Bulletin of the Chemists and Technologists 2017 of Bosnia and Herzegovina 49 Print ISSN: 0367-4444 UDC: __________________________ 1-8 Online ISSN: 2232-7266 Original Scientific Paper Phytochemical screening, quantitative determination of phenolic compounds, and antioxidative activity of Ostrya carpinifolia Starčević, M., Subašić, M., Pustahija, F.* aFaculty of Forestry, University of Sarajevo, Zagrebačka 20, 71000 Sarajevo, Bosnia and Herzegovina Abstract: Ostrya carpinifolia is an interesting and suitable species for reforestation and Article info landscaping. For the first time ever, phenolic profile, antioxidative and antimicrobial activity of O. Received: 20/07/2017 Accepted: 02/11/2017 carpinifolia was done in this study. Aqueous and methanol extracts of the aerial parts were analyzed using either fast screening methods of secondary metabolites, and UV/VIS spectrophotometry for Keywords: determination of polyphenolic contents and antioxidant activity (DPPH). Antimicrobial activity of Antimicrobial activity, Balkans, methanol extracts was investigated using the disc diffusion method against a selected nine Betulaceae, microorganisms. Phytochemical tests confirm the presence of cardiac glycosides, coumarins, DPPH, European hop-hornbeam, emodins, flavonoids, tannins, terpenes, terpenoids and steroids, while anthocyanins, fatty acids and Secondary metabolites saponins were absent in all aqueous extracts. Leucoanthocyanins were observed only in the stem extract. Methanol extracts of leaves contain the highest level of total phenolics and flavonoids (35.574 and 30.908 mg CE g-1 DW, respectively), while the inflorescences extracts were the richest with total proanthocyanidins and phenolic acids (19.165 mg CE and 9.342 mg CAE g-1 DW, respectively). All methanol extracts showed very strong antioxidative activity, where the lowest -1 activity was recorded for inflorescences (IC50: 0.242 mg mL ) and the highest for stem (IC50: 0.107 -1 mg mL ). Analyzed extracts showed no antimicrobial activity against the test organisms. ANOVA *Corresponding author: E-mail: [email protected] indicated the presence of significant differences between the total phenolics and flavonoids and Phone: 00-387-33-812490 DPPH (p<0.05). Duncan’s test confirmed the presence of statistically significant and very high Fax: 00-387-33-812488 positive correlation (R=0.989) between total phenolics and phenolic acids contents. Obtained results indicate the necessity of further research of European hop-hornbeam. INTRODUCTION their biosynthesis (Bennet and Wallsgrove, 1994; Croteau, Kutchan and Lewis, 2000; Boudet, 2007; Plants have an enormous ability to synthesize different Edreva, Velikova, Tsonev, et al., 2008; Acquaviva, bioactive organic compounds (i.e. secondary Menichini, Ragusa, et al., 2012; Bartwal, Mall, Lohani, metabolites, SMs) that are not directly involved in their et al., 2013; Lattanzio, 2013; Murkovic, 2016). growth, development and reproduction but in absence According to available data, more than 200,000 different they may be harmful or even fatal for plant's SMs have been reported, and this number is still survivability. Secondary metabolites have essential roles continually increasing (Verpoorte, van der Heijden, ten in stabilizing cellular structure, plant adjustment and Hoopen, et al., 1999; Hadacek, 2002; Edreva et al., defense reacting under biotic and abiotic stresses. They 2008; Springob and Kutchan, 2009; Bartwal et al., are included in different physiology processes: 2013). Among them, the most abundant group of SMs pigmentation, pollination, seed germination, signaling, with wide array of roles in plants are phenolic and resistance mechanisms under suboptimal conditions. compounds including phenolic acids, tannins, lignins The main characteristics of SMs are large chemical and most diverse flavonoids (Bartwal et al., 2013) with diversity, complexity and structure heterogeneity, as also increasing biological interest of proanthocyanidins as the the presence of diverse mechanisms and pathways of most widespread bioflavonoids (Škerget, Kotnik, 2 Starčević et al. Hadolin, et al., 2005). Namely, in recent years, phenolics Bacillus subtilis subsp. spizizenii ATCC 6633™, and phenolic-related compounds gained huge importance Enterococcus faecalis ATCC 19433, and Bacillus and attention in medicine, pharmacology, food vulgatus ATCC 8482), Gram-negative bacteria processing and cosmetics industry, due to their (Salmonella abony NCTC 6017TM, Escherichia coli antioxidant, antimicrobial and anti-inflammatory ATCC 8739TM, and Pseudomonas aeruginosa ATCC properties (Oliviera, Sousa, Morais, et al., 2008; Abou- 9027) and fungus Candida albicans (ATCC 10231). The Zeid, Bidak and Gohar, 2014; Ashraf, Sarfraz, Rashid, et bacterial strains