastsSaccharomycescerevisiaeand Candida albicans with inhibition zones of 20 0.6 mm and 10 0.6 mm respectively. TheactivityofthatextractagainstPseudomonasaeruginosawaslowcomparedto ciprofloxacinusedascontrol.However,itshowedahighantifungalactivityagainstCandida albicans when compared to the control nystatin. The antifungal activity displayed against candida albicanswashigherthanwiththeotheryeastSaccharomycescerevisiae.Thisextractwasnot active against the rest of the microorganisms tested. ThehexanefractionexhibitedanantibacterialeffectagainstPseudomonasaeruginosa with an inhibition zone of 22 0.6 mm. An antifungal effect against Candida albicans has been shownwithazoneofinhibitionof280.6mm.Thisfractionwasnotactiveatthesame concentrationagainstothermicroorganismstested.Theactivityoftheextractagainst Pseudomonas aeruginosa was low compared to the antibiotic used as control; but that was higher thantheactivityofthecrudemethanolicextract.Thisfractionexhibitedalsoanantifungal activityagainstCandidaalbicanshigherthanthecontrolnystatinandthanthecrudemethanol extract. In phytochemical studies performed on this fraction were identified terpenoids. Accordingtoliterature,theactivityofthesecompoundsisrelatedtoitslipidsolubility which allows them to interact with the biomembranes of microorganisms, causing rupture of the 25 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. cells. The solubility of thesecompounds in the biomembranesenables their interaction with ion channels,carriermolecules,membranesreceptors,whichleadstochangesinconformationand loss of function (Lopez, 2010). ThechloroformfractionalsoshowedanantibacterialeffectagainstPseudomonas aeruginosa with a zone of inhibitionof23 0.6mmand a bacteriostatic effect againstShigella flexneriandSalmonellatyphimurium.ItwasalsoshownantifungalactivityagainstCandida albicanswithazoneofinhibitionof250.6mm.Thisfractionwasnotactiveatthesame concentrationagainstothermicroorganismstested.Theactivityoftheextractagainst Pseudomonas aeruginosa was low compared to the control. However, the activity of this fraction wassuperiortotheactivityofthecrudemethanolextractandthehexanefraction.Thisfraction alsoshowedantifungalactivityagainstCandidaalbicanshigherthanthecontrol,thecrude methanol extract, and the hexane fraction.Inphytochemicaltestsperformedonthisfractionbyprecipitationmethods(Mayerand Dragendorff) and thin layer chromatography were identified alkaloids. The presence of alkaloids in this plant have been reported by Odhiambo et al (2010) as the active constituents present in the extract CH2Cl2: CH3OH (1:1) against Aspergillus niger. TheethylacetatefractionexhibitedanantibacterialactivityagainstPseudomonas aeruginosawithaninhibitionzoneof250.0mmandabacteriostaticeffectagainstShigella flexneriandSalmonellatyphimurium.ThisfractionwasalsoactiveagainsttheyeastsCandida albicansandSaccharomycescerevisiaewithzonesofinhibitionof240.60.3and20mm respectively. This fraction was not active at the same concentration against other microorganisms tested.TheantibacterialactivityagainstPseudomonasaeruginosaofthisfractionwaslow comparedtothecontrol,butgreaterthantheactivityofthecrudemethanolextract,thehexane andchloroformfractions.ThisfractionwasmoreactiveagainstCandidaalbicansthanagainst the yeast Saccharomyces cerevisiae. It also showed antifungal activity against Candida albicans higherthanthecontrolnystatin,crudemethanolextract,hexaneandchloroformfractions. Phytochemicaltestsrealizedonthisfractionshowedthepresenceofalkaloids,coumarinsand saponins that may be associated with antimicrobial activity shown by this fraction. The butanol fraction showed antibacterial activityagainst Pseudomonasaeruginosawith azoneofinhibitionof200.1mm,alowactivityagainstStaphylococcusaureusanda bacteriostaticeffectagainstSalmonellatyphimurium.Thisfractionalsoshowedantifungal 26 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. activityagainstCandidaalbicanswithazoneofinhibitionof230.6mmandamoderate activity against the yeast Saccharomyces cerevisiae with a zone of inhibition of 13 0.6 mm. TheactivityofthisfractionagainstPseudomonasaeruginosawaslowcomparedtothe antibioticusedascontrol;butitwashigherthanthecrudemethanolextract.Italsoshowed