In vitro antifungal activities of leaf extracts of Lippia alba ( Verbenaceae ) against clinically important yeast species

Introduction: There are few studies reporting the antifungal activities of Lippia alba extracts. Methods: A broth microdilution assay was used to evaluate the antifungal effects of Lippia alba extracts against seven yeast species of Candida and Cryptococcus. The butanol fraction was investigated by gas chromatography-mass spectrometry. Results: The butanol fraction showed the highest activity against Candida glabrata. The fraction also acted synergistically with itraconazole and fl uconazole against C. glabrata. The dominant compounds in the butanol fraction were 2,2,5-trimethyl-3,4-hexanedione, 3,5-dimethyl-4-octanone and hexadecane. Conclusions: The butanol fraction may be a good candidate in the search for new drugs from natural products with antifungal activity.

Plants exhibit antifungal properties, and several compounds, such as coumarins, terpenes and fl avonoids 1 , could be responsible for these activities.The incidence of invasive opportunistic mycoses has increased in immunosuppressed patients, including those undergoing organ transplantation or hematopoietic stem cell therapy and those suffering from cancer or acquired immunodeficiency syndrome (AIDS) 2 .Clinical Candida species remain the most important cause of opportunistic mycoses worldwide.The majority of invasive infections due to Candida spp.are attributed to several species, including Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis and Candida krusei 2 .Cryptococcus neoformans is the etiological agent of cryptococcosis and is responsible for up to 30% of deaths in AIDS patients 3 .Cryptococcus gattii causes cryptococcal infections mostly in immunocompetent individuals 4 .Despite some effective treatment options, such mycoses are associated with high morbidity 2 .To overcome this problem, identifying new compounds, especially natural products, that exert antifungal activities, is very important.
Lippia alba (Mill.)N.E.Brown (Verbenaceae) is a shrub widely distributed throughout South America and is popularly known as cidreira or false melissa.In traditional popular medicine, the tea from its leaves is largely utilized as a tranquilizer but is also used to treat gastrointestinal and respiratory infections 5 .Several studies have reported on the antimicrobial activity of the essential oil of L. alba, but there are few reports in the literature on the extracts from this species.In the present work, the extract and fractions obtained from the leaves of L. alba were evaluated in respect to their antifungal activities against seven clinically important yeast species.We also tested the synergistic interaction of the butanol fraction with commercial antifungal drugs.
Leaves of L. alba were collected in the City of Carmopólis de Minas, State of Minas Gerais (MG), Brazil, in March 2011.A voucher specimen (BHCB 147243) was deposited at the Instituto de Ciências Biológicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.The fresh leaf material (216.73g) was extracted by cold maceration in 95% ethanol (Vetec, Brazil) over a period of 10 days at room temperature.The extract was then fi ltered and concentrated in a rotary evaporator at 40°C under reduced pressure to yield the ethanol extract (Et, 5.83g).Part of this extract (2.05g) was dissolved in EtOH/H 2 O (7:3) and then partitioned successively with hexane, dichloromethane, ethyl acetate and butanol (Vetec, Brazil; 15mL, 3 times with each solvent), resulting in 1. 13 For screening of antifungal activities, the ethanol extract and fractions of L. alba (dissolved in dimethylsulfoxide) were diluted to a fi nal concentration of 2,000μg/mL for use in the antifungal assay.The yeast species were grown at 37°C in Sabouraud (Himedia, India) media.After 24h under these Oliveira GT et al -In vitro antifungal activities of Lippia alba conditions, a yeast inoculum was prepared by diluting the cell suspensions appropriately in RPMI (Roswell Park Memorial Institute) 1640 (supplemented with 2% glucose).A total of 50µl of yeast inoculum was added to each well of a 96-well plate and adjusted to 1.0 × 10 6 cells/mL.After adding 25µL of the extract or control solution and 25µL of each medium to attain the desired concentrations, the plates were incubated at 37°C for 24h for Candida or 48h for Cryptococcus.As an indicator of microorganism growth, 10µL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (Sigma, USA) (dissolved in sterile water at 5mg/mL) was added to each well and incubated at 37°C for 4h.The results are expressed as the percent inhibition with respect to the controls without drugs 6 .Amphotericin B (2µg/mL) (Sigma, USA) was used as a positive control.All assays were performed in triplicate.
