FABAD J. Pharm. Sci., 32, 59-63, 2007 RESEARCH ARTICLE Free Radical Scavenging and Antimicrobial Activities of Three Geranium Species Growing in Turkey Didem fiÖHRETO⁄LU*°, M. Koray SAKAR*, Melike EK‹ZO⁄LU**, Meral ÖZALP** Free Radical Scavenging and Antimicrobial Activities of Türkiye’de Yetiflen Üç Geranium Turunun Serbest Radikal Three Geranium Species Growing in Turkey Supurucu ve Antimikrobiyal Aktiviteleri Summary Özet Antimicrobial and free radical scavenging activities of EtOAc, Bu çal›flmada, Geranium glaberrimum Boiss et Heldr. Geranium n-BuOH and the remaining H2O extracts of aerial parts ofstepporum Davis and Geranium psilostemon Ledeb.’ in toprak Geranium glaberrimum Boiss et Heldr. Geranium stepporum üstü k›s›mlar›ndan haz›rlanan EtOAc, n-BuOH ve kalan H2O Davis and Geranium psilostemon Ledeb were investigated in this ekstrelerinin antimikrobiyal ve serbest radikal süpürücü aktiviteleri study. Broth microdilution and spectrophotometric DPPH methods araflt›r›lm›flt›r. Antimikrobiyal aktivite testi için s›v› mikrodilüsyon were used to test antimicrobial and free radical scavenging yöntemi, serbest radikal süpürücü aktivite tayini için activity, respectively. All tested extracts showed free radical spektrofotometrik DPPH yöntemi kullan›lm›flt›r. Tüm ekstreler scavenging activity. The EtOAc extracts of all studied plants were serbest radikal süpürücü aktivite göstermifltir. Araflt›r›lan found to possess the highest antimicrobial and free radical ekstrelerin EtOAc fazlar› en yüksek antimikrobiyal ve serbest scavenging activity. Among the tested extracts, the EtOAc extract radikal süpürücü aktivite göstermifllerdir. Test edilen ekstreler of G. psilostemon is considered the most active against the tested aras›nda G. psilostemon’ un EtOAc ekstresi mikroorganizmalara microorganisms. All EtOAc extracts were shown to possess higher karfl› en yüksek etkili ekstre olarak belirlenmifltir. Tüm EtOAc free radical scavenging activity than that of ascorbic acid at 50 ekstreleri 50 µg/mL’ da askorbik asitten daha yüksek serbest µg/mL. radikal süpürücü aktiviteye sahiptir. Key Words: Geranium, Geranium glaberrimum, GeraniumAnahtar Kelimeler: Geranium, Geranium glaberrimum, Geranium stepporum, Geranium psilostemon, Geraniaceae, antimicrobial stepporum, Geranium psilostemon, Geraniaceae, antimikrobiyal activity, free radical scavenging activity. aktivite, serbest radikal süpürücü aktivite Received : 02.04.2009 Revised : 21.05.2009 Accepted : 11.06.2009 INTRODUCTION The genus Geranium L. comprises almost 400 species in activity against influenza A and B viruses (3-11). Leaves temperate areas and tropical mountains throughout most of some Geranium species are consumed as food in western of the world (1). Some Geranium species are used in Anatolia (12). Phytochemical investigations in our traditional medicine as antidiabetic, hemostatic, laboratory on G. stepporum resulted in the isolation of antihemorrhoidal, antidiarrheic and as a remedy for some flavonoid glycosides (13). Based on the traditional tonsillitis, cough, whooping cough, urticaria, dysentery, usage and previous research on Geranium species in Turkey kidney pain, and gastrointestinal ailments (2-4). There are and worldwide, the present activity screening study was evidences indicating various properties of Geranium plants performed on the extracts prepared with ethy(l) acetate such as antiprotozoal, antileishmanial, antiinflammatory, (EtOAc), n-butanol (n-BuOH), and water (H2O) from the α-glucosidase and HIV reverse transcriptase inhibitory, aerial parts of G. glaberrimum, G. stepporum and G. antioxidant, and antibacterial; they have also shown antiviral psilostemon in order to evaluate their possible antimicrobial *Hacettepe University, Faculty of Pharmacy, Department of Pharmacognosy, Ankara, Turkey **Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, Ankara, Turkey ° Corresponding author e-mail: [email protected] 59 fiöhreto¤lu, Sakar, Ekizo¤lu, Özalp and free radical scavenging activities. Broth microdilution Table 1. Yield of extracts method recommended by the Clinical and Laboratory Extract Yield (%) Standards Institute (CLSI, formerly NCCLS) was used to G. glaberrimum PE (GG-PE) 0.92 determine antimicrobial activity(14,15). DPPH (2,2- EtOAc (GG- EtOAc) 3.50 n-BuOH (GG- n-BuOH) 3.75 diphenyl-1-picrylhydrazyl) radical was used as radical for H2O (GG- H2O) 14.21 free radical scavenging activity (16). G. stepporum PE (GS-PE) 0.85 EtOAc (GS- EtOAc) 2.85 n-BuOH (GS- n-BuOH) 5.43 MATERIALS and METHODS H2O (GS- H2O) 16.32 G. psilostemon PE (GP-PE) 0.91 EtOAc (GP- EtOAc) 3.92 Plant Material n-BuOH (GP- n-BuOH) 3.22 G. glaberrimum Boiss et Heldr. was collected between H2O (GP- H2O) 12.24 Akseki and Seydiflehir, 36 km from Akseki, 1700-1750 m, in May 