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Further in vitro evaluation of antiradical and antimicrobial activities of phytol a
a
b
b
b
B. Pejin , A. Savic , M. Sokovic , J. Glamoclija , A. Ciric , M. b
a
Nikolic , K. Radotic & M. Mojovic
c
a
Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, Belgrade 11030, Serbia b
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Department of Plant Physiology – Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade 11060, Serbia c
Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia Published online: 14 Jan 2014.
To cite this article: B. Pejin, A. Savic, M. Sokovic, J. Glamoclija, A. Ciric, M. Nikolic, K. Radotic & M. Mojovic (2014) Further in vitro evaluation of antiradical and antimicrobial activities of phytol, Natural Product Research: Formerly Natural Product Letters, 28:6, 372-376, DOI: 10.1080/14786419.2013.869692 To link to this article: http://dx.doi.org/10.1080/14786419.2013.869692
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Natural Product Research, 2014 Vol. 28, No. 6, 372–376, http://dx.doi.org/10.1080/14786419.2013.869692
Further in vitro evaluation of antiradical and antimicrobial activities of phytol B. Pejina*, A. Savica, M. Sokovicb, J. Glamoclijab, A. Ciricb, M. Nikolicb, K. Radotica and M. Mojovicc*
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a
Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, Belgrade 11030, Serbia; bDepartment of Plant Physiology – Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade 11060, Serbia; cFaculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia (Received 28 August 2013; final version received 19 November 2013) The antiradical activity of phytol was evaluated by electron paramagnetic resonance towards hydroxyl radical (zOH), superoxide anion radical (zO2 2 ), methoxy radical (zCH2OH), carbon-dioxide anion radical (zCO2 2 ), as well as towards nitric-oxide radical (zNO) and 2,2-diphenyl-1-picrylhydrazyl (zDPPH) radical. It reduced the production of all tested radicals showing more promising activity against zCO2 2 , zCH2OH and zDPPH radicals (56%, 50% and 48%, respectively) in comparison with zNO, zO2 2 and zOH radicals (38%, 23% and 15%, respectively). The antimicrobial activity of phytol was evaluated by the microdilution method against eight bacterial and eight fungal strains. To varying degrees, it was proven to be active against all tested bacteria and fungi (MIC 0.003– 0.038 mg/mL and MBC 0.013 – 0.052 mg/mL, MIC 0.008 – 0.016 mg/mL and MFC 0.090 – 0.520 mg/mL, respectively). According to the obtained results, medical foods containing phytol may support development of new therapies for heart disease. Keywords: phytol; reactive oxygen and nitrogen species; DPPH; EPR; microorganisms; medical foods
1. Introduction Cardiovascular diseases are the major cause of death worldwide according to the World Health Organization, with the projection to increase from 17.3 million people in 2008 to more than 23.3 million people in 2030 (WHO 2013). Traditional Chinese medicine suggests that mosses of Rhodobryum species (Bryaceae) can cure heart disorders as crude drugs (Ding 1982). Preliminary analysis of the moss Rhodobryum ontariense volatiles has indicated diterpene phytol (3,7,11,15-tetramethyl-2-hexadecen-l-ol; Figure 1) as the main chemical constituent (31.95%) (Pejin et al. 2011). Such an abundance of phytol has not previously been reported in ¨ zdemir et al. 2009). essential oils of other mosses (Zhao et al. 1998; Saritas et al. 2001; O Phytol is an active ingredient in formulations that lower serum levels of triglycerides and/or cholesterol. It can be administered to patients with disease conditions related to increased levels of cholesterol or triglycerides such as type II diabetes, obesity or other patients at risk of cardiovascular diseases due to elevated cholesterol levels. At the same time, phytol can be administered to healthy individuals to maintain normal levels of serum cholesterol (Olofsson et al. 2011; Elmazar et al. 2013). This terpene molecule is able to reduce the production of free
*Corresponding authors. Email: [email protected], [email protected], [email protected], mojovic@
gmail.com q 2014 Taylor & Francis
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Figure 1. Phytol.