The minimal inhibitory concentration (MIC) was obtained from the broth microdilution tests performed in accordance with the guidelines in the Clinical and Laboratory Standards Institute (CLSI) document M27-A3 7 .Suspensions from the cultures of the Candida and Cryptococcus species were prepared according to the CLSI document M27-A3 7 and the modifi cations suggested by Johann et al. 1 to obtain a fi nal inoculum of 2.5 x 10 3 cells/mL.Amphotericin B (Sigma, USA), fl uconazole (Pfi zer Pharmaceutical, USA) and itraconazole (Jansen Pharmaceutical, Belgium) were included as positive controls; the stock solutions of amphotericin B and itraconazole were prepared in dimethylsulfoxide, and fl uconazole was prepared in water.Twofold serial dilutions were prepared exactly as outlined in CLSI document M27-A3 7 .All assays were performed in triplicate.
Eight serial dilutions of the butanol fraction (1.9 to 250µg/ mL) and amphotericin B (0.008 to 1µg/mL) were prepared with the same solvents used in the MIC test.Fifty-microliter aliquots of each dilution of the butanol fraction were added to ninety-sixwell plates in a vertical orientation, and fi fty microliters of each amphotericin B dilution was added in a horizontal orientation so that each well on the plate contained various concentrations of each sample (amphotericin B and butanol fraction).After the addition of 100µl of C. glabrata inoculum (the same as the one used in the MIC test) to each well, the plate was cultured for 48h at 37ºC.The fractional inhibitory concentration (FIC) of amphotericin B was calculated as the MIC of amphotericin B in the presence of the butanol fraction divided by the MIC of amphotericin B alone.The FIC of the butanol fraction was calculated in the same fashion.The fractional inhibitory concentration index (FICI) was calculated by adding both FICs.With this method, FICI ≤ 0.5 indicated synergistic activities, 0.5 < FICI < 2.0 defi ned additive or indifferent effects, and FICI > 2.0 described antagonistic effects 8 .This analysis was also performed for the butanol fraction with fl uconazole (0.062 to 8µg/mL) and itraconazole (0.015 to 2µg/mL).All assays were performed in triplicate.
Gas chromatography-mass spectrometry (GC-MS) of the butanol fraction was performed on a Shimadzu model QP5050A instrument equipped with a DB-5 column (30m x 0.25mm, 0.25µm).The initial temperature of the column was 80°C for 1min, which was then increased at a rate of 7°C/min to 300°C and held for 5min; the injector temperature was kept at 250°C (split 1:20), and the detector temperature was kept at 260 °C.Helium (He) was used as the carrier gas, with a linear fl owrate of 39.3mL/min (115.4Kpa).For each analysis, 1µL of the sample was injected into the GC.The scan range was from 50 to 500m/z at a scan rate of 0.50 scan/s.The solvent delay was 2.5min.The compounds were identifi ed by a mass spectral database search (NIST) (National Institute of Standards and Technology), followed by matching of MS data and expressed as relative percentages of each compound, calculated by internal normalization of the chromatographic peak area.All volatile compounds showing mass spectra with match factors ≥ 90% were put on a positive list of tentatively identifi ed metabolites.
The antifungal effect of the ethanol extract and fractions of L. alba was screened against seven yeast species of clinical interest at a concentration of 2,000µg/mL (Figure 1).C. krusei was the species most sensitive to the ethanol extract  and fractions, with inhibition rates ranging from 57% to 88%.The ethanol extract and hydroalcoholic fraction were also active against the yeast species.The butanol fraction showed the highest growth inhibition against C. glabrata (95%) compared with the other fractions and with the ethanol extract of L. alba.
