Mihajilov-Krstev, T. et al.  Antimicrobial and antioxydant potential… BIOLOGICA NYSSANA 6 (2)  December 2015: 55-58 Mihajilov-Krstev, T. et al.  Antimicrobial and antioxydant potential… 55 Original Article Received: 15 July 2015 Revised: 12 August 2015 Accepted: 01 October 2015 Antimicrobial and antioxidant potential of wild growing Silene baccifera (L.) Roth. (Caryophyllaceae) fruits juice Tatjana Mihajilov-Krstev1*, Bojan Zlatković1, Marija Ilić2, Vesna Stankov-Jovanović2, Violeta Mitić2 1University of Niš, Faculty of Sciences and Mathematics, Department of Biology and Ecology, Višegradska 33, 18000 Niš, Serbia 2University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia * E-mail: tatjanamk@pmf.ni.ac.rs Abstract: Mihajilov-Krstev, T., Zlatković, B., Ilić, M., Stankov-Jovanović, V., Mitić, V.: Antimicrobial and antioxidant potential of wild growing Silene baccifera (L.) Roth (Caryophyllaceae) fruits juice. Biologica Nyssana, 6 (2), December 2015: 55-58. Silene baccifera is widespread plant species in Europe, Asia and North Africa, registered in the list of medicinal plants of India. Insufficiently known, biological activities of juice obtained from fresh fruits of this plant were investigated in this study. Antimicrobial activity of juice was tested against pathogenic gastrointestinal microbial strains, using microwell-dilution method, while antioxidant properties were evaluated employing DPPH and total phenolic and flavonoid content assays. To our knowledge, this is the first study of the juice from fruits of this plant species. Key words: Silene baccifera, fruit juice, antimicrobial, antioxidant activity Apstrakt: Mihajilov-Krstev, T., Zlatković, B., Ilić, M., Stankov-Jovanović, V., Mitić, V.: Antimikrobni i antioksidativni potencijal soka plodova divljerastuće vrste Silene baccifera (L.) Roth (Caryophyllaceae). Biologica Nyssana, 6 (2), December 2015: 55-58. Silene baccifera predstavlja rasprostranjenu biljnu vrstu na području Evrope, Azije i Severne Afrike. Registrovana je na listi lekovitih biljnih vrsta Indije. U ovom radu je ispitivana biološka aktivnost soka dobijenog ceđenjem svežih plodova pomenute vrste. Antimikrobna aktivnost je testirana mikrodilucionom metodom protiv patogena gastrointestinalnog trakta. Antioksidantna aktivnost je procenjena DPPH metodom i određivanjem ukupnog sadržaja fenola i flavonoida. Prema našem saznanju, ovo je prva takva studija soka plodova ove biljne vrste. Key words: Silene baccifera, sok od plodova, antimikrobna, antioksidativna aktivnost Introduction Silene baccifera (L.) Roth. (syn. Cucubalus baccifer L.) is widely spread plant species in the area of Europe, Asia and North Africa, especially in the regions with a temperate climate (G a j i ć , 1970). It is perennial plant with long, prostrate or ascending (50-150 cm), well-branched stems and white or 6 (2) • December 2015: 55-58 BIOLOGICA NYSSANA 6 (2)  December 2015: 55-58 Mihajilov-Krstev, T. et al.  Antimicrobial and antioxydant potential… 56 greenish nodding flowers sorted in lateral brunches of the inflorescence. Comparatively large (up to 15 mm in diameter), subglobose, fleshy, blackish-red berries are distinctly exerted from the calyx during the ripening period. Blossoms appear in June to August, fruits ripen from July to October (K o n r a d v o n W e i h e , 1972). Silene baccifera is common plant species that usually grows along roadsides and weedy places in the human settlements. This plant species is registered in the List of Indian Medicinal Plants (www.docslide.us) and is used in traditional medicine for treatment of nephritis, hydropsy, bone-fractures, pulmonary tuberculosis and scrofula (C h e n g et al., 2001). To the best of the author’s knowledge, there is no literature or field data on its use in official either traditional medicine in Serbia or Balkan countries. The chemical composition of the underground part of the S. baccifera has been poorly