radicals in in vitro conditions as well, attributing the activity to its hydroxyl group (de Menezes Patrı´cio Santos et al. 2013). Since reactive oxygen species (ROS) and reactive nitrogen species (RNS) are directly linked to heart disorders (Cai & Harrison 2000), antioxidant agents can be used for the treatment of the relevant oxidative pathologies by neutralising ROS and RNS, chelating catalytic metals and acting as their scavengers (Gulcin et al. 2003). A large body of evidence implicates a number of microbial strains in the pathogenesis of infective endocarditis (IE), an inflammation of the inner tissue of the heart, such as its valves (Kumar et al. 2007; Mitchell et al. 2007). Staphylococcus aureus is among the most common and most virulent aetiologic pathogens in cases of IE (Yeaman & Bayer 2000). The bacteria Bacillus cereus, Micrococcus luteus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Enterobacter cloacae as well as the fungi Aspergillus spp. and Penicillium spp. are also among the microorganisms which can cause IE (DelRossi et al. 1980; Sheinman et al. 1985; Steen et al. 1992; Tunkel et al. 1992; Dawson et al. 2011; Khan et al. 2011; Lauridsen et al. 2011; Miltiadous & Elisaf 2011; Palomares et al. 2011). The aim of this study was to investigate in vitro antiradical (towards six radical species) and antimicrobial (on 16 human pathogenic strains) activities of phytol in a medical foods context, with stress on cardiovascular diseases.
2. Results and discussion 2.1. Antiradical activity Phytol lowered production of hydroxyl radicals (zOH) by 15%, superoxide anion radicals (zO2 2 ) by 23% and nitric oxide radicals (zNO) by 38% (Figure S1, available in online). These results are in good agreement with the previous study of de Menezes Patrı´cio Santos et al. (2013) which indicated anti-hydroxyl and anti-nitric oxide radical activity of the compound tested. Endothelium-dependent vascular relaxation is associated with enhanced degradation of zNO by ROS (Cai & Harrison 2000), the process related to numerous diseases including hypertension, diabetes and heart failure. The role of antioxidants is to enhance endothelium-dependent vasodilatation in coronary and forearm circulation. Superoxide is capable of reacting with zNO, thus the reduction of zO2 2 production can have positive effects through superoxide radical inactivation of endothelium-derived relaxing factor (Laurindo et al. 1991). Phytol showed even better antiradical activity against carbon dioxide anion radicals (zCO2 2 ) and methoxy radicals (zCH2OH) radicals (56% and 50%, respectively) (Figure S1). Although not relevant to physiological conditions, 2,2-diphenyl-1-picrylhydrazyl radical (zDPPH) test is quite frequently used in food research for determination of antioxidative capacities of various samples (Anesini et al. 2008). The examined compound reduced the amount of zDPPH radical by 48%, thus confirming its antiradical potential. To the best of our knowledge, this is the first report of in vitro antiradical activity of phytol determined by electron paramagnetic resonance (EPR) spectroscopy.
2.2. Antimicrobial activity Phytol exhibited a strong antibacterial activity with MIC 0.003 –0.038 mg/mL and MBC 0.013 – 0.052 mg/mL (Table S1, available in online). The most sensitive bacteria was L. monocytogenes,
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while the most resistant was M. flavus. It proved to be more effective in comparison with both positive controls, streptomycin and ampicillin. This compound showed significant antifungal activity as well (MIC 0.008 – 0.016 mg/mL and MFC 0.090 – 0.520 mg/mL) (Table S2). The most sensitive fungus was T. viride, while the most resistant was A. fumigatus. Phytol was more effective than both bifonazole and ketoconazole, used as a positive control. Available reports of antimicrobial activity of phytol are very scarce. Rajab et al. (1998) reported an MIC value of 2 mg/mL against Mycobacterium tuberculosis for (E)-phytol, (Z)phytol, commercially available 2:1 mixture of (E) and (Z)-phytol and (3R,S,7R,11R)phytanol, respectively. The derivatives (E)-phytol acetate, a mixture of the (2S,3S)- and (2R,3R)-isomers of (E)-phytol epoxide and (3R,S,7R,11R)-phytanic acid displayed lower activities with MICs of 8 mg/mL, 16 mg/mL and . 128 mg/mL, respectively. The activities of (E)-phytol, (Z)-phytol and (3R,S,7R,11R)-phytanol were found to be in the same range as ethambutol, a clinically useful drug with an MIC in the range 0.95 –3.80 mg/mL. In addition, the novel ester (E)-phytol (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, isolated from the diatom Navicula delognei f. elliptica Lobban, showed antibacterial activity against S. aureus (zone of inhibition . 4 mm), S. epidermidis (zone of inhibition . 2 mm), S. typhimurium (zone of inhibition . 2 mm) and Proteus vulgaris (zone of inhibition noticeable) (Findlay & Patil 1984). According to the report of Togashi et al. (2010) on antibacterial activity of terpene alcohols with aliphatic carbon chains of various lengths, the carbon chain length of C10 – C12 is the most appropriate for the activity. This is the first report of antimicrobial activity of phytol against majority of microorganisms tested, with the exception of S. aureus (Inoue et al. 2005).
