Mycopathologia (2011) 172:117–124 DOI 10.1007/s11046-011-9404-z
Evaluation of Antifungal Activity of Medicinal Plant Extracts Against Oral Candida albicans and Proteinases Jose´ Francisco Ho¨fling • Rita Ca´ssia Mardegan • Paula Cristina Anibal • Vivian Fernandes Furletti Mary Ann Foglio
•
Received: 24 September 2009 / Accepted: 16 February 2011 / Published online: 16 March 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Proteinases produced by Candida albicans are one kind of virulence factor expressed that contribute to adherence and invasion of host tissue. Proteinase inhibitor of human immunodeficiency virus in experimental candidiasis suggested reduction in fungal infection, and medicinal plants could be a source of alternative agent to prevent diseases. In this study, we investigated the production of proteinases by C. albicans from clinical isolates and the action of plant extracts against strains of C. albicans and its synthesized proteinases, comparing with antifungal fluconazole and amphotericin B and proteinase inhibitors pepstatin A, amprenavir, and ritonavir. The results reported here showed that these extracts have a certain kind of action and that the search for new antifungal agents could be found at the plants.
J. F. Ho¨fling (&) Departamento de Diagno´stico Oral, Faculdade de Odontologia de Piracicaba, FOP/UNICAMP Universidade Estadual de Campinas, Caixa Postal 52, Piracicaba, SP 13414-903, Brazil e-mail:
[email protected] R. C. Mardegan P. C. Anibal V. F. Furletti Departamento de Diagno´stico Oral pela Faculdade de Odontologia de Piracicaba, FOP/UNICAMP, Piracicaba, SP 13414-903, Brazil M. A. Foglio Coordenadora da Divisa˜o de Fitoquı´mica do, CPQBA/ UNICAMP, CP 6171, Campinas, SP 13081-970, Brazil
Keywords Antifungal Candida Medicinal plants Protease inhibitors Proteinases
Introduction The human oral cavity has been regarded as a unique environment that offers a variety of ecologic niches to the microbial colonization. Fungi of the genus Candida cohorts different epithelial surface, including the oral mucosa, being part of the resident microbiota [28]. Although Candida albicans is the major medically important species and etiologic agent of many fungal infections occurring in oral cavity, other species such as C. tropicalis, C. parapsilosis, C. krusei, C kefyr, C. glabrata, C. guilliermondii, C. lusitaneae, and C. dubliniensis have been isolated [11, 43]. The mechanisms determining the pathogenicity of genera Candida involve intrinsic factors of the species, such as adherence to tissue, dimorphism, cellular wall composition, and the production of toxins and proteolytic enzymes [18, 27, 31]. Proteinases produced by C. albicans may be secreted or presented as aspartyl proteinase associated with cellular membrane, predominantly expressed in most of the pathogenic species of the genera Candida [39], degrading epithelial surfaces and digesting host proteins to promote a nitrogen source to the cell, exerting an important role from the initial stage to the fungal infections, contributing to the adhesion and invasion of the host tissue [6, 22, 38].
123
118
The search for new drugs, with antifungal activity, has been a constant preoccupation of the researchers, toward pathological studies caused by these microorganisms. However, no conventional treatments have revealed good results at the antifungal therapies. Studies in vitro suggest that protease inhibitors of human immunodeficiency virus (HIV) may show inhibitory properties in relation to C. albicans factors [7]. Studies with animals revealed the action of the protease inhibitors saquinavir and amprenavir in reducing the virulence of these microorganisms in experimental candidiasis [26, 36], suggesting reduction in the fungal infection incidence in HIV-infected patients. The antifungal potential of the protease inhibitor of HIV belongs to the same class as Candida spp. aspartate protease, which is an important virulence factor of the fungi [21, 26]. On the other hand, and with the same purposes, empiric knowledge about plants with medicinal properties has been accumulated through centuries, always following human evolution through the time [16]. Medicinal plants are frequently used to treat many diseases and recently have been intensively studied as an alternative agent to prevent diseases such as oral pathogenies [8, 32]. Approximately 50% of new chemical molecules from natural products found between 2000 and 2006 have shown their importance in the development of drugs for the treatment of infectious diseases [29]. Studies on some plants revealed their antimicrobial activity. Among them, we found Casearia sylvestris Sw [2, 3], Rosmarinus officinalis L [14, 19, 25, 40, 42], Mentha piperita L [15, 24], Tabebuia avellanedae (Panizza 1997), Arrabidaea chica [33, 46], and Arctium lappa [10, 35]. The aim of this study was to evaluate the antifungal activity of these plant extracts against strains of Candida spp. and their action on the proteolytic activity of proteinases produced by clinical oral C. albicans.