investigated. Previously isolated constituents from the whole plant were oligosaccharides (C o u r t o i s & A r i y o s h i , 1960; 1962), tocopherol and tocotrienol (I v a n o v & A i t z e t m u e l l e r , 1998) and phytoecdysterones and cucubalugenin A (C h e n g et al., 2001a; 2001b). To the best of our knowledge, antioxidant and antimicrobial activity of this plant species were not investigated. In this paper, the first results of antioxidant and antimicrobial activity of the juice obtained from fresh fruits of S. baccifera are presented. Material and methods Plant material Ripe fruits of wild growing Silene baccifera were collected on the territory of Serbia (Kamenica village, Stara planina, E Serbia) and identified by Dr Bojan Zlatković. A voucher specimens, under the acquisition number 6859, were deposited at the herbarium collection of Department of Biology and Ecology, Faculty of Science and Mathematics in Niš (HMN). Preparation of juice Cleaned, ripe fruits were tap washed, followed by washing with distilled water and subsequent drying. The fruits were separated from the seeds, cut in small pieces, arranged in clean plastic containers and frozen. Melted at room temperature, the softened material was grounded in mechanical blender to a fine mash prior to filtration and the juice extraction. Antimicrobial assay Microbial strains. In vitro antimicrobial activity of the tested samples have been investigated against the commonest human gastrointestinal pathogenic microbial strains: Salmonella enteritidis (ATCC 13076), Shigella sonnei (ATCC 25931), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 9027), Listeria monocytogenes (ATCC 7644), Bacillus cereus (ATCC 10786), Staphylococcus aureus (ATCC 6538) and Candida albicans (ATCC 10031) using microwell-dilution method. Microwell dilution method. Determination of the minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) was carried out according to the method described by CLSI (2005) with some modifications. An overnight culture of tested strains were used for the preparation of 0.5 McFarland standard turbidity suspension (corresponding 108 CFU/mL). A serial doubling dilutions of tested juice were prepared in the range 500.00-0.25 µL/mL, in a 96/well microtiter plate with inoculated Mueller-Hinton broth (MHB) for bacteria and Sabouraud dextrose broth (SDB) for yeast. The final volume was 100 µL and the final concentration of bacterial cells was 106 CFU/mL in each well. The plates were incubated for 24 h at 37 °C. Chloramphenicol at concentrations ranging from 10.00-0.002 µg/mL was used as positive control. All determinations were performed in triplicate. Microbial growth was determined by adding 20 μL of 0.5% triphenyl tetrazolium chloride (TTC) aqueous solution. MIC was defined as the lowest concentration of juice at which microorganisms showed no visible growth. In order to determine MMC, broth was taken from each well and inoculated on Mueller-Hinton agar (MHA) for bacteria and Sabouraud dextrose agar (SDA) for yeast. The plates were incubated for 24 h at 37°C. The MMC is defined as the lowest concentration of the juice at which 99.9% of inoculated microorganisms were killed. Antioxidant assay The antioxidant properties of the tested juice were evaluated employing DPPH and total phenolic and flavonoid content assays. All the assays were carried out in triplicate and average value was considered. 1,1-diphenyl-2-picrylhydrasyl (DPPH) radical scavenging assay. Relatively stable organic radical DPPH has been widely used in the determination of antioxidant activity of single compounds as well as the different plant extracts (K u l i š i ć , 2004). The DPPH˙-assay was performed as described (S t o j a n o v i ć , 2010). Samples of juice methanol solution (10 μL, 232.4, 232.5 and 232.6 mg/mL respectively) were mixed with 90 μmol/L DPPH˙in methanol (1.0 mL), and these BIOLOGICA NYSSANA 6 (2)  December 2015: 55-58 Mihajilov-Krstev, T. et al.  