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Composition and biological activity of the essential oil from leaves of Plinia cerrocampanensis, a new source of α-bisabolol
Bioresource Technology 101 (2010) 2510–2514
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Composition and biological activity of the essential oil from leaves of Plinia cerrocampanensis, a new source of a-bisabolol Roser Vila a, Ana Isabel Santana b, Renato Pérez-Rosés a, Anayansi Valderrama c, M. Victoria Castelli d, Sergio Mendonca e, Susana Zacchino d, Mahabir P. Gupta b, Salvador Cañigueral a,* a
Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panama, Panama, Republic of Panama Instituto Conmemorativo Gorgas en Estudio de Salud, Panama, Republic of Panama d Laboratorio de Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina e Laboratorio de Microbiología, Mestrado Profissional em Farmácia, Universidade Bandeirante de Sa˘o Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 28 October 2009 Accepted 5 November 2009 Available online 16 December 2009 Keywords: Plinia cerrocampanensis Essential oil a-Bisabolol Antimicrobial activity Aedes aegypti
a b s t r a c t The essential oil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae), obtained by hydrodistillation, was analysed by GC–FID and GC–MS. Forty components, representing more than 91% of the oil, were identified. Oxygenated sesquiterpenes represented the main fraction with a-bisabolol (42.8%) as the major constituent, making this plant a new and good source of this substance. Biological activity of the essential oil was evaluated against several bacterial and fungal strains as well as larvae from Aedes aegypti. The highest activity was found against Staphylococcus aureus, Pseudomonas aeruginosa, Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum with MIC values from 32 to 125 lg/ml. The essential oil also showed potent inhibitory and bactericidal activities against three H. pylori strains, with MIC and MBC values of 62.5 lg/ml, and caused 100% mortality of A. aegypti larvae at a concentration of 500 lg/ml. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Plants provide a multitude of flavours and fragrances which have found their way into everyday life. According to different authors, approximately 3000 plant species contain essential oils, from which only 300 are commercially important. Essential oils and some of their constituents are used not only in pharmaceutical products for their therapeutic activities but also in agriculture, as food preservers and additives for human or animal use, in cosmetics and perfumes, and other industrial fields. In many cases, they serve as plant defence mechanisms against predation by microorganisms, insects, and herbivores (Bakkali et al., 2008). The complex composition of the essential oils and the variety of chemical structures of their constituents are responsible of a wide range of biological activities many of which are of increasing interest in the fields of human and animal health. Particularly, many essential oils and their constituents have traditionally been used for their antimicrobial activity which has long been recognized. In addition, some of them may be useful in the control of mosquito larvae that are responsible of the transmission of several diseases
* Corresponding author. Tel.: +34 93 4024531; fax: +34 93 4035982. E-mail address: [email protected] (S. Cañigueral). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.11.021
such as malaria, dengue fever or yellow fever, that are among the greatest health problems in the world (Cheng et al., 2003, 2009). The need of new anti-infective agents due to the emergence of multiple antibiotic resistances has lead to the search of new sources of potential antimicrobials (Carson and Riley, 2003). Among them, the plant kingdom offers a wide range of biodiversity of great value for the pharmaceutical industry. Within the framework of our ongoing research on aromatic flora from Panama, in view of potentiate the use of its natural resources, the present work deals with the study of the essential oil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae), in particular its chemical composition and the evaluation of its biological activity against several bacterial and fungal strains, as well as against larvae from Aedes aegypti. P. cerrocampanensis, which has recently been described as a new species of the genus Plinia, is a tree that grows between 800 and 1000 m of altitude in the surroundings of Cerro Campana (Republic of Panama), reaching a height of about 8 m (Barrie, 2004). Until now no data on its chemical constituents or its biological activity are available in the scientific literature, although the composition of the essential oils (Apel et al., 2006; Pino et al., 2002, 2003) and the xanthine oxidase inhibitory activity of hydro-alcoholic extracts (Theoduloz et al., 1988) of other Plinia sp. have been reported previously.
R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514
2. Methods 2.1. Plant material Fresh leaves of P. cerrocampanensis Barrie were collected in the National Park ‘‘Altos de Campana” (N08°410 09.800 ; W079°560 04.400 ), Province of Panama (Republic of Panama) in February 2005. A voucher specimen No. FLORPAN 6623 is deposited in the Herbarium of the University of Panama (PMA). 2.2. Isolation and analysis of the essential oil The essential oil was obtained from 100 g of fresh leaves by hydrodistillation, using the standard apparatus described in the European Pharmacopeia (Council of Europe, 2005). Analysis of the oil was carried out by GC–FID and GC–MS using two fused silica capillary columns (30 m 0.25 mm i.d.; 0.25 lm film thickness) of different stationary phases: Supelcowax™ 10 and methylsilicone SE-30. GC–FID analyses were performed on a Hewlett–Packard 6890 instrument, equipped with a HP ChemStation data processor software, using the following analytical conditions: carrier gas, Helium; flow rate, 1 ml/min; oven temperature programmed from 60–220 °C at 4 °C/min, 220 °C (10 min); injector temperature, 250 °C; detector temperature, 270 °C; split ratio 1:80. The essential oil was injected undiluted (0.1 ll). Mass spectra were obtained with a computerized system constituted by a GC Hewlett–Packard 6890 coupled to a mass selective detector Hewlett– Packard 5973N, using the same analytical conditions as above. Mass spectra were taken over m/z 35–400, using an ionizing voltage of 70 eV. Identification of components was achieved by means of their GC retention indices in two stationary phases, determined in relation to a homologous series of fatty acid methyl esters, and by comparison of fragmentation patterns in the mass spectra with those stored in our own library, in the GC–MS database and with literature data (Adams, 1995; McLafferty, 1993). Quantification of each compound was performed on the basis of their GC peak areas on the two columns, using the normalisation procedure without corrections for response factor.
