438
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Composition and Activity against Oral Pathogens of the Essential Oil of Melampodium divaricatum (Rich.) DC. by Raquel Regina Duarte Moreira* a ), Gilmarcio Zimmermann Martins a ), Vincius Teixeira Botelho a ), Luis Eduardo dos Santos a ), Carlos Cavaleiro b ), Lgia Salgueiro b ), Ge´ssica Andrade c ), and Carlos Henrique Gomes Martins c ) a ) Departamento de Princpios Ativos Naturais e Toxicologia, Faculdade de Cieˆncias Farmaceˆuticas – UNESP – Universidade Estadual Paulista, Rodovia Araraquara, Jau´ Km 1 s/n Cep: 14901-502, Campus Ville, Araraquara, Sa˜o Paulo, Brasil (phone: þ 55-16-33016992; fax: þ 55-16-33016980; e-mail: [email protected]) b ) Centro de Estudos Farmaceˆuticos, Faculdade de Farma´cia, Universidade de Coimbra, Portugal c ) Laborato´rio de Pesquisas em Microbiologia Aplicada, LaPeMA – Unifran - Universidade de Franca, Franca, Sa˜o Paulo, Brasil
The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 mg/ml were obtained against Streptococcus sobrinus (90 mg/ml), Lactobacillus casei (30 mg/ml), S. mutans (20 mg/ml), and S. mitis (18 mg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 mg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.
Introduction. – Dental caries and periodontal diseases are associated with oral pathogens, mainly bacteria [1]. Both the cariogenic and periodontopathic bacteria present in the oral cavity form an adherent, structurally and functionally organized biofilm [2]. Strategies to control caries include inhibition of the biofilm development, i.e., prevention of the attachment of cariogenic bacteria, delivery of effective antimicrobials, or enhancement of the host defenses [3]. Mechanical methods of oral hygiene such as brushing and flossing are the most commonly applied approaches for routine denture biofilm control [4] [5]. However, studies have indicated that most people fail to maintain a sufficient level of biofilm control by brushing only, so that chemotherapeutic mouth rinses may play a key role as adjuncts to the daily tooth care [6]. Several plant derivatives, including crude extracts, essential oils, and pure compounds, have been evaluated with respect to their antimicrobial effects against oral pathogens [7 – 10]. These derivatives have attracted the interest of research groups, since they may be employed in the development of new mouth rinses for oral hygiene. However, reports on the antimicrobial activity of natural products against oral pathogens are still scarce. 2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
439
As part of our ongoing research on the antimicrobial activities of Brazilian plants and bioactive natural compounds [11] [12], and considering previous reports documenting the antimicrobial activity of essential oils against oral pathogens [13] [14], the in vitro antimicrobial activity of the essential oil of Melampodium divaricatum (Rich.) DC. (Asteraceae) was investigated. It is an herbaceous species (80 – 160 cm high) naturally occurring in the northeastern regions of Brazil, locally known as falsacaleˆndula, flor-amarela, estrelinha, flor-de-ouro, pica˜o-de-praia, fel-de-terra, or salsa-da-praia [15] [16]. It is appreciated in the local traditional medicine for its healing, diaphoretic, and diuretic proprieties as well as for its usefulness in the treatment of leucorrhea [15]. Investigations of the chemistry of M. divaricatum resulted in the identification of flavonoids [17], coumarins [18], pyrrolizidine alkaloids [19], and terpenoids [20]. To the best of our knowledge, there are no published studies concerning the composition of the essential oil of M. divaricatum, although its antimicrobial activity was reported [21]. Hence, the chemical composition of the essential oil isolated from the aerial parts of M. divaricatum and its antimicrobial activity against oral pathogens was evaluated here. Results and Discussion. – Essential-Oil Composition. The essential oil isolated by hydrodistillation from the aerial parts of M. divaricatum was a yellow liquid with an intense woody scent. In total, 26 constituents were identified, accounting for 92.1% of the whole oil composition (Table 1). Sesquiterpene hydrocarbons (84.3%) and oxygenated sesquiterpenes (7.8%) predominated, while monoterpenes and phenylpropanoids were present only at trace amounts. The principal constituents identified were (E)-caryophyllene (1, 56.0%), germacrene D (2, 12.7%), bicyclogermacrene (3, 9.2%), caryophyllene oxide (4, 3.0%), a-humulene (5, 1.9%), spathulenol (6, 1.9%), caryophylla-4(14),8(15)-dien-5-ol (7, 1.5%), and b-elemene (8, 1.4%; Fig.). Among them, only caryophyllene oxide and spathulenol have previously been detected as
Figure. Major compounds isolated from the essential oil of Melampodium divaricatum. 1, (E)Caryophyllene (content ¼ 56.0%); 2, germacrene D (12.7%); 3, bicyclogermacrene (9.2%); 4, caryophyllene oxide (3.0%); 5, a-humulene (1.9%); 6, spathulenol (1.9%); 7, caryophylla-4(14),8(15)dien-5-ol (1.5%); 8, b-elemene (1.4%).