Antimicrobial and antioxydant potential… 57 solutions were diluted up to 4.0 mL. After shaking mixtures vigorously, they were stored in darkness for 60 min at room temperature and the absorbances were measured at 515 nm (Perkin-Elmer Lambda 15 UV-VIS spectrophotometer). Radical scavenging activity of the samples was calculated applying following equation: DPPH RSC (%) = 100 (A0−A1/A0) Where: A0-absorbance of the blank; A1- absorbance of the sample. Total phenolic content determination. The total phenolic concentration was determined spectrophotometrically according to modified Folin- Ciocalteu method (D i M a j o , 2008). To the samples of juice (100 µL) portions of 1 mL of Folin- Ciocalteu reagent (purchased from “Mol” Belgrade, Serbia) and 4 mL of sodium carbonate (20% v/v) were added and diluted with distilled water up to 20 mL. The mixture was allowed to stand at room temperature in dark place for 30 min and the absorbance of the solution at 750 nm was measured with a Perkin Elmer Lambda 15 UV/VIS spectrophotometer. The total phenolic concentrations were calculated from a calibration curve using gallic acid as a standard. Gallic acid was provided by Sigma Aldrich (Darmstadt, Germany). Data were expressed as gallic acid equivalents per 1 mg of juice. The levels of total infusions in juice determined according to the Folin–Ciocalteu method are not absolute measurements of the amounts of phenolic materials, but are in fact based on their chemical reducing capacity relative to an equivalent reducing capacity of gallic acid. Determination of total flavonoid content. The amount of total flavonoids was determined with aluminium chloride (AlCl3) colorimetric assay according to a known method (R i c e - E v a n s et al., 1996). Briefly, 0.5 mL of juice was made up to a final volume of 1 mL with reaction medium (MeO/H2O/CH3COOH=14:5:1). Prepared solution was mixed with AlCl3 reagent (4 mL, 133 mg of AlCl3x6H2O and 400 mg of CH3COONa dissolved in 100 mL H2O). After 5 min, the absorbance level was measured versus prepared reagent blank (containing the same chemicals, except for the sample) at 430 nm (Perkin-Elmer Lambda 15 UV-VIS spectrophotometer). Total flavonoid content was calculated on the basis of the calibration curve of rutin and expressed by mg rutin/g dry extract. The total flavonoid assay was measured in triplicate. Results and discussion Freshly drained juice from the fruits have shown antimicrobial activity on all tested strains of patogenic microbes (Tab. 1). The obtained minimal inhibitory concentrations were in the range 31.2-500.0 µL/mL, and minimal microbicidal concentrations ranged from 125.0-500.0 µL/mL. The tested juice exhibited the highest activity against P. aeruginosa and S. enteritidis (MIC/MMC=31.2/125 and 62.5/125 µL/mL, respectively). Also, good effect was observed against E. coli (MIC=MMC=125 µL/mL). The juice was the least active against S. aureus and C. albicans (MIC=MMC=500 µL/mL). Table 1. Antimicrobial activity of juice from S. baccifera fruits and reference antibiotics (Tested strains: SB - S. baccifera fruits juice, MIC/MMC in µL/mL; A - Antibiotic, MIC/MMC in µg/mL; bacterial strains: Se – Salmonella enteritidis; Ss - Shigella sonnei, Ec - Escherichia coli, Pa - Pseudomonas aeruginosa, Lm - Listeria monocytogenes, Bc - Bacillus cereus, Sa - Staphylococcus aureus; Ca – Candida albicans) Tested strains Se Ss Ec Pa Lm Bc Sa Ca SB 62.5/125 250/250 125/125 31.2/125 250/500 125/500 500/500 500/500 A 0.035* 0.035* 0.035* 0.078* 0.035* 0.035* 0.035* 0.035** *Chloramphenicol, **Nystatin Table 2. The antioxidant activity of juice from S. baccifera fruits Tested samples RSA (EC 50 u µg/mL)** PPC (gallic acid equivalents, µg/mg of dry matter) PC (rutin equivalents, µg/mg of dry matter) Silene baccifera* 0.65 6.00 15.18 *juice was prepared by dissolving fruit in water; ** EC 50 for BHT as a standard is 0.63 µg/mL BIOLOGICA NYSSANA 6 (1)  September 2015: 55-58 Mihajilov-Krstev, T. et al.  