Mycopathologia (2012) 174:61–67 DOI 10.1007/s11046-011-9519-2
Chemical Composition and Antifungal Activity of Essential Oils and Supercritical CO2 Extracts of Apium nodiflorum (L.) Lag. Andrea Maxia • Danilo Falconieri • Alessandra Piras • Silvia Porcedda • Bruno Marongiu • Maria Assunta Frau • Maria J. Gonc¸alves • Ce´lia Cabral Carlos Cavaleiro • Lı´gia Salgueiro
•
Received: 7 September 2011 / Accepted: 9 December 2011 / Published online: 12 January 2012 Ó Springer Science+Business Media B.V. 2012
Abstract Aerial parts of Apium nodiflorum collected in Portugal and Italy were submitted to hydrodistillation; also a supercritical fluid extract was obtained from Italian plants. The extracts were analyzed by GC and GC/MS. Both essential oils, obtained from Portuguese and Italian plants, posses high content of phenylpropanoids (51.6 vs. 70.8%); in the former, the percentage split in myristicin (29.1%) and dillapiol (22.5%), whereas in the latter, the total percentage is only of dillapiol (70.8%). The co-occurrence of myristicin and dillapiol is frequent because dillapiol results from enzymatic methoxylation of myristicin. Antimicrobial activity of phenylpropanoids has been
A. Maxia M. A. Frau Dipartimento della Scienza della Vita e dell’AmbienteMacrosezione di Botanica e Orto Botanico, Universita` degli Studi di Cagliari, Viale Sant’ Ignazio, 09123 Cagliari, Italy D. Falconieri A. Piras S. Porcedda B. Marongiu Dipartimento di Scienze Chimiche, Universita` degli Studi di Cagliari, Cittadella Universitaria di Monserrato, SS 554, Km 4.500, 09042 Cagliari, Italy M. J. Gonc¸alves C. Cabral C. Cavaleiro L. Salgueiro Centro de Estudos Farmaceˆuticos, Faculdade de Farmacia, Universidade de Coimbra, 3000-548 Coimbra, Portugal L. Salgueiro (&) Laborato´rio de Farmacognosia, Faculdade de Farma´cia da Universidade de Coimbra, Po´lo das Cieˆncias da Sau´de, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal e-mail:
[email protected]
patented, what suggest the potential of plants with high amounts of these compounds. Minimal inhibitory concentration (MIC) and minimal lethal concentration, determined according to NCCLS, were used to evaluate the antifungal activity of the essential oils against yeasts, Aspergillus species and dermatophytes. Essential oils exhibited higher antifungal activity than other Apiaceae against dermatophytes, with MIC ranging from 0.04 to 0.32 ll/ml. These results support the potential of A. nodiflorum oil in the treatment of dermatophytosis and candidosis. Keywords Apium nodiflorum Essential oil Antifungal activity Dermatophytosis Candidosis
Introduction Dermatophytes are keratinophilic and keratinolytic fungi. Their high affinity for the keratinized tissues makes them responsible for dermatophytosis, which are superficial mycosis affecting human skin, nails, hair or beard [1]. Candida spp. are microorganisms, frequently human commensal, which can cause an infection known as candidosis, in several different anatomical sites. It usually occurs in cases where there are changes in host microbiota or immunosuppression [2]. Members of the genus Aspergillus are responsible for a wide variety of diseases called aspergillosis, but immunosuppression is generally a prerequisite for systemic Aspergillus infections in humans. The most
