1. International Network Environmental Management Conflicts http://www.igetecon.org/revista/index.php/inicio/indexQualitative Phytochemical Screening and Antimicrobial Activity Evaluation of the Bulb Extracts of Gladiolus psittacinus Hook (Iridaceae) François Munyemana PhD, Professor Auxiliar, Departamento de Química, Universidade Eduardo Mondlane, Maputo, Moçambique E-mail: firstname.lastname@example.org ou email@example.com Ana Paula Mondego Mestre, Assistente, Curso de Farmácia, Instituto Superior de Ciências e Tecnologia de Moçambique, Maputo, Moçambique Paulo Cumbane Licenciado, Assistente Estagiário, Curso de Farmácia, Instituto Superior de Ciências e Tecnologia de Moçambique, Maputo, Moçambique AbstractIn Mozambique, the bulb of Gladiolus psittacinus Hook is used in treating diarrhea, dysentery,gonorrhea, renal and rheumatic pains, etc. In this study, was evaluated the antimicrobial activityof different extracts and fractions of dried and fresh bulb against 6 bacterial strains:Staphylococcus aureus ATTC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniaeATCC 15380, Pseudomonas aeruginosa ATCC 27953, Shigella flexneri ATCC 12022 and 2fungi: Candida albicans and Saccharomyces cerevisiae, and the interaction Ciprofloxacin –Extract. Most of the bulb extracts and fractions showed strong inhibitory activity against Candidaalbicans, Saccharomyces cerevisiae and Pseudomonas aeruginosa. The aqueous extract revealedantagonism with ciprofloxacin while the juice (from the fresh bulb) showed additive effect.Keywords: Gladiolus psittacinus; Iridaceae; Antimicrobial; Bulb. Introduction Since the antiquity, the man uses natural resources such as vegetables, for variouspurposes, mainly food and medicine. In this constant man-environment interaction, the need hasbecome an important factor in the development of folk medicine. Plants have been used as thebasis of many traditional medicine systems throughout the world for thousands of years. Theycontinue to provide mankind with new remedies. Every region has its own history of traditionalmedicine, for example traditional Chinese medicine, Arabic traditional medicine and Africantraditional medicine.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
2. 15Qualitative Phytochemical Screening… Such practices are traditional because they are deeply rooted in a specific socio-culturalcontext, which varies from one community to another. Each community has its own particularapproach to health and disease even at the level of ethno-pathogenic perceptions of diseases andtherapeutic behavior (Rukangira; Ochoa; Ocampo; Muñoz, 2001). In Africa, herbal medicine gained popularity as alternative and complementary therapies,largely as a result of cultural traditions and excessive cost of modern medicines. Traditionalremedies made from plants play an important role in the health of millions of people. Low-income people such as subsistence farmers, people of small isolated villages and nativecommunities 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 havebeen a valuable source of products for maintaining human health, becoming potential candidatesfor many applications in the pharmaceutical industry. Medicinal plants are the best sources toobtain a variety of drugs (Santos; Ferreira; Damiao, 2010). Mozambique, with its rich and varied flora, is one of the countries where people relymuch on the use of traditional knowledge and medicinal plants to treat various diseases.Gladiolus psittacinus Hook (synonyms in southern Africa collections: Gladiolus natalensis andGladiolus dalenii) is an herbaceous and bulbous plant belonging to the family Iridaceae whichcomprises about 1800 species grouped in 88 genus. Of these genus, Gladiolus and Iris are themost representative with about 260 and 250 species respectively (Machado, 2007 & Manning,2004). The greatest economic importance of the Iridaceae family resides in ornamentals andmedicinal use. Phytochemical study of several species of the family Iridaceae has led to the isolation ofseveral secondary metabolites with high biological activity, 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. The plants of the genus Gladiolus are used in different pharmacopoeias for treatingvarious diseases. In some regions in southern Africa plants of the genus Gladiolus are used totreat a variety of diseases, including acute diarrhea, intestinal parasitic infections, asthma,diabetes, constipation, impotency, headaches, relieve rheumatic pains and hemorrhoids.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
