- Email: [email protected]
Extracts from Alternanthera maritima as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT)
Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Extracts from Alternanthera maritima as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT) Adriana Gasparetto a, Tadia F. Lapinski a, Stella R. Zamuner b, Sonia Khouri c, Leandro P. Alves d, Egberto Munin d,*, Marcos J. Salvador e a
Universidade Comunitária Regional de Chapecó (UNOCHAPECÓ), Av. Sen. Attílio Fontana, 591-E, 89809-000 – Cx Postal: 747 – Chapecó – SC, Brazil Faculdade de Ciências Aplicadas (FCA) – UNICAMP, Rua Pedro Zacarias 1300, 13484-350 Limeira, SP, Brazil Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil d Universidade Camilo Castelo Branco – UNICASTELO, Rodovia Presidente Dutra, Km 138, Núcleo do Parque Tecnológico, 12247-004 São José dos Campos, SP, Brazil e Instituto de Biologia, Departamento de Biologia Vegetal, Universidade Estadual de Campinas (UNICAMP), Cx Postal 6109, 13083-970 Campinas, SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 June 2009 Received in revised form 16 January 2010 Accepted 18 January 2010 Available online 25 January 2010 Keywords: PDT PACT Photosensitizer
a b s t r a c t This study investigated the effect of photodynamic antimicrobial chemotherapy (PACT) using extracts from Alternanthera maritima on the viability of Candida dubliniensis. Human infections constitute a great health problem. Several antifungal drugs are currently available, but their uses are limited by a number of factors, such as low potency, poor solubility, microbial resistance, and drug toxicity. Therefore, the search for new and more effective antimicrobial agents and the development of alternative therapies, such as PACT, are necessary. Crude hexane and ethanol extracts of A. maritima were produced. The prepared extracts presented absorption at 650–700 nm. For bioassays, 50 lL of culture medium, 50 lL of extract (25 mg/mL) or control, and 5 lL of a suspension of the microorganism to be tested (C. dubliniensis ATCC 778157 or ATCC 777, 107 CFU/mL) were placed in a sterile 96-well microtiter plate (well cross section = 0.38 cm2). The contents of each well were irradiated with a 685-nm diode laser with an output power of 35 mW, which was distributed through the well cross section yielding an energy dosage of 28 J/cm2. In each assay (n = 6), one plate was subjected to irradiation, and one was not. For each active sample, the number of colony-forming units per milliliter (CFU/mL) was obtained, and data were analyzed by the Tukey test. The chemical compositions of the extracts were determined by chromatographic and spectroscopic techniques. The results suggest inhibition of the growth of C. dubliniensis when irradiated with a diode laser in the presence of hexane and ethanol extracts from A. maritima as photosensitizers. Laser irradiation alone or crude extracts at 25 mg/mL did not significantly reduce the number of CFU/ mL. Steroids, triterpenes, and flavonoids were identified in the analyzed extracts. In conclusion, the photoactivation of crude hexane and ethanol extracts of A. maritima by red laser radiation at 685 nm promoted an antimicrobial effect, showing that these natural products can be used as photosensitizers in PACT. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Human infections, particularly those involving mucosal surfaces, constitute a great health problem. Dermatophytes, Candida spp., and bacteria are among the most frequent pathogens. Candida dubliniensis is an opportunistic pathogen that has been difficult to characterize, mainly due to phenotypic similarity to Candida albicans species. C. dubliniensis, described in 1995 [1], was first associated with oral candidiasis in immunodepressed patients with human immunodeficiency virus (HIV). More recently, it has been recognized as the cause of superficial and systemic infections in
* Corresponding author. Tel./fax: +55 12 3905 4401. E-mail addresses: [email protected], [email protected] (E. Munin). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.01.009
immunodepressed patients who do not have HIV [2]. The occurrence of alterations in host defense mechanisms and the loss of anatomical barriers through burns, ulcerations, or invasive procedures facilitate the appearance of infections by these fungi. Moreover, the resistance of strains of C. dubliniensis to some antifungal agents has been reported [3]. Nowadays, several antifungal drugs are available, but their use is limited by a number of factors, such as low potency, poor solubility, microbial resistance, and high drug toxicity. Therefore, research has focused on new natural antimicrobial agents, especially antifungal agents, aiming to minimize common problems caused by available antimicrobial agents. Thus, it is recognized that research on natural products and the search for new therapeutic alternatives for antimicrobials should be encouraged [4–6].
16
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
Natural products produced by plant species have attracted researchers in several fields. The compounds that stand out are those derived from ‘‘secondary metabolism,” in light of their possible therapeutic application. In industrialized countries, 25% of prescribed and registered medicines contain active natural compounds or derivatives (extracts or pure or chemically modified compounds). Up to 1985, 119 potential drugs have been described from at least 80 plant species [7]. The family Amaranthaceae comprises many species that are used in traditional folk medicines for the treatment of several diseases, such as infections and inflammation [8,9]. Alternanthera maritima (Gomphreneae, Amaranthaceae) is an herbaceous plant, commonly found on the sandy beaches of the Brazilian east coast. In previous bioassay-based phytochemical studies, the hexane and ethanol extracts of A. maritima were found to have antibacterial, antifungal, and antiprotozoal activity, and it was possible to identify which fractions or major compounds were responsible for these activities. Steroids, saponins, and flavonoids were identified in the bioactive extracts [9–11]. An alternative for treating fungal infections is photodynamic antimicrobial chemotherapy (PACT) [12,13], which consists of the association of a photosensitizer agent, administered topically or systemically, with a light source. The photo-induced reaction promotes the destruction of the target organism by oxidation mechanisms that lead to microbial membrane lysis and protein degradation [5,12,14]. Since photodynamic therapy (PDT) is quite efficient in inducing cellular death in experimental tests, the application of such a method for the inhibition of pathogenic microorganisms provides a promising therapeutic alternative. In this way, topical PDT can be employed for treating infections, providing a less toxic alternative when compared with other topical antimicrobial treatments [15]. Thus, the present study aimed to contribute to the search for new photosensitizers with potential for use in light-associated treatment of fungal infections. In this investigation, extracts of A. maritima were evaluated as photosensitizers by testing the viability of C. dubliniensis strains after PACT. This study is part of a broader collection of research surveying potential bioactive extracts and natural photosensitizers in Brazilian plants.
2. Materials and methods Aerial parts of A. maritima (Mart.) St. Hil were collected at Restinga de Marica, Rio de Janeiro, RJ, Brazil and identified by Professor Dr. Josafá Carlos de Siqueira (Pontifícia Universidade Católica, Rio de Janeiro, RJ, Brazil). A type specimen was deposited at the Herbarium of the Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto/USP, SP, Brazil (register number SPFR 02968). For the preparation of crude extracts, air-dried powdered plant material (aerial parts, 2.3 kg) was obtained by exhaustive maceration at room temperature, using hexane and ethanol, successively, at a mass/solvent ratio of 1:20 (mass/volume). After maceration, the biomass was filtered, and the solvents were removed on a rotary evaporator (below 40 °C) under reduced pressure, to obtain the hexane (AMPAH, yield 10 g) and ethanol (AMPAE, yield 86 g) crude extracts. The absorption spectrum in the 400–700 nm range was measured to determine the excitation region of the crude hexane and ethanol extracts of A. maritima. The extracts were evaluated at concentrations of 25 mg/mL. The absorption spectra were taken with a 1/4-m spectrometer (Oriel Instruments, model MS257), furnished with a 300 line per mm diffraction grating. A CCD detector with 1024 256 pixels was connected at the detector output port of the monochromator.