Antimicrobial and antioxydant potential… 58 The obtained results of antioxidant activity of juices are summarized and presented in Tab. 2. Comparative study of antimicrobial and antioxidant activities of the fresh juices obtained from the fruits of plant species: Viburnum lantana, Viburnum opulus, Sambucus nigra, Cornus sanguinea and Paliurus spina-christi, wild growing on the territory of Serbia, showed that all tested juices had inhibitory concentrations in the range from 15.6- 500.0 μL/mL and microbicidal concentrations from MBC/MFC=62.5-500.0 μL/mL (M i h a j i l o v - K r s t e v et al., 2011). Generally, better activity than S. baccifera juice had only the juices from V. opulus and C. sanguinea. Also, the juice of this species demonstrated the best antioxidant activity in comparison to the mentioned juices. Conclusion The obtained results showed that S. baccifera fruit juice presents a natural source of antimicrobial and antioxidant components that can be applied in prevention and treatment of gastrointestinal diseases and oxidative stress in humans. In future studies, the chemical composition and biological activity of different extracts of certain parts of the plant should be investigated, as well as their cytotoxicity and acute toxicity. Acknowledgements. The financial support of this work was provided by Ministry of Education and Science of the Republic of Serbia (project 172047). References Cheng, Y.X., Zhou, J., Tan, N.H., Ding, D.T. 2001a: Indian medical plants in traditionl medicine. Acta Botanica Sinica, 43:31. Cheng, Y.X., Zhou J., Dai, H.F., Ding, D.T. 2001b: Cucubalugenin A, a new triterpenoid from Cucubalus baccifer. Fitoterapia, 72: 848-849. CLSI, 2005: Clinical and Laboratory Standards Institute, Wayne, PA. M100-S22 Courtois, J.E., Ariyoshi, U. 1960: Saccharose galactosides from the roots of Cucubalus baccifer (Caryophyllaceae). Study of their structure. Bulletin de la Société de Chimie Biologique, 42:737-51. Courtois, J.E., Ariyoshi, U. 1962: The galactosides of saccharose in the roots of the carnation. Bulletin de la Société de Chimie Biologique, 44:23-30. Di Majo, D., Guardia, M.L., Giammanco, S., Neve, L.L., Giammanco, M. 2008: The antioxidant capacity of red wine in relationship with its polyphenolic constituents. Food Chemistry, 111:45–49. Gajić, M. 1970: Cucubalus L. [In. Gajić, M.: Caryophyllaceae Juss.]. In: Josifović, M. (ed.): Flora Srbije 2:240-241. Srpska akademija nauka i umetnosti, Beograd. Ivanov, S.A., Aitzetmuller, K. 1998: Undersuchungen über die tocopherol-und- tocotrienol Zusammensetzung der samenlipide einiger arten der Bulagarischen Flora. Fett/Lipid, 100: 348-352. Konrad von Weihe, H. 1972: Illustrierte Flora. Deutschland und angrenzende Gebiete. Gefäßkryptogamen und Blütenpflanzen. Begründet von August Garcke. 23. Auflage. Paul Parey, Berlin/Hamburg. Kulišić, T., Radonić, A., Katalinić, V., Miloš, M. 2004: Use of different methods for testing antioxidative activity of oregano essential oil. Food Chemistry, 85:633–640. Mihajilov-Krstev, T., Zlatković, B., Ilić, M., Stankov-Jovanović, V., Mitić, V. 2011: Comparative study of antibacterial and activities activities of wild growing fruits juices. Book of Abstracts of International Conference ''Medicinal and Aromatic Plants in Generating of New Values in 21st Century''. Academy of Sciences and Arts of Bosnia and Herzegovina, Special Edition Vol. CXL, Department of Natural Sciences and Mathematics, Sarajevo, 9-12 November, 223. Rice-Evans, C.A., Miller, N.J., Paganga, G. 1996: Structure antioxidant activity relationshipe of flavonoids and fenolic acids. Free radical in biology and medicine, 20: 933-956. Stojanović, G., Stojanović, I., Stankov-Jovanović, V., Mitić, V., Kostić, D. 2010: Reducing power and radical scavenging activity of four Parmeliaceae species. Open Life Sciences, 5(6):808–813. www.docslide.us