3. 16Qualitative Phytochemical Screening… 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 anddysentery it is an infusion that can be administered orally or enema. As sedative, the plant is usedto calm patients with mental disorders in case of extreme agitation: two drops of fresh juice of thebulb are applied in the nostrils of the patient. In the present study was carried out the qualitative phytochemical screening of the bulbsextracts of Gladiolus psittacinus Hook grown in Mozambique and evaluated the antimicrobialactivity of different extracts and fractions of dried and fresh bulb against gram-positive, gram-negative bacteria and fungi. Materials and MethodsPlant Material and Extraction Fresh samples of Gladiolus psittacinus Hook bulbs were collected in Chiboene, Moambadistrict, Maputo province in July 2006 and in Bilene district, Gaza province in March 2011. Thesamples authentication was made in the herbarium of the Department of Biology – EduardoMondlane University and in the herbarium of the Botany Department – Institute of agriculturalResearch of Mozambique (voucher specimen Nr. 48529).Samples preparationFresh Juice:After collection, the fresh bulbs were washed, cut into small pieces and mechanically crushedwith the help of a centrifuge juice in order to extract the fresh juice.Dried powdered bulb:The fresh bulbs were washed, cut into small pieces and were dried in the oven for 7 days at atemperature of about 50 ˚C and then ground with the help of an electric grinder.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
4. 17Qualitative Phytochemical Screening…Preparation of extracts Crude extracts were prepared by maceration, percolation or Soxhlet extraction usingfollowing solvents: Methanol 90%, Methanol, Dichloromethane – methanol (1:1), Distilledwater. Fractions were obtained by solvent-solvent partition with increasing polarities (hexane orpetroleum ether, Chloroform, ethyl acetate, n-butanol).Extraction of dried sample by percolation 300 g of dried powdered bulb was percolated for 4 days in a 500 ml percolator using assolvent a mixture of methanol and water (9:1). The resulting extract (3 l) was filtered with suctionon a borosilicate glass funnel fitted with a filter membrane and stored at 4 ° C in a fridge for 48hours for precipitation of the fatty material. The extract with precipitated material was left at room temperature for 1 h and thendecanted and filtrated with Whatman ® filter paper (No. 1) and concentrated under reducedpressure in a rotary evaporator BUCHI ® at 40 ° C until the formation of a pasty mass (methanolextract). 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 andthen submitted to liquid / liquid partition using solvents with increasing polarities (n-hexane,chloroform, ethyl acetate and n-butanol). The hexane fraction (HEX) was obtained by liquid / liquid partition using 3x100 ml of n-hexane. And then separated into a 500 ml flask and concentrated in a rotary evaporator underreduced pressure at 40oC. The chloroform fraction (CHL) was also obtained by liquid / liquidpartition with 3x100ml of chloroform. And then separated into a 500 ml flask and concentratedto dryness in a rotary evaporator at 40o C. The hydromethanolic solution was transferred to around bottom flask of 500 ml and concentrated until elimination of methanol. The aqueous extract was further entirely subjected to liquid / liquid partition with 3x100ml of ethyl acetate, followed by 3x100 ml of n-Butanol, yielding three fractions: ethyl acetatefraction (EA), n-butanol fraction (Bu) and aqueous fraction (Aq.Fr.) respectively. The twoorganic fractions were also concentrated in a rotary evaporator under reduced pressure at 40 ° C.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
5. 18Qualitative Phytochemical Screening…All fractions were stored in dry desiccator until constant weight is obtained, and after 7 days theyield 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 (30mm x 100 mm) and subjected to Soxhlet extraction with 400 ml of the mixture dichloromethane -methanol (1:1). After 24 hours the extract was filtered by suction on a glass funnel with amembrane filter, then through a filter paper, and concentrated in rotary evaporator at low pressureuntil dryness. The dried extract was placed in a desiccator until obtaining constant weight, andafter 7 days the yield of extraction was determinedExtraction of the fresh sample by Soxhlet 30 g of the fresh bulb, pre-treated with distilled water and cut into small fragments, wasmilled in Kenwood blender and transferred quantitatively to the cellulose cartridge. Thereafter,the material was extracted in a Soxhlet using as solvent 400 ml of distilled water. After 24 hours,the extract was recovered from the flask, cooled to room temperature and filtered with cottonwool in glass funnel, followed by filter paper and taken to carry out biological testing, after apreliminary 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 andmilled in Kenwood blender. The resulting sample was transferred to a 250 ml Beaker and filteredwith cotton wool in a glass funnel. The filtrate was centrifuged and the resulting extract waspurified with cellulose acetate filter (GVS Filter Technology, USA) porosity of 0.45 mm anddiameter of 25 mm, and transferred to a sterile glass container and taken to carry out biologicaltesting.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
