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Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity Zorica Šarac a,n, Jelena S. Matejić b, Zorica Z. Stojanović-Radić a, Jovana B. Veselinović c, Ana M. Džamić d, Srdjan Bojović e, Petar D. Marin d a
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Department of Pharmacy, Faculty of Medicine, University of Niš, Boulevard Dr Zorana Đinđića 81, 18000 Niš, Serbia c Department of Chemistry, Faculty of Medicine, University of Niš, Boulevard Dr Zorana Đinđića 81, 18000 Niš, Serbia d Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia e Institute for Biological Research “Siniša Stanković”, University of Belgrade, Boulevard Despota Stefana 142, 11060 Belgrade, Serbia b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 June 2014 Accepted 19 August 2014
In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50 ¼25.08 mg/mL and VitC ¼0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/ mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. & 2014 Published by Elsevier Ltd.
Keywords: Pinus nigra Needle terpenes Biological activity Bacillus cereus Molecular docking
1. Introduction Pinus (Pinaceae), with more than 200 generally recognized species, is the largest extant genus of Pinopsida [1]. Economically, pines are one of the most important tree species valued for wood, paper, resins, charcoal, food (particularly seeds), and ornamentals [2]. Moreover, most pines are considered as significant source of structurally diverse, bioactive compounds, and have provided contributions to the discovery of pharmaceutical agents and other biomedical applications [3]. For instance, turpentine have a long tradition of remedial utilization as topical rubefacient for the treatment of rheumatic diseases while pine bark extract is used in anti-aging cosmetics [4]. However, essential oils from Pinus species have been reported to have a variety of therapeutic properties. According to Dervendzi [5] pine needle oils are mostly used in folk medicine for the healing of respiratory infections
n
Corresponding author. Tel.: þ 381 18 533 015; fax: þ 381 18 533 014. E-mail address: [email protected] (Z. Šarac).
accompanied by cough, bronchitis, bronchial asthma, emphysema, tracheitis, sinusitis, laryngitis, pharyngitis, and influenza. Moreover, pine oils are widely used as flavoring additives for food and beverages. In various household cleaning products they are well known as scenting and disinfectant agents. Also, these oils are used in pharmaceutical industry as aroma or natural source of intermediates in the synthesis of perfume components [6]. The European black pine (Pinus nigra J.F.Arnold) is a tertiary relict belonging to the group of Mediterranean pines [7]. It is one of the most widespread and polymorphic conifers in Europe with highly fragmented distribution range that extends from North Africa through the northern Mediterranean and eastwards to the Black Sea. In the Flora of Serbia [8], two subspecies of black pine (ssp. nigra and ssp. pallasiana) are distinguished and within them several varieties (var. nigra, var. zlatiborica, var. gocensis, and var. banatica). However, recent studies on the chemotaxonomy of P. nigra in Serbia, based on both the n-alkane [9] and terpene [10] variability, recognized only three distinct chemotypes (nigra, pallasiana i banatica) within Serbian black pine populations. According to Šarac et al. [10], the major components in the needle
http://dx.doi.org/10.1016/j.compbiomed.2014.08.022 0010-4825/& 2014 Published by Elsevier Ltd.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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Table 1 Chemical composition of the needle essential oils of three infraspecific taxa of Pinus nigra from Serbia [10]. Entry
Compound
Content [%]a P. nigra taxa Location
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
a b
Average content [%]b ssp. nigra Zmajevački potok
ssp. pallasiana Jarešnik
var. banatica Lazareva reka
Tricyclene α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene Δ3-Carene α-Terpinene Limonene (E)-β-Ocimene γ-Terpinene Terpinolene trans-Verbenol α-Terpineol Linalool acetate Bornyl acetate trans-Verbenyl acetate trans-Pinocarvyl acetate Myrtenyl acetate δ-Elemene α-Terpinyl acetate α-Cubebene α-Ylangene α-Copaene Geranyl acetate β-Bourbonene β-Cubebene β-Elemene (E)-Caryophyllene β-Copaene 6,9-Guaiadiene α-Humulene (E)-β-Farnesene γ-Muurolene Germacrene D β-Selinene Phenylethyl 3-methylbutanoate γ-Amorphene Bicyclogermacrene α-Muurolene Germacrene A γ-Cadinene δ-Cadinene trans-Cadina-1,4-diene α-Cadinene Germacrene D-4-ol Caryophyllene oxide 1,7-Diepi-α-Cedrenal T-Cadinol α-Cadinol Germacra-4(15),5,10(14)-trien-1-alpha-ol Oplopanone Unknown components
0.16 (0.07) 0.41 (0.30) 45.93 (10.46) 0.95 (0.29) 0.31 (0.18) 6.90 (7.04) 0.88 (0.21) 0.00 (0.00) 0.03 (0.12) 0.00 (0.00) 2.16 (1.98) 0.86 (0.47) 0.00 (0.00) 0.57 (0.41) 0.00 (0.00) 0.00 (0.00) 0.05 (0.13) 0.63 (0.52) 0.03 (0.07) 0.01 (0.03) 0.03 (0.10) 0.00 (0.00) 0.48 (0.95) 0.00 (0.00) 0.00 (0.00) 0.02 (0.05) 0.00 (0.00) 0.02 (0.06) 0.00 (0.00) 0.03 (0.11) 8.13 (3.47) 0.02 (0.07) 0.00 (0.00) 1.24 (0.52) 0.01 (0.04) 0.34 (0.38) 27.50 (5.89) 0.01 (0.03) 0.12 (0.60) 0.15 (0.26) 0.19 (0.88) 0.08 (0.11) 0.09 (0.33) 0.25 (0.32) 0.61 (0.52) 0.00 (0.00) 0.00 (0.01) 0.57 (1.44) 0.08 (0.33) 0.00 (0.00) 0.03 (0.11) 0.03 (0.13) 0.00 (0.01) 0.02 (0.09) 0.05 (0.02)
0.40 (0.61) 0.41 (0.40) 42.33 (8.59) 1.95 (2.64) 0.26 (0.18) 5.15 (5.90) 0.45 (0.32) 0.01 (0.03) 0.70 (2.65) 0.00 (0.02) 3.08 (5.61) 0.27 (0.42) 0.00 (0.02) 0.20 (0.22) 0.01 (0.03) 0.00 (0.00) 0.04 (0.11) 0.62 (1.17) 0.01 (0.03) 0.00 (0.00) 0.00 (0.02) 0.02 (0.07) 0.14 (0.27) 0.01 (0.04) 0.00 (0.00) 0.03 (0.04) 0.02 (0.09) 0.04 (0.05) 0.04 (0.06) 0.01 (0.03) 7.43 (2.70) 0.10 (0.18) 0.02 (0.07) 1.07 (0.41) 0.00 (0.00) 0.37 (0.34) 30.59 (11.15) 0.02 (0.08) 0.00 (0.00) 0.00 (0.00) 0.62 (1.41) 0.22 (0.35) 0.05 (0.18) 0.37 (0.44) 0.69 (0.55) 0.00 (0.00) 0.00 (0.00) 1.93 (4.77) 0.04 (0.07) 0.06 (0.13) 0.01 (0.06) 0.02 (0.07) 0.13 (0.22) 0.00 (0.00) 0.05 (0.01)
0.18 (0.06) 0.79 (0.34) 50.83 (7.39) 0.92 (0.36) 0.33 (0.05) 3.10 (2.31) 1.21 (0.26) 0.00 (0.00) 0.09 (0.31) 0.01 (0.02) 5.04 (2.82) 0.51 (0.38) 0.01 (0.02) 0.75 (0.30) 0.00 (0.00) 0.05 (0.07) 0.12 (0.08) 0.57 (0.61) 0.00 (0.00) 0.00 (0.00) 0.09 (0.09) 0.00 (0.00) 0.83 (0.36) 0.00 (0.00) 0.01 (0.03) 0.07 (0.07) 0.00 (0.00) 0.08 (0.09) 0.05 (0.06) 0.00 (0.00) 7.31 (0.66) 0.06 (0.07) 0.00 (0.00) 1.09 (0.11) 0.00 (0.00) 0.60 (0.45) 23.69 (7.29) 0.00 (0.00) 0.00 (0.00) 0.15 (0.14) 0.00 (0.00) 0.14 (0.12) 0.00 (0.00) 0.35 (0.30) 0.84 (0.59) 0.01 (0.02) 0.01 (0.02) 0.01 (0.03) 0.02 (0.04) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.08 (0.08)
0.26 (0.41) 0.48 (0.37) 45.40 (9.60) 1.35 (1.75) 0.29 (0.17) 5.47 (6.03) 0.77 (0.39) 0.00 (0.02) 0.31 (1.71) 0.00 (0.02) 3.08 (4.07) 0.55 (0.51) 0.00 (0.02) 0.46 (0.39) 0.00 (0.02) 0.01 (0.03) 0.06 (0.12) 0.61 (0.85) 0.02 (0.05) 0.00 (0.02) 0.03 (0.08) 0.01 (0.04) 0.41 (0.69) 0.01 (0.02) 0.00 (0.01) 0.03 (0.05) 0.01 (0.05) 0.04 (0.07) 0.02 (0.05) 0.02 (0.07) 7.69 (2.81) 0.06 (0.13) 0.01 (0.05) 1.14 (0.43) 0.00 (0.03) 0.47 (0.43) 28.03 (8.89) 0.01 (0.05) 0.05 (0.38) 0.09 (0.19) 0.33 (1.08) 0.15 (0.25) 0.06 (0.24) 0.32 (0.37) 0.69 (0.54) 0.00 (0.01) 0.00 (0.01) 1.02 (3.24) 0.05 (0.22) 0.02 (0.08) 0.02 (0.08) 0.02 (0.10) 0.05 (0.15) 0.01 (0.06) 0.06 (0.04)
Total [%]
100
100
100
100
Contents are given as percentages (mean 7 SD) of the total terpene content. Average contents of the indicated populations.
