Bioresource Technology 101 (2010) 2510–2514

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Bioresource Technology

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Composition and biological activity of the essential oil from leaves of Pliniacerrocampanensis, 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, Spainb Centro de Investigaciones Farmacognósticas de la Flora Panameña (CIFLORPAN), Facultad de Farmacia, Universidad de Panama, Panama, Republic of Panamac Instituto Conmemorativo Gorgas en Estudio de Salud, Panama, Republic of Panamad Laboratorio de Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentinae Laboratorio de Microbiología, Mestrado Profissional em Farmácia, Universidade Bandeirante de Sao Paulo, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 28 July 2009Received in revised form 28 October 2009Accepted 5 November 2009Available online 16 December 2009

Keywords:Plinia cerrocampanensisEssential oila-BisabololAntimicrobial activityAedes aegypti

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.11.021

* Corresponding author. Tel.: +34 93 4024531; fax:E-mail address: s.canigueral@ub.edu (S. Cañiguera

The essential oil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae), obtained by hydrodistil-lation, 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%) asthe 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 wellas larvae from Aedes aegypti. The highest activity was found against Staphylococcus aureus, Pseudomonasaeruginosa, Microsporum gypseum, Trichophyton mentagrophytes and Trichophyton rubrum with MIC valuesfrom 32 to 125 lg/ml. The essential oil also showed potent inhibitory and bactericidal activities againstthree H. pylori strains, with MIC and MBC values of 62.5 lg/ml, and caused 100% mortality of A. aegyptilarvae at a concentration of 500 lg/ml.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Plants provide a multitude of flavours and fragrances whichhave found their way into everyday life. According to differentauthors, approximately 3000 plant species contain essential oils,from which only 300 are commercially important. Essential oilsand some of their constituents are used not only in pharmaceuticalproducts for their therapeutic activities but also in agriculture, asfood preservers and additives for human or animal use, in cosmet-ics and perfumes, and other industrial fields. In many cases, theyserve as plant defence mechanisms against predation by microor-ganisms, insects, and herbivores (Bakkali et al., 2008).

The complex composition of the essential oils and the variety ofchemical structures of their constituents are responsible of a widerange of biological activities many of which are of increasing inter-est in the fields of human and animal health. Particularly, manyessential oils and their constituents have traditionally been usedfor their antimicrobial activity which has long been recognized.In addition, some of them may be useful in the control of mosquitolarvae that are responsible of the transmission of several diseases

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+34 93 4035982.l).

such as malaria, dengue fever or yellow fever, that are among thegreatest health problems in the world (Cheng et al., 2003, 2009).

The need of new anti-infective agents due to the emergence ofmultiple antibiotic resistances has lead to the search of newsources of potential antimicrobials (Carson and Riley, 2003).Among them, the plant kingdom offers a wide range of biodiversityof great value for the pharmaceutical industry.

Within the framework of our ongoing research on aromaticflora from Panama, in view of potentiate the use of its natural re-sources, the present work deals with the study of the essentialoil from fresh leaves of Plinia cerrocampanensis Barrie (Myrtaceae),in particular its chemical composition and the evaluation of its bio-logical activity against several bacterial and fungal strains, as wellas against larvae from Aedes aegypti.

P. cerrocampanensis, which has recently been described as a newspecies of the genus Plinia, is a tree that grows between 800 and1000 m of altitude in the surroundings of Cerro Campana (Republicof Panama), reaching a height of about 8 m (Barrie, 2004). Untilnow no data on its chemical constituents or its biological activityare available in the scientific literature, although the compositionof the essential oils (Apel et al., 2006; Pino et al., 2002, 2003) andthe xanthine oxidase inhibitory activity of hydro-alcoholic extracts(Theoduloz et al., 1988) of other Plinia sp. have been reportedpreviously.

R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514 2511

2. Methods

2.1. Plant material

Fresh leaves of P. cerrocampanensis Barrie were collected in theNational Park ‘‘Altos de Campana” (N08�41009.80 0; W079�56004.40 0),Province of Panama (Republic of Panama) in February 2005. A vou-cher specimen No. FLORPAN 6623 is deposited in the Herbarium ofthe University of Panama (PMA).