Materials and Methods Selected Plants Selected plants were indicated by Prof. Dr. Valter Radame´s Accorsi and collected from the experimental
123
Mycopathologia (2011) 172:117–124
field of the Research Center for Chemistry, Biology and Agriculture, State University of Campinas (CPQBA/UNICAMP), Brazil. Voucher specimens were deposited at the State University of Campinas Herbarium (UEC) and were identified by Prof. Dr. Jorge Yoshio Tamashiro (Table 1). Preparation of Extracts Each selected plant (5 g) was extracted with 600 ml of dichloromethane (Labsynth PA) with a Dispersive Extratur (QuimisÒ Q-252-28 model) and was thereafter filtered. The vegetal residue was re-extracted with methanol (Labsynth PA). Solvents were evaporated under reduced pressure and dried using a rotary evaporator (BuchiÒ R-200 model) [20]. Crude extracts were monitored by chromatography in thin layer in silicagel chromatoplaques (60 F254 Merck 1.05554), eluting in systems of solvents using gradients of dichloromethane and methanol, according to the crude extract polarity. The dried vegetal crude extracts were dissolved in 5% DMSO in distilled sterile water, filtered through a 0.22-lm membrane filter (TPP), and stored at 4°C until further use. Microorganism Strains The test organisms used were C. albicans CBS-562, from the Microbiology and Immunology Laboratory at FOP/UNICAMP. A total of 200 C. albicans isolated were obtained from oral cavity of 20 volunteers from the Faculty Clinic, 100 isolates were from 10 healthy children aged 24–36 months (Ethic Committee 021/2002), and 100 isolates from 10 adults with periodontal disease who aged 30–60 years (Ethic Committee 008/2003). The samples were identified through the chromogenic media
Table 1 Identification, plant parts, and voucher specimen of the studied plants Medicinal plant
Plant parts Voucher
Arctium lappa (Hill) Bernh.
Leaves
1,265
Mentha piperita L.
Leaves
1,253
Rosmarinus officinalis L.
Leaves
1,264
Arrabidaea chica (Bonpl.) B. Verl.
Leaves
1,254
Tabebuia avellanedae Lor. Ex Griseb. Bark
1,256
Casearia sylvestris Sw.