6. 19Qualitative Phytochemical Screening…Solvent-Solvent Partition of the fresh juice In that extraction, 20 g of fresh juice was treated with methanol (600ml) for 24 hours.After filtration, the filtrate was evaporated to dryness in rotary evaporator, leading to a yellowresidue after total evaporation of the solvent. This residue was dissolved in distilled water (20ml)and then proceeded to the successive extractions (3 times respectively), first with the solventpetroleum ether (PE), then with chloroform (CHL), followed by ethyl acetate (EA ) and finallywith n-butanol (Bu) with equal volumes of 100ml each. Petroleum ether (PE), chloroform (CHL),ethyl acetate (EA) and n-butanol (Bu) fractions were concentrated on the rotary evaporator togive petroleum ether residue ( white ), chloroform residue (pale yellow), ethyl acetate residue(yellow and butanolic residue (intense yellow 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,steroids and triterpenoids, amino acids, proteins and carbohydrates adopting the proceduresdescribed by Gomes & Silva (2007), Duarte; Fonte & Santos (2010) and Khan et al (2010).Antimicrobial assay Bacterial cultures reference standard (ATCC) and the yeast Candida albicans (isolatedfrom vaginal discharge) were obtained from the Microbiology Laboratory of the Central Hospitalof Maputo. The yeast Saccharomyces cerevisiae was obtained from commercial packaging yeast.The tests were performed using a standard method recommended for this purpose (NCCLS). Sixbacterial strains were used in the tests, Staphylococcus aureus (ATTC 25923), Escherichia coli(ATCC 25922), Klebsiella pneumoniae (ATCC 15380), Pseudomonas aeruginosa (ATCC27953), Shigella flexneri (ATCC 12022) and Salmonella typhimurium (ATCC 14028). Theculture media used were Nutrient broth, Sabouraud Dextrose Broth and Mueller – Hinton Agar.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
7. 20Qualitative Phytochemical Screening…Bacterial sensitivity test by disc diffusion method The assay was performed using the disk diffusion method where each sterile disk ofWatman filter paper of 6.0 mm in diameter was impregnated with 10 µl aliquots of the extract atconcentrations of 1 mg / disk to the total extract and the respective fractions. The aqueous extract and juice were placed in Petri dishes containing sterile paper discsand allowed to soak for two hours prior to testing. The suspensions containing the bacteria wereprepared separately suspending a colony of each microorganism taken from stock samples withthe aid of a platinum loop in test tubes containing nutrient broth and incubated for 24 hours at 37oC. After were adjusted according to the turbidity of 0.5 in McFarland scale, by visualcomparison with a standard tube containing a standardized suspension.The adjusted suspensions were separately dispersed in a Petri dish containing Mueller – HintonAgar. The Petri dish was divided into four equal portions and placed aseptically one disc of eachimpregnated with plant extract in the center of each division.Plates were also placed in the disks impregnated with ciprofloxacin, DMSO and pure distilledwater respectively. The plates were incubated for 24 h at 37 ° C and then the reading of thediameter of zones of inhibition. All tests were done in triplicate.Fungal sensitivity test by disc diffusion method The suspensions of the yeasts Candida albicans and Saccharomyces cerevisiae wereprepared separately by suspending a colony of each microorganism in test tubes containingSabouraud Dextrose Agar (SDB) incubated at 37 ° C for 48 hours, then standardized according tothe turbidity 0 5 in McFarland scale for visual comparison with standard test tube. The dispersionof the suspension in the plate containing the Mueller – Hinton Agar, was cleaned with sterilecotton swab across size of Petri dish (90 mm) up to obtain a uniform inoculum. The yeastSaccharomyces cerevisiae was isolated by suspending 1 g of yeast in 20 ml of sugar solution.Was allowed to stand for five minutes to allow the activation of yeast and subsequently taken 200µl of it to a test tube containing 15 ml of SDB and incubated for 48 h. Assays were performedInternational Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
