African Journal of Microbiology Research Vol. 4 (11) pp. 1154-1161, 4 June, 2010 Available online http://www.academicjournals.org/ajmr ISSN 1996-0808 © 2010 Academic Journals Full Length Research Paper Phenolic profiles, antimicrobial and antioxidant activity of the various extracts of Crocus species in Anatolia Gulumser Acar1*, Nazime Mercan Dogan1, Mehmet Emin Duru2 and Ibrahim Kıvrak2 1Department of Biology, Faculty of Science and Arts, Pamukkale University, 20017, Kinikli, Denizli, Turkey. 2Department of Chemistry, Faculty of Science and Arts, Mula University, 48000, Mula, Turkey. Accepted 6 May, 2010 The phenolic profile and quantitative composition of methanol extracts of Crocus baytopiorum which was endemic species in Denizli, Turkey was detected by high performance liquid chromatography (HPLC-DAD). The HPLC analysis of phenolic compounds in methanol extract of C. baytopiorum showed that p-Coumaric acid, apigenin-glucoside, rosmarinic acid, quercetin and kampferol were present. Also, the methanol, ethyl acetate and hexane extracts from Crocus biflorus, C. baytopiorum and Crocus flavus subp. dissectus were investigated for their in vitro antimicrobial and antioxidant activities in the present study. Ethyl acetate and methanol extracts have demonstrated significant antimicrobial activities against tested micro organisms Escherichia coli ATCC 35218, Pseudomonas aeruginosa NRRL B-23, Klebsiella pneumoniae ATCC 27736, Yersinia enterecolitica RSKK 1501, Proteus vulgaris RSKK 96026, Bacillus cereus RSKK 863, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Micrococcus luteus NRRL B-4375 and Candida albicans (clinical isolate). The methanol extract of C. flavus subsp. dissectus had maximum activities on Yersinia enterocolitica RSKK 1501. Minimum inhibition concentrations of plant extracts have investigated on Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and Bacillus cereus RSKK 863. The Minimum inhibition concentration (MIC) of samples ranged from 0.10 to 20.48 mg/ml. In terms of radical scavenging activity and antioxidant activity, in the concentration of 2 mg/ml of methanol extract of C. flavus (92.67 and 89.32% respectively) displayed inhibition equal to in the concentration of 0.8 mg/ml butylated hydroxytoluene (BHA) used as standard antioxidant (91.45 and 89.78%, respectively). Positive correlations were found between total phenolic content in the Crocus extracts and their antioxidant activities. Thus, it is envisaged that Crocus species may have potential for acting as natural antioxidants. Key words: Crocus, antimicrobial activity, antioxidant activity, HPLC, phenolic compounds. INTRODUCTION Crocus is the largest genus of Iridaceae. This genus is used in industry with a wide range of uses from medicine, represented in Turkish flora by 70 taxa (Guner et al., to textile dye and to culinary adjunct (Lozano et al., 2000; Davis, 1984). The species Crocus baytopiorum is 1999). Studies have shown that crocin which is the endemic to Denizli which is the province of Turkey. It is compound of C. sativus has antioxidant effects (Zheng et distributed Honaz Mountain, 2000 m. Saffron plant al., 2007) and may have cardio protective effect (Goyal et (Crocus sativus L.) is a vegetative propagated crop and is al., 2010). During the last few years, the anti-tumoural currently used as a source of food additives, colorants properties of crude saffron stigma extracts, both in vitro and as a component of traditional medicines (Escribano and in vivo, have also been demonstrated (Escribano et et al., 1999). Saffron, which is obtained from the dried al., 1999). Due to the large production of this plant and stigmas of Crocus sativus is the most expensive spice the use of its stigma only, it is very important to find some other uses of saffron (Vahidi et al., 2002). In addition, researchers have been interested in isolating biologically active compounds isolated from plant species for the *Corresponding author. E-mail: [email protected]. Tel: elimination of pathogenic micro-organisms (Essawi and +90 258 296 36 72. Srour, 2000). In this respect, the antimicrobial potential of Acar et al. 1155 Jatropha podagrica (hook) (Euphorbiaceae) was subp. dissectus T. Baytop and Mathew on several investigated in 2008. The researchers were found to be pathogen micro-organisms, as there is a significant lack 15 mg/ml of minimum inhibitory concentration (MIC) of information on such activities in literature. value. At this concentration stem bark extract showed remarkable antibacterial activity as compared to stem extract and their zone of inhibition compared with MATERIALS AND METHODS standard antibiotics (Bhaskarwar et al., 2008). Also, a lot of plants used in traditional medicine today antimicrobial Plant materials