1,257
Leaves
Mycopathologia (2011) 172:117–124
CHROMagar-Candida (BD, Paris, France) followed by morphological and biochemical tests of fermentation and carbohydrate assimilation. Proteinase Test Proteinase-producing strains were selected according to Price et al. [37], with some modifications. Clinical isolates were inoculated in tubes containing 5 ml of YPD broth (Merck, German) media and incubated for 18 h at 37°C. After that, aliquots of 1.5 ml from the culture were transferred to Eppendorf and centrifuged at 3,000 rpm for 5 min at 4°C. The cellular pellets were resuspended in saline solution (NaCl 0.9%) and centrifuged twice in the same conditions to remove the rest of the media. Concentration of the strain suspension was standardized using MacFarland 0.5 (1 9 106 UFC/ml), and 1 ll of the suspension was inoculated in equidistant points on the proteinase agar, measuring the enzymatic activity according to Price et al. [37]. Proteolytic Activity of Candida albicans Proteinase A total of 20 isolates (10 from children and 10 from adults) showing high indices of proteinase production were selected. Isolates were cultured for 48 h at 37°C in Sabouraud/glucose (Merck, German). C. albicans proteinases were inducted according to Korting et al. [26], with some modifications. Aliquots of 100 ml of inductor proteinase (BSA/YNB—Merck, German) broth were incubated for 7 days at 27°C in a shaker at 150 rpm. After the period of incubation, UFC/ml was determined and the yeasts cells were removed with centrifugation at 1,500g for 30 min at 4°C. The supernatant was adjusted to a pH 6.5 and freezed to -20°C, after sterilization by filtration. Then, 0.5 ml of the proteinase extract was added to 0.5 ml of 2% BSA (buffer HCl/sodium citrate 0.2 M) and to 0.5 ml of each vegetal extract at different concentrations (2.5, 1.5, 0.5 mg/ml). Tests with protease inhibitor drugs such as Amprenavir (APV— AgeneraseÒ/Glaxo Smith Kline), Ritonavir (RTV— NorvirÒ/Abbott), and Pepstatin A (Sigma) were conducted at the same concentrations. Experimental controls were vegetal extracts, protease inhibitor drugs (diluents DMSO 5% in distilled sterile water), and the substances used as diluents of the protease inhibitor drugs (15% DMSO in distilled sterile water).
119
For the tests, the material was incubated for 60 min at 37°C. After the incubation, the reactions were interrupted with 0.5 ml of trichloroacetic acid (TCA—Merck, German) and kept in ice. For each reagent mixture, an additional control was prepared, adding 20% TCA in distilled sterile water before the incubation, which was determined by the initial time (T0) to obtain proteolytic activity indices. After the addition of TCA, all specimens were centrifuged at 3,000 rpm for 30 min at 4°C. Next, aliquots of 160 ll of each sample plus 40 ll of Coomassie Brilliant Blue (G-250, Bio-Rad) were added to microplates. Peptides produced because of proteolytic activity were not precipitated by TCA and bound to dye. The quantity of peptides in the supernatant was measured in automatic microplate reader at 595 nm of absorbance and related to proteolytic activity. To calculate the proteolytic activity, the following formula was used: absorbance value of sample measured at the final time, after 60 min, less the absorbance value in T0. The activity was calculated from the variation (increase) of 0.1 unit for 60 min at 595 nm.
Results To select the strains of C. albicans with high proteolytic activity, the proteinase test was performed with 100 samples from the buccal cavity of healthy children and 100 samples from the buccal cavity of adults. To quantify the proteinase production, the following indices were used: 0 for no enzymatic activity, 1 for positive enzymatic activity, and 2 for strains with high positive enzymatic activity. After the preliminary tests, 50 strains of C. albicans with high enzymatic activity (Indices 2) from buccal cavity of healthy children and 50 samples from the buccal cavity of adults with periodontal disease were chosen for the tests with vegetal material (Table 2). Besides these clinical isolates, strain C. albicans CBS-562 was used. These clinical samples were tested against fluconazole and amphotericin B antifungal susceptibility. Most of the isolates from children and adults showed sensibility with 75 and 69% of inhibition, respectively, and were classified as susceptibility-dependent dose in 21% of children and 18% of adult samples.
123
120
Mycopathologia (2011) 172:117–124
Table 2 Antifungal activity of the vegetal extracts against clinical isolates of C. albicans (minimum inhibitory concentration—MIC lg/ml) MIC in clinical isolates of children (n = 50) and adults (n = 50) Plants
