1B GJMPR July 2016 - aensi

Global Journal ofMedicinal Plant Research 2016. 4(3): 1-5 ISSN: 2074-0883 Journal home page: http://www.aensiweb.com/GJMPR/

RESARCH ARTICLE

Antimicrobial Activity of Five Different Extracts of Gladiolus italicus Miller from Turkey 1Sevil

Toroğlu, 2Sayım Aktürk, 2Sadık Dinçerand 3Dilek Keskin

1Kahramanmaraş

Sütcü İmam University, Department of Biology, Faculty of Arts and Science, Kahramanmaras 46100- Kahramanmaras, Turkey, 2Cukurova University, Department of Biology, Faculty of Arts and Science, Balcalı 01330- Adana, Turkey, 3Adnan Menderes University, Cine Vocational High School, , 09500- Cine-Aydin, Turkey

ABSTRACT In the present study, the antimicrobial activities of the extracts of methanol, DMSO, ethylacetate, n-Hexan and chloroform G. italicus MILLERwere studied by disc diffusion method. These extracts were tested against fourteen bacteria and one fungi, which revealed various levels of antimicrobial activity. DMSO extracts of flowers of the plant showed the best antimicrobial activity against VRE( 9mm50 μl-1). DMSO extracts of leaves of it showed the best antimicrobial activity against E. coli 9539994( 10mm50 μl-1). DMSO extracts of branches of itshowed the best antimicrobial activity against E. coli 9539994, Methicillin-resistant S aureus (MRSA), S. aureus ATTC 6538, S. aureus ATTC 29213 (9mm 50 µl-1). DMSO extracts of corms of itshowed the best antimicrobial activity against E. coli 9539994, Methicillin-resistant S aureus (MRSA) (BaL) and E. cloacea(9mm 50 µl-1). The methanol, ethylacetate, n-Hexan and chloroform extracts of different parts of this plant showed no inhibition zones all tested bacteria and fungi. But, DMSO extracts of different parts of this plant showed moderate antimicrobial and antifungal activities. Key words: Iridaceae,Gladiolus italicusMILLER, Antimicrobial activity, Plant extracts, Aladağlar mountains (Yahyalı/KayseriTurkey). Address for Correspondence: Sevil Toroğlu,Kahramanmaraş Sütcü İmam University, Department of Biology, Faculty of Arts and Science, Kahramanmaras 46100Kahramanmaras, Turkey, E-mail:[email protected]

Received 3 July 2016; accepted 16 September 2016; published 30 September 2016 INTRODUCTION The emergence and spread of antimicrobial resistance is a growing problem in both developing and developed countries and threatens to become a global health crisis (Shinwari et al., 2009). Multiple microbial resistances among Gram-negative microorganisms have been a long term and well recognized problem with enteric infections (Saeed & Tariq, 2007). New strategy for the containment of resistance needs to be developed, applied and evaluated. The most important approaches of solving this problem has lead to the screening of several medicinal plants for their potential antimicrobial activity (Keskin & Toroglu., 2011; Shinwari et al., 2012; Sivasankaridevi et al., 2013; Bisignano et al., 2013; Walter et al., 2011). Gladiolus L. (Iridaceae) is a large genus that occurs in Africa, the Mediterranean basin and Western asia, a member of Iridaceae, subfamily Crocoideae and comprises approximately 260 species (Ameh et al., 2010). Nine Gladiolus species have grown in various regions of Turkey, four of which are endemic to the country (Uzen, 1999). Gladiolus anatolicus (Boiss.) Stapf., G. humilis Stapf., G. micranthus Stapf., G. halophilus Boiss. and Heldr., G. italicus Miller, G. antakiensis A.P. Hamilton, G. kotschyanus Boiss., G. illyricus W. Koch., G. Open Access Journal Published BY AENSI Publication © 2016 AENSI Publisher All rights reserved This work is licensed under the Creative Commons Attribution International License (CC BY).http://creativecommons.org/licenses/by/4.0/

ToCite ThisArticle:Sevil Toroğlu,Sayım Aktürk, Sadık Dinçer and Dilek Keskin,Antimicrobial Activity of Five Different Extracts of Gladiolus italicus Miller from Turkey. Glob. J. Med. Plant Res, 4(3): 1-5, 2016