8. 21Qualitative Phytochemical Screening…using the same procedure followed for antibacterial tests, with the exception of the positivecontrol, having been used in this nystatin.Interaction Extract – Ciprofloxacin The aqueous extract (Aq.Extr.) and juice were evaluated for the ability to interact withciprofloxacin. Disks were initially impregnated to saturation with the extracts and dried in sterilemedium for 30 minutes. Thereafter foi taken 10 uL of ciprofloxacin 2 mg / ml and was also addedon the disks containing extracts and aseptically transferred to Petri dishes previously inoculatedwith bacteria and incubated for 24 h at 37 ° C. All tests were done in triplicates and the respectivepercentages of interaction determined using the following formula: Interaction% = (Di-Dii) / Diix100 where: Di: mean zone of inhibition of ciprofloxacin combined with extract. Dii: mean zoneof inhibition of ciprofloxacin alone. Results and discussionPhytochemical Screening The phytochemical tests carried out on the extracts, fractions and fresh juice of the bulb,allowed the identification of the following metabolites: alkaloids, anthraquinones, flavonoids,coumarins, saponins, hydrolysable tannins, steroids and triterpenoids, amino acids, proteins andreducing sugars. The phytochemical screening results showed that there is not much difference betweenthe chemical composition of juice and powdered dry bulb, but in practice it has been proved to beeasier to work with the dry powdered bulb than the juice in particular during the solvent / solventpartition. The extraction solvent / solvent of the juice extracted from the fresh bulb and powdereddry bulb allowed to obtain less complex fractions aiming semi-purification of substances throughtheir polarity and solubility.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
9. 22Qualitative Phytochemical Screening… The results of the phytochemical screening of the crude extracts are summarized in thetable 1, and the results of the phytochemical screening of the fractions from solvent-Solventpartition were summarized in table 2.Table 1: Phytochemical tests of Crude extracts Dichloromethane- Aqueous Methanol 90% Methanol(1:1) extract (24h Juice Phytoconstituents Extract ( fresh Extract Soxhlet fresh (fresh bulb) bulb) (24h soxhlet dried bulb) bulb)Alkaloids + + + +Anthraquinones + + - -Coumarins - + - -Flavonoids + + - -Saponins + + - +Tannins + + - -Amino acids + + + +Proteins - - - +Reducing Sugars - + + ++: present; -: absentTable 2: Phytochemical tests of fractions from solvent – solvent Partition Juice (Fresh Bulb) Powdered dry bulb Phytoconstituents PE/HEX CHL EA Bu PE/HEX CHL EA BuAlkaloids - + - - - + + -Anthraquinones - - + - - + + -Coumarins - + + - - + + -Flavonoids - + + + - + + +Iridoids - - - - - - - -Saponins - - - + - - + +Steroids/Terpenoids + + + + + + + ++: present, -: absent, PE: petroleum ether fraction, HEX: hexane fraction, CHL: chloroformicfraction, EA: ethyl acetate fraction, Bu: butanolic fraction.Results of antimicrobial assay The results of the antimicrobial screening realized in this study with extracts and fractionsless complex of Gladiolus psittacinus Hook against Escherichia coli, Pseudomonas aeruginosa,Staphylococcus aureus, Shigella flexneri, Salmonella typhimurium, Klebsiella pneumoniae,Candida albicans and Saccharomyces cerevisiae showed interesting antimicrobial activities ofthis plant as shown in table 3a and 3b.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
10. 23Qualitative Phytochemical Screening…Table 3a. Results of antimicrobial activity tests mean diameter of inhibition zone (mm). Micro- NT CIP MET HEX CHL EA BU organisms Candida 18±0,6 20±0,6 28±0,6 25±0,6 24±0,6 23±0,6 albicans E.coli 32±0,6 - - - - - ATCC25922 P. aeruginosa 32±0,0 18±0,6 22±1,0 23±0,3 25±0,0 20±0,1 ATCC 27953 K. pneumoniae 30±1,0 - - - - - ATCC 15380 S.aureus 32±0,6 - - - - 7±0,0 ATCC 25923 Saccharomyces 10±0,6 - - 20±0,3 13±0,6 cerevisiae S. typhimurium 32±0,0 BE - BE BE BE ATCC 14028 Shigella flexneri 33±0,6 - - BE BE - ATCC 12022(_ ± _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) No zoneof inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT )3.2 cµg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction;CHL: chloroform fraction; EA: ethyl acetate fraction; BU: butanol fraction; Aq.frac.: aqueous fraction;Aq.Extr.: aqueous extract; BE - bacteriostatic activity. The yeast Saccharomyces cerevisiae had notcontrol.Table 3b. Results of antimicrobial activity tests (cont.)Mean diameter of inhibition zone (mm) CH2Cl2-Micro-organisms NT CIP