3. Experimental 3.1. General Phytol was used as received from Sigma-Aldrich, Munich, Germany (97%, mixture of isomers), while spin-trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, purchased from Enzo Life Sciences (Lausen, Switzerland), was purified according to the procedure proposed by Jackson et al. (2002). EPR spectra were recorded using a Varian E104-A X-band EPR spectrometer (Varian, Palo Alto, CA, USA) at room temperature.
3.2. Antiradical activity Hydroxyl radicals, methoxy radicals, carbon dioxide anion radicals and superoxide anion radicals were generated as previously published (Savic´ & Mojovic´ 2012). Nitric oxide radicals were generated according to Katayama et al. (2001). Anti zDPPH radical activity was determined as previously published (Godevac et al. 2008). In all tested systems the final concentration of phytol was 0.1 mg/mL, while the incubation time was 10 min.
3.3. Antimicrobial activity The bacteria tested were obtained from the Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade. The antibacterial assay was carried out by a microdilution method (Hanel & Raether 1988; Espinel-Ingroff 2001). The fungi tested were obtained from the same laboratory. The antifungal assay was carried out by a modified microdilution method (Booth 1971; Hanel & Raether 1988; Espinel-Ingroff 2001).
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4. Conclusion Additional investigations of bioactivity of phytol are necessary, first of all due to its presence in numerous food products. Indeed, the medical foods containing this compound and/or its derivatives may inspire new therapies in heart medicine.
Supplementary material Supplementary material relating to this article is available online, alongside Figure S1 and Tables S1 and S2.
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Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Research grants Nos 41005, 173017, 173032 and 173040). The authors gratefully thank Milena Dimitrijevic (MSc) and Irena Brajkovic (MSc candidate) for their technical assistance.
References Anesini C, Ferraro GE, Filip R. 2008. Total polyphenol content and antioxidant capacity of commercially available tea (Camellia sinensis) in Argentina. J Agric Food Chem. 56:9225–9229. Booth C. 1971. Fungal culture media. In: Norris JR, Ribbons DW, editors. Methods in microbiology. London: Academic Press. p. 49– 94. Cai H, Harrison DG. 2000. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circul Res. 87:840–844. Dawson NL, Brumble LM, Pritt BS, Yao JD, Echols JD, Alvarez S. 2011. Left-sided Pseudomonas aeruginosa endocarditis in patients without injection drug use. Medicine (Baltimore). 90:250–255. DelRossi AJ, Morse D, Spagna PM, Lemole GM. 1980. Successful management of Penicillium endocarditis. J Thorac Cardiovasc Surg. 80:945–947. de Menezes Patrı´cio Santos CC, Salvadori MS, Mota VM, Costa LM, de Almeida AAC, de Oliviera GAL, de Almeida RN. 2013. Antinociceptive and antioxidant activities of phytol in vivo and in vitro models. Neurosci J., doi:10. 1155/2013/949452. Ding H. 1982. Medicinal spore-bearing plants of China. Shangai: Shangai Press. Elmazar MM, El-Abhar HS, Schaalan MF, Farag NA. 2013. Phytol/phytanic acid and insulin resistance: potential role of phytanic acid proven by docking simulation and modulation of biochemical alterations. PLoS ONE. 8:e45638. Espinel-Ingroff A. 2001. Comparison of the E-test with the NCCLS M38-P method antifungal susceptibility testing of common and emerging pathogenic filamentous fungi. J Clin Microbiol. 39:1360–1367. Findlay JA, Patil AD. 1984. Antibacterial constituents of the diatom Navicula delognei. J Nat Prod. 47:815–818. Godevac D, Vujisic D, Ignjatovic A, Spasojevic I, Vajs V. 2008. Evaluation of antioxidant capacity of Allium ursinum L. volatile oil and its effect on membrane fluidity. Food Chem. 107:1692–1700. Gulcin I, Oktay M, Kirecci E, Kufrevioglu OI. 2003. Screening of antioxidant and antimicrobial activities of anise (Pimpinella anisum L.) seed extracts. Food Chem. 83:371–382. Hanel H, Raether W. 1988. A more sophisticated method of determining the fungicidal effect of water-insoluble preparations with a cell harvester, using miconazole as an example. Mycoses. 