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common forms are allergic bronchopulmonary aspergillosis, pulmonary aspergilloma and invasive aspergillosis [3]. The increasing impact of these infections, the limitations encountered in their treatment (e.g. resistance, side-effects and high toxicity), and the rising overprescription and overuse of conventional antifungal all stimulate a search for alternative natural drugs [4, 5]. Essential oils are good candidates, as they have been traditionally used for centuries due to their antifungal properties [6]. These properties have been confirmed by several studies [7–13] Therefore, essential oils is one of the most promising groups of natural products for the development of broad-spectrum, safer and cheaper antifungal agents. Celery (Apium graveolens L.) is a very old spice native in various countries and also cultivated in Europe. This species is traditionally used as antirheumatic, diuretic, antispasmodic and antihypertensive, and also as a natural source of food flavoring [14]. A. graveolens is the best known member of the Apium genus (Apiaceae or Umbelliferae). Apium nodiflorum (L.) Lag. (syns. Sium nodiflorum L. & Helosciadium nodiflorum (L.) W.D.J. Koch) belongs to the family Apiaceae. A. nodiflorum is commonly known as ‘‘fool’s watercress’’, in Portugal is known by ‘‘rabac¸as’’ and in Italy is known by ‘‘sedanina d’acqua’’, ‘‘gorgalestro’’, ‘‘erba canella’’ or ‘‘cannizelle’’. It is a glabrous, prostrate perennial herb up to 100 cm long, typically found in shallow streams throughout the Mediterranean region [15]. Apium nodiflorum is used in Italy as a culinary herb boiled or in salads [16]. In Italy, generally, it is traditionally used in digestive disorders, dysfunctions of the gastrointestinal or respiratory tract, as depurative, in the treatment of cough and inflammation, and in postpartum disorders in veterinary medicine [17]. In Abruzzo region (Central Italy), extracts from hypogenous parts are used as diuretics, and a decoction of leaves is used against stomachache [18]. A single study was published on the chemical composition of A. nodiflorum essential oil from Abruzzo region (Italy) and its inhibitory activity against Helicobacter pilori. This oil was dominated by limonene, p-cymene, myristicin and b-pinene [19]. Various studies in plants of the Apiaceae family, reporting the chemical composition and biological activities of their essential oils, pointed to significative antifungal activity [20–22]. Considering the lack of
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studies testing the antifungal activity of A. nodiflorum, the aims of the present work were the evaluation of the chemical composition and antifungal activity against yeasts and filamentous fungi of essential oils obtained from Portuguese and Italian plants. Supercritical fluid extract from Italian plants was also studied.
Materials and Methods Plant Material Aerial parts of Apium nodiflorum were collected from two different sites: Monte dei Sette Fratelli (Sardinia, Italy) and Penacova (Portugal). Voucher specimens were deposited in the Herbarium of the Dipartimento de Scienze Botaniche e Orto Botanico, Universita` degli Studi di Cagliari (CAG n. 575), and Herbarium of Medicinal Plants, Faculty of Pharmacy, University of Coimbra (L. Salgueiro 620). Plant material was air-dried at 40°C with forced ventilation for 2 days. Before utilization, matter was ground with a Malavasi mill (Bologna, Italy) taking care to avoid overheating. Both samples from Portugal and Italy were subjected to hydrodistillation; Italian plants were also subjected to supercritical fluid extraction. Hydrodistillation Hydrodistillation was performed during 4 h in a circulatory Clevenger-type apparatus up to exhaustion of the oil contained in the matrix, according to the procedure described in the European Pharmacopoeia [23]. Apparatus for SFE The supercritical CO2 extractions were performed in a laboratory apparatus equipped with a 320 cm3 extraction vessel operating in the single-pass CO2 mode, through a fixed bed of ground material 20 cm high. About 300 g A. nodiflorum were charged at each run. A single separator allowed discharge of the extract at the desired time. In this section, the temperature was maintained at a fixed value by means of a water thermostatted system and using a heating ribbon wrapped around the pipe exiting the separator. A high pressure diaphragm pump, Lewa-model EL 1, with a maximum capacity of 6 kg/h, pumped liquid CO2 at
