European Journal of Medicinal Chemistry 52 (2012) 66e69

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European Journal of Medicinal Chemistry

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Original article In vitro antimycoplasmal activity of bergamia and its major components

Pio Maria Furneri a,*, Luigi Mondello b, Giuseppina Mandalari c, Donatella Paolino d, Paola Dugo b, Adriana Garozzo a, Giuseppe Bisignano c a Department of Bio-Medical Sciences, Microbiology section, University of Catania, Via Androne 81, 91524 Catania, Italy b Department of Pharmacochemistry, University of Messina, Viale Annunziata, 98100 Messina, Italy c Department of Pharmacobiology, University of Messina, Viale Annunziata, 98100 Messina, Italy d Department of Sperimental Medicine, University "Magna Grecia" of Catanzaro, Viale Europa, I-88100 Catanzaro, Italy article info abstract

Article history: Forty-two strains of Mycoplasma hominis (including PG21), 2 strain of Mycoplasma fermentans (Pg18 and Received 27 June 2011 K7), 1 strain of Mycoplasma pneumoniae (strain m129) were investigated for their susceptibilities to Citrus Received in revised form bergamia essential oil and to its major components (, linalyl acetate and linalool). C. bergamia 1 March 2012 essential oil inhibited mycoplasmas at concentrations from 0.5 to 1% (MIC value as % v/v). M. hominis Accepted 2 March 2012 showed MIC values of 0.5% and MIC values of 1%; M. pneumoniae showed a MIC value of 0.5% while Available online 11 March 2012 50 90 M. fermentans strains were inhibited by MIC values of 1%. M. pneumoniae and M. hominis shared the same susceptibility to linalyl acetate, with MIC values of 0.015% (corresponding to MIC and MIC for Keywords: 50 90 Mycoplasma M. hominis); M. fermentans strains were less susceptible with MIC values of 0.12%. Among the major Susceptibility components tested, linalool showed higher activity against M. pneumoniae and M. fermentans (MIC values Bergamot oil of 0.015 and 0.06%, respectively) but was less active against M. hominis (MIC50 and MIC90 values of both Limonene 1%); limonene was active against M. pneumoniae (MIC value of 0.03%) but was less active against Linalyl acetate M. fermentans (MIC values of 1%) and M. hominis (both MIC50 and MIC90 values of 4%). The results Linalool indicated that C. bergamia essential oil and its major components had shown an interesting in vitro antimycoplasmal activity. Ó 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction important raw material for the cosmetic and food industries [9,13e16] One interesting characteristic of this plant is the growth The anti-infective activity of essential oils is well known, and site, 90% of which is localized in a narrow strip of land in Calabria, their importance in phytotherapy has been described elsewhere Italy. Like other citrus oils, it is made up of a mixture of mono- [1e12]. Their efficacy is due to: 1) a good antibacterial and anti- and sesquiterpene hydrocarbons and their oxygenated fungal activity for both human and plant pathogens; 2) a wide derivatives (volatile fraction), and a non-volatile residue. Volatile range of activity; 3) a lower insurgence of resistance, probably due components make up approximately 93e96% by weight of to their mechanisms of action. bergamot oil, while the non-volatile residue represents the Bergamot oil, the trivial name of essential oil obtained from remaining 4e7% [15e19]. The latter has been, in the last years, Citrus bergamia Risso or Poiteau [13e15], is obtained by cold a relevant issue for researchers because of its photosensitizing pressing the peel of the bergamot fruit [13e19]. C. bergamia is effect on the skin, mainly due to the presence of and cultivated mainly for its valuable essential oil, which constitutes an [20e22]. Furthermore, Furthermore, as a natural odour fixative, the non-volatile fraction is responsible for the overall oil attributes of bergamot oil and thus affects its industrial * Corresponding author. Dipartimento di Scienze Bio-Mediche, Sez. di Micro- use [23]. biologia, Via Androne 81, 95124 Catania, Italy. Tel.: þ39 0952504705; fax: þ39 It is well known that bergamot fruits, compared to other 0952504753. congeners, are more resistant to plant parasites. Bergamot oil has E-mail addresses: [email protected] (P.M. Furneri), [email protected] been used as an antiseptic folk remedy for the treatment of infec- (L. Mondello), [email protected] (G. Mandalari), [email protected] (D. Paolino), [email protected] (P. Dugo), [email protected] (A. Garozzo), [email protected] tious skin diseases in Italy since the 16th century [24]. Recently, it (G. Bisignano). has been used in douches and hip baths for urinary and vaginal