440
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 1. Composition of the Essential Oil Isolated from the Aerial Parts of Melampodium divaricatum Compound name and class a )
RIapolar b )
RIpolar c )
Content [%] d )
1,8-Cineole Limonene Citronellal Terpinene-4-ol Eugenol a-Copaene d-Elemene b-Bourbonene b-Elemene (8) ( E )-Caryophyllene (1) Selina-3,6-diene b-Funebrene ( E )-a-Bergamotene a-Patchoulene a-Humulene (5) Germacrene D (2) b-Selinene Bicyclogermacrene (3) g-Cadinene d-Cadinene ( E )-Nerolidol Spathulenol (6) Caryophyllene oxide (4) Caryophylla-4(14),8(15)-dien-5-ol (7) t-Muurolol a-Cadinol
1019 1019 1129 1158 1324 1369 1328 1375 1383 1410 1417 1430 1430 1435 1440 1467 1472 1482 1497 1507 1543 1552 1556 1607 1614 1627
n.d. n.d 1480 1597 2159 1487 n.d. 1519 n.d. 1597 n.d. 1604 n.d. n.d. 1663 1702 1717 1727 1754 1754 2038 2111 1969 2296 n.d. 2223
tr tr tr tr tr tr 0.2 0.8 1.4 56.0 0.4 0.4 0.7 0.1 1.9 12.7 0.1 9.2 0.1 0.3 0.4 1.9 3.0 1.5 0.5 0.5
Monoterpene hydrocarbons Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenylpropanoids
tr 84.3 7.8 tr
Total identified a
92.1 b
) Compounds are listed in order of their elution on the apolar SPB-1 column. ) RIapolar : Retention index determined on the apolar SPB-1 column relative to a series of n-alkanes (C8 – C23 ). c ) RIpolar : Retention index determined on the polar SupelcoWax-10 column relative to a series of n-alkanes (C8 – C23 ); n.d., not determined. d ) Values are means (n ¼ 3); tr, traces.
metabolites of M. divaricatum in a CHCl3 extract of air-dried stems and leaves, together with guaianol and the oxygenated diterpene kolavenol [20]. To the best of our knowledge, this is the first report on the composition of M. divaricatum essential oil. Antimicrobial Activity against Oral Pathogens. The in vitro antimicrobial activity of the M. divaricatum oil and of some of its components are summarized in Table 2. The minimum inhibitory concentrations (MICs) of the oil ranged from 18 to > 400 mg/ml. At concentrations below 100 mg/ml, the essential oil inhibited the growth of four of the seven oral pathogens tested, viz., Streptococcus mitis (MIC ¼ 18 mg/ml), S. mutans (MIC ¼ 20 mg/ml), Lactobacillus casei (MIC ¼ 30 mg/ml), and S. sobrinus (MIC ¼ 90 mg/
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
441
Table 2. Growth Inhibitory Activity of the Melampodium divaricatum Essential Oil, Its Major Components 1, 4, and 5, and the Positive Control Chlorhexidine against Seven Oral Pathogens Oral pathogen
Enterococcus faecalis Lactobacillus casei Streptococcus mitis Streptococcus mutans Streptococcus sanguinis Streptococcus sobrinus Streptococcus salivarius
Minimum inhibitory concentration ( MIC) [mg/ml] Chlorhexidine
Essential oil
1
4
5
14.75 3.69 14.75 0.92 7.74 1.84 7.38
> 400 30 18 20 300 90 300
> 400 > 400 200 > 400 > 400 > 400 > 400
> 400 > 400 > 400 > 400 > 400 > 400 > 400
> 400 > 400 > 400 > 400 > 400 > 400 > 400
ml). Enterococcus faecalis, S. salivarius, and S. sanguinis were susceptible to the essential oil only at concentrations above 300 mg/ml. These are interesting results, particularly those concerning the growth inhibition of S. mitis and S. mutans. Indeed, the two bacterial strains belong to the principal causative organisms of dental caries for which few natural inhibitors are known [22]. The high content of caryophyllene derivatives in the essential oil (60.5%) and the published biocide activity of this kind of compounds [23 – 26] lead us to investigate the antimicrobial activity of (E)-caryophyllene, caryophyllene oxide, as well as the structurally related compound a-humulene. (E)-Caryophyllene is a characteristic compound of the essential oils of several Asteraceae species [27 – 30], among them Bidens sulphurea, whose oil proved to be active against S. mitis [14]. However, the MIC values of the tested oil components were higher than 200 mg/ml, revealing that they were ineffective against the tested bacterial strains. These results suggest that minor compounds of the M. divaricatum essential oil might play an important role in the bioactivity of the oil and that synergic effects among the oil components might also explain its remarkable activity. Conclusions. – The results demonstrated that M. divaricatum essential oil can be used either as an antimicrobial agent to treat infections by microorganisms responsible for dental caries and periodontal diseases or as a source of new active compounds with antimicrobial proprieties. Further studies are needed to identify the active compounds among the minor constituents, to characterize synergic effects, to elucidate the mechanisms of action, and to discard toxic effects. The authors thank the FUNDUNESP (Brazil) and the FundaÅa˜o para a Cieˆncia e Tecnologia (Portugal, project PEst-OE/SAU/UI0177/2011) for their financial support.