essential oils of the infraspecific taxa of P. nigra from Serbia were α-pinene, germacrene D, (E)-caryophyllene, and β-pinene as nonpolar and germacrene D-4-ol as polar compound (Table 1). One of the most studied properties of essential oils are their antimicrobial and antioxidant activity, important for both food preservation and control of human and animal diseases of microbial origin. Although the chemical composition and variability of the essential oils of black pine have been intensively studied in many parts of its habitat [10–15], etc., there is little information
about biological activities of P. nigra oils. According to the literature data, antimicrobial and antioxidant properties of P. nigra essential oils have been examined only for endemic Dalmatian black pine (P. nigra ssp. dalmatica) [3,16] and Crimean black pine (P. nigra ssp. pallasiana) from Turkey [17,18]. It is well known that essential oils possess antimicrobial activity owing to their lipophylicity and, thus, easy incorporation into bacterial membranes. For this reason, the most common mode of their action is membrane permeability alteration, which leads to
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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changes in membrane electrochemical potential and functionality. There are also other mechanisms of antimicrobial action reported for essential oils, such as RNA synthesis [19], inhibition of enzymes [20,21] where one of the promising ones is inhibition of different enzymes, especially those specific only for bacteria and widespread in bacterial kingdom. Recently, FtsZ (filamenting temperature sensitive strain Z), a 40-kDa protein became a highly promising target for new antibacterial drugs because of its central role in bacterial cell division [22]. The inhibition of FtsZ can lead to antibacterial activity which would be non-selective against bacteria (wide spectrum of activity). At the same time, the active compound would not disturb the structure of eucaryotic tubulin due to its evolutionary distance from FtsZ [23]. The aim of the present study was to define and compare the in vitro antibacterial, antifungal, and antioxidant activities of the essential oils isolated from needles of three infraspecific taxa of P. nigra (ssp. nigra, ssp. pallasiana, and var. banatica) from Serbia and to relate them with their chemical properties. Also, molecular docking studies were performed in order to evaluate whether some essential oil’s components can inhibit FtsZ and in this way contribute to antimicrobial activity.
2. Experimental sections 2.1. Plant material For this research three infraspecies taxa of P. nigra Arn. from Serbia were selected: ssp. nigra, ssp. pallasiana, and var. banatica. Twigs with needles from the lowest third of the tree crown were collected in late summer to early fall 2009 from three native populations: I—Mt. Tara (Zmajevački potok) (P. nigra ssp. nigra), II—Mt. Crnook (Jarešnik) (P. nigra ssp. pallasiana), and III—Lazareva reka (P. nigra var. banatica). The needles were stored in labeled (sample plot, date of collection, and age of the needles) polyethylene bags and samples were put into a fridge immediately after collection, and then stored at 201. Voucher specimens are deposited with the Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia. 2.2. Isolation of essential oils Two-year-old needles, stored until extraction in a freezer at 20 1C, were cut into pieces of 2–3 mm length, and extracted with pentane (1 g of needles per ml of solvent). The extracts were kept at 4–6 1C for 24 h and then filtered. After evaporation of the solvent, the obtained essential oil samples were subjected to subsequent analysis. 2.3. Evaluation of DPPH scavenging activity The antioxidant activity of the essential oils was studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method as described by Blois [24] with some modifications. Namely, 300 μL of a methanolic solution of investigated oils (the oils concentrations are between 1.00 and 30.00 mg/mL) were mixed with 2700 μL methanolic solution of DPPH radical (concentration of 0.04 mg/mL). After 30 min incubation in the dark at room temperature, the decrease in absorbance of the remaining DPPH radical was measured at 517 nm (A1) on a Shimadzu, UV–Visible PC 1650 spectrophotometer. Methanol (A0) was used as a blank solution, while Vitamin C and BHA were tested as standards. Each experiment was carried out in triplicate. Reduction of absorption of the DPPH solution was calculated in the following way: Percentage of absorption decrease (517 nm)¼(A0A1) 100/A0
3
Sample concentrations necessary to decrease the absorbance of DPPH by 50% (IC50) were obtained from the absorption of curves DPPH solution at 517 nm for each compound and standard antioxidant. Data analysis was performed with Origin 7.0 software. 2.4. Evaluation of ABTS radical scavenging activity The radical-scavenging activity of essential oils for the ABTS (2,20 -azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined according to the method of Miller and Rice-Evans with some modifications [25]. The ABTS þ radical cation was generated by mixing 19.2 mg of ABTS with 5 mL of potassium persulfate (2.46 mM) and kept for 12–16 h in the dark at room temperature. The ABTS þ solution (1 mL) was diluted with 100– 110 mL water to give an absorbance of 0.7 70.02 units at 734 nm using spectrophotometer (Shimadzu, UV–Visible PC 1650). New ABTS þ solution was prepared for each analysis. Antioxidant or standard solutions, 75 μL, were mixed with 3 mL of diluted ABTS þ solution. After 30 min incubation at 30 1C, the absorbance was measured at 734 nm. Modifications of the method are the following: instead of ethanol (95%), water was used as a blank and instead of Trolox, Vitamin C was used for obtaining calibration curve. ABTS radical scavenging activity in essential oils was calculated from the Vitamin C (VitC) calibration curve (0–2 mg/L) and expressed as Vitamin C (VitC)/g of dry extract. All measurements were performed in triplicate and were expressed as average of three analyses 7 standard deviation. 2.5. Antibacterial test The antimicrobial activity was evaluated against a panel of microorganisms, including Gram-positive Staphylococcus aureus (ATCC 6538), Sarcina lutea (ATCC 9431), Bacillus cereus (ATCC 10876), Gram-negative Proteus mirabilis (ATCC 12453), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), yeast Candida albicans (ATCC 10231) and mold Aspergillus niger (ATCC 16404). Cultures of bacterial species were maintained on Nutrient agar and fungal cultures on Sabouraud dextrose agar at appropriate optimal temperature (37 and 30 1C, respectively). Antimicrobial activity was tested using broth microdilution method, where minimum inhibitory concentrations (MIC) determination was performed by a serial dilution method in 96 well microtitre plates. Bacterial species were cultured at 37 1C in Mueller Hinton agar for bacteria and Sabouraud dextrose agar for fungi (30 C). After 18 h of cultivation, bacterial suspensions were made in Mueller Hinton broth and their turbidity was standardized using McFarland Densitometer (DEN-1B, Biosan). The final density of bacterial and yeast’s inoculi were 5 105. Suspension of the mold was made in Sabouraud dextrose broth and its turbidity was confirmed by viable counting in a Thoma chamber. Final size of the fungal inoculum was 1 104. Stock solutions of the essential oils were made in dimethylsulfoxide (DMSO, 10%) and than serially diluted (the diluting factor 2). The final concentration of the tested oils in the media ranged from 20.00 to 0.009 mg/mL. The highest concentration of the solvent (DMSO) in any well was 5% (v/v), which was previously confirmed as concentration which does not affect the growth of the tested cells. After making dilutions of the essential oils, the inoculum was added to all wells and the plates were incubated at 37 1C during 24 h (bacteria) or at 30 1C for 48 h (fungal strains). Chloramphenicol and nystatin served as positive controls, while the solvent (5% DMSO) was used as a negative control. To ensure medium sterility, each plate contained one non-inoculated well without antimicrobial agent. MIC was defined as the lowest concentration of the oil that inhibited visible growth of the cell cultures. All experiments were done in duplicate.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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2.6. Molecular docking 3D structures of the compounds for docking simulation were constructed using MarvinSketch 6.1.0, 2013, ChemAxon (http:// www.chemaxon.com). Geometry optimization was carried out by employing MMFF94 molecular force field [26]. E. coli FtsZ homology model, built from the X-ray crystal structure of M. jannaschii (PDB:1FSZ, 2.8 Å) [27] was used as target enzyme. The compounds were docked into enzyme binding sites using the MolegroVirtual Docker (MVD) [28]. The Molegro Virtual Docker (MVD v. 2013.6.0.1.) software was employed for docking ligands to the rigid enzyme model for identification of hydrogen bonds and hydrophobic interactions between residues at the active site. The binding site was computed with a grid resolution of 0.3 Å. The MolDock SE as a search algorithm was used with the number of runs was set to 100. The parameters of docking procedure were: population size 50, maximum number of iterations 1500, energy threshold 100.00 and maximum number of steps 300. The number of generated poses was 10. The estimation of ligand–receptor interactions was described by the MVD-related scoring functions: MolDock Score, Rerank Score, Hbond Score, Similarity Score, Docking Score. The ligand was docked into computed cavity instead ligand from 1FSZ using the MolDock Optimizer algorithm and its interactions were monitored using detailed energy estimates. A maximum population of 100 and maximum interactions of 10,000 were used for each run and the 5 best poses were retained.