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and bacterial density adjusted to 6 108 CFU. Viability control was done by Gram staining and colony count. MIC and MBC determinations: broth microdilution was performed in brucella broth supplemented with 2% fetal calf serum (Cultilab, Brasil) and 0.2% DMSO. Two fold dilutions of essential oil ranging from 1000 to 7.8 lg/ml were used. The standardized inoculum was diluted to achieve a final inoculum concentration of approximately 6 106 CFU per well. The microplates were incubated at 37 °C under microaerophilic conditions. All assays were performed in duplicate using amoxicillin (ranging from 16 to 0.125 lg/ml) as positive internal control. The microplates were aseptically examined for the presence of turbidity after 72 h of incubation, and MIC were the lowest concentration of essential oil that inhibited detectable bacterial growth. After that, 2 ll of each sample were spread onto brucella-sheep blood agar plates in order to determine the MBC. These plates were monitored for the presence of bacterial growth after 48–72 h of incubation, and MBC were the lowest concentration that killed at least 99.9% of the original inoculum. Amoxicillin was used as a positive control. 2.5. Antifungal activity
The antibacterial activity was assayed against Escherichia coli (ATCC 9637), Klebsiella pneumoniae (ATCC 10031), Mycobacterium smegmatis (ATCC 607), Pseudomonas aeruginosa (ATCC 27853), Salmonella gallinarum (ATCC 9184) and Staphylococcus aureus (ATCC 6538), following the method described by Mitscher et al. (1971), using streptomycin sulphate as positive control. The essential oil was first dissolved in DMSO (1000 lg/ml) and the bacteriostatic activity was determined by measuring the minimum inhibitory concentration (MIC) from diluted aqueous samples of 500, 250, 125, 62.5, 31.25, 15.6 and 7.8 lg/ml. All the experiments were performed in triplicate and the results are expressed as mean values.
Antifungal activity was assayed against several yeasts and filamentous fungi strains from the American Type Culture Collection (ATCC, Rockville, MD, USA) and Centro de Referencia en Micología CEREMIC (C, Facultad de Ciencias Bioquímicas y Farmacéuticas, Rosario, Argentina): Candida albicans (ATCC 1023), C. tropicalis (C 131), Saccharomyces cerevisiae (ATCC 9763), Cryptococcus neoformans (ATCC 32264), Aspergillus flavus (ATCC 9170), A. fumigatus (ATCC 26934), A. niger (ATCC 9029), Trichophyton rubrum (C 110), Trichophyton mentagrophytes (ATCC 9972) and Microsporum gypseum (C 115). MIC values were determined using broth dilution techniques as described by the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards) for yeasts (M27-A2) (NCCLS, 2002a) as well for filamentous fungi (M38-A) (NCCLS, 2002b) in microtiters of 96 wells. RPMI1640 (Sigma, St. Louis, Mo, USA) buffered to a pH 7.0 with MOPS was used. The starting inocula were approximately 1 103 to 5 103 CFU/ml. Microtiter trays were incubated at 35 °C for yeasts and hyalohyphomycetes and at 28–30 °C for dermatophytes in a moist, dark chamber. MIC values were recorded at 48 h for yeasts and at a time according to the control fungus growth, for the other fungi. The susceptibility of the standard drugs ketoconazole, terbinafine and amphotericin B was defined as the lowest concentration of drug which resulted in total inhibition of fungal growth. For the assay, the essential oil of P. cerrocampanensis was twofold diluted with RPMI from 1000 to 0.98 lg/ml (final volume = 100 ll) and a final DMSO concentration 61%. A volume of 100 ll of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. The MIC was defined as the minimum inhibitory concentration of the essential oil which resulted in total inhibition of the fungal growth.