31:148–154. Inoue Y, Hada T, Shiraishi A, Hirose K, Hamashima H, Kobayashi S. 2005. Biphasic effects of geranylgeraniol, terpenone, and phytol on the growth of Staphylococcus aureus. Antimicrob Agents Chemother. 49:1770– 1774. Jackson SK, Liu KJ, Liu M, Timminis GS. 2002. Detection and removal of contaminating hydroxylamines from the spin trap DEPMPO, and re-evaluation of its use to indicate nitrone radical cation formation and S(N)1 reactions. Free Rad Biol Med. 32:228–232. Katayama Y, Soh N, Maeda M. 2001. A new strategy for the design of molecular probes for investigating endogenous nitric oxide using an EPR or fluorescent technique. Eur J Chem Phys Phys Chem. 2:655–661. Khan JA, Ali B, Masood T, Ahmed F, Sial JA, Balooch ZH. 2011. Salmonella typhi infection: a rare cause of endocarditis. J Coll Physicians Surg Pak. 21:559–560. Kumar D, Cawley JJ, Irizarry-Alvarado JM, Alvarez A, Alvarez S. 2007. Case of Staphylococcus schleiferi subspecies coagulans endocarditis and metastatic infection in an immune compromised host. Transpl Infect Dis. 9:336–338. Lauridsen TK, Arpi M, Fritz-Hansen T, Frimodt-Møller N, Bruun NE. 2011. Infectious endocarditis caused by Escherichia coli. Scand J Infect Dis. 43:545–546.
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Laurindo FR, da Luz PL, Uint L, Rocha TF, Jaeger RG, Lopes EA. 1991. Evidence for superoxide radical-dependent coronary vasospasm after angioplasty in intact dogs. Circulation. 83:1705– 1715. Miltiadous G, Elisaf M. 2011. Native valve endocarditis due to Microccocus luteus: a case report and review of the literature. J Med Case Rep. 5:251. Mitchell RS, Kumar V, Robbins SL, Abbas AK, Fausto N. 2007. Robbins basic pathology. Philadelphia, PA: Saunders/ Elsevier. Olofsson P, Hultqvist M, Holmdahl R. 2011. Phytol as a cholesterol lowering agent. United States Patent Application Publication, appl. no. 12/921,169. ¨ zdemir T, Yayli N, Cansu TB, Volga C, Yayli N. 2009. Essential oil in mosses (Brachythecium salebrosum, O Eurhynchium pulchellum and Plagiomnium undulatum) grown in Turkey. Asian J Chem. 21:5505 –5509. Palomares JC, Bernal S, Marin M, Holgado VP, Castro C, Novales WP, Martin E. 2011. Molecular diagnosis of Aspergillus fumigatus endocarditis. Diagn Microbiol Infect Dis. 70:534–537. Pejin B, Vujisic LJ, Sabovljevic M, Tesevic V, Vajs V. 2011. Preliminary data on essential oil composition of the moss Rhodobryum ontariense (Kindb.) Kindb. Crypt Bryol. 32:113–117. Rajab MS, Cantrell CL, Franzblau SG, Fischer NH. 1998. Antimycobacterial activity of (E)-phytol and derivatives: a preliminary structure–activity study. Planta Med. 64:2–4. Saritas Y, Sonwa MM, Iznaguen H, Koning WA, Muhle H, Mues R. 2001. Volatile constituents in mosses (Musci). Phytochemistry. 57:443–457. Savic´ GA, Mojovic´ M. 2012. Free radicals identification from the complex EPR signals by applying higher order statistics. Anal Chem. 84:3398–3402. Sheinman BD, Evans T, Sage R. 1985. Listeria monocytogenes endocarditis. Postgrad Med J. 61:51–52. Steen MK, Bruno-Murtha LA, Chaux G, Lazar H, Bernard S, Sulis C. 1992. Bacillus cereus endocarditis: report of a case and review. Clin Infect Dis. 14:945–946. Togashi N, Hamashima H, Shiraishi A, Inoue Y, Takano A. 2010. Antibacterial activities against Staphylococcus aureus of terpene alcohols with aliphatic carbon chains. J Essent Oil Res. 22:263–269. Tunkel AR, Fisch MJ, Schlein A, Scheld WM. 1992. Enterobacter endocarditis. Scand J Infect Dis. 24:233–240. World Health Organization. 2013. Available from: http://www.who.int/cardiovascular_diseases/en/ Yeaman MR, Bayer AS. 2000. Staphylococcus aureus, platelets, and the heart. Curr Infect Dis. 2:281–298. Zhao J, Song Q, Wang J. 1998. Preliminary chemical analysis on the volatile oils of some mosses. Ziran Kexue Ban. 22:254–256.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Further in vitro evaluation of antiradical and antimicrobial activities of phytol a