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Sevil Toroğluet al., 2016/Global Journal of Medicinal Plant Research, 4(3): 1-5, 2016

atroviolaceus Boiss. (Erol et al., 2006). Many medicinal usage of Gladiolus species were known, for example treating infections of the skin, gut, urogenital system, upper respiratory tract, treating gonorrhea, dysentery, meningitis, malaria, diarrhoea, ulcers and HIV related fungal infections and other infectious conditions (Nguedia et al., 2004; Ameh et al., 2010; Odhiambo et al., 2010). The antimicrobial effect of some species of Gladiolus has been studied by previous researchers (Nguedia et al., 2004; Ameh et al., 2010; Odhiambo et al., 2010; Kahriman et al., 2012). As far as we know, this is the first study,the antimicrobial activity of Gladiolus italicus MILLERfive different extracts against to eight bacteria and one fungus were reported in this study from Turkey. MATERIALS AND METHODS Plant collection and preparation of extracts: G. italicusMILLER was collected by Dr. E. Toroğlu in Aladağlar Mountain (Yahyalı/Kayseri-Turkey), Gökoluk Yayla at an altitude of 1600-2000 meter during the flowering stage in July 2006. The plant was identified by Dr. M. Çenet. Voucher specimen was deposited in the Herbarium of the Department of Biology, Osmaniye Korkut Ata University, Osmaniye, Turkey (Voucher no: 1809 OKUH). Morphological characters (numbers of leaves, vascularisation and width of the leaf’s surface and number of flower and flowers’ colour and properties of leaves and flowers) of G. italicuswere determined using stereo microscope (Nikon SMZ 1000 model) and the present results were compared with the previous information in the flora of Turkey (Davis et al., 1982). The plant parts (flowers, leaves, branches and corms) were washed with sterile distilled water and then broken into small pieces. 20 g of this plant was extracted with 150 mL of methanol, DMSO, ethylacetate nHexan and chloroform extracts (Merck, Darmstadt) for 24 h by Soxhlet appartaus (Keskin & Toroglu, 2011; Toroglu & Cenet, 2013). Prepared extracts were dried at 30oC using a rotary evaporator until amount of each extracts was 1 mL. Microorganisms and media: Fourteen bacteria (1-Escherichia coli K-12 (Standard), 2-Staphylococcus aureus ATTC 29213 (Standard), 3-Bacillus subtilis B-354 (Standard), 4-Pseudomonas aeruginosa (Clinic), 5-Klebsiella spp.1 (Clinic), 6Escherichia coli 9539994 (Standard), 7- Staphylococcus aureus ATTC 6538 (Standard), 8StaphylococcusepidermidisB-4268 (Standard), 9-Vancomycin-resistant Enterococcus (VRE) (Clinic), 10Methicillin-resistant Staphylococcus aureus (MRSA) (Clinic), 11-Enterobacter cloacea (Natural), 12Staphylococcusepidermidis (Clinic), 13- Escherichia coli (Clinic), 14- Pseudomonas aeruginosa (Natural)) were obtained from the Biology Department of Cukurova University, Science and Arts Faculty. Cultures of these bacteria were grown in Nutrient Broth (NB) (Difco) at 37±0.1oC for 24 h. Cultures of one fungus (Candida albicans (Clinic). were grown in Sabouraud Dextrose Broth (SDB) (Difco) at 25±0.1oC for 24 h. Antimicrobial assay: The disc assay described by Bauer et al. (1966) was used. Each of the extracts individually were injected into empty sterlized antibiotic discs having a diameter of 6 mm (Schleicher and Schül No:2668, Germany) in the amount of 50 μL. Discs injected with pure methanol, DMSO, ethylacetate n- Hexan and chloroform served as negative controls. The bacteria were incubated in Nutrient Broth (NB) (Difco) at 37±0.1oC for 24 hr, and the fungi were incubated in Sabouraud Dextrose Broth (SDB) (Difco) at 25±0.1oC for 24 hr. The tested bacteria and fungi were injected into petri dishes (9 cm) in the amount of 0.01ml (106 ml-1 for the bacteria and 105 ml-1 for the fungi) (Anon., 2000), 15 ml of Muller-Hinton agar (MHA, Oxoid) and Saboraud Dextrose Agar (SDA) (sterilized in a flask and cooled to 45-50oC) were distributed homogeneously (Collins et al., 1989). Discs injected with each of the extracts were applied on the solid agar medium by pressing slightly. The treated petri dishes were placed at 4oC for 1-2 hr and then the injected plates with bacteria were incubated at 37±0.1oC for 18-24 hr, plates inoculated with fungi were incubated at 25±0.1oC for 48 hr (Collins et al., 1989; Bradshaw, 1992; Toroglu, 2007). Ampicillin (30 µg/disc) and Nystatin 100 Units (10 μg/disc) discs were used as standard antibiotics (as positive control). After incubation, all plates were observed for zones of growth inhibition, and the diameters of these zones were measured in millimeters. The experiments were conducted three times. RESULTS AND DISCUSSION The antimicrobial activity screening for five different solvent extracts of G. italicus MILLER. was tested in vitro using disc diffusion method with the microorganisms as given in Table 1. DMSO, methanol, ethylacetate, n-Hexan and chloroform used as negative controls against the all tested microorganism and they did not show antimicrobial activity. Possessed antimicrobial activities of extracts of flowers, leaves, branches and corms as shown in Table 1.

