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Antibacterial mechanisms of cinnamon and its constituents: A review
Accepted Manuscript Antibacterial mechanisms of cinnamon and its constituents: A review N.G. Vasconcelos, J. Croda, S. Simionatto PII:
S0882-4010(18)30566-7
DOI:
10.1016/j.micpath.2018.04.036
Reference:
YMPAT 2915
To appear in:
Microbial Pathogenesis
Received Date: 29 March 2018 Revised Date:
17 April 2018
Accepted Date: 19 April 2018
Please cite this article as: Vasconcelos NG, Croda J, Simionatto S, Antibacterial mechanisms of cinnamon and its constituents: A review, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2018.04.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibacterial Mechanisms of Cinnamon and its Constituents: a Review
Vasconcelos, N. G.a,b, Croda, J. c,d, and Simionatto, S.*a a
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Research Laboratory of Health Sciences, Federal University of Grande Dourados - UFGD, Dourados, Mato Grasso do Sul, Brazil; b Universitary Hospital of Dourados, Federal University of Grande Dourados - UFGD, Dourados, Mato Grosso do Sul, Brazil; c Oswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, Brazil; d Federal University of Mato Grosso do Sul – UFMS, Campo Grande, Mato Grosso do Sul, Brazil.
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*Address correspondence to this author at the Research Laboratory of Health Sciences, Federal University of Grande Dourados - UFGD, Rodovia Dourados - Itahum km 12, Cidade Universitária, CEP: 79804970, Dourados, Mato Grosso do Sul, Brazil. E-mail: [email protected].
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Abstract: BACKGROUND: In the current healthcare environment, an alarming rise in multi-drug resistant bacterial infections has led to a global health threat. The lack of new antibiotics has created a need for developing alternative strategies. OBJECTIVE: Understanding the antibacterial mechanisms of Cinnamon and its constituents is crucial to enhance it as a potential new source of antibiotic. The objective of this review is to provide a compilation of all described mechanisms of antibacterial action of Cinnamon and its constituents and synergism with commercial antibiotics in order to better understand how Cinnamon and its constituents can collaborate as alternative treatment to multi-drug resistant bacterial infections. METHODS: The relevant references on antibacterial activities of cinnamon and its constituents were searched. Meanwhile, the references were classified according to the type of mechanism of action against bacteria. Relationships of cinnamon or its constituents and antibiotics were also analyzed and summarized. RESULTS: Cinnamon extracts, essential oils, and their compounds have been reported to inhibit bacteria by damaging cell membrane; altering the lipid profile; inhibiting ATPases, cell division, membrane porins, motility, and biofilm formation; and via anti-quorum sensing effects. CONCLUSION: This review describes the antibacterial effects of cinnamon and its constituents, such as cinnamaldehyde and cinnamic acid, against pathogenic Gram-positive and Gram-negative bacteria. The review also provides an overview of the current knowledge of the primary modes of action of these compounds as wells the synergistic interactions between cinnamon or its constituents with known antibacterial agents. This information will be useful in improving the effectiveness of therapeutics based on these compounds.
Running Title: Antibacterial Mechanism of Cinnamon Keywords: antimicrobial activity, cinnamon, mechanisms of action, multi-drug resistance, synergism, trans-cinnamaldehyde.
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have demonstrated the instability of TC when exposed to air, as the reactive unsaturated aldehyde is readily oxidized to cinnamic acid, which causes volatile loss and the instability of TC [23]. In addition, in vivo, decomposition might also occur before it is able to perform bactericidal activity, as absorbed TC can rapidly and irreversibly become cinnamic acid through enzyme catalysis; thus, it is unstable in the blood [24]. With regard to the minor components, an antibacterial effect has been reported for methoxycinnamaldehyde, whereas coumarin, cinnamyl acetate, and benzaldehyde were shown to have low or no antibacterial effect [25,26]. It is known that the mixture of different constituents can result in synergistic or additive effects [27]; despite being present at low levels or having no antibacterial effect when used alone, it is possible that these minor components could increase the effect of TC or have other targets in bacterial cells. Owing to this richness in composition, plantderived products and their components have a variety of targets; in particular, they attack the membrane, cytoplasm, and in certain situations, may completely alter the morphology of the cells. This review describes the antibacterial mechanisms of action of cinnamon against pathogenic bacteria.
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1. INTRODUCTION Bacterial infections have become a major healthcare challenge owing to the rise and dissemination of multi-drug resistant bacteria, which has led to increased interest in the development of new antimicrobial agents. Plant extracts, oils, and their derivative compounds are known to be active against a wide variety of microorganisms and have been used for hundreds of years to combat pathogens such as bacteria, fungi, and viruses [1]. Many studies have described the antimicrobial effects of cinnamon on both Gram-positive and Gram-negative bacteria [2,3,4]. Cinnamon is a native plant of Sri Lanka and a tropical Asian spice [5] obtained from the inner bark of several trees from the genus Cinnamomum [6]. There are various cinnamon species, which are important popular spices used worldwide, not only for cooking, but also in traditional and modern medicines [7]. The barks and leaves are commonly used to treat various disorders and are known to exert antibacterial [8], antifungal [9], antioxidant [10], antidiabetic [11], anti-inflammatory [12,13], nematicidal [14], insecticidal [15] and anticancer [16] effects. Overall, approximately 250 species have been identified in the Cinnamomum genus, with trees found all over the world [17]. The most important volatile oils of cinnamon are from C. burmannii, C. camphora, C. cassia, C. osmophloeum, C. verum, and C. zeylanicum; pharmaceutical manufacturers use oils both of Ceylon cinnamon (cinnamon oil) and Chinese cinnamon (cassia oil) without distinction between them [5]. Cinnamon, like other plants, has a wide variety of secondary metabolites that exhibit antibacterial properties [18,19]. Secondary metabolites, in contrast to primary metabolites, are not essential for the survival of the plant; instead, they are defensive compounds against competitors and pathogens [20]. These compounds include cinnamaldehyde, cinnamate, cinnamic acid [17,21], and a wide range of essential oils, such as transcinnamaldehyde, cinnamyl acetate, eugenol, Lborneol, camphor, caryophyllene oxide, bcaryophyllene, L-bornyl acetate, E-nerolidol, αcubebene, α-terpineol, terpinolene, and α-thujene [12,13]. The amount and presence of each compound vary depending on the part of the plant [7]. Trans-cinnamaldehyde (TC), an unsaturated aldehyde, was shown to be responsible for antibacterial activity, with the acrolein group (α,βunsaturated carbonyl moiety) in these molecules being essential for the activity [22]. Some studies
2. MATERIAL AND METHODS The present review manuscript considered the literature published prior to April 2018 on antibacterial activity of extracts, essential oils and natural products isolated from different parts of Cinnamomum species and synergism actions when Cinnamon or its constituents were associated to commercial antibiotics. Therefore, only literature available in databases such as Google Scholar and PubMed was reviewed. This review only considered peer-reviewed research papers with impact factor. The keywords used to search were: antibacterial, antimicrobial, mechanisms, Cinnamon, Cinnamomum, trans-cinnamaldeyde, synergism, and multi-drug-resistance. 3. MECHANISMS OF ANTIBACTERIAL ACTION 3.1. Alterations in cell membrane and its lipid profile Usually, Gram-negative bacteria are more resistant to plant extracts, oils, and their constituents than Gram-positive bacteria, because the cell wall of Gram-negative bacteria is more