2.4. Anti-Helicobacter pylori activity
2.6. Larvicidal activity
Stock cultures of H. pylori 26695 (ATCC 700392), J99 (ATCC 700824) and SS1 (Sidney Strain 1) were reactivated on Columbia agar plates (CA) (Merck, Germany) supplemented with 10% defibrinated sheep blood (BBV, Brazil), 10 mg/L vancomycin, 20 mg/L nalidixic acid, 2 mg/L amphotericin B and 40 mg/L 2,3,5-triphenyltetrazolium chloride (Sigma, Germany), and cultured at 37 °C in a humidified 12% CO2 incubator (Revco, USA). The identity of the colonies was confirmed by Gram staining and oxidase, catalase and urease production. The colonies were suspended in PBS (pH 7.2)
For this purpose, larvae of A. aegypti (Culicidade: Diptera) of III and IV Stage obtained after 6–8 days post oviposition were used. The insectarium was kept at temperature of 22.5–25 °C, relative humidity of 80% and with a photoperiod of 12:12 h. The larvae were fed with granulated yeast suspended in water (1:4). The essential oils were dissolved in ethanol and were tested at a final concentration of 1, 100 and 500 ppm in triplicate. Water and alcohol were used as control. For each replicate, 20 individuals of mosquito species were placed in a foam container. After 24 h, per
2.3. Antibacterial activity
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cent mortality was calculated. Tetramethrin with a LC100 value of 0.25 ± 0.01 lg/ml was used as a positive control. 3. Results and discussion The fresh leaves of P. cerrocampanensis gave by hydrodistillation an essential oil yield of 0.64% (v/w). Qualitative and quantitative analysis of the oil by GC and GC–MS allowed the identification of forty components, representing more than 91% of the total sample. The oil was very rich in oxygenated sesquiterpenes (65.9%), especially a-bisabolol (42.8%), bisabolol oxide B (10.3%) and trans-nerolidol (9.4%). Among monoterpenes, linalool (10.3%) was found in the highest percentage. Further details of the essential oil composition are shown in Table 1. Table 1 Composition of the essential oil from leaves of Plinia cerrocampanensis. Constituentsa
RI–CWb
RI–SEc
%d
a-Pinene
112 204 220 225 237 271 323 337 378 380 399 434 439 447 457 462 474 483 489 510 521 544 615 623 656 662 676 700 710 763 824 – – – – – – – – –
209 251 255 266 246 224 270 278 287 296 323 – 475 329 487 492 494 477 – – – – 518 – 562 568 – 579 555 593 640 208 243 260 281 428 445 458 489 508
0.2e,f,g 0.4e,f,g 0.1e,f,g 0.5e,f,g 1.1e,f,g 0.2e,f,g 0.6e,f,g 0.7e,f,g 10.3e,f,g 0.1e,f,g 1.1e,f,g 0.3e,f 0.1e,f,g 1.2e,f,g 0.1e,f,g 4.8e,f,g 0.6e,f,g 0.8e,f,g 0.1e,f 0.3e,f 0.7e,f 0.1e,f 9.4e,f,g te,f 10.3e,f,g 1.4e,f,g 0.5e,f 42.8e,f,g 0.6e,f,g 0.5e,f,g 0.4e,f,g 0.1e,g te,g 0.1e,g 0.1e,g 0.1e,g 0.2e,g 0.1e,g 0.3e,g 0.3e,g
Limonene cis-Ocimene c-Terpinene p-Cymene 6-Methyl-5-hepten-2-one trans-Linalool oxide cis-Linalool oxide Linalool 4-Acetyl-1-methyl-1-cyclohexene Terpinen-4-ol Estragole a-Amorphene a-Terpineol a-Muurolene b-Bisabolene d-Cadinene a-Curcumene Nerol Calameneneh Geraniol a-Calacorene trans-Nerolidol Cubenol a-Bisabolol oxide B b-Bisabolol 10-epi-Cadinol a-Bisabolol b-Eudesmol E,E-Farnesol a-Bisabolol oxide A Benzaldehyde d-3-Carene trans-b-Ocimene Terpinolene a-Copaene a-Cedrene trans-a-Bergamotene cis-a-Bisabolene trans-a-Bisabolene
This composition pattern is quite unusual among essential oils from leaves of other Plinia sp. previously reported. Although most of them are also characterized by the predominance of oxygenated sesquiterpenes (Apel et al., 2006; Pino et al., 2003), only the one from P. cordifolia showed remarkable percentages of compounds with a bisabolane nucleus, such as a-bisabolol oxide A (28.0%), a-bisabolol oxide B (7.0%) and a-bisabolol (5.8%) (Apel et al., 2006). Results on the antibacterial activity of the essential oil of P. cerrocampanensis are summarized in Table 2. It exhibited the strongest activity against P. aeruginosa and S. aureus with MIC values of 62.5 and 125 lg/ml, respectively. This essential oil also showed potent inhibitory and bactericidal activities against three H. pylori strains (including SS1, traditionally used for in vivo assays), with MIC and MBC values of 62.5 lg/ml. H. pylori, a gastric pathogen whose infection is associated with chronic superficial gastritis, peptic ulceration and gastric cancer, chronically infects more than half of the world’s population. To be effective, therapies require the use of more than one antimicrobial in combination. Unfortunately, increased primary resistance to recommended antibiotics modifies the therapy effectiveness and negatively affects its eradication, requiring the search for new strategies (Ortiz Godoy et al., 2003). Therefore, the oil of P. cerrocampanensis stands out as a new contribution in this search. MIC values towards several yeasts and fungi strains are shown in Table 3. Dermatophytes were the most sensitive ones, particularly T. mentagrophytes, T. rubrum and M. gypseum with MIC values of 32, 62.5 and 125 lg/ml, respectively. All these activities can be related to the major constituents, mainly a-bisabolol (Kedzia, 1991; Szalontai et al., 1976), linalool (Pattnaik et al., 1997; Sonboli et al., 2006) and nerolidol (Kubo et al., 1992) whose antimicrobial properties have been previously reported. Particularly, nerolidol has been recently found to inhibit the hyphal growth of T. mentagrophytes causing destruction and disorganization of organelles in the fungal cytoplasm (Park et al., 2009). A. aegypti is the major vector of dengue and yellow fever which have experimented a recrudescence due to the increasing resistance of mosquitoes to current commercial insecticides. Although yellow fever has been reasonably brought under control with its vaccine, no vaccine is available for dengue. Several secondary metabolite plant products have been successfully assayed for their larvicidal activity against A. aegypti, particularly essential oils and their constituents. (Barreira Cavalcanti et al., 2004; Urano Carvalho et al., 2003). In the present work, the essential oil of P. cerrocampanensis showed, at concentrations of 100 and 500 lg/ml, 53% and 100% mortality of A. aegypti larvae, respectively, constituting a potential alternative to the conventional chemical control. Table 2 Antibacterial activity of the essential oil of Plinia cerrocampanensis. Bacteria
EOPCa
Streptomycin sulphate MIC
Amoxicillin
>1000 >1000 >1000
6.25 3.12 12.50
– – –
62.5 >1000 125 62.5/62.5 62.5/62.5 62.5/62.5
1.56 3.12 12.50 – – –
– – – 0.125/0.5 0.125/0.5 0.125/0.5
b
Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified
MIC / MBCc
2.5 14.7 7.8 65.9 0.7 91.6
Escherichia coli Klebsiella pneumoniae Mycobacterium smegmatis Pseudomonas aeruginosa Salmonella gallinarum Staphylococcus aureus Helicobacter pylori 26695 Helicobacter pylori J99 Helicobacter pylori SS1
a
Components are listed in increasing order according to their retention indices in Supelcowax™ 10 except the last nine constituents, which were only detected in methylsilicone. b RI–CW: retention indices in Supelcowax™10 column. c RI–SE: retention indices in methylsilicone (SE-30) column. d t: traces (60.05). e Identification method: GC–MS. f Identification method: retention index in Supelcowax™10. g Identification method: retention index in methylsilicone. h Isomer not assigned.