Extract
Children (%) MIC—lg/ml
Adults (%) MIC—lg/ml
B500
500–1,500
C1,500
B500
500–1,500
C1,500
DC
*
18
82
*
*
100
MT
*
*
100
*
*
100
DC
*
68
32
*
62
38
MT
*
*
100
*
*
100
T. avellanedae Lor. Ex Griseb.
DC
*
12
88
*
2
98
A. chica (Bonpl.) B. Verl.
MT DC
* *
* *
100 100
* *
* *
100 100
MT
4
96
*
*
96
4
DC
*
38
62
*
90
10
MT
*
*
100
*
*
100
DC
*
92
8
*
4
96
MT
*
*
100
*
*
100
R. officinalis L. M. piperita L.
A. lappa (Hill) Bernh. C. sylvestris Sw.
DC Dichloromethane Extract, MT Methanol Extract * MIC values C 2,500 lg/ml
Fluconazol resistance was seen in 4% of children samples and 13% of adults samples, respectively. Susceptibility to amphotericin B is observed in the isolates of 92% of children and 89% of adult samples. Resistance was observed in 8% of healthy children and 11% of adults with periodontal disease. Among samples that were resistant to fluconazole and amphotericin B, all showed MIC higher than 2,250 lg/ml, representing a weak inhibition in relation to dichloromethane and methanol extracts tested. The best inhibitory concentration of proteolytic activity for all tested extracts was 2.5 mg/ml, as it can be observed in Table 3 and Fig. 1. The extracts that presented better inhibition in relation to proteolytic activity of C. albicans proteinase were A. chica (dichloromethane extract), M. piperita (methanol extract), M. piperita (dichloromethane extract), C. sylvestris (methanol extract), C. sylvestris (dichloromethane extract), T. avellanedae (dichloromethane extract), R. officinalis (dichloromethane extract) and later R. officinalis (methanol extract), in the sequence T. avellanedae (methanol extract), A. chica (methanol extract), and finally A. lappa (dichloromethane and methanol extracts) that revealed weak inhibitory activity against secreted aspartyl protease (SAP). One of the control drugs used in this experiment was the protease inhibitory drug, pepstatin A, with
123
inhibitory activity of the proteinases in a concentration of 100 lg/ml, which inhibited the proteolytic activity of the strains in children and adults in 92% and 90%, respectively (medium), and the strain CBS562 in 94%. The other tested concentrations, 25 and 50 lg/ml, also revealed inhibitory activity but with lower indices when compared to the concentration of 100 lg/ml (Fig. 2). Other drugs known as proteinase inhibitors were analyzed. Amprenavir at 100 lg/ml inhibited proteolytic activity in 86, 90, and 88% of proteinases from children, adults, and CBS-562, respectively. Proteinases from children and adults were inhibited in 79 and 81%, respectively, and CBS-562 in 79% with ritonavir drug. Inhibition at concentrations of 25 and 50 lg/ml was observed but in low amount in relation to the major concentration (100 lg/ml).
Discussion Proteinase production by the strain of C. albicans may vary, depending on the invasion of the strain and colonization of host. Proteolytic activity of C. albicans strains from two groups of volunteers, of healthy children (n = 100) and adults with periodontal disease (n = 100), showed that the majority of C.
Mycopathologia (2011) 172:117–124
121
Table 3 Activity of vegetal extracts to inhibit proteins produced by clinical isolates of C. albicans and strain CBS-562 Proteinase inhibition of C. albicans (%) of vegetal extracts Plants
Extract Children (n = 10) MIC—mg/ml Adults (n = 10) MIC—mg/ml CBS-562 MIC—mg/ml 0.5