2006. G. stepporum Davis was collected fromThin Layer Chromatography E¤risö¤üt, P›narbafl›, Kayseri in May 2006 by D. fiöhreto¤lu. TLC analyses were carried out on pre-coated aluminium G. psilostemon Ledeb was collected in Hamsiköy, Trabzon sheets (Merck Art. 5554). CHCl3-MeOH-H2O (80:20:2, in August 2006 by D. fiöhreto¤lu. A voucher specimen (H. 70:30:3; 61:32:7) were used for the development of the Duman, 9661) of G. glaberrimum has been deposited at plates. Plates were examined by UV fluorescence and the Herbarium of the Department of Biology, Faculty of spraying 10% H2SO4 or 3% FeCl3/MeOH, followed by Science and Literature, University of Gazi, Ankara, Turkey heating at 100°C for 1-2 min. (GAZI). Voucher specimens of G. stepporum and G. psilostemon were deposited in the Herbarium of Hacettepe Antimicrobial Activity University Faculty of Pharmacy, Ankara, Turkey (HUEF Test organisms: Plant extracts were tested against two 06001, 06003, respectively). standard Gram-positive bacteria: Staphylococcus aureus ATCC 29213 (American Type Culture Collection) and Preparation of Extracts Enterococcus faecalis ATCC 29212; two standard Gram- Air-dried and powdered aerial parts of the plant material negative bacteria: Escherichia coli ATCC 25922 and (20 g) were extracted with MeOH:H2O (8:2) mixture (3x Pseudomonas aeruginosa ATCC 27853; and three fungi: 200 mL) for 5 h at 40°C and concentrated to dryness under Candida albicans ATCC 90028, C. krusei ATCC 6258 reduced pressure. An aliquot of the concentrated crude and C. parapsilosis ATCC 22019. extract was suspended in H2O (100 mL) and partitioned with petroleum ether (40-60°C) (PE) (3x 100 ml), EtOAc Antimicrobial activity test: Broth microdilution method (x 100 mL) and n-BuOH (5x 100 ml), individually. PE, recommended by the CLSI was used to determine the EtOAc, and n-BuOH extracts, as well as the remaining antimicrobial activity (14,15). Antibacterial activity test H2O phase, were concentrated to dryness and lyophilized was performed in Mueller-Hinton broth (MHB, Difco in vacuo. The yielded extracts are given in Table 1. Laboratories, Detroit, MI, USA); for antifungal test, RPMI- 1640 medium with L-glutamine (ICN-Flow, Aurora, OH, Crude extracts were first subjected to phytochemical analysis USA), buffered with MOPS buffer (ICN-Flow, Aurora, by thin layer chromatography (TLC) and then activity tests OH, USA) was used. The inoculum densities were were applied. approximately 5x105 cfu/mL and 0.5-2.5x103 cfu/mL for bacteria and fungi, respectively. Chemicals TLC Plates: Silica gel 60 F254 20x20 Merck (Art. 5554); Each plant extract was dissolved in sterile distilled water. Organic Solvents: Methanol, Chloroform: Carlo Erba, Final two-fold concentrations were prepared in the wells Petroleum Ether, n-Butanol, Ethyl acetate: Merck; DPPH: of the microtiter plates, between 1024-1 µg/mL. Ampicillin Fluka; Ampicillin: Mustafa Nevzat; Fluconazole: Pfizer. and fluconazole were used as reference antibiotics for bacteria and fungi, respectively (64-0.0625 µg/mL). 60 FABAD J. Pharm. Sci., 32, 59-63, 2007 Microtiter plates were incubated at 35°C for 18-24 h for almost all tested Gram-positive bacteria and fungi. While bacteria and 48 h for fungi. After the incubation period, none of the tested extracts showed activity against E. coli, minimum inhibitory concentration (MIC) values were S. aureus was the most susceptible organism among those defined as the lowest concentration of the extracts that tested at the tested concentrations. GP-EtOAc was the most inhibits the visible growth of the microorganisms. effective against C. krusei among the tested extracts. DPPH radical scavenging activity: Experiments were carried Table 2. Antimicrobial activities of different extracts of Geranium glaberrimum, G. stepporum and G. out according to slightly modified procedure (16). Briefly, psilostemon 3 mL solutions of the proper extract dilution (25, 50, 100 Test MIC (µg/mL) and 200 µg/mL) were added to a 1 mL solution of 1.5 x material Bacteria Fungi 10-5 M DPPH radical solution in methanol. The absorbance S. E. E. P. C. C. C. was measured at 517 nm after 30 min of incubation at aureus faecalis coli aeruginosa albicans krusei parapsilosis room temperature. Decreased DPPH solution absorbance ATCC ATCC ATCC ATCC ATCC ATCC ATCC indicates an increase in the DPPH radical scavenging 29213 29212 25922 27853 90028 6258 22019 GG-EtOAc 32 256 ‡1024 >1024 512 512 1024 activity. The percent of radical scavenging activity was GG-n-BuOH ‡1024 1024 1024 1024 512 512 1024 calculated as the ratio of the absorption of the sample GG-H2O ‡1024 1024 ‡1024 ‡1024 1024 1024 1024 relative to the control DPPH solution by the following GS-EtOAc 64 512 ‡1024 ‡1024 1024 1024 ‡1024 GS-n-BuOH ‡1024 ‡1024 ‡1024 ‡1024 ‡1024