a
MIC/MBC
EOPC: essential oil of P. cerrocampanensis. MIC: minimum inhibitory concentration (lg/ml). c MBC: minimum bactericidal concentration (lg/ml). MBC was only determined against Helicobacter pylori. b
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R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514 Table 3 Antifungal activity of the essential oil of Plinia cerrocampanensis. EOPCa MICb
Amphotericin B MIC
Ketoconazole MIC
Terbinafine MIC
Yeasts Candida albicans Candida tropicalis Cryptococcus neoformans Saccharomyces cerevisiae
>250 >250 >250 >250
0.78 1.56 0.78 0.78
6.25 6.25 1.56 3.12
– – – –
Filamentous fungi Aspergillus flavus Aspergillus fumigatus Aspergillus niger
>250 >250 >250
0.78 3.12 0.78
6.25 12.5 6.25
– – –
Dermatophytes Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
125 32 62.5
0.25 0.75 0.75
0.50 0.25 0.25
0.04 0.02 0.01
Fungi
a b
EOPC: essential oil of P. cerrocampanensis. MIC: minimum inhibitory concentration (lg/ml).
The essential oil of P. cerrocampanensis contains a 42% of abisabolol, its major constituent, being a potential industrial source of this interesting compound. a-Bisabolol, a well known monocyclic unsaturated sesquiterpene alcohol which is one of the main active principles of chamomile (Chamomilla recutita), is widely used in cosmetic preparations due to its anti-inflammatory activity and low toxicity (Habersang et al., 1979; Hempel and Hirschelmann, 1998; Isaac, 1979; Jellinek, 1984; Madhavan, 1999; Yakovlev and von Schlichtegroll, 1969). Furthermore, both a-bisabolol and nerolidol, also one of the main constituents of the essential oil (9.4%), may increase dermal absorption of other substances by more than 20-fold, being useful vehicles for other drugs (Cornwell and Barry, 1994). These two sesquiterpenes are also able to enhance bacterial permeability and susceptibility to clinically important antibiotic compounds (Brehm-Stecher and Johnson, 2003). Moreover, a-bisabolol has been found to be a promising inducer of apoptosis in highly malignant glioma cells (Cavalieri et al., 2004). Finally, it is interesting to highlight that linalool, the major monoterpene of the oil of P. cerrocampanensis (10.3%), could also enhance the activities of a-bisabolol in the oil, since it has shown anti-inflammatory, anti-hyperalgesic and anti-nociceptive effects in several animal models, which have been ascribed to different mechanisms of action (Peana et al., 2002, 2004, 2006a,b; Re et al., 2000). 4. Conclusions In conclusion, the essential oil from leaves of P. cerrocampanensis from Panama is an outstanding new source of a-bisabolol, a compound highly appreciated in the pharmaceutical and cosmetic industry. In addition, it shows interesting antimicrobial and larvicidal activities which make this essential oil a potential industrial resource of new products. Acknowledgements Authors are grateful to National Secretariat for Science, Technology, and Innovation (SENACYT) of Panama, Project No. 112004 and the Organization of American States for financial support to CIFLORPAN. Also, thanks are due to Cristina Minguillon and Antoni Riera (University of Barcelona) for helping to confirm the identification of a-bisabolol by means chiral chromatography. R. Pérez-Rosés was supported by the Generalitat de Catalunya (Education and Universities Department) and the European Social Fund. S. Zacchino is grateful to ANPCyT (PICT R 260). M.V. Castelli acknowledges CONICET.