antifungalactivityagainstCandidaalbicanshigherthanthecontrolnystatinandthecrude methanolextract.Thephytochemicaltestsofthisfractionmadepossibletheidentificationof saponins. The presence of these compounds in the extracts of this plant has also been evidenced by Odhiambo; Siboe; Lukhoba; Dossaji, (2010). TheextractCH2Cl2:CH3OH(1:1)showedantibacterialactivityagainstPseudomonas aeruginosawithaninhibitionzoneof151.0mm.Italsoshowedactivityagainstcandida albicans and Saccharomyces cerevisiae with zones of inhibition of 21 0.3 mm and 12 0.6 mm respectively.TheactivityoftheextractagainstPseudomonasaeruginosawaslowcomparedto theantibioticusedascontrol.Furthermore,theiractivityagainstCandidaalbicanswashigher than the control. TheactivitydemonstratedbytheextractagainstCandidaalbicanswashigherthanthat displayedagainstSaccharomycescerevisiae.PhytochemicalTestscarriedoutonthisextract madepossibletheidentificationofalkaloids,aminoacids,anthraquinones,flavonoids,tannins and saponins.Thequinoliniccompoundsarecapableofformingirreversiblecomplexeswith nucleophilic amino acids in proteins, often leading in inactivating proteins and consequent loss of function,whichexplainsthehighantimicrobialeffectofthesecompounds(Lopez,2010).The biological role of tannins in plants has been investigated and it is believed that they are involved in chemical defense of plantsagainst attack byherbivores and against pathogens (Oliveira et al, 2007).Notsurprisingly,extractscontainingtanninsshowactivityagainstmicroorganisms. Flavonoidsarecompoundssynthesizedbytheplantsinresponsetomicrobialinfection(Dixon; Dey;Lamb,1983), soit isnot surprising to demonstrate antimicrobial activity in vitro against a broadrangeofmicroorganisms.Thisactivityisprobablyduetotheabilitytoformcomplexes with extracellular soluble proteins and the bacterial cell wall. The more lipophilic flavonoids can also disrupt microbial membranes. In studies realized by Odhiambo et al (2010), was also identified the presence of alkaloids intheextractCH2Cl2CH3OH(1:1)whichwereattributedthepotentantifungalactivity 27 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. demonstratedbythisextractagainstAspergillusniger.Theactivityexhibitedbythisextract against Candida albicans can probably also be associated with these compounds identified.The juice from the fresh bulb showed an antibacterial activity against Escherichia coli and Pseudomonas aeruginosa with inhibition zones of 19 0.4 and 12 1.0 mm respectively. It was alsoactiveagainstcandidaalbicansandSaccharomycescerevisiaewithrespectivezonesof inhibition of 16 0.3 and 15 0.0 mm.Theactivity of theextract against Escherichia coliand Pseudomonas aeruginosa was low compared to the control. Its activity against Candida albicans was also lower than control.Phytochemicaltestscarriedoutonthisextract,havemadepossibletheidentificationof aminoacids,proteins,alkaloidsandsaponins.Theactivityshownbythisextractmaybe associated with these compounds. TheaqueousextractofthefreshbulbwasactiveagainstPseudomonasaeruginosaand Escherichia coliwith the respective zones of inhibition of 16 0.1and 14 0.3mm.However was inactive against the rest of the tested bacteria. No antifungal effect was demonstrated in this extract.TheactivityoftheextractagainstEscherichiacoliandPseudomonasaeruginosawas low compared to the antibiotic used as control. Phytochemicaltestscarriedoutonthisextractmadepossibletheidentificationof alkaloids.ThestudyrealizedbyAmehetal(2011),reportedthepresenceofalkaloidsinthe aqueousextractofthebulbofGladiolusobtainedbythesamemethod.However,theabove extractwasfoundtobeslightlyactiveagainstPseudomonasaeruginosaandAspergillusniger and inactive against Candida albicans. ThebacteriaEscherichiacoliwassusceptibletoaqueousextractandjuice,whichare generally used in traditional medicine for the treatment of diarrhea, gonorrhea and other intestinal disturbances. This activitymayexplain its traditional use in the treatment of diarrhea of various origins. TheresultsoftheinteractionExtract-Ciprofloxacinshowanadditiveeffectforthe combinationJuice+CiprofloxacinincaseofE.coli,S.flexneri,K.pneumoniaeandP. aeruginosa; Indifference in case of S. aureus and S. typhimurium. In the case of the combination of Ciprofloxacin+ aqueousextract, theantagonistic effect was observedforallmicroorganisms tested. The results of the interaction Extract Ciprofloxacin are summarized in table 4. 