3. Results and discussion 3.1. Antioxidative activity 3.1.1. DPPH scavenging activity The effect of antioxidant on DPPH radical scavenging was conceived to their hydrogen-donating ability. Mixing a solution of DPPH with that of the investigated compounds that can donate a hydrogen atom, generates a reduced form of diphenylpicrylhydrazine, which is characterized by the loss of its violet color [29]. DPPH radical scavenging capacities of the tested oils are shown in Table 2. The parameter IC50 (“efficient concentration” value) was defined as the concentration of substrate that causes 50% loss of the DPPH activity (color) which means that a lower IC50 value indicates higher antioxidant activity. The needle essential oils were found to be active with an IC50 value for P. nigra ssp. nigra of 25.596 mg/mL of solution, for P. nigra ssp. pallasiana 28.677 mg/ mL of solution and the IC50 value for P. nigra var. banatica 25.080 mg/mL of solution. The IC50 values for the synthetic antioxidants BHA and Vitamin C were 0.093 mg/mL and 0.054 mg/mL, respectively. According to the data obtained, essential oil of var. banatica possessed the strongest antioxidant activity compared to other taxa. Table 2 Antioxidant activity of Pinus nigra essential oils using DPPH scavenging method and antioxidant capacities by ABTS assay. Each value in the table was obtained by calculating the average of three analysis 7 standard deviation. Essential oils
P. nigra ssp. nigra P. nigra ssp. pallasiana P. nigra var. banatica BHA Vitamin C
DPPH assay
ABTS assay
IC50 (mg/mL)
Concentration (mg/mL)
Results
25.596 28.677 25.080 0.093 0.054
10 10 10 0.1 _
0.58 70.005 0.46 70.026 0.6770.052 2.66 70.005 _
3.1.2. ABTS scavenging activity Similar to DPPH, the decolorization of ABTS radical reflects the capacity of an antioxidant species to donate electron or hydrogen atoms to inactivate this radical cation [30]. The results from the ABTS assay are shown in Table 2. The amount ranged from 0.46 to 0.67 mg VitC/g of essential oils of P. nigra infraspecies taxa. The highest activity was identified in P. nigra var. banatica and the lowest in P. nigra ssp. pallasiana (with 10 mg/mL extract concentration). P. nigra ssp. nigra essential oil (with 10 mg/mL extract concentration) had approximately the same values. Comparison of DPPH and ABTS assays for estimating antioxidant potential shows low antioxidative activity of P. nigra essential oils. According to Šarac et al. [10], the average chemical profile of the main terpene components (contents4 5%) in the needle essential oils of P. nigra infraspecific taxa from Serbia was α-pinene c germacrene D c 4 (E)-caryophyllene 4 β-pinene (where, according to Petrakis et al. [31], 4, c , and c 4 represent differences of 1.1–5.0%, 5.1–15.0%, and more than 15.1%, resp.) (Table 1). The four most abundant terpene components in Serbian P. nigra taxa were also present in the needle oils of black pine from the Dalmatian islands (P. nigra ssp. dalmatica), but in different relative order [16]. This essential oil was also found to have weak antioxidant properties tested by three different methods (DPPH, FRAP, and TBARS). Although the mentioned study used different experimental protocol in DPPH analysis, the results are comparable for oils of ssp. dalmatica with the herein studied var. banatica (IC50 ¼ 23.6 and 25.08 mg/mL, respectively). Moreover, the needle oil of Crimean black pine (P. nigra ssp. pallasiana) from Turkey which contains α-pinene, β-pinene, and germacrene D as dominant components did not show significant antioxidative activity in FRAP assay [18]. A comparison of the results of the present and previous papers indicates that low antioxidative activity of black pine essential oils might be related to the predominance of mono- and sesquiterpene hydrocarbons which do not contain phenol groups. Among plant secondary metabolites, polyphenols are known to have a high scavenging ability on free radicals due the presence of hydroxyl substituents and their aromatic structure [32]. 3.2. Antimicrobial activity Using a broth microdilution method, the isolated essential oils were screened for their in vitro antimicrobial activity against six bacterial (three Gram-positive and three Gram-negative) and two fungal strains. The results of the MIC determination are presented in Table 3. Among the tested strains, one fungal (Aspergilus niger) and two bacterial (S. aureus and B. cereus) showed sensitivity against all three essential oils. Considering bacteria tested, Grampositive strains showed higher susceptibility, where B. cereus was the most sensitive one. Among the tested fungal strains, A. niger showed higher sensitivity to the action of the oils with active concentrations of 5.00 and 10.00 mg/mL. Tested oils have shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where P. nigra var. banatica exhibited the highest and P. nigra ssp. nigra the lowest antimicrobial action. P. nigra ssp. nigra essential oil showed the lowest antimicrobial potential among the tested oils, with exhibited activity against only two bacterial and one fungal strain. Minimal inhibitory concentrations of this oil were within the concentration range from 20.00 to 2.50 mg/mL. The highest activity was obtained against Gram-positive B. cereus, where 2.50 mg/mL was necessary to achieve inhibition of the growth. Essential oil of P. nigra ssp. pallasiana showed higher activity than the one obtained by ssp. nigra, where only two (Gram-negative and therefore less sensitive to antimicrobial agents) strains were completely resistant to the highest tested concentration. Active inhibitory
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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Table 3 Minimum inhibitory activities of the P. nigra ssp. nigra, P. nigra ssp. pallasiana and P. nigra var. banatica needle essential oils. Bacterial/fungal strain
Gþ
G
Fungi
a b
Staphylococcus aureus Sarcina lutea Bacillus cereus Proteus mirabilis Esherichia coli Pseudomonas aeruginosa Candida albicans Aspergillus niger
ATCC number
P. nigra ssp. nigra mg/mL MIC
P. nigra ssp. pallasiana mg/mL MIC
P. nigra var. banatica mg/mL MIC
Positive control mg/mL
6538 9341 10876 12453 8739 9027 10231 16404
20.00 420.00 2.50 420.00 420.00 420.00 420.00 10.00
10.00 20.00 1.50 420.00 420.00 20.00 10.00 5.00
20.00 10.00 0.62 20.00 420.00 10.00 5.00 5.00
0.19a 0.78a 0.19a 0.04a 0.19a 0.19a 0.9b 1.9b
Chloramphenicol. Nystatin.