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permeability, potentially reaching a critical level to induce cell death [2]. Analysis of DNA, RNA, and protein leakage provided additional evidence of antibacterial mechanism of TC and C. zeylanicum bark essential oil against Porphyromonas gingivalis, a Gram-negative anaerobic bacterium that is one of the key causative pathogens in chronic periodontitis, it was observed irreversible damage to the bacterial membrane by propidium iodide uptake assay and confocal microscopy images [37]. All these findings indicate that the membrane is the first target of TC. However, the mechanism of action of TC is not isolated, but instead involves a series of events on the cell surface and within the cytoplasm. 3.2. Inhibition of ATPase In cellular respiration, the electron transport chain generates a transmembrane proton gradient necessary for adenosine triphosphate (ATP) synthesis, performed by multiple enzymes with ATPase activity, including ATP-dependent transport proteins and the F1F0-ATPase complex [38,39]. The F1F0-ATPase complex is a reversible proton-translocating pump that may extrude protons from the cytoplasm by use of energy from ATP hydrolysis and such proton efflux enhances the proton gradient and assists in the regulation of cytoplasmic pH [40]. The F-class pumps are found in bacterial plasma membranes, mitochondria, and chloroplasts [41]. The inhibition of membranebound or membrane-embedded enzymes by TC may result in changes in the protein conformation, which causes the inhibition of ATPase activity, the inhibition of other enzymes, and altered bacterial growth [42]. TC downregulated F1F0-ATPase, thereby inhibiting ATP synthesis in Cronobacter sakazakii [43]. The treatment of E. coli and Listeria monocytogenes with TC caused a disruption of the proton motive force with rapid depletion of ATP and prevented an increase in cellular ATP concentration, which suggested that ATPase inhibition may play a significant role in reducing the growth rate at the sub-lethal concentrations [44,45]. At high concentrations, TC also acts as an ATPase inhibitor [18]. The effects of both cinnamon water extract and TC on mitochondrial F1F0-ATPase in rat liver are similar to those observed in bacterial cells: they dissipate the proton gradient, uncouple the mitochondria, stimulate ATP hydrolysis, lower the level of ATP, and consequently damage mitochondrial functions, which may be attributable to either the release of protons into the matrix or the interaction of cinnamon and TC with the sulfhydryl groups of
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complex [28]. Porin proteins serve as hydrophilic transmembrane channels for small hydrophilic solutes, which easily pass through the outer membrane of Gram-negative bacteria; however, it is hard for hydrophobic antibiotics to penetrate the cell and this is one reason that makes Gramnegative bacteria more resistant [29]. The Grampositive bacteria cell wall allows hydrophobic molecules to easily penetrate and act on both the cell wall and in the cytoplasm [18]. TC, a commonly studied phenylpropene, is synthesized from the amino acid precursor phenylalanine in plants. TC contains hydrophobic interactions and a six-carbon aromatic phenol group, which constitutes a small part of essential oils [30]. Molecules rich in phenolics are able to pass through the phospholipid bilayer of bacterial cell walls and bind to proteins to prevent them from performing their normal functions [31]. The alteration of membrane permeability and the loss of functional proteins transporting molecules and ions perturbs the microbial cell. This subsequently leads to cytoplasmic coagulation, denaturation of several enzymes and proteins, and the loss of metabolites and ions [32]. Studies have reported contrasting information on the membrane-perturbing activity of TC. A sub-lethal concentration of TC did not affect the integrity of the membrane in Escherichia coli, but inhibited the growth and bioluminescence of Photobacterium leiognathi, which suggests that TC gains access to the periplasm and perhaps to the cytoplasm [33]. At a lethal concentration, TC was shown to perturb the cell membrane and is capable of altering the lipid profile of the membrane [34]. E. coli cells possessed a reduced negative charge after exposure to cinnamon bark essential oil and bacterial cells experienced irreversible membrane damage caused by the acidification and protein denaturation of the cell membrane owing to the accumulation of the components of the oil, which allowed the access of antibiotics to PBPs (penicillin-binding proteins) and the induction of cell death [4]. C. cassia essential oil, as well as TC, induced an immediate increase in the rigidity of the cytoplasmic membrane at the surface and at its core in Listeria innocua strains; the intensity depended on the quantity of TC accumulated in the membrane [2]. An increase in bacterial membrane rigidity after the addition of TC to growth media was attributed to a large increase in the proportion of saturated fatty acids in the membrane phospholipids. This modification of fatty acids was probably a compensatory effect to maintain the membrane structure because of the fluidifying effect of TC [35,36]. The accumulation of numerous molecules inside the cell can perturb its selective
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In general, bypassing the cell membrane is a prerequisite for any solute to exert bactericidal activity, including cinnamon extracts or essential oil. The cell structure of Gram-positive bacteria allows hydrophobic molecules to penetrate relatively easily to the cytoplasm. The cell wall of Gram-negative bacteria is more complex, containing an outer membrane and lipopolysaccharides (LPS), conferring greater resistance to hydrophobic natural extracts or essential oils. Hydrophilic molecules can pass through easily because of the abundant protein porins on Gram-negative bacterial cell membranes, to which they have affinity [18]. TC has the ability to decrease the gene expression of the membrane porins OmpA, OmpC, and OmpR, and amino acid transporters that disrupt active transport and diffusion through the bacterial cell membrane [43,53]. Several outer membrane porins were found to be upregulated after exposure to osmotic stress and desiccation in C. sakazakii [54]. However, porins such as the Omps described above, which are involved in the transport of osmoprotectants across the cytoplasmic membrane of bacteria under hyperosmotic stress, were found to be downregulated by TC [55]. TC can reduce resistance to osmotic stress and resistance to desiccation in C. sakazakii and it may be attributable to the decreased ompC and ompR gene expression, which are required for substrate transport across the membrane [53]. All these data reaffirmed the proposed mechanism that TC could disrupt the cytoplasmic membrane and inhibit active transport across it [56]. There are few published studies that correlate the antibacterial action of TC with membrane porins. The measurement of the antibacterial effect of TC on porin-deficient bacterial mutants would be helpful for the exploration of the targets of TC in this action. 3.5. Inhibition of motility and biofilm formation Motility plays a critical role in host-microbial interactions. Bacterial colonization and virulence comprises multiple events, including signal transduction, chemotaxis, and flagellar movement [57]. In addition, the development of complex structures in biofilms may be regulated by cellular motility [58]. Biofilm formation is a form of coordinated collective behavior; its development proceeds through stages of adhesion, proliferation, microcolony formation, and maturation, in order to favor bacterial survival [59]. Biofilm growth provides many advantages, such as protection from desiccation, antimicrobials, metabolic cooperation, and increased genetic diversity via horizontal gene transfer [60].