1.5
2.5
0.5
1.5
2.5
0.5
1.5
DC
5
8
15
5
7
15
7
11
13
MT
6
7
13
7
8
13
7
9
13
DC
8
20
28
9
19
29
9
18
27
MT
10
18
32
10
18
31
9
18
32
T. avellanedae Lor. Ex Griseb. DC
6
11
18
6
10
19
5
10
18
R. officinalis L. M. piperita L.
A. chica (Bonpl.) B. Verl. A. lappa (Hill) Bernh. C. sylvestris Sw.
2.5
MT
4
6
13
4
6
12
5
7
14
DC
18
21
36
16
20
33
19
22
43
MT
6
8
12
6
8
9
7
10
13
DC
4
7
8
4
7
8
5
7
8
MT
4
6
8
4
6
7
5
7
8
DC MT
8 9
16 17
19 21
8 8
17 17
19 21
7 13
17 17
20 20
DC dichloromethane extract, MT methanol extract Fig. 1 Inhibitory activity (%) of C. albicans proteinases from buccal cavity of healthy children (n = 10) and samples from the buccal cavity of adults with periodontal disease (n = 10) by extracts (dichloromethane and methanol) of all plants analyzed. DC dichloromethane extract, MT methanol extract
Fig. 2 Inhibitory activity (%) of C. albicans protease from clinical samples of buccal cavity of children, adults with periodontal disease, and strain CBS-562 obtained with the proteinase inhibitors of pepstatin A, amprenavir, and ritonavir. Concentrations in lg/ml
123
122
albicans are active producers of proteinases, as 94 and 95% of healthily children (n = 100) and adults with periodontal disease, respectively, were positive in relation to the production of this enzyme. These results are similar to those obtained by Penha and Bezerra [34], who analyzed C. albicans samples of oral cavity and observed that 100% of them were proteinase producers. Different patterns of proteinase production by strains of C. albicans from adults belong to two groups of caries-active and caries-free showed that 89.4 and 98% of the strains have positive enzymatic activity, respectively [27]; these results indicate that proteinases are relevant virulence factors in some types of infections caused by C. albicans, and the inhibition of this enzyme reveals a protector effect to the host [13, 22]. Studies of Cassone et al. [7] demonstrated the inhibition of aspartyl protease (SAP) secretion by indinavir, ritonavir, and pepstatin A, with a higher inhibition of pepstatin A, which is a specific SAP inhibitor. Production of SAP, in particular SAP2, is essential to the pathogenicity of the yeast Candida in experimental vaginal models infection in rats [12]. Studies of Beansejour et al. [4] showed that SAP production may be essential for inflammatory answer and oral infection caused by Candida. In general, the ability to secret one or more of this kind of enzyme may be the key to the virulence factor in experimental mucosal infections caused by Candida. Antifungal drugs that inhibit systemic infections in use do not completely satisfy the medical necessity because of problems such as spectrum, potency, security, and pharmacokinetic properties of the available agents. Considering the increase in the incidence of systemic fungal infection and the consequent increase in the mortality, an appropriate choice of antifungal therapy is necessary, besides an effective prophylaxis and the development of drugs that increase the response to the immunocompromised organisms. Nowadays, there is an increased interest in searching for new antifungal compounds that function as selective and low toxic. Many substances with antifungal effect are known to be a conventional medical treatment. The widespread use of antifungal agents has been followed by an increase in antifungal resistance and by a noticeable shift toward non-albicans species with relative resistance to fluconazole and itraconazole [41]. In the last decades, however, it has been demonstrated an
123
Mycopathologia (2011) 172:117–124
increase in the interest in alternative medicine and in natural therapies. These substances were used to treat diverse pathologies in the last decades. In the last years, it has been considerably amplified [10]. Studies with proteinase inhibitors are being the purpose of new therapies in the control of candidiasis [21], including the use of medicinal plants. Nowadays, no conventional treatments have revealed good results in antifungal therapies. Studies in vitro suggest that HIV protease inhibitors may show inhibitory properties in relation to the virulence factors of C. albicans [7]. Studies with protease inhibitors also demonstrated the potential of this drug to inhibit the adhesion of this yeast to epithelial cells [5]. Major compounds that could produce inhibitory effects on strains of Candida may be tannins (A. chica and R. officinalis), anthocyanins (A. chica), flavonoids (A. chica and R. officinalis), naphthoquinones (T. avellanedae), menthol and menthone (M. piperita), diterpenoid casearvestrin (C. sylvestris), and terpenoid b-eudesmol (A. lappa), which are known to have antimicrobial properties against microorganisms [1, 9, 17, 23, 24, 30, 44, 45]. Vegetal extracts of Arrabidaea chica (methanol) and Arctium lappa and Casearia sylvestris (dichloromethane), at the concentrations between 500 and 1,500 lg/ml, showed activity against clinical isolates of C. albicans, while other extracts were active at concentration above 1,500 lg/ml (Table 2). Inhibition of proteolytic activity of C. albicans proteinases by A. chica dichloromethane extract, Mentha piperita and Casearia sylvestris, and Mentha piperita and Casearia sylvestris methanol extracts (at concentration 1,500 lg/ml) revealed effective activity in this concentration, even though in other concentrations it has been lower. These results, when compared with those obtained by protease inhibitors pepstatin A, amprenavir, and ritonavir, were not similar to those obtained with vegetal extracts. However, A. chica (methanol extract) and A. lappa, M. piperita, and C. sylvestris (dichloromethane extracts) showed important antifungal activity in vitro against a variety of species of Candida, suggesting to be a promissory antifungal agents. Utilization of proteinase inhibitor agents of Candida spp. from vegetal extracts may represent, in a near future, an important strategy to control and prevent infections, as candidiasis in mucosa, frequently observed in immunocompromised patients.