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the desired flow rate. CO2 was then heated to the extraction temperature in a thermostatted oven controlled by a PID controller, model 2116 (Eurotherm). Extractions were carried out in a semi-batch mode: batch charging of vegetable matter and continuous flow solvent. The carbon dioxide flow was monitored by a calibrated rotameter (Sho-rate, model 1355) located after the separator. Total CO2 delivered during a run was measured by a dry test meter. Temperatures and pressures along the extraction apparatus were measured by thermocouple and Bourdon-tube test gauges, respectively. The pressure was regulated by high pressure valves under manual control. Experiments were carried out at 90 bar and 40°C in the extraction vessel, at 90 bar and -10°C in the first separator and at 20 bar and 15°C in the second one. GC and GC–MS Analysis of Essential Oil Analysis of the volatile extracts was carried out by gas chromatography (GC) and by gas chromatographymass spectrometry (GC–MS). Analytical GC was carried out in a gas chromatograph (Agilent, Model 7890A, Palo Alto, CA), equipped with a flame ionization detector (FID), an autosampler (Agilent, Model 7683B), Agilent HP5 fused silica column (5% phenyl-methylpolysiloxane), 30 m 9 0.25 mm i.d., film thickness 0.25 lm and a Agilent ChemStation software system. Oven temperature was settled at 60°C, raising at 3°C/min to 250°C and then held 20 min at 250°C; injector temperature: 250°C; carrier gas: helium at 1.0 ml/min; splitting ratio 1:10; detectors temperature: 300°C. GC–MS analyses were carried out in a gas chromatograph (Agilent, Model 6890N, Palo Alto, CA) equipped with a split/splitless injector, an autosampler Agilent model 7683 and an Agilent HP5 fused silica column; 5% phenyl-methylpolysiloxane, 30 m 9 0.25 mm i.d., film thickness 0.25 lm. GC conditions used were as follows: programmed heating from 60 to 250°C at 3°C/min followed by 20 min under isothermal conditions. The injector was maintained at 250°C. Helium was the carrier gas at 1.0 ml/min; the sample (1 ll) was injected in the split mode (1:10). The GC was fitted with a quadrupole mass spectrometer, MS, Agilent model 5973 detector. MS conditions were as follows: ionization energy 70 eV, electronic impact ion source temperature 200°C, quadrupole temperature 150°C, scan rate 3.2 scan/s and mass range 30–480 units. Software adopted to handle mass
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spectra and chromatograms was a ChemStation. NIST 02 and LIBR (TP) Mass Spectra Libraries were used as references [24, 25]. Samples were run in chloroform with a dilution ratio of 1:100. Compounds were identified by matching their mass spectra and retention indices with those reported in the literature. Moreover, whenever possible, identification has been confirmed by injection of pure compounds. Percentage of individual components was calculated based on GC peak areas without FID response factor correction. Antifungal Activity Evaluation Fungal Strains The antifungal activity of the oils was evaluated against yeasts and filamentous fungi strains (Aspergillus spp. and dermatophytes). Yeasts: three ATCC (American Type Culture Collection) type strains (Candida albicans ATCC 10231, C. parapsilosis ATCC 90018, C. tropicalis ATCC 13803); one CECT (Coleccio´n Espan˜ola de Cultivos Tipo) type strain (Cryptococcus neoformans CECT 1078); and two clinical strains isolated from recurrent cases of vulvovaginal candidiasis (Candida guillermondii MAT23 and C. krusei H9). Aspergillus species: two ATCC type strains (Aspergillus niger ATCC 16404 and A. fumigatus ATCC 46645) and one clinical strain isolated from bronchial secretions (A. flavus F44). Dermatophytes: three dermatophyte clinical strains isolated from nails and skin (Epidermophyton floccosum FF9, Trichophyton mentagrophytes FF7, Microsporum canis FF1); and four CECT (Coleccio´n Espan˜ola de Cultivos Tipo) type strains (Trichophyton rubrum CECT 2794, M. gypseum CECT 2908, T. mentagrophytes var. interdigitale CECT 2958, T. verrucosum CECT 2992). Candida parapsilosis ATCC 90018 was used as control. The fungal isolates were identified by standard microbiology methods and stored on Sabouraud broth with glycerol at –70°C. Prior to antifungal susceptibility testing, each isolate was inoculated on Sabouraud agar to ensure optimal growth characteristics and purity. Antifungal Activity Methods A macrodilution broth method was used to determine the Minimal Inhibitory Concentrations (MIC) and
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Minimal Lethal Concentrations (MLC), according to NCCLS reference documents M27-A2 [26] and M38A [27] for yeasts and filamentous fungi, respectively. The serial doubling dilution of each essential oil was prepared in dimethyl sulfoxide (DMSO), with concentrations ranging from 0.02 to 20 ll/ml. Final concentration of DMSO never exceeded 2%. Recent cultures of each strain were used to prepare the cell suspension adjusted to 1–2 9 103 cells per ml for yeasts, and 1–2 9 104 cells per ml for filamentous fungi. The concentration of cells was confirmed by viable count on Sabouraud agar. The test tubes were incubated aerobically at 35°C for 48/72 h (Candida spp. and Aspergillus spp./Cryptococcus neoformans) and at 30°C for 7 days (dermatophytes), and MICs were determined. To evaluate MLCs, aliquots (20 ll) of broth were taken from each negative tube after MIC reading and cultured in Sabouraud dextrose agar plates. Plates were then incubated at 35°C for 48 h (Candida spp. and Aspergillus spp.) and 72 h for Cryptococcus neoformans, and 30°C for 7 days (dermatophytes). In addition, two reference antifungal compounds, amphotericin B (Fluka) and fluconazole (Pfizer), were used to control the sensitivity of the tested microorganisms. All tests were performed in RPMI medium. For each strain tested, both growth conditions and sterility of the medium were checked in two control tubes. The innocuity of the DMSO was also checked at the highest tested concentration. All experiments were performed in triplicate and repeated, if the results differed.
Results The essential oils of Apium nodiflorum from Portugal and Italy obtained by hydrodistillation were obtained in yields of 1 and 1.1%, respectively (v/w). The identified compounds found in both essential oils, and their percentages are listed by order of their elution on a polydimethylsiloxane column (Table 1). In total, thirty-two compounds were identified accounting for 99.3 and 99.1%, in Portuguese and Italian essential oils, respectively. The oils were characterized mainly by phenylpropanoids (51.6 vs. 70.8%) and monoterpene hydrocarbons (42.2 vs. 20.9%), although with significant quantitative differences depending on the origin of plants. Portuguese
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Mycopathologia (2012) 174:61–67 Table 1 Retention index and chromatographic area percentages of compounds identified in Apium nodiflorum volatile oil extracted by SFE at 90 bar 40°C: Italy (SFE-1); and by Hydrodistillation: Italy (HD-1); Portugal (HD-2) IR
Compound
SFE-1
HD-1
HD-2
938
a-pinene
tr.
0.1
0.3
976
Sabinene
tr.
tr.
0.1
980
b-Pinene
1.6
3.1
3.6
992
Myrcene
0.2
0.3
0.6
1027
o-Cymene
0.1
0.6
1.9
1032 1040
Limonene cis-Ocimene
8.4 0.3
14.4 0.6
16.7 7.5
1051
trans-Ocimene
tr.
0.1
0.3
1061
c-Terpinene
0.6
1.1
4.0
1090
Terpinolene
0.3
0.5
7.2
1186
p-Cymen-8-ol
0.1
tr.
0.2
1376
a-Copaene
0.1
0.1
0.1
1419
b-Caryophyllene
0.7
1.0
1.4
1453
a-Humulene
tr.
0.1
0.1
1470
Dauca-5,8-diene
0.1
0.1
–
1477
c-Muurolene
tr.
tr.