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membrane proteins [46]. In addition, the dissipation of membrane potential across the mitochondrial membrane, such as that caused by TC, would increase ATP hydrolysis by uncoupling oxidation from phosphorylation [41]. These uncouplers are reversed by thiols [46]. TC was found to target thiols of cysteine residues on protein molecules [47], leading to possible destabilization of ATP activity. TC also inhibited the expression of the ATP synthase alpha chain protein in Salmonella Typhimurium, a component of the ATP synthase protein complex involved in production of ATP [48]. To more accurately determine the interactions of TC with membrane enzymes and with ATPase activity, or identify specific or non-specific interactions, studies on the kinetics of purified enzymes are necessary. 3.3. Inhibition of cell division Bacterial cell division is regulated by FtsZ, a prokaryotic homolog of tubulin. Guanosine triphosphate (GTP)-dependent polymerization makes FtsZ into filaments, a highly dynamic polymeric structure known as the Z-ring, which is assembled at the midcell and constricts the cell envelope, finally separating the mother cell into two daughter cells [49]. TC is able to inhibit GTPdependent FtsZ polymerization, decrease the in vitro assembly reaction and bundling of FtsZ, perturb the Z-ring morphology in vivo, and reduce the frequency of the Z-ring per unit of cell length of E. coli in a dose-dependent manner [50]. The confocal microscopy of live E. coli cells showed that TC specifically targeted Z-ring spatial arrangement, dissipating the Z-rings and reducing the frequency of Z-ring formation by almost half [50]. It was also shown that TC was capable of inhibiting cell separation by binding a region of FtsZ in Bacillus cereus [18]. In addition, it was hypothesized by using an in silico docking model that TC binds to FtsZ at the C-terminal region involving the T7 loop, which perturbs cytokinetic Z-ring formation and inhibits assembly dynamics [50]. Another study with B. cereus showed that without TC treatment, the bacteria appeared as well-separated rods, and when treated, the cells appeared as elongated, filamentous structures in which the daughter cells did not appear to be separated from the previous generation because the septa formation was incomplete [33]. FtsZ is a promising target for new antibacterial drugs because of its prokaryotic specificity, its evolutionary distance from eukaryotic tubulin, and great importance in bacterial cell division [51,52]. 3.4. Inhibition of membrane porins
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exposure may be associated to the disruption of biofilm structure [48]. In health and industrial areas, these microbial biofilms constitute a substantial challenge. Bacterial biofilms are more resistant to antibiotics, host defenses, and contribute to bacterial persistence in chronic infections through medical devices [72]. Therefore, the direct or indirect antibiofilm and anti-quorum sensing action of TC on pathogenic bacteria would be of great importance and the here reported proteins provide potential and interesting targets for exploring new control strategies for biofilms. 3.6. Anti-quorum sensing effect Quorum sensing (QS) is an intercellular communication system used by both Grampositive and Gram-negative bacteria based on the secretion and detection of external signal molecules. QS also influences motility and biofilm formation [73]. Plants that live in constantly wet environments, which are ideal for biofilm formation, face the similar problems in their stem and barks. It is expected that these plants will have compound that interrupt QS and inhibit biofilm formation [74]. The expression of bcsA and luxR, both involved in QS, is downregulated by TC [43]. TC also interfered with an autoinducer of the QS system and biofilm formation without inhibiting bacterial growth in Vibrio spp. [70]. TC inhibited QS in E. coli [67], P. aeruginosa [70], and S. pyogenes [65]. Another study used an E. coli biosensor that showed a reduction in bioluminescence in the presence of QS inhibitors (in this case, C. verum bark essential oil) to confirm the possible anti-QS effect of the essential oil. They proposed that the main mode of action of C. verum essential oil occurred through the disruption of the bacterial membrane at both lethal and sublethal concentrations, which subsequently increased the nonspecific mobility of the antibiotic into the bacterial cell; this indicated not only membrane permeabilizing activity, but also anti-quorum sensing effects [4]. The search for new methods to target QS in bacteria is an interesting strategy for the development of new antibiotics [75]. This unique bacterial community makes anti-QS compounds a promising way to overcome bacterial pathogenicity without causing any selective pressure for the introduction of resistance [4].
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When the effect of sub-inhibitory concentrations of TC on C. sakazakii was investigated, a significant reduction in both motility and biofilm formation was observed. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) results confirmed that TC downregulated the genes associated with the flagellar apparatus (fliD, flgJ, motA, and motB) and biofilm formation was reduced via the downregulation of motility genes [43]. TC reduced biofilm formation by the inhibition of exopolysaccharide synthesis (cellulose), flagellar formation and function, and cell-to-cell signaling through reduced gene expression [43,61]. TC is also effective for the prevention and inactivation of biofilms of uropathogenic E. coli on two kinds of urinary catheter surfaces (polystyrene and latex), which represent a major challenge to the health care industry [62]. TC was reported to exert antibiofilm effects on methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis [63,64], Streptococcus pyogenes [65], Burkholderia spp. [66], E. coli, Pseudomonas sp. [67], Listeria sp. [68], Salmonella sp. [48,69], and Vibrio spp. [70]. Biofilm formation is associated with quorum sensing activity [43]. Another study showed biofilm inhibition by bark oil and TC, which was partially caused by the downregulation of quorum sensing systems, and observed an inhibition in the production of a quorum sensing molecule (PQS – Pseudomonas quinolone signal) in a Pseudomonas aeruginosa strain [71]. Proteomic analysis performed in S. Typhimurium to gain further insight into the physiological changes that occur in biofilms compared with free-living cells when exposed to TC showed several proteins up or downregulated. 50S ribosomal protein L3 and 30S ribosomal protein S2 were both not expressed after treatment, and are both involved in biofilm protein biosynthesis. Conjugal transfer nickase/helicase TraI, involved in DNA metabolism and elongation factor G involved in protein synthesis were upregulated in biofilm cells compared with planktonic cells. These proteins were not expressed in biofilm cells that were exposed to TC. TC also induced the downregulation of peroxiredoxin. Once bacterial biofilms are formed, cells must deal with oxidative stress, and protective proteins need to be expressed for bacterial survival. Peroxiredoxin is involved in bacterial defenses against toxic peroxides, TC acts then, reducing oxidative stress responses and making bacterial cells in biofilms more susceptible to peroxides and other chemical agents. Serine hydroxymethyltransferase was the most strongly downregulated protein, it is associated with biofilm matrix component production. The TC
4. SYNERGISTIC EFFECTS OF CINNAMON AND ITS COMPOUNDS WITH ANTIBACTERIAL AGENTS
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biofilms may occurs as the essential oil damages the biofilms and permits the easy access of antibiotics to the bacterial cell [83]. Synergism of cinnamic acid and amikacin, ampicillin, ciprofloxacin, erythromycin, or vancomycin against E. coli and S. aureus and the combination of cinnamic acid with ciprofloxacin against P. aeruginosa have been reported [84]. Cinnamic acid is, as already described, a phenolic component of cinnamon that has been used to augment the activity of various antibiotics against Mycobacterium avium [85] and displays synergistic effects with several anti-tuberculosis drugs against Mycobacterium tuberculosis, such asamikacin, clofazimine, isoniazid, and rifampin [86]. The structural similarity of trans-cinnamic acid to phenylalanine, a component in the glycopeptidolipid antigen of the outer cell wall of M. avium, was hypothesized to be the mechanism for this inhibitory activity. However, M. tuberculosis does not synthesize these antigens. Thus, other sites are apparently also being targeted by trans-cinnamic acid [85,86]. Most studies showed synergy in vitro, but did not fully investigate the underlying mechanism; in most cases, it may be a result of multi-target effects. Research into antimicrobial combinations may provide new ways of combating the everincreasing burden of multi-drug resistant bacterial infections. To improve the efficacy of antibiotics, it is necessary to study the modes of action that improve the diffusion of antibiotics and bypass the bacterial membrane barrier, which is responsible for the general antibiotic resistance in bacteria [87]. Although there are a considerable number of studies of the antimicrobial action of cinnamon or its constituents in literature, few have investigated its effects on multi-resistant bacteria [3,4,78]. Studies about the most concerning pathogen in a “real world” health scenario, carbapenemaseproducing Gram-negative bacteria, are scarce. The accurate detection of the genotype of carbapenemases and other resistance enzymes, the mechanisms of bacterial resistance, and the mechanisms of new antimicrobial agents will help to minimize the spread and evolution of resistance, reduce the imprudent use of antibiotics, and possibly allow future control of infections caused by these extremely concerning pathogens.