Mycopathologia (2011) 172:117–124
So the search for new antifungal agents to an alternative therapy—to the elaboration of new pharmaceuticals—must be part of continuous searches with the objective to identify possible crude vegetal extracts, essential oils, and bioactive compounds with inhibitory activity against Candida and synthesized proteinases by these microorganisms. Acknowledgments This study was supported by the Research Foundation for the State of Sa˜o Paulo (Fundac¸a˜o de Apoio a` Pesquisa do Estado de Sa˜o Paulo—FAPESP) and CAPES.
References 1. Alcerito T, Barbo FE, Negri G, Santos DYAC, Meda CI, Young MCM, Cha´vez D, Blatt CTT. Folicular epicuticular wax of Arrabidaea brachypoda: flavonoids and antifungal activity. Biochem Syst Ecol. 2002;30:677–83. 2. Almeida-Alves TM, Ribeiro FL, Kloss H, Zani CL. Polygodial, the fungitoxic component from the Brazilian medicinal plant Polygonum punctatum. Mem Inst Oswaldo Cruz. 2001;96(6):831–3. 3. Arantes A, Carvalho-Eda S, Medeiros EA, Farhat CK, Mantese OC. Pedriatic risk of mortality and hospital infection. Infect Control Hosp Epidemiol. 2004;25(9):783–5. 4. Beansejour A, Grenier D, Goulet JP, Deslaurirs N. Proteolitic activation of the interleukin 1b precursor by Candida albicans. Infect Immun. 1998;66:676–81. 5. Bektic´ J, Lell CP, Fuchs A, Stoiber H, Speth C, Lass-Flo¨rl C, Zepelin MB, Dierich MP, Wu¨rzner R. HIV protease inhibitors attenuate adherence of Candida albicans to epithelial cells in vitro. FEMS Immnuol Med Microbiol. 2001;31(1):65–71. 6. Borg M, Ruchel R. Expression of extracellular acid proteinase by proteolitic Candida spp during experimental infection of oral mucosa. Infect Immun. 1998;56(3):626–31. 7. Cassone A, De Bernardis F, Torosantucci A, Tacconelli E, Tumbarello M, Cauda R. In vitro and in vivo anticandidal activity of human immunodeficiency virus protease inhibitors. J Infect Dis. 1999;180:448–53. 8. Chandrasekaran M, Venkatesalu V. Antibacterial and antifungal activity of Syzygium jambolonum seeds. J Ethopharmacol. 2004;91:105–8. 9. Cordeiro CHG, Sacramento LVS, Correˆa MA, Pizzolitto AC, Bauab TM. Ana´lise farmacogno´stica e atividade antibacteriana de extratos vegetais empregados em formulac¸a˜o para a higiene bucal. Rev Bras Cieˆncias Farmaceˆuticas. 2006;42(3):395–404. 10. Correa AF, Andrade LR, Soares MJ. Elemental composition of acidocalcisomes of Trypanosoma cruzi bloodstream trypomastigote forms. Parasitol Res. 2002;88(10):875–80. 11. Costa CR, Lemos JA, Passos XS, Arau´jo CR, Cohen AJ, Souza LKH, Silva MRR. Species distribution and antifungal susceptibility profile of oral Candida isolates from HIV-infected patients in the antiretroviral therapy era. Mycopathologia. 2006;162:45–50.