Experimental Part Plant Material. The aerial parts of Melampodium divaricatum (Rich.) DC. were collected in the garden, Horto de Plantas Medicinais e To´xicas, of the Faculdade de Cieˆncias Farmaceˆuticas - UNESP in Araraquara, Sa˜o Paulo state, Brazil (218 48’ 51.4’’ S and 488 12’ 5.1’’ W). A voucher specimen (HRCB 35294) has been deposited with the Herba´rio do Instituto de Biocieˆncias - UNESP in Rio Claro, Sa˜o Paulo state, Brazil.
442
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Essential-Oil Isolation. The essential oil was obtained by hydrodistillation of the aerial parts for 4 h in a modified Clevenger apparatus according to the procedure described in the European Pharmacopoeia [31]. GC Analysis. The GC analyses were carried out using a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a HP GC ChemStation Rev. A.05.04 datahandling system, a single injector, and two flame-ionization detection (FID) systems. A Graphpak divider (Agilent Technologies, part number 5021 – 7148) was used for simultaneous sampling to two Supelco (Bellefonte, PA, USA) fused-silica cap. columns (30 m 0.20 mm i.d., film thickness 0.20 mm) with stationary phases of different polarity, i.e., an apolar SPB-1 (polydimethylsiloxane), and a polar Supelcowax-10 (polyethylene glycol) column. The oven temp. was programmed rising from 70 to 2208 at 38/min and then held isothermal at 2208 for 15 min; injector temp., 2508; FID temp., 2508; carrier gas, He (adjusted to a linear velocity of 30 cm/s); split ratio, 1 : 40. The calculation of the relative contents of the individual components was based on GC-FID raw-data areas without FID response-factor correction. GC/MS Analysis. The GC/MS analyses were carried out using a Hewlett-Packard 6890 gas chromatograph equipped with a HP1 fused-silica cap. column (polydimethylsiloxane, 30 m 0.25 mm i.d., film thickness 0.25 mm) and a Hewlett-Packard 5973 mass selective detector (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00. The GC parameters were as described above (cf. GC Analysis); interface temp., 2508; MS source temp., 2308; MS quadrupole temp., 1508; ionization energy, 70 eV; ionization current, 60 mA; scan range, 35 – 350 amu; scans, 4.51. Compound Identification. The identification of the individual essential-oil components was based on the comparison of i) their retention indices (RIs), determined on both SPB-1 and Supelcowax-10 columns by linear interpolation rel. to the retention times (tR ) of a series of n-alkanes (C8 – C23 ), with those of reference samples included in the home-made laboratory database of the Centro de Estudos Farmaceˆuticos, Faculty of Pharmacy, University of Coimbra, and ii) the acquired mass spectra with reference spectra compiled in the home-made laboratory database and the commercial Wiley and NIST mass-spectral libraries [32] and literature data [33] [34]. Antimicrobial Activity. The minimum inhibitory concentrations (MICs) of the crude essential oil, (E)-caryophylene (Chromadex), a-humulene (Chromadex), and Caryophylene oxide (95%, Sigma) were determined with the broth microdilution method [35] in 96-well microplates. The following standard strains from the American Type Culture Collection (ATCC) were used: Enterococcus faecalis (ATCC 4082), Streptococcus salivarius (ATCC 25975), Streptococcus sobrinus (ATCC 33478), Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Lactobacillus casei (ATCC 11578). Individual 24-h colonies from blood agar (Difco Labs, Detroit, USA) were suspended in 10.0 ml of tryptic soy broth (Difco). The standardization of each microorganism suspension was carried out by adjusting the transmittance at l 625 nm to 81, equivalent to 0.5 McFarland scale (1.5 108 CFU/ml), using a Femto spectrophotometer (Sa˜o Paulo, Brazil). The suspensions were then diluted to a final concentration of 5 105 CFU/ml. The samples were dissolved in DMSO (Merck, Darmstadt, Germany) at a concentration of 1 mg/ml and were then diluted with tryptic soy broth to obtain concentrations in the range of 20 – 400 mg/ml. The final DMSO concentration was 5% (v/v), and DMSO solns. (concentrations ranging from 1 – 5%) were used as negative control. One inoculated well was included, to control the adequacy of the broth for organism growth, and one noninoculated well free of antimicrobial agent was included, to ensure medium sterility. Two-fold serial dilutions of chlorhexidine (Sigma) made in tryptic soy broth to achieve concentrations ranging from 59.0 – 0.115 mg/ml were used as positive control. The microplates (96 wells) were sealed with Parafilm and incubated at 378 for 24 h. After incubation, 30 ml of a 0.02% aq. soln. of resazurin (Sigma, St. Louis, MO, USA) were added to each microplate well, to indicate the microorganism viability [36]. The MIC value was determined as the lowest concentration of sample capable of inhibiting microorganism growth. Three replicates were made for each sample and microorganism.