DMSO Aq.Frac. Juice Aq.Extr. CH3OH Candida albicans 18±0,6 - 21±0,3 - 16±0,3 -E.coli ATCC25922 32±0,6 - - - 19±0,4 16±0,1 P. aeruginosa 32±0,0 - 15±1,0 - 12±1,0 14±0,3 ATCC 27953 K. pneumoniae 30±1,0 - - - - - ATCC 15380 S.aureus 32±0,6 - - - - - ATCC 25923 Saccharomyces - 12±0,3 - 15±0,0 - cerevisiae S. typhimurium 32±0,0 - - - - - ATCC 14028 Shigella flexneri 33±0,6 - - - - - ATCC 12022(_ ± _): Mean and standard deviation of triplicates; concentrations of extracts - 100 mg / disc, (-) : No zoneof inhibition was observed; Positive Control : Ciprofloxacin (CIP) 2 mg / disc for bacteria and nystatin (NT )3.2 cµg / disk for C. albicans; DMSO: solvent control; MET: crude methanol extract; HEX: hexane fraction;CHL: chloroform fraction; EA: ethyl acetate fraction; BU: butanol fraction; Aq.Frac.: aqueous fraction;Aq.Extr.: aqueous extract; BE: bacteriostatic activity. The yeast Saccharomyces cerevisiae had notcontrol.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
11. 24Qualitative Phytochemical Screening… Preparations of the bulb of Gladiolus psittacinus Hook are used in traditional medicinefor the treatment of various diarrheal diseases. The presence of secondary metabolites activeagainst microbial pathogens in the extracts of this plant has been reported. Ameh et al (2011) demonstrated antimicrobial activity against Pseudomonas aeruginosaand Aspergillus niger of the aqueous extract of Gladiolus bulb. The phytochemical screening ofthe same extract resulted in the identification of some metabolites namely: alkaloids, tannins,cardiotonic glycosides, flavonoids and carbohydrates. Nguedia et al (2004) showed antifungal activity of the hydroethanolic extract of the bulbof Gladiolus gregasius Baker against Candida albicans and Candida krusei. However, the sameextract was not active against Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris,Staphylococcus aureus and Streptococcus faecalis. The phytochemical screening revealed thepresence of saponins, glycosides, polyphenols, phenols, triterpenes and steroids. The crude methanolic extract exhibited an antibacterial effect against Pseudomonasaeruginosa with an inhibition zone of 18 ± 0.6 mm and a bacteriostatic effect against Salmonellatyphimurium. An antifungal effect was shown against the yeasts Saccharomyces cerevisiae andCandida albicans with inhibition zones of 20± 0.6 mm and 10 ± 0.6 mm respectively. The activity of that extract against Pseudomonas aeruginosa was low compared tociprofloxacin used as control. However, it showed a high antifungal activity against Candidaalbicans when compared to the control nystatin. The antifungal activity displayed against candidaalbicans was higher than with the other yeast Saccharomyces cerevisiae. This extract was notactive against the rest of the microorganisms tested. The hexane fraction exhibited an antibacterial effect against Pseudomonas aeruginosawith an inhibition zone of 22 ± 0.6 mm. An antifungal effect against Candida albicans has beenshown with a zone of inhibition of 28 ± 0.6 mm. This fraction was not active at the sameconcentration against other microorganisms tested. The activity of the extract againstPseudomonas aeruginosa was low compared to the antibiotic used as control; but that was higherthan the activity of the crude methanolic extract. This fraction exhibited also an antifungalactivity against Candida albicans higher than the control nystatin and than the crude methanolextract. In phytochemical studies performed on this fraction were identified terpenoids. According to literature, the activity of these compounds is related to its lipid solubilitywhich allows them to interact with the biomembranes of microorganisms, causing rupture of theInternational Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
12. 25Qualitative Phytochemical Screening…cells. The solubility of these compounds in the biomembranes enables their interaction with ionchannels, carrier molecules, membranes receptors, which leads to changes in conformation andloss of function (Lopez, 2010). The chloroform fraction also showed an antibacterial effect against Pseudomonasaeruginosa with a zone of inhibition of 23 ± 0.6 mm and a bacteriostatic effect against Shigellaflexneri and Salmonella typhimurium. It was also shown antifungal activity against Candidaalbicans with a zone of inhibition of 25 ± 0.6 mm. This fraction was not active at the sameconcentration against other microorganisms tested. The activity of the extract againstPseudomonas aeruginosa was low compared to the control. However, the activity of this fractionwas superior to the activity of the crude methanol extract and the hexane fraction. This fractionalso showed