123 12. De Bernardis F, Arancia S, Morelli L, et al. Evidence that members of the secretory aspartyl proteinases gene family (SAP), in particular SAP2, are virulence factors for Candida vaginitis. J Infect Dis. 1999;179:201–8. 13. De Bernardis F. Aspartyl proteinases of Candida albicans and their role in pathogenicity. Med Mycol. 2001;39:303–13. 14. Djilas SM, Markov SL, Cetkovic DD, Canadanovic-Brunet JM, Cetkovic GS, Tumbas VT. Antimicrobial and free radical scavenging activities of Teucrium montanum. Fitoterapia. 2006;13(2):114–9. 15. Duarte MC, Figueira GM, Sartoratto A, Rehder VL, Delarmelina C. Anti-Candida activity of Brazilian medicinal plants. J Ethnopharmacol. 2005;97(2):305–11. 16. Ferro D. Fitoterapia: conceitos clı´nicos. Sa˜o Paulo: Ateneu; 2006. 17. Gershon H. Fungitoxicity of 1, 4-naphtoquinones to Candida albicans. Can J Microbiol. 1975;21:1317–21. 18. Ghannoum MA, Abbu-Elteen KH. Pathogenicity determinants of Candida. Mycoses. 1990;33(6):265–82. 19. Gulluce M, Sokmen M, Daferera D, Agar G, Ozkan H, Kartal N, et al. In vitro antibacterial, antifungal and antioxidant activities of the essential oil and methanol extracts of herbal parts and callus culture of Satureja hortensis L. J Agric Food Chem. 2003;51(14):3958–65. 20. Ha¨rma¨la P, Vuorela H, Hiltunen R, Nyiredy Sz, Sticher O, To¨rnquist K et al. Strategy for the isolation and identification of coumarins with calcium antagonistic properties from the roots of Angelica archangelica. Phytochem Anal. 1992; 3:42. 21. Hoegl L, Korting HC, Klebe G. Inhibitors of asrpartic proteases in human diseases: molecular modeling comes of age. Pharmazie. 1999;54(5):319–29. 22. Hube B, Naglik J. Candida albicans proteinases: resolving the mystery of a gene family. Microbiology. 2001;147(8):1997–2005. 23. Hussein SAM, Barakat HH, Merfort I, Nawwar MAM. Tannins from the leaves of Punica granatum. Phytochemistry. 1997;45:819–23. 24. Iscan G, Kirimer N, Ku¨rkcu¨oglu M, Baser KHC, Demirci F. Antimicrobial screening of Mentha piperita essencial oils. J Agric Food Chem. 2002;50:3943–6. 25. Kariott A, Vrahimi-Hadjilouca T, Droushiotis D, Rancic a, Hadjipavlou-Litina D, Skaltsa H. Analysis of the essential oil of Origanum dubium growing Wild in Cyprus. Investigation of its antioxidant capacity and antimicrobial activity. Planta Med. 2006; 72(14):1330–4. 26. Korting HC, Schaller M, Eder G, Hamm G, Bohmer U, Hube B. Effects of the human immunodeficiency virus (HIV) proteinase inhibitors saquinavir and indinavir on in vitro activities of secreted aspartyl proteinases of Candida albicans isolates from HIV-infected patients. Antimicrob Agents Chemother. 1999;43(8):2038–42. 27. Mardegan RC, Klein MI, Golvea MB, Rodrigues JAO, Gonc¸alves RB, Ho¨fling JF. Biotyping and genotyping diversity among oral Candida albicans strains from caries free and caries activity healthy children. Braz J Microbiol. 2006;37:26–32. 28. McCullough MJ, Ross BC, Reade PC. Candida albicans: a review of its history, taxonomy, epidemiology, virulence attributes, and methods of strain differentiation. Int Oral Maxillofac Surg. 1996;25(2):136–44.