0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmech.2012.03.005 P.M. Furneri et al. / European Journal of Medicinal Chemistry 52 (2012) 66e69 67 infections. Bergamot oil shows a high antibacterial and antifungal Table 1 activity by inhalation [25] and is therefore useful for treating can- Quantitative (g% w/w) results for the Citrus bergamia oils analyzed. didoses of the mucous membranes and staphylococcus infections Peak Compound Min Max [4e6,9,13]. The contained in bergamot oil are able to expel 1 Tricyclene 0.002 0.005 toxins in organs such as liver and kidneys. Sesquiterpenes are also 2 a-Thujene 0.192 0.314 known to be antiseptic and anti-inflammatory [Carolyn Gross, 3 a-Pinene 0.728 1.370 www.treatableandbeatable.com]. The and alcohols of 4 Camphene 0.026 0.036 5 Sabinene 0.760 1.178 bergamot reduce depression and anxiety by calming and soothing 6 b-Pinene 4.535 6.004 the nervous system. Studies have shown that bergamot balances 7 Myrcene 0.610 1.256 the activity of the hypothalamus which is stimulated by stress and 8 Octanal þ a-Phellandrene 0.070 0.126 transmits a stress reaction to the glands in the body, resulting in 9 de3-Carene 0.002 0.005 10 a-Terpinene 0.102 0.193 nervous tension and mental strain [26]. 11 p-Cymene 0.186 0.326 Chromatography is the primary technique for the analysis of 12 Limonene 25.645 33.690 essential oils. GC has beenproved to be the most suitable tools for 13 (Z)-b-ocimene 0.019 0.036 the identification and quantitation of the volatile constituents, 14 (E)-b-ocimene 0.135 0.280 15 g-Terpinene 7.351 9.645 while HPLC is more powerful for that of non-volatile components þ e 16 Cis-sabinene hydrate octanol 0.026 0.054 [16 19]. 17 Terpinolene 0.246 0.440 The aim of this study was to investigate the in vitro anti- 18 Linalool 5.497 13.238 mycoplasmal activity of C. bergamia essential oil and some of its 19 Nonanal 0.022 0.075 components (limonene, linalyl acetate and linalool). 20 Cis-limonene oxide 0.002 0.008 21 Trans-limonene oxide 0.003 0.012 22 Citronellal 0.012 0.024 2. Results 23 Terpinen-4-ol 0.016 0.034 24 a-Terpineol 0.044 0.096 25 Decanal 0.042 0.065 All the compounds identified by GC/MSand their minimum and 26 Octyl acetate 0.085 0.112 maximum contents (g% w/w) are listed in Table 1. The results 27 Nerol 0.047 0.063 obtained show that limonene (25.645e33.690%), linalyl acetate 28 Citronellol 0.002 0.005 (20.136e27.055%), linalool (5.497e13.238%), b-pinene 29 Neral 0.162 0.268 30 Linalyl acetate 20.136 27.055 e e (4.535 6.004%) and g-terpinene (7.351 9.645%) are the main 31 Geranial 0.259 0.428 volatile components. Compared to other citrus oils, bergamot oil is 32 Perilla 0.012 0.021 characterized by a higher presence of linalool and linalyl acetate, 33 Isobornyl acetate 0.009 0.035 components that show peculiar ranges wider than those present in 34 Undecanal 0.012 0.023 35 Nonyl acetate 0.007 0.026 other common citrus oils (e.g. lemon oil). 