2.2. Isolation and analysis of the essential oil

The essential oil was obtained from 100 g of fresh leaves byhydrodistillation, using the standard apparatus described in theEuropean Pharmacopeia (Council of Europe, 2005).

Analysis of the oil was carried out by GC–FID and GC–MS usingtwo fused silica capillary columns (30 m � 0.25 mm i.d.; 0.25 lmfilm thickness) of different stationary phases: Supelcowax™ 10and methylsilicone SE-30. GC–FID analyses were performed on aHewlett–Packard 6890 instrument, equipped with a HP ChemSta-tion data processor software, using the following analytical condi-tions: carrier gas, Helium; flow rate, 1 ml/min; oven temperatureprogrammed from 60–220 �C at 4 �C/min, 220 �C (10 min); injectortemperature, 250 �C; detector temperature, 270 �C; split ratio 1:80.The essential oil was injected undiluted (0.1 ll). Mass spectra wereobtained with a computerized system constituted by a GC Hew-lett–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 volt-age of 70 eV.

Identification of components was achieved by means of their GCretention indices in two stationary phases, determined in relationto a homologous series of fatty acid methyl esters, and by compar-ison of fragmentation patterns in the mass spectra with thosestored in our own library, in the GC–MS database and with litera-ture data (Adams, 1995; McLafferty, 1993). Quantification of eachcompound was performed on the basis of their GC peak areas onthe two columns, using the normalisation procedure without cor-rections for response factor.

2.3. Antibacterial activity

The antibacterial activity was assayed against Escherichia coli(ATCC 9637), Klebsiella pneumoniae (ATCC 10031), Mycobacteriumsmegmatis (ATCC 607), Pseudomonas aeruginosa (ATCC 27853), Sal-monella gallinarum (ATCC 9184) and Staphylococcus aureus (ATCC6538), following the method described by Mitscher et al. (1971),using streptomycin sulphate as positive control. The essential oilwas first dissolved in DMSO (1000 lg/ml) and the bacteriostaticactivity was determined by measuring the minimum inhibitoryconcentration (MIC) from diluted aqueous samples of 500, 250,125, 62.5, 31.25, 15.6 and 7.8 lg/ml. All the experiments were per-formed in triplicate and the results are expressed as mean values.

2.4. Anti-Helicobacter pylori activity

Stock cultures of H. pylori 26695 (ATCC 700392), J99 (ATCC700824) and SS1 (Sidney Strain 1) were reactivated on Columbiaagar plates (CA) (Merck, Germany) supplemented with 10% defi-brinated sheep blood (BBV, Brazil), 10 mg/L vancomycin, 20 mg/Lnalidixic acid, 2 mg/L amphotericin B and 40 mg/L 2,3,5-triphenyl-tetrazolium chloride (Sigma, Germany), and cultured at 37 �C in ahumidified 12% CO2 incubator (Revco, USA). The identity of the col-onies was confirmed by Gram staining and oxidase, catalase andurease production. The colonies were suspended in PBS (pH 7.2)

and bacterial density adjusted to 6 � 108 CFU. Viability controlwas done by Gram staining and colony count.

MIC and MBC determinations: broth microdilution was per-formed in brucella broth supplemented with 2% fetal calf serum(Cultilab, Brasil) and 0.2% DMSO. Two fold dilutions of essentialoil ranging from 1000 to 7.8 lg/ml were used. The standardizedinoculum was diluted to achieve a final inoculum concentrationof approximately 6 � 106 CFU per well. The microplates were incu-bated at 37 �C under microaerophilic conditions. All assays wereperformed in duplicate using amoxicillin (ranging from 16 to0.125 lg/ml) as positive internal control. The microplates wereaseptically examined for the presence of turbidity after 72 h ofincubation, and MIC were the lowest concentration of essentialoil that inhibited detectable bacterial growth. After that, 2 ll ofeach sample were spread onto brucella-sheep blood agar platesin order to determine the MBC. These plates were monitored forthe presence of bacterial growth after 48–72 h of incubation, andMBC were the lowest concentration that killed at least 99.9% ofthe original inoculum. Amoxicillin was used as a positive control.