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
443
REFERENCES [1] M. A. Botelho, N. A. Nogueira, G. M. Bastos, S. G. Fonseca, T. L. Lemos, F. J. Matos, D. Montenegro, J. Heukelbach, V. S. Rao, G. A. Brito, Braz. J. Med. Biol. Res. 2007, 40, 349. [2] P. D. Marsh, BMC Oral Health 2006, 6, S1. [3] P. Gilbert, T. Maira-Litran, A. J. McBain, A. H. Rickard, F. W. Whyte, Adv. Microb. Physiol. 2002, 46, 203. [4] N. Sharma, C. H. Charges, M. C. Lynch, J. Qaqish, J. A. McGuire, J. G. Galustians, L. D. Kumar, J. Am. Dent. Assoc. 2004, 135, 496. [5] L. Barnett, J. Am. Dent. Assoc. 2006, 137, 16S. [6] A. Santos, J. Clin. Periodontol. 2003, 5, 13. [7] H. F. Abd El Rahman, N. Skaug, G. W. Francis, Saudi Dent. J. 2002, 14, 26. [8] S. Prabuseenivasan, M. Jayakumar, S. Ignacimuthu, BMC Complement. Altern. Med. 2006, 6, 39. [9] M. L. A. Silva, H. Coimbra, A. C. Pereira, V. A. Souza, T. C. Lima, E. S. Costa, A. H. C. Vinholis, V. A. Royo, R. Silva, A. A. Silva Filho, W. R. Cunha, N. A. J. C. Furtado, Phytother. Res. 2007, 21, 420. [10] J. L. Rios, M. C. Recio, J. Ethnopharmacol. 2005, 100, 80. [11] G. F. Oliveira, N. A. J. C. Furtado, A. A. Silva Filho, C. H. G. Martins, J. K. Bastos, W. R. Cunha, M. L. A. Silva, Braz. J. Microbiol. 2007, 38, 381. [12] L. C. S. Cunha, M. L. A. Silva, N. A. J. C. Furtado, A. H. C. Vinho´lis, C. H. G. Martins, A. A. SilvaFilho, W. R. Cunha, Z. Naturforsch., C 2007, 62, 668. [13] K. Takarada, R. Kimizuka, N. Takahashi, K. Honma, K. Okuda, T. Kato, Oral Microbiol. Immunol. 2004, 19, 61. [14] G. P. Aguiar, C. E. Carvalho, H. J. Dias, E. B. Reis, M. H. G. Martins, K. A. L. Wakabayashi, M. Groppo, C. H. G. Martins, W. R. Cunha, A. E. M. Crotti, Nat. Prod. Res. 2013, 27, 1668. [15] M. F. Agra, P. F. Freitas, J. M. Barbosa-Filho, Rev. Bras. Farmacogn. 2007, 17, 114. [16] H. Lorenzi, Plantas Daninhas do Brasil: Terrestres, Aqua´ticas, Parasitas e To´xicas, 3rd edn., Instituto Plantarum, Nova Odessa, SP, 2002. [17] J. Hther, C. M. Passreiter, V. Wray, G. Willuhn, Phytochemistry 1999, 51, 979. [18] J. Borges-del-Castillo, A. I. Martinez-Martir, F. Rodriguez-Luis, J. C. Rodriguez-Ubis, P. VazquezBueno, Phytochemistry 1984, 23, 859. [19] J. Schngel, C. M. Passreiter, Biochem. Syst. Ecol. 2000, 28, 705. [20] T. D. Hubert, D. F. Wiemer, Phytochemistry 1985, 24, 1197. [21] G. P. Pelissari, R. C. L. R. Pietro, R. R. D. Moreira, Rev. Bras. Farmacogn. 2010, 20, 70. [22] M. Saleem, M. Nazir, M. S. Ali, H. Hussain, Y. S. Lee, N. Riaz, A. Jabbar, Nat. Prod. Rep. 2010, 27, 238. [23] D. Yang, L. Michel, J. P. Chaumont, J. Millet-Clerc, Mycopathologia 1999, 148, 79. [24] K. K. Macleod, H. B. Rasmussen, Phytochemistry 1999, 50, 105. [25] B. Demirci, K. H. C. Bas¸er, F. Demirci, M. T. Hamann, J. Nat. Prod. 2000, 63, 902. [26] C. Cavaleiro, M. J. GonÅalves, D. Serra, G. Santoro, F. Tomi, A. Bighelli, L. Salgueiro, J. Casanova, J. Pharm. Biomed. 2011, 54, 619. [27] R. K. Joshi, Nat. Prod. Commun. 2013, 8, 225. [28] R. K. Joshi, Nat. Prod. Commun. 2013, 8, 401. [29] M. Kazemi, S. Akhavani, Asian J. Chem. 2013, 25, 2985. [30] R. S. Verma, R. C. Padalia, A. Chauhan, Natl. Acad. Sci. Lett. 2013, 36, 447. [31] Council of Europe, European Pharmacopoeia, 3rd edn., Strasbourg, 1997. [32] Registry, 8th edn., with NIST 05 MS Spectra, Revision D.06.00, Agilent Technologies, Wiley, 2007. [33] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy, Allured Publishing Corporation, Carol Stream, IL, USA, 1995. [34] D. Joulain, W. Koenig, The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998. [35] Clinical and Laboratory Standards Institute (CLSI), Document M7-A7, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 7th edn., CLSI, Wayne, PA, 2006, Vol. 23, No. 2.