concentrations of this oil were in the range from 1.50 to 20.00 mg/ mL. The most sensitive strain of the tested panel was B. cereus, while P. aeruginosa and S. lutea were the most resistant ones among the strains inhibited by its antimicrobial action (MIC¼ 20.00 mg/mL). The third tested essential oil, isolated from the needles of P. nigra var. banatica showed the highest antimicrobial activity, where concentrations from 0.62 to 20.00 mg/mL inhibited the growth of both bacteria and fungi. This oil was active against all tested strains except against Gram-negative bacteria E. coli, which was also completely resistant to other two tested essential oils. The lowest active concentration of this oil inhibited the growth of B. cereus, which makes it, once again, the most sensitive one among the tested strains. Previous investigations on this subject were very poor, with only one paper reporting antimicrobial activity of endemic dalmatian black pine (P. nigra ssp. dalmatica) needle essential oil [3]. In the mentioned study, it was determined that it possess both antimicrobial and antifungal activity in the range from 0.03 to 3.20% (v/v). Although the previous study did not tested the same set of strains, we are able to compare the results for some strains at the species level. B. cereus, which was the most sensitive one in the present study (MIC in the range 0.62–2.50 mg/mL), showed comparable results with the previous research (MIC ¼ 2.30 mg/ mL). On the other hand, the most susceptible strain in the previous study was S. aureus (MIC at 0.3 mg/mL) which had MIC in the range 10–20.00 mg/mL. In both studies, E. coli showed the highest resistance, where concentrations above 20.00 mg/mL were necessary to inhibit its growth. Opposite results are reported in the case of fungal organisms, where C. albicans showed higher susceptibility then A. niger to the oil of P. nigra, which was not the case in our results. Considering A. niger, the results are comparable for oils of P. nigra ssp. dalmatica with the herein studied P. nigra ssp. pallasiana and var. banatica (4.6, 5 and 5 mg/mL, respectively). Similarities of the previous and present results, such as higher sensitivity of the Gram-positive strains and completely resistance of Gram-negative enterobacteria can also be noted. Mono- and sesquiterpene hydrocarbons, which dominated in the essential oils of P. nigra, have been generally considered as volatile components with low antimicrobial activity due to their low hydrogen bonding capacity and water solubility [33]. Nevertheless, one fungal (A. niger) and two bacterial strains (S. aureus and B. cereus) showed sensitivity against essential oils of all three P. nigra taxa. Essential oil of var. banatica, which is characterized by the same main compounds as other two taxa oils showed the highest antimicrobial activity. This can be attributed to synergistic effect of some minor components such as thujene, myrcene and camphene, which are already reported as antimicrobials [34–37]. The oil of var. banatica exhibited the highest contents of these components compared to other two taxa (Table 1).
Fig. 1. Chemical structures of investigated compounds: (A) germacrene D-4-ol and (B) germacrene D.
3.3. Docking studies To determine potential compounds that are responsible for alternative mechanism of action by inhibiting FtsZ, molecular docking simulations were performed [38]. The antimicrobial activity of essential oils is related to their chemical composition and the amount of the single chemical components. Further, the mechanism of antibacterial action of chemical compounds is related to their structure. Terpenes such as limonene, α-pinene, β-pinene, γ-terpinene δ 3-carene, (þ )-sabinene and α-terpinene had very low or no antimicrobial activity against 25 genera of bacteria [39]. On the other hand, the addition of hydroxyl group can lead to increase of antibacterial activity. For example, carvacrol is more effective than p-cymene [39,40]. Since changes in molecular structures, like hydroxyl group addition, can enhance antibacterial activity of terpenes, one of aims of this research was the determination of differences between terpenes and terpenoids in the mode of antibacterial activity. Terpenoids with aldehide or alcoholic moieties can interact with membrane incorporated proteins by changing their conformation and, thus, their functionality [41]. Considering the fact that Germacrene D-4-ol is the most abundant terpenoid in the essential oils of P. nigra taxa, germacrene D is selected among the non polar constituents to be tested in molecular docking studies. For this reason, germacrene D and germacrene D-4-ol docking inside FtsZ, a prokaryotic homolog of tubulin was performed. Structures of germacrene D and germacrene D-4-ol are presented at Fig. 1. Number, bond length and bond energy of hydrogen bonds formed between ligand and enzyme have an important role in ligand effect on investigated activity. It was observed from in silico studies that only germacrene D-4-ol forms hydrogen bonds with Ala-97 (2.56365 Å) and Thr-135 (3.16367 Å). However, because hydrogen bond length is different, bond with Ala-97 is more significant one. All formed hydrogen bonds between germacrene D-4-ol and FtsZ binding pocket are presented in Fig. 2.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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The surface diagram of the docked molecules into the binding pocket of FtsZ is presented in Fig. 3. Main interactions between docked molecules and the binding pocked are hydrophobic.
Two dimensional representation of the best docking pose for both molecules inside enzyme binding pocket are shown at Fig. 4 [42]. These results point to additional mechanism of antimicrobial action, expressed by tested essential oils, which targets bacterial enzyme FtsZ. On the other hand, the main antimicrobial mode of action is probably membrane structure alteration and permeabilization caused by main compounds such as α-pinene, germacrene D and (E)-caryophyllene. In this way, synergistic activity between major and minor compounds present in the tested oils is proposed. Future studies should be focused on confirmation of this synergistic activity trough testing of antimicrobial activity of the pure major components and their combinations with germacrene-D-4-ol.
4. Conclusion
Fig. 2. Docked germacrene D-4-ol showing hydrogen bonding interactions with FtsZ.
This is the first report on the antimicrobial and antioxidant properties of the needle essential oils of P. nigra ssp. nigra, P. nigra var. banatica, and P. nigra ssp. pallasiana from Serbia. The black pine essential oils showed weak DPPH and ABTS scavenging effects. In the antimicrobial assays, one fungal (A. niger) and two bacterial strains (S. aureus and B. cereus) showed sensitivity against
Fig. 3. Surface diagram showing docked molecules (A) germacrene D-4-ol and (B) germacrene D with FtsZ.
Fig. 4. Two dimensional representation of the best docking pose for (A) germacrene D-4-ol and (B) germacrene D inside FtsZ binding pocket.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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essential oils of all three P. nigra taxa. Considering bacterial strains tested, Gram-positive strains showed higher susceptibility, where B. cereus was the most sensitive one. Docking simulation of germacrene D-4-ol and germacrene D inside FtsZ was performed and it was determined that germacrene D-4-ol interacts with FtsZ binding pocket. In this way, additional mechanism of antimicrobial action and synergistic activity between major and minor compounds present in the tested oils are proposed.
[17] [18]
[19]
[20]
Conflict of interest statement None.
[21]
[22] [23]
Acknowledgements This research was supported by a grant from the Ministry of Education and Science of the Republic of Serbia (project 173029).