2.5. Antifungal activity

Antifungal activity was assayed against several yeasts and fila-mentous fungi strains from the American Type Culture Collection(ATCC, Rockville, MD, USA) and Centro de Referencia en MicologíaCEREMIC (C, Facultad de Ciencias Bioquímicas y Farmacéuticas,Rosario, Argentina): Candida albicans (ATCC 1023), C. tropicalis (C131), Saccharomyces cerevisiae (ATCC 9763), Cryptococcus neofor-mans (ATCC 32264), Aspergillus flavus (ATCC 9170), A. fumigatus(ATCC 26934), A. niger (ATCC 9029), Trichophyton rubrum (C 110),Trichophyton mentagrophytes (ATCC 9972) and Microsporum gypse-um (C 115).

MIC values were determined using broth dilution techniques asdescribed by the Clinical and Laboratory Standards Institute (CLSI,formerly National Committee for Clinical and Laboratory Stan-dards) for yeasts (M27-A2) (NCCLS, 2002a) as well for filamentousfungi (M38-A) (NCCLS, 2002b) in microtiters of 96 wells. RPMI-1640 (Sigma, St. Louis, Mo, USA) buffered to a pH 7.0 with MOPSwas used. The starting inocula were approximately 1 � 103 to5 � 103 CFU/ml. Microtiter trays were incubated at 35 �C for yeastsand hyalohyphomycetes and at 28–30 �C for dermatophytes in amoist, dark chamber. MIC values were recorded at 48 h for yeastsand at a time according to the control fungus growth, for the otherfungi. The susceptibility of the standard drugs ketoconazole, terbi-nafine and amphotericin B was defined as the lowest concentrationof drug which resulted in total inhibition of fungal growth.

For the assay, the essential oil of P. cerrocampanensis was two-fold diluted with RPMI from 1000 to 0.98 lg/ml (final vol-ume = 100 ll) and a final DMSO concentration 61%. A volume of100 ll of inoculum suspension was added to each well with theexception of the sterility control where sterile water was addedto the well instead. The MIC was defined as the minimum inhibi-tory concentration of the essential oil which resulted in total inhi-bition of the fungal growth.

2.6. Larvicidal activity

For this purpose, larvae of A. aegypti (Culicidade: Diptera) of IIIand IV Stage obtained after 6–8 days post oviposition were used.The insectarium was kept at temperature of 22.5–25 �C, relativehumidity of 80% and with a photoperiod of 12:12 h. The larvaewere fed with granulated yeast suspended in water (1:4).

The essential oils were dissolved in ethanol and were tested at afinal concentration of 1, 100 and 500 ppm in triplicate. Water andalcohol were used as control. For each replicate, 20 individuals ofmosquito species were placed in a foam container. After 24 h, per

2512 R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514

cent mortality was calculated. Tetramethrin with a LC100 value of0.25 ± 0.01 lg/ml was used as a positive control.

3. Results and discussion

The fresh leaves of P. cerrocampanensis gave by hydrodistillationan essential oil yield of 0.64% (v/w). Qualitative and quantitativeanalysis of the oil by GC and GC–MS allowed the identification offorty components, representing more than 91% of the total sample.The oil was very rich in oxygenated sesquiterpenes (65.9%), espe-cially a-bisabolol (42.8%), bisabolol oxide B (10.3%) and trans-ner-olidol (9.4%). Among monoterpenes, linalool (10.3%) was found inthe highest percentage. Further details of the essential oil compo-sition are shown in Table 1.

Table 1Composition of the essential oil from leaves of Plinia cerrocampanensis.