444
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
[36] T. S. Porto, R. Rangel, N. A. J. C. Furtado, T. C. Carvalho, C. H. G. Martins, R. C. S. Veneziani, F. B. Costa, A. H. C. Vinholis, W. R. Cunha, V. C. G. Heleno, S. R. Ambrosio, Molecules 2009, 14, 191. Received September 30, 2013
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Composition and Activity against Oral Pathogens of the Essential Oil of Melampodium divaricatum (Rich.) DC. by Raquel Regina Duarte Moreira* a ), Gilmarcio Zimmermann Martins a ), Vincius Teixeira Botelho a ), Luis Eduardo dos Santos a ), Carlos Cavaleiro b ), Lgia Salgueiro b ), Ge´ssica Andrade c ), and Carlos Henrique Gomes Martins c ) a ) Departamento de Princpios Ativos Naturais e Toxicologia, Faculdade de Cieˆncias Farmaceˆuticas – UNESP – Universidade Estadual Paulista, Rodovia Araraquara, Jau´ Km 1 s/n Cep: 14901-502, Campus Ville, Araraquara, Sa˜o Paulo, Brasil (phone: þ 55-16-33016992; fax: þ 55-16-33016980; e-mail: [email protected]) b ) Centro de Estudos Farmaceˆuticos, Faculdade de Farma´cia, Universidade de Coimbra, Portugal c ) Laborato´rio de Pesquisas em Microbiologia Aplicada, LaPeMA – Unifran - Universidade de Franca, Franca, Sa˜o Paulo, Brasil
The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 mg/ml were obtained against Streptococcus sobrinus (90 mg/ml), Lactobacillus casei (30 mg/ml), S. mutans (20 mg/ml), and S. mitis (18 mg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 mg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.
Introduction. – Dental caries and periodontal diseases are associated with oral pathogens, mainly bacteria [1]. Both the cariogenic and periodontopathic bacteria present in the oral cavity form an adherent, structurally and functionally organized biofilm [2]. Strategies to control caries include inhibition of the biofilm development, i.e., prevention of the attachment of cariogenic bacteria, delivery of effective antimicrobials, or enhancement of the host defenses [3]. Mechanical methods of oral hygiene such as brushing and flossing are the most commonly applied approaches for routine denture biofilm control [4] [5]. However, studies have indicated that most people fail to maintain a sufficient level of biofilm control by brushing only, so that chemotherapeutic mouth rinses may play a key role as adjuncts to the daily tooth care [6]. Several plant derivatives, including crude extracts, essential oils, and pure compounds, have been evaluated with respect to their antimicrobial effects against oral pathogens [7 – 10]. These derivatives have attracted the interest of research groups, since they may be employed in the development of new mouth rinses for oral hygiene. However, reports on the antimicrobial activity of natural products against oral pathogens are still scarce. 2014 Verlag Helvetica Chimica Acta AG, Zrich
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
439
As part of our ongoing research on the antimicrobial activities of Brazilian plants and bioactive natural compounds [11] [12], and considering previous reports documenting the antimicrobial activity of essential oils against oral pathogens [13] [14], the in vitro antimicrobial activity of the essential oil of Melampodium divaricatum (Rich.) DC. (Asteraceae) was investigated. It is an herbaceous species (80 – 160 cm high) naturally occurring in the northeastern regions of Brazil, locally known as falsacaleˆndula, flor-amarela, estrelinha, flor-de-ouro, pica˜o-de-praia, fel-de-terra, or salsa-da-praia [15] [16]. It is appreciated in the local traditional medicine for its healing, diaphoretic, and diuretic proprieties as well as for its usefulness in the treatment of leucorrhea [15]. Investigations of the chemistry of M. divaricatum resulted in the identification of flavonoids [17], coumarins [18], pyrrolizidine alkaloids [19], and terpenoids [20]. To the best of our knowledge, there are no published studies concerning the composition of the essential oil of M. divaricatum, although its antimicrobial activity was reported [21]. Hence, the chemical composition of the essential oil isolated from the aerial parts of M. divaricatum and its antimicrobial activity against oral pathogens was evaluated here. Results and Discussion. – Essential-Oil Composition. The essential oil isolated by hydrodistillation from the aerial parts of M. divaricatum was a yellow liquid with an intense woody scent. In total, 26 constituents were identified, accounting for 92.1% of the whole oil composition (Table 1). Sesquiterpene hydrocarbons (84.3%) and oxygenated sesquiterpenes (7.8%) predominated, while monoterpenes and phenylpropanoids were present only at trace amounts. The principal constituents identified were (E)-caryophyllene (1, 56.0%), germacrene D (2, 12.7%), bicyclogermacrene (3, 9.2%), caryophyllene oxide (4, 3.0%), a-humulene (5, 1.9%), spathulenol (6, 1.9%), caryophylla-4(14),8(15)-dien-5-ol (7, 1.5%), and b-elemene (8, 1.4%; Fig.). Among them, only caryophyllene oxide and spathulenol have previously been detected as
Figure. Major compounds isolated from the essential oil of Melampodium divaricatum. 1, (E)Caryophyllene (content ¼ 56.0%); 2, germacrene D (12.7%); 3, bicyclogermacrene (9.2%); 4, caryophyllene oxide (3.0%); 5, a-humulene (1.9%); 6, spathulenol (1.9%); 7, caryophylla-4(14),8(15)dien-5-ol (1.5%); 8, b-elemene (1.4%).