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Synergy between antimicrobial agents is based on the principle that the formulation may enhance efficacy, reduce toxicity, decrease adverse side effects, increase bioavailability, lower the required dose, and reduce the advent of antimicrobial resistance [76]. New antimicrobial combination drugs that include natural product combinations have emerged as a research priority [77]. C. zeylanicum essential oil functions synergistically with amikacin, gentamicin, imipenem, and meropenem against Acinetobacter baumannii [78] and showed promise in combination with colistin, a drug currently used for the treatment of carbapenemase-producing Gram-negative bacterial infection [3]. C. burmannii essential oil functioned synergistically with gentamicin against S. epidermidis [79] and C. verum essential oil demonstrated the potential to reverse a TEM-1 beta-lactamase gene carrier E. coli resistance to piperacillin [4]. The scanning electron micrograph images confirmed that treated bacterial cell surface was considerably structurally discrepant from the untreated cells, attributed to cell membrane disruption, thereby causing lysis of the bacterial cell wall followed by loss of intracellular dense material. The cells treated with C. verum essential oil alone showed a corrugated surface, whereas treatment in combination with piperacillin showed large damage with changes in size and shape. Furthermore, the treated cells possessed a reduced negative charge and it was suggested that the damage was caused by the acidification and protein denaturation of the cell membrane that resulted from an accumulation of the components of the essential oil [4]. TC decreased the minimal inhibitory concentration (MIC) of clindamycin for Clostridium difficile by 16-fold [80]. The possible mechanism arose from the inhibition of a multidrug efflux pump system (CdeA) identified in this bacterium. TC also functioned synergistically with ampicillin, bacitracin, clindamycin, erythromycin, novobiocin, penicillin, streptomycin, sulfamethoxazole, and tetracycline for several Gram-negative and Gram-positive species [80,81,82]. A synergistic effect of TC and streptomycin was described against L. monocytogenes and Salmonella typhimurium. The synergistic combinations had stronger antibiofilm activities than the individual components and caused a reduction in live bacteria. Fluorescence microscopy images demonstrated that the architecture of the biofilms of both strains displayed very few scattered cell aggregates. As it is already known that TC can inhibit the QS system and influence the expression of biofilmrelated genes, the combination treatment of antibiotics and cinnamon essential oil to bacteria
CONCLUSION It has been shown that resistance to crude extracts occurs more infrequently than resistance to single compounds. Plants have developed various metabolic mechanisms for the production of structurally and functionally diverse compounds to overcome emerging resistance of pathogens.
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and proton motive force of Listeria innocua. Can. J. Microbiol., 2015, 61(4), 263–271. Utchariyakiat, I.; Surassmo, S.; Jaturanpinyo, M.; Khuntayaporn, P.; Chomnawang, M.T. Efficacy of cinnamon bark oil and cinnamaldehyde on antimultidrug resistant Pseudomonas aeruginosa and the synergistic effects in combination with other antimicrobial agents. BMC Complement. Alternat. Med., 2016, 16, 158. Yap, P.S.; Krishnan, T.; Chan, K.G.; Lim, S.H. Antibacterial mode of action of Cinnamomum verum bark essential oil, alone and in combination with piperacillin, against a multi-drug-resistant Escherichia coli strain. J. Microbiol. Biotechnol., 2015, 25(8), 1299–1306. Jayaprakasha, G.K.; Rao, L.J.M. Chemistry, biogenesis, and biological activities of Cinnamomum zeylanicum. Crit. Rev. Food Sci. Nutr., 2011, 51, 547–562. Shreaz, S.; Wani, W.A.; Behbehani, J.M.; Raja, V.; Irshad, M.; Karched, M.; Ali, I.; Siddiqi, W.A.; Hun, L.T. Cinnamaldehyde and its constituents, a novel class of antifungal agents. Fitoterapia, 2016, 12, 11631. Rao, P.V.; Gan, S.H. Cinnamon: A multifaceted medicinal plant. Evid. Based Complement. Alternat. Med., 2014, 2014, 642942. Melo, A.D.; Amaral, A.F.; Schaefer, G.; Luciano, F.B.; de Andrade, C.; Costa, L.B.; Rostagno, M.H. Antimicrobial effect against different bacterial strains and bacterial adaptation to essential oils used as feed additives. Can. J. Vet. Res., 2015, 79(4), 285–289. Wang, S.Y.; Chen, P.F.; Chang, S.T. Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi. Bioresource Tech., 2005, 96(7), 813–818. Mathew, S.; Abraham, T.E. Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chem., 2006, 94(4), 520–528. Lu, Z.; Jia, Q.; Wang, R.; Wu, X.; Wu, Y.; Huang, C.; Li, Y. Hypoglycemic acivities of A and B-type procyanidin oligomer-rich extracts from different Cinnamon barks. Phytomedicine, 2011, 18(4), 298– 302. [18] Nazzaro, F.; Fratianni, F.; De Martino, L.; Coppola, R.; De Feo, V. Effect of Essential Oils on Pathogenic Bacteria. Pharmaceuticals, 2013, 6, 1451–1474. Tung, Y.T.; Chua, M.T.; Wang, S.Y.; Chang, S.T. Antiinflammation activities of essential oil and its constituents from indigenous cinnamon (Cinnamomum osmophloeum) twigs. Bioresource Tech., 2008, 99(9), 3908–3913. Tung, Y.T.; Yen, P.L.; Lin, C.Y.; Chang, S.T. Antiinflammatory activities of essential oils and their constituents from different provenances of indigenous cinnamon (Cinnamomum osmophloeum) leaves. Pharma Biol., 2010, 48(10), 1130–1136. Kong, J.O.; Lee, S.M.; Moon, Y.S.; Lee, S.G.; Ahn, Y.J. Nematicidal activity of cassia and cinnamon oil compounds and related compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae). J. Nematol., 2007, 39(1), 31– 36. Cheng, S.S.; Liu, J.Y.; Huang, C.G.; Hsui, Y.R.; Chen, W.J.; Chang, S.T. Insecticidal activities of leaf essential oils from Cinnamomum osmophloeum against three mosquito species. Bioresource Tech., 2009, 100(1), 457–464. Koppikar, S.J.; Choudhari, A.S.; Suryavanshi, S.A.; Kumari, S.; Chattopadhyay, S.; Kaul-Ghanekar, R. Aqueous cinnamon rxtract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human
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Thus, the use of cinnamon extracts and essential oils could be beneficial for human health and considered as an alternative agent for antimicrobial therapy, medical applications, and antibacterial supplement in health products. In addition, it might reduce the cost of medicines; further, no acute or chronic toxicity, no mutagenicity or genotoxicity, and no carcinogenicity have been detected in mammalian studies. However, further research to achieve a better understanding of the mechanisms of action, in vivo experiments, and clinical trials on cinnamon oil or its active compounds are still necessary to determine the pharmacodynamics and pharmacokinetics. The accurate detection of resistance genes, particular those in carbapenemases, is essential for infection control and the management of antibiotic therapies. As currently available antibiotics may be not sufficient for the treatment of all types of pathogens, we highlight the need to research novel treatment alternatives, not only against antibioticsensitive bacteria, but more importantly, against the multi-resistant strains.
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LIST OF ABBREVIATIONS ATP: adenosine triphosphate; GTP: guanosine triphosphate; LPS: lipopolysaccharides; MIC: minimal inhibitory concentration; PBP: penicillinbinding proteins; PQS: Pseudomonas quinolone signal; QS: Quorum sensing; RT-qPCR: Reverse transcriptase quantitative polymerase chain reaction; TC: tran-cinnamaldehyde.
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ACKNOWLEDGEMENTS This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number: 407791/20132] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant number: 88887.103413/2015-01]. NGV performed the literature research and was the major contributor in writing the manuscript. SS oriented the research. JC oriented the research. All authors read and approved the final manuscript.