0.1
1480
Germacrene D
1.0
0.6
2.9
1495
Bicyclogermacrene
0.1
0.1
0.3
1509
b-Bisabolene
tr.
0.1
–
1521
Myristicin
–
–
29.1
1524
b-Sesquiphellandrene
1.0
1.2
–
1576 1581
Spathulenol Caryophyllene oxide
0.2 0.3
0.5 0.7
0.2 0.2
1591
Salvial-4(14)-en-1-one
tr.
tr.
tr
1610
b-Atlantol
tr.
0.1
tr.
1623
Dillapiol
57.5
70.8
22.5
1646
3-iso-Thujopsanone
tr.
0.1
–
1688
Eudesma-4(15),7-dien-1b-ol
0.1
0.1
tr.
1846
Neophytadiene
7.1
0.9
0.2
2099
(Z)-Falcarinol
6.8
0.9
tr.
2135
Phytol
12.7
0.8
–
Total identified
99.3
99.1
99.3
Monoterpene hydrocarbons
11.5
20.9
42.2
Oxygen-containing monoterpenes
0.1
0.0
0.2
Sesquiterpene hydrocarbons
2.9
3.3
4.9
Oxygen-containing sesquiterpenes
7.4
2.4
0.4
Phenylpropanoids Diterpene hydrocarbons
57.6 7.1
70.8 0.9
51.6 0.2
Oxygen-containing diterpenes
12.7
0.8
–
tr. traces
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Table 2 Antifungal activity (MIC and MLC) of Apium nodiflorum oil against yeasts, dermatophytes and Aspergillus spp. Apium nodiflorum (Italy)
Apium nodiflorum (Portugal)
Fluconazole
Amphotericin B
MICa
MLCa
MICa
MLCa
MICb
MLCb
MICc
MLCc
Candida albicans ATCC 10231
1.25
2.5
0.64
1.25
1
[128
N.Tc
N.T
Candida tropicalis ATCC 13803
1.25
2.5
0.64
1.25
4
[128
N.T
N.T
Candida krusei H9
1.25
1.25–2.5
1.25
5
64
64–128
N.T
N.T
Candida guillermondii MAT23
0.64
1.25
0.32
0.64
8
8
N.T
N.T
Candida parapsilosis ATCC 90018
1.25
5
1.25
[20
\1
\1
N.T
N.T
Cryptococcus neoformans CECT 1078
0.16
0.32
0.32
0.32
16
128
N.T
N.T
T. mentagrophytes FF7
0.16
0.16
0.16
0.16
16–32
32–64
N.T
N.T
Microsporum canis FF1
0.04
0.04
0.04
0.04–0.08
128
128
N.T
N.T
Trichophyton rubrum CECT 2794
0.08
0.16
0.16
0.32
16
64
N.T
N.T
M. gypseum CECT 2908
0.08
0.08–0.16
0.08
0.16
128
[128
N.T
N.T
Epidermophyton floccosum FF9 T. mentagrophytes var. interdigitale CECT 2958
0.08 0.16
0.08 0.32
0.16 0.16
0.16 0.32
16 128
16 C128
N.T N.T
N.T N.T
T. verrucosum CECT 2992
0.32
0.64
0.32
0.64
[128
[128
N.T
N.T
A. niger ATCC 16404
1.25
10
2.5
10
N.T
N.T
1–2
4
A. fumigatus ATCC 46645
2.5
10
0.32
10
N.T
N.T
2
4
A. flavus F44
5
[10
0.32
5
N.T
N.T
2
8
Results were obtained from 3 independent experiments performed in duplicate a
MIC and MLC were determined by a macrodilution method and expressed in ll/ml (V/V)
b
MIC and MLC were determined by a macrodilution method and expressed in lg/ml (W/V)
c
Not tested
sample is mainly compounded by myristicin (29.1%), dillapiol (22.5%) and limonene (16.7%), whereas Italian sample has dillapiol as main compound (70.8%) followed by limonene (14.4%). Supercritical CO2 extracts, obtained from the Italian plants, are mainly constituted by limonene (8.4%), dillapiol (57.5%), neophytadiene (7.1%), (Z)falcarinol (6.6%) and phytol (12.7%). Evaluation of MIC and MLC of the oils showed a variability of inhibition among all the fungal strains tested, being specially active against dermatophytes with MIC values ranging from 0.04 to 0.32 ll/ml in both essential oils. Also for Cryptococcus neoformans, they showed a significant activity with MIC values ranging from 0.16 to 0.32 ll/ml. For Aspergillus spp., the oils are less effective particularly the Italian oil. The results of the antifungal tests are summarized in Table 2.