antifungal activity against Candida albicans higher than the control, the crudemethanol extract, and the hexane fraction. In phytochemical tests performed on this fraction by precipitation methods (Mayer andDragendorff) and thin layer chromatography were identified alkaloids. The presence of alkaloidsin this plant have been reported by Odhiambo et al (2010) as the active constituents present in theextract CH2Cl2: CH3OH (1:1) against Aspergillus niger. The ethyl acetate fraction exhibited an antibacterial activity against Pseudomonasaeruginosa with an inhibition zone of 25 ± 0.0 mm and a bacteriostatic effect against Shigellaflexneri and Salmonella typhimurium. This fraction was also active against the yeasts Candidaalbicans and Saccharomyces cerevisiae with zones of inhibition of 24 ± 0.6 ± 0.3 and 20 mmrespectively. This fraction was not active at the same concentration against other microorganismstested. The antibacterial activity against Pseudomonas aeruginosa of this fraction was lowcompared to the control, but greater than the activity of the crude methanol extract, the hexaneand chloroform fractions. This fraction was more active against Candida albicans than againstthe yeast Saccharomyces cerevisiae. It also showed antifungal activity against Candida albicanshigher than the control nystatin, crude methanol extract, hexane and chloroform fractions.Phytochemical tests realized on this fraction showed the presence of alkaloids, coumarins andsaponins that may be associated with antimicrobial activity shown by this fraction. The butanol fraction showed antibacterial activity against Pseudomonas aeruginosa witha zone of inhibition of 20 ± 0.1 mm, a low activity against Staphylococcus aureus and abacteriostatic effect against Salmonella typhimurium. This fraction also showed antifungalInternational Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
13. 26Qualitative Phytochemical Screening…activity against Candida albicans with a zone of inhibition of 23 ± 0.6 mm and a moderateactivity against the yeast Saccharomyces cerevisiae with a zone of inhibition of 13 ± 0.6 mm. The activity of this fraction against Pseudomonas aeruginosa was low compared to theantibiotic used as control; but it was higher than the crude methanol extract. It also showedantifungal activity against Candida albicans higher than the control nystatin and the crudemethanol extract. The phytochemical tests of this fraction made possible the identification ofsaponins. The presence of these compounds in the extracts of this plant has also been evidencedby Odhiambo; Siboe; Lukhoba; Dossaji, (2010). The extract CH2Cl2: CH3OH (1:1) showed antibacterial activity against Pseudomonasaeruginosa with an inhibition zone of 15 ± 1.0 mm. It also showed activity against candidaalbicans and Saccharomyces cerevisiae with zones of inhibition of 21 ± 0.3 mm and 12 ± 0.6 mmrespectively. The activity of the extract against Pseudomonas aeruginosa was low compared tothe antibiotic used as control. Furthermore, their activity against Candida albicans was higherthan the control. The activity demonstrated by the extract against Candida albicans was higher than thatdisplayed against Saccharomyces cerevisiae. Phytochemical Tests carried out on this extractmade possible the identification of alkaloids, amino acids, anthraquinones, flavonoids, tanninsand saponins. The quinolinic compounds are capable of forming irreversible complexes withnucleophilic amino acids in proteins, often leading in inactivating proteins and consequent loss offunction, which explains the high antimicrobial effect of these compounds (Lopez, 2010). Thebiological role of tannins in plants has been investigated and it is believed that they are involvedin chemical defense of plants against attack by herbivores and against pathogens (Oliveira et al,2007). Not surprisingly, extracts containing tannins show activity against microorganisms.Flavonoids are compounds synthesized by the plants in response to microbial infection (Dixon;Dey; Lamb, 1983), so it is not surprising to demonstrate antimicrobial activity in vitro against abroad range of microorganisms. This activity is probably due to the ability to form complexeswith extracellular soluble proteins and the bacterial cell wall. The more lipophilic flavonoids canalso disrupt microbial membranes. In studies realized by Odhiambo et al (2010), was also identified the presence of alkaloidsin the extract CH2Cl2 – CH3OH (1:1) which were attributed the potent antifungal activityInternational Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