28 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. Table 4 - Results of the interaction Extract - Ciprofloxacin Mean diameter of the inhibition zone (mm) Microorganism Distilled water CIPJuiceJuice+CIPAq.Extr. CIP+ Aq.Extr. E.coli ATCC25922 -310,6190,6330,6160,0280,3 S.aureus ATCC 25923 -330.3-330,6-302,6 Shigella flexneri ATCC 12022 -321,0-341,0-300,6 S. typhimurium ATCC 14028 -320.3-320,3-310,6 K. pneumoniae ATCC 15380 -290,0-301,6271,0 P. aeruginosa ATCC 27953 -301,0280.5340,6191,0250,3 Purifieddistilledwater:Negativecontrol;CIP:Ciprofloxacin-positivecontrol;CIP+Aq.Extr.: ciprofloxacin combined with aqueous extract; Juice + CIP: ciprofloxacin combined with juice. Diameters of inhibition of ciprofloxacin when tested in combination with the juice show a slightincrease.OnlyinthecaseofStaphylococcusaureusandSalmonellatyphimurium,the combinationshowedindifference.However,theaqueousextracthadanoppositeeffect,the diametersofinhibitionofthecontrolshowedareductionwhenusedincombinationwiththe aqueous extract for all tested microorganisms. AccordingtoNwze&Eze(2009),theconventionsuggeststouseadditivewhenthe percentage of the increase of the diameter of inhibition of the plant extract combination with the antibioticislessthanorequalto19%;indifferent,whenthediameterofinhibitionofthe antibiotic combination with the extract is equal to the diameter of inhibition when theantibiotic is tested alone, and there is an antagonistic effect in case of loss of inhibition diameter when the antibiotic control is used in combination with the extract.Thisworkwasbasedonthesameconvention.Anadditiveeffectwasobservedinthe following microorganisms: Escherichia coli with 6.45% addition, Shigella flexneri with addition of6.25%,Klebsiellapneumoniaewith3.45%ofadditionandPseudomonasaeruginosawith additionof13.33%whenthejuicecombinedwithciprofloxacin.Moreover,thebacteria Salmonella typhimurium and S. aureus were indifferent to the combination. The combination of theantibioticwiththeaqueousextractshowedanantagonisticeffectforallthemicroorganisms tested. 29 Qualitative Phytochemical Screening International Network Environmental Management Conflicts, Santa Catarina Brasil, 2(1), pp. 14-31, jan. 2013. It is important to note that the action of medicinal plants can never be fully understood by analyzing its components separately. Proponents of this theory argue that the properties are due to interactionsbetweenmultiplecomponents(Wink,2008).Forexample,thesaponinspresentin many medicinal plants, enhance the absorption and activity of other substances. The saponins of Sapindusmukorossiincreasetheintestinalabsorptionofcertainnaturalantibiotics.Therefore, positiveinteractionfoundinthecombinationofthejuicewithciprofloxacincanbeassociated withthepresenceofsaponinsintheplant,whichcanessentiallyincreasetheabsorptionof ciprofloxacin through the biological membranes of the bacteria, thereby enhancing its effect. Conclusion Qualitative phytochemical analysis of the extracts and fractions of the bulbrevealed the presenceof:alkaloids,anthraquinones,tannins,saponins,flavonoids,terpenoids,steroids, cumarins, reducing sugar, aminoacids and proteins. Most of the bulb extracts and fractions showed strong inhibitory activity against Candida albicans,SaccharomycescerevisiaeandPseudomonasaeruginosa.Thedeterminationofthe interactionCiprofloxacin-aqueousextractandCiprofloxacin-juicerevealedanantagonism between ciprofloxacin and the aqueous extract and an additive effect between Ciprofloxacin and juice.Phytochemicalandantimicrobialtestresultsobtainedinthisworkincombinationwith thepreviouslyreportedinliteraturebydifferentauthorsvalidatetosomeextenttheuseofthe bulbofGladioluspsittacinusHookintraditionalmedicineintreatingdiseasesofmicrobial origin, thus demonstrating the potential of this plant. References Ameh, S., Obodozie, O., Olorunfemi, P., Okoliko, I., Ochekpe, N. (2011). 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