Discussion and Conclusion Essential oil composition depends upon intrinsic and extrinsic factors affecting the plant such as genetic information [28] and ecological conditions [29]. As reported in previous works concerning chemical composition of Umbelliferae, the content of phenylpropanoids changes appreciably depending on the growing region and the phenotype [30–32]. Both essential oils, obtained from Portuguese and Italian plants, posses high content of phenylpropanoids (51.6 vs. 70.8%); in the former, the percentage split in myristicin (29.1%) and dillapiol (22.5%), whereas in the latter, the total percentage is only of dillapiol (70.8%). The co-occurrence of myristicin and dillapiol is frequent because dillapiol results from enzymatic methoxylation of myristicin [32, 33]. Considering the common biogenetic pathway of these two compounds,
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it could be inferred that these differences are related to different environmental conditions, e.g. soil and climate variations [34]. Due to the high content in dillapiol in Italian plants (70.8%), it was decided to test the efficiency of supercritical fluid extraction in the obtaining of phenylpropanoids. A volatile oil was extracted by means of supercritical fluid with CO2 at 90 bar and 40°C. The yield was 0.2%, and the extract revealed quantitative differences in the composition when compared with that of distilled essential oil. As example, SFE volatile oil from Italian plants contains lower amounts of phenylpropanoids (57.6 vs. 70.8%) and monoterpene hydrocarbons (11.5 vs. 20.9%), whereas contains higher quantity of diterpenes as neophytadiene (7.1 vs. 0.9%) and phytol (12.7 vs. 0.8%). Antimicrobial activity of phenylpropanoids has been patented [35], which suggest the potential of plants where these compounds are present in high amounts in their essential oils. In most studies where myristicin and dillapiol are compared regarding their biological activities, dillapiol has a more potent effect than myristicin [36–38]. Also, unwanted carcinogenic and genotoxic properties decreased with further methoxylations of the aromatic nucleus in the order: safrole [ myristicin [ apiol [ dillapiol [39, 40]. This makes the essential oil provenant from Italian Apium nodiflorum more desirable for in vivo studies because of the absence of myristicin and a very high amount of dillapiol (70.8%). Essential oils exhibited very high antifungal activity specially against dermatophytes with MICs ranging from 0.04 to 0.32 ll/ml in both essential oils. This activity is significantly higher than in other Apiaceae [20–22]. For yeasts and Aspergillus strains, the antifungal activity of Portuguese oil is more effective, with MIC values of 0.32–2.5 ll/ml. According to some authors, the occurrence of myristicin and dilapiol could have a synergistic effect, which could explain the relatively higher antifungal activity against these strains of the Portuguese sample compared with the Italian [36, 40, 41]. Frequently, Aspergillus are less sensitive to the effect of essential oils, so these results, specially MICs of 0.32 ll/ml for Aspergillus fumigatus and A. flavus (common agents of aspergillosis), reveal the fungistatic activity for these strains.
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These results support the potential of A. nodiflorum essential oil in the treatment of dermatophytosis and candidosis. Acknowledgments This work was supported by CEF/ POCI2010/FEDER.
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