a
b
b
b
B. Pejin , A. Savic , M. Sokovic , J. Glamoclija , A. Ciric , M. b
a
Nikolic , K. Radotic & M. Mojovic
c
a
Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, Belgrade 11030, Serbia b
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Department of Plant Physiology – Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade 11060, Serbia c
Faculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia Published online: 14 Jan 2014.
To cite this article: B. Pejin, A. Savic, M. Sokovic, J. Glamoclija, A. Ciric, M. Nikolic, K. Radotic & M. Mojovic (2014) Further in vitro evaluation of antiradical and antimicrobial activities of phytol, Natural Product Research: Formerly Natural Product Letters, 28:6, 372-376, DOI: 10.1080/14786419.2013.869692 To link to this article: http://dx.doi.org/10.1080/14786419.2013.869692
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
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This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2014 Vol. 28, No. 6, 372–376, http://dx.doi.org/10.1080/14786419.2013.869692
Further in vitro evaluation of antiradical and antimicrobial activities of phytol B. Pejina*, A. Savica, M. Sokovicb, J. Glamoclijab, A. Ciricb, M. Nikolicb, K. Radotica and M. Mojovicc*
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a
Department of Life Sciences, Institute for Multidisciplinary Research, University of Belgrade, Kneza Viseslava 1, Belgrade 11030, Serbia; bDepartment of Plant Physiology – Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade, Bulevar Despota Stefana 142, Belgrade 11060, Serbia; cFaculty of Physical Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade 11158, Serbia (Received 28 August 2013; final version received 19 November 2013) The antiradical activity of phytol was evaluated by electron paramagnetic resonance towards hydroxyl radical (zOH), superoxide anion radical (zO2 2 ), methoxy radical (zCH2OH), carbon-dioxide anion radical (zCO2 2 ), as well as towards nitric-oxide radical (zNO) and 2,2-diphenyl-1-picrylhydrazyl (zDPPH) radical. It reduced the production of all tested radicals showing more promising activity against zCO2 2 , zCH2OH and zDPPH radicals (56%, 50% and 48%, respectively) in comparison with zNO, zO2 2 and zOH radicals (38%, 23% and 15%, respectively). The antimicrobial activity of phytol was evaluated by the microdilution method against eight bacterial and eight fungal strains. To varying degrees, it was proven to be active against all tested bacteria and fungi (MIC 0.003– 0.038 mg/mL and MBC 0.013 – 0.052 mg/mL, MIC 0.008 – 0.016 mg/mL and MFC 0.090 – 0.520 mg/mL, respectively). According to the obtained results, medical foods containing phytol may support development of new therapies for heart disease. Keywords: phytol; reactive oxygen and nitrogen species; DPPH; EPR; microorganisms; medical foods
1. Introduction Cardiovascular diseases are the major cause of death worldwide according to the World Health Organization, with the projection to increase from 17.3 million people in 2008 to more than 23.3 million people in 2030 (WHO 2013). Traditional Chinese medicine suggests that mosses of Rhodobryum species (Bryaceae) can cure heart disorders as crude drugs (Ding 1982). Preliminary analysis of the moss Rhodobryum ontariense volatiles has indicated diterpene phytol (3,7,11,15-tetramethyl-2-hexadecen-l-ol; Figure 1) as the main chemical constituent (31.95%) (Pejin et al. 2011). Such an abundance of phytol has not previously been reported in ¨ zdemir et al. 2009). essential oils of other mosses (Zhao et al. 1998; Saritas