440
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Table 1. Composition of the Essential Oil Isolated from the Aerial Parts of Melampodium divaricatum Compound name and class a )
RIapolar b )
RIpolar c )
Content [%] d )
1,8-Cineole Limonene Citronellal Terpinene-4-ol Eugenol a-Copaene d-Elemene b-Bourbonene b-Elemene (8) ( E )-Caryophyllene (1) Selina-3,6-diene b-Funebrene ( E )-a-Bergamotene a-Patchoulene a-Humulene (5) Germacrene D (2) b-Selinene Bicyclogermacrene (3) g-Cadinene d-Cadinene ( E )-Nerolidol Spathulenol (6) Caryophyllene oxide (4) Caryophylla-4(14),8(15)-dien-5-ol (7) t-Muurolol a-Cadinol
1019 1019 1129 1158 1324 1369 1328 1375 1383 1410 1417 1430 1430 1435 1440 1467 1472 1482 1497 1507 1543 1552 1556 1607 1614 1627
n.d. n.d 1480 1597 2159 1487 n.d. 1519 n.d. 1597 n.d. 1604 n.d. n.d. 1663 1702 1717 1727 1754 1754 2038 2111 1969 2296 n.d. 2223
tr tr tr tr tr tr 0.2 0.8 1.4 56.0 0.4 0.4 0.7 0.1 1.9 12.7 0.1 9.2 0.1 0.3 0.4 1.9 3.0 1.5 0.5 0.5
Monoterpene hydrocarbons Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Phenylpropanoids
tr 84.3 7.8 tr
Total identified a
92.1 b
) Compounds are listed in order of their elution on the apolar SPB-1 column. ) RIapolar : Retention index determined on the apolar SPB-1 column relative to a series of n-alkanes (C8 – C23 ). c ) RIpolar : Retention index determined on the polar SupelcoWax-10 column relative to a series of n-alkanes (C8 – C23 ); n.d., not determined. d ) Values are means (n ¼ 3); tr, traces.
metabolites of M. divaricatum in a CHCl3 extract of air-dried stems and leaves, together with guaianol and the oxygenated diterpene kolavenol [20]. To the best of our knowledge, this is the first report on the composition of M. divaricatum essential oil. Antimicrobial Activity against Oral Pathogens. The in vitro antimicrobial activity of the M. divaricatum oil and of some of its components are summarized in Table 2. The minimum inhibitory concentrations (MICs) of the oil ranged from 18 to > 400 mg/ml. At concentrations below 100 mg/ml, the essential oil inhibited the growth of four of the seven oral pathogens tested, viz., Streptococcus mitis (MIC ¼ 18 mg/ml), S. mutans (MIC ¼ 20 mg/ml), Lactobacillus casei (MIC ¼ 30 mg/ml), and S. sobrinus (MIC ¼ 90 mg/
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
441
Table 2. Growth Inhibitory Activity of the Melampodium divaricatum Essential Oil, Its Major Components 1, 4, and 5, and the Positive Control Chlorhexidine against Seven Oral Pathogens Oral pathogen
Enterococcus faecalis Lactobacillus casei Streptococcus mitis Streptococcus mutans Streptococcus sanguinis Streptococcus sobrinus Streptococcus salivarius
Minimum inhibitory concentration ( MIC) [mg/ml] Chlorhexidine
Essential oil
1
4
5
14.75 3.69 14.75 0.92 7.74 1.84 7.38
> 400 30 18 20 300 90 300
> 400 > 400 200 > 400 > 400 > 400 > 400
> 400 > 400 > 400 > 400 > 400 > 400 > 400
> 400 > 400 > 400 > 400 > 400 > 400 > 400
ml). Enterococcus faecalis, S. salivarius, and S. sanguinis were susceptible to the essential oil only at concentrations above 300 mg/ml. These are interesting results, particularly those concerning the growth inhibition of S. mitis and S. mutans. Indeed, the two bacterial strains belong to the principal causative organisms of dental caries for which few natural inhibitors are known [22]. The high content of caryophyllene derivatives in the essential oil (60.5%) and the published biocide activity of this kind of compounds [23 – 26] lead us to investigate the antimicrobial activity of (E)-caryophyllene, caryophyllene oxide, as well as the structurally related compound a-humulene. (E)-Caryophyllene is a characteristic compound of the essential oils of several Asteraceae species [27 – 30], among them Bidens sulphurea, whose oil proved to be active against S. mitis [14]. However, the MIC values of the tested oil components were higher than 200 mg/ml, revealing that they were ineffective against the tested bacterial strains. These results suggest that minor compounds of the M. divaricatum essential oil might play an important role in the bioactivity of the oil and that synergic effects among the oil components might also explain its remarkable activity. Conclusions. – The results demonstrated that M. divaricatum essential oil can be used either as an antimicrobial agent to treat infections by microorganisms responsible for dental caries and periodontal diseases or as a source of new active compounds with antimicrobial proprieties. Further studies are needed to identify the active compounds among the minor constituents, to characterize synergic effects, to elucidate the mechanisms of action, and to discard toxic effects. The authors thank the FUNDUNESP (Brazil) and the FundaÅa˜o para a Cieˆncia e Tecnologia (Portugal, project PEst-OE/SAU/UI0177/2011) for their financial support.
Experimental Part Plant Material. The aerial parts of Melampodium divaricatum (Rich.) DC. were collected in the garden, Horto de Plantas Medicinais e To´xicas, of the Faculdade de Cieˆncias Farmaceˆuticas - UNESP in Araraquara, Sa˜o Paulo state, Brazil (218 48’ 51.4’’ S and 488 12’ 5.1’’ W). A voucher specimen (HRCB 35294) has been deposited with the Herba´rio do Instituto de Biocieˆncias - UNESP in Rio Claro, Sa˜o Paulo state, Brazil.