After the screen, all fraction activities were evaluated in the MIC tests.The butanol fraction showed the highest activity against C. glabrata, with a MIC value of 62.5µg/ mL, and was also active against the other yeast species tested, C. albicans, C. krusei, C. gattii and C. neoformans.However, for the other species, the MIC value was 2,000µg/mL (Table 1).The ethanol extract and ethyl acetate fraction showed activity against C. krusei, with a MIC value of 1,000µg/mL.The ethanol extract also presented activity against C. parapsilosis and C. neoformans with a MIC of 1,000µg/mL, and against C. albicans, C. glabrata and C. tropicalis, with a MIC of 2,000µg/mL.
The hydroalcoholic fraction was active against C. albicans, C. glabrata, C. krusei, C. parapsilosis, C. gattii and C. neoformans, with a MIC of 2,000µg/mL.The dichloromethane fraction displayed activity only against C. krusei, with a MIC of 2,000µg/mL.In contrast, the hexane fraction was inactive against all the yeast species studied.Notably, the ethanol extract and fractions of L. alba were less potent than amphotericin B, fl uconazole and itraconazole.
The activity of the butanol fraction against C. glabrata, but not for other Candida species or Cryptococcus, could be explained because these yeast species are phylogenetically different.C. glabrata is a species more phylogenetically related to Saccharomyces cerevisiae than other pathogenic Candida species 9 .The specifi c activity of the butanol fraction against C. glabrata also suggests a selectivity of the natural products present in this fraction.
The antifungal activities observed in the present work have been confi rmed by other studies on L. alba extracts, being used against C. albicans, C. krusei, and C. tropicalis.To the best of our knowledge, this study is the fi rst to show the antifungal activity of the ethanol extract and fractions of leaves of L. alba against C. glabrata and C. gattii.
The hexane and chloroform extracts from the leaves and fl owers of L. alba showed activity against C. albicans 10 .The hydroalcoholic and methanol extracts obtained from the leaves of L. alba exerted effects against C. krusei, with MIC values of 125 and 165.2µg/mL, respectively 11,12 .The hydroalcoholic extract also showed activity against C. tropicalis, with a MIC of 1,000µg/mL 11 .
The effect against Candida species is interesting, mainly because of the innate resistance of C. krusei to fl uconazole.Candida glabrata shows less susceptibility than other Candida species, and the development of resistance to fl uconazole among the clinical strains of C. albicans, C. parapsilosis and Candida tropicalis, hampers the treatment of these infections 13 .The activity against C. neoformans is of particular importance, as this yeast is the major cause of meningitis in AIDS patients and has been identifi ed as the fourth most common cause of life-threatening infection in AIDS patients 14 .
To explore the possibility of developing more effective combination therapies, the butanol fraction from the leaves of L. alba was combined with amphotericin B, itraconazole and fl uconazole and tested against C. glabrata.Table 2 shows the additive or indifferent effect of combining the butanol fraction with amphotericin B, and the synergistic effect of this fraction with itraconazole and fluconazole.These results are very interesting because C. glabrata exhibits intrinsic resistance against azole antifungal drugs 15 .
In this study, we showed that the butanol fraction from the leaves of L. alba, combined with fl uconazole and itraconazole, had a synergistic effect against C. glabrata and, therefore, could be an alternative in the treatment of candidemia caused by this yeast species.However, additional studies are needed to evaluate the toxicity of this fraction and the in vivo action of fl uconazole and itraconazole combined with the butanol fraction against this opportunistic yeast.

FIGURE 1 -
FIGURE 1 -Growth inhibition of seven yeast species of clinical interest by ethanol extract and fractions of Lippia alba at concentration of 2,000μg/mL.

TABLE 1 -
Minimal inhibitory concentration activity of ethanol extract and fractio ns of Lippia alba against seven clinically important yeast species.