Constituentsa RI–CWb RI–SEc %d

a-Pinene 112 209 0.2e,f,g

Limonene 204 251 0.4e,f,g

cis-Ocimene 220 255 0.1e,f,g

c-Terpinene 225 266 0.5e,f,g

p-Cymene 237 246 1.1e,f,g

6-Methyl-5-hepten-2-one 271 224 0.2e,f,g

trans-Linalool oxide 323 270 0.6e,f,g

cis-Linalool oxide 337 278 0.7e,f,g

Linalool 378 287 10.3e,f,g

4-Acetyl-1-methyl-1-cyclohexene 380 296 0.1e,f,g

Terpinen-4-ol 399 323 1.1e,f,g

Estragole 434 – 0.3e,f

a-Amorphene 439 475 0.1e,f,g

a-Terpineol 447 329 1.2e,f,g

a-Muurolene 457 487 0.1e,f,g

b-Bisabolene 462 492 4.8e,f,g

d-Cadinene 474 494 0.6e,f,g

a-Curcumene 483 477 0.8e,f,g

Nerol 489 – 0.1e,f

Calameneneh 510 – 0.3e,f

Geraniol 521 – 0.7e,f

a-Calacorene 544 – 0.1e,f

trans-Nerolidol 615 518 9.4e,f,g

Cubenol 623 – te,f

a-Bisabolol oxide B 656 562 10.3e,f,g

b-Bisabolol 662 568 1.4e,f,g

10-epi-Cadinol 676 – 0.5e,f

a-Bisabolol 700 579 42.8e,f,g

b-Eudesmol 710 555 0.6e,f,g

E,E-Farnesol 763 593 0.5e,f,g

a-Bisabolol oxide A 824 640 0.4e,f,g

Benzaldehyde – 208 0.1e,g

d-3-Carene – 243 te,g

trans-b-Ocimene – 260 0.1e,g

Terpinolene – 281 0.1e,g

a-Copaene – 428 0.1e,g

a-Cedrene – 445 0.2e,g

trans-a-Bergamotene – 458 0.1e,g

cis-a-Bisabolene – 489 0.3e,g

trans-a-Bisabolene – 508 0.3e,g

Monoterpene hydrocarbons 2.5Oxygenated monoterpenes 14.7Sesquiterpene hydrocarbons 7.8Oxygenated sesquiterpenes 65.9Others 0.7Total identified 91.6

a Components are listed in increasing order according to their retention indices inSupelcowax™ 10 except the last nine constituents, which were only detected inmethylsilicone.

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.

This composition pattern is quite unusual among essential oilsfrom leaves of other Plinia sp. previously reported. Although mostof them are also characterized by the predominance of oxygenatedsesquiterpenes (Apel et al., 2006; Pino et al., 2003), only the onefrom P. cordifolia showed remarkable percentages of compoundswith 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. cer-rocampanensis are summarized in Table 2. It exhibited the stron-gest activity against P. aeruginosa and S. aureus with MIC valuesof 62.5 and 125 lg/ml, respectively. This essential oil also showedpotent inhibitory and bactericidal activities against three H. pyloristrains (including SS1, traditionally used for in vivo assays), withMIC and MBC values of 62.5 lg/ml. H. pylori, a gastric pathogenwhose infection is associated with chronic superficial gastritis,peptic ulceration and gastric cancer, chronically infects more thanhalf of the world’s population. To be effective, therapies require theuse of more than one antimicrobial in combination. Unfortunately,increased primary resistance to recommended antibiotics modifiesthe 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 contri-bution in this search.

MIC values towards several yeasts and fungi strains are shownin Table 3. Dermatophytes were the most sensitive ones, particu-larly T. mentagrophytes, T. rubrum and M. gypseum with MIC valuesof 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 (Kuboet al., 1992) whose antimicrobial properties have been previouslyreported. Particularly, nerolidol has been recently found to inhibitthe hyphal growth of T. mentagrophytes causing destruction anddisorganization of organelles in the fungal cytoplasm (Park et al.,2009).

A. aegypti is the major vector of dengue and yellow fever whichhave experimented a recrudescence due to the increasing resis-tance of mosquitoes to current commercial insecticides. Althoughyellow fever has been reasonably brought under control with itsvaccine, no vaccine is available for dengue. Several secondarymetabolite plant products have been successfully assayed for theirlarvicidal activity against A. aegypti, particularly essential oils andtheir constituents. (Barreira Cavalcanti et al., 2004; Urano Carvalhoet al., 2003). In the present work, the essential oil of P. cerrocam-panensis showed, at concentrations of 100 and 500 lg/ml, 53%and 100% mortality of A. aegypti larvae, respectively, constitutinga potential alternative to the conventional chemical control.

Table 2Antibacterial activity of the essential oil of Plinia cerrocampanensis.