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Pino, J.A., Bello, A., Urquiola, A., Correa, T., Rosado, A., 2002. Essential oil of Plinia rubrinervis Urb from Cuba. J. Essent. Oil Res. 14, 372. Pino, J.A., Bello, A., Urquiola, A., Aguero, J., 2003. Chemical composition of the leaf oil of Plinia dermatodes. J. Essent. Oil Res. 15, 23–24. Re, L., Baroci, S., Mencarelli, A., Vivani, C., Paolucci, G., Scarpantonio, A., Rinaldi, L., Mosca, E., 2000. Linalool modifies the nicotinic receptor-ion channel kinetics at the mouse neuromuscular junction. Pharmacol. Res. 42, 177–181. Sonboli, A., Babakhani, B., Mehrabian, A.R., 2006. Antimicrobial activity of six constituents of essential oil from Salvia. Z. Naturforsch C – J. Biosci. 61, 160– 164. Szalontai, M., Verzar-Petri, G., Florian, E., 1976. Data on the antifungal effect of the biologically active components of Matricaria chamomilla L. Acta Farm. Hung. 46, 232–247. Theoduloz, C., Franco, L., Ferro, E., Schmeda Hirschmann, G., 1988. Xanthine oxidase inhibitory activity of Paraguayan Myrtaceae. J. Ethnopharmacol. 24, 179–183. Urano Carvalho, A.F., Maciel Melo, V.M., Aragão Craveiro, A., Lacerda Machado, M.I., Braga Bantim, M., Fontenele Rabelo, E., 2003. Larvicidal activity of the essential oil from Lippia sidoides Cham. against Aedes aegypti Linn. Mem. Inst. Oswaldo Cruz Rio de Janeiro 98, 569–571. Yakovlev, V., von Schlichtegroll, A., 1969. Anti-inflammatory activity of ()-abisabolol, an essential oil component of chamomile oil. Arzneimittel–Forsch. 19, 615–616.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Composition and biological activity of the essential oil from leaves of Plinia cerrocampanensis, a new source of a-bisabolol Roser Vila a, Ana Isabel Santana b, Renato Pérez-Rosés a, Anayansi Valderrama c, M. Victoria Castelli d, Sergio Mendonca e, Susana Zacchino d, Mahabir P. Gupta b, Salvador Cañigueral a,* a
Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panama, Panama, Republic of Panama Instituto Conmemorativo Gorgas en Estudio de Salud, Panama, Republic of Panama d Laboratorio de Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina e Laboratorio de Microbiología, Mestrado Profissional em Farmácia, Universidade Bandeirante de Sa˘o Paulo, Brazil b c
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 28 October 2009 Accepted 5 November 2009 Available online 16 December 2009 Keywords: Plinia cerrocampanensis Essential oil a-Bisabolol Antimicrobial activity Aedes aegypti
a b s t r a c t The essential oil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae), obtained by hydrodistillation, was analysed by GC–FID and GC–MS. Forty components, representing more than 91% of the oil, were identified. Oxygenated sesquiterpenes represented the main fraction with a-bisabolol (42.8%) as the major constituent, making this plant a new and good source of this substance. Biological activity of the essential oil was evaluated against several bacterial and fungal strains as well as larvae from Aedes aegypti. The highest activity was found against Staphylococcus aureus, Pseudomonas aeruginosa, Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum with MIC values from 32 to 125 lg/ml. The essential oil also showed potent inhibitory and bactericidal activities against three H. pylori strains, with MIC and MBC values of 62.5 lg/ml, and caused 100% mortality of A. aegypti larvae at a concentration of 500 lg/ml. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Plants provide a multitude of flavours and fragrances which have found their way into everyday life. According to different authors, approximately 3000 plant species contain essential oils, from which only 300 are commercially important. Essential oils and some of their constituents are used not only in pharmaceutical products for their therapeutic activities but also in agriculture, as food preservers and additives for human or animal use, in cosmetics and perfumes, and other industrial fields. In many cases, they serve as plant defence mechanisms against predation by microorganisms, insects, and herbivores (Bakkali et al., 2008). The complex composition of the essential oils and the variety of chemical structures of their constituents are responsible of a wide range of biological activities many of which are of increasing interest in the fields of human and animal health. Particularly, many essential oils and their constituents have traditionally been used for their antimicrobial activity which has long been recognized. In addition, some of them may be useful in the control of mosquito larvae that are responsible of the transmission of several diseases
* Corresponding author. Tel.: +34 93 4024531; fax: +34 93 4035982. E-mail address: [email protected] (S. Cañigueral). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.11.021
such as malaria, dengue fever or yellow fever, that are among the greatest health problems in the world (Cheng et al., 2003, 2009). The need of new anti-infective agents due to the emergence of multiple antibiotic resistances has lead to the search of new sources of potential antimicrobials (Carson and Riley, 2003). Among them, the plant kingdom offers a wide range of biodiversity of great value for the pharmaceutical industry. Within the framework of our ongoing research on aromatic flora from Panama, in view of potentiate the use of its natural resources, the present work deals with the study of the essential oil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae), in particular its chemical composition and the evaluation of its biological activity against several bacterial and fungal strains, as well as against larvae from Aedes aegypti. P. cerrocampanensis, which has recently been described as a new species of the genus Plinia, is a tree that grows between 800 and 1000 m of altitude in the surroundings of Cerro Campana (Republic of Panama), reaching a height of about 8 m (Barrie, 2004). Until now no data on its chemical constituents or its biological activity are available in the scientific literature, although the composition of the essential oils (Apel et al., 2006; Pino et al., 2002, 2003) and the xanthine oxidase inhibitory activity of hydro-alcoholic extracts (Theoduloz et al., 1988) of other Plinia sp. have been reported previously.