442
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
Essential-Oil Isolation. The essential oil was obtained by hydrodistillation of the aerial parts for 4 h in a modified Clevenger apparatus according to the procedure described in the European Pharmacopoeia [31]. GC Analysis. The GC analyses were carried out using a Hewlett-Packard 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a HP GC ChemStation Rev. A.05.04 datahandling system, a single injector, and two flame-ionization detection (FID) systems. A Graphpak divider (Agilent Technologies, part number 5021 – 7148) was used for simultaneous sampling to two Supelco (Bellefonte, PA, USA) fused-silica cap. columns (30 m 0.20 mm i.d., film thickness 0.20 mm) with stationary phases of different polarity, i.e., an apolar SPB-1 (polydimethylsiloxane), and a polar Supelcowax-10 (polyethylene glycol) column. The oven temp. was programmed rising from 70 to 2208 at 38/min and then held isothermal at 2208 for 15 min; injector temp., 2508; FID temp., 2508; carrier gas, He (adjusted to a linear velocity of 30 cm/s); split ratio, 1 : 40. The calculation of the relative contents of the individual components was based on GC-FID raw-data areas without FID response-factor correction. GC/MS Analysis. The GC/MS analyses were carried out using a Hewlett-Packard 6890 gas chromatograph equipped with a HP1 fused-silica cap. column (polydimethylsiloxane, 30 m 0.25 mm i.d., film thickness 0.25 mm) and a Hewlett-Packard 5973 mass selective detector (Agilent Technologies) operated by HP Enhanced ChemStation software, version A.03.00. The GC parameters were as described above (cf. GC Analysis); interface temp., 2508; MS source temp., 2308; MS quadrupole temp., 1508; ionization energy, 70 eV; ionization current, 60 mA; scan range, 35 – 350 amu; scans, 4.51. Compound Identification. The identification of the individual essential-oil components was based on the comparison of i) their retention indices (RIs), determined on both SPB-1 and Supelcowax-10 columns by linear interpolation rel. to the retention times (tR ) of a series of n-alkanes (C8 – C23 ), with those of reference samples included in the home-made laboratory database of the Centro de Estudos Farmaceˆuticos, Faculty of Pharmacy, University of Coimbra, and ii) the acquired mass spectra with reference spectra compiled in the home-made laboratory database and the commercial Wiley and NIST mass-spectral libraries [32] and literature data [33] [34]. Antimicrobial Activity. The minimum inhibitory concentrations (MICs) of the crude essential oil, (E)-caryophylene (Chromadex), a-humulene (Chromadex), and Caryophylene oxide (95%, Sigma) were determined with the broth microdilution method [35] in 96-well microplates. The following standard strains from the American Type Culture Collection (ATCC) were used: Enterococcus faecalis (ATCC 4082), Streptococcus salivarius (ATCC 25975), Streptococcus sobrinus (ATCC 33478), Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Streptococcus sanguinis (ATCC 10556), and Lactobacillus casei (ATCC 11578). Individual 24-h colonies from blood agar (Difco Labs, Detroit, USA) were suspended in 10.0 ml of tryptic soy broth (Difco). The standardization of each microorganism suspension was carried out by adjusting the transmittance at l 625 nm to 81, equivalent to 0.5 McFarland scale (1.5 108 CFU/ml), using a Femto spectrophotometer (Sa˜o Paulo, Brazil). The suspensions were then diluted to a final concentration of 5 105 CFU/ml. The samples were dissolved in DMSO (Merck, Darmstadt, Germany) at a concentration of 1 mg/ml and were then diluted with tryptic soy broth to obtain concentrations in the range of 20 – 400 mg/ml. The final DMSO concentration was 5% (v/v), and DMSO solns. (concentrations ranging from 1 – 5%) were used as negative control. One inoculated well was included, to control the adequacy of the broth for organism growth, and one noninoculated well free of antimicrobial agent was included, to ensure medium sterility. Two-fold serial dilutions of chlorhexidine (Sigma) made in tryptic soy broth to achieve concentrations ranging from 59.0 – 0.115 mg/ml were used as positive control. The microplates (96 wells) were sealed with Parafilm and incubated at 378 for 24 h. After incubation, 30 ml of a 0.02% aq. soln. of resazurin (Sigma, St. Louis, MO, USA) were added to each microplate well, to indicate the microorganism viability [36]. The MIC value was determined as the lowest concentration of sample capable of inhibiting microorganism growth. Three replicates were made for each sample and microorganism.