Bacteria EOPCa Streptomycinsulphate

Amoxicillin

MICb/MBCc

MIC MIC/MBC

Escherichia coli >1000 6.25 –Klebsiella pneumoniae >1000 3.12 –Mycobacterium

smegmatis>1000 12.50 –

Pseudomonas aeruginosa 62.5 1.56 –Salmonella gallinarum >1000 3.12 –Staphylococcus aureus 125 12.50 –Helicobacter pylori 26695 62.5/62.5 – 0.125/0.5Helicobacter pylori J99 62.5/62.5 – 0.125/0.5Helicobacter pylori SS1 62.5/62.5 – 0.125/0.5

a EOPC: essential oil of P. cerrocampanensis.b MIC: minimum inhibitory concentration (lg/ml).c MBC: minimum bactericidal concentration (lg/ml). MBC was only determined

against Helicobacter pylori.

Table 3Antifungal activity of the essential oil of Plinia cerrocampanensis.

Fungi EOPCa Amphotericin B Ketoconazole TerbinafineMICb MIC MIC MIC

YeastsCandida albicans >250 0.78 6.25 –Candida tropicalis >250 1.56 6.25 –Cryptococcus neoformans >250 0.78 1.56 –Saccharomyces cerevisiae >250 0.78 3.12 –

Filamentous fungiAspergillus flavus >250 0.78 6.25 –Aspergillus fumigatus >250 3.12 12.5 –Aspergillus niger >250 0.78 6.25 –

DermatophytesMicrosporum gypseum 125 0.25 0.50 0.04Trichophyton mentagrophytes 32 0.75 0.25 0.02Trichophyton rubrum 62.5 0.75 0.25 0.01

a EOPC: essential oil of P. cerrocampanensis.b MIC: minimum inhibitory concentration (lg/ml).

R. Vila et al. / Bioresource Technology 101 (2010) 2510–2514 2513

The essential oil of P. cerrocampanensis contains a 42% of a-bisabolol, its major constituent, being a potential industrial sourceof this interesting compound. a-Bisabolol, a well known monocy-clic unsaturated sesquiterpene alcohol which is one of the main ac-tive principles of chamomile (Chamomilla recutita), is widely usedin cosmetic preparations due to its anti-inflammatory activityand low toxicity (Habersang et al., 1979; Hempel and Hirschel-mann, 1998; Isaac, 1979; Jellinek, 1984; Madhavan, 1999; Yakov-lev and von Schlichtegroll, 1969). Furthermore, both a-bisabololand nerolidol, also one of the main constituents of the essentialoil (9.4%), may increase dermal absorption of other substances bymore than 20-fold, being useful vehicles for other drugs (Cornwelland Barry, 1994). These two sesquiterpenes are also able to en-hance bacterial permeability and susceptibility to clinically impor-tant antibiotic compounds (Brehm-Stecher and Johnson, 2003).Moreover, a-bisabolol has been found to be a promising inducerof apoptosis in highly malignant glioma cells (Cavalieri et al.,2004). Finally, it is interesting to highlight that linalool, the majormonoterpene of the oil of P. cerrocampanensis (10.3%), could alsoenhance the activities of a-bisabolol in the oil, since it has shownanti-inflammatory, anti-hyperalgesic and anti-nociceptive effectsin several animal models, which have been ascribed to differentmechanisms of action (Peana et al., 2002, 2004, 2006a,b; Reet al., 2000).

4. Conclusions

In conclusion, the essential oil from leaves of P. cerrocampanen-sis from Panama is an outstanding new source of a-bisabolol, acompound highly appreciated in the pharmaceutical and cosmeticindustry. In addition, it shows interesting antimicrobial and larvi-cidal activities which make this essential oil a potential industrialresource of new products.

Acknowledgements

Authors are grateful to National Secretariat for Science, Tech-nology, and Innovation (SENACYT) of Panama, Project No. 11-2004 and the Organization of American States for financial supportto CIFLORPAN. Also, thanks are due to Cristina Minguillon andAntoni Riera (University of Barcelona) for helping to confirm theidentification of a-bisabolol by means chiral chromatography. R.Pérez-Rosés was supported by the Generalitat de Catalunya (Edu-cation and Universities Department) and the European Social Fund.S. Zacchino is grateful to ANPCyT (PICT R 260). M.V. Castelliacknowledges CONICET.

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