et al. 2001; O Phytol is an active ingredient in formulations that lower serum levels of triglycerides and/or cholesterol. It can be administered to patients with disease conditions related to increased levels of cholesterol or triglycerides such as type II diabetes, obesity or other patients at risk of cardiovascular diseases due to elevated cholesterol levels. At the same time, phytol can be administered to healthy individuals to maintain normal levels of serum cholesterol (Olofsson et al. 2011; Elmazar et al. 2013). This terpene molecule is able to reduce the production of free
*Corresponding authors. Email: [email protected], [email protected], [email protected], mojovic@
gmail.com q 2014 Taylor & Francis
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Figure 1. Phytol.
radicals in in vitro conditions as well, attributing the activity to its hydroxyl group (de Menezes Patrı´cio Santos et al. 2013). Since reactive oxygen species (ROS) and reactive nitrogen species (RNS) are directly linked to heart disorders (Cai & Harrison 2000), antioxidant agents can be used for the treatment of the relevant oxidative pathologies by neutralising ROS and RNS, chelating catalytic metals and acting as their scavengers (Gulcin et al. 2003). A large body of evidence implicates a number of microbial strains in the pathogenesis of infective endocarditis (IE), an inflammation of the inner tissue of the heart, such as its valves (Kumar et al. 2007; Mitchell et al. 2007). Staphylococcus aureus is among the most common and most virulent aetiologic pathogens in cases of IE (Yeaman & Bayer 2000). The bacteria Bacillus cereus, Micrococcus luteus, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli and Enterobacter cloacae as well as the fungi Aspergillus spp. and Penicillium spp. are also among the microorganisms which can cause IE (DelRossi et al. 1980; Sheinman et al. 1985; Steen et al. 1992; Tunkel et al. 1992; Dawson et al. 2011; Khan et al. 2011; Lauridsen et al. 2011; Miltiadous & Elisaf 2011; Palomares et al. 2011). The aim of this study was to investigate in vitro antiradical (towards six radical species) and antimicrobial (on 16 human pathogenic strains) activities of phytol in a medical foods context, with stress on cardiovascular diseases.
2. Results and discussion 2.1. Antiradical activity Phytol lowered production of hydroxyl radicals (zOH) by 15%, superoxide anion radicals (zO2 2 ) by 23% and nitric oxide radicals (zNO) by 38% (Figure S1, available in online). These results are in good agreement with the previous study of de Menezes Patrı´cio Santos et al. (2013) which indicated anti-hydroxyl and anti-nitric oxide radical activity of the compound tested. Endothelium-dependent vascular relaxation is associated with enhanced degradation of zNO by ROS (Cai & Harrison 2000), the process related to numerous diseases including hypertension, diabetes and heart failure. The role of antioxidants is to enhance endothelium-dependent vasodilatation in coronary and forearm circulation. Superoxide is capable of reacting with zNO, thus the reduction of zO2 2 production can have positive effects through superoxide radical inactivation of endothelium-derived relaxing factor (Laurindo et al. 1991). Phytol showed even better antiradical activity against carbon dioxide anion radicals (zCO2 2 ) and methoxy radicals (zCH2OH) radicals (56% and 50%, respectively) (Figure S1). Although not relevant to physiological conditions, 2,2-diphenyl-1-picrylhydrazyl radical (zDPPH) test is quite frequently used in food research for determination of antioxidative capacities of various samples (Anesini et al. 2008). The examined compound reduced the amount of zDPPH radical by 48%, thus confirming its antiradical potential. To the best of our knowledge, this is the first report of in vitro antiradical activity of phytol determined by electron paramagnetic resonance (EPR) spectroscopy.