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2. Methods 2.1. Plant material Fresh leaves of P. cerrocampanensis Barrie were collected in the National Park ‘‘Altos de Campana” (N08°410 09.800 ; W079°560 04.400 ), Province of Panama (Republic of Panama) in February 2005. A voucher specimen No. FLORPAN 6623 is deposited in the Herbarium of the University of Panama (PMA). 2.2. Isolation and analysis of the essential oil The essential oil was obtained from 100 g of fresh leaves by hydrodistillation, using the standard apparatus described in the European Pharmacopeia (Council of Europe, 2005). Analysis of the oil was carried out by GC–FID and GC–MS using two fused silica capillary columns (30 m 0.25 mm i.d.; 0.25 lm film thickness) of different stationary phases: Supelcowax™ 10 and methylsilicone SE-30. GC–FID analyses were performed on a Hewlett–Packard 6890 instrument, equipped with a HP ChemStation data processor software, using the following analytical conditions: carrier gas, Helium; flow rate, 1 ml/min; oven temperature programmed from 60–220 °C at 4 °C/min, 220 °C (10 min); injector temperature, 250 °C; detector temperature, 270 °C; split ratio 1:80. The essential oil was injected undiluted (0.1 ll). Mass spectra were obtained with a computerized system constituted by a GC Hewlett–Packard 6890 coupled to a mass selective detector Hewlett– Packard 5973N, using the same analytical conditions as above. Mass spectra were taken over m/z 35–400, using an ionizing voltage of 70 eV. Identification of components was achieved by means of their GC retention indices in two stationary phases, determined in relation to a homologous series of fatty acid methyl esters, and by comparison of fragmentation patterns in the mass spectra with those stored in our own library, in the GC–MS database and with literature data (Adams, 1995; McLafferty, 1993). Quantification of each compound was performed on the basis of their GC peak areas on the two columns, using the normalisation procedure without corrections for response factor.
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and bacterial density adjusted to 6 108 CFU. Viability control was done by Gram staining and colony count. MIC and MBC determinations: broth microdilution was performed in brucella broth supplemented with 2% fetal calf serum (Cultilab, Brasil) and 0.2% DMSO. Two fold dilutions of essential oil ranging from 1000 to 7.8 lg/ml were used. The standardized inoculum was diluted to achieve a final inoculum concentration of approximately 6 106 CFU per well. The microplates were incubated at 37 °C under microaerophilic conditions. All assays were performed in duplicate using amoxicillin (ranging from 16 to 0.125 lg/ml) as positive internal control. The microplates were aseptically examined for the presence of turbidity after 72 h of incubation, and MIC were the lowest concentration of essential oil that inhibited detectable bacterial growth. After that, 2 ll of each sample were spread onto brucella-sheep blood agar plates in order to determine the MBC. These plates were monitored for the presence of bacterial growth after 48–72 h of incubation, and MBC were the lowest concentration that killed at least 99.9% of the original inoculum. Amoxicillin was used as a positive control. 2.5. Antifungal activity
The antibacterial activity was assayed against Escherichia coli (ATCC 9637), Klebsiella pneumoniae (ATCC 10031), Mycobacterium smegmatis (ATCC 607), Pseudomonas aeruginosa (ATCC 27853), Salmonella gallinarum (ATCC 9184) and Staphylococcus aureus (ATCC 6538), following the method described by Mitscher et al. (1971), using streptomycin sulphate as positive control. The essential oil was first dissolved in DMSO (1000 lg/ml) and the bacteriostatic activity was determined by measuring the minimum inhibitory concentration (MIC) from diluted aqueous samples of 500, 250, 125, 62.5, 31.25, 15.6 and 7.8 lg/ml. All the experiments were performed in triplicate and the results are expressed as mean values.
Antifungal activity was assayed against several yeasts and filamentous fungi strains from the American Type Culture Collection (ATCC, Rockville, MD, USA) and Centro de Referencia en Micología CEREMIC (C, Facultad de Ciencias Bioquímicas y Farmacéuticas, Rosario, Argentina): Candida albicans (ATCC 1023), C. tropicalis (C 131), Saccharomyces cerevisiae (ATCC 9763), Cryptococcus neoformans (ATCC 32264), Aspergillus flavus (ATCC 9170), A. fumigatus (ATCC 26934), A. niger (ATCC 9029), Trichophyton rubrum (C 110), Trichophyton mentagrophytes (ATCC 9972) and Microsporum gypseum (C 115). MIC values were determined using broth dilution techniques as described by the Clinical and Laboratory Standards Institute (CLSI, formerly National Committee for Clinical and Laboratory Standards) for yeasts (M27-A2) (NCCLS, 2002a) as well for filamentous fungi (M38-A) (NCCLS, 2002b) in microtiters of 96 wells. RPMI1640 (Sigma, St. Louis, Mo, USA) buffered to a pH 7.0 with MOPS was used. The starting inocula were approximately 1 103 to 5 103 CFU/ml. Microtiter trays were incubated at 35 °C for yeasts and hyalohyphomycetes and at 28–30 °C for dermatophytes in a moist, dark chamber. MIC values were recorded at 48 h for yeasts and at a time according to the control fungus growth, for the other fungi. The susceptibility of the standard drugs ketoconazole, terbinafine and amphotericin B was defined as the lowest concentration of drug which resulted in total inhibition of fungal growth. For the assay, the essential oil of P. cerrocampanensis was twofold diluted with RPMI from 1000 to 0.98 lg/ml (final volume = 100 ll) and a final DMSO concentration 61%. A volume of 100 ll of inoculum suspension was added to each well with the exception of the sterility control where sterile water was added to the well instead. The MIC was defined as the minimum inhibitory concentration of the essential oil which resulted in total inhibition of the fungal growth.