14. 27Qualitative Phytochemical Screening…demonstrated by this extract against Aspergillus niger. The activity exhibited by this extractagainst Candida albicans can probably also be associated with these compounds identified. The juice from the fresh bulb showed an antibacterial activity against Escherichia coli andPseudomonas aeruginosa with inhibition zones of 19 ± 0.4 and 12 ± 1.0 mm respectively. It wasalso active against candida albicans and Saccharomyces cerevisiae with respective zones ofinhibition of 16 ± 0.3 and 15 ± 0.0 mm. The activity of the extract against Escherichia coli andPseudomonas aeruginosa was low compared to the control. Its activity against Candida albicanswas also lower than control. Phytochemical tests carried out on this extract, have made possible the identification ofamino acids, proteins, alkaloids and saponins. The activity shown by this extract may beassociated with these compounds. The aqueous extract of the fresh bulb was active against Pseudomonas aeruginosa andEscherichia coli with the respective zones of inhibition of 16 ± 0.1 and 14 ± 0.3 mm. Howeverwas inactive against the rest of the tested bacteria. No antifungal effect was demonstrated in thisextract. The activity of the extract against Escherichia coli and Pseudomonas aeruginosa waslow compared to the antibiotic used as control. Phytochemical tests carried out on this extract made possible the identification ofalkaloids. The study realized by Ameh et al (2011), reported the presence of alkaloids in theaqueous extract of the bulb of Gladiolus obtained by the same method. However, the aboveextract was found to be slightly active against Pseudomonas aeruginosa and Aspergillus nigerand inactive against Candida albicans. The bacteria Escherichia coli was susceptible to aqueous extract and juice, which aregenerally used in traditional medicine for the treatment of diarrhea, gonorrhea and other intestinaldisturbances. This activity may explain its traditional use in the treatment of diarrhea of variousorigins. The results of the interaction Extract-Ciprofloxacin show an additive effect for thecombination Juice + Ciprofloxacin in case of E. coli, S. flexneri, K. pneumoniae and P.aeruginosa; Indifference in case of S. aureus and S. typhimurium. In the case of the combinationof Ciprofloxacin + aqueous extract, the antagonistic effect was observed for all microorganismstested. The results of the interaction Extract – Ciprofloxacin are summarized in table 4.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
15. 28Qualitative Phytochemical Screening…Table 4 - Results of the interaction Extract - CiprofloxacinMean diameter of the inhibition zone (mm) Distilled CIP+ Microorganism CIP Juice Juice+CIP Aq.Extr. water Aq.Extr. E.coli - 31±0,6 19±0,6 33±0,6 16±0,0 28±0,3 ATCC25922 S.aureus - 33±0.3 - 33±0,6 - 30±2,6 ATCC 25923 Shigella flexneri - 32±1,0 - 34±1,0 - 30±0,6 ATCC 12022 S. typhimurium - 32±0.3 - 32±0,3 - 31±0,6 ATCC 14028 K. pneumoniae - 29±0,0 - 30±1,6 27±1,0 ATCC 15380 P. aeruginosa - 30±1,0 28±0.5 34±0,6 19±1,0 25±0,3 ATCC 27953Purified distilled water: Negative control; CIP: Ciprofloxacin - positive control; 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 aslight increase. Only in the case of Staphylococcus aureus and Salmonella typhimurium, thecombination showed indifference. However, the aqueous extract had an opposite effect, thediameters of inhibition of the control showed a reduction when used in combination with theaqueous extract for all tested microorganisms. According to Nwze & Eze (2009), the convention suggests to use additive when thepercentage of the increase of the diameter of inhibition of the plant extract combination with theantibiotic is less than or equal to 19%; indifferent, when the diameter of inhibition of theantibiotic combination with the extract is equal to the diameter of inhibition when the antibioticis tested alone, and there is an antagonistic effect in case of loss of inhibition diameter when theantibiotic control is used in combination with the extract. This work was based on the same convention. An additive effect was observed in thefollowing microorganisms: Escherichia coli with 6.45% addition, Shigella flexneri with additionof 6.25%, Klebsiella pneumoniae with 3.45% of addition and Pseudomonas aeruginosa withaddition of 13.33% when the juice combined with ciprofloxacin. Moreover, the bacteriaSalmonella typhimurium and S. aureus were indifferent to the combination. The combination ofthe antibiotic with the aqueous extract showed an antagonistic effect for all the microorganismstested.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