2.2. Antimicrobial activity Phytol exhibited a strong antibacterial activity with MIC 0.003 –0.038 mg/mL and MBC 0.013 – 0.052 mg/mL (Table S1, available in online). The most sensitive bacteria was L. monocytogenes,
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while the most resistant was M. flavus. It proved to be more effective in comparison with both positive controls, streptomycin and ampicillin. This compound showed significant antifungal activity as well (MIC 0.008 – 0.016 mg/mL and MFC 0.090 – 0.520 mg/mL) (Table S2). The most sensitive fungus was T. viride, while the most resistant was A. fumigatus. Phytol was more effective than both bifonazole and ketoconazole, used as a positive control. Available reports of antimicrobial activity of phytol are very scarce. Rajab et al. (1998) reported an MIC value of 2 mg/mL against Mycobacterium tuberculosis for (E)-phytol, (Z)phytol, commercially available 2:1 mixture of (E) and (Z)-phytol and (3R,S,7R,11R)phytanol, respectively. The derivatives (E)-phytol acetate, a mixture of the (2S,3S)- and (2R,3R)-isomers of (E)-phytol epoxide and (3R,S,7R,11R)-phytanic acid displayed lower activities with MICs of 8 mg/mL, 16 mg/mL and . 128 mg/mL, respectively. The activities of (E)-phytol, (Z)-phytol and (3R,S,7R,11R)-phytanol were found to be in the same range as ethambutol, a clinically useful drug with an MIC in the range 0.95 –3.80 mg/mL. In addition, the novel ester (E)-phytol (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, isolated from the diatom Navicula delognei f. elliptica Lobban, showed antibacterial activity against S. aureus (zone of inhibition . 4 mm), S. epidermidis (zone of inhibition . 2 mm), S. typhimurium (zone of inhibition . 2 mm) and Proteus vulgaris (zone of inhibition noticeable) (Findlay & Patil 1984). According to the report of Togashi et al. (2010) on antibacterial activity of terpene alcohols with aliphatic carbon chains of various lengths, the carbon chain length of C10 – C12 is the most appropriate for the activity. This is the first report of antimicrobial activity of phytol against majority of microorganisms tested, with the exception of S. aureus (Inoue et al. 2005).
3. Experimental 3.1. General Phytol was used as received from Sigma-Aldrich, Munich, Germany (97%, mixture of isomers), while spin-trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, purchased from Enzo Life Sciences (Lausen, Switzerland), was purified according to the procedure proposed by Jackson et al. (2002). EPR spectra were recorded using a Varian E104-A X-band EPR spectrometer (Varian, Palo Alto, CA, USA) at room temperature.
3.2. Antiradical activity Hydroxyl radicals, methoxy radicals, carbon dioxide anion radicals and superoxide anion radicals were generated as previously published (Savic´ & Mojovic´ 2012). Nitric oxide radicals were generated according to Katayama et al. (2001). Anti zDPPH radical activity was determined as previously published (Godevac et al. 2008). In all tested systems the final concentration of phytol was 0.1 mg/mL, while the incubation time was 10 min.
3.3. Antimicrobial activity The bacteria tested were obtained from the Mycological Laboratory, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade. The antibacterial assay was carried out by a microdilution method (Hanel & Raether 1988; Espinel-Ingroff 2001). The fungi tested were obtained from the same laboratory. The antifungal assay was carried out by a modified microdilution method (Booth 1971; Hanel & Raether 1988; Espinel-Ingroff 2001).
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4. Conclusion Additional investigations of bioactivity of phytol are necessary, first of all due to its presence in numerous food products. Indeed, the medical foods containing this compound and/or its derivatives may inspire new therapies in heart medicine.
Supplementary material Supplementary material relating to this article is available online, alongside Figure S1 and Tables S1 and S2.
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Acknowledgements This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Research grants Nos 41005, 173017, 173032 and 173040). The authors gratefully thank Milena Dimitrijevic (MSc) and Irena Brajkovic (MSc candidate) for their technical assistance.
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