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
443
REFERENCES [1] M. A. Botelho, N. A. Nogueira, G. M. Bastos, S. G. Fonseca, T. L. Lemos, F. J. Matos, D. Montenegro, J. Heukelbach, V. S. Rao, G. A. Brito, Braz. J. Med. Biol. Res. 2007, 40, 349. [2] P. D. Marsh, BMC Oral Health 2006, 6, S1. [3] P. Gilbert, T. Maira-Litran, A. J. McBain, A. H. Rickard, F. W. Whyte, Adv. Microb. Physiol. 2002, 46, 203. [4] N. Sharma, C. H. Charges, M. C. Lynch, J. Qaqish, J. A. McGuire, J. G. Galustians, L. D. Kumar, J. Am. Dent. Assoc. 2004, 135, 496. [5] L. Barnett, J. Am. Dent. Assoc. 2006, 137, 16S. [6] A. Santos, J. Clin. Periodontol. 2003, 5, 13. [7] H. F. Abd El Rahman, N. Skaug, G. W. Francis, Saudi Dent. J. 2002, 14, 26. [8] S. Prabuseenivasan, M. Jayakumar, S. Ignacimuthu, BMC Complement. Altern. Med. 2006, 6, 39. [9] M. L. A. Silva, H. Coimbra, A. C. Pereira, V. A. Souza, T. C. Lima, E. S. Costa, A. H. C. Vinholis, V. A. Royo, R. Silva, A. A. Silva Filho, W. R. Cunha, N. A. J. C. Furtado, Phytother. Res. 2007, 21, 420. [10] J. L. Rios, M. C. Recio, J. Ethnopharmacol. 2005, 100, 80. [11] G. F. Oliveira, N. A. J. C. Furtado, A. A. Silva Filho, C. H. G. Martins, J. K. Bastos, W. R. Cunha, M. L. A. Silva, Braz. J. Microbiol. 2007, 38, 381. [12] L. C. S. Cunha, M. L. A. Silva, N. A. J. C. Furtado, A. H. C. Vinho´lis, C. H. G. Martins, A. A. SilvaFilho, W. R. Cunha, Z. Naturforsch., C 2007, 62, 668. [13] K. Takarada, R. Kimizuka, N. Takahashi, K. Honma, K. Okuda, T. Kato, Oral Microbiol. Immunol. 2004, 19, 61. [14] G. P. Aguiar, C. E. Carvalho, H. J. Dias, E. B. Reis, M. H. G. Martins, K. A. L. Wakabayashi, M. Groppo, C. H. G. Martins, W. R. Cunha, A. E. M. Crotti, Nat. Prod. Res. 2013, 27, 1668. [15] M. F. Agra, P. F. Freitas, J. M. Barbosa-Filho, Rev. Bras. Farmacogn. 2007, 17, 114. [16] H. Lorenzi, Plantas Daninhas do Brasil: Terrestres, Aqua´ticas, Parasitas e To´xicas, 3rd edn., Instituto Plantarum, Nova Odessa, SP, 2002. [17] J. Hther, C. M. Passreiter, V. Wray, G. Willuhn, Phytochemistry 1999, 51, 979. [18] J. Borges-del-Castillo, A. I. Martinez-Martir, F. Rodriguez-Luis, J. C. Rodriguez-Ubis, P. VazquezBueno, Phytochemistry 1984, 23, 859. [19] J. Schngel, C. M. Passreiter, Biochem. Syst. Ecol. 2000, 28, 705. [20] T. D. Hubert, D. F. Wiemer, Phytochemistry 1985, 24, 1197. [21] G. P. Pelissari, R. C. L. R. Pietro, R. R. D. Moreira, Rev. Bras. Farmacogn. 2010, 20, 70. [22] M. Saleem, M. Nazir, M. S. Ali, H. Hussain, Y. S. Lee, N. Riaz, A. Jabbar, Nat. Prod. Rep. 2010, 27, 238. [23] D. Yang, L. Michel, J. P. Chaumont, J. Millet-Clerc, Mycopathologia 1999, 148, 79. [24] K. K. Macleod, H. B. Rasmussen, Phytochemistry 1999, 50, 105. [25] B. Demirci, K. H. C. Bas¸er, F. Demirci, M. T. Hamann, J. Nat. Prod. 2000, 63, 902. [26] C. Cavaleiro, M. J. GonÅalves, D. Serra, G. Santoro, F. Tomi, A. Bighelli, L. Salgueiro, J. Casanova, J. Pharm. Biomed. 2011, 54, 619. [27] R. K. Joshi, Nat. Prod. Commun. 2013, 8, 225. [28] R. K. Joshi, Nat. Prod. Commun. 2013, 8, 401. [29] M. Kazemi, S. Akhavani, Asian J. Chem. 2013, 25, 2985. [30] R. S. Verma, R. C. Padalia, A. Chauhan, Natl. Acad. Sci. Lett. 2013, 36, 447. [31] Council of Europe, European Pharmacopoeia, 3rd edn., Strasbourg, 1997. [32] Registry, 8th edn., with NIST 05 MS Spectra, Revision D.06.00, Agilent Technologies, Wiley, 2007. [33] R. P. Adams, Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy, Allured Publishing Corporation, Carol Stream, IL, USA, 1995. [34] D. Joulain, W. Koenig, The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998. [35] Clinical and Laboratory Standards Institute (CLSI), Document M7-A7, Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, 7th edn., CLSI, Wayne, PA, 2006, Vol. 23, No. 2.
444
CHEMISTRY & BIODIVERSITY – Vol. 11 (2014)
[36] T. S. Porto, R. Rangel, N. A. J. C. Furtado, T. C. Carvalho, C. H. G. Martins, R. C. S. Veneziani, F. B. Costa, A. H. C. Vinholis, W. R. Cunha, V. C. G. Heleno, S. R. Ambrosio, Molecules 2009, 14, 191. Received September 30, 2013