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S0882-4010(18)30566-7
DOI:
10.1016/j.micpath.2018.04.036
Reference:
YMPAT 2915
To appear in:
Microbial Pathogenesis
Received Date: 29 March 2018 Revised Date:
17 April 2018
Accepted Date: 19 April 2018
Please cite this article as: Vasconcelos NG, Croda J, Simionatto S, Antibacterial mechanisms of cinnamon and its constituents: A review, Microbial Pathogenesis (2018), doi: 10.1016/ j.micpath.2018.04.036. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Antibacterial Mechanisms of Cinnamon and its Constituents: a Review
Vasconcelos, N. G.a,b, Croda, J. c,d, and Simionatto, S.*a a
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Research Laboratory of Health Sciences, Federal University of Grande Dourados - UFGD, Dourados, Mato Grasso do Sul, Brazil; b Universitary Hospital of Dourados, Federal University of Grande Dourados - UFGD, Dourados, Mato Grosso do Sul, Brazil; c Oswaldo Cruz Foundation, Campo Grande, Mato Grosso do Sul, Brazil; d Federal University of Mato Grosso do Sul – UFMS, Campo Grande, Mato Grosso do Sul, Brazil.
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*Address correspondence to this author at the Research Laboratory of Health Sciences, Federal University of Grande Dourados - UFGD, Rodovia Dourados - Itahum km 12, Cidade Universitária, CEP: 79804970, Dourados, Mato Grosso do Sul, Brazil. E-mail: [email protected].
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Abstract: BACKGROUND: In the current healthcare environment, an alarming rise in multi-drug resistant bacterial infections has led to a global health threat. The lack of new antibiotics has created a need for developing alternative strategies. OBJECTIVE: Understanding the antibacterial mechanisms of Cinnamon and its constituents is crucial to enhance it as a potential new source of antibiotic. The objective of this review is to provide a compilation of all described mechanisms of antibacterial action of Cinnamon and its constituents and synergism with commercial antibiotics in order to better understand how Cinnamon and its constituents can collaborate as alternative treatment to multi-drug resistant bacterial infections. METHODS: The relevant references on antibacterial activities of cinnamon and its constituents were searched. Meanwhile, the references were classified according to the type of mechanism of action against bacteria. Relationships of cinnamon or its constituents and antibiotics were also analyzed and summarized. RESULTS: Cinnamon extracts, essential oils, and their compounds have been reported to inhibit bacteria by damaging cell membrane; altering the lipid profile; inhibiting ATPases, cell division, membrane porins, motility, and biofilm formation; and via anti-quorum sensing effects. CONCLUSION: This review describes the antibacterial effects of cinnamon and its constituents, such as cinnamaldehyde and cinnamic acid, against pathogenic Gram-positive and Gram-negative bacteria. The review also provides an overview of the current knowledge of the primary modes of action of these compounds as wells the synergistic interactions between cinnamon or its constituents with known antibacterial agents. This information will be useful in improving the effectiveness of therapeutics based on these compounds.
Running Title: Antibacterial Mechanism of Cinnamon Keywords: antimicrobial activity, cinnamon, mechanisms of action, multi-drug resistance, synergism, trans-cinnamaldehyde.
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have demonstrated the instability of TC when exposed to air, as the reactive unsaturated aldehyde is readily oxidized to cinnamic acid, which causes volatile loss and the instability of TC [23]. In addition, in vivo, decomposition might also occur before it is able to perform bactericidal activity, as absorbed TC can rapidly and irreversibly become cinnamic acid through enzyme catalysis; thus, it is unstable in the blood [24]. With regard to the minor components, an antibacterial effect has been reported for methoxycinnamaldehyde, whereas coumarin, cinnamyl acetate, and benzaldehyde were shown to have low or no antibacterial effect [25,26]. It is known that the mixture of different constituents can result in synergistic or additive effects [27]; despite being present at low levels or having no antibacterial effect when used alone, it is possible that these minor components could increase the effect of TC or have other targets in bacterial cells. Owing to this richness in composition, plantderived products and their components have a variety of targets; in particular, they attack the membrane, cytoplasm, and in certain situations, may completely alter the morphology of the cells. This review describes the antibacterial mechanisms of action of cinnamon against pathogenic bacteria.
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1. INTRODUCTION Bacterial infections have become a major healthcare challenge owing to the rise and dissemination of multi-drug resistant bacteria, which has led to increased interest in the development of new antimicrobial agents. Plant extracts, oils, and their derivative compounds are known to be active against a wide variety of microorganisms and have been used for hundreds of years to combat pathogens such as bacteria, fungi, and viruses [1]. Many studies have described the antimicrobial effects of cinnamon on both Gram-positive and Gram-negative bacteria [2,3,4]. Cinnamon is a native plant of Sri Lanka and a tropical Asian spice [5] obtained from the inner bark of several trees from the genus Cinnamomum [6]. There are various cinnamon species, which are important popular spices used worldwide, not only for cooking, but also in traditional and modern medicines [7]. The barks and leaves are commonly used to treat various disorders and are known to exert antibacterial [8], antifungal [9], antioxidant [10], antidiabetic [11], anti-inflammatory [12,13], nematicidal [14], insecticidal [15] and anticancer [16] effects. Overall, approximately 250 species have been identified in the Cinnamomum genus, with trees found all over the world [17]. The most important volatile oils of cinnamon are from C. burmannii, C. camphora, C. cassia, C. osmophloeum, C. verum, and C. zeylanicum; pharmaceutical manufacturers use oils both of Ceylon cinnamon (cinnamon oil) and Chinese cinnamon (cassia oil) without distinction between them [5]. Cinnamon, like other plants, has a wide variety of secondary metabolites that exhibit antibacterial properties [18,19]. Secondary metabolites, in contrast to primary metabolites, are not essential for the survival of the plant; instead, they are defensive compounds against competitors and pathogens [20]. These compounds include cinnamaldehyde, cinnamate, cinnamic acid [17,21], and a wide range of essential oils, such as transcinnamaldehyde, cinnamyl acetate, eugenol, Lborneol, camphor, caryophyllene oxide, bcaryophyllene, L-bornyl acetate, E-nerolidol, αcubebene, α-terpineol, terpinolene, and α-thujene [12,13]. The amount and presence of each compound vary depending on the part of the plant [7]. Trans-cinnamaldehyde (TC), an unsaturated aldehyde, was shown to be responsible for antibacterial activity, with the acrolein group (α,βunsaturated carbonyl moiety) in these molecules being essential for the activity [22]. Some studies
2. MATERIAL AND METHODS The present review manuscript considered the literature published prior to April 2018 on antibacterial activity of extracts, essential oils and natural products isolated from different parts of Cinnamomum species and synergism actions when Cinnamon or its constituents were associated to commercial antibiotics. Therefore, only literature available in databases such as Google Scholar and PubMed was reviewed. This review only considered peer-reviewed research papers with impact factor. The keywords used to search were: antibacterial, antimicrobial, mechanisms, Cinnamon, Cinnamomum, trans-cinnamaldeyde, synergism, and multi-drug-resistance. 3. MECHANISMS OF ANTIBACTERIAL ACTION 3.1. Alterations in cell membrane and its lipid profile Usually, Gram-negative bacteria are more resistant to plant extracts, oils, and their constituents than Gram-positive bacteria, because the cell wall of Gram-negative bacteria is more