were cultured in Müeller Hinton agar al., 2015; Hofmann, Nebehaj and Albert, 2016). while fungus was cultured on Sabouraud dextrose agar. Ostrya carpinifolia Scop. (European hop-hornbeam), the only native species of genus in Europe and endemic to Aqueous extracts preparation temperate West Eurasia, is a small to medium-sized Per 5 g of ground plant materials were placed in to the broadleaved deciduous tree (Pasta, de Rigo and glass beakers and 50 mL of distilled water was added in Caudullo, 2016). In terms of forestry, it is very each. After 30 min of incubation at 55°C in water bath interesting and suitable species for the reforestation of the extracts were cooled, centrifuged for 20 min at 1800 many degraded sites and landscaping (Weber, 2011; rpm (Centric 322 B, Techtnica), and filtered through Ivetić, Devetaković, Davorija, et al., 2015; Pasta et al., Whatman No.1 filter papers. 2016). Although, to date, chemical composition and biological activities of this species have been scarcely Preliminary phytochemicals screening studied (Wollenweber, 1975), according to published Preliminary fast phytochemical standard tests for the data about other Ostrya species, O. carpinifolia can also screening and qualitative identification of bioactive be a source of natural phytochemicals with beneficial chemical constituents of plant aqueous extracts of O. effects in broad arrays (Matsuki and Koike, 2006; carpinifolia were carried out by modified methods of Barbehenn, Weir and Salminen, 2008; Park, Kim, Ko, et Trease and Evans (2002), Savithramma, Linga Rao and al., 2010; Kim, Park and Lim, 2010). Suhrulatha (2011), and Subhashini Devi, Satyanarayana Based on the lack of information and keeping the above and Tarakeswara Naidu (2014). Leaves, stem and facts in view, the aims of this study are: 1) qualitative inflorescences extracts were screened for 12 and quantitative analysis of aqueous and methanol phytochemical constituents: anthocyanins, cardiac extracts of different aerial parts of Ostrya carpinifolia glycosides, coumarins, emodins, fatty acids, flavonoids, and 2) evaluation of antioxidant and antimicrobial leucoanthocyanins, saponins, steroids, tannins, terpenes, activities of methanol extracts of European hop- and terpenoids. hornbeam. Methanol extracts preparation MATERIAL AND METHODS Grinded plant materials (0.5 g) were extracted with 25 mL of 80% methanol. The extraction process was carried Plant material out in two replicates (twice with 12 mL and adjusted to Plant material (leaves, stems, inflorescences) was 25 mL) using both times an ultrasonic bath (Elma sonic collected from healthy trees of O. carpinifolia in its S 60 H) for 30 min. Extracts were then centrifuged at natural population near Sarajevo (43˚51’19’’ N, 1800 rpm for 10 min. The obtained supernatants were 18˚27’27’’ E, 595 m ASL, S-SW), in May 2015. stored in plastic tubes at 5°C for further analysis. Different plant parts were separated immediately and The Shimadzu UV-mini 1240 spectrophotometer was packed into paper bags. In the laboratory, the plant used for determination of polyphenolic contents and materials were washed with running tap water, rinsed antioxidant activity of all methanol extracts. All samples with distilled water and naturally air dried during 10 were analyzed in triplicates. days at room temperature in a dry, shaded and well- aerated place. Then, dried materials were grounded into Determination of total phenolic content powder using mixer and stored in plastic bags, at room Total phenolic content of the methanol extracts was temperature, until extraction processes. determined using modified Folin-Ciocalteu method (Wolfe, Wu and Liu, 2003). An aliquot of the methanol Chemicals and reagents extract (20μL) was mixed with distilled water followed Folin-Ciocalteu’s reagent, catechin, caffeic acid, DPPH by addition of 100 μL of Folin-Ciocalteu’s reagent and (1,1-diphenyl-2-picryl-hydrazyl), sodium carbonate, 300 μL of freshly prepared sodium carbonate solution absolute methanol, and aluminium chloride were (7.5%). After mixing, the tubes were incubated for 30 purchased from Sigma-Aldrich (Steinheim, Germany), min at 45°C in water bath until blue color development. while Amoxicillin, Müeller Hinton and Sabouraud The absorbance of the resulting solvents was measured dextrose agars were purchased from HiMedia Chemicals at 765 nm. Total phenolic content was expressed as mg Ltd. (Mumbai, India). All other used chemicals and of catechin equivalents per gram of dry plant sample (mg solvents were of analytical grade. CE g-1). Tested microorganisms Determination of total flavonoids Antimicrobial activity was tested against a panel of Total flavonoid content was determined by the modified microorganisms: Gram-positive bacteria method of Ordoñez,