2.4. Anti-Helicobacter pylori activity
2.6. Larvicidal activity
Stock cultures of H. pylori 26695 (ATCC 700392), J99 (ATCC 700824) and SS1 (Sidney Strain 1) were reactivated on Columbia agar plates (CA) (Merck, Germany) supplemented with 10% defibrinated sheep blood (BBV, Brazil), 10 mg/L vancomycin, 20 mg/L nalidixic acid, 2 mg/L amphotericin B and 40 mg/L 2,3,5-triphenyltetrazolium chloride (Sigma, Germany), and cultured at 37 °C in a humidified 12% CO2 incubator (Revco, USA). The identity of the colonies was confirmed by Gram staining and oxidase, catalase and urease production. The colonies were suspended in PBS (pH 7.2)
For this purpose, larvae of A. aegypti (Culicidade: Diptera) of III and IV Stage obtained after 6–8 days post oviposition were used. The insectarium was kept at temperature of 22.5–25 °C, relative humidity of 80% and with a photoperiod of 12:12 h. The larvae were fed with granulated yeast suspended in water (1:4). The essential oils were dissolved in ethanol and were tested at a final concentration of 1, 100 and 500 ppm in triplicate. Water and alcohol were used as control. For each replicate, 20 individuals of mosquito species were placed in a foam container. After 24 h, per
2.3. Antibacterial activity
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cent mortality was calculated. Tetramethrin with a LC100 value of 0.25 ± 0.01 lg/ml was used as a positive control. 3. Results and discussion The fresh leaves of P. cerrocampanensis gave by hydrodistillation an essential oil yield of 0.64% (v/w). Qualitative and quantitative analysis of the oil by GC and GC–MS allowed the identification of forty components, representing more than 91% of the total sample. The oil was very rich in oxygenated sesquiterpenes (65.9%), especially a-bisabolol (42.8%), bisabolol oxide B (10.3%) and trans-nerolidol (9.4%). Among monoterpenes, linalool (10.3%) was found in the highest percentage. Further details of the essential oil composition are shown in Table 1. Table 1 Composition of the essential oil from leaves of Plinia cerrocampanensis. Constituentsa
RI–CWb
RI–SEc
%d
a-Pinene
112 204 220 225 237 271 323 337 378 380 399 434 439 447 457 462 474 483 489 510 521 544 615 623 656 662 676 700 710 763 824 – – – – – – – – –
209 251 255 266 246 224 270 278 287 296 323 – 475 329 487 492 494 477 – – – – 518 – 562 568 – 579 555 593 640 208 243 260 281 428 445 458 489 508
0.2e,f,g 0.4e,f,g 0.1e,f,g 0.5e,f,g 1.1e,f,g 0.2e,f,g 0.6e,f,g 0.7e,f,g 10.3e,f,g 0.1e,f,g 1.1e,f,g 0.3e,f 0.1e,f,g 1.2e,f,g 0.1e,f,g 4.8e,f,g 0.6e,f,g 0.8e,f,g 0.1e,f 0.3e,f 0.7e,f 0.1e,f 9.4e,f,g te,f 10.3e,f,g 1.4e,f,g 0.5e,f 42.8e,f,g 0.6e,f,g 0.5e,f,g 0.4e,f,g 0.1e,g te,g 0.1e,g 0.1e,g 0.1e,g 0.2e,g 0.1e,g 0.3e,g 0.3e,g
Limonene cis-Ocimene c-Terpinene p-Cymene 6-Methyl-5-hepten-2-one trans-Linalool oxide cis-Linalool oxide Linalool 4-Acetyl-1-methyl-1-cyclohexene Terpinen-4-ol Estragole a-Amorphene a-Terpineol a-Muurolene b-Bisabolene d-Cadinene a-Curcumene Nerol Calameneneh Geraniol a-Calacorene trans-Nerolidol Cubenol a-Bisabolol oxide B b-Bisabolol 10-epi-Cadinol a-Bisabolol b-Eudesmol E,E-Farnesol a-Bisabolol oxide A Benzaldehyde d-3-Carene trans-b-Ocimene Terpinolene a-Copaene a-Cedrene trans-a-Bergamotene cis-a-Bisabolene trans-a-Bisabolene
This composition pattern is quite unusual among essential oils from leaves of other Plinia sp. previously reported. Although most of them are also characterized by the predominance of oxygenated sesquiterpenes (Apel et al., 2006; Pino et al., 2003), only the one from P. cordifolia showed remarkable percentages of compounds with a bisabolane nucleus, such as a-bisabolol oxide A (28.0%), a-bisabolol oxide B (7.0%) and a-bisabolol (5.8%) (Apel et al., 2006). Results on the antibacterial activity of the essential oil of P. cerrocampanensis are summarized in Table 2. It exhibited the strongest activity against P. aeruginosa and S. aureus with MIC values of 62.5 and 125 lg/ml, respectively. This essential oil also showed potent inhibitory and bactericidal activities against three H. pylori strains (including SS1, traditionally used for in vivo assays), with MIC and MBC values of 62.5 lg/ml. H. pylori, a gastric pathogen whose infection is associated with chronic superficial gastritis, peptic ulceration and gastric cancer, chronically infects more than half of the world’s population. To be effective, therapies require the use of more than one antimicrobial in combination. Unfortunately, increased primary resistance to recommended antibiotics modifies the therapy effectiveness and negatively affects its eradication, requiring the search for new strategies (Ortiz Godoy et al., 2003). Therefore, the oil of P. cerrocampanensis stands out as a new contribution in this search. MIC values towards several yeasts and fungi strains are shown in Table 3. Dermatophytes were the most sensitive ones, particularly T. mentagrophytes, T. rubrum and M. gypseum with MIC values of 32, 62.5 and 125 lg/ml, respectively. All these activities can be related to the major constituents, mainly a-bisabolol (Kedzia, 1991; Szalontai et al., 1976), linalool (Pattnaik et al., 1997; Sonboli et al., 2006) and nerolidol (Kubo et al., 1992) whose antimicrobial properties have been previously reported. Particularly, nerolidol has been recently found to inhibit the hyphal growth of T. mentagrophytes causing destruction and disorganization of organelles in the fungal cytoplasm (Park et