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In our study, only DMSO extracts of the plant showed moderate antimicrobial activity against majority of the microorganisms. But, the methanol, ethylacetate, n-Hexan and chloroform extracts of the plant showed no antimicrobial activity against against the all tested microorganism. As for the antimicrobial activity of flowers of the plant (GIF), the DMSO extracts of GIF showed the best antimicrobial activity against VRE( 9mm50 μl-1). When it comes to antimicrobial activity of leaves of the plant (GIL), the DMSO extracts of GIL showed the best antimicrobial activity against E. coli 9539994( 10mm50 μl-1). With regard to antimicrobial activity of branches of the plant (GIB), the DMSO extracts of GIB showed the best antimicrobial activity against E. coli 9539994, Methicillin-resistant S aureus (MRSA), S. aureus ATTC 6538, S. aureus ATTC 29213 (9mm 50 µl-1). As regard as, antimicrobial activity of corms of the plant (GIC), the DMSO extracts of GIC showed the best antimicrobial activity against E. coli 9539994, Methicillin-resistant S aureus (MRSA) and E. cloacea(9mm 50 µl-1). In previous studies about antimicrobial activity of Gladiolus species, Odhiambo et al. (2010) reported that Dichloromethane and methanol extracts of Gladiolus dalenii showed antifungal activity against Aspergillus niger. But Steenkamp et al. (2007) reported that no inhibition zones of water and methanol bulb extracts of this Gladiolusdalenii van Geel against S. aureus and S. epidermidis. SimilarlyFawole et al. (2009) showed that ethanol extracts were active against C. albicans but inactive against E. coli. Ameh et al. (2010) reported that aqueous extracts of corms of Gladiolus showed antibacterial activities against Pseudomonas aeruginosa and Aspergillus niger. Assob et al. (2011) reported that hexane, ethylacetate and methanol extracts of Gladiolus gregasius showed only mild antibacterial and antifungal activities (on Candida albicans and Candida krusei). But methanolic extract of G. gregasius showed no inhibition zone P. mirabilis, S. flexneri, P. aeruginosa and K. pneumonia. In contrast, Nguedia et al. (2004) informed that bulb extract of this plant was fungicidal with no antibacterial activity. Ncube et al. (2008) showed that harvesting periods of species of Gladiolus, and used solvents for extraction of the this plant can change antimicrobial activity. The observed antibacterial activity is attributed to the presence of bioactive compounds in the extracts of plants tested. Plants containing active compounds are able to inhibit the microbial growth and also the presence of these bioactive compounds in crude extracts is known to confer antibacterial activity against disease-causing microorganisms (Farnsworth, 1982) and offer protection to plants themselves against pathogenic microbial infections (De & Ifeoma, 2002). They are widely recognised as sources of active antimicrobial metabolites (McGaw et al., 2008). Especially, alkaloids, tannins, saponins, cardiac glycosides, flavonoids and carbohydrate are known to have broad spectrum of pharmacological activities, including antimicrobial properties (Ameh et al., 2010). Table 1 insert here please: Table 1:Antimicrobial activity of five different solvent extracts of Gladiolus italicus Mıller (Iridaceae) flowers, leaves, branches and corms. Inhibition zone (mm)* (50µl/disc) Standard Control Microorganisms Flowers Leaves Branches Corms antibiotics Discs (µg/disc) A B C D E A B C D E A B C D E A B C D E Amp30 N10 ABCDE 1. Escherichia coli KNT 0 0 8 0 0 0 0 9 0 0 0 0 8 0 0 0 0 7 0 0 0 11 12 2. Staphylococcus aureus 0 7 0 0 0 0 9 0 0 0 0 9 0 0 0 0 8 0 0 0 11 NT 0 ATTC 29213 3. Bacillus subtilis B0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 18 NT 0 354 4. Pseudomonas 0 8 0 0 0 0 8 0 0 0 0 8 0 0 0 0 8 0 0 0 12 NT 0 aeruginosa 5. Klebsiella spp.1 0 0 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NT 0 6. Escherichia coli 0 7 0 0 0 0 10 0 0 0 0 9 0 0 0 0 9 0 0 0 0 NT 0 9539994 7. Staphylococcus aureus 0 8 0 0 0 0 9 0 0 0 0 9 0 0 0 0 8 0 0 0 11 NT 0 ATTC 6538 8. Staphylococcusepide rmidis 0 0 0 0 0 0 9 0 0 0 0 8 0 0 0 0 7 0 0 0 11 NT 0 B-4268 9. Vancomycinresistant 0 9 0 0 0 0 9 0 0 0 0 8 0 0 0 0 7 0 0 0 12 NT 0 Enterococcus (VRE) 10. Methicillin-resistant 0 0 0 0 0 0 0 0 0 0 0 9 0 0 0 0 9 0 0 0 0 NT 0 Staphylococcus