16. 29Qualitative Phytochemical Screening… It is important to note that the action of medicinal plants can never be fully understood byanalyzing its components separately. Proponents of this theory argue that the properties are due tointeractions between multiple components (Wink, 2008). For example, the saponins present inmany medicinal plants, enhance the absorption and activity of other substances. The saponins ofSapindus mukorossi increase the intestinal absorption of certain natural antibiotics. Therefore,positive interaction found in the combination of the juice with ciprofloxacin can be associatedwith the presence of saponins in the plant, which can essentially increase the absorption ofciprofloxacin through the biological membranes of the bacteria, thereby enhancing its effect. Conclusion Qualitative phytochemical analysis of the extracts and fractions of the bulb revealed thepresence of: 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 Candidaalbicans, Saccharomyces cerevisiae and Pseudomonas aeruginosa. The determination of theinteraction Ciprofloxacin - aqueous extract and Ciprofloxacin - juice revealed an antagonismbetween ciprofloxacin and the aqueous extract and an additive effect between Ciprofloxacin andjuice. Phytochemical and antimicrobial test results obtained in this work in combination withthe previously reported in literature by different authors validate to some extent the use of thebulb of Gladiolus psittacinus Hook in traditional medicine in treating diseases of microbialorigin, thus demonstrating the potential of this plant. ReferencesAmeh, S., Obodozie, O., Olorunfemi, P., Okoliko, I., Ochekpe, N. (2011). Potentials of gladioluscorms as an antimicrobial agent in food processing and traditional medicine. Journal ofMicrobiology And Antimicrobials, 3(1), 8-12.Dixon, R.A., Dey, P.M., Lamb, C.J. (1983). Advances In enzymology and relates areas ofmolecular biology. New York: John Wlley & Sons.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
17. 30Qualitative Phytochemical Screening…Duarte, M., Fonte, N., & Santos, C. (2010). Farmacognosia: manual prático. Curitiba:Universidade Federal do Paraná.Gomes, E., Silva, O. (2007). Plantas medicinais e fitoterapia: técnicas laboratoriais. Maputo.Khan, R., Zakir, M., Afaq, S. H., Latif, A., & Khan, A. U. (2010). Activity of solvent extracts ofprosopis spicigera, zingiber officinale and trachyspermum ammi against multidrug resistantbacterial and fungal strains. Journal of Infection in Developing Countries, 3(5), 292-300.López, P.V. (2010). Bioprospecção de extractos de croton urucurana baill e seus fungosendofíticos. (Tese de Mestrado, 2010). Programa de Pós Graduçao em Microbiologia,Universidade Federal do Paraná, 138.Machado, C. (2007). Iridaceae. Herbário da Universidade de Coimbra. Coimbra: Universidadede Coimbra. Retrieved on July 31, 2012 ofhttp://www.uc.pt/herbario_digital/flora_PT/familias/Iridaceae/.Manning, J. (2004). Iridaceae. Herbário da Universidade de Coimbra. Coimbra: Universidade deCoimbra. retrieved on June 28, 2011 of http://www.plantzafrica.com/planthij/Iridaceae.htm.Nguedia, C. A., Etoa, F.X., Benga, V.P., Lontsi, D., Kuete, Y., Moyou, R. (2004). Anti-candidalproperty and acute toxicity of gladiolus gregasius baker (Iridaceae). Medical & PharmaceuticalTrade , 13, 149-159.Nweze, E.I., Eze, E. E. (2009). Justification for the use of ocimum gratissimum l in herbalmedicine and its interaction with disc antibiotics. BMC Complementary and AlternativeMedicine, 9(37), 1-6.Odhiambo, J., Siboe, G., Lukhoba, C., Dossaji, S. (2010). Antifungal activity of crude extracts ofgladiolus dalanii van geel (Iridaceae). Afr. J. Trad. Cam, 7(1), 53-58.Oliveira, C., Schenkel, E.M., Gosmann, G., Auller, L., Palazzo, G.J., Ros, P. (2007).Farmacognosia: da planta ao medicamento. Porto Alegre: UFRGS.Rojas, J.J., Ochoa, V. J., Ocampo, S.A., Muñoz, J.F. (2006). Screening for antimicrobial activityof ten medicinal plants used in Colombian folkloric medicine: a possible alternative in thetreatment of non-nosocomial infections. BMC Complementary and Alternative Medicine, 6, 2.Rukangira, E., (2001). Medicinal plants and traditional medicine in Africa: constraints andchallenges. Sustainable Development International, 4, 179-184.Santos, S.C., Ferreira, F.S., Damiao, A.O., Damiao, A.O. (2010). Avaliação da atividadeantibacteriana dos extratos de Avicennia schaueriana Stapf & Leechm. ex Moldenke,Verbenaceae. Brazilian Journal of Pharmacognosy , 20(1), 124-129.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.
18. 31Qualitative Phytochemical Screening…Wink, M. (2003). Evolution of Secondary metabolites from an ecological and molecularphytogenetic perspective. Phytochemistry , 64, 3-19.International Network Environmental Management Conflicts, Santa Catarina – Brasil, 2(1), pp. 14-31, jan. 2013.