123
124 29. Newman DJ, Cragg GM. Natural products as source of new drugs over the last 25 years. J Nat Prod. 2007;70:461–77. 30. Oberlies NH, Burgess JP, Navarro HA, Pinos RE, Fairchild CR, Peterson RW, Soejarto DD, Farnsworth NR, Kinghorn AD, Wani MC, Wall ME. Novel bioactive clerodane diterpenoids from the Leaves and Twigs of Casearia Sylvestris. J Nat Prod. 2002;65(2):95–9. 31. Odds FC. Candida infections in AIDS patients. Int J STD AIDS. 1992;3(3):157–60. 32. Pai MR, Acharya LD, Udupa N. Evaluation of antiplaque activity of Azadirachta indica leaf extract gel–a 6-week clinical study. J Ethnopharmacol. 2004;90:99–103. 33. Pauletti PM, Castro-Gamboa I, Silva DHS, Young MCM, Tomazela DM, Eberlin MN, et al. New antioxidant C-glucosylxanthones from the stems of Arrabidaea samydoides. J Nat Prod. 2003;66:1384–7. 34. Penha CV, Bezerra LM. Concanavaliin A-binding cell wall antigens of Sporotrix schenckii: a serological study. Med Mycol. 2000;38(1):1–7. 35. Pereira AO, Cartucho DJ, Duarte AS, Gil MH, Cabrita AM, Patrı´cio JA, et al. Immobilisation of cardosin A in chitosan sponges as a novel implant for drug delivery. Curr Drug Discov Technol. 2005;2(4):231–8. 36. Pichova I, Pavlickowa L, Dostal J, Dolejsi E, HruskovaHeidingsfeldova O, Weber J, et al. Secreted aspartic proteases of Candida lusitaniae–inhibition with peptidomimetic inhibitors. Eur J Biochem. 2001;268:2669–77. 37. Price MF, Wilkinson ID, Gentry LO. Plate method for detection of phospholipase activity in Candida albicans. Sabouraudia. 1982;20(1):7–14.
123
Mycopathologia (2011) 172:117–124 38. Ray TL, Payne CD. Scanning electron microscopy of epidermal adherence and cavitation in murine candidiasis: a role for Candida acid proteinase. Infect Immun. 1988;56(8):1942–9. 39. Ray TL, Payne CD, Morrow BJ. Candida albicans acid proteinase: characterization and role in candidiasis. Adv Exper Med Biol. 1991;306:173–83. 40. Shokeen, P, Ray K, Bala M, Tandon V. Preliminary studies on activity of Ocimum sanctym, Drynaria quercifolia and Annona squamosa against Neisseria gonorrhoeae. Sex Transm Dis. 2005; 32(2):106–11. 41. Sojakova M, Liptajova D, Borovsky M, Subik J. Fluconazole and itraconazole susceptibility of vaginal yeast isolates from Slovakia. Mycopathologia. 2004;157:163–9. 42. Soylu EM, Soylu S, Kurt S. Antimicrobial activities of the essential oils of various plants against tomato late blights disease agent Phytophtora infestans. Mycopathologia. 2006;161(12):119–28. 43. Stenderup A. Oral mycology. Acta Odontol Scand. 1990; 48(1):3–10. 44. Wagner H, Kreher B, Lotter H, Hamburger MO, Cordell GA. Structure determination of new isomeric naphtha [2, 3-b] furan-4, 9-diones from Tabebuia avellanedae by the selective-INEPT technique. Helv Chim Acta. 1989;72:659–67. 45. Yayli N, Yasar A, Gu¨llec¸ C, Usla A, Kolayli S, Coskuncelebi K, Karaoglu S. Composition and antimicrobial activity of essential oils from Centaurea sessilis and Centaurea armena. Phytochemistry. 2005; 66:1741–5. 46. Zorn B, Garcia-Pin˜eres AJ, Castro V, Murillo R, Mora G, Merfort I. 3-Desoxyanthocyanidins from Arrabideae chica. Phytochemistry. 2001;56:831–5.