al., 2009). A. aegypti is the major vector of dengue and yellow fever which have experimented a recrudescence due to the increasing resistance of mosquitoes to current commercial insecticides. Although yellow fever has been reasonably brought under control with its vaccine, no vaccine is available for dengue. Several secondary metabolite plant products have been successfully assayed for their larvicidal activity against A. aegypti, particularly essential oils and their constituents. (Barreira Cavalcanti et al., 2004; Urano Carvalho et al., 2003). In the present work, the essential oil of P. cerrocampanensis showed, at concentrations of 100 and 500 lg/ml, 53% and 100% mortality of A. aegypti larvae, respectively, constituting a potential alternative to the conventional chemical control. Table 2 Antibacterial activity of the essential oil of Plinia cerrocampanensis. Bacteria
EOPCa
Streptomycin sulphate MIC
Amoxicillin
>1000 >1000 >1000
6.25 3.12 12.50
– – –
62.5 >1000 125 62.5/62.5 62.5/62.5 62.5/62.5
1.56 3.12 12.50 – – –
– – – 0.125/0.5 0.125/0.5 0.125/0.5
b
Monoterpene hydrocarbons Oxygenated monoterpenes Sesquiterpene hydrocarbons Oxygenated sesquiterpenes Others Total identified
MIC / MBCc
2.5 14.7 7.8 65.9 0.7 91.6
Escherichia coli Klebsiella pneumoniae Mycobacterium smegmatis Pseudomonas aeruginosa Salmonella gallinarum Staphylococcus aureus Helicobacter pylori 26695 Helicobacter pylori J99 Helicobacter pylori SS1
a
Components are listed in increasing order according to their retention indices in Supelcowax™ 10 except the last nine constituents, which were only detected in methylsilicone. b RI–CW: retention indices in Supelcowax™10 column. c RI–SE: retention indices in methylsilicone (SE-30) column. d t: traces (60.05). e Identification method: GC–MS. f Identification method: retention index in Supelcowax™10. g Identification method: retention index in methylsilicone. h Isomer not assigned.
a
MIC/MBC
EOPC: essential oil of P. cerrocampanensis. MIC: minimum inhibitory concentration (lg/ml). c MBC: minimum bactericidal concentration (lg/ml). MBC was only determined against Helicobacter pylori. b
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R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514 Table 3 Antifungal activity of the essential oil of Plinia cerrocampanensis. EOPCa MICb
Amphotericin B MIC
Ketoconazole MIC
Terbinafine MIC
Yeasts Candida albicans Candida tropicalis Cryptococcus neoformans Saccharomyces cerevisiae
>250 >250 >250 >250
0.78 1.56 0.78 0.78
6.25 6.25 1.56 3.12
– – – –
Filamentous fungi Aspergillus flavus Aspergillus fumigatus Aspergillus niger
>250 >250 >250
0.78 3.12 0.78
6.25 12.5 6.25
– – –
Dermatophytes Microsporum gypseum Trichophyton mentagrophytes Trichophyton rubrum
125 32 62.5
0.25 0.75 0.75
0.50 0.25 0.25
0.04 0.02 0.01
Fungi
a b
EOPC: essential oil of P. cerrocampanensis. MIC: minimum inhibitory concentration (lg/ml).
The essential oil of P. cerrocampanensis contains a 42% of abisabolol, its major constituent, being a potential industrial source of this interesting compound. a-Bisabolol, a well known monocyclic unsaturated sesquiterpene alcohol which is one of the main active principles of chamomile (Chamomilla recutita), is widely used in cosmetic preparations due to its anti-inflammatory activity and low toxicity (Habersang et al., 1979; Hempel and Hirschelmann, 1998; Isaac, 1979; Jellinek, 1984; Madhavan, 1999; Yakovlev and von Schlichtegroll, 1969). Furthermore, both a-bisabolol and nerolidol, also one of the main constituents of the essential oil (9.4%), may increase dermal absorption of other substances by more than 20-fold, being useful vehicles for other drugs (Cornwell and Barry, 1994). These two sesquiterpenes are also able to enhance bacterial permeability and susceptibility to clinically important antibiotic compounds (Brehm-Stecher and Johnson, 2003). Moreover, a-bisabolol has been found to be a promising inducer of apoptosis in highly malignant glioma cells (Cavalieri et al., 2004). Finally, it is interesting to highlight that linalool, the major monoterpene of the oil of P. cerrocampanensis (10.3%), could also enhance the activities of a-bisabolol in the oil, since it has shown anti-inflammatory, anti-hyperalgesic and anti-nociceptive effects in several animal models, which have been ascribed to different mechanisms of action (Peana et al., 2002, 2004, 2006a,b; Re et al., 2000). 4. Conclusions In conclusion, the essential oil from leaves of P. cerrocampanensis from Panama is an outstanding new source of a-bisabolol, a compound highly appreciated in the pharmaceutical and cosmetic industry. In addition, it shows interesting antimicrobial and larvicidal activities which make this essential oil a potential industrial resource of new products. Acknowledgements Authors are grateful to National Secretariat for Science, Technology, and Innovation (SENACYT) of Panama, Project No. 112004 and the Organization of American States for financial support to CIFLORPAN. Also, thanks are due to Cristina Minguillon and Antoni Riera (University of Barcelona) for helping to confirm the identification of a-bisabolol by means chiral chromatography. R. Pérez-Rosés was supported by the Generalitat de Catalunya (Education and Universities Department) and the European Social Fund. S. Zacchino is grateful to ANPCyT (PICT R 260). M.V. Castelli acknowledges CONICET.
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