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Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity Zorica Šarac a,n, Jelena S. Matejić b, Zorica Z. Stojanović-Radić a, Jovana B. Veselinović c, Ana M. Džamić d, Srdjan Bojović e, Petar D. Marin d a
Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia Department of Pharmacy, Faculty of Medicine, University of Niš, Boulevard Dr Zorana Đinđića 81, 18000 Niš, Serbia c Department of Chemistry, Faculty of Medicine, University of Niš, Boulevard Dr Zorana Đinđića 81, 18000 Niš, Serbia d Institute of Botany and Botanical Garden “Jevremovac”, Faculty of Biology, University of Belgrade, Studentski trg 16, 11000 Belgrade, Serbia e Institute for Biological Research “Siniša Stanković”, University of Belgrade, Boulevard Despota Stefana 142, 11060 Belgrade, Serbia b
art ic l e i nf o
a b s t r a c t
Article history: Received 15 June 2014 Accepted 19 August 2014
In the current work, in vitro antioxidant, antibacterial, and antifungal activites of the needle terpenes of three taxa of Pinus nigra from Serbia (ssp. nigra, ssp. pallasiana, and var. banatica) were analyzed. The black pine essential oils showed generally weak antioxidative properties tested by two methods (DPPH and ABTS scavenging assays), where the highest activity was identified in P. nigra var. banatica (IC50 ¼25.08 mg/mL and VitC ¼0.67 mg (vitamin C)/g when tested with the DPPH and ABTS reagents, respectively). In the antimicrobial assays, one fungal (Aspergilus niger) and two bacterial strains (Staphylococcus aureus and Bacillus cereus) showed sensitivity against essential oils of all three P. nigra taxa. The tested oils have been shown to possess inhibitory action in the range from 20.00 to 0.62 mg/ mL, where var. banatica exhibited the highest and ssp. nigra the lowest antimicrobial action. In order to determine potential compounds that are responsible for alternative mode of action, molecular docking simulations inside FtsZ (a prokaryotic homolog of tubulin) were performed. Tested compounds were the most abundant terpenoid (germacrene D-4-ol) and its structurally similar terpene (germacrene D), both present in all three essential oils. It was determined that the oxygenated form of the molecule creates stable bonds with investigated enzyme FtsZ, and that this compound, through this mechanism of action participates in the antimicrobial activity. & 2014 Published by Elsevier Ltd.
Keywords: Pinus nigra Needle terpenes Biological activity Bacillus cereus Molecular docking
1. Introduction Pinus (Pinaceae), with more than 200 generally recognized species, is the largest extant genus of Pinopsida [1]. Economically, pines are one of the most important tree species valued for wood, paper, resins, charcoal, food (particularly seeds), and ornamentals [2]. Moreover, most pines are considered as significant source of structurally diverse, bioactive compounds, and have provided contributions to the discovery of pharmaceutical agents and other biomedical applications [3]. For instance, turpentine have a long tradition of remedial utilization as topical rubefacient for the treatment of rheumatic diseases while pine bark extract is used in anti-aging cosmetics [4]. However, essential oils from Pinus species have been reported to have a variety of therapeutic properties. According to Dervendzi [5] pine needle oils are mostly used in folk medicine for the healing of respiratory infections
n
Corresponding author. Tel.: þ 381 18 533 015; fax: þ 381 18 533 014. E-mail address: [email protected] (Z. Šarac).
accompanied by cough, bronchitis, bronchial asthma, emphysema, tracheitis, sinusitis, laryngitis, pharyngitis, and influenza. Moreover, pine oils are widely used as flavoring additives for food and beverages. In various household cleaning products they are well known as scenting and disinfectant agents. Also, these oils are used in pharmaceutical industry as aroma or natural source of intermediates in the synthesis of perfume components [6]. The European black pine (Pinus nigra J.F.Arnold) is a tertiary relict belonging to the group of Mediterranean pines [7]. It is one of the most widespread and polymorphic conifers in Europe with highly fragmented distribution range that extends from North Africa through the northern Mediterranean and eastwards to the Black Sea. In the Flora of Serbia [8], two subspecies of black pine (ssp. nigra and ssp. pallasiana) are distinguished and within them several varieties (var. nigra, var. zlatiborica, var. gocensis, and var. banatica). However, recent studies on the chemotaxonomy of P. nigra in Serbia, based on both the n-alkane [9] and terpene [10] variability, recognized only three distinct chemotypes (nigra, pallasiana i banatica) within Serbian black pine populations. According to Šarac et al. [10], the major components in the needle
http://dx.doi.org/10.1016/j.compbiomed.2014.08.022 0010-4825/& 2014 Published by Elsevier Ltd.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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Table 1 Chemical composition of the needle essential oils of three infraspecific taxa of Pinus nigra from Serbia [10]. Entry
Compound
Content [%]a P. nigra taxa Location
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55
a b
Average content [%]b ssp. nigra Zmajevački potok
ssp. pallasiana Jarešnik
var. banatica Lazareva reka
Tricyclene α-Thujene α-Pinene Camphene Sabinene β-Pinene Myrcene α-Phellandrene Δ3-Carene α-Terpinene Limonene (E)-β-Ocimene γ-Terpinene Terpinolene trans-Verbenol α-Terpineol Linalool acetate Bornyl acetate trans-Verbenyl acetate trans-Pinocarvyl acetate Myrtenyl acetate δ-Elemene α-Terpinyl acetate α-Cubebene α-Ylangene α-Copaene Geranyl acetate β-Bourbonene β-Cubebene β-Elemene (E)-Caryophyllene β-Copaene 6,9-Guaiadiene α-Humulene (E)-β-Farnesene γ-Muurolene Germacrene D β-Selinene Phenylethyl 3-methylbutanoate γ-Amorphene Bicyclogermacrene α-Muurolene Germacrene A γ-Cadinene δ-Cadinene trans-Cadina-1,4-diene α-Cadinene Germacrene D-4-ol Caryophyllene oxide 1,7-Diepi-α-Cedrenal T-Cadinol α-Cadinol Germacra-4(15),5,10(14)-trien-1-alpha-ol Oplopanone Unknown components
0.16 (0.07) 0.41 (0.30) 45.93 (10.46) 0.95 (0.29) 0.31 (0.18) 6.90 (7.04) 0.88 (0.21) 0.00 (0.00) 0.03 (0.12) 0.00 (0.00) 2.16 (1.98) 0.86 (0.47) 0.00 (0.00) 0.57 (0.41) 0.00 (0.00) 0.00 (0.00) 0.05 (0.13) 0.63 (0.52) 0.03 (0.07) 0.01 (0.03) 0.03 (0.10) 0.00 (0.00) 0.48 (0.95) 0.00 (0.00) 0.00 (0.00) 0.02 (0.05) 0.00 (0.00) 0.02 (0.06) 0.00 (0.00) 0.03 (0.11) 8.13 (3.47) 0.02 (0.07) 0.00 (0.00) 1.24 (0.52) 0.01 (0.04) 0.34 (0.38) 27.50 (5.89) 0.01 (0.03) 0.12 (0.60) 0.15 (0.26) 0.19 (0.88) 0.08 (0.11) 0.09 (0.33) 0.25 (0.32) 0.61 (0.52) 0.00 (0.00) 0.00 (0.01) 0.57 (1.44) 0.08 (0.33) 0.00 (0.00) 0.03 (0.11) 0.03 (0.13) 0.00 (0.01) 0.02 (0.09) 0.05 (0.02)
0.40 (0.61) 0.41 (0.40) 42.33 (8.59) 1.95 (2.64) 0.26 (0.18) 5.15 (5.90) 0.45 (0.32) 0.01 (0.03) 0.70 (2.65) 0.00 (0.02) 3.08 (5.61) 0.27 (0.42) 0.00 (0.02) 0.20 (0.22) 0.01 (0.03) 0.00 (0.00) 0.04 (0.11) 0.62 (1.17) 0.01 (0.03) 0.00 (0.00) 0.00 (0.02) 0.02 (0.07) 0.14 (0.27) 0.01 (0.04) 0.00 (0.00) 0.03 (0.04) 0.02 (0.09) 0.04 (0.05) 0.04 (0.06) 0.01 (0.03) 7.43 (2.70) 0.10 (0.18) 0.02 (0.07) 1.07 (0.41) 0.00 (0.00) 0.37 (0.34) 30.59 (11.15) 0.02 (0.08) 0.00 (0.00) 0.00 (0.00) 0.62 (1.41) 0.22 (0.35) 0.05 (0.18) 0.37 (0.44) 0.69 (0.55) 0.00 (0.00) 0.00 (0.00) 1.93 (4.77) 0.04 (0.07) 0.06 (0.13) 0.01 (0.06) 0.02 (0.07) 0.13 (0.22) 0.00 (0.00) 0.05 (0.01)
0.18 (0.06) 0.79 (0.34) 50.83 (7.39) 0.92 (0.36) 0.33 (0.05) 3.10 (2.31) 1.21 (0.26) 0.00 (0.00) 0.09 (0.31) 0.01 (0.02) 5.04 (2.82) 0.51 (0.38) 0.01 (0.02) 0.75 (0.30) 0.00 (0.00) 0.05 (0.07) 0.12 (0.08) 0.57 (0.61) 0.00 (0.00) 0.00 (0.00) 0.09 (0.09) 0.00 (0.00) 0.83 (0.36) 0.00 (0.00) 0.01 (0.03) 0.07 (0.07) 0.00 (0.00) 0.08 (0.09) 0.05 (0.06) 0.00 (0.00) 7.31 (0.66) 0.06 (0.07) 0.00 (0.00) 1.09 (0.11) 0.00 (0.00) 0.60 (0.45) 23.69 (7.29) 0.00 (0.00) 0.00 (0.00) 0.15 (0.14) 0.00 (0.00) 0.14 (0.12) 0.00 (0.00) 0.35 (0.30) 0.84 (0.59) 0.01 (0.02) 0.01 (0.02) 0.01 (0.03) 0.02 (0.04) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.00 (0.00) 0.08 (0.08)
0.26 (0.41) 0.48 (0.37) 45.40 (9.60) 1.35 (1.75) 0.29 (0.17) 5.47 (6.03) 0.77 (0.39) 0.00 (0.02) 0.31 (1.71) 0.00 (0.02) 3.08 (4.07) 0.55 (0.51) 0.00 (0.02) 0.46 (0.39) 0.00 (0.02) 0.01 (0.03) 0.06 (0.12) 0.61 (0.85) 0.02 (0.05) 0.00 (0.02) 0.03 (0.08) 0.01 (0.04) 0.41 (0.69) 0.01 (0.02) 0.00 (0.01) 0.03 (0.05) 0.01 (0.05) 0.04 (0.07) 0.02 (0.05) 0.02 (0.07) 7.69 (2.81) 0.06 (0.13) 0.01 (0.05) 1.14 (0.43) 0.00 (0.03) 0.47 (0.43) 28.03 (8.89) 0.01 (0.05) 0.05 (0.38) 0.09 (0.19) 0.33 (1.08) 0.15 (0.25) 0.06 (0.24) 0.32 (0.37) 0.69 (0.54) 0.00 (0.01) 0.00 (0.01) 1.02 (3.24) 0.05 (0.22) 0.02 (0.08) 0.02 (0.08) 0.02 (0.10) 0.05 (0.15) 0.01 (0.06) 0.06 (0.04)
Total [%]
100
100
100
100
Contents are given as percentages (mean 7 SD) of the total terpene content. Average contents of the indicated populations.