The chemical compositions of the hexane and ethanol extracts were determined by gas chromatography (HRGC) and mass spectrometry (ESI–MS), respectively, with comparative analysis using isolated standards. An aliquot (10 mg) of the hexane extract was resuspended in analytical-grade chloroform (3 mL) and percolated through a sep-pak column (Alltech, silica-gel 200 mg, 3 mL). The column was eluted with hexane (10 mL) and chloroform (10 mL). Fractions were collected and evaporated to dryness at room temperature. The chloroform phase was analyzed by HRGC (in duplicate) on a Hewlett–Packard model 5890 Series II Gas Chromatograph with a split injector at 330 °C. The injected volume was 2 lL. Hydrogen was employed as the carrier gas at an average linear velocity of 44 cm/s (HP-50) or 42 cm/s (HP-1). HP-50 (crosslinked 50% phenyl–methyl–silicone, 30 m 0.25 mm 0.25 mm) and HP-1 (cross-linked methyl–silicone, 30 m 0.25 mm 0.25 mm) capillary columns were employed. For HP-50, the column temperature was 280 °C (isothermic). For HP-1, the temperature program was set at 250 °C, held for 12 min, and then increased at 6 °C/min to 280 °C and held at this temperature for 30 min. Data were processed on a Hewlett–Packard model 3395 injector. The 26 authentic standards of various triterpenes and sterols used were obtained from several plant species, which were studied in our laboratory. The ethanol extract (10 mg) was resuspended in methanol/water (7:3, v/v, 3 mL) and percolated through a sep-pak column (Alltech, C-18, 200 mg, 3 mL). The column was eluted with analytical-grade methanol (10 mL) and chloroform (10 mL). The chloroform phase was analyzed by HRGC, while the methanol phase was analyzed by ESI–MS. The ESI–MS spectra were acquired in a Quattro LC quadrupole mass spectrometer fitted with an electrospray interface operating in negative ion mode (Micromass, United Kingdom). The source and desolvation temperatures were 70 °C and 100 °C, respectively. Cone voltage was 30 V. The parent ion was compared with previously isolated standards of flavonoids [8–11]. For the execution of the bioassays, the following microorganism standard strains were used: C. dubliniensis ATCC 777 and C. dubliniensis ATCC 778157. These strains were cultivated for 48 h at 37 °C in Sabouraud dextrose agar (Merck). After this period, a sample of colonies was removed from the surface of the agar plate and suspended in sterile physiological solution (0.85% NaCl) at a density of 1–5 107 viable colony-forming units per milliliter (CFU/mL), obtained using the Neubauer’s chamber method of exclusion of the vital dye, methylene blue [16]. The hexane and ethanol crude extracts of A. maritima were evaluated for antimicrobial activity in vitro. Sensitivity tests were performed by a modified agar-well diffusion method (well technique with double layers) and by the method of microdilution [17,18], according to Salvador et al. [11]. The inoculum size of each tested strain was standardized according to the Clinical and Laboratory Standard Institute [18]. Subsequently, aliquots of 20 lL of each test-sample solution were applied to 5.0-mm diameter wells. Crude extract solutions were prepared in propyleneglycol/sterile distilled water (5:95) at 100 mg/mL. After incubation at 37 °C for 24 or 48 h, the inhibition zone corresponding to the halo (h) formed from the well edge to the beginning of the region of microbial growth was measured in mm. In the tests, ketoconazole (0.2 mg/mL) was used as a positive control, and propyleneglycol/ sterile distilled water (5:95) as a negative control. For the extracts that showed activity at the 100 mg/mL concentration, determination of the minimal inhibitory concentration (MIC) was carried out, using the method of microdilution in 96-well plates [17]. The MIC was determined in mg/mL. The sensitivity tests were performed in duplicate for each evaluated microorganism strain, and the final results were presented as arithmetic averages. For the sensitization of C. dubliniensis strains, the hexane and ethanol crude extracts of A. maritima were used as photosensitizers
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
at sub-inhibitory concentrations (25 mg/mL). At this concentration, the solution of both extracts presented a brown color and was transparent, with clear substrate. The extract solutions were prepared in propyleneglycol/sterile distilled water (5:95, v/v). Methylene blue (0.05 mg/ml) was used as the PDT positive control, and the solvent (propyleneglycol/sterile distilled water, 5:95, v/v) was used as a negative control. The solution of methylene blue was prepared by dissolving the powder (Synth, São Paulo, Brazil) in physiological solution (0.85% NaCl). Then, the solution was filtered through a sterile filter membrane (0.22 lm, Millipore, São Paulo, Brazil). The employed light source was a 685-nm diode laser with 35 mW of output power (Model Photon Lase, DMC, São Carlos, Brazil). The laser beam illuminated an area of 0.38 cm2, and the irradiation time was 5 min, resulting in an energy dosage of 28 J/ cm2 for each sample. A dull and dark screen with an aperture having a diameter coincident with the well size was used to minimize light spreading (aperture area and well diameter = 0.38 cm2). For evaluating the crude hexane and ethanol extracts of A. maritima as natural photosensitizers, a procedure for in vitro photosensitization assays was carried out, according to the method described by de Souza et al. [16]. To each well of a sterile 96-well flat-bottomed microtitulation plate, 50 lL of Sabouraud dextrose broth, 50 lL of extract (at 25 mg/mL) or control solution, and 5 lL of microorganism suspension were added. This amount of the microorganism solution is equivalent to the standard 0.5 on the MacFarland scale, which corresponds to approximately 107 CFU/mL. Then, the plate containing the samples was agitated and incubated in the dark for 5 min at room temperature. After this period, the contents of each well were irradiated. The irradiation of the samples was performed under aseptic conditions in a laminar air flow chamber. As positive controls, methylene blue photosensitizer diluted in sterile saline (0.05 mg/mL), as well as the antifungal ketoconazole (0.2 mg/ mL), were used. As negative controls, the diluent (propyleneglycol/sterile distilled water, 5:95), the inoculum (fungi incubated in sterile physiological solution), and the culture medium alone were used. The experiments were performed in duplicate for each microorganism tested, and three experiments were carried out on three different days (total replicates, n = 6). In each assay, one plate was subjected to irradiation (once, over the entire 0.38 cm2 well area), and one plate was not irradiated (control plate, wrapped in aluminum foil and not exposed to any light). After irradiation, the plates were incubated at 37 °C for 24 or 48 h. After the incubation period, serial dilutions of 102 and 103 were obtained from each sample in sterile physiological solution, and aliquots of 10 lL were added in duplicate to Sabouraud dextrose broth (Difco, Detroit, USA). After incubation at 37 °C for 24 or 48 h, the number of CFU/mL was determined for each bioactive drug test, and data were subjected to statistical analysis. The percent reduction of microbial growth for each species was calculated for the studied samples. Throughout the experiment, the samples were manipulated in the dark, under aseptic conditions. Results are expressed as means (with the relative standard deviation, %RSD). Statistical differences between treatments were analyzed by the Tukey test, considering the CFU/mL values of each bioactive drug test in the photosensitization assays compared with the inoculum-only control group. The level of significance was set at a p-value < 0.05.