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permeability, potentially reaching a critical level to induce cell death [2]. Analysis of DNA, RNA, and protein leakage provided additional evidence of antibacterial mechanism of TC and C. zeylanicum bark essential oil against Porphyromonas gingivalis, a Gram-negative anaerobic bacterium that is one of the key causative pathogens in chronic periodontitis, it was observed irreversible damage to the bacterial membrane by propidium iodide uptake assay and confocal microscopy images [37]. All these findings indicate that the membrane is the first target of TC. However, the mechanism of action of TC is not isolated, but instead involves a series of events on the cell surface and within the cytoplasm. 3.2. Inhibition of ATPase In cellular respiration, the electron transport chain generates a transmembrane proton gradient necessary for adenosine triphosphate (ATP) synthesis, performed by multiple enzymes with ATPase activity, including ATP-dependent transport proteins and the F1F0-ATPase complex [38,39]. The F1F0-ATPase complex is a reversible proton-translocating pump that may extrude protons from the cytoplasm by use of energy from ATP hydrolysis and such proton efflux enhances the proton gradient and assists in the regulation of cytoplasmic pH [40]. The F-class pumps are found in bacterial plasma membranes, mitochondria, and chloroplasts [41]. The inhibition of membranebound or membrane-embedded enzymes by TC may result in changes in the protein conformation, which causes the inhibition of ATPase activity, the inhibition of other enzymes, and altered bacterial growth [42]. TC downregulated F1F0-ATPase, thereby inhibiting ATP synthesis in Cronobacter sakazakii [43]. The treatment of E. coli and Listeria monocytogenes with TC caused a disruption of the proton motive force with rapid depletion of ATP and prevented an increase in cellular ATP concentration, which suggested that ATPase inhibition may play a significant role in reducing the growth rate at the sub-lethal concentrations [44,45]. At high concentrations, TC also acts as an ATPase inhibitor [18]. The effects of both cinnamon water extract and TC on mitochondrial F1F0-ATPase in rat liver are similar to those observed in bacterial cells: they dissipate the proton gradient, uncouple the mitochondria, stimulate ATP hydrolysis, lower the level of ATP, and consequently damage mitochondrial functions, which may be attributable to either the release of protons into the matrix or the interaction of cinnamon and TC with the sulfhydryl groups of
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complex [28]. Porin proteins serve as hydrophilic transmembrane channels for small hydrophilic solutes, which easily pass through the outer membrane of Gram-negative bacteria; however, it is hard for hydrophobic antibiotics to penetrate the cell and this is one reason that makes Gramnegative bacteria more resistant [29]. The Grampositive bacteria cell wall allows hydrophobic molecules to easily penetrate and act on both the cell wall and in the cytoplasm [18]. TC, a commonly studied phenylpropene, is synthesized from the amino acid precursor phenylalanine in plants. TC contains hydrophobic interactions and a six-carbon aromatic phenol group, which constitutes a small part of essential oils [30]. Molecules rich in phenolics are able to pass through the phospholipid bilayer of bacterial cell walls and bind to proteins to prevent them from performing their normal functions [31]. The alteration of membrane permeability and the loss of functional proteins transporting molecules and ions perturbs the microbial cell. This subsequently leads to cytoplasmic coagulation, denaturation of several enzymes and proteins, and the loss of metabolites and ions [32]. Studies have reported contrasting information on the membrane-perturbing activity of TC. A sub-lethal concentration of TC did not affect the integrity of the membrane in Escherichia coli, but inhibited the growth and bioluminescence of Photobacterium leiognathi, which suggests that TC gains access to the periplasm and perhaps to the cytoplasm [33]. At a lethal concentration, TC was shown to perturb the cell membrane and is capable of altering the lipid profile of the membrane [34]. E. coli cells possessed a reduced negative charge after exposure to cinnamon bark essential oil and bacterial cells experienced irreversible membrane damage caused by the acidification and protein denaturation of the cell membrane owing to the accumulation of the components of the oil, which allowed the access of antibiotics to PBPs (penicillin-binding proteins) and the induction of cell death [4]. C. cassia essential oil, as well as TC, induced an immediate increase in the rigidity of the cytoplasmic membrane at the surface and at its core in Listeria innocua strains; the intensity depended on the quantity of TC accumulated in the membrane [2]. An increase in bacterial membrane rigidity after the addition of TC to growth media was attributed to a large increase in the proportion of saturated fatty acids in the membrane phospholipids. This modification of fatty acids was probably a compensatory effect to maintain the membrane structure because of the fluidifying effect of TC [35,36]. The accumulation of numerous molecules inside the cell can perturb its selective
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In general, bypassing the cell membrane is a prerequisite for any solute to exert bactericidal activity, including cinnamon extracts or essential oil. The cell structure of Gram-positive bacteria allows hydrophobic molecules to penetrate relatively easily to the cytoplasm. The cell wall of Gram-negative bacteria is more complex, containing an outer membrane and lipopolysaccharides (LPS), conferring greater resistance to hydrophobic natural extracts or essential oils. Hydrophilic molecules can pass through easily because of the abundant protein porins on Gram-negative bacterial cell membranes, to which they have affinity [18]. TC has the ability to decrease the gene expression of the membrane porins OmpA, OmpC, and OmpR, and amino acid transporters that disrupt active transport and diffusion through the bacterial cell membrane [43,53]. Several outer membrane porins were found to be upregulated after exposure to osmotic stress and desiccation in C. sakazakii [54]. However, porins such as the Omps described above, which are involved in the transport of osmoprotectants across the cytoplasmic membrane of bacteria under hyperosmotic stress, were found to be downregulated by TC [55]. TC can reduce resistance to osmotic stress and resistance to desiccation in C. sakazakii and it may be attributable to the decreased ompC and ompR gene expression, which are required for substrate transport across the membrane [53]. All these data reaffirmed the proposed mechanism that TC could disrupt the cytoplasmic membrane and inhibit active transport across it [56]. There are few published studies that correlate the antibacterial action of TC with membrane porins. The measurement of the antibacterial effect of TC on porin-deficient bacterial mutants would be helpful for the exploration of the targets of TC in this action. 3.5. Inhibition of motility and biofilm formation Motility plays a critical role in host-microbial interactions. Bacterial colonization and virulence comprises multiple events, including signal transduction, chemotaxis, and flagellar movement [57]. In addition, the development of complex structures in biofilms may be regulated by cellular motility [58]. Biofilm formation is a form of coordinated collective behavior; its development proceeds through stages of adhesion, proliferation, microcolony formation, and maturation, in order to favor bacterial survival [59]. Biofilm growth provides many advantages, such as protection from desiccation, antimicrobials, metabolic cooperation, and increased genetic diversity via horizontal gene transfer [60].
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membrane proteins [46]. In addition, the dissipation of membrane potential across the mitochondrial membrane, such as that caused by TC, would increase ATP hydrolysis by uncoupling oxidation from phosphorylation [41]. These uncouplers are reversed by thiols [46]. TC was found to target thiols of cysteine residues on protein molecules [47], leading to possible destabilization of ATP activity. TC also inhibited the expression of the ATP synthase alpha chain protein in Salmonella Typhimurium, a component of the ATP synthase protein complex involved in production of ATP [48]. To more accurately determine the interactions of TC with membrane enzymes and with ATPase activity, or identify specific or non-specific interactions, studies on the kinetics of purified enzymes are necessary. 3.3. Inhibition of cell division Bacterial cell division is regulated by FtsZ, a prokaryotic homolog of tubulin. Guanosine triphosphate (GTP)-dependent polymerization makes FtsZ into filaments, a highly dynamic polymeric structure known as the Z-ring, which is assembled at the midcell and constricts the cell envelope, finally separating the mother cell into two daughter cells [49]. TC is able to inhibit GTPdependent FtsZ polymerization, decrease the in vitro assembly reaction and bundling of FtsZ, perturb the Z-ring morphology in vivo, and reduce the frequency of the Z-ring per unit of cell length of E. coli in a dose-dependent manner [50]. The confocal microscopy of live E. coli cells showed that TC specifically targeted Z-ring spatial arrangement, dissipating the Z-rings and reducing the frequency of Z-ring formation by almost half [50]. It was also shown that TC was capable of inhibiting cell separation by binding a region of FtsZ in Bacillus cereus [18]. In addition, it was hypothesized by using an in silico docking model that TC binds to FtsZ at the C-terminal region involving the T7 loop, which perturbs cytokinetic Z-ring formation and inhibits assembly dynamics [50]. Another study with B. cereus showed that without TC treatment, the bacteria appeared as well-separated rods, and when treated, the cells appeared as elongated, filamentous structures in which the daughter cells did not appear to be separated from the previous generation because the septa formation was incomplete [33]. FtsZ is a promising target for new antibacterial drugs because of its prokaryotic specificity, its evolutionary distance from eukaryotic tubulin, and great importance in bacterial cell division [51,52]. 3.4. Inhibition of membrane porins
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exposure may be associated to the disruption of biofilm structure [48]. In health and industrial areas, these microbial biofilms constitute a substantial challenge. Bacterial biofilms are more resistant to antibiotics, host defenses, and contribute to bacterial persistence in chronic infections through medical devices [72]. Therefore, the direct or indirect antibiofilm and anti-quorum sensing action of TC on pathogenic bacteria would be of great importance and the here reported proteins provide potential and interesting targets for exploring new control strategies for biofilms. 3.6. Anti-quorum sensing effect Quorum sensing (QS) is an intercellular communication system used by both Grampositive and Gram-negative bacteria based on the secretion and detection of external signal molecules. QS also influences motility and biofilm formation [73]. Plants that live in constantly wet environments, which are ideal for biofilm formation, face the similar problems in their stem and barks. It is expected that these plants will have compound that interrupt QS and inhibit biofilm formation [74]. The expression of bcsA and luxR, both involved in QS, is downregulated by TC [43]. TC also interfered with an autoinducer of the QS system and biofilm formation without inhibiting bacterial growth in Vibrio spp. [70]. TC inhibited QS in E. coli [67], P. aeruginosa [70], and S. pyogenes [65]. Another study used an E. coli biosensor that showed a reduction in bioluminescence in the presence of QS inhibitors (in this case, C. verum bark essential oil) to confirm the possible anti-QS effect of the essential oil. They proposed that the main mode of action of C. verum essential oil occurred through the disruption of the bacterial membrane at both lethal and sublethal concentrations, which subsequently increased the nonspecific mobility of the antibiotic into the bacterial cell; this indicated not only membrane permeabilizing activity, but also anti-quorum sensing effects [4]. The search for new methods to target QS in bacteria is an interesting strategy for the development of new antibiotics [75]. This unique bacterial community makes anti-QS compounds a promising way to overcome bacterial pathogenicity without causing any selective pressure for the introduction of resistance [4].
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When the effect of sub-inhibitory concentrations of TC on C. sakazakii was investigated, a significant reduction in both motility and biofilm formation was observed. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) results confirmed that TC downregulated the genes associated with the flagellar apparatus (fliD, flgJ, motA, and motB) and biofilm formation was reduced via the downregulation of motility genes [43]. TC reduced biofilm formation by the inhibition of exopolysaccharide synthesis (cellulose), flagellar formation and function, and cell-to-cell signaling through reduced gene expression [43,61]. TC is also effective for the prevention and inactivation of biofilms of uropathogenic E. coli on two kinds of urinary catheter surfaces (polystyrene and latex), which represent a major challenge to the health care industry [62]. TC was reported to exert antibiofilm effects on methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis [63,64], Streptococcus pyogenes [65], Burkholderia spp. [66], E. coli, Pseudomonas sp. [67], Listeria sp. [68], Salmonella sp. [48,69], and Vibrio spp. [70]. Biofilm formation is associated with quorum sensing activity [43]. Another study showed biofilm inhibition by bark oil and TC, which was partially caused by the downregulation of quorum sensing systems, and observed an inhibition in the production of a quorum sensing molecule (PQS – Pseudomonas quinolone signal) in a Pseudomonas aeruginosa strain [71]. Proteomic analysis performed in S. Typhimurium to gain further insight into the physiological changes that occur in biofilms compared with free-living cells when exposed to TC showed several proteins up or downregulated. 50S ribosomal protein L3 and 30S ribosomal protein S2 were both not expressed after treatment, and are both involved in biofilm protein biosynthesis. Conjugal transfer nickase/helicase TraI, involved in DNA metabolism and elongation factor G involved in protein synthesis were upregulated in biofilm cells compared with planktonic cells. These proteins were not expressed in biofilm cells that were exposed to TC. TC also induced the downregulation of peroxiredoxin. Once bacterial biofilms are formed, cells must deal with oxidative stress, and protective proteins need to be expressed for bacterial survival. Peroxiredoxin is involved in bacterial defenses against toxic peroxides, TC acts then, reducing oxidative stress responses and making bacterial cells in biofilms more susceptible to peroxides and other chemical agents. Serine hydroxymethyltransferase was the most strongly downregulated protein, it is associated with biofilm matrix component production. The TC
4. SYNERGISTIC EFFECTS OF CINNAMON AND ITS COMPOUNDS WITH ANTIBACTERIAL AGENTS
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biofilms may occurs as the essential oil damages the biofilms and permits the easy access of antibiotics to the bacterial cell [83]. Synergism of cinnamic acid and amikacin, ampicillin, ciprofloxacin, erythromycin, or vancomycin against E. coli and S. aureus and the combination of cinnamic acid with ciprofloxacin against P. aeruginosa have been reported [84]. Cinnamic acid is, as already described, a phenolic component of cinnamon that has been used to augment the activity of various antibiotics against Mycobacterium avium [85] and displays synergistic effects with several anti-tuberculosis drugs against Mycobacterium tuberculosis, such asamikacin, clofazimine, isoniazid, and rifampin [86]. The structural similarity of trans-cinnamic acid to phenylalanine, a component in the glycopeptidolipid antigen of the outer cell wall of M. avium, was hypothesized to be the mechanism for this inhibitory activity. However, M. tuberculosis does not synthesize these antigens. Thus, other sites are apparently also being targeted by trans-cinnamic acid [85,86]. Most studies showed synergy in vitro, but did not fully investigate the underlying mechanism; in most cases, it may be a result of multi-target effects. Research into antimicrobial combinations may provide new ways of combating the everincreasing burden of multi-drug resistant bacterial infections. To improve the efficacy of antibiotics, it is necessary to study the modes of action that improve the diffusion of antibiotics and bypass the bacterial membrane barrier, which is responsible for the general antibiotic resistance in bacteria [87]. Although there are a considerable number of studies of the antimicrobial action of cinnamon or its constituents in literature, few have investigated its effects on multi-resistant bacteria [3,4,78]. Studies about the most concerning pathogen in a “real world” health scenario, carbapenemaseproducing Gram-negative bacteria, are scarce. The accurate detection of the genotype of carbapenemases and other resistance enzymes, the mechanisms of bacterial resistance, and the mechanisms of new antimicrobial agents will help to minimize the spread and evolution of resistance, reduce the imprudent use of antibiotics, and possibly allow future control of infections caused by these extremely concerning pathogens.
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Synergy between antimicrobial agents is based on the principle that the formulation may enhance efficacy, reduce toxicity, decrease adverse side effects, increase bioavailability, lower the required dose, and reduce the advent of antimicrobial resistance [76]. New antimicrobial combination drugs that include natural product combinations have emerged as a research priority [77]. C. zeylanicum essential oil functions synergistically with amikacin, gentamicin, imipenem, and meropenem against Acinetobacter baumannii [78] and showed promise in combination with colistin, a drug currently used for the treatment of carbapenemase-producing Gram-negative bacterial infection [3]. C. burmannii essential oil functioned synergistically with gentamicin against S. epidermidis [79] and C. verum essential oil demonstrated the potential to reverse a TEM-1 beta-lactamase gene carrier E. coli resistance to piperacillin [4]. The scanning electron micrograph images confirmed that treated bacterial cell surface was considerably structurally discrepant from the untreated cells, attributed to cell membrane disruption, thereby causing lysis of the bacterial cell wall followed by loss of intracellular dense material. The cells treated with C. verum essential oil alone showed a corrugated surface, whereas treatment in combination with piperacillin showed large damage with changes in size and shape. Furthermore, the treated cells possessed a reduced negative charge and it was suggested that the damage was caused by the acidification and protein denaturation of the cell membrane that resulted from an accumulation of the components of the essential oil [4]. TC decreased the minimal inhibitory concentration (MIC) of clindamycin for Clostridium difficile by 16-fold [80]. The possible mechanism arose from the inhibition of a multidrug efflux pump system (CdeA) identified in this bacterium. TC also functioned synergistically with ampicillin, bacitracin, clindamycin, erythromycin, novobiocin, penicillin, streptomycin, sulfamethoxazole, and tetracycline for several Gram-negative and Gram-positive species [80,81,82]. A synergistic effect of TC and streptomycin was described against L. monocytogenes and Salmonella typhimurium. The synergistic combinations had stronger antibiofilm activities than the individual components and caused a reduction in live bacteria. Fluorescence microscopy images demonstrated that the architecture of the biofilms of both strains displayed very few scattered cell aggregates. As it is already known that TC can inhibit the QS system and influence the expression of biofilmrelated genes, the combination treatment of antibiotics and cinnamon essential oil to bacteria
CONCLUSION It has been shown that resistance to crude extracts occurs more infrequently than resistance to single compounds. Plants have developed various metabolic mechanisms for the production of structurally and functionally diverse compounds to overcome emerging resistance of pathogens.
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and proton motive force of Listeria innocua. Can. J. Microbiol., 2015, 61(4), 263–271. Utchariyakiat, I.; Surassmo, S.; Jaturanpinyo, M.; Khuntayaporn, P.; Chomnawang, M.T. Efficacy of cinnamon bark oil and cinnamaldehyde on antimultidrug resistant Pseudomonas aeruginosa and the synergistic effects in combination with other antimicrobial agents. BMC Complement. Alternat. Med., 2016, 16, 158. Yap, P.S.; Krishnan, T.; Chan, K.G.; Lim, S.H. Antibacterial mode of action of Cinnamomum verum bark essential oil, alone and in combination with piperacillin, against a multi-drug-resistant Escherichia coli strain. J. Microbiol. Biotechnol., 2015, 25(8), 1299–1306. Jayaprakasha, G.K.; Rao, L.J.M. Chemistry, biogenesis, and biological activities of Cinnamomum zeylanicum. Crit. Rev. Food Sci. Nutr., 2011, 51, 547–562. Shreaz, S.; Wani, W.A.; Behbehani, J.M.; Raja, V.; Irshad, M.; Karched, M.; Ali, I.; Siddiqi, W.A.; Hun, L.T. Cinnamaldehyde and its constituents, a novel class of antifungal agents. Fitoterapia, 2016, 12, 11631. Rao, P.V.; Gan, S.H. Cinnamon: A multifaceted medicinal plant. Evid. Based Complement. Alternat. Med., 2014, 2014, 642942. Melo, A.D.; Amaral, A.F.; Schaefer, G.; Luciano, F.B.; de Andrade, C.; Costa, L.B.; Rostagno, M.H. Antimicrobial effect against different bacterial strains and bacterial adaptation to essential oils used as feed additives. Can. J. Vet. Res., 2015, 79(4), 285–289. Wang, S.Y.; Chen, P.F.; Chang, S.T. Antifungal activities of essential oils and their constituents from indigenous cinnamon (Cinnamomum osmophloeum) leaves against wood decay fungi. Bioresource Tech., 2005, 96(7), 813–818. Mathew, S.; Abraham, T.E. Studies on the antioxidant activities of cinnamon (Cinnamomum verum) bark extracts, through various in vitro models. Food Chem., 2006, 94(4), 520–528. Lu, Z.; Jia, Q.; Wang, R.; Wu, X.; Wu, Y.; Huang, C.; Li, Y. Hypoglycemic acivities of A and B-type procyanidin oligomer-rich extracts from different Cinnamon barks. Phytomedicine, 2011, 18(4), 298– 302. [18] Nazzaro, F.; Fratianni, F.; De Martino, L.; Coppola, R.; De Feo, V. Effect of Essential Oils on Pathogenic Bacteria. Pharmaceuticals, 2013, 6, 1451–1474. Tung, Y.T.; Chua, M.T.; Wang, S.Y.; Chang, S.T. Antiinflammation activities of essential oil and its constituents from indigenous cinnamon (Cinnamomum osmophloeum) twigs. Bioresource Tech., 2008, 99(9), 3908–3913. Tung, Y.T.; Yen, P.L.; Lin, C.Y.; Chang, S.T. Antiinflammatory activities of essential oils and their constituents from different provenances of indigenous cinnamon (Cinnamomum osmophloeum) leaves. Pharma Biol., 2010, 48(10), 1130–1136. Kong, J.O.; Lee, S.M.; Moon, Y.S.; Lee, S.G.; Ahn, Y.J. Nematicidal activity of cassia and cinnamon oil compounds and related compounds toward Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae). J. Nematol., 2007, 39(1), 31– 36. Cheng, S.S.; Liu, J.Y.; Huang, C.G.; Hsui, Y.R.; Chen, W.J.; Chang, S.T. Insecticidal activities of leaf essential oils from Cinnamomum osmophloeum against three mosquito species. Bioresource Tech., 2009, 100(1), 457–464. Koppikar, S.J.; Choudhari, A.S.; Suryavanshi, S.A.; Kumari, S.; Chattopadhyay, S.; Kaul-Ghanekar, R. Aqueous cinnamon rxtract (ACE-c) from the bark of Cinnamomum cassia causes apoptosis in human
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Thus, the use of cinnamon extracts and essential oils could be beneficial for human health and considered as an alternative agent for antimicrobial therapy, medical applications, and antibacterial supplement in health products. In addition, it might reduce the cost of medicines; further, no acute or chronic toxicity, no mutagenicity or genotoxicity, and no carcinogenicity have been detected in mammalian studies. However, further research to achieve a better understanding of the mechanisms of action, in vivo experiments, and clinical trials on cinnamon oil or its active compounds are still necessary to determine the pharmacodynamics and pharmacokinetics. The accurate detection of resistance genes, particular those in carbapenemases, is essential for infection control and the management of antibiotic therapies. As currently available antibiotics may be not sufficient for the treatment of all types of pathogens, we highlight the need to research novel treatment alternatives, not only against antibioticsensitive bacteria, but more importantly, against the multi-resistant strains.
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LIST OF ABBREVIATIONS ATP: adenosine triphosphate; GTP: guanosine triphosphate; LPS: lipopolysaccharides; MIC: minimal inhibitory concentration; PBP: penicillinbinding proteins; PQS: Pseudomonas quinolone signal; QS: Quorum sensing; RT-qPCR: Reverse transcriptase quantitative polymerase chain reaction; TC: tran-cinnamaldehyde.
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ACKNOWLEDGEMENTS This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) [grant number: 407791/20132] and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant number: 88887.103413/2015-01]. NGV performed the literature research and was the major contributor in writing the manuscript. SS oriented the research. JC oriented the research. All authors read and approved the final manuscript.
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