essential oils of the infraspecific taxa of P. nigra from Serbia were α-pinene, germacrene D, (E)-caryophyllene, and β-pinene as nonpolar and germacrene D-4-ol as polar compound (Table 1). One of the most studied properties of essential oils are their antimicrobial and antioxidant activity, important for both food preservation and control of human and animal diseases of microbial origin. Although the chemical composition and variability of the essential oils of black pine have been intensively studied in many parts of its habitat [10–15], etc., there is little information
about biological activities of P. nigra oils. According to the literature data, antimicrobial and antioxidant properties of P. nigra essential oils have been examined only for endemic Dalmatian black pine (P. nigra ssp. dalmatica) [3,16] and Crimean black pine (P. nigra ssp. pallasiana) from Turkey [17,18]. It is well known that essential oils possess antimicrobial activity owing to their lipophylicity and, thus, easy incorporation into bacterial membranes. For this reason, the most common mode of their action is membrane permeability alteration, which leads to
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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changes in membrane electrochemical potential and functionality. There are also other mechanisms of antimicrobial action reported for essential oils, such as RNA synthesis [19], inhibition of enzymes [20,21] where one of the promising ones is inhibition of different enzymes, especially those specific only for bacteria and widespread in bacterial kingdom. Recently, FtsZ (filamenting temperature sensitive strain Z), a 40-kDa protein became a highly promising target for new antibacterial drugs because of its central role in bacterial cell division [22]. The inhibition of FtsZ can lead to antibacterial activity which would be non-selective against bacteria (wide spectrum of activity). At the same time, the active compound would not disturb the structure of eucaryotic tubulin due to its evolutionary distance from FtsZ [23]. The aim of the present study was to define and compare the in vitro antibacterial, antifungal, and antioxidant activities of the essential oils isolated from needles of three infraspecific taxa of P. nigra (ssp. nigra, ssp. pallasiana, and var. banatica) from Serbia and to relate them with their chemical properties. Also, molecular docking studies were performed in order to evaluate whether some essential oil’s components can inhibit FtsZ and in this way contribute to antimicrobial activity.
2. Experimental sections 2.1. Plant material For this research three infraspecies taxa of P. nigra Arn. from Serbia were selected: ssp. nigra, ssp. pallasiana, and var. banatica. Twigs with needles from the lowest third of the tree crown were collected in late summer to early fall 2009 from three native populations: I—Mt. Tara (Zmajevački potok) (P. nigra ssp. nigra), II—Mt. Crnook (Jarešnik) (P. nigra ssp. pallasiana), and III—Lazareva reka (P. nigra var. banatica). The needles were stored in labeled (sample plot, date of collection, and age of the needles) polyethylene bags and samples were put into a fridge immediately after collection, and then stored at 201. Voucher specimens are deposited with the Institute for Biological Research “Siniša Stanković”, University of Belgrade, Serbia. 2.2. Isolation of essential oils Two-year-old needles, stored until extraction in a freezer at 20 1C, were cut into pieces of 2–3 mm length, and extracted with pentane (1 g of needles per ml of solvent). The extracts were kept at 4–6 1C for 24 h and then filtered. After evaporation of the solvent, the obtained essential oil samples were subjected to subsequent analysis. 2.3. Evaluation of DPPH scavenging activity The antioxidant activity of the essential oils was studied using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method as described by Blois [24] with some modifications. Namely, 300 μL of a methanolic solution of investigated oils (the oils concentrations are between 1.00 and 30.00 mg/mL) were mixed with 2700 μL methanolic solution of DPPH radical (concentration of 0.04 mg/mL). After 30 min incubation in the dark at room temperature, the decrease in absorbance of the remaining DPPH radical was measured at 517 nm (A1) on a Shimadzu, UV–Visible PC 1650 spectrophotometer. Methanol (A0) was used as a blank solution, while Vitamin C and BHA were tested as standards. Each experiment was carried out in triplicate. Reduction of absorption of the DPPH solution was calculated in the following way: Percentage of absorption decrease (517 nm)¼(A0A1) 100/A0
3
Sample concentrations necessary to decrease the absorbance of DPPH by 50% (IC50) were obtained from the absorption of curves DPPH solution at 517 nm for each compound and standard antioxidant. Data analysis was performed with Origin 7.0 software. 2.4. Evaluation of ABTS radical scavenging activity The radical-scavenging activity of essential oils for the ABTS (2,20 -azinobis-3-ethylbenzothiazoline-6-sulphonate) radical cation was determined according to the method of Miller and Rice-Evans with some modifications [25]. The ABTS þ radical cation was generated by mixing 19.2 mg of ABTS with 5 mL of potassium persulfate (2.46 mM) and kept for 12–16 h in the dark at room temperature. The ABTS þ solution (1 mL) was diluted with 100– 110 mL water to give an absorbance of 0.7 70.02 units at 734 nm using spectrophotometer (Shimadzu, UV–Visible PC 1650). New ABTS þ solution was prepared for each analysis. Antioxidant or standard solutions, 75 μL, were mixed with 3 mL of diluted ABTS þ solution. After 30 min incubation at 30 1C, the absorbance was measured at 734 nm. Modifications of the method are the following: instead of ethanol (95%), water was used as a blank and instead of Trolox, Vitamin C was used for obtaining calibration curve. ABTS radical scavenging activity in essential oils was calculated from the Vitamin C (VitC) calibration curve (0–2 mg/L) and expressed as Vitamin C (VitC)/g of dry extract. All measurements were performed in triplicate and were expressed as average of three analyses 7 standard deviation. 2.5. Antibacterial test The antimicrobial activity was evaluated against a panel of microorganisms, including Gram-positive Staphylococcus aureus (ATCC 6538), Sarcina lutea (ATCC 9431), Bacillus cereus (ATCC 10876), Gram-negative Proteus mirabilis (ATCC 12453), Pseudomonas aeruginosa (ATCC 9027), Escherichia coli (ATCC 8739), yeast Candida albicans (ATCC 10231) and mold Aspergillus niger (ATCC 16404). Cultures of bacterial species were maintained on Nutrient agar and fungal cultures on Sabouraud dextrose agar at appropriate optimal temperature (37 and 30 1C, respectively). Antimicrobial activity was tested using broth microdilution method, where minimum inhibitory concentrations (MIC) determination was performed by a serial dilution method in 96 well microtitre plates. Bacterial species were cultured at 37 1C in Mueller Hinton agar for bacteria and Sabouraud dextrose agar for fungi (30 C). After 18 h of cultivation, bacterial suspensions were made in Mueller Hinton broth and their turbidity was standardized using McFarland Densitometer (DEN-1B, Biosan). The final density of bacterial and yeast’s inoculi were 5 105. Suspension of the mold was made in Sabouraud dextrose broth and its turbidity was confirmed by viable counting in a Thoma chamber. Final size of the fungal inoculum was 1 104. Stock solutions of the essential oils were made in dimethylsulfoxide (DMSO, 10%) and than serially diluted (the diluting factor 2). The final concentration of the tested oils in the media ranged from 20.00 to 0.009 mg/mL. The highest concentration of the solvent (DMSO) in any well was 5% (v/v), which was previously confirmed as concentration which does not affect the growth of the tested cells. After making dilutions of the essential oils, the inoculum was added to all wells and the plates were incubated at 37 1C during 24 h (bacteria) or at 30 1C for 48 h (fungal strains). Chloramphenicol and nystatin served as positive controls, while the solvent (5% DMSO) was used as a negative control. To ensure medium sterility, each plate contained one non-inoculated well without antimicrobial agent. MIC was defined as the lowest concentration of the oil that inhibited visible growth of the cell cultures. All experiments were done in duplicate.