activity have been identified (Zampini et al., 2009; Oke et Plant samples were collected in an area around Denizli, particularly al., 2009; Mboss et al., 2010). on Mount Honaz at 2000 m height by the authors of this paper in Antibiotics have been used for the treatment of March and April. Dr. Ali CELIK further identified all of the collected infectious diseases for a long time. But, antimicrobial plants. Specimens of the plants were preserved in the herbarium at resistance, among pathogen bacteria, against drugs used Pamukkale University. Plants were dried in the shade and ground into a powder material using an appropriate seed mill. in the treatment of human infection is increasing. This situation has forced scientists to search for new antimicrobial substances from various plants which are Preparation of the crude extract the good sources of novel antimicrobial chemothera- peutic agents (Karaman et al., 2003). Plant products Extracts of plant materials were prepared using solvents of varying have also been known to possess potential for food polarity. About 200 g of dry powdered plant material was extracted with hexane (HE), followed by ethyl acetate (ETA) and methanol preservation (Baratta et al., 1998). Oxidation is essential (MeOH) in a soxhlet apparatus (6 h for each solvent). All solvents to many living organisms for the production of energy to were purchased from Merck. The extracts were evaporated under fuel biological processes. However, oxygen free radicals reduced pressure and dried using rotary evaporator. Dried extracts and other reactive oxygen species that are continuously were stored in labelled sterile screw capped bottles at -20°C. produced in vivo, result in cell deaths and tissue damage. Oxidation-related damage caused by free radicals may Micro-organisms be related to aging and diseases, such as atherosclerosis, diabetes, cancer and cirrhosis (Halliwell The following nine strains of bacteria and one strain of yeast were and Gutteridge, 1984). Although almost all organisms used as test micro-organisms respectively: Escherichia coli ATCC possess antioxidant defence and repair systems, which 35218, Pseudomonas aeruginosa NRRL B-23, Klebsiella have evolved to protect them against oxidative damage, pneumoniae ATCC 27736, Yersinia enterecolitica RSKK 1501, Proteus vulgaris RSKK 96026, Bacillus cereus RSKK 863, Bacillus these systems are insufficient to prevent the damage subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, entirely (Simic, 1988). However, antioxidant supplements Micrococcus luteus NRRL B-4375 and Candida albicans (clinical or foods containing antioxidants may be used to help the isolate). These micro-organisms were obtained from Microbiology human body reduce oxidation-related damage (Yanga et Laboratory of Pamukkale University Biology Department. al., 2002). Synthetic antioxidants have been used in stabilisation of foods. The most commonly used synthetic Antimicrobial screening antioxidants are butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylated Two different methods were employed for determining the hydroxyquinone (TBHQ), which are applied in fat and oily antimicrobial activities of Crocus species: Agar-well diffusion foods to prevent oxidative deterioration (Löliger, 1991). method for the extracts and (MIC) analysis. Originally, BHA appeared to have tumour-initiating as well as tumour promoting action. Recently, it has been established that tumour formation appears to involve only Agar-well diffusion method tumour promotion caused by BHA and BHT (Botterweck The antimicrobial activity of the samples was assayed by the Agar- et al., 2000). For this reason, governmental authorities well diffusion method (Perez et al., 1990). All the aforementioned and consumers are concerned about the safety of their micro-organisms were incubated at 37 ± 0.1°C (30 ± 0.1°C for only food and about the potential effects of synthetic additives M. luteus NRRL B-4375) for 24 h by inoculation into Nutrient broth. on health (Reische et al., 1998). Antimicrobial agents, C. albicans was incubated YEPD in broth at 28 ± 0.1°C for 48 h. The culture suspensions were prepared and adjusted by using 0.5 including food preservatives have been used to inhibit food Mc Farland turbidity standard tubes. Nutrient Agar (NA; g/L: beef borne bacteria and extend the shelf life of processed extract 1, peptone 5, yeast extract 2, NaCl 5, agar 17) and YEPD food. Many naturally occurring extracts like essential oils, Agar (g/L: peptone 20, yeast extract 10, dextrose 20) were poured herbs and spices have been shown to possess into each sterilised Petri dish (10 x 100 mm diameter) after injecting antimicrobial functions and could serve as a source for cultures (100 µl) of bacteria and yeast and