17
extracts was 50 mg/mL for both C. dubliniensis strains that were studied in this work. Fig. 1 shows the measured absorption spectra for the methylene blue photosensitizer (positive control), for the solvent, propyleneglycol/sterile distilled water (5:95, negative control), and for the ethanol (AMPAE) and hexane (AMPAH) crude extracts. The crude extracts showed light absorption in the spectral region between 600 and 700 nm. From the obtained MIC values, a study protocol was established using lower MIC concentrations for photosensitization experiments. The standardized inocula for microorganism tests were prepared to equal 0.5 on the MacFarland scale, and these were also used for the inoculum control (initial inoculum) in photosensitization experiments (Table 1). The methylene blue (MB, 0.05 mg/mL) photosensitizer, used as positive control for PDT, did not exhibit any toxic effect on C. dubliniensis ATCC 777 or C. dubliniensis ATCC 778157, when not exposed to laser excitation. However, after photoactivation, MB was able to inhibit the growth of the two strains studied. These results are in agreement with the results of Souza et al. [16]. The mixture containing propyleneglycol and sterile distilled water (5:95), used as negative control, did not inhibit the growth of the microorganisms in any of the plates (non-irradiated or irradiated group); the microorganisms grew and proliferated. The antifungal drug standard used (ketoconazole, 0.2 mg/mL) totally inhibited yeast growth in the absence of light, as well as when submitted to laser radiation. When inoculated alone into the culture medium, the microorganisms (inocula alone) proliferated in both laser-irradiated and non-irradiated plates (Table 1). The hexane and ethanol extracts of A. maritima (25 mg/mL) were not active against the studied microorganisms in the absence of irradiation (reduction in CFU/mL was 0%). However, when sensitized with laser radiation (685 nm), these extracts presented biocidal effects and considerably reduced the viable number of CFU/ mL for the yeast strains studied. After photoactivation, the hexane extract caused reductions of 99.8% and 98.9% in the number of CFU/mL of the C. dubliniensis ATCC 778157 and ATCC 777 strains, respectively, while the ethanol extract caused reductions of 98.2% and 99.8% for C. dubliniensis ATCC 778157 and C. dubliniensis ATCC 777, respectively (Table 1, Fig. 2). In order to verify the chemical composition of the bioactive crude extracts from A. maritima, analyses were carried out using HRGC and ESI–MS techniques. Table 2 contains a summary of the HRGC results of extracted and identified sterols and triterpenes. These structures were unequivocally confirmed by co-injection of authentic standards and identified by relative retention values. ESI–MS represents a powerful detection and characterization technique, which has been successfully applied to the determina-
3. Results Preliminary experiments were performed with crude extracts of A. maritima to determine the amount of mass to be used in the extraction process. These extracts were submitted to an evaluation of antimicrobial activity, and the MIC of the hexane and ethanol
Fig. 1. Absorption spectra of hexane (AMPAH) and ethanol (AMPAE) extracts of A. maritima, methylene blue, and the solvent (propyleneglycol/water, 5:95).
18
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
Table 1 Antimicrobial activity (CFU/mL) of A. maritima hexane (AMPAH) and ethanol (AMPAE) extracts and control samples in the presence (L) or absence (L+) of laser irradiation. Microorganism
Non-irradiated group (L) Means of microorganism numbers expressed in CFU/mLa AMPAH L 25 mg/mL
AMPAE L 25 mg/mL
Inoculum control L
Negative control L
Positive control PDT L
Positive control L
Initial inoculum size
Candida dubliniensis ATCC 778157 Candida dubliniensis ATCC 777
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
0b 0b
1.14 107 (0.30)a 1.70 106 (0.10)a
Microorganism
Microorganism irradiated group (L+) Means of microorganism numbers expressed in CFU/mLa
Candida dubliniensis ATCC 778157 Candida dubliniensis ATCC 777
AMPAH L + 25 mg/mL
AMPAE L + 25 mg/mL
Inoculum control L+
Negative control L+
Positive control PDT L+
Positive control L+
Initial inoculum size
2.20 104 (1.29)d 1.86 104 (2.10)d
2.10 105 (1.50)d 2.00 104 (2.05)d
>1.0 107 >1.0 106
>1.0 107 >1.0 106
0c 0c
0c 0c
1.14 107 (0.30)b 1.70 106 (0.10)b
a Data expressed as means, n = 6, with relative standard deviations (%RSDs). Positive control: ketoconazole (0.20 mg/mL) for yeast; positive control for PDT: methylene blue (0.05 mg/mL); negative control: propyleneglycol/distilled sterilized water (5:95). b–d Statistical significance b – c – d, Tukey’s test, p < 0.05.
Table 2 Triterpenes and sterols identified by HRGC (HP-50 column) in the hexane and ethanol extracts of A. maritima. Standards
RRa
AMPAH extract
AMPAE extract
Campesterol Stigmasterol b-Sitosterol Stigmast-7en-3b-ol Spinasterol Taraxerone Epitaraxerol Taraxerol b-Amyrin a-Amyrin Lupeol b-Friedelanol Friedelin Pseudotaraxasterol Taraxasterol 11-Oxours-12-ene 11-Oxoolean-12-ene Taraxerol acetate b-Amyrin acetate a-Amyrin acetate Lupeol acetate Bauerenyl acetate 11a, 12a-Oxidetaraxeryl acetate b-Friedelanol acetate a-Amyrinonil acetate b-Amyrinonil acetate
1238 1308 1453 1513 1683 1628 1656 1698 1768 1992 2052 2491 2724 2479 2570 3160 3586 1872 1930 2154 2234 2508 2810 2871 3653 4126
+ + + + + + + + +
+ + + + + + +
+: Compound detected. : Compound not detected. a RR = relative retention compared to cholesterol (internal standard).
Fig. 2. Number of colony-forming units per milliliter (CFU/mL) of indicated strains treated with A. maritima crude extracts either in the presence (L+) or absence (L) of irradiation with a laser diode. Statistics: , – , Tukey test, p < 0.05.
tion of compounds present in materials from a variety of natural product sources. The specific application of LC-ESI–MS natural product mixture analysis is a procedure known as dereplication, and this process is rapid, precise, and efficient [19]. In order to verify the correct identification of the flavonoids present in the etha-
nol extract of A. maritima, standard flavonoids previously isolated in our laboratory were used. The following flavonoids were identified in the ethanol extract by ESI–MS analysis: quercetin – ESI–MS: m/z = 301 [MH], 3-methoxy quercetin – ESI–MS: m/z = 315 [MH], vitexin/isovitexin – ESI–MS: m/z = 431 [MH], 200 -O-a L-rhamnopyranosyl-vitexin – ESI–MS: m/z = 577 [MH] , acacetin 8-C-[a-L-rhamnopyranosyl-(1 ? 2)-b-D-glucopyranoside] – ESI– MS: m/z = 609 [MH], 200 -O-b-D-glucopyranosyl-vitexin – ESI– MS: m/z = 593 [MH], rutin – ESI–MS: m/z = 301 [MH], isorhamnetin, 3-O-a-L-rhamnosyl-(1 ? 6)-b-D-galactopyranoside/isorhamnetin, and 3-O-a-L-rhamnosyl-(1 ? 6)-b-L-glucopyranoside – ESI–MS: m/z = 623 [MH]. 4. Discussion The prevalence of Candida species in systemic infections, together with the increase in resistance to traditional antifungal
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
drugs is troubling [20]. The incidence of infections by pathogenic fungi is increasing due to more common occurrence of hospital infections in immunodepressed patients. Currently, many antimicrobials, and in particular antifungal drugs, have shown limited use for a number of reasons, such as low potency, poor solubility, microbial resistance, and drug toxicity [11,20]. In the present study, the application of one session of PDT to cultures of C. dubliniensis using plant extracts as photosensitizing agents showed satisfactory results. Consequently, the use of PDT employing the hexane and ethanol extracts of A. maritima as photosensitizers constitutes a potential alternative for the treatment of superficial infections, particularly those caused by C. dubliniensis. However, these studies are preliminary, and more studies with these extracts are needed, including PDT tests with other microorganisms. Data from de Souza et al. [16] indicate that PDT with an energy dose of 28 J/cm2 at 685 nm in the presence of methylene blue (0.1 mg/mL) as a photosensitizer reduced the number of CFU/mL of the yeast strains Candida krusei, C. albicans, C. dubliniensis, and Candida tropicalis by 88.6%, 84.8%, 91.6%, and 82.3%, respectively. Our results, using a similar experimental protocol for PDT and the same energy dosage of 28 J/cm2 at 685 nm in the presence of hexane and ethanol extracts of A. maritima (25 mg/mL) as photosensitizers, demonstrate that the number of CFU/mL of two distinct strains of C. dubliniensis was also significantly reduced (Fig. 2). With regard to the photodynamic antimicrobial activity of A. maritima extracts, there is a need to identify and isolate the active photosensitizing agents present in these extracts. It is important to consider the possibility that, in a synergistic way, more than one compound may be involved in the observed biological activity, as the extract is a complex mixture of compounds. In order to verify the chemical composition of the crude hexane and ethanol extracts from A. maritima, chromatographic (HRGC) and spectroscopic (ESI– MS) analyses were carried out. Steroids, triterpenes, and polyphenolic compounds such as flavonoids were detected, and these results are in agreement with the literature [11]. Salvador et al. [11], in a phytochemical study of antimicrobial and antiprotozoal activities, documented the presence of flavonoids (particularly aglycone and O- and C-glycosides) as the main constituents of the ethanol extract of A. maritima (aerial parts), together with the presence of saponins and steroids. In the hexane extract, fatty acids, steroids, and triterpenoids were identified. Moreover, in preliminary cytotoxicity evaluation, the ethanol extract of A. maritima and isolated flavonoids were not toxic to opsonized, zymosanstimulated human neutrophils [9]. No study for the evaluation of photosensitizing agents present in extracts of A. maritima has previously been reported. However, further studies are needed to verify if these identified secondary metabolites are photoactive and are contributing to the photosensitizing effect in PACT seen for the crude extracts of A. maritima. On the other hand, it is important to emphasize that PDT does not substitute for conventional antimicrobial therapy. However, it does represent one more alternative for topical therapy that is more selective than systemic therapy, which could reduce unwanted side effects of the administration of systemic drugs. It is an option for rapid local treatment, with the advantages of low cost and low or non-existent overdose risk [12]. Microbial resistance can be avoided by the use of PDT, and the appearance of fungal resistance to the treatment is improbable, since microbial inactivation is mediated by the oxygen singlet [12,16]. Moreover, PDT does not present drug interactions, especially as it is used in local therapy [21–23]. These advantages encourage further detailed in vitro and in vivo studies with natural products for possible application as natural photosensitizers in antimicrobial PDT.