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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2.6. Molecular docking 3D structures of the compounds for docking simulation were constructed using MarvinSketch 6.1.0, 2013, ChemAxon (http:// www.chemaxon.com). Geometry optimization was carried out by employing MMFF94 molecular force field [26]. E. coli FtsZ homology model, built from the X-ray crystal structure of M. jannaschii (PDB:1FSZ, 2.8 Å) [27] was used as target enzyme. The compounds were docked into enzyme binding sites using the MolegroVirtual Docker (MVD) [28]. The Molegro Virtual Docker (MVD v. 2013.6.0.1.) software was employed for docking ligands to the rigid enzyme model for identification of hydrogen bonds and hydrophobic interactions between residues at the active site. The binding site was computed with a grid resolution of 0.3 Å. The MolDock SE as a search algorithm was used with the number of runs was set to 100. The parameters of docking procedure were: population size 50, maximum number of iterations 1500, energy threshold 100.00 and maximum number of steps 300. The number of generated poses was 10. The estimation of ligand–receptor interactions was described by the MVD-related scoring functions: MolDock Score, Rerank Score, Hbond Score, Similarity Score, Docking Score. The ligand was docked into computed cavity instead ligand from 1FSZ using the MolDock Optimizer algorithm and its interactions were monitored using detailed energy estimates. A maximum population of 100 and maximum interactions of 10,000 were used for each run and the 5 best poses were retained.
3. Results and discussion 3.1. Antioxidative activity 3.1.1. DPPH scavenging activity The effect of antioxidant on DPPH radical scavenging was conceived to their hydrogen-donating ability. Mixing a solution of DPPH with that of the investigated compounds that can donate a hydrogen atom, generates a reduced form of diphenylpicrylhydrazine, which is characterized by the loss of its violet color [29]. DPPH radical scavenging capacities of the tested oils are shown in Table 2. The parameter IC50 (“efficient concentration” value) was defined as the concentration of substrate that causes 50% loss of the DPPH activity (color) which means that a lower IC50 value indicates higher antioxidant activity. The needle essential oils were found to be active with an IC50 value for P. nigra ssp. nigra of 25.596 mg/mL of solution, for P. nigra ssp. pallasiana 28.677 mg/ mL of solution and the IC50 value for P. nigra var. banatica 25.080 mg/mL of solution. The IC50 values for the synthetic antioxidants BHA and Vitamin C were 0.093 mg/mL and 0.054 mg/mL, respectively. According to the data obtained, essential oil of var. banatica possessed the strongest antioxidant activity compared to other taxa. Table 2 Antioxidant activity of Pinus nigra essential oils using DPPH scavenging method and antioxidant capacities by ABTS assay. Each value in the table was obtained by calculating the average of three analysis 7 standard deviation. Essential oils
P. nigra ssp. nigra P. nigra ssp. pallasiana P. nigra var. banatica BHA Vitamin C
DPPH assay
ABTS assay
IC50 (mg/mL)
Concentration (mg/mL)
Results
25.596 28.677 25.080 0.093 0.054
10 10 10 0.1 _
0.58 70.005 0.46 70.026 0.6770.052 2.66 70.005 _
3.1.2. ABTS scavenging activity Similar to DPPH, the decolorization of ABTS radical reflects the capacity of an antioxidant species to donate electron or hydrogen atoms to inactivate this radical cation [30]. The results from the ABTS assay are shown in Table 2. The amount ranged from 0.46 to 0.67 mg VitC/g of essential oils of P. nigra infraspecies taxa. The highest activity was identified in P. nigra var. banatica and the lowest in P. nigra ssp. pallasiana (with 10 mg/mL extract concentration). P. nigra ssp. nigra essential oil (with 10 mg/mL extract concentration) had approximately the same values. Comparison of DPPH and ABTS assays for estimating antioxidant potential shows low antioxidative activity of P. nigra essential oils. According to Šarac et al. [10], the average chemical profile of the main terpene components (contents4 5%) in the needle essential oils of P. nigra infraspecific taxa from Serbia was α-pinene c germacrene D c 4 (E)-caryophyllene 4 β-pinene (where, according to Petrakis et al. [31], 4, c , and c 4 represent differences of 1.1–5.0%, 5.1–15.0%, and more than 15.1%, resp.) (Table 1). The four most abundant terpene components in Serbian P. nigra taxa were also present in the needle oils of black pine from the Dalmatian islands (P. nigra ssp. dalmatica), but in different relative order [16]. This essential oil was also found to have weak antioxidant properties tested by three different methods (DPPH, FRAP, and TBARS). Although the mentioned study used different experimental protocol in DPPH analysis, the results are comparable for oils of ssp. dalmatica with the herein studied var. banatica (IC50 ¼ 23.6 and 25.08 mg/mL, respectively). Moreover, the needle oil of Crimean black pine (P. nigra ssp. pallasiana) from Turkey which contains α-pinene, β-pinene, and germacrene D as dominant components did not show significant antioxidative activity in FRAP assay [18]. A comparison of the results of the present and previous papers indicates that low antioxidative activity of black pine essential oils might be related to the predominance of mono- and sesquiterpene hydrocarbons which do not contain phenol groups. Among plant secondary metabolites, polyphenols are known to have a high scavenging ability on free radicals due the presence of hydroxyl substituents and their aromatic structure [32]. 3.2. Antimicrobial activity Using a broth microdilution method, the isolated essential oils were screened for their in vitro antimicrobial activity against six bacterial (three Gram-positive and three Gram-negative) and two fungal strains. The results of the MIC determination are presented in Table 3. Among the tested strains, one fungal (Aspergilus niger) and two bacterial (S. aureus and B. cereus) showed sensitivity against all three essential oils. Considering bacteria tested, Grampositive strains showed higher susceptibility, where B. cereus was the most sensitive one. Among the tested fungal strains, A. niger showed higher sensitivity to the action of the oils with active concentrations of 5.00 and 10.00 mg/mL. Tested oils have shown to possess inhibitory action in the range from 20.00 to 0.62 mg/mL, where P. nigra var. banatica exhibited the highest and P. nigra ssp. nigra the lowest antimicrobial action. P. nigra ssp. nigra essential oil showed the lowest antimicrobial potential among the tested oils, with exhibited activity against only two bacterial and one fungal strain. Minimal inhibitory concentrations of this oil were within the concentration range from 20.00 to 2.50 mg/mL. The highest activity was obtained against Gram-positive B. cereus, where 2.50 mg/mL was necessary to achieve inhibition of the growth. Essential oil of P. nigra ssp. pallasiana showed higher activity than the one obtained by ssp. nigra, where only two (Gram-negative and therefore less sensitive to antimicrobial agents) strains were completely resistant to the highest tested concentration. Active inhibitory
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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Table 3 Minimum inhibitory activities of the P. nigra ssp. nigra, P. nigra ssp. pallasiana and P. nigra var. banatica needle essential oils. Bacterial/fungal strain
Gþ
G
Fungi
a b
Staphylococcus aureus Sarcina lutea Bacillus cereus Proteus mirabilis Esherichia coli Pseudomonas aeruginosa Candida albicans Aspergillus niger
ATCC number
P. nigra ssp. nigra mg/mL MIC
P. nigra ssp. pallasiana mg/mL MIC
P. nigra var. banatica mg/mL MIC
Positive control mg/mL
6538 9341 10876 12453 8739 9027 10231 16404
20.00 420.00 2.50 420.00 420.00 420.00 420.00 10.00
10.00 20.00 1.50 420.00 420.00 20.00 10.00 5.00
20.00 10.00 0.62 20.00 420.00 10.00 5.00 5.00
0.19a 0.78a 0.19a 0.04a 0.19a 0.19a 0.9b 1.9b
Chloramphenicol. Nystatin.