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aureus (MRSA) 11. Enterobacter 0 0 0 0 0 0 8 0 0 0 0 8 0 0 0 0 9 0 0 0 0 NT 0 cloacea 12. Staphylococcus 0 8 0 0 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NT 0 epidermidis 13. Escherichia coli 0 0 0 0 0 0 8 0 0 0 0 0 0 0 0 0 8 0 0 0 0 NT 0 14. Pseudomonas 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NT 0 aeruginosa 15. Candida albicans 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 NT 18 0 A: Methanol, B: DMSO, C: Ethylacetate D: n- Hexan E: Chloroform; Amp (30 µg/disc), Nystatin 100 Units (10 µg/disc) NT: Not tested discs

Conclusıon: Present study depicts that degree of antimicrobial activity depends on type of extracts, and part of the plant and compounds of it. In our study, only DMSO extracts of this plant showed antimicrobial activity against tested microorganisms. Soluble compounds of this plant in DMSO solvent can cause antimicrobial activities. Characterization of compounds of the plant can require for antimicrobial activities and chemistry. The millenarian use of these plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases. ACKNOWLEDGEMENT The authors thank to Dr. E. Toroğlu for collecting plant used in this study and to Dr. M. Cenet for identifying this plant used in this study. REFERENCES Ameh, S.J., N.A. Ochekpe, E.I. Okolikoand P.O. Olorunfemi, 2010. Basis for Ethnomedical use of Gladiolus corm (Family: Iridaceae) in West Africa. Asian J. Exp. Biol. Sci., 1(4): 902-906. Anonymous, 2000. National commitee for clinical laboratory standarts. Performance Standarts for Antimicrobial Disc Suspectibility Tests,7th edition. Approved Standart M2-A7 NCCLS, Pennsyvania, USA. Assob, J.N.C., H.F.L.F. Kamga, D.S. Nsagha, A.L. Njunda, P.F. NDE, E.A. Asongalem, A.J. Njouendou, B. Sandjon andV.B. Penlap, 2011. Antimicrobial and toxicological activities of five medicinal plant species from Cameroon Traditional Medicine. BMC Complementary & Alternative Medicine, 70: 1-11. Bauer, A.W., W.M.M. Kirby, J.C. Sherrisand M. Turck,1966. Antibiot ic susceptibility testing by a standardized single disc method. Am. J. Clin. Pathol., 45: 493-496. Bisignano, C., A. Filocamo, R.M. Faulksand G. Mandalari, 2013. In vitro antimicrobial activity of pistachio (Pistacia vera L.) polyphenols. FEMS Microbiol. Lett., 341: 62-67. Bradshaw, L.J., 1992. Laboratory Microbiology, 4th edition. Saunders College Publishing. Fort worth, Philadelphia, USA. Collins, C.H., P.M. Lyne and J.M. Grange, 1989. Microbiological methods. 6thEdn. pp. 410. Butterworths, London. Davis, P.H., R.R. Mill and K. Tan. 1982. Flora of Turkey and the east Aegean Islands. Edinburgh Univ. Pres., 8: 441-450. De, N. and E. Ifeoma. 2002. Antimicrobial effects of components of the bark extracts of neem (Azadirachta indica A. juss). J. Technol. Dev.,8: 23-28. Erol, O., E. Uzen and O. Kucuker, 2006. Preliminary SEM Observations on the Seed Testa Structure of Gladiolus L. species from Turkey. Int. J. Botany., 2(2): 125-127. FarnsworthA AC., 1982. The role of ethnopharmacology drug development from plants. England Ciba: John Wiley and Sons, pp: 2-10. Fawole, A, O.A., J.F. Finnie Van and J.T. Staden, 2009. Antimicrobial activity and mutagenic effects of twelve traditional medicinal plants used to treat ailments related to the gastro-intestinal tract in South Africa. S. Afr. J. Botany., 75: 356-362. Kahriman, N., M. Yucel, B. Yaylı, T. Aslan, S.A. Karaoglu and N. Yaylı, 2012. Chemical composition and antimicrobial activity of the volatile of Gladiolus atroviolaceus Boiss. Asian J. Chem., 24(4): 1461-1464. Keskin, D. and S. Toroglu,2011. Studies on antimicrobial activities of solvent extracts of different commercial spices. Journal of Environmental Biology, 32(2-3): 251-256. Mcgaw, L.J., N.J.J. Lall, M. Meyer and J.N. Eloff, 2008. The potential of South African plants against Mycobacterium infections. Journal of Ethnopharmacology, 119(3): 482-500. Ncube, N.S., A.J. Afolayan and A.I. Okoh,2008. Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. African J. Biotech., 7(12): 1797-1806.

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