19
5. Conclusions Our data demonstrate that the photoactivation of hexane and ethanol extracts of A. maritima aerial parts by laser irradiation at 685 nm produce an antimicrobial effect against C. dubliniensis strains and that the extracts of A. maritima are promising as natural photosensitizers in photodynamic therapy. Despite the positive results, further investigations are necessary to confirm the potential of these natural products for application as photosensitizers in photodynamic antimicrobial chemotherapy. Acknowledgments The authors are grateful to FAPESP, CAPES, CNPq and FAEPEXUNICAMP for financial support; to Professor Josafá Carlos de Siqueira for the identification of plant material and to Guilherme R. Teodoro for technical assistance in the antimicrobial experiments. References [1] D. Sullivan, D. Coleman, Candida dubliniensis: characteristics and identification, J. Clin. Microbiol. 36 (1998) 329–334. [2] M.E. Brandt, L.H. Harrison, M. Pass, A.N. Sofair, S. Huie, R.K. Li, C.J. Morrison, D.W. Warnock, R.A. Hajjeh, Candida dubliniensis fungemia: the first four cases in North America, Emerg. Infect. Dis. 6 (2000) 46–49. [3] M.C. Dignani, J.S. Solomkin, E.J. Anaissie, Candida, in: E.J. Anaissie, M.R. McGinnis, M.A. Pfaller (Eds.), Clinical Mycology, Churchill Livingstone, New York, 2003. [4] V. Carre, O. Gaud, I. Sylvain, O. Bourdon, M. Spiro, J. Biais, R. Granet, P. Krausz, M. Guilloton, Fungicidal properties of meso-arylglycosylporphyrins: influence of sugar substituents on photoinduced damage in the yeast Saccharomyces cerevisiae, J. Photochem. Photobiol. B 48 (1999) 57–62. [5] L.M. Giroldo, M.P. Felipe, M.A. de Oliveira, E. Munin, L.P. Alves, M.S. Costa, Photodynamic antimicrobial chemotherapy (PACT) with methylene blue increases membrane permeability in Candida albicans, Lasers Med. Sci. 24 (2009) 109–112. [6] E. Munin, L.M. Giroldo, L.P. Alves, M.S. Costa, Study of germ tube formation by Candida albicans after photodynamic antimicrobial chemotherapy (PACT), J. Photochem. Photobiol. B 88 (2007) 16–20. [7] G.A. Cordell, Biodiversity and drug discovery – a symbiotic relationship, Phytochemistry 55 (2000) 463–480. [8] M.J. Salvador, E.O. Ferreira, S.U. Mertens-Talcott, W.V. Castro, V. Butterweck, H. Derendorf, D.A. Dias, Isolation and HPLC quantitative analysis of antioxidant flavonoids from Alternanthera tenella Colla, Z. Naturforschung 61c (2006) 19– 25. [9] J.G. Souza, R.R. Tomei, A. Kanashiro, L.M. Kabeya, A.E.C.S. Azzolini, D.A. Dias, M.J. Salvador, Y.M. Lucisano-Valim, Ethanolic crude extract and flavonoids isolated from Alternanthera maritima: neutrophil chemiluminescence inhibition and free radical scavenging activity, Z. Naturforschung 62c (2007) 339–347. [10] M.J. Salvador, D.A. Dias, Flavone C-glycosides from Alternanthera maritima (Mart.) St. Hil. (Amaranthaceae), Bio-chem. Syst. Ecol. 32 (2004) 107–110. [11] M.J. Salvador, O.L.A.D. Zucchi, R.C. Candido, I.Y. Ito, D.A. Dias, Alternanthera maritima (Amaranthaceae), Pharm. Biol. 42 (2004) 138–148. [12] M. Wainwright, Photodynamic antimicrobial chemotherapy, J. Antimicrob. Chemother. 42 (1998) 13–28. [13] R.C. Souza, J.C. Junqueira, R.D. Rossoni, C.A. Pereira, E. Munin, A.O. Jorge, Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue malachite green and low-power laser irradiation alone against Candida albicans, Lasers Med. Sci. (2009), doi:10.1007/s10103-009-0706-z. [14] K.A. Salva, Photodynamic therapy: unapproved uses, dosages or indications, Clin. Dermatol. 20 (2002) 571–581. [15] J.R. Perussi, Inativação fotodinâmica de microorganismos, Quím. Nova 30 (2007) 1–7. [16] S.C. Souza, J.C. Junqueira, I. Balducci, C.Y. Koga-Ito, E. Munin, A.O.C. Jorge, Photosensitization of different Candida species by low power laser light, J. Photochem. Photobiol. B. 83 (2006) 34–38. [17] M.E. Stefanello, A.C. Cervi, I.Y. Ito, M.J. Salvador, A. Wisniewski Jr., E.L. Simionatto, Chemical composition and antimicrobial activity of essential oils of Eugenia chlorophylla (Myrtaceae), J. Essent. Oil Res. 20 (2008) 75–78. [18] National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi. Proposed Standard M38-P. National Committee for Clinical Laboratory Standard, Wayne, 1998, 28p. [19] M.A. Strege, High-performance liquid chromatography-electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening, J. Chromatogr. B 725 (1999) 67–78. [20] J.M. Bliss, C.E. Bigelow, T.H. Foster, C.G. Haidaris, Susceptibility of Candida species to photodynamic effects of photofrin, Antimicrob. Agents Chemother. 48 (2004) 2000–2006.
20
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
[21] M.L. Embleton, S.P. Nair, B.D. Cookson, M. Wilson, Selective lethal photosensitization of methicillin-resistant Staphylococcus aureus using an IgG-tin (IV) chlorin e6 conjugate, J. Antimicrob. Chemother. 50 (2002) 857– 864. [22] S. Karrer, R.M. Szeimies, S. Ernst, C. Abels, W. Baumler, M. Landthaler, Photodynamic inactivation of Staphylococci with 5-aminolaevulinic acid or photofrin, Lasers Med. Sci. 14 (1999) 54–61.
[23] M.C. Teichert, J.W. Jones, M.N. Usacheva, M.A. Biel, Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model, Oral Surg., Oral Med., Oral Pathol., Oral Radiol. Endodont. 93 (2002) 155–160.