concentrations of this oil were in the range from 1.50 to 20.00 mg/ mL. The most sensitive strain of the tested panel was B. cereus, while P. aeruginosa and S. lutea were the most resistant ones among the strains inhibited by its antimicrobial action (MIC¼ 20.00 mg/mL). The third tested essential oil, isolated from the needles of P. nigra var. banatica showed the highest antimicrobial activity, where concentrations from 0.62 to 20.00 mg/mL inhibited the growth of both bacteria and fungi. This oil was active against all tested strains except against Gram-negative bacteria E. coli, which was also completely resistant to other two tested essential oils. The lowest active concentration of this oil inhibited the growth of B. cereus, which makes it, once again, the most sensitive one among the tested strains. Previous investigations on this subject were very poor, with only one paper reporting antimicrobial activity of endemic dalmatian black pine (P. nigra ssp. dalmatica) needle essential oil [3]. In the mentioned study, it was determined that it possess both antimicrobial and antifungal activity in the range from 0.03 to 3.20% (v/v). Although the previous study did not tested the same set of strains, we are able to compare the results for some strains at the species level. B. cereus, which was the most sensitive one in the present study (MIC in the range 0.62–2.50 mg/mL), showed comparable results with the previous research (MIC ¼ 2.30 mg/ mL). On the other hand, the most susceptible strain in the previous study was S. aureus (MIC at 0.3 mg/mL) which had MIC in the range 10–20.00 mg/mL. In both studies, E. coli showed the highest resistance, where concentrations above 20.00 mg/mL were necessary to inhibit its growth. Opposite results are reported in the case of fungal organisms, where C. albicans showed higher susceptibility then A. niger to the oil of P. nigra, which was not the case in our results. Considering A. niger, the results are comparable for oils of P. nigra ssp. dalmatica with the herein studied P. nigra ssp. pallasiana and var. banatica (4.6, 5 and 5 mg/mL, respectively). Similarities of the previous and present results, such as higher sensitivity of the Gram-positive strains and completely resistance of Gram-negative enterobacteria can also be noted. Mono- and sesquiterpene hydrocarbons, which dominated in the essential oils of P. nigra, have been generally considered as volatile components with low antimicrobial activity due to their low hydrogen bonding capacity and water solubility [33]. Nevertheless, one fungal (A. niger) and two bacterial strains (S. aureus and B. cereus) showed sensitivity against essential oils of all three P. nigra taxa. Essential oil of var. banatica, which is characterized by the same main compounds as other two taxa oils showed the highest antimicrobial activity. This can be attributed to synergistic effect of some minor components such as thujene, myrcene and camphene, which are already reported as antimicrobials [34–37]. The oil of var. banatica exhibited the highest contents of these components compared to other two taxa (Table 1).
Fig. 1. Chemical structures of investigated compounds: (A) germacrene D-4-ol and (B) germacrene D.
3.3. Docking studies To determine potential compounds that are responsible for alternative mechanism of action by inhibiting FtsZ, molecular docking simulations were performed [38]. The antimicrobial activity of essential oils is related to their chemical composition and the amount of the single chemical components. Further, the mechanism of antibacterial action of chemical compounds is related to their structure. Terpenes such as limonene, α-pinene, β-pinene, γ-terpinene δ 3-carene, (þ )-sabinene and α-terpinene had very low or no antimicrobial activity against 25 genera of bacteria [39]. On the other hand, the addition of hydroxyl group can lead to increase of antibacterial activity. For example, carvacrol is more effective than p-cymene [39,40]. Since changes in molecular structures, like hydroxyl group addition, can enhance antibacterial activity of terpenes, one of aims of this research was the determination of differences between terpenes and terpenoids in the mode of antibacterial activity. Terpenoids with aldehide or alcoholic moieties can interact with membrane incorporated proteins by changing their conformation and, thus, their functionality [41]. Considering the fact that Germacrene D-4-ol is the most abundant terpenoid in the essential oils of P. nigra taxa, germacrene D is selected among the non polar constituents to be tested in molecular docking studies. For this reason, germacrene D and germacrene D-4-ol docking inside FtsZ, a prokaryotic homolog of tubulin was performed. Structures of germacrene D and germacrene D-4-ol are presented at Fig. 1. Number, bond length and bond energy of hydrogen bonds formed between ligand and enzyme have an important role in ligand effect on investigated activity. It was observed from in silico studies that only germacrene D-4-ol forms hydrogen bonds with Ala-97 (2.56365 Å) and Thr-135 (3.16367 Å). However, because hydrogen bond length is different, bond with Ala-97 is more significant one. All formed hydrogen bonds between germacrene D-4-ol and FtsZ binding pocket are presented in Fig. 2.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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The surface diagram of the docked molecules into the binding pocket of FtsZ is presented in Fig. 3. Main interactions between docked molecules and the binding pocked are hydrophobic.
Two dimensional representation of the best docking pose for both molecules inside enzyme binding pocket are shown at Fig. 4 [42]. These results point to additional mechanism of antimicrobial action, expressed by tested essential oils, which targets bacterial enzyme FtsZ. On the other hand, the main antimicrobial mode of action is probably membrane structure alteration and permeabilization caused by main compounds such as α-pinene, germacrene D and (E)-caryophyllene. In this way, synergistic activity between major and minor compounds present in the tested oils is proposed. Future studies should be focused on confirmation of this synergistic activity trough testing of antimicrobial activity of the pure major components and their combinations with germacrene-D-4-ol.
4. Conclusion
Fig. 2. Docked germacrene D-4-ol showing hydrogen bonding interactions with FtsZ.
This is the first report on the antimicrobial and antioxidant properties of the needle essential oils of P. nigra ssp. nigra, P. nigra var. banatica, and P. nigra ssp. pallasiana from Serbia. The black pine essential oils showed weak DPPH and ABTS scavenging effects. In the antimicrobial assays, one fungal (A. niger) and two bacterial strains (S. aureus and B. cereus) showed sensitivity against
Fig. 3. Surface diagram showing docked molecules (A) germacrene D-4-ol and (B) germacrene D with FtsZ.
Fig. 4. Two dimensional representation of the best docking pose for (A) germacrene D-4-ol and (B) germacrene D inside FtsZ binding pocket.
Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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essential oils of all three P. nigra taxa. Considering bacterial strains tested, Gram-positive strains showed higher susceptibility, where B. cereus was the most sensitive one. Docking simulation of germacrene D-4-ol and germacrene D inside FtsZ was performed and it was determined that germacrene D-4-ol interacts with FtsZ binding pocket. In this way, additional mechanism of antimicrobial action and synergistic activity between major and minor compounds present in the tested oils are proposed.
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Conflict of interest statement None.
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Acknowledgements This research was supported by a grant from the Ministry of Education and Science of the Republic of Serbia (project 173029).
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Please cite this article as: Z. Šarac, et al., Biological activity of Pinus nigra terpenes—Evaluation of FtsZ inhibition by selected compounds as contribution to their antimicrobial activity, Comput. Biol. Med. (2014), http://dx.doi.org/10.1016/j.compbiomed.2014.08.022i
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