36 Methyl geranoate 0.005 0.016 The in vitro susceptibilities are shown in Table 2. C. bergamia 37 d-Elemene 0.003 0.013 essential oil inhibited mycoplasmas at concentrations from 0.5 to 38 a-Terpinyl acetate 0.151 0.215 39 Citronellyl acetate 0.013 0.042 1% (MIC value as % v/v). Mycoplasma hominis showed MIC50 values of 0.5% and 1% for MIC . Mycoplasma pneumoniae showed a MIC 40 Neryl acetate 0.215 0.378 90 41 Geranyl acetate 0.182 0.265 value of 0.5% while Mycoplasma fermentans strains were inhibited 42 Decyl acetate 0.013 0.025 by MIC values of 1%. 43 Sesquithujene 0.009 0.017 M. pneumoniae and M. hominis shared the same susceptibility to 44 b-Caryophyllene 0.216 0.380 45 Trans-a-Bergamotene 0.205 0.319 linalyl acetate, with MIC values of 0.015% (i.e. the MIC50 and MIC90 46 a-Humulene 0.026 0.035 for M. hominis); M. fermentans strains were less susceptible with 47 Cis-b-farnesene þ (Z)-b-santalene 0.043 0.058 MIC values of 0.12%. 48 Germacrene D 0.049 0.095 Linalool showed higher activity against M. pneumoniae and 49 Bicyclogermacrene 0.032 0.068 M. fermentans with MIC values of 0.015 and 0.06%, respectively, 50 b-Bisabolene 0.386 0.406 51 (Z)-g-Bisabolene 0.002 0.005 while M. hominis showed a lower susceptibility with both MIC 50 52 b-Sesquifellandrene 0.003 0.007 and MIC90 values of 1%. 53 (E)-g-Bisabolene 0.004 0.008 Limonene was active against the M. pneumoniae strain with MIC 54 (E)-a-Nerolidol 0.013 0.027 values of 0.03%; M. fermentans strains were less susceptible with 55 Trans-sesquisabinene hydrate 0.027 0.031 MIC values of 1%, while M. hominis showed a lower susceptibility 56 Norbornanol 0.006 0.023 57 Campherenol 0.009 0.017 with both MIC and MIC values of 4% and 8%, respectively. 50 90 58 a-Bisabolol 0.009 0.022 59 Nootkatone 0.013 0.055 3. Discussion The treatment of mycoplasma infections is often achieved with Mycoplasmas, the smallest no-cell-wall free- living prokaryotes, tetracyclines. Although M. pneumoniae is susceptible to macrolides belong to the class of Mollicutes [31]. The highest number of human and M. hominis and M. fermentans aresusceptible to 16-membered pathogenic mycoplasmas belong to the Mycoplasma genus. Among macrolides, M. hominis and M. fermentans are naturally resistant the 17 species isolated from man, most of which are commensal, to erythromycin and 14-membered macrolides [30]. only M. pneumoniae, M. hominis, and less frequently M. fermentans, The in vitro activities of essential oil and natural phenols against are pathogenic for humans [32]. M. pneumoniae is a respiratory mycoplasmas have been studied by our group. Oleuropein [32] and pathogen, although it is involved in other diseases of other organs its derivative hydroxytyrosol [33] as well as the essential oil of and apparatuses [33]. M. hominis is involved in many diseases of the Melaleuca alternifolia [7] all showed good antimycoplasmal genital tract, while M. fermentans has been involved as a cofactor of activity. AIDS, as pathogen in immunocompromised patient, and in the As suggested by Trombetta et al. [34] and Sikkema et al. [35], the etiology of rheumatoid arthritis [32]. mechanism of action of terpenes is associated to a damage of the 68 P.M. Furneri et al. / European Journal of Medicinal Chemistry 52 (2012) 66e69

Table 2 In vitro activity of C. bergamia essential oil and its derivatives (MIC as % v/v).

Microoganisms (no.) Essential oil Linalyl acetate Linalool Limonene

a a a a a a a a MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 MIC50 MIC90 M. pneumoniae (1)a e 0.5a 0.015a e 0.015a e 0.03a M. fermentans (2)a e 1a e 0.12a e 0.06a e 1a M. hominis (42) 0.5 1 0.015 0.015 1 1 4 8

The value referred to M. pneumoniae and M. fermentans were reported as MIC values, no MIC50 or MIC90 values were calculated. a MIC50: The concentration of given drug inhibiting 50% of the strains; MIC90: The concentration of given drug inhibiting 90% of the strains. plasma membrane stability [12]. Linalyl acetate has been associated Class VP 4.3 Chromatography Workstation software (Shimadzu, to perturbation of the lipid component of citoplasmatic membrane Italy) was used. Qualitative results were obtained by means of of bacteria producing change in permeability and loss of intracel- a Shimadzu QP 5050A GC/MS, fitted with a Shimadzu AOC-20i lular content [34]. autosampler. Interface temperature: 230 C. Mass range: 40e400 Limone, a hydrocarbon, is classified as a cyclic monoterpene, m/z. Scan speed: 4000 amu/sec. Ionization energy: 1.50 kV. Gas- and has been associated with damage of the energetic pathway at chromatographic conditions were the same as above reported. Data the level of the respiratory chain [35]. This feature is, probably, acquisition was performed by a Shimadzu Class-5000 Chromatog- correlated with the lower activity of limonene against M. hominis. raphy Workstation software (Shimadzu, Italy) [18,19,27,28]. In fact, M. hominis gains ATP through the arginine hydrolysis pathway [36] and, like all mycoplasmas NADH oxidase is localized 5.2. Strains and drugs in the cytoplasma [37]. Moreover, mycoplasmas lack many systems such as tricarboxylic acid cycle, quinones and cytochromes, the Forty-two strains of M. hominis (including PG21), two strains of electron transport system is reduced, and ATP is produced by M. fermentans (Pg18 and K7), and one strain of M. pneumoniae substrate-level phosphorylation instead of the electron transport (M129) were obtained from the collection of the Dept. Bio-Medical chain [31]. The fact that M. pneumoniae and M. fermentans were Sciences, at the University of Catania. Mycoplasmas were grown in susceptible to limone was probably due to the impairment of those SP-4 and maintained frozen (80 C) until assayed against the drug fi membrane functions involving sugar uptake (ie. Glucose speci c [7,29]. Limonene, linalool and linalyl acetate were purchased from fi PTS or Fructose speci c) [35,36]. SigmaeAldrich. The determination of the enantiomeric distribution Linalool, a terpene alcohol, was reported to be associated to on these components was not carried out. antimicrobial, antiviral, and anti-inflammatory activities [38].Itis possible to postulate that its antimycoplasmal action was due to its interaction with plasma membrane [34,35]. 5.3. Susceptibility testing procedure Plant products have found industrial applications as nutra- ceuticals and/or functional foods as well as for dietary, pharma- The MIC was determined by a broth microdilution assay as fi ceutical, preservative and cosmetic purposes [39]. Furthermore, the previously described [7,29]. The MIC was de ned as the lowest in vitro antimicrobial activity of natural compounds could provide concentration of antibiotic (as % v/v) that inhibited a colour change with potentially useful information for developing novel antibiotics in the broth by a given strain of mycoplasma at the time when the [1e13]. colour of the control tube changed (i.e., when the pH of the medium Our discoveries and those reported by others [6,9,13,40] for decreased from 7.5 to 7.0 for M. pneumoniae, M. fermentans or bergamot essential oil as active products against both Gram- increased from 6.0 to 6.5 for M. hominis). The required incubation e positive and Gram-negative bacteria, yeast, and dermathophytes times were 24 48 h for M. hominis and M. fermentans and from 3 to [6,9,13,40] suggested that C. bergamia oil might be a source for 5 days for M. pneumoniae [30]. Each mycoplasma strain was tested developing novel antibiotics. six times against each antimicrobial agent. The strains were tested six additional times, on different days with all drugs to ensure the 4. Conclusion reproducibility of the results. A positive control (growth) consisting of organisms in broth, a negative control (sterility) consisting of broth with the highest concentrations of the control drug consist- C. bergamia and its derivatives have shown an interesting in vitro ing of broth with the highest concentrations of the drug were antimycoplasmal activity. Appropriate studies are now needed to included for each mycoplasmal strain tested [7,29,30]. determine whether this in vitro activity will translate into in vivo activity. References 5. Material and methods [1] K.A. Hammer, C.F. Carson, T.V. Riley, Antimicrobial activity of essential oils and other plant extracts, J. Appl. Microbiol. 86 (1999) 985e990. 5.1. Gas chromatographic analysis of cold pressed bergamot oil [2] M.M. Cowan, Plant products as antimicrobial agents, Clin. Microbiol. Rev. 12 (1999) 564e582. 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