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Extracts from Alternanthera maritima as natural photosensitizers in photodynamic antimicrobial chemotherapy (PACT) Adriana Gasparetto a, Tadia F. Lapinski a, Stella R. Zamuner b, Sonia Khouri c, Leandro P. Alves d, Egberto Munin d,*, Marcos J. Salvador e a
Universidade Comunitária Regional de Chapecó (UNOCHAPECÓ), Av. Sen. Attílio Fontana, 591-E, 89809-000 – Cx Postal: 747 – Chapecó – SC, Brazil Faculdade de Ciências Aplicadas (FCA) – UNICAMP, Rua Pedro Zacarias 1300, 13484-350 Limeira, SP, Brazil Universidade do Vale do Paraíba (UNIVAP), Av. Shishima Hifumi, 2911, 12244-000 São José dos Campos, SP, Brazil d Universidade Camilo Castelo Branco – UNICASTELO, Rodovia Presidente Dutra, Km 138, Núcleo do Parque Tecnológico, 12247-004 São José dos Campos, SP, Brazil e Instituto de Biologia, Departamento de Biologia Vegetal, Universidade Estadual de Campinas (UNICAMP), Cx Postal 6109, 13083-970 Campinas, SP, Brazil b c
a r t i c l e
i n f o
Article history: Received 29 June 2009 Received in revised form 16 January 2010 Accepted 18 January 2010 Available online 25 January 2010 Keywords: PDT PACT Photosensitizer
a b s t r a c t This study investigated the effect of photodynamic antimicrobial chemotherapy (PACT) using extracts from Alternanthera maritima on the viability of Candida dubliniensis. Human infections constitute a great health problem. Several antifungal drugs are currently available, but their uses are limited by a number of factors, such as low potency, poor solubility, microbial resistance, and drug toxicity. Therefore, the search for new and more effective antimicrobial agents and the development of alternative therapies, such as PACT, are necessary. Crude hexane and ethanol extracts of A. maritima were produced. The prepared extracts presented absorption at 650–700 nm. For bioassays, 50 lL of culture medium, 50 lL of extract (25 mg/mL) or control, and 5 lL of a suspension of the microorganism to be tested (C. dubliniensis ATCC 778157 or ATCC 777, 107 CFU/mL) were placed in a sterile 96-well microtiter plate (well cross section = 0.38 cm2). The contents of each well were irradiated with a 685-nm diode laser with an output power of 35 mW, which was distributed through the well cross section yielding an energy dosage of 28 J/cm2. In each assay (n = 6), one plate was subjected to irradiation, and one was not. For each active sample, the number of colony-forming units per milliliter (CFU/mL) was obtained, and data were analyzed by the Tukey test. The chemical compositions of the extracts were determined by chromatographic and spectroscopic techniques. The results suggest inhibition of the growth of C. dubliniensis when irradiated with a diode laser in the presence of hexane and ethanol extracts from A. maritima as photosensitizers. Laser irradiation alone or crude extracts at 25 mg/mL did not significantly reduce the number of CFU/ mL. Steroids, triterpenes, and flavonoids were identified in the analyzed extracts. In conclusion, the photoactivation of crude hexane and ethanol extracts of A. maritima by red laser radiation at 685 nm promoted an antimicrobial effect, showing that these natural products can be used as photosensitizers in PACT. Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction Human infections, particularly those involving mucosal surfaces, constitute a great health problem. Dermatophytes, Candida spp., and bacteria are among the most frequent pathogens. Candida dubliniensis is an opportunistic pathogen that has been difficult to characterize, mainly due to phenotypic similarity to Candida albicans species. C. dubliniensis, described in 1995 [1], was first associated with oral candidiasis in immunodepressed patients with human immunodeficiency virus (HIV). More recently, it has been recognized as the cause of superficial and systemic infections in
* Corresponding author. Tel./fax: +55 12 3905 4401. E-mail addresses: [email protected], [email protected] (E. Munin). 1011-1344/$ - see front matter Ó 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2010.01.009
immunodepressed patients who do not have HIV [2]. The occurrence of alterations in host defense mechanisms and the loss of anatomical barriers through burns, ulcerations, or invasive procedures facilitate the appearance of infections by these fungi. Moreover, the resistance of strains of C. dubliniensis to some antifungal agents has been reported [3]. Nowadays, several antifungal drugs are available, but their use is limited by a number of factors, such as low potency, poor solubility, microbial resistance, and high drug toxicity. Therefore, research has focused on new natural antimicrobial agents, especially antifungal agents, aiming to minimize common problems caused by available antimicrobial agents. Thus, it is recognized that research on natural products and the search for new therapeutic alternatives for antimicrobials should be encouraged [4–6].
16
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
Natural products produced by plant species have attracted researchers in several fields. The compounds that stand out are those derived from ‘‘secondary metabolism,” in light of their possible therapeutic application. In industrialized countries, 25% of prescribed and registered medicines contain active natural compounds or derivatives (extracts or pure or chemically modified compounds). Up to 1985, 119 potential drugs have been described from at least 80 plant species [7]. The family Amaranthaceae comprises many species that are used in traditional folk medicines for the treatment of several diseases, such as infections and inflammation [8,9]. Alternanthera maritima (Gomphreneae, Amaranthaceae) is an herbaceous plant, commonly found on the sandy beaches of the Brazilian east coast. In previous bioassay-based phytochemical studies, the hexane and ethanol extracts of A. maritima were found to have antibacterial, antifungal, and antiprotozoal activity, and it was possible to identify which fractions or major compounds were responsible for these activities. Steroids, saponins, and flavonoids were identified in the bioactive extracts [9–11]. An alternative for treating fungal infections is photodynamic antimicrobial chemotherapy (PACT) [12,13], which consists of the association of a photosensitizer agent, administered topically or systemically, with a light source. The photo-induced reaction promotes the destruction of the target organism by oxidation mechanisms that lead to microbial membrane lysis and protein degradation [5,12,14]. Since photodynamic therapy (PDT) is quite efficient in inducing cellular death in experimental tests, the application of such a method for the inhibition of pathogenic microorganisms provides a promising therapeutic alternative. In this way, topical PDT can be employed for treating infections, providing a less toxic alternative when compared with other topical antimicrobial treatments [15]. Thus, the present study aimed to contribute to the search for new photosensitizers with potential for use in light-associated treatment of fungal infections. In this investigation, extracts of A. maritima were evaluated as photosensitizers by testing the viability of C. dubliniensis strains after PACT. This study is part of a broader collection of research surveying potential bioactive extracts and natural photosensitizers in Brazilian plants.
2. Materials and methods Aerial parts of A. maritima (Mart.) St. Hil were collected at Restinga de Marica, Rio de Janeiro, RJ, Brazil and identified by Professor Dr. Josafá Carlos de Siqueira (Pontifícia Universidade Católica, Rio de Janeiro, RJ, Brazil). A type specimen was deposited at the Herbarium of the Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto/USP, SP, Brazil (register number SPFR 02968). For the preparation of crude extracts, air-dried powdered plant material (aerial parts, 2.3 kg) was obtained by exhaustive maceration at room temperature, using hexane and ethanol, successively, at a mass/solvent ratio of 1:20 (mass/volume). After maceration, the biomass was filtered, and the solvents were removed on a rotary evaporator (below 40 °C) under reduced pressure, to obtain the hexane (AMPAH, yield 10 g) and ethanol (AMPAE, yield 86 g) crude extracts. The absorption spectrum in the 400–700 nm range was measured to determine the excitation region of the crude hexane and ethanol extracts of A. maritima. The extracts were evaluated at concentrations of 25 mg/mL. The absorption spectra were taken with a 1/4-m spectrometer (Oriel Instruments, model MS257), furnished with a 300 line per mm diffraction grating. A CCD detector with 1024 256 pixels was connected at the detector output port of the monochromator.
The chemical compositions of the hexane and ethanol extracts were determined by gas chromatography (HRGC) and mass spectrometry (ESI–MS), respectively, with comparative analysis using isolated standards. An aliquot (10 mg) of the hexane extract was resuspended in analytical-grade chloroform (3 mL) and percolated through a sep-pak column (Alltech, silica-gel 200 mg, 3 mL). The column was eluted with hexane (10 mL) and chloroform (10 mL). Fractions were collected and evaporated to dryness at room temperature. The chloroform phase was analyzed by HRGC (in duplicate) on a Hewlett–Packard model 5890 Series II Gas Chromatograph with a split injector at 330 °C. The injected volume was 2 lL. Hydrogen was employed as the carrier gas at an average linear velocity of 44 cm/s (HP-50) or 42 cm/s (HP-1). HP-50 (crosslinked 50% phenyl–methyl–silicone, 30 m 0.25 mm 0.25 mm) and HP-1 (cross-linked methyl–silicone, 30 m 0.25 mm 0.25 mm) capillary columns were employed. For HP-50, the column temperature was 280 °C (isothermic). For HP-1, the temperature program was set at 250 °C, held for 12 min, and then increased at 6 °C/min to 280 °C and held at this temperature for 30 min. Data were processed on a Hewlett–Packard model 3395 injector. The 26 authentic standards of various triterpenes and sterols used were obtained from several plant species, which were studied in our laboratory. The ethanol extract (10 mg) was resuspended in methanol/water (7:3, v/v, 3 mL) and percolated through a sep-pak column (Alltech, C-18, 200 mg, 3 mL). The column was eluted with analytical-grade methanol (10 mL) and chloroform (10 mL). The chloroform phase was analyzed by HRGC, while the methanol phase was analyzed by ESI–MS. The ESI–MS spectra were acquired in a Quattro LC quadrupole mass spectrometer fitted with an electrospray interface operating in negative ion mode (Micromass, United Kingdom). The source and desolvation temperatures were 70 °C and 100 °C, respectively. Cone voltage was 30 V. The parent ion was compared with previously isolated standards of flavonoids [8–11]. For the execution of the bioassays, the following microorganism standard strains were used: C. dubliniensis ATCC 777 and C. dubliniensis ATCC 778157. These strains were cultivated for 48 h at 37 °C in Sabouraud dextrose agar (Merck). After this period, a sample of colonies was removed from the surface of the agar plate and suspended in sterile physiological solution (0.85% NaCl) at a density of 1–5 107 viable colony-forming units per milliliter (CFU/mL), obtained using the Neubauer’s chamber method of exclusion of the vital dye, methylene blue [16]. The hexane and ethanol crude extracts of A. maritima were evaluated for antimicrobial activity in vitro. Sensitivity tests were performed by a modified agar-well diffusion method (well technique with double layers) and by the method of microdilution [17,18], according to Salvador et al. [11]. The inoculum size of each tested strain was standardized according to the Clinical and Laboratory Standard Institute [18]. Subsequently, aliquots of 20 lL of each test-sample solution were applied to 5.0-mm diameter wells. Crude extract solutions were prepared in propyleneglycol/sterile distilled water (5:95) at 100 mg/mL. After incubation at 37 °C for 24 or 48 h, the inhibition zone corresponding to the halo (h) formed from the well edge to the beginning of the region of microbial growth was measured in mm. In the tests, ketoconazole (0.2 mg/mL) was used as a positive control, and propyleneglycol/ sterile distilled water (5:95) as a negative control. For the extracts that showed activity at the 100 mg/mL concentration, determination of the minimal inhibitory concentration (MIC) was carried out, using the method of microdilution in 96-well plates [17]. The MIC was determined in mg/mL. The sensitivity tests were performed in duplicate for each evaluated microorganism strain, and the final results were presented as arithmetic averages. For the sensitization of C. dubliniensis strains, the hexane and ethanol crude extracts of A. maritima were used as photosensitizers
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
at sub-inhibitory concentrations (25 mg/mL). At this concentration, the solution of both extracts presented a brown color and was transparent, with clear substrate. The extract solutions were prepared in propyleneglycol/sterile distilled water (5:95, v/v). Methylene blue (0.05 mg/ml) was used as the PDT positive control, and the solvent (propyleneglycol/sterile distilled water, 5:95, v/v) was used as a negative control. The solution of methylene blue was prepared by dissolving the powder (Synth, São Paulo, Brazil) in physiological solution (0.85% NaCl). Then, the solution was filtered through a sterile filter membrane (0.22 lm, Millipore, São Paulo, Brazil). The employed light source was a 685-nm diode laser with 35 mW of output power (Model Photon Lase, DMC, São Carlos, Brazil). The laser beam illuminated an area of 0.38 cm2, and the irradiation time was 5 min, resulting in an energy dosage of 28 J/ cm2 for each sample. A dull and dark screen with an aperture having a diameter coincident with the well size was used to minimize light spreading (aperture area and well diameter = 0.38 cm2). For evaluating the crude hexane and ethanol extracts of A. maritima as natural photosensitizers, a procedure for in vitro photosensitization assays was carried out, according to the method described by de Souza et al. [16]. To each well of a sterile 96-well flat-bottomed microtitulation plate, 50 lL of Sabouraud dextrose broth, 50 lL of extract (at 25 mg/mL) or control solution, and 5 lL of microorganism suspension were added. This amount of the microorganism solution is equivalent to the standard 0.5 on the MacFarland scale, which corresponds to approximately 107 CFU/mL. Then, the plate containing the samples was agitated and incubated in the dark for 5 min at room temperature. After this period, the contents of each well were irradiated. The irradiation of the samples was performed under aseptic conditions in a laminar air flow chamber. As positive controls, methylene blue photosensitizer diluted in sterile saline (0.05 mg/mL), as well as the antifungal ketoconazole (0.2 mg/ mL), were used. As negative controls, the diluent (propyleneglycol/sterile distilled water, 5:95), the inoculum (fungi incubated in sterile physiological solution), and the culture medium alone were used. The experiments were performed in duplicate for each microorganism tested, and three experiments were carried out on three different days (total replicates, n = 6). In each assay, one plate was subjected to irradiation (once, over the entire 0.38 cm2 well area), and one plate was not irradiated (control plate, wrapped in aluminum foil and not exposed to any light). After irradiation, the plates were incubated at 37 °C for 24 or 48 h. After the incubation period, serial dilutions of 102 and 103 were obtained from each sample in sterile physiological solution, and aliquots of 10 lL were added in duplicate to Sabouraud dextrose broth (Difco, Detroit, USA). After incubation at 37 °C for 24 or 48 h, the number of CFU/mL was determined for each bioactive drug test, and data were subjected to statistical analysis. The percent reduction of microbial growth for each species was calculated for the studied samples. Throughout the experiment, the samples were manipulated in the dark, under aseptic conditions. Results are expressed as means (with the relative standard deviation, %RSD). Statistical differences between treatments were analyzed by the Tukey test, considering the CFU/mL values of each bioactive drug test in the photosensitization assays compared with the inoculum-only control group. The level of significance was set at a p-value < 0.05.
17
extracts was 50 mg/mL for both C. dubliniensis strains that were studied in this work. Fig. 1 shows the measured absorption spectra for the methylene blue photosensitizer (positive control), for the solvent, propyleneglycol/sterile distilled water (5:95, negative control), and for the ethanol (AMPAE) and hexane (AMPAH) crude extracts. The crude extracts showed light absorption in the spectral region between 600 and 700 nm. From the obtained MIC values, a study protocol was established using lower MIC concentrations for photosensitization experiments. The standardized inocula for microorganism tests were prepared to equal 0.5 on the MacFarland scale, and these were also used for the inoculum control (initial inoculum) in photosensitization experiments (Table 1). The methylene blue (MB, 0.05 mg/mL) photosensitizer, used as positive control for PDT, did not exhibit any toxic effect on C. dubliniensis ATCC 777 or C. dubliniensis ATCC 778157, when not exposed to laser excitation. However, after photoactivation, MB was able to inhibit the growth of the two strains studied. These results are in agreement with the results of Souza et al. [16]. The mixture containing propyleneglycol and sterile distilled water (5:95), used as negative control, did not inhibit the growth of the microorganisms in any of the plates (non-irradiated or irradiated group); the microorganisms grew and proliferated. The antifungal drug standard used (ketoconazole, 0.2 mg/mL) totally inhibited yeast growth in the absence of light, as well as when submitted to laser radiation. When inoculated alone into the culture medium, the microorganisms (inocula alone) proliferated in both laser-irradiated and non-irradiated plates (Table 1). The hexane and ethanol extracts of A. maritima (25 mg/mL) were not active against the studied microorganisms in the absence of irradiation (reduction in CFU/mL was 0%). However, when sensitized with laser radiation (685 nm), these extracts presented biocidal effects and considerably reduced the viable number of CFU/ mL for the yeast strains studied. After photoactivation, the hexane extract caused reductions of 99.8% and 98.9% in the number of CFU/mL of the C. dubliniensis ATCC 778157 and ATCC 777 strains, respectively, while the ethanol extract caused reductions of 98.2% and 99.8% for C. dubliniensis ATCC 778157 and C. dubliniensis ATCC 777, respectively (Table 1, Fig. 2). In order to verify the chemical composition of the bioactive crude extracts from A. maritima, analyses were carried out using HRGC and ESI–MS techniques. Table 2 contains a summary of the HRGC results of extracted and identified sterols and triterpenes. These structures were unequivocally confirmed by co-injection of authentic standards and identified by relative retention values. ESI–MS represents a powerful detection and characterization technique, which has been successfully applied to the determina-
3. Results Preliminary experiments were performed with crude extracts of A. maritima to determine the amount of mass to be used in the extraction process. These extracts were submitted to an evaluation of antimicrobial activity, and the MIC of the hexane and ethanol
Fig. 1. Absorption spectra of hexane (AMPAH) and ethanol (AMPAE) extracts of A. maritima, methylene blue, and the solvent (propyleneglycol/water, 5:95).
18
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
Table 1 Antimicrobial activity (CFU/mL) of A. maritima hexane (AMPAH) and ethanol (AMPAE) extracts and control samples in the presence (L) or absence (L+) of laser irradiation. Microorganism
Non-irradiated group (L) Means of microorganism numbers expressed in CFU/mLa AMPAH L 25 mg/mL
AMPAE L 25 mg/mL
Inoculum control L
Negative control L
Positive control PDT L
Positive control L
Initial inoculum size
Candida dubliniensis ATCC 778157 Candida dubliniensis ATCC 777
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
>1.0 107 >1.0 106
0b 0b
1.14 107 (0.30)a 1.70 106 (0.10)a
Microorganism
Microorganism irradiated group (L+) Means of microorganism numbers expressed in CFU/mLa
Candida dubliniensis ATCC 778157 Candida dubliniensis ATCC 777
AMPAH L + 25 mg/mL
AMPAE L + 25 mg/mL
Inoculum control L+
Negative control L+
Positive control PDT L+
Positive control L+
Initial inoculum size
2.20 104 (1.29)d 1.86 104 (2.10)d
2.10 105 (1.50)d 2.00 104 (2.05)d
>1.0 107 >1.0 106
>1.0 107 >1.0 106
0c 0c
0c 0c
1.14 107 (0.30)b 1.70 106 (0.10)b
a Data expressed as means, n = 6, with relative standard deviations (%RSDs). Positive control: ketoconazole (0.20 mg/mL) for yeast; positive control for PDT: methylene blue (0.05 mg/mL); negative control: propyleneglycol/distilled sterilized water (5:95). b–d Statistical significance b – c – d, Tukey’s test, p < 0.05.
Table 2 Triterpenes and sterols identified by HRGC (HP-50 column) in the hexane and ethanol extracts of A. maritima. Standards
RRa
AMPAH extract
AMPAE extract
Campesterol Stigmasterol b-Sitosterol Stigmast-7en-3b-ol Spinasterol Taraxerone Epitaraxerol Taraxerol b-Amyrin a-Amyrin Lupeol b-Friedelanol Friedelin Pseudotaraxasterol Taraxasterol 11-Oxours-12-ene 11-Oxoolean-12-ene Taraxerol acetate b-Amyrin acetate a-Amyrin acetate Lupeol acetate Bauerenyl acetate 11a, 12a-Oxidetaraxeryl acetate b-Friedelanol acetate a-Amyrinonil acetate b-Amyrinonil acetate
1238 1308 1453 1513 1683 1628 1656 1698 1768 1992 2052 2491 2724 2479 2570 3160 3586 1872 1930 2154 2234 2508 2810 2871 3653 4126
+ + + + + + + + +
+ + + + + + +
+: Compound detected. : Compound not detected. a RR = relative retention compared to cholesterol (internal standard).
Fig. 2. Number of colony-forming units per milliliter (CFU/mL) of indicated strains treated with A. maritima crude extracts either in the presence (L+) or absence (L) of irradiation with a laser diode. Statistics: , – , Tukey test, p < 0.05.
tion of compounds present in materials from a variety of natural product sources. The specific application of LC-ESI–MS natural product mixture analysis is a procedure known as dereplication, and this process is rapid, precise, and efficient [19]. In order to verify the correct identification of the flavonoids present in the etha-
nol extract of A. maritima, standard flavonoids previously isolated in our laboratory were used. The following flavonoids were identified in the ethanol extract by ESI–MS analysis: quercetin – ESI–MS: m/z = 301 [MH], 3-methoxy quercetin – ESI–MS: m/z = 315 [MH], vitexin/isovitexin – ESI–MS: m/z = 431 [MH], 200 -O-a L-rhamnopyranosyl-vitexin – ESI–MS: m/z = 577 [MH] , acacetin 8-C-[a-L-rhamnopyranosyl-(1 ? 2)-b-D-glucopyranoside] – ESI– MS: m/z = 609 [MH], 200 -O-b-D-glucopyranosyl-vitexin – ESI– MS: m/z = 593 [MH], rutin – ESI–MS: m/z = 301 [MH], isorhamnetin, 3-O-a-L-rhamnosyl-(1 ? 6)-b-D-galactopyranoside/isorhamnetin, and 3-O-a-L-rhamnosyl-(1 ? 6)-b-L-glucopyranoside – ESI–MS: m/z = 623 [MH]. 4. Discussion The prevalence of Candida species in systemic infections, together with the increase in resistance to traditional antifungal
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
drugs is troubling [20]. The incidence of infections by pathogenic fungi is increasing due to more common occurrence of hospital infections in immunodepressed patients. Currently, many antimicrobials, and in particular antifungal drugs, have shown limited use for a number of reasons, such as low potency, poor solubility, microbial resistance, and drug toxicity [11,20]. In the present study, the application of one session of PDT to cultures of C. dubliniensis using plant extracts as photosensitizing agents showed satisfactory results. Consequently, the use of PDT employing the hexane and ethanol extracts of A. maritima as photosensitizers constitutes a potential alternative for the treatment of superficial infections, particularly those caused by C. dubliniensis. However, these studies are preliminary, and more studies with these extracts are needed, including PDT tests with other microorganisms. Data from de Souza et al. [16] indicate that PDT with an energy dose of 28 J/cm2 at 685 nm in the presence of methylene blue (0.1 mg/mL) as a photosensitizer reduced the number of CFU/mL of the yeast strains Candida krusei, C. albicans, C. dubliniensis, and Candida tropicalis by 88.6%, 84.8%, 91.6%, and 82.3%, respectively. Our results, using a similar experimental protocol for PDT and the same energy dosage of 28 J/cm2 at 685 nm in the presence of hexane and ethanol extracts of A. maritima (25 mg/mL) as photosensitizers, demonstrate that the number of CFU/mL of two distinct strains of C. dubliniensis was also significantly reduced (Fig. 2). With regard to the photodynamic antimicrobial activity of A. maritima extracts, there is a need to identify and isolate the active photosensitizing agents present in these extracts. It is important to consider the possibility that, in a synergistic way, more than one compound may be involved in the observed biological activity, as the extract is a complex mixture of compounds. In order to verify the chemical composition of the crude hexane and ethanol extracts from A. maritima, chromatographic (HRGC) and spectroscopic (ESI– MS) analyses were carried out. Steroids, triterpenes, and polyphenolic compounds such as flavonoids were detected, and these results are in agreement with the literature [11]. Salvador et al. [11], in a phytochemical study of antimicrobial and antiprotozoal activities, documented the presence of flavonoids (particularly aglycone and O- and C-glycosides) as the main constituents of the ethanol extract of A. maritima (aerial parts), together with the presence of saponins and steroids. In the hexane extract, fatty acids, steroids, and triterpenoids were identified. Moreover, in preliminary cytotoxicity evaluation, the ethanol extract of A. maritima and isolated flavonoids were not toxic to opsonized, zymosanstimulated human neutrophils [9]. No study for the evaluation of photosensitizing agents present in extracts of A. maritima has previously been reported. However, further studies are needed to verify if these identified secondary metabolites are photoactive and are contributing to the photosensitizing effect in PACT seen for the crude extracts of A. maritima. On the other hand, it is important to emphasize that PDT does not substitute for conventional antimicrobial therapy. However, it does represent one more alternative for topical therapy that is more selective than systemic therapy, which could reduce unwanted side effects of the administration of systemic drugs. It is an option for rapid local treatment, with the advantages of low cost and low or non-existent overdose risk [12]. Microbial resistance can be avoided by the use of PDT, and the appearance of fungal resistance to the treatment is improbable, since microbial inactivation is mediated by the oxygen singlet [12,16]. Moreover, PDT does not present drug interactions, especially as it is used in local therapy [21–23]. These advantages encourage further detailed in vitro and in vivo studies with natural products for possible application as natural photosensitizers in antimicrobial PDT.
19
5. Conclusions Our data demonstrate that the photoactivation of hexane and ethanol extracts of A. maritima aerial parts by laser irradiation at 685 nm produce an antimicrobial effect against C. dubliniensis strains and that the extracts of A. maritima are promising as natural photosensitizers in photodynamic therapy. Despite the positive results, further investigations are necessary to confirm the potential of these natural products for application as photosensitizers in photodynamic antimicrobial chemotherapy. Acknowledgments The authors are grateful to FAPESP, CAPES, CNPq and FAEPEXUNICAMP for financial support; to Professor Josafá Carlos de Siqueira for the identification of plant material and to Guilherme R. Teodoro for technical assistance in the antimicrobial experiments. References [1] D. Sullivan, D. Coleman, Candida dubliniensis: characteristics and identification, J. Clin. Microbiol. 36 (1998) 329–334. [2] M.E. Brandt, L.H. Harrison, M. Pass, A.N. Sofair, S. Huie, R.K. Li, C.J. Morrison, D.W. Warnock, R.A. Hajjeh, Candida dubliniensis fungemia: the first four cases in North America, Emerg. Infect. Dis. 6 (2000) 46–49. [3] M.C. Dignani, J.S. Solomkin, E.J. Anaissie, Candida, in: E.J. Anaissie, M.R. McGinnis, M.A. Pfaller (Eds.), Clinical Mycology, Churchill Livingstone, New York, 2003. [4] V. Carre, O. Gaud, I. Sylvain, O. Bourdon, M. Spiro, J. Biais, R. Granet, P. Krausz, M. Guilloton, Fungicidal properties of meso-arylglycosylporphyrins: influence of sugar substituents on photoinduced damage in the yeast Saccharomyces cerevisiae, J. Photochem. Photobiol. B 48 (1999) 57–62. [5] L.M. Giroldo, M.P. Felipe, M.A. de Oliveira, E. Munin, L.P. Alves, M.S. Costa, Photodynamic antimicrobial chemotherapy (PACT) with methylene blue increases membrane permeability in Candida albicans, Lasers Med. Sci. 24 (2009) 109–112. [6] E. Munin, L.M. Giroldo, L.P. Alves, M.S. Costa, Study of germ tube formation by Candida albicans after photodynamic antimicrobial chemotherapy (PACT), J. Photochem. Photobiol. B 88 (2007) 16–20. [7] G.A. Cordell, Biodiversity and drug discovery – a symbiotic relationship, Phytochemistry 55 (2000) 463–480. [8] M.J. Salvador, E.O. Ferreira, S.U. Mertens-Talcott, W.V. Castro, V. Butterweck, H. Derendorf, D.A. Dias, Isolation and HPLC quantitative analysis of antioxidant flavonoids from Alternanthera tenella Colla, Z. Naturforschung 61c (2006) 19– 25. [9] J.G. Souza, R.R. Tomei, A. Kanashiro, L.M. Kabeya, A.E.C.S. Azzolini, D.A. Dias, M.J. Salvador, Y.M. Lucisano-Valim, Ethanolic crude extract and flavonoids isolated from Alternanthera maritima: neutrophil chemiluminescence inhibition and free radical scavenging activity, Z. Naturforschung 62c (2007) 339–347. [10] M.J. Salvador, D.A. Dias, Flavone C-glycosides from Alternanthera maritima (Mart.) St. Hil. (Amaranthaceae), Bio-chem. Syst. Ecol. 32 (2004) 107–110. [11] M.J. Salvador, O.L.A.D. Zucchi, R.C. Candido, I.Y. Ito, D.A. Dias, Alternanthera maritima (Amaranthaceae), Pharm. Biol. 42 (2004) 138–148. [12] M. Wainwright, Photodynamic antimicrobial chemotherapy, J. Antimicrob. Chemother. 42 (1998) 13–28. [13] R.C. Souza, J.C. Junqueira, R.D. Rossoni, C.A. Pereira, E. Munin, A.O. Jorge, Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue malachite green and low-power laser irradiation alone against Candida albicans, Lasers Med. Sci. (2009), doi:10.1007/s10103-009-0706-z. [14] K.A. Salva, Photodynamic therapy: unapproved uses, dosages or indications, Clin. Dermatol. 20 (2002) 571–581. [15] J.R. Perussi, Inativação fotodinâmica de microorganismos, Quím. Nova 30 (2007) 1–7. [16] S.C. Souza, J.C. Junqueira, I. Balducci, C.Y. Koga-Ito, E. Munin, A.O.C. Jorge, Photosensitization of different Candida species by low power laser light, J. Photochem. Photobiol. B. 83 (2006) 34–38. [17] M.E. Stefanello, A.C. Cervi, I.Y. Ito, M.J. Salvador, A. Wisniewski Jr., E.L. Simionatto, Chemical composition and antimicrobial activity of essential oils of Eugenia chlorophylla (Myrtaceae), J. Essent. Oil Res. 20 (2008) 75–78. [18] National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi. Proposed Standard M38-P. National Committee for Clinical Laboratory Standard, Wayne, 1998, 28p. [19] M.A. Strege, High-performance liquid chromatography-electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening, J. Chromatogr. B 725 (1999) 67–78. [20] J.M. Bliss, C.E. Bigelow, T.H. Foster, C.G. Haidaris, Susceptibility of Candida species to photodynamic effects of photofrin, Antimicrob. Agents Chemother. 48 (2004) 2000–2006.
20
A. Gasparetto et al. / Journal of Photochemistry and Photobiology B: Biology 99 (2010) 15–20
[21] M.L. Embleton, S.P. Nair, B.D. Cookson, M. Wilson, Selective lethal photosensitization of methicillin-resistant Staphylococcus aureus using an IgG-tin (IV) chlorin e6 conjugate, J. Antimicrob. Chemother. 50 (2002) 857– 864. [22] S. Karrer, R.M. Szeimies, S. Ernst, C. Abels, W. Baumler, M. Landthaler, Photodynamic inactivation of Staphylococci with 5-aminolaevulinic acid or photofrin, Lasers Med. Sci. 14 (1999) 54–61.
[23] M.C. Teichert, J.W. Jones, M.N. Usacheva, M.A. Biel, Treatment of oral candidiasis with methylene blue-mediated photodynamic therapy in an immunodeficient murine model, Oral Surg., Oral Med., Oral Pathol